Re-Imagining The Feed Industry

Proceedings of Alltech’s 20th Annual Symposium
Nutritional Biotechnology in the Feed and Food Industries

Re-imagining the Feed Industry
Natural Technologies for Food Production
C an we re-invent feeding animals? Feed companies? Ingredients and nutrient supplies? Can
we change how food animal products and crops are perceived by, and marketed to,
consumers?
The theme of the 20th Annual Alltech Symposium focuses on doing exactly that: re-defining
how we feed animals and re-imagining our agribusinesses to create the compelling force
behind an exciting future.
The basic and applied research is done. Natural feeding strategies can reduce environmental
impact, replace antibiotics, make producers more competitive and change how consumers
view and value food animal products. There is a shift in the dynamic of the industry as well.
Change is no longer something ‘happening to’ the feed industry. Change is something the
industry makes happen.
Nutritional
Biotechnology in the
Feed and Food Industries
Lyons &
Jacques Edited by T.P. Lyons
2004 and K.A. Jacques
Contents i

Contents iii
Nutritional Biotechnology in the Feed

and Food Industries
Proceedings of
Alltech’s Twentieth Annual Symposium
Edited by TP Lyons and KA Jacques

iv Contents
Nottingham University Press

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NOTTINGHAM
First published 2004

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ISBN 1-904761-27-5
Typeset by Nottingham University Press, Nottingham

Printed and bound by Bath Press, Bath, England
Contents v
Table of contents
Re-imagining the feed industry: focus on price, perception and policy 1

T. Pearse Lyons
Alltech Inc., Nicholasville, Kentucky, USA
Future of the feed/food industry: re-inventing animal feed 11

Charles V. Maxwell
Animal Science Department, University of Arkansas, Fayetteville, Arkansas, USA
Glycomics: putting carbohydrates to work for animal and human health 27

Kyle E. Newman
Venture Laboratories, Inc., Lexington, Kentucky, USA
Focus on Poultry
Selenium sources and selenoproteins in practical poultry production 35

Frank W. Edens and Kymberly M. Gowdy
Department of Poultry Science, North Carolina State University, Raleigh, North Carolina, USA
Alternatives to antibiotics in poultry production: responses, practical experience and 57

recommendations
Peter R. Ferket
Facing the realities of poultry health and performance without antibiotics in Europe 69
1 2 1
G.G. Mateos , J.M. Gonzalez-Alvarado and R. Lázaro
1
Departamento de Producción Animal, Universidad Politécnica de Madrid, Madrid, Spain
2
Departamento de Agrobiología. Universidad Autónoma de Tlaxcala, Tlaxcala, México
Reproductive responses to Sel-Plex® organic selenium in male and female broiler breeders: 81
impact on production traits and hatchability
Robert A. Renema
Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton,
Alberta, Canada
Optimizing the replacement of pronutrient antibiotics in poultry nutrition 93

Gordon D. Rosen
Pronutrient Services Ltd., Wimbledon, London, United Kingdom
Comparative aspects of Fusarium mycotoxicoses in broiler chickens, laying hens and turkeys 103
and the efficacy of a polymeric glucomannan mycotoxin adsorbent: Mycosorb®
Trevor K. Smith, Shankar R. Chowdhury and H.V.L.N. Swamy
Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada
vi Contents
Pig Science
Creating technical and educational forums that help pig producers meet performance and 113
economic goals: the Premier Pig Program™
William H. Close1 and Kim Turnley2
1
Close Consultancy, Wokingham, Berkshire, UK
2
Alltech Inc., Melbourne, Victoria, Australia
Successful feed companies in the future 121

Jim Hedges
Hubbard Feeds Inc., Mankato, Minnesota, USA
The role of selenium and Sel-Plex® in sow reproduction 131

Don Mahan
Department of Animal Sciences, The Ohio State University, Columbus, Ohio USA
Adding value to pork for producers and consumers: enhancing omega-3 DHA and selenium 141
content of meat
Paul Penny
JSR Genetics Ltd, Southburn, Driffield, United Kingdom
Reducing the environmental impact of swine production through nutritional means 149
K.J. Stalder1, W.J. Powers1, J.L. Burkett1, and J.L. Pierce2
1
Department of Animal Science, Iowa State University, Ames, Iowa, USA
2
North American Biosciences Center, Alltech, Inc., Nicholasville, Kentucky, USA
Nucleotides and young animal health: can we enhance intestinal tract development and 159
immune function?
C.D. Mateo and H.H. Stein
Department of Animal and Range Sciences, South Dakota State University, Brookings,
South Dakota, USA
Dairy and Beef Cattle
Evaluating inoculants for forage crops in Argentine beef and milk grazing systems: 171
effects on silage quality and system profitability
Leandro O. Abdelhadi
El Encuentro Livestock farm, Research & Extension in Ruminant Nutrition, Alltech Argentina, Cnel.
Brandsen, Argentina
Optigen® 1200: controlled release of non-protein nitrogen in the rumen 179

1 1 2 1
Veysel Akay , Jeff Tikofsky , Corwin Holtz and Karl A. Dawson
1
North American Biosciences Center, Alltech Inc., Nicholasville, Kentucky, USA
2
Comsen Dairy Consultation, LLC, Dryden, New York, USA
Contents vii
Dairy nutrition models: their forms and applications 187

William Chalupa, Ray Boston and Robert Munson
School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania, USA
Gastrointestinal development in dairy calves 195

S. I. Kehoe and A. J. Heinrichs
Dairy and Animal Science Department, The Pennsylvania State University, University Park,
Pennsylvania, USA
Meeting the educational needs of dairy clientele in 2020 205

Michael F. Hutjens
Department of Animal Sciences, University of Illinois, Urbana, Illinois, USA
Redefining selenium nutrition using organic selenium (Sel-Plex®): defining maximal 211
acceptable tissue residues in beef
C.J. Richards and H.D. Loveday
Animal Science Department, University of Tennessee, Knoxville, Tennessee, USA
Rumen acidosis: modeling ruminant response to yeast culture 221

D. Sauvant, S. Giger-Reverdin and P. Schmidely
INAPG Département des Sciences Animales – UMR INRA–INAPG Physiologie de la Nutrition et
Alimentation, Paris, France
The top ten most frequently-asked questions about mycotoxins, cattle and dairy food products 231
L.W. Whitlow1 and W.M. Hagler, Jr.2
1
Department of Animal Science, North Carolina State University, Raleigh, North Carolina, USA
2
Food, Nutrition and Health
All in good taste: creating natural savory flavorings from yeast 257
John Diehl
John Diehl Consulting Services, Darien, Illinois, USA
One university’s search for intelligence in a universe of foods for wellness 265
Suanne J. Klahorst
California Institute of Food and Agricultural Research, University of California, Davis, USA
How does diet influence health – could the food chain benefit from a more proactive approach 275
to clinical nutrition issues?
John C. MacRae
Rowett Research Institute, Bucksburn, Aberdeen, United Kingdom
Functional components of the cell wall of Saccharomyces cerevisiae: applications for yeast 283
glucan and mannan
Colm A. Moran
viii Contents
Food animal agriculture: a few issues that will impact our future food supply 297
Joe M. Regenstein
Cornell Kosher Food Initiative, Department of Food Science, Cornell University, Ithaca, New York, USA
Mycotoxins in the food chain: a look at their impact on immunological responses 305
Raghubir P. Sharma
Department of Physiology and Pharmacology, The University of Georgia, Athens, Georgia, USA
Antioxidant activity of hydrolyzed whey, soy, and yeast proteins 315

Youling L. Xiong,1 Lin Wang,1 E. Aida Peña-Ramos,2 and Changtzheng Wang3
1
Department of Animal Sciences, University of Kentucky, Lexington, Kentucky, USA
2
Animal Derived Food Department, CIAD, Hermosillo, Sonora, Mexico
3
Human Nutrition Program, Kentucky State University, Frankfort, Kentucky, USA
Equine Topics
A novel, knowledge-based concept for performance diagnosis and training adjustment in horses 325
Matthias Bojer
Institut für Natursport und Ökologie, Abteilung Reitsport, Deutsche Sporthochschule Köln, Germany
Physiology and feed formulation: the proper role of carbohydrates in the equine diet 331
Stephen Duren1 and Kathryn Watts2
1
Performance Horse Nutrition, LLC, Weiser, Idaho, USA
2
Rocky Mountain Research and Consulting, Center, Colorado, USA
Relevance of the NRC to today’s horse industry 337

Kevin H. Kline
Department of Animal Sciences, University of Illinois, Champaign-Urbana, Illinois, USA
Bone biomechanics: a review of the influences of exercise and nutritional management on bone 345
modeling in the growing and exercising horse
John R. Kohnke
John Kohnke Consultancy Pty Ltd, Rouse Hill, New South Wales, Australia
Of caterpillars and horses: recent scientific progress on the cause of Mare Reproductive 355
Loss Syndrome
Karen J. McDowell
Department of Veterinary Science, University of Kentucky, Lexington, Kentucky, USA
Modern Agronomics
The pre-and postharvest application potential for Crop-SetTM and ISR 2000TM on citrus 361
John P. Bower
Horticultural Science, University of KwaZulu-Natal, Pietermaritzburg, South Africa
Contents ix
Citrus fruit size and quality: response to Crop-Set™ in North America 369
Lawrence J. Marais and John G. Frank
Improcrop Inc., Nicholasville, Kentucky, USA
Integrated efficacy of several Improcrop compounds on bacterial wilt of tomato plants 375
under greenhouse conditions
Pingsheng Ji1, Tim Momol1 and Necip Tosun2
1
North Florida Research and Education Center, IFAS, University of Florida, Gainesville, FL, USA
2
Plant Protection Department, Faculty of Agriculture, Ege University, Izmir, Turkey
Alternatives against Alternaria: controlling brown spot on Murcott tangors 379

Tim Johnston1, Lawrence J. Marais2 and L.W. Timmer1
1
University of Florida, IFAS, Citrus Research and Education Center, Lake Alfred, Florida, USA
2
Seed and soil treatments with a natural fungicide product against some fungal and bacterial 383
diseases of vegetables
Necip Tosun and Huseyin Turkusay
Department of Plant Protection, Faculty of Agriculture, Ege University, Bornova–Izmir, Turkey
Abiotic stresses and plant activators 387

1 1 1 2
Ismail Türkan , Tijen Demiral , A. Hediye Sekmen and Necip Tosun
1
Department of Biology, Faculty of Science, Ege University, Izmir, Turkey
2
Department of Plant Protection, Faculty of Agriculture, Ege University, Izmir, Turkey
Advances in Aquaculture
Opportunities and dilemmas in molecular aquaculture genetics 393

Roger W. Doyle
Genetic Computation Limited, Halifax, Nova Scotia, Canada
Feeds for the future: the importance of better broodstock and larval nutrition in successful 407
aquaculture
D.F. Fegan
Alltech Inc., Bangkok, Thailand
European finfish culture: current status, recent advances and future perspectives 421
John W. Sweetman
Ecomarine Ltd, Cephalonia, Greece
Fish meal and fish oil use in aquaculture: global overview and prospects for substitution 433
Albert G.J. Tacon
SEALAB Aquaculture Laboratory, Hawaiian Institute of Marine Biology, University of Hawaii
at Manoa, USA
x Contents
Creating alternative protein sources for aquafeeds using applied enzyme technologies 449
S. L. Woodgate
PDM Group Ltd, Greenleigh, Kelmarsh Rd, Clipston, United Kingdom
From farm to fork: the challenges that fish farming faces as a responsible supplier of global food 457
Rohana P. Subasinghe
Fisheries Department, FAO, Rome
Companion Animals
USA poultry meal: quality issues and concerns in pet foods 467
Greg Aldrich
Pet Food & Ingredient Technology, Inc., Topeka Kansas, USA
The role of yeasts in companion animal nutrition 475

Kelly S. Swanson and George C. Fahey, Jr
The expanding pet food industry: where are the opportunities? 485
Tim Phillips
PETFOOD INDUSTRY Magazine, Watt Publishing Co., Mt. Morris, Illinois, USA
Using nutritional genomics to study canine obesity and diabetes 495

Kelly S. Swanson
A peek into the new NRC for dogs and cats 503
Angele Thompson
Thompson PetTech, New Providence, New Jersey, USA
The importance of antioxidant protection: demonstrating and branding benefits in pet food 509
Lucy Tucker
Alltech Inc., Stamford, Lincolnshire, United Kingdom
A changing landscape: the pet food market in Europe 517

Jürgen Zentek
Institute of Nutrition, Department of Veterinary Public Health and Food Science, Veterinary
University of Vienna, Austria
Index of topics 523

T.P. Lyons 1
Re-imagining the feed industry: focus on price, perception and policy

T. PEARSE LYONS
Alltech Inc., Nicholasville, Kentucky, USA
Global agricultural is in turmoil. World trade barriers we use feed ingredient raw materials, and the other
are coming down. The EU Common Agricultural is through improved animal health.
Policy and the US supports may become a thing of
the past, opening up markets and leveling the playing
field. BSE cases in Canada and the US have thrown Success Factor 1
these industries into a quandary, while southeast Asia Lowering production costs
is reeling from the impact of avian flu.
Natural feed technologies such as those offered by Raw materials Utilize wider availability of cereals, protein sources
Alltech have never been more in favor. Increasing New generation SSF enzymes to improve efficiency
acceptance of natural feeding strategies reflects the
realization that there is no going back to previous Improved Herd longevity: cows, sows
methods in today’s consumer-oriented markets. At a animal health More piglets weaned
recent roundtable discussion sponsored by Alltech in More high quality chicks per breeder
the aquaculture sector, one attendee described the Reduce heath costs
event as “the most exciting discussion in which I have
participated in the past 10 years”. Such is the
enthusiasm in these markets. We believe that every
cloud has a silver lining, but for agriculture the THE KOJI PROCESS OF SOLID STATE
difficult times of these past years can only be turned FERMENTATION: LOWERING THE COST OF
around if we embrace change and recognize the three CONVERTING FEED TO MEAT AND EGGS
key determinants of success: Price competitiveness,
Nature ensures utilization of its abundant feedstuffs
Perception of the consumer, and Policies we can
by placing microbes and animals in symbiosis. In
depend on to guide us now and in the future.
both ruminants and monogastrics, rumen or hindgut
microbes digest the structural carbohydrates in fiber
to release energy and ultimately to provide protein
Success Factor No. 1: from microbial cells for animal use. Without the
Price competitiveness microbe’s ability to produce enzyme arrays, the host
animal could not make much use of a vegetable-
How can other markets compete given the size of based diet.
US and Brazilian farms? How can a small country Alltech harnessed this symbiosis with a unique
maintain its position? To be successful we must adopt fermented koji, which is sold under the name of
new technologies, which is how Brazil made such VegproTM SSF. In the koji fermentation the enzyme-
remarkable strides forward in a comparatively short producing microbes are grown on substrates similar
period of time. to those that food animals consume, which induces
What are some of the new technologies that can the microbe to produce the spectrum of enzymes most
make food animal production more price- appropriate for the job. When added to poultry and
competitive? The two most important are pig diets containing oilseed meals such as soya and
technologies that improve the efficiency with which
2 Re-imagining the feed industry
canola, seven enzyme activities in VegproTM SSF lactation and 20,000 per lifetime, this means a
interact to boost release of energy and protein. replacement cost of $0.06/kg of milk - nearly 24%
Brazilian experience in formulating poultry and pig of the total selling price of milk! Imagine any
diets with VegproTM SSF has shown that savings of company devoting 20-25% of the sales to replacing
as much as $10-15 per tonne are possible. As global the equipment! They could not survive, and nor can
reliance on shorter supplies of vegetable proteins we. If an extra lactation can be achieved, replacement
increases, this technology could be crucial (Table 1). cost drops to $0.04/kg or 17% of the total cost
Why? Because the enzymes release the energy in soya (Table 3).
and improve protein digestibility. A summary of
experience with VegproTM SSF in South America is Table 3. Calculated cost of replacing a cow based on two or three
found in Table 2. lactations.
Replacement heifer cost, USD 1400

Table 1. The soybean challenge: usage and production growing at Culled cow price, USD 200
different rates. Milk per lactation, kg 10,000
2001/2002 2001/2002 2003 Milk price, USD/kg 0.20
(Million bushels) (Million tonnes) Change (%) Replacement cost of the cow, USD/kg milk
Longevity: 2 lactations 0.06
Production Longevity: 3 lactations 0.04
United States 2,890 78.6 -11
Brazil 1,598 43.5 +10
Argentina 1,084 29.5 +12
China 556 15.1 +9 Sel-Plex® impact on health
Total 6,751 183.7 +6
Cows and sows are culled for reasons of health and
Use projection for 2004-2005
China +15%
reproduction, both of which are at risk when selenium
Russia +25% status is marginal; and it is this specific area where
Sel-Plex®, organic selenium produced by yeast, can
help. Selenium in Sel-Plex® is present in the ideal
LOWER PRODUCTION COSTS BY IMPROVING
ratio of selenoamino acids. When mastitis/MMA
ANIMAL HEALTH
impact is reduced, and selenium needs for
At a recent presentation on the future of American reproduction are met, commercial experience with
dairy farming, Dr. Steve Koenig pointed out that Sel-Plex is that herd longevity can be increased,
animal health is the key to success in the future. While however an extra lactation is just one of the benefits
he used the dairy farm to illustrate his points, they noted.
could be applied to any animal production system. Sel-Plex ® has implications for health and
An example illustrating the impact improved animal reproductive efficiency in all food animal species
health has on productivity is cow longevity. Dairy (Table 4). For sows, commercial and university
cows average only two lactations in several regions reports have indicated more pigs born alive and more
of the US, while 60% of all sows are culled after pigs weaned; and a review of poultry data in refereed
only three parities. Given that peak milk production publications alone demonstrates increased number of
in the dairy cow and peak sow productivity are well chicks hatched (2-4) per broiler breeder hen.
after these ages, the amount of lost production is Furthermore, improvements in health make the switch
astounding. At an average of 10,000 kg of milk per to Sel-Plex® easy. Is this new? No. Dr. Don Mahan
Table 2. More than 50 experiments and 120 companies confirm that the koji enzyme response has a major impact on production economics.
Results
Weight (kg) Feed conversion
Country No. of birds Age (days) Control Vegpro Control Vegpro
Argentina 4000 49 2.56 2.57 2.14 2.15
4000 51 2.75 2.87 2.08 2.02
3200000 53 2.64 2.64 2.23 2.24
5000 49 3.43 3.45 1.79 1.79
Brazil 3292 42 2.07 2.05 1.95 1.94
Ecuador 1200 42 2.50 2.63 1.58 1.51
Peru 65600 49 2.70 2.79 2.03 1.92
95000 47 2.67 2.72 2.08 2.02
T.P. Lyons 3
at Ohio State predicted this in 1995! The issue is as response differs between selenium yeast sources.
ever – not whether the new technology will lower French researchers noted that the profile of selenium
costs – it can – but whether the will to make the compounds differs among commercial selenium
change in order to reap the benefits exists. Organic sources (Figure 1). Reasons might include differing
selenium – Sel-Plex® – has truly redefined selenium growth media, pH and temperature conditions and(or)
nutrition and indeed vitamin E and ‘antioxidant’ yeast strain. As such, data generated from a product
supplementation in general. However, there can be manufactured by one process cannot be extrapolated
no half measures. Full replacement of sodium to another. This why in clearing ‘selenium yeast’ for
selenite at all stages of life is required. Health is a use in the US following review of Sel-Plex®, FDA
lifelong requirement. defined an allowable product as one made precisely
by this process. In effect, the regulators are holding
Table 4. Sel-Plex® impact on herd health and productivity. all new products to the standard set by Sel-Plex®.
Dairy cattle Cow longevity in the herd: lifetime yield
Fewer days open Key Success Factor No. 2:
Fewer services/conception
No Se injections needed Perceptions of the consumer
Reduced retained placenta incidence
Reduced mastitis incidence/impact Overcoming the negative perception of the consumer
Lower SCC is more difficult to achieve than a reduction in costs.
Calf livability
Supranutritional vitamin E levels unneeded
Due to a litany of scares – BSE, Foot and Mouth
Disease and dioxin contaminations – the public is
Beef cattle Meat quality
Calf livability
often suspicious of modern agriculture.
Feed efficiency
Pigs Sow longevity in the herd: lifetime productivity
An extra 0.5-1 pigs weaned/litter
Reduced MMA Success Factor 2
Piglet health at birth and weaning
Changing consumer perceptions
Chickens Breeders
More settable eggs produced
Fertility (male and female) Animal feed contains antibiotics used in human medicine
Hatchability
More chicks per hen
Animal feeds contain recycled 'dangerous' animal proteins
Agriculture pollutes soil and water
Broilers
Reduced mortality/culls Meat, milk and eggs are not 'healthy' foods
Improved uniformity
Feed efficiency
Meat quality
Layers The recent mad cow scare in the US illustrates how
Egg production reluctant as an industry we are to change, and perhaps
Egg quality
Shell quality validates consumer skepticism and the demand for
greater scrutiny. Carol Tucker Forman, director of
the Food Policy Institute of the Consumer Federation
Knowledge about organic selenium is accumulating of America, was quoted as saying “the damage to the
at an incredible rate in all disciplines, but agriculture American meat industry, and therefore the feed
is the sector able to take greatest advantage of it. industry, costs infinitely more than anything US
Still, it is always best to remember how much we cattlemen would have to pay to do things right”. But
still do not know! Not long ago science only knew doing things ‘right’ is not something we are always
of the role of selenium in glutathione peroxidase perceived to excel at. Least cost formulations
(GSH-Px). Now we know there are six forms of occasionally overrule common sense, and it seems
GSH-Px, and 30-50 selenoproteins. Likewise, we incredible that in a time when markets are asking for
now know that there are a wide range of total transparency and traceability that one would
selenocompounds in plants and yeast, and failure to leave anything to chance, much less take unnecessary
discount the importance of any of them because we risks. As we marvel at the apparent “repeating of the
do not today know their function would be absurd. European BSE mistakes” in the US, we remind
Nature rarely makes things for no reason. Modern ourselves that the perception is that many of our
analytical techniques have revealed one reason problems originate from what and how we feed
Water soluble
Not all selenium yeasts are alike
Polysaccharide
bound Fraction Yeast A Yeast B Yeast C
% of total Se
Protein bound Water Soluble 12 28 22
Polysaccharide-bound 15 26 72
Residual protein Protein-bound 18 40 4
A bound
Residual protein-bound 39 4 0
B Residual Residual hydrolysable 16 2 2
C hydrolyzable
Figure 1. Differing proportions of selenium in various fractions of three commercial selenium yeast sources (adapted from
Encinar et al., 2003).
livestock. This should not be the case; there are a responses are modulated (as opposed to stimulated
number of alternatives in use in all sectors of the directly), leaving the animal more prepared when
industry. Let’s briefly evaluate ways in which natural exposed to pathogens.
feeding programs combat perceptions of food animal The message with Bio-Mos® is that animal health,
agriculture. beginning with gut health, is the key to success.
PERCEPTION 1: ALL ANIMAL FEED CONTAINS

GROWTH-PROMOTING ANTIBIOTICS PERCEPTION: AGRICULTURE POLLUTES
Less true each year. While there were never antibiotics The latest restriction to be placed on animal
in ‘all’ feeds, even those sectors where inclusion was production in an increasing number of markets is
routine such as grow-finish pigs and broiler diets are the mandated reduction in dietary copper and zinc
steadily eliminating AGPs and have replaced them with in order to prevent accumulation in soil profiles
natural products and programs that promote health (Figure 2). Supranutritional levels have traditionally
and growth. been included in monogastric diets, especially those
Bio-Mos® was introduced to the marketplace at fed pigs, to reduce enteric disorders. Mandated
Alltech’s 1992 international feed industry symposium. reductions, however, allow only nutritional
The past 14 years have seen numerous successful trials minimums at a time when many are questioning
and in the past 12 months meta-analysis summaries whether such levels are adequate to meet demands
of the data in studies with weanling pigs, broilers and of modern genetic lines.
turkeys have been published (Pettigrew 2003; Hooge,
2003a; Hooge, 2003b). One researcher working on
modeling approaches to use in evaluating Bio-Mos®
confirmed that he has found nearly 300 publications Old New
in this area (G. Rosen, personal communication). The
Cu
resounding conclusion: the product is stable in feed,
Pigs: 35 - 175 ppm 25 ppm
acceptable to the consumer, and works as well if not
better than AGPs in comparison studies and on All species
commercial farms. Analysis of the broiler data show Fe: 1250 250 - 750 ppm
a 2% improvement in FCR, a 2% improvement in Mn: 250 100 - 150 ppm
weight gain, and a 20% decrease in mortality. It clearly Zn: 250 150-250 ppm (species-dependent)
has lived up to its motto: Bio-Mos ®: Performs. Co: 10 2 ppm
Promise. Its mode of action targets intestinal health
and immune modulation. The mannan fraction of Bio- Figure 2. Changes in trace mineral allowances for food
Mos® carbohydrates provides a ‘decoy’ to which animal diets in Europe.
pathogens adhere, thereby avoiding intestinal
epithelial colonization, which in turn leads to healthier Can animal health and productivity survive with
villi and more absorption of nutrients. Immune reductions of critical trace minerals to 20-30% of
T.P. Lyons 5
their current levels? The answer is yes, providing Feeding essential trace metals in the form of
that dietary trace minerals are supplied in forms best Bioplexes circumvents these problems by a)
suited to the intestinal environment and absorptive completely avoiding the risk of hydroxy poly-
mechanisms. Before reaching the site of absorption merization reactions, and b) speeding the rate of
(the enterocyte membrane) ingested minerals first passage of the metal ion across the negatively charged
encounter an unstirred water layer and then a mucus mucus layer by presenting it in a reduced charge or
layer with an intense negative charge (Figure 3). This electrically neutral form (Figure 4).
means that though the enterocyte membrane is very When dietary trace minerals are in this form, the
thin, the mineral must first traverse two layers, which nutritional minimums mandated by environmental
are orders of magnitude thicker than the absorptive laws are able to meet the needs of modern, highly
surface itself. For inorganic metal ions such as Cu, prolific genetic lines. In studies comparing Bioplex™
Zn, Mn and Fe, an immediate danger is so-called and inorganic zinc for grow-finish pigs Fremaut
‘hydroxy-polymerization’ whereby the increasing pH (2003) demonstrated that Bioplex™ Zn supplied at
in the small intestine, and particularly in the unstirred 30% of the inorganic Zn level resulted in improved
water layer, causes them to form large insoluble metal daily gain while the environmental goal of reduced
hydroxides that cannot be absorbed. excretion was accomplished.
The negatively charged mucus layer presents
another barrier against the passage of inorganic metal
ions and evolved as a protective mechanism against
toxic elements such as aluminum (Al3+). Because of
the intense negatively charged nature of this layer,
the strength of metal cation binding can be described
as follows; M3+ > M2+ > M+ (where M represents a
metal ion). Essentially, toxic elements such as Al3+
are bound so tightly that they rarely manage to
traverse this layer and are sloughed off as the layer is
replaced. As the charge on the metal ion decreases,
inorganic metal ions (which have avoided hydroxy
polymerization) may traverse the layer, but at Bioplexes:
Peptide protection from:
relatively slow rates. This is basically why ferric iron
(Fe 3+) must first be reduced to ferrous iron (Fe2+) Forming insoluble complexes
The negatively charged mucus layer
before it can be absorbed.
Figure 4. General structure of a Bioplex trace mineral.
Gut lumen
Unstirred water layer (600 µm)
Unstirred
Increasing pH causes inorganic ions to 'hydroxy-polymerize',
water layer forming non-absorbable complexes that are excreted.
Mucus
Mucus layer (50-100 µm)
Negative charge protects enterocytes against highly charged
Enterocytes or toxic ions such as Al+3. This is why Fe+2 is better absorbed
lining the villi than Fe+3, and why Bioplexes are more easily absorbed
than inorganic ions!
Villus height 600 µm
Blood
Figure 3. Barriers to absorption of highly charged inorganic cations: formation of unabsorbable hydroxy polymers in the unstirred
water layer and adherence to the negatively-charged mucus layer.
PERCEPTION: RENDERED ANIMAL BY- Alltech is essentially a yeast biorefinery (Figure 6),
PRODUCTS IN FEED – DO WE HAVE AN constantly examining ways of utilizing yeast or their
ALTERNATIVE? components. In applying the biorefinery concept to
our use of yeast; so another high quality protein for
While the antibiotic issue can be put aside safely with animal feeds arises. In addition to a wide range of
a tried and proven replacement, and bioplexing allows specialty yeast applications from animal feeds to
lower trace mineral levels, this cannot be said of ethanol, processes that utilize cell wall fractions in
animal by-products. In the US alone, 35 million cattle production of Bio-Mos® and Mycosorb® yield a form
are processed every year. What could we possibly do of yeast extract, which includes the highly nutritious
with the waste protein and fat? Europe has grappled cell contents. It is this extract that is processed into
with this problem, but if the US reduces its use of NuProTM, a yeast protein high in nucleotides with
animal by-products, the impact on protein prices will application in a broad spectrum of specialty diets,
be enormous. particularly those for neonates of all species.
The lesson of NuPro™, however, is not just that
New plant and yeast protein sources: The possible new proteins are available in increasing
‘Biorefinery’ quantities; the message is that innovative research
The nutritional, cost and environmental problems of and process results in innovative products if we think
not recycling animal by-products has no simple outside the box and develop new technologies.
solution, but perhaps the ‘biorefinery concept’, at
work in the rapidly expanding fuel ethanol industry,
PERCEPTION: ‘MYCOTOXINS ARE NOT IN
can provide a useful alternative protein source. Fuel
ANIMAL FEEDS SO WE ARE DOING NOTHING
ethanol is produced in either the grain dry milling or
ABOUT THEM’
wet-milling process, using a variety of starch and
sugar substrates across the globe. Grain dry mills Like other food safety issues, mycotoxins are a
currently produce ethanol, distiller’s dried grains with subject that consumers can be expected to be
solubles (DDGS) and CO2. Removal of the starch increasingly familiar with in upcoming years.
for fermentation to ethanol leaves the protein, Regulators are extending guidelines to include
minerals and fat concentrated in co-products currently mycotoxins other than aflatoxin as science provides
used in animal feeding, primarily ruminants but more and more information about these toxins.
increasingly in monogastrics. With ~30% CP, energy The increasing scientific information about toxin
equal to the original grain owing to concentration of chemistry and function provides us an advantage,
fat and ~0.7% phosphorus (90% of which is however, since it gives us an ability to solve the
available), these co-products have much to offer the problem. Knowledge about mycotoxin structural
food animal industry in terms of addressing a protein chemistry provides clues useful in building adsorbents.
shortage, but can we improve them further? The The 3-dimensional structure of yeast cell wall glucan,
‘biorefinery’ approach to processing starch/sugar the starting material for Mycosorb ® , can be
sources says yes! manipulated to optimize toxin-cell wall interaction
Dry mill ethanol plants using corn produce about making a ‘glucan web’ to prevent toxins from
30 kgs of DDGS for each 100 kgs of corn ground. A affecting the animal or its products (Figure 7).
‘biorefinery’, in contrast to an ‘ethanol plant’
integrates process streams such that a number of
products are produced, with ethanol being only one
of potentially many. Options for further processing
of spent grains and solubles include secondary
fermentations to increase protein content, boost lysine
content as much as 3-fold and decrease the indigestible
fiber. Enzymatic hydrolysis of DDG and/or solubles
is another approach to add flexibility. Ethanol
producers seeking to expand the market for distillery
co-products have begun integrating processes that ‘re-
ferment’ a portion of the solubles and spent grains to
provide specialty ruminant products such as VA101
Figure 5). Such directions go well beyond simply
upgrading a ‘by-product’. Figure 7. Three dimensional structure of yeast cell glucan.
T.P. Lyons 7
Feedstock
Milling,
processing,
cooking
ETHANOL
CO2 Fermentor
Distillation
Whole stillage
Separation, Solids Solubles

Inositol Centrifuge Evaporator
Refining
Solids Organic acids

Glycerol
CO2 Secondary
Fermentation Dryer
Plant oils
Extraction,
Proteins Enzymatic
Fatty acids processing
Pharmaceuticals Specialty Distillers DDGS Condensed

feed wet distillers
Heterologous proteins ingredients grains solubles
Figure 5. From distillery to biorefinery.
Viable Yeast-
yeast products biosynthesized products
®
FERMENTATION Sel-Plex
BioChromeTM
Yea-Sacc1026® Fuel ethanol

yeasts
Yeast component products

Cell solubles stream Cell solubles stream
NuProTM
Mannan stream Glucan stream
Separation
® ®
Bio-Mos Mycosorb
Figure 6. A yeast biorefinery.

Comparing commercially available adsorbents has A key step in defining those policies is deciding where
become a necessity for feed manufacturers. Table 5 we stand with regard to change: are we going to be
contains a 7-point guideline for evaluating such proactive or reactive? Is it something that is going to
products. happen to us or will it be something we make happen?
Table 5. 7-point comparison for mycotoxin adsorbents.
1. Can the product adsorb a wide range of toxins?
CHANGE IS CONSTANT
2. Is inclusion rate sufficiently low (ie. 0.5-2.0 kg/t)?
3. Is the adsorbent stable in the pH range of the GIT? Change is inevitable in the dynamic animal feed
4. Is adsorption capacity high (will it not be overwhelmed at market, and failure to change has been the death knell
high toxin concentrations)? of many enterprises. Once we accept that change is a
5. Is adsorption affinity high (is it effective at low toxin constant, our main decisions revolve around how to
concentrations since mycotoxins are often toxic at low
concentrations)? deal with change. We can either embrace change and
6. Is adsorption sufficiently rapid? move forward, or we can ignore it until change is
7. Are there in vivo data that show protection of production forced upon us.
animals against toxins? Two large companies whose strategies for change
are apparent to us all are McDonald’s ® and
Mycosorb®, with its low inclusion rate and structure Starbucks ®. The fast-food industry ‘re-invented’
adapted to adsorb a range of mycotoxins including eating out; and for years seemed immune to recession.
aflatoxin, zearalenone, T-2 and DON, is rapidly Recently they have begun to feel the pinch as they
becoming the adsorbent of choice global. Protected have watched consumers ‘re-invent’ what is ‘good’
by three patents, Mycosorb® has unlimited potential about food. As a result McDonald’s® stopped buying
as we learn more about its structure and how beef produced using antibiotic growth promoters, they
modifications can increase adsorption of both known refuse genetically modified potatoes, and in Great
and newly-identified mycotoxins. Again, the Britain have begun to provide organic milk. US
appliance of science to solve a practical problem. McDonald’s® franchises offer ‘Atkins-friendly’ meals
The fact is that the technology is available to prevent for the growing number of carbohydrate-counting
mycotoxins from having an impact at even the animal customers. Is this a case of McDonald’s ® being
level, which means that toxins from this source need proactive about changing menus, or are they being
not threaten food safety in either perception or reality. reactive when forced to change?
Starbucks® ‘re-invented’ stopping for a cup of coffee
Key Success Factor No. 3: with huge success, but now they have begun to add
Designing policies for the future: ‘Fair Trade’ and environmentally friendly products.
With Conservation International they have
transparency and innovation collaborated on a project to encourage sustainable
Even if we adequately address price competitiveness agricultural practices and biodiversity through the
and consumer perception, in order to be sustainable production of shade-grown coffee, which follows the
we need policies that maintain transparency and spur Institution of Coffee Purchasing’s guidelines. Is
innovation in both products and business strategies. Starbucks listening to the consumer or is Starbucks®
being proactive?
Success Factor 3 The changes in McDonald’s® and Starbucks® are
examples of transparency and proactive efforts to offer
Designing a sustainable policy products modern customers are interested in buying.
for the future They want customers to know of their commitments
to food quality, safety and sustainable agricultural
Meet change Listen and act
practices. Is our industry just as proactive? Have we
head-on Take on new technologies before competitors do
lost sight of what Dan Glickman (former US
Make transparency standard
Differentiation Avoid the 'sameness' trap
Secretary of Agriculture) advised at the Alltech
Choose exceptional, passionate people
International Feed Industry Symposium in 2000 –
Innovation Creative products “Tell us what you want and we will grow it”?
Creative R&D strategies
T.P. Lyons 9
AVOIDING THE SAMENESS TRAP: companies (and individuals) from failures. Another
DIFFERENTIATE WITH PEOPLE AND PRODUCTS important element of the future viability of our
industry will be our ability to give consumers not
In order to be sustainable, companies must avoid the only what they want, but more importantly what they
‘sameness trap’ described in Funky Business by did not realize they wanted. The new competition
Nordström and Ridderstråle (2000). They describe will take place not only in terms of market share, but
an oversupplied world of similar companies, more importantly in newly created markets.
employing similar people, with similar educational Innovation, while a term vastly overused, is a
backgrounds, coming up with similar ideas, competency that Alltech and all companies need to
producing similar things, with similar prices and excel at in order to prosper.
similar quality. Does this sound like our industry? It I was once asked how it could be possible to take a
does, and it underscores our need to differentiate. commodity item like milk and make it unique – a
We need to create new solutions to problems and in value-added product. Is it simple? No. Is it possible?
doing so create profit and success for ourselves and Absolutely. We created a slogan: ‘A milk for all ages’.
our partners. We can make our companies, and hence For the young, a lactoferrin-rich milk for the lactose-
our industry, different and make them stand out in intolerant. For teenagers, perhaps higher calcium
the industry. levels for growing bones; while low fat, high omega-
3 and high cholesterol-blocking statin might form
The people factor: exceptional people help part of ‘milk for middle ages’. For all ages,
companies differentiate enrichment with selenium through Sel-Plex® in the
‘Our people make the difference’ must be more than cow’s diet to fight against cancer. A Korean company
a well-meaning cliché. Many business commentators went further and changed the name from milk to
believe that we are entering an era where the ‘war SELK to emphasize selenium enrichment.
for talent’ is the most important battle that will be
fought. When land was the important asset, countries
battled for it, now that talent is the important asset
for business success, companies will battle for talent.
Paul Allaire, former CEO at Xerox®, calls it “the
brawl with no rules”.
What kinds of talents are we looking for? It is one
of the fundamental roles of the leader that he/she
develop the talent around him/her. Inside rapidly-
growing Alltech the need to ensure that the next
generation of leaders is in place has been acute. We
have key questions to ask potential employees – the
most important of which is: What are you passionate
about? There is no right or wrong answer, it is simply
important to find people with the energy and drive
for accomplishment. We have successfully made the
transition from a small local player into a medium-
sized global enterprise. The next challenge for our
people and for our industry involves becoming the
industry standard bearer. Part of our future success
will be due to recruiting talented and diverse
individuals from across the world, including a greater
proportion of women, a group whose skills and
management styles have been underutilized in
agribusiness.
FOSTER INNOVATION
In the ‘over-supplied world’ described by Nordström The size of a company is irrelevant when it comes to
and Ridderstråle, ideas are what separate successful innovation. The tiny New Zealand dairy co-operative,
Tatua, with only 30,000 cows is still the world’s most References
profitable, largely due to the value-added dairy
products it offers such as lactoferrin for infant Encinar, J.R., M. ?liwka-Kaszyñska, A. Polatajko,
formulas and lactoperoxidase as a natural sterilant. V. Vacchina and J. Szpunar. 2003. Methodological
Alltech’s Bioscience Centers, where scientists advances for selenium speciation analysis in yeast.
complete research toward MSc and PhD degrees while Analyt. Chim. Acta 500:171-183.
working with Alltech’s research group, are at the hub Fremaut, D. 2003. Trace mineral proteinates in
of our innovation. We support these scientists’ efforts modern pig production: reducing mineral excretion
and encourage creative thinking. Over time, 9 PhD without sacrificing performance. In: Nutritional
and 42 MSc students contributed to the research on Biotechnology in the Feed and Food Industries,
Yea-Sacc1026®, now the world’s No. 1 natural rumen Proceedings of Alltech’s 19th Annual Symposium
modifier, which is the reason we understand its mode (K.A. Jacques and T.P. Lyons, eds). Nottingham
of action so well. A good example of the impact of University Press, UK, pp. 171.
this work is the recently obtained EU approval for
Hooge, D.M. 2004a. Meta-analysis of broiler chicken
Yea-Sacc1026® in horses. In the US alone we support
pen trials evaluating dietary mannan
work being conducted by 36 doctoral candidates and
oligosaccharide, 1993-2003. Intl J. Poult. Sci.
have 135 ongoing projects in Europe.
3(3):163-174.
Hooge, D.M. 2004b. Turkey pen trials with dietary
mannan oligosaccharide: meta-analysis, 1993-2003.
Summary
Intl J. Poult. Sci. 3(3):179-188.
Re-imagining the feed industry means re-imagining Nordström, K.A. and J. Ridderstråle. 2000. Funky
our companies: our goals and what we stand for, our Business - Talent makes capital dance. Financial
people and the corporate environment we create. We Times Prentice Hall, New Jersey.
must ask and answer carefully the questions ‘Are we Pettigrew, J.E. 2000. Mannan oligosaccharides’
fostering the creativity we need to carry the company effects on performance reviewed. Feedstuffs 52
into the future? Do our products and research (December 25).
directions address industry needs for price
competitiveness and consumer perception? Are the
policies sustainable?
At Alltech, we recognize the importance of ongoing
discussion of these questions in building a dynamic The Alltech 'Big 6'
corporate culture. It has allowed us to focus on core
competencies to develop a ‘Big 6’ list of product Yea-Sacc1026® Viable yeast culture for cattle & horses
directions while giving us the freedom to find ways Bio-Mos® Mannan oligosaccharide,
to expand to a ‘Big 8’ or ‘Big 10’. non-AGP growth promoter
Another result of this corporate dynamic is the Mycosorb® Reduce mycotoxin
growing role of the Bioscience Centers as hubs of impact
innovation, both in scientific exploration and in the
structure of modern corporate agricultural research-
our relationships with other research groups at Sel-Plex® Selenium
universities and institutes. in the 'food' form
The process is exciting; and it is providing products
that have increasing importance across the world in VegproTM SSF enzyme for vegetable proteins
the areas of animal health, performance and
reproductive efficiency, and consumer perception of BioplexesTM Trace mineral proteinates for all species
food animal products. Clearly decisions we make
surrounding Price, Perception and Policy define
ultimately where each of our companies will be in
10, 20 or 30 years’ time.
C.V. Maxwell 11
Future of the feed/food industry: re-inventing animal feed

CHARLES V. MAXWELL
Animal Science Department, University of Arkansas, Fayetteville, Arkansas, USA
Challenges and opportunities facing the international Three key issues asked of the industry today are 1)
feed/food industry have never been greater than those what are animals consuming, 2) how are animals
faced by these industries today. It has become apparent being cared for, and 3) has the animal been sick?
that anything less than a very proactive approach to The traditional protein business chain from vegetable
addressing current challenges may not be sufficient. to animal protein has changed dramatically.
Current international events are leading feed Traditionally, this has been a production-based model
companies to attempt to move from a traditional role from the farmer to the consumer with little oversight.
as a ‘feed company’ to becoming an integral part of The reality today is that the consumer is providing
the ‘Food Supply Chain’. This is driven essentially oversight to the retailer who then places constraints
by the fact that although one cannot ensure that food on the production chain to conform to specific
company products eaten by consumers are safe, as a standards of production. Overall confidence in food
prominent player in the early part of the food chain, safety was down in the early 1990s, tended to rise
it is essential that the feed industry ensure that the and peaked in the mid 1990s and has declined since
feedstuff supply chain is not the problem. (Figure 1).
This new reality has led feed companies to initiation Another issue being addressed by the multinational
of the concepts of oversight, control, and overall feed companies is globalization. Given all the issues
security of their component of the food chain. Feed being addressed internationally, specific feeds need
companies are, out of necessity, now becoming ‘Food to be modified to fit specific country labeling. This
Safety Guardians’. The commitment by the feed presents tremendous difficulties in developing
company to provide customers with products of the branded products with international acceptance.
highest quality has been deemed vital to the success Solutions to these issues are exacerbated by the
of the business. Therefore, feed companies must multitude of regulatory hurdles that must be
become early leaders in assuring that all products are overcome. These include differences in the use of
Hazard Analysis and Critical Control Point (HACCP) antibiotics, animal proteins, genetically modified
certified. In addition, it is essential that feed companies materials, and feed additives. Companies that supply
become registered in the ISO 9001 quality standard inputs are required to think as globally as processors
or adopt a similar standard. HACCP is a systematic and retailers.
approach to identifying and preventing contamination These issues were in the implementation stage prior
of food and food products during the manufacturing to the discovery of BSE in North America. All
process. ISO certification is an internationally components of the feed/food industries are being
recognized quality management system that affected by this discovery. This is leading to the rapid
emphasizes integrity throughout the manufacturing development and implementation of a national
process, using standardized and verifiable procedures identification program in the US. The ID program
in all aspects of operations from product design will likely bring rapid adoption of Radio Frequency
through manufacturing and distribution. The same Identification Devices (RFID) in production and
level of concern has progressed through the livestock processing sectors of the food supply chain and will
and poultry production chain to include producers, match the drive of Wal-Mart’s RFID technology in
integrators, processors, and retailers.
12 Future of the feed/food industry: re-inventing animal feed
85
80
Percentage
75
70
65
1991 1992 1993 1994 1995 1996 1997 1998 1999 2000
Figure 1. Overall confidence in food safety: percentage of consumers completely or mostly confident
(Food Marketing Institute, 1991-2000).
the retail sector. There will be public pressure for projected to decrease over that same time period.
the US government to fund the development and Thus, it is imperative to improve production
implementation of the national animal ID program, efficiency in both the livestock and crop sectors to
perceived as essential for traceability in a be able to meet the rising demands. The other huge
comprehensive food safety program. The need for area is the need to revamp production agriculture so
traceability has been the greatest cost impediment to that all components can be brought back to consistent
the adoption and implementation of country-of-origin profitability.
labeling (COOL). The Manitoba Pork Council has
initiated a pilot swine traceability study as part of a Table 1. Students enrolled in swine production in top 10 swine
national effort to identify the most cost efficient producing statesa.
method of tracing swine movement in Canada. State Fall 1998/ Fall 2002/ Change
The lack of qualified workers has been and continues Spring 1999 Spring 2003 (%)
to be a major constraint for those associated with the
integrated livestock and poultry industries. A survey Iowa 97 75 ↓ 22
North Carolina 64 30 ↓ 53
of students enrolled in swine production in the top Minnesota 12 0 ↓100
swine-producing US states was conducted by Duane Illinois 15 14 ↓ 6
Reese (Table 1). This study indicates that interest Indiana 30 18 ↓ 40
among students had declined in 9 out of the top 10 Nebraska 11 6 ↓ 45
Missouri 25 22 ↓ 12
swine-producing states over the last five years, with Oklahoma 37 30 ↓ 15
an average decline of 28% in the number of students Kansas 47 48 ↑ 2
enrolled. Many other states reported that a lack of Ohio 14 10 ↓ 28
interest among students is resulting in swine Total 352 253 ↓ 28
production not being offered or offered on alternate a
Reese, University of Nebraska, personal communication
years. Similarly, poultry production is only offered
by a limited number of universities and interest in a Evaluation for technology development
poultry production major is low. It is worth noting
that this lack of interest comes at a time when world
by the livestock industry
meat demands are expected to increase as developing The swine and poultry industries have made
countries have more disposable income, with a tremendous progress through the years in terms of
projected increase of 50% by the year 2025 (Elam, genetics, nutrition, husbandry and health. Advances
2004). At the same time, the acreage of row crops is in production and management have provided the
C.V. Maxwell 13
marketplace with a high volume, low cost animal smaller effect on profitability. A 3-4% gain in feed
protein. Historically the livestock industries have been efficiency is almost too small to measure, but the
mostly concerned about commodity production that economic impact on profitability in the integrated
works best in a least cost, most efficient production industry is huge.
model. There are two schools of thought regarding Although the challenges are great, much progress
technology evaluation for large systems. The typical is being made in providing alternatives that could
approach that has served the integrated livestock benefit the feed/food industries tremendously. The
industry well in the past is the cost analysis system fact that growth-promoting levels of antibiotics are
that is used by Agrimetrics and Agristats. The no longer permitted in Europe and the possibility of
technique used is a comparison-based cost analysis restrictions being imposed elsewhere has led to a
that compares the cost-of-gain of one company with plethora of studies investigating replacements. These
the cost-of-gain of other comparable enterprises in a studies offer the potential of a better understanding
manner that keeps the actual companies involved in of the relationship between the microbiota in the
the analysis confidential. These tools have been largely environment and improved livestock performance as
used to drive systems to ‘least cost’ production with well as alternative strategies to control the threat of
little regard to optimal cost and maximal profit. Some specific microorganisms. This may result in improved
have argued that this system may be overly simplistic performance over that observed with growth-
and does not effectively address the concept of value promoting levels of antibiotics. Similarly, studies to
returned/cost of input. For example, the most value replace specific animal proteins may lead to a better
out of a feeding program may result in a higher cost understanding of factors associated with reduced
of gain if the increased cost results in improved performance with plant proteins in neonatal animals.
performance. The other system is to look at returns
over feed costs or a return over input cost. Data
analysis services are available that may more Relationship between the gut microbiota
effectively address where improvements may be made and performance in swine
using a more broad-based cost/benefit analysis. The
approach taken by MetaFarms Inc. is to conduct an The gastrointestinal tract of the pig harbors a
analysis of ‘Process Enablers’, which affect a number metabolically active microbiota that stimulates the
of specific parameters monitored in an enterprise. normal maturation of host tissues and provides key
This leads to a continuing evaluation of the impact defense functions (Gaskins, 2001). Several recent
that specific processes have on parameters being examples of improved post-weaning performance in
measured rather than a single focus on cost of gain. the young pig suggest that much of the improvement
One example of this type of analysis is the effect of observed in nutritional studies may be through an
ractopamine use in a swine production enterprise on impact on the intestinal microbiota. A good argument
performance. Ractopamine added 10 lbs of weight can be made that the improved performance observed
per pig sold, which improved the bottom line by $1.50 in the young pig as a result of feeding plasma protein,
to $2.00/pig. Although cost of gain is improved in complex diets, antibiotics or acidifiers might be an
pigs fed ractopamine, the improvement is minimal indirect effect of altering the gut microbiota.
compared to the value of improved gain and lean Similarly, the positive effects of popular management
yield; but ractopamine may not be considered unless strategies such as segregated early weaning (SEW)
one analyzes technologies outside a simple cost of may be mediated through reductions in exposure to
gain model. That leaves us questioning how we should pathogens.
evaluate technology. Perhaps the effect a technology Segregated early weaning reduces the incidence of
has on both the cost side of the equation as well as a number of pathogens, thus reducing immunological
the revenue side should be evaluated. It is essential stress, which results in improved growth and higher
that models are developed that evaluate profitability, efficiency of feed utilization (reviewed by Maxwell,
not just cost of production. 1999). This strategy has been successful in reducing
It is also interesting to note that once all the factors the number of pathogens, but has not been successful
with huge effects on performance and efficiency have in eliminating all pathogens. The premise is that pigs
been implemented, this leaves the livestock industries are removed from the sow while their immunity, as
with the unenviable task of attempting to determine a consequence of maternal antibodies, is still high.
impacts of products and(or) systems that have a much This maternally derived passive immunity will
prevent vertical transfer of indigenous pathogens. Pigs be totally equipped to deal with such pathogenic
reared in isolation have been shown to have reduced challenges.
immunological stress (Johnson, 1997) resulting in Recently there has been a concern about the use of
improved growth and efficiency of feed utilization. antibiotics in animal production in part due to
This is consistent with observations in our research antimicrobial resistant bacteria. Over the past two
at the University of Arkansas to determine if decades, probiotics (direct-fed microbials), which
differences in immune stimulation can explain include Lactobacillus cultures, have been used as an
performance differences in conventional vs off-site alternative to antibiotics in animal production (Jin et
reared pigs. A total 432 weanling barrows (19 ± 2 al., 1998). Lactobacilli are normal inhabitants of the
day of age) were obtained from a local commercial gastrointestinal tract of pigs. Their beneficial role in
company from a single source. One-half the barrows the intestinal tract has been attributed to their ability
from litters were selected for the off-site nursery to survive the digestive process, attach to the epithelial
study (6 pigs/pen) with the remaining pigs staying in lining of the intestinal tract, produce lactic acid and
the conventional nursery facilities (approximately 18 other microbial compounds and prevent colonization
pigs/pen). Pigs were weighed and serum samples by pathogens via competitive exclusion (Savage,
obtained via venipuncture on days 0, 14, and 34 post- 1987). To investigate weaning-induced changes
weaning from a total of 72 pre-selected pigs. The within the enteric system, two experiments were
pigs were placed in the conventional facilities (a conducted to determine the effect of milk
minimum of 1 pig/litter was sampled) and an off- supplementation with Lactobacillus brevis (1E-1) on
site nursery (a minimum of two pigs in each of 36 pre- and post-weaning pig performance, and intestinal
pens was sampled). Serum α1-acid glycoprotein microflora. The 1E-1 isolate was sampled from the
concentrations were determined by a single radial intestinal tracts of 10 healthy pigs and five pigs with
immunodiffusion method using a commercial kit scours, and it was reported that healthy pigs had
(porcine α1-acid glycoprotein plate, Development higher levels of lactobacilli, with the majority of
Technologies International, Inc., Frederick, MD). isolates identified as Lactobacillus brevis (Parrott et
Pigs reared in the off-site nursery were 0.89 kg al., 1994). In each experiment, litters were allotted
heavier (P<0.01) at day 14 post-weaning and 2.40 to two treatments at farrowing: either a control milk
kg heavier (P<0.01) at 34 days post-weaning. In supplement, or the control containing 1E-1 via an
addition, serum α1-acid glycoprotein concentration in-line system using a cup dispenser for each litter.
was elevated (P<0.01) in pigs reared in the Coliforms and E. coli were enumerated from
conventional nursery. This suggests that reduced esophageal, duodenal, jejunal, and ileal regions of
performance in a conventional nursery may be the enteric tracts in Experiment 1. Pigs receiving 1E-
associated with the immunological stress associated 1 had lower (P<0.05) jejunal E. coli populations
with production under these conditions. pre-weaning and post-weaning compared to pigs
The swine industry is implementing early weaning provided only milk supplement (Table 2). Ileal E.
for efficient and economical pig production (Wilson, coli populations were lower (P<0.02) during the post-
1995; Patience et al., 1997). The obvious consequence weaning period for pigs receiving 1E-1 compared to
of weaning is the abrupt change in diet from sow’s pigs provided milk replacer without 1E-1. The
milk to solid feed and a change in the environment. administration of 1E-1 prior to weaning may deter
There is reduced feed intake during the first week the detrimental alterations in the microbial population
and associated adverse changes in the animal’s gut that occur at weaning as has been observed in pigs
anatomy and physiology such as villus atrophy, fed zinc oxide (Katouli et al., 1999). During the pre-
deeper crypts, and infiltration of the villus tip by weaning period (birth to weaning), administration
immature enterocytes (Spring, 1999). Villous atrophy of 1E-1 tended to increase weight gain (Figure 2,
means that there is less absorptive area available for P<0.06). During the first five days post-weaning,
nutrient uptake and deeper crypts represent a large pigs fed 1E-1 prior to weaning had greater ADG
tissue turnover (Spring, 1999). The intestinal (Table 3, 277 vs 194 g/d; P<0.05) compared to pigs
microflora can be adversely affected during weaning, provided only milk replacer, and overall ADG was
resulting in higher numbers of potentially pathogenic improved in pigs fed milk replacer with 1E-1. Pigs
acid-intolerant coliforms and a decline of favorable previously fed milk replacer with 1E-1 were 1.93 kg
lactobacilli (Bolduan, 1999). In addition, since the heavier at the completion of the 28-day study when
piglets are young, their immune systems might not compared to those receiving the milk replacer alone
(Figure 3).
C.V. Maxwell 15
Table 2. Pre- and post-weaning mean E. coli populations in the A second study to confirm these results has recently
jejunum and ileum of pigs (CFU/g log10). been completed. Beginning at farrowing, pigs were
Pre-wean Post-wean provided milk supplementation either with or without
Control 1E-1 Control 1E-1 the addition of Lactobacillus brevis (1E-1) via an in-
Experiment 1 line system using a cup dispenser for each litter. These
Mean E. coli, CFU/g (log10) treatments were continued during the nursery period,
Jejunum 5.53a 3.42b 7.10a 4.80b in which pigs that were administered 1E-1 via milk
Ileum 5.91 4.71 6.63 a 4.96b supplementation continued to receive 1E-1 through
a,b
the watering system. Pigs supplemented with 1E-1
Pre- and post-wean means within a row with different letters
had greater ADG (P<0.05) during Phase 2 and in the
differ significantly (P<0.05)
overall nursery period (day 0 to 38), greater ADFI
Table 3. Effect of Lactobacillus brevis (1E-1) during lactation on (P<0.05) during Phase 3 and the overall nursery
subsequent nursery pig performance (Experiment 1). period, and tended to have increased gain:feed
Treatment (P<0.10) during Phase 3 (Table 4). In this study,
Milk Milk + L. brevis (1E-1) 1E-1 supplementation resulted in a 1.58 kg
improvement (P<0.01) in body weight at the end of
Phase 1 (day 0-5)
ADG, g 194b 277a the six-week nursery period compared to pigs not
ADFI, g 91 109 receiving 1E-1 (Figure 4). These data indicate that
Phase 1 (day 0-14) 1E-1 supplementation pre-weaning improves nursery
ADG, g 211 258 performance and may provide a healthier intestinal
ADFI, g 211 250
environment.
Overall (day 0-28) Studies that I have summarized involve classical
ADG, g 355 388
ADFI, g 435 461 culture techniques. We are adapting procedures that
Gain:feed 0.83 0.85 allow quantitative determination of all the primary
Weight, kg microbiological species in the gut microbiota using
Initial 5.50 c 6.31d molecular techniques (Figure 5). The development
Phase 1 8.26 9.88
Phase 2 15.19 17.12 of molecular-based methods that by-pass the need
for culturing bacteria has led to a renaissance in
ab
Letters that differ within the same column are different (P< 0.02). microbial ecology studies. Molecular methods that
cd
Letters that differ within the same column are different (P<0.09). utilize the Polymerase Chain Reaction (PCR)
exponentially amplify copies of bacterial DNA to
literally produce a billion copies of the original
template without growing the bacteria. These
300
P<0.06
250
200
Daily gain (g)
150
100
50
0
Control L. brevis
Figure 2. Effect of milk supplementation with Lactobacillus brevis (1E-1) on daily gain from birth to weaning.
20
19
18
17
BW (kg)
16
15
14
13
12
Control L. brevis
Figure 3. Effect of milk replacer supplementation with Lactobacillus brevis (1E-1) on final nursery weight (Trial 1-28 days, kg).
Table 4. Effect of Lactobacillus brevis (1E-1) during lactation on every known bacterium. This amplified mix of gene
subsequent nursery pig performance (Experiment 2). sequences can be separated, sequenced and compared
Trait Milk Milk + P-value to the 16S rRNA/DNA database presently containing
L. brevis (1E-1) taxonomic data of over 85,000 bacterial sequences.
These studies suggest that the impact of gut
ADG, kg microbes on performance in young pigs is greater
Phase 1 0.092 0.100 0.51
Phase 2 0.286 0.332 0.014 than once thought. Other researchers have
Phase 3 0.507 0.535 0.26 documented enhanced performance in growing/
Phase 1-3 0.307 0.336 0.05 finishing pigs associated with improved health and
ADFI, kg management. Pigs with minimal disease due to SEW,
Phase 1 0.125 0.128 0.13 which were fed a series of non-limiting diets and
Phase 2 0.304 0.347 0.17 reared in pens of three pigs (2.23 m2/pig), achieved
Phase 3 0.563 0.622 0.03
Phase 1-3 0.343 0.380 0.04 104 kg at 136 days of age and 120 kg at 151 days of
age (Schinckel et al., 1995). Pigs raised on the
Feed:gain
Phase 1 1.632 1.482 0.57 original commercial farm, conventionally weaned
Phase 2 1.123 1.079 0.64 with all-in, all-out production, required 184 days to
Phase 3 1.109 1.180 0.14 attain 104 kg live weight. This dramatic effect of
Phase 1-3 1.141 1.145 0.93 health is likely mediated by inflammatory cytokines
Weight, kg or other systemic inflammatory effects in response
Initial 7.86 7.88 0.09 to bacterial toxins. The overall effect is to down
Phase 1 8.73 8.88 0.24
Phase 2 12.72 13.55 0.013 regulate muscle synthesis and growth. Schinckel et
Phase 3 19.87 21.45 <0.01 al. (2003) presented a review of current research on
muscle endocrine, and immune regulation of growth.
methodologies have created the first opportunity for

ecologists to gain a true representation of the microbial Bio-Mos®
world as it would exist in natural environments. To
examine the bacterial ecology of known and unknown Although prohibited in many European countries,
bacteria, the most often used method is to PCR the addition of antibiotic growth promoters to swine
amplify the 16S rRNA/DNA genes from a mixed diets remains a common practice in the US,
bacterial community. These highly conserved genes particularly to the diets of newly weaned pigs.
encode a portion of the bacterial ribosome found in However, there has been increasing pressure on the
C.V. Maxwell 17
22
P<0.01
BW (kg) 20
18
16
14
Control L. brevis
Figure 4. Effect of milk replacer supplementation with Lactobacillus brevis (1E-1) on final nursery weight (Trial 2-42 days).
1 2 5' 3'
3' 5'
5' 3'
3' 5' 5' 3'
Extract DNA PCR with a 3' 5'

from community fluorescently labeled
16sRNA forward primer
3
Restriction digest
5 4 of PCR product
5' A D 3'
C
Relative Flourescence
A 3' 5'
B 5' B E 3'
A F
B 3' 5'
C C F
E 5' 3'
D 3' 5'
Recognition of Fragment separation

labeled fragments in sequencing gel (size)
Figure 5. Procedural outline to quantitatively determine all species in a mixed culture (adapted from Liu et al., 1997).
livestock industry to decrease or discontinue these to positively alter immune function (Berg, 1998;
additions because of the potential development of Turner et al., 2001).
antibiotic resistance. The need for alternative methods Supplementation of swine diets with mannan
to improve growth and efficiency of livestock oligosaccharides derived from the yeast cell wall of
production and to modulate the animal’s natural Saccharomyces cerevisiae has the potential to provide
ability to fight disease has prompted the scientific an alternative to growth-promoting antibiotics.
investigation of several feed additives and their ability Mannan-based supplements have the ability to alter
the microbial population in the intestinal tract. This (P<0.05) in the phagocytic capacity of jejunal lamina
modification seems to be accomplished by the ability propria macrophages compared to pigs fed the basal
of mannans to attach to mannose-binding proteins diet (P<0.05, Table 6). Data compiled from five
on the cell surface of some strains of bacteria, thereby experiments conducted at the University of Arkansas
preventing these bacteria from colonizing the conclude that Bio-Mos® supplementation improves
intestinal tract by interfering with the binding of weight gain and feed efficiency in nursery pigs.
carbohydrate residues on epithelial cell surfaces Although the function of lymphocytes derived from
(Spring et al., 2000). Mannans have also been peripheral blood was not affected by Bio-Mos ®
reported to alter immune function in swine (Kim et supplementation, Bio-Mos ® did enhance innate
al., 2000), and this may be an additional mechanism immune function in the gastrointestinal system.
by which mannans improve growth performance.
The effects of Bio-Mos® on pig performance and Table 5. Summary of the effect of Bio-Mos® on growth perfor-
immunocompetence was evaluated in five nursery mance in 5 trials.
pig trials conducted at the University of Arkansas. A Bio-Mos® (kg/t)
total of 412 pigs were included in the evaluation with
0 0.2 0.3 P-value
82 total observations (38 pens fed the basal diet, 15
pens fed 0.2% Bio-Mos®, and 29 pens fed 0.3% Bio- ADG, g
Mos®). In four of the five trials, Phase 1, Phase 2, Phase 1c 147 + 9 168 + 1 6 166 +1 0 0.26
and Phase 3 were defined as day 0 to 10 after weaning, Phase 2 372 + 1 2 369 + 2 2 391 +1 4 0.56
Phase 3d 461 + 1 4 516 + 2 3 501 +1 8 0.07
day 10 to 24 after weaning, and day 24 to 38 after
weaning, respectively. The fifth trial consisted of a Feed:gain
Phase 1d 1.709 + 0.067a 1.388 + 0.126b 1.493 + 0.080b 0.02
14-day Phase 1, and a 7-day Phase 2. During Phase Phase 2 1.437 + 0.085 1.428 + 0.160 1.195 + 0.104 0.20
1, Bio-Mos® supplementation improved (P<0.02) Phase 1-2f 1.379 + 0.025a 1.375 + 0.046ab 1.273 + 0.030b 0.03
feed efficiency compared to pigs fed the basal diet, Phase 3d 1.700 + 0.028a 1.586 + 0.046b 1.610 + 0.036ab 0.03
whereas improvement in ADG approached a,b
Means in a row with no letters in common differ (P<0.05).
significance (Table 5, P=0.11). Pigs supplemented c
Contrast: 0 vs 0.2 + 0.3% Bio-Mos®; P = 0.11
with 0.3% Bio-Mos® had improved feed efficiency d
Contrast: 0 vs 0.2 + 0.3% Bio-Mos®; P<0.05
for the overall Phase 1 and 2 periods (P<0.03) when e
Contrast: 0 vs 0.2 + 0.3% Bio-Mos®; P<0.10
compared to those fed the control diet. During the
f
Contrast: 0.2% vs. 0.3% Bio-Mos®; P<0.10
first week of Phase 3, ADG (P<0.05) and feed
efficiency (P<0.05) were improved in pigs fed Bio- Our data indicate that Bio-Mos® alters the proportion
Mos® when compared to pigs fed the basal diet. There and function of leukocytes isolated from the
were no differences (P>0.20) in lymphocyte peripheral blood as well as the gastrointestinal tract
proliferation between pigs fed Bio-Mos® and those of the weanling pig. One of the questions raised by
fed the basal diet when data from the five trials were the results of these studies with Bio-Mos® is; how
combined. However, evaluation of immune assays can a non-digestible feed additive alter immune
conducted in the fifth trial revealed that Bio-Mos®- responses in the gastrointestinal tract and systemically
supplemented pigs had a greater proportion of in the pig? One possible mechanism is via the uptake
lymphocytes in the peripheral blood (P<0.03), an of Bio-Mos® from the lumen of the gastrointestinal
increase (P<0.10) in the proportion of macrophages tract by M cells of Peyer’s patches. Peyer’s patches
in the jejunal lamina propria, and an increase are organized lymphoid follicles located along the
luminal surface of the small intestine. Dispersed
Table 6. Immune responses to Bio-Mos® supplementation to nursery pig diets.
Control Bio-Mos ® SEM P=
Leukocyte, %
Neutrophils 52.8 45.3 2.7 0.08
Lymphocytes 42.8 50.7 2.0 0.03
Macrophage phagocytosis (lamina propria)
Average No. SRBC phagocytosed 2.31 2.63 0.11 0.05
CD14+ macrophages, % 6.9 13.5 1.6 0.10
C.V. Maxwell 19
throughout the epithelial layer of the Peyer’s patch combination of consistent quality and competitive
are specialized epithelial cells, termed M price. Demand for plasma protein is high and supply
(membranous) cells, which function to pinocytose is limited, therefore plasma is an expensive protein
and transport macromolecules from the intestinal source for nursery diets. Also, regulatory constraints
lumen into the subepithelial tissue, delivering that prohibit the use of plasma protein in many
antigenic molecules to leukocytes within the Peyer’s countries may affect the use of bovine plasma in the
patch. The extraction of Bio-Mos® from the lumen US. Similarly, increased demand and decreased
of the small intestine by the M cells of the Peyer’s supply of fish meal has resulted in increased price
patch and its exposure to the immune cells located volatility and relatively high current prices.
there, may be the impetus for a cascade of Preventing intestinal damage or atrophy
immunomodulatory events that develop and enhance immediately post-weaning caused by reduced feed
immune function, both locally in the gastrointestinal intake and lack of stimulation of the intestinal
tract as well as systemically, as cells migrate out of epithelium by ingested particles has been suggested
the gastrointestinal tissue into the periphery. as important in maintaining growth performance in
Because mannan oligosaccharides have been nursery pigs (Cera et al., 1988; Dunsford et al.,
documented to alter bacterial populations within the 1989). However, there are many other factors,
intestinal tract (Spring et al., 2000), another including removal of beneficial factors from sow’s
explanation for the alterations in immune function milk, diet form, stress, invasion by microorganisms,
observed in these studies may be from changes or introduction of allergenic compounds in the
elicited in the enteric microbial population by the nursery diet, that may also contribute to intestinal
presence of mannans in the luminal environment of atrophy. Glutamic acid and nucleotides may be
the intestinal tract. The microflora present in the important nutrient sources for maintaining gut
gastrointestinal tract are known to be a factor in the integrity during the early nursery period.
development of the young pig’s immune system, both NuProTM 2000 is a protein source high in crude
enterically and systemically (Gaskins, 1997), and the protein (51 to 55%) and digestible amino acids that
alteration of these microbial populations by Bio-Mos® has potential as a possible alternative protein source
could have an impact on the progression of immune in nursery diets. NuProTM is also high in glutamic
system development. acid and is an excellent source of nucleotides. Several
animal-based specialty feed ingredients have been
developed to compete against the animal plasma and
NuProTM 2000 fish meal market share. However, NuPro TM is a
vegetable-based peptide product which may have
Pigs produced in conventional intensively managed greater international market appeal compared to
swine production systems are routinely weaned at 19 products originating from animal by-products and
to 21 days of age and as early as 10 to 14 days of age the high level of nucleotides may be uniquely
in off-site SEW systems. At this age, pigs are very beneficial to the early-weaned pig
sensitive to the source of dietary protein. Many dietary A study has been completed at the University of
proteins produce allergic reactions in which diarrhea, Arkansas involving a total of 216 pigs to evaluate
reduced growth and increased mortality can occur the efficacy of feeding NuPro™ as an alternative to
(Bimbo and Crowther, 1992). Various protein sources plasma protein in nursery pig diets (9 pens/treatment).
have been tested in early-weaned pig diets in an Three dietary treatments were fed from day 0 to 7
attempt to overcome these problems and to decrease after weaning (Phase 1) and day 7 to 21 after weaning
diet cost. Spray-dried plasma protein is a protein (Phase 2) and were comprised of 1) a basal diet
source that has consistently been shown to improve consisting of a complex nursery diet containing spray-
performance of early-weaned pigs when included in dried plasma protein devoid of NuProTM, 2) the basal
Phase 1 (day 0 to 14 post-weaning) diets at the diet with 50% of the plasma protein replaced by
expense of dried skim milk (Hansen et al., 1993; NuProTM, and 3) the basal diet with 100% of the
Kats et al., 1994; de Rodas et al., 1995), soybean plasma protein replaced by NuProTM. During Phase
meal (Fakler et al., 1993; Coffey and Cromwell, 3 (day 21 to 42 after weaning) a common diet was
1995; de Rodas et al., 1995), and whey (Hansen et fed to groups previously receiving Treatments 1 and
al., 1993). Select grade menhaden fish meal has also 2. Half of the pigs previously fed Treatment 3 were
been a widely utilized protein source due to a fed the common diet received by the Treatment
groups 1 and 2; while the other half were fed a diet to the feed/food industries. Although inorganic
containing 1.3% NuProTM during the first week of selenium (sodium selenite) has routinely been added
Phase 3 (day 21 to 28) followed by the common diet to most animal diets, research has shown that about
for the remainder of the phase (day 28 to 42). During 60% of it is excreted in the urine. Organic selenium
Phases 1 and 2, no significant differences were (selenium enriched yeast) is an effective source of
observed among the four dietary treatments with selenium as it is more effectively retained in muscle,
regard to ADG, ADFI, or G:F (Table 7). During the milk, and fetal tissues than inorganic selenium and
first week of Phase 3, pigs previously fed the basal less is excreted. Accumulation in tissues provides a
diet containing plasma protein and fed NuProTM at selenium reserve that can be used under conditions
the 50% replacement level had lower (P=0.07) ADFI of stress for additional synthesis of selenoproteins
than pigs previously fed NuPro TM at the 100% essential for counteracting adverse effects of free
replacement level and pigs fed NuProTM at 1.3% of radicals. Organic selenium is also transferred into
the diet during the first week of Phase 3. This study the egg more efficiently and into embryonic tissues
indicates that NuProTM maybe used as an alternative in mammals via improved placental transfer when
to spray-dried plasma protein in nursery pig diets, compared to sodium selenite. This provides the young
and the removal of NuProTM from the diet does not animal with higher selenium stores, which can
result in decreased feed intake as is often the case promote improved disease resistance. In addition to
with the removal of plasma protein. This study the benefits to livestock species, higher tissue levels
indicates that NuPro™ may be an effective of organic selenium may offer health benefits to
replacement for plasma protein. consumers who choose Se-enriched animal products.
Although somewhat controversial, increased
selenium intake has been associated with reductions
Organic selenium from yeast in cancer risks in epidemiological studies (Vogt et
al., 2003), animal models (Popova, 2002) and
Organic selenium offers several major opportunities chemopreventive studies (Combs et al., 2001; Clark
Table 7. Efficacy of NuProTM in early-weaned pig diets.
Group 1 Group 2 Group 3 Group 4
Trait Control 50% 100% 100% SE P>F
NuProTM NuProTM NuProTM
ADG, g
Phase 1 47 45 31 35 10 0.60
Phase 2 399 402 429 404 11 0.19
Phase 1-2 282 283 296 281 9 0.58
Phase 3 623 590 626 621 12 0.17
Phase 1-3 451 437 461 450 8 0.20
ADFI, g
Phase 1 118 113 99 116 7 0.25
Phase 2 473 475 497 465 15 0.47
Phase 1-2 355 354 365 349 11 0.78
Phase 3 1009 962 1037 1002 19 0.08
Phase 1-3 680 658 701 675 13 0.18
Gain:feed
Phase 1 0.393 0.376 0.266 0.272 0.077 0.53
Phase 2 0.845 0.850 0.868 0.874 0.012 0.25
Phase 1-2 0.795 0.799 0.816 0.810 0.011 0.54
Phase 3 0.615 0.615 0.603 0.613 0.008 0.71
Phase 1-3 0.662 0.665 0.658 0.663 0.006 0.87
Weight, kg
Initial 6.45 6.45 6.45 6.45 0.005 0.86
Phase 1 6.78 6.77 6.67 6.69 0.07 0.62
Phase 2 12.37 12.39 12.67 12.35 0.18 0.57
Phase 3 25.44 24.79 25.81 25.51 0.34 0.22
Group 1: days 1-21, basal diet; days 21-42 common diet.
Group 2: days 1-21, basal with 50% NuProTM replacement; days 21-42 common.
Group 3: days 1-21, basal with 10% NuProTM replacement; days 21-42 common.
Group 4: days 1-21, basal with 100% NuProTM replacement; days 21-28 1.3% NuProTM; days 28-42 common.
C.V. Maxwell 21
and Marshall, 2001). Should additional studies disposal system and allowed the accumulation of the
underway prove conclusive, this presents the livestock two types of manures for application on watersheds.
and poultry industries with an opportunity to provide Table 8 provides the average total and soluble
additional health benefits to the public consuming phosphorus analysed in the holding ponds. The 24.8%
animal products. reduction in total phosphorus is consistent with the
magnitude of reduction observed in phosphorus
balance studies with pigs to determine the magnitude
Environmental impact of concentrated of reduction of phosphorus expected by feeding
animal feeding units reduced phosphorus diets with added phytase. The
magnitude of reduction in soluble phosphorus was
Another major problem facing the feed/food industry only 8.95%, suggesting that a higher percentage of
is the concentration of nutrients in modern livestock the phosphorus from pigs fed phytase was in the soluble
and poultry production systems. Northwest Arkansas form. This is consistent with other observations that
contains the headwaters for two scenic rivers and is phytase increases soluble phosphorus in manure.
also the location of a major concentration of animal
production, primarily poultry. Disposal of the Table 8. Phosphorus concentration in holding ponds (mg/L).
concentrated animal waste, which accumulates in Item Normal P Phytase P Reduction (%)
efficient production systems, in a manner that
minimizes odor and optimizes nutrient utilization is Total P 289.7 217.9 24.8
an increasing problem facing the livestock and poultry Soluble P 138.4 126.0 8.95
industries in our state. Animal waste can be a valuable N = 6 samples per manure source
resource as an alternative source of fertilizer nitrogen
(N), phosphorus (P), and potassium (K) in The reduced risk of phosphorus runoff from
maintaining and restoring soil productivity. In fact, watersheds receiving manure from phytase-treated
by improving ground cover, runoff volume and pig diets in relation to manure from pigs fed normal
erosion may also be reduced. Conversely, application phosphorus, non-phytase diets was also demonstrated.
of animal manure at rates greater than a crop can Concern over water quality near animal production
utilize has been shown to result in nitrate (NO3) facilities is primarily with regard to transport of
movement through the soil into ground water and excessive amounts of N and(or) P from the animal
can result in an excessive rise in soil P levels, leading waste.
to increased phosphorus runoff. This can be a problem A third watershed evaluated the efficacy of
since phosphorus is normally the limiting nutrient aluminum chloride (AlCl3) addition to swine manure
for eutrophication in freshwater systems. Odor and on runoff. Shreve et al. (1995), Moore et al. (1995)
nutrient problems can both be exacerbated by and Smith et al. (2001) recommended treatment of
excessive nutrient buildup in lagoons/holding ponds manure with aluminum chloride as a means of
that have not been dewatered in a timely manner. reducing both P and NH3 losses. Runoff of nutrients
With the initial population of the new University was compared to a watershed that received no manure
of Arkansas 2000 head/year finishing facility, a or fertilizer. The watershed sites were designated:
decision was made to demonstrate the use of dietary
phytase addition to substantially reduce phosphorus 1. No manure or fertilizer application
production in swine manure without affecting swine
performance or profitability. Facilities were 2. Phytase manure: Low P diet, high N, but low P
constructed to permit production of two types of loading, lower risk of P runoff.
manure that was stored in holding ponds. Pigs placed 3. Normal manure: Normal P diet , high N and P
in half of the pens received normal phosphorus diets loading on pasture, high risk of P runoff.
devoid of phytase and pigs placed in the other half
of the facility received diets with reduced phosphorus 4. Phytase manure: Low P diet, high N, but low P
supplemented with phytase. The holding ponds were loading, aluminum chloride added to reduce
managed by emptying the shallow pit under the pigs soluble phosphorus and lower risk of P runoff.
on each diet on a weekly basis and recharging the pit
with effluent from the top of the holding pond. This Manure was transported from the respective holding
simulated the management of a pull-plug waste basins and applied to two separate pastures in multiple
applications at rates equivalent to a target of 150 lb or watersheds fertilized with phytase manure or
N/acre/year. Manure from pigs fed the reduced phytase manure with AlCl3 (Table 11). This total mass
phosphorus diet with added phytase was also treated of P loss in the watershed fertilized with normal
with aluminum chloride by adding 0.75% aluminum manure also represented the greatest percentage of
chloride to the manure prior to application. This was applied total P lost among the watersheds (5.4 vs 3.6
added to a third watershed at the same application and 4.9% for the watersheds treated with phytase
rate used in the other watersheds. A fourth watershed manure and phytase with AlCl 3, respectively).
had no manure or fertilizer added. A total of three Application of manure from pigs fed phytase, with
applications were made during the project. A ‘Small or without treatment with AlCl3, reduced the mass of
In-field Runoff Collector’ system was used to collect soluble and total P runoff. In fact, the watershed
runoff water. Samples from each watershed and storm treated with phytase manure produced the lowest total
event were composited and analyzed for total Kjeldahl P and percentage of soluble and total P runoff among
N, total P, soluble P, NH3-N, NO3-N, copper and zinc. the watersheds, even lower than that observed in the
Total runoff data are presented in Table 9. The unfertilized watershed. It should be noted, however,
watershed that received no manure or fertilizer that runoff volumes were variable between
produced the greatest total runoff with 191,344 liters. watersheds, which had an impact on the total mass
This might be expected since reduced forage cover of nutrients lost from the runoff events. When
may increase runoff. The watershed receiving the comparing the mass of nutrients applied to that which
normal phosphorus manure was next with 179,028 was lost through runoff, it is important to note that
liters followed by the watershed receiving AlCl3 the vast majority of nutrients remained within the
treated manure from pigs fed phytase (162,418 liters). watershed. In general, more than 90% of the nutrients
The watershed receiving untreated manure from pigs applied remained in the watershed. The exceptions
fed phytase had the lowest total runoff of 112,826 to this are the percentage of soluble P lost from the
liters. watershed receiving the normal P manure (12.7%)
and the percentage of soluble P lost from the
Table 9. Total runoff. watershed receiving the phytase manure with AlCl3
Treatment (Liters) (60.6%). A higher percentage of soluble nutrients
Watershed 1 No manure 191,344 were lost through runoff, because the soluble fraction
Watershed 2 Phytase 112,826 is fairly dynamic, and is also more susceptible to
Watershed 3 Normal P 179,028 runoff losses than the total fraction. There was more
Watershed 4 Phytase+AlCl3 162,418 soluble P removed from the phytase manure with
AlCl3 watershed than was applied from the manure,
most likely due to the natural loss of soil P as seen in
The total nutrients applied to the watersheds in the
the unfertilized watershed. The N lost from these
three applications are presented in Table 10. The
watersheds was a very small fraction of what was
application of total N was approximately 150 lbs of
applied, ranging from 0.6% to 1.2%.
total N/acre/year. The addition of aluminum chloride
to the swine manure also substantially reduced the Table 11. Mass of nutrients lost from watersheds and percentage
soluble phosphorus at the time of manure application, of applied nutrients lost.
as expected.
Treatment Soluble P Total P Total N
Table 10. Nutrients applied to soil from manure by treatment (lb/acre) (%) (lb/acre) (%) (lb/acre) (%)
(lb/ac).a
Treatment Soluble P Total P Total N Unfertilized 0.89 1.26 1.84
Phytase diet 0.92 7.2 1.12 3.6 1.39 0.6
Unfertilized n/a n/a n/a Normal diet 1.30 12.7 1.65 5.4 2.38 1.2
Phytase diet 12.7 31.5 230 Phytase + A1C13 0.97 60.6 1.31 4.9 2.28 1.0
Normal diet 10.2 30.4 205
Phytase + AlCl3 1.6 26.6 240
a
Represents three applications over 1.5 years. Zinc concentrations from runoff were very low
(Figure 6). Manure had no apparent impact on metal
The mass of soluble and total P lost from the watershed runoff when applied as a fertilizer resource to the
fertilized with normal manure was greater when watersheds. Copper concentrations in runoff were also
compared to the runoff in the unfertilized watershed very low, in the ppb range, and were not affected by
the addition of manure to the watersheds (Table 12).
C.V. Maxwell 23
0.14
0.12
0.10
Zinc (mg/L)
0.08
0.06
0.04
0.02
0.00
Unfertilized Phytase Normal Phytase with
AlCl3
Figure 6. Effect of manure fertilization on zinc concentration in runoff.
Table 12. Concentration of copper lost in runoff by treatment. Table 13. Farm phosphorus balance.
Treatment Cu (µg/L) lbs %
1. Unfertilized 2.5 Total P delivered in feed 8,222

2. Phytase Diet 2.3 P removed in pigs marketed 2,507 30.5
3. Normal Diet 3.6 P in manure spread 1,616 19.6
4. Phytase + A1C13 3.5 Residual 4,099 49.8
Table 14. Farm nitrogen balance.

One of the objectives of this project was to conduct a
lbs %
phosphorus and nitrogen budget for the farm. A total
of 8,222 lb of phosphorus was delivered to the farm Total N delivered in feed 40,839
and an estimated 2,507 lbs (30.5%) was removed in N removed in pigs marketed 11,680 28.60
N in manure spread 3,655 8.94
pigs marketed or retained in pigs kept as replacement
Residual 25,504 62.45
breeding stock (Table 13). A total of 1,616 lbs
(19.6%) was spread on 88 acres for an average
application rate of over 12 lbs of total P/acre/yr, This study demonstrates that even with judicious
which exceeds the P needed for forage production management, phosphorus in the soil accumulates with
for grazing or hay. The amount of total N delivered application of swine manure based on plant
to the farm was 40,839 lb (Table 14). An estimated requirements for N in forage-based systems.
11,608 lbs (28.60%) was removed in pigs marketed Construction of new production facilities should only
or retained in pigs kept as replacement breeding stock. be considered after development of nutrient
A total of 3,655 lb. (8.94%) was spread on 88 acres management plans ensuring application of nutrients
for an average maximum application of 41.50 lbs of that do not exceed crop needs. Technologies to further
N/acre. If one obtained the expected ammonia loss reduce phosphorus in manure would reduce the land
from volatilization of 25%, then the actual applied base needed for concentrated animal production
N would be about 31 lb/acre, which is probably below facilities.
the crop needs for either the bermuda or fescue In areas where nutrient excesses exist, progress is
pastures where manure was applied. The residual N being made in developing technologies to address
is most likely much less than the calculated residual the problem and some are even receiving praise for
since ammonia volatilization from the production delivering both environmental and economic benefits.
facility and holding ponds is likely to be substantial. Singled out in the popular press recently is the manure
management system in the Chino Basin in Southern
California, which utilizes a digester to convert dairy Combs, Jr., G. F., C. L. Clark and B. W. Turnbull.
manure into fertilizer and methane used for power 2001. An analysis of cancer prevention by selenium.
generation. Smithfield Foods is constructing a $20 Biofactors 14: 153-9.
million system to convert swine manure into liquid de Rodas, B. Z., K. S. Sohn, C. V. Maxwell, and L.
methanol to be used in the production of biodiesel. J. Spicer. 1995. Plasma protein for pigs weaned at
Success of these systems is critical for maintaining 19 to 24 days of age: effect on performance and
good relationships in communities with large livestock plasma insulin-like growth factor I, growth
production facilities. Why would every state not want hormone, insulin, and glucose concentrations. J.
to have a $7 billion poultry industry like Arkansas? Anim. Sci. 73:3657.
Dunsford, B. R., D. A. Knabe and W. E. Haensly.
1989. A comparison of dietary soybean meal on
Conclusions the microscopic anatomy of the small intestine in
In summary, it appears there will be increased the early-weaned pig. J. Anim. Sci. 67:1855-1863.
emphasis on food safety that will require dramatic Elam, T. E., 2004. Meeting growing meat demand
changes in the way national and international feed/ for the future while protecting environment will
food companies operate. This has benefits not only be a challenge. Feedstuffs. 76(No. 4):23-28.
from a food safety standpoint, but as we develop a Fakler, T. M., C. M. Adams, and C. V. Maxwell.
better understanding of controlling microorganisms 1993. Effect of dietary fat source on performance
in the environment, we may also tremendously and fatty acid absorption in the early-weaned pig.
improve animal health and performance and should J. Anim. Sci. 71(Suppl. 1):174(Abstr.).
have a number of alternatives to antibiotics. The Gaskins, H. R. 1997. Immunological aspects of host/
impact of an animal’s interaction with microorganisms microbiota interactions at the intestinal epithelium.
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greater than once thought. Finally, it is evident that (Eds.), Gastrointestinal Microbes and Host
environmental and animal welfare issues will have Interactions, Vol. 2, Chapman and Hall, New York,
increasing influence on decision making. pp. 537-587.
Gaskins, H. R.. 2001. Intestinal bacteria and their
influence on swine growth In: Swine Nutrition 2nd
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oil: Current uses. J. Am. Oil Chem. Soc. 69:221. L. Weeden. 1993. Evaluation of animal protein
Bolduan, G. 1999. Feeding weaner pigs without in-
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Symposium. (T.P. Lyons and K.A. Jacques (eds). P Growth performance, intestinal microbial
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Johnson, R.W. 1997. Explanation for why sick pigs
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K.E. Newman 27
Glycomics: putting carbohydrates to work for animal and human health

KYLE E. NEWMAN
Venture Laboratories, Inc., Lexington, Kentucky, USA
Introduction
In October of 2001 bioterrorism came to the United scientists studying proteins and lipids by being
States in the form of letters containing anthrax spores. associated with these compounds and subsequently
Imagine a time in the very near future when simply needing to be stripped away in order to truly
eating a carbohydrate fraction from yeast can protect understand the function of the protein. However, it
you from such an attack. It is not as far-fetched as turns out that the glycosylation of these compounds
you may think. A recent trial in mice found that mice can define their function or serve to stabilize them.
receiving yeast glucans for 1 week prior to anthrax A good example of this stabilization is industrial-
infection had double the survival rate of grade enzymes, where shelf-life and heat stability
unsupplemented animals. As a therapeutic agent, mice have been enhanced by glycosylation of the protein.
receiving yeast glucans had a 90% survival rate Unlike amino acids or nucleic acids that have a
compared to 30% survival for control animals certain predictability to their structure, there is no
(Kournikakis et al., 2003). simple code for determining the structure of complex
In February of 2003, the magazine Technology sugars. The biological diversity of these compounds
Reviews identified glycomics as one of the “10 can be easily demonstrated by examining the
Emerging Technologies That Will Change the World.” difference between α- and ß-bonded (1-4) glucose
Glycomics is defined as the characterization of the units (Figure 1). When these two glucose units are
sugars and the structure of these sugars that make up bound in the α configuration, the resulting
a cell. We are all aware of the quest to define the compound, amylose, is easily degraded by starch-
human genome (genomics: the full DNA degrading enzymes found in saliva. Conversely,
complement) that was recently completed. Many are ß-bonded (1-4) glucose represents cellobiose, a
aware of proteomics, which is the study of the full compound that is not degraded by any mammalian
set of proteins encoded by a genome, but very few enzyme system. This exemplifies the difference in
people are aware of glycomics. Putting these sciences biological activity of the same two glucose molecules
in perspective, genomics was child’s play compared bound together at the same site with the only
to the undertaking of proteomics, which is dwarfed difference being the type of bond between them.
in comparison to glycomics. Compared to DNA or amino acid linkages, which
are somewhat linear, imagine the complexity when
you consider that a hexose molecule (like glucose)
New roles for carbohydrates has six binding sites, can branch and the bonds can
orient in different ways (as seen in Figure 1). The
At one time, it was thought that there were three possibilities for oligo- or polysaccharides can be a
main roles of carbohydrates in biological systems. bit overwhelming.
The most obvious role of sugar is as an energy source Complex carbohydrates have become a prominent
or storage component for energy reserves. The second research topic with the realization that distinct
function is as a structural component such as cellulose carbohydrate structures can have very specific
or chitin. The third function seemed to be to confound biological activities. One need only imagine the
28 Glycomics: putting carbohydrates to work for animal and human health
CH2OH CH2OH H OH
CH2OH
O O H O OH
H H H H OH H
O
α-D GLUCOSE α-D GLUCOSE β-D GLUCOSE β-D GLUCOSE
OH H O OH H O OH H H H H H
O OH
β-(1-4) linkage
O
α-(1-4) linkage
H OH H OH H OH CH2OH
Amylose Cellobiose
Figure 1. α- and ß-bonded (1-4) glucose units.
diverse nature of carbohydrate chemistry to differentiation from bone marrow in supplemented

understand that the opportunities for novel compounds mice prevented the femoral bone loss that is normally
with unique biological activity are boundless. In fact, attributed to estrogen deficiency (Nishizaki et al.,
the diversity and complexity of these compounds has 2000). The most striking finding of this study is that
kept investigators from fully understanding them until the improvements seen were not due to boosting
recently. Carbohydrates and oligosaccharides are also estrogen levels (the normal therapy for menopause-
now being utilized as a nutritional means of enhancing induced osteoporosis). This could be of enormous
immune function. benefit since the usual therapy to prevent osteoporosis
Trehalose is a disaccharide of two glucose is supplemental estrogen, which has been linked with
molecules linked by α(1,1) linkages (Figure 2). This certain cancers (Stalhberg et al., 2004).
compound is a non-reducing sugar that does not react
with amino acids or proteins, making it unaffected
by Maillard reactions. When incorporated into Carbohydrate-related diseases
materials prior to freezing, trehalose has shown an
ability to prevent ice crystallization damage to the The role of carbohydrates in health and disease is
molecule. This phenomenon has been exploited to coming out of a blur and into focus, but we are only
increase the shelf life of materials ranging from foods observing the infancy of this field of study. In 2002,
to probiotic preparations. The role of trehalose is a Harvard researcher proposed that an immune
not limited to extending shelf life. It has been shown response to the complex carbohydrate glycosamin-
that mice supplemented with 2% trehalose had oglycan (GAG) is a potential cause of rheumatoid
reduced symptoms of Huntington disease (Tanaka et arthritis (Wang and Roehrl, 2002). Rheumatoid
al., 2004). Huntington’s disease is a rare, hereditary arthritis is a systemic autoimmune disease of
neurological illness characterized by sporadic and connective tissue; and GAGs are a major component
involuntary muscle movements. The disease affects of that tissue. This study was unique in that it
approximately 1 in 10,000 people. demonstrated a direct link between human disease,
carbohydrate antigens and the immune system.
Congenital disorders of glycosylation (CDG), also
CH2OH H OH known as ‘carbohydrate-deficient glycoprotein
H
O
H H H syndrome,’ are a group of inherited disorders where
OH H
many glycoproteins are deficient in the carbohydrate
OH
OH H
O
HOH2C fraction of the compound. Adults and children with
OH
O
CDG have varying degrees of disabilities such as
H OH H speech and cognitive difficulties, poor balance and
Figure 2. Trehalose, a non-reducing disaccharide. impaired motor skills. Several human diseases are a
result of faulty carbohydrate metabolism, the most
Trehalose has also been examined as a possible therapy common of these being diabetes, a condition
in osteoporosis. Ovariectomized mice receiving 100 characterized by abnormally high levels of blood
mg/kg of trehalose had suppressed osteoclast glucose from a failure in glucose transport from the
differentiation compared to unsupplemented blood into the cells. Another condition related to
ovariectomized mice. The improved osteoclast errors in carbohydrate metabolism is Tay-Sachs
disease, an inherited disorder caused by a recessive
K.E. Newman 29
defect in the gene encoding for hexosaminidase A. proteases (MASP and MASP2), which ultimately lead
This leads to an unusual accumulation of ganglioside to antibody independent activation of the classical
GM2 in the central nervous system. Characteristics pathway of the immune system (Roitt et al., 1998).
of the disease include blindness, seizures, a Bacterial infection is due in many cases to the ability
degeneration of motor and mental function with early of the bacteria to recognize host cell surface sugars
death in childhood. While most of the defined diseases and use specific receptors that allow them to attach,
of sugar metabolism are relatively rare, many of the colonize, and in the case of pathogens, cause disease
so-called genetic diseases with unknown causes may in the animal. Mannose-specific adhesins (the binding
be caused by errors in glycosylation because of the entity on the surface of bacterial cells) are utilized
importance of glycosylation in cell-to-cell interaction by many gastrointestinal pathogens as a means of
and the role of glycomics in the immune system. attachment to the gut epithelium. One way to prevent
pathogens from causing disease is to prevent them
from attaching to the epithelial cells in the gut. Early
Carbohydrates, cell-to-cell studies using mannose in the drinking water of broiler
communications and defense against chicks demonstrated that this therapy could reduce
colonization rate of Salmonella typhimurium.
pathogens Purified mannose and a complex sugar called mannan
Carbohydrates are important surface entities of oligosaccharide (MOS) have been successfully used
animal cells that function in a variety of ways to to prevent bacterial attachment to the host animal by
influence cell-to-cell communication, impact the providing the bacteria a mannose-rich receptor that
immune system and allow bacterial attachment to the serves to occupy the binding sites on the bacteria and
host. These complexed molecules project from the prevent colonization in the animal.
cell surface and form the antigenic determinants of Several studies have been conducted examining the
certain cell types. One of the classical examples of role of mannans and their derivatives on binding of
this antigenicity is blood type in humans. The ABO pathogens to epithelial cells in the gastrointestinal
blood group antigens are glycoproteins on red blood tract. E. coli with mannose-specific lectins did not
cells. Small differences in the terminal sugar residues attach to mammalian cells when mannose was present
distinguish the A and B blood-group antigens (Kuby, (Salit and Gotschlich, 1977). Spring and coworkers
1994; Figure 3) Mannose binding protein (MBP) is (2000) used a chick model to demonstrate that MOS
an integral part of the immune system. MBP in the (Bio-Mos ® ) could significantly reduce the
serum can bind to terminal mannose groups on the colonization of Salmonella and E. coli. Animal trials
surface of bacteria and interact with two serine in other species show similar benefits in reducing
Galactose
Glucose
Lipid or protein
Fucose N-acetylglucosamine
O antigen
N-acetylgalactosamine
A antigen B antigen
Figure 3. Differences in the terminal sugar residues distinguish the A and B blood group antigens.
pathogen concentrations. In dogs, as well as in poultry, by the virus that frees the virus from the sialic acid.
reductions in fecal clostridial concentrations have also By tying up this enzyme, the virus cannot easily
been noted with Bio-Mos® supplementation (Finucane spread and infect other cells (Schmidt, 2002). There
et al., 1999; Strickling, 1999). are also data examining a novel anti-human
Fructo-oligosaccharides (FOS) have been immunodeficiency virus (HIV) protein. This protein,
investigated for nutritional manipulation of the called actinohivin, binds to a glycoprotein on various
gastrointestinal tract to inhibit pathogens. The principle HIV strains and simian immunodeficiency virus
behind the use of FOS involves the structure and (SIV) inhibiting viral entry into cells by binding to
bonding of the fructose molecules. Purified this envelope glycoprotein. Further investigation
preparations of FOS have been shown to provide a showed that only yeast mannan can inhibit the
nutrient source for beneficial bacteria such as binding of actinohivin to these viruses. These results
bifidobacteria and certain lactobacilli. By supporting demonstrate that the mannose saccharide chains of
the growth of the beneficial bacteria it is thought that the virus glycoprotein are the molecular targets of
this will provide an in situ competitive exclusion (CE) the anti-HIV activity of actinohivin (Chiba et al.,
effect, thus improving animal health. However, it 2004). Sulfated galactomannans also demonstrate in
seems important that the concentration of non- vitro and in vivo activity against the flaviviruses,
complexed fructose molecules be kept to a minimum yellow fever virus and dengue virus (Ono et al.,
in order for this oligosaccharide to be successful. 2003). West Nile virus has also gained a strong
Oyarzabal and coworkers (1995) found that foothold in the United States, affecting birds, horses
Salmonella spp. could not use a purified FOS and man. N-linked sugars with mannose residues on
preparation for growth but were able to utilize a the cell membrane protein were found to be
commercial preparation of FOS. The authors suggest important in West Nile virus binding to the cell (Chu
the use of lactic acid bacteria in combination with and Ng, 2003).
FOS as a feasible approach to control Salmonella. The future of the science of glycomics seems
Other studies have demonstrated a reduction in enormous at this time. While mannan oligosaccharide
Salmonella concentrations in birds challenged with is currently being used to improve health and
S. typhimurium with and without FOS and a CE production of animals, there are enormous
culture. FOS alone had little effect on Salmonella possibilities to use other sugars as possible agents
exclusion when FOS was administered after infection, against pathogen infection. Table 1 shows a partial
but FOS in combination with a defined CE product summary of scientific studies examining bacterial
had an additive effect on Salmonella exclusion adhesins.
especially when used as a prophylactic prior to
infection (Bailey et al., 1991). However consistent Table 1. Carbohydrate adhesins of various bacterial strains1.
response of animals to FOS supplementation is a Bacterial strain Expressing mannose Other carbohydrate
problem and may affect other as yet undefined adhesins (% of adhesins
interactions. Waldroup and coworkers (1993) found that those examined)
supplementing broilers with 0.375% FOS had few Campylobacter coli 0 Glucose
consistent effects on production parameters or carcass Campylobacter jejuni2 0 Glucose
Clostridium spp.2 0 Galactose, glucose,
Salmonella concentrations. These authors also caution lactose
of possible antagonism between FOS and BMD. Edwardsiella ictaluri 100 Not known
Human data for FOS are much more consistent. Enterobacter cloacae 100 Not known
Hidaka et al. (1986) found that consumption of 8 g Escherichia coli2 53 Fucose, galactose,
glucose
FOS/day increased numbers of bifidobacteria, Fusobacterium spp. 0 Galactose, lactose,
improved blood lipid profiles and suppressed raffinose
putrefactive substances in the intestine. Haemophilus influenzae 0 Galactose, glucose
Glycomics also plays a vital role in viral diseases. Klebsiella pneumoniae 100 Glucose
Salmonella spp.2 64 Fucose, galactose
The influenza virus infects by first attaching to a cell Serratia marcescens 100 Not known
surface carbohydrate called sialic acid. This Shigella spp. 75 Fucose
attachment ‘opens the door’ of the cell and allows the Streptococcus bovis 0 Glucose
virus to replicate within. The commercial drugs Streptococcus suis 0 Galactose
Tamiflu and Relenza shorten the duration of the flu 1
Summarized from Mirelman et al., 1980; 1986; Ofek et al.,
by binding to the active site of an enzyme produced 2003
2
In vivo data have shown reductions in these populations with
Bio-Mos®.
K.E. Newman 31
Conclusions microbial lectins and agglutinins. In: Microbial

Lectins and Agglutins-Properties and Biological
To say that carbohydrates are involved in virtually Activity. (D. Mirelman, ed). John Wiley & Sons,
every aspect of biology is not an understatement. Inc. NY.
Finding ways to exploit this knowledge is the current Nishizaki, Y., C. Yoshizane, Y. Toshimori, N. Arai,
challenge. The vast array of possibilities that exist S. Akamatsu, T. Hanaya, S. Arai, M. Ikeda and M.
with polysaccharide structure and function make Kurimoto. 2000. Disaccharide-trehalose inhibits
glycomics a science that may well pass on to future bone resorption in ovariectomized mice. Nut. Res.
generations. We have only scratched the surface, but
20:653-664.
we have a better understanding of arthritis, how the
Ofek, I., D.L. Hasty, R.J. Doyle. 2003. In: Bacterial
immune system works in identifying invasion, how
Adhesion to Animal Cells and Tissues. ASM Press.
certain congenital disorders debilitate and we have
used our limited knowledge to take advantage of the Washington, D.C.
‘sweet tooth’ of pathogens to control infection. It Ono, L., W. Wollinger, I.M. Rocco, T.L. Coimbra,
brings a whole new meaning to ‘a spoonful of sugar P.A. Gorin, M.R. Sierakowski. 2003. In vitro and
helps the medicine go down’. in vivo antiviral properties of sulfated
galactomannans against yellow fever virus (BeH111
strain) and dengue 1 virus (Hawaii strain). Antiviral
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Bailey, J.S., L.C. Blankenship and N.A. Cox. 1991. 1995. Fructooligosaccharide utilization by
Effect of fructooligosaccharide on Salmonella Salmonellae and potential direct-fed-microbial
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70:2433-2438. Oyofo, B.A., J.R. DeLoach, D.E. Corrier, J.O.
Chiba, H., J. Inokoshi, H. Nakashima, S. Omura and Norman, R.L. Ziprin, and H.H. Mollenhauer.
H. Tanaka. 2004. Actinohivin, a novel anti-human 1989a. Prevention of Salmonella typhimurium
immunodeficiency virus protein from an colonization of broilers with D-mannose. Poultry
actinomycetes, inhibits viral entry to cells by Sci. 68:1357-1360.
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Chu, J.J. and M.L. Ng. 2003. Characterization of a London.
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that is involved in West Nile virus binding and 146:1182-1194.
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oligosaccharide on the composition of the Newman. 2000. The effects of dietary mannan
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Tashiro. 1986. Effects of fructooligosaccharides on 79:205-211.
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Focus on Poultry
POULTRY
F.W. Edens and K.M. Gowdy 35
Selenium sources and selenoproteins in practical poultry production

FRANK W. EDENS AND KYMBERLY M. GOWDY
Introduction
Nutritional needs of food animals must be met by Among the ultramicronutrients, selenium may hold
provision of nutrients from plants, soil, and even the distinction of being the most difficult to
from prey animals. Many of the potential nutrients understand. It can exist in four valence states: -2
are minerals that exist in chemical complexes that (hydrogen selenide, sodium selenide, dimethyl
are not readily available. This condition is selenium, trimethyl selenium, and selenoamino acids
advantageous for animals because many of the such as selenomethionine), 0 (elemental selenium),
minerals can be toxic, but nutritionists have +4 (selenium dioxide, selenous acid, and sodium
demonstrated that a large number of minerals, either selenite), and +6 (selenic acid and sodium selenate),
as macronutrients or as micronutrients, are required and depending upon its valence state and water
for normal growth and development of animals solubility, the gastrointestinal absorption rate can be
including humans. The macronutrients consist of affected. Selenium can be toxic in valence states –2,
minerals such as calcium, phosphorus, sodium, and +4 and +6, but in appropriate trace levels in feed or
potassium because these are involved in structural drinking water, selenium in valence states –2, +4 and
integrity of the body and in homeostatic mechanisms. +6 can also serve in several essential roles in
Micronutrients, often called trace nutrients, include maintenance of the body’s homeostatic condition.
minerals such as magnesium, manganese, zinc, iron,
copper, and there are ultramicronutrient minerals such
as molybdenum, selenium, iodine, cobalt, chromium Forms and availability of selenium used
and even vanadium. The trace minerals as either in animal production
micro- or ultramicronutrients function as parts of
proteins, hormones, enzymes, or as co-factors that Selenium supplementation of poultry rations is now
activate specific enzymes. a routine procedure. Since 1974, when the US Food
Nature’s provision of nutrients to the inhabitants and Drug Administration (FDA) approved selenium
of the earth is a process that can be very variable. as a feed supplement, sodium selenite has become
Although common foodstuffs such as corn, wheat, the traditional source for dietary supplemental
barley, soybeans, or oats are now grown around the selenium for poultry and livestock (Leeson and
world, they are not the same world wide. In the case Summers, 1991). Other inorganic sources of selenium
of foodstuffs that provide selenium, some can be are sodium selenate and calcium selenate (Echevarria
enriched as the result of high concentrations of et al., 1988a,b). Poultry obtain all of their selenium
selenium in the soil. In contrast, some foodstuffs can through their feed. The commonly used feed ingredients,
have very low levels of selenium because the soil in such as cereal products (Burk, 1976) and meals of fish,
which they are grown had very low concentrations. poultry and meat (Levander 1986; Cai et al., 1995),
This situation requires that supplemental selenium contain selenium almost exclusively as organic
be provided via the manufactured feed to ensure compounds such as the naturally occurring selenoamino
optimal performance of food production animals. acids (selenomethionine and selenocysteine). The
selenoamino acids are incorporated into protein,
36 Selenium sources, selenoproteins and practical poultry production
principally as selenomethionine and selenocysteine, The availability of selenium from different sources
and constitute 50 to 80% of the total selenium in such as selenomethionine or inorganic selenium from
plants and grains (Butler and Peterson, 1967). selenite or selenate may be affected by numerous
Estimates of bioavailability of selenium for poultry factors. Cantor and Johnson (1985) concluded that
vary considerably depending upon which criteria are selenium was made more available in diets that were
used for the evaluation. Scott and Thompson (1971) low in protein, possibly as a result of decreased total
determined that there was a linear relationship in methionine in the diet. The kind and quality of fish
tissue deposition of dietary inorganic selenium up to meal that has been used in poultry diets also influences
0.3 mg/kg. Cantor et al. (1975a,b) have reported the availability of selenium for poultry (Miller et
diverse and variable results when exudative diathesis al., 1972; Whitacre and Latshaw, 1982). Furthermore,
or pancreatic fibrosis were used to evaluate selenium intestinal absorption of selenium in foodstuffs varies
bioavailability. In both studies, plant-based selenium by species, and bioavailability varies according to
sources were superior to inorganic sources, but there the form that is eaten (Gabrielsen and Opstevedt,
were low protective values associated with plant- 1980; Douglas et al., 1981; Ringdal et al., 1985; Wen
based organic selenium in terms of protection against et al., 1997; Schen et al., 1997). Schen et al. (1997)
development of exudative diathesis (Cantor et al., reported that selenomethionine and sodium selenate
1975a). In contrast, Osman and Latshaw (1976) are more diffusible than selenocysteine and sodium
reported that selenomethionine was at least equivalent selenite, explaining the higher absorption rate for
to sodium selenite in the protection of the chick selenomethionine and sodium selenate. However,
against exudative diathesis. Cantor et al., (1975b) utilization of selenium from sodium selenate can be
reported high availability of selenomethionine in quite variable because a significant fraction may be
protection of the chick against pancreatic fibrosis. lost in urine before it can be incorporated into body
Echevarria et al. (1988a,b) indicated that sodium tissues (Xia et al., 1992).
selenite, sodium selenate, and calcium selenate are Bioavailability of selenium has been estimated by
absorbed equally well by chicks based upon tissue a tissue residue approach by numerous investigators.
distributions of selenium after short term feeding Miller et al. (1972) found selenomethionine to be
trials with high levels of the inorganic selenium more available to chicks than selenium from sodium
sources. Echevarria et al. (1988a,b) demonstrated that selenite or selenium from fish products. Cantor et
selenium concentrations ranged from highest to lowest al. (1982) observed significantly more selenium from
in kidney, liver, muscle, and plasma, respectively. selenomethionine in muscles of turkey poults than
Increasing levels of dietary inorganic selenium were selenium from inorganic selenium sources. However,
associated with greater tissue concentrations. The Cantor and Tarino (1982) showed that availability
increasing tissue concentrations of selenium were of selenium from selenite was greater than from
time dependent with liver showing weekly increases selenomethionine when plasma glutathione peroxidase
in selenium concentrations whereas selenium reached (GSH-Px) activity was used an indicator. Moksnes
a plateau within seven days of feeding in kidney, and Norheim (1986) also demonstrated greater tissue
muscle, and plasma. In a study conducted with rats, concentrations of selenium and GSH-Px activity in
Vinson and Bose (1987) found that organic selenium selenomethionine-fed chickens compared with sodium
from yeast was more available to blood and liver selenite-fed chickens. There is no question that
proteins than from either sodium selenite or an amino selenomethionine will increase tissue concentrations
acid-selenium chelate (Table 1). of selenium as compared with selenium deposition
from inorganic selenium.
Table 1. Bioavailability of selenium forms in the inorganic sodium When the FDA approved selenium supplements for
selenite, amino acid chelate, and organic selenomethionine in yeast poultry and swine in 1974, the animal industries
(Vinson and Bose, 1987).
settled on the use of inorganic selenium forms rather
Form of selenium Tissue Bioavailability, %
than the organic selenoamino acids. The primary
Sodium selenite Blood 100 reason for that decision was based on cost of the
Chelated amino acid Blood 60
Yeast Blood 138
selenium supplements and lack of information on
selenomethionine. The inorganic forms were cheap
Sodium selenite Liver 100
Chelated amino acid Liver 88 and the organic forms were expensive. Without a
Yeast Liver 147 doubt, the use of inorganic selenium supplements in
feeds has improved the performance of all classes of
commercial poultry. In modern high-yielding poultry, Schrauzer (2001) has reported that there are quality
which have higher metabolic rates and different concerns associated with many selenium supplements.
nutritional needs compared with poultry from 30 Some yeast products do not contain organic selenium.
years ago, there is a need to reassess nutrient Instead they are made with inorganic sodium selenite
requirements. Inorganic selenium has some problems or sodium selenate. Some feed supplements do not
associated with its use. Among those problems are tell which form of selenium is provided. Some
the minimal levels of selenium in meat proteins and products are made with selenium proteinates or
the potential for toxicity if too high a dietary level selenoamino acid chelates. Schrauzer also pointed out
of inorganic selenium is provided to chickens. Thus, that vitamin supplements for humans are often
a need to revisit organic selenium as a feed formulated with vitamin C and that over time, the
supplement for poultry is apparent. selenium is reduced to elemental selenium and is not
After many years of laboratory and field research, available.
a source for natural organic selenium (Sel-Plex®, Thus, it is clear that the compounders and
Alltech Inc., Nicholasville, KY), was approved for commercial nutritionists have had a difficult task in
use in the poultry industry in the US by the FDA making an informed decision concerning the
(Federal Register, 2000; 2002). Sel-Plex® provides appropriate product to use for supplementation of
a broad spectrum of selenium compounds (Kelly and organic selenium. There are many companies around
Power, 1995), but selenomethionine in the selenium- the world that produce organic selenium yeast
enriched yeast cellular protein component is the products. Each manufacturer has claimed superior
primary form of selenium in Sel-Plex®. The organic performance of food animals fed their product.
selenium profile in Sel-Plex® is similar to the organic However, little is known about speciation of selenium
selenium profile in plants and grains (Kelly and in selenized yeast products. In a recently published
Power, 1995). The organic selenium in Sel-Plex® is article, investigators, using state-of-the-art
readily available and will be absorbed actively methodologies, studied the fractionation of selenium
(Mahan, 1994; 1995) from the intestine via the Na+- in yeast into different classes of chemical species and
dependent methionine transport system (Spencer and determined the true speciation of protein-incorporated
Blau, 1962) while sodium selenite is absorbed selenium in individual yeast proteins characterized
passively by diffusion from the intestinal tract by unique amino acid sequences (Encinar et al.,
(McConnell and Cho, 1965; Schrauzer, 2001). 2003). In this study, they used three commercial yeast
Even though organic selenium in Sel-Plex® is a products. They quantified water soluble selenium,
superior source of selenium for poultry production water insoluble polysaccharide-bound selenium,
(Edens, 1996; 2002; Edens and Sefton, 2002; Edens water-insoluble protein-bound selenium, residual
et al., 2002), sodium selenite is still used as the protein-bound selenium, and residual hydrolysable
principal source of selenium in animal feeds. This selenium (Table 2). Based on these percentage values,
seems paradoxical because sodium selenite has a it is apparent that the profile of selenocompounds
documented pro-oxidant influence in all animals differs markedly, which indicates that all sources of
tested including humans (Hafeman et al., 1974; ‘selenium yeast’ are not alike. Thus, as one begins to
Csallany and Menken, 1986; Spallholz, 1997; Terada think about and then select a yeast product for dietary
et al., 1999). organic selenium supplementation for food producing
animals, it is important to know about the availability
of the selenium in the product.
Selection of the best form of organic
Table 2. Selenium species in three selenized yeast products1.
selenium as a feed supplement
Selenium species in yeast fractions A B C
Based on natural availability of organic selenium in (%) (%) (%)
plant–based foodstuffs and its superior bioavailability,
it should be easy to decide to provide organic feed Water soluble Se 11.5 28.0 21.0
Water insoluble polysaccharide-bound Se 15.5 27.0 72.0
supplements, rather than the pro-oxidative sodium Water insoluble protein-bound Se 19.0 38.0 4.0
selenite, to food producing animals. However, the Residual protein-bound Se 38.0 6.0 1.0
choices among selenium supplements available to Residual hydrolysable Se 16.0 1.0 1.0
feed compounders and commercial nutritionists are 1
Percentage values based on Encinar et al., 2003
rather large and diverse in their composition.
Why is selenium an essential nutrient? associated with selenium deficiency. Selenium

deficiency can manifest itself in many diseases and
The essentiality of selenium as an ultramicronutrient dysfunctions such as liver necrosis, muscular
in the daily nutrition of mammals was demonstrated dystrophy, microangiopathy, exudative diathesis,
by Schwarz and Foltz (1957). Their discovery that pancreatic fibrosis, poor feathering, retained
selenium deficient rats would suffer hepatic necrosis placenta, mastitis, cystic ovaries, general
was rapidly followed by the discovery that selenium unthriftiness, Keshan disease, Kashin-Beck disease,
was essential in poultry (Schwarz et al., 1957). At cancer, numerous heart diseases, immune
Lederle Laboratories, Pearl River, New York, deficiencies, reduced fecundity, and many others and
Patterson et al. (1957) made an independent can affect humans and food production animals alike.
observation that exudative diathesis in chickens was These listed problems show us that no animal is
prevented when selenium was added to the feed. It exempt from selenium deficiency syndromes.
was not until 1973 that the beneficial effects of Therefore, one can confidently state that selenium
selenium could be ascribed a biochemical function is essential and must be provided if natural intake is
when Rotruck et al. (1973) and Flohé et al. (1973) below optimal requirements.
discovered that it was an essential component of an
important antioxidant enzyme, glutathione peroxidase
(GSH-Px). Continued study of GSH-Px has shown Selenium in body proteins
that there are at least six isoenzyme forms in various
organs and tissues in mammalian species (Table 3). Schrauzer (2003) reported that selenomethionine is
Corresponding evidence is not available in avian incorporated into body proteins in place of
species, but empirical data from studies with selenium methionine, providing a means for reversible storage
in poultry suggest that several functional GSH-Px of selenium in organs and tissues. This property of
isoenzymes and other selenoproteins are also present selenomethionine is not shared with any other
in avian species. This would suggest that at least for selenoamino acid, which points to a specific
GSH-Px and some of the other selenoproteins, the physiological function of selenomethionine
active selenocysteine site on the enzyme/protein has (Schrauzer, 2003). In fact, Schrauzer (2003) has
been conserved. Conservation of active sites may not asserted that all needed metabolic forms of selenium
be limited to vertebrate animals. Indeed, in a parasite, can be produced from selenomethionine, and it,
the blood fluke Schistasoma mansoni, phospholipid- therefore, meets all the criteria for an essential amino
hydroperoxide GSH-Px has been discovered (Maiorino acid. The situation associated with the use of
et al., 1996). Thus, selenium has a special function in inorganic selenium is different because inorganic
antioxidant control mechanisms in animal species. selenium from selenite or selenate results in very
Mertz (1987) suggested that selenium is unique limited nonspecific insertion of selenocysteine into
among the essential trace elements especially in the protein but serves as a substrate for synthesis of
manner in which its deficiency is expressed. Mertz selenocysteine for specific insertion into
(1987) stated that a selenium deficiency in mammals selenoproteins (Hawkes et al., 1985a,b; 2003).
was likely to be manifest in second generation progeny. Selenium from sodium selenite or selenate might
Selenium deficient rats with clinical signs die within also form a selenotrisulfide (-S-Se-S-) bond (Ilian
a few weeks if they become vitamin E deficient and Whanger, 1989) that is subject to rapid oxidation
(Schwarz, 1951), but if rats in the first generation of and release from protein. The selenotrisulfide bond
selenium deficiency are force-fed high levels of fat, is not formed with selenomethionine.
they die within a few hours (Schwarz, 1954). These Cummins and Martin (1967) and Latshaw and
observations by Schwarz and by Mertz pointed to the Osman (1976) have demonstrated that selenium
complex interaction between selenium and vitamin E derived from selenite was easily released from
and established that there was a need for adequate animal protein subjected to alkaline dialysis whereas
levels of both in diets of humans and food animals. selenium from selenomethionine was retained as a
A survey of the literature readily reveals that part of the protein. Ort and Latshaw (1978) have
selenium, through the selenoproteins, influences shown that even when toxic levels of
numerous physiological and biochemical functions that selenomethionine or sodium selenite are fed to laying
are necessary for maintenance of homeostasis in all hens, egg levels and body tissue levels return to
animals. Edens (1996) listed a number of problems normal within two to four weeks after cessation of
feeding toxic levels of selenite selenium, but a longer stem-loop structure in the 3´ non-translated region
time is required for birds fed selenomethionine. of the mRNA that designates selenocysteine insertion
Selenium from selenite, therefore, appears to interact at the UGA codon instead of chain termination (Berry
loosely with the cysteine thiol group and selenium in et al., 1991a,b; 1993).
selenomethionine is molecularly integrated into In order for selenocysteine to be incorporated into
protein. The stored form of organic selenium as specific selenoproteins, there is a requirement for
selenomethionine is in a non-functional state, i.e. not selenocysteine-ß-lyase reaction with free
used immediately for formation of biologically selenocysteine that causes the release of selenide in
functional selenoproteins (Mahan, 1994; 1995). Since the presence of reducing agents (Sunde, 1990; Burk,
selenium in selenomethionine is better retained than 1991). Another selenoenzyme, selenophosphate
inorganic selenium, the ultimate metabolism of synthetase, using selenide and serine as substrates,
organic selenium with liberation of selenide that phosphorylates selenide to form selenophosphate. The
enters the pathway for selenocysteine synthesis for selenophosphate is made available to a unique seryl-
incorporation into functional selenoproteins appears tRNASEC that is recognized by selenocysteine synthetase.
to be more efficient. As a consequence of the large Under the influence of selenocysteine synthetase, that
pool of stored selenium in protein, in times of converts seryl-tRNASEC to selenocysteyl-tRNASEC, the
oxidative stress, body protein can be degraded rapidly selenium in selenophosphate is co-translationally
providing more than adequate concentrations of incorporated into selenocysteine. Then, selenocysteyl-
organic selenium that can be used for synthesis of tRNA[Ser]SEC, which recognizes the specific UGA
specific selenoproteins. codon in the selenoprotein-mRNA, inserts the new, co-
Constant provision of a readily available source of translationally synthesized selenocysteine into the
organic selenium from body proteins for synthesis specific selenoprotein (Burk, 1991). The base triplet
of selenoproteins is a unique and continuous process. UGA that normally functions as a stop codon
Mitch and Goldberg (1996) have shown that most (Amberg et al., 1996) encodes this process of
proteins are degraded within a few hours after their selenocysteine insertion at its appropriate site in the
synthesis. Functional proteins such as enzymes have peptide. The selenocysteine insertion also requires a
a more rapid degradation rate than do structural specific mRNA, an elongation factor, GTP, and the
proteins such as muscle protein. Consumption of diets selenocysteine insertion sequence (SECIS) that all
containing organic selenium results in a high rate of interact at the ribosome to read the UGA
selenomethionine incorporation in muscle protein. selenocysteine codon (Low and Berry, 1996).
As the muscle protein is replaced, the stored Thus, organic selenium must be converted from its
selenomethionine is released into the free amino acid original organic form (-2 valence) to the inorganic
pool from which it is made available for selenoprotein selenide (also -2 valence) form then back to an
synthesis. In times of oxidative stress, the proteasome, organic form (-2 valence) to fulfill its biological
a cytosolic organelle, increases the rate of protein function (Arthur, 1997). This conversion is crucial
degradation and increases amino acid availability for with regard to synthesis of selenoproteins because it
synthesis of other proteins such as the selenoproteins. has been reported that 30 to 80% of the selenium in
In fast growing animals such as the high-yielding the body may be selenocysteine (Hawks et al., 1985a).
commercial broiler lines, it is important to have a Nevertheless, selenomethionine is a highly available
readily available source of selenium to be used in substrate for many proteins and can substitute non-
selenoprotein synthesis. specifically for methionine in their structure (Daniels,
1996). In cases of selenomethionine supplementation
to feeds, it can be demonstrated that 40 to 50% of
Selenium incorporation into total body selenium, as selenomethionine, can be
selenoproteins found in muscle protein (Daniels, 1996). Natural
selenocysteine also can be substituted non-specifically
The interest in the antioxidant properties of selenium for cysteine in many proteins, but it is not
has been facilitated by the fact that the active site(s) incorporated directly into specific selenoproteins
on the various selenoproteins, which often act in (Sunde, 1990; Daniels, 1996).
antioxidant systems, contain selenocysteine- the Animals cannot synthesize selenomethionine, the
proclaimed 21 st amino acid. The locus of primary selenoamino acid, directly from selenite or
selenocysteine in selenoproteins involves use of a selenate forms of inorganic selenium (Cummins and
Martin, 1967; Sunde, 1990). Selenoamino acids are because it also expresses a monomeric GSH-Px in its
acquired naturally from plants and grains and animal tissues (Miyazaki and Motoi, 1992; 1996). Finally,
protein eaten by the animals. Selenocysteine can be thioredoxin reductase (TrxR) activity has been
found in animals fed inorganic selenium such as reported for the chicken (Smith and Levander, 2001;
selenite and selenate. In animals consuming selenium 2002), and it was determined that the activity of
rich foodstuffs, selenomethionine is present in high chicken TrxR is very much less than mammalian
concentrations and is easily converted to selenocysteine TrxR. Tissue distribution of chicken TrxR has not
via cystethionase (Esaki et al., 1981). been determined yet, but preliminary data will be
presented herein. The presence of other selenoproteins
can be inferred from current and previous studies;
Selenium functions through but based on the literature, it is possible that the
selenoproteins chicken will express selenoproteins similar to
mammals.
In mammals there are at least 18 known (Hatfield Edens (2001) reported that broiler chickens given
and Gladyshev, 2003) and possibly more genes that a Sel-Plex® supplement in the feed were more resistant
encode selenoproteins (Behne et al., 1997). Recent to enteropathogenic E. coli enteric infection than
evidence strongly suggests that there are at least 25 chickens that had no supplemental selenium. The
selenoproteins in the mammalian selenoproteome broilers given Sel-Plex® had body weights and feed
(Kryukov et al., 2003), and this would suggest more conversions that were similar to the birds with no
genes that encode selenoproteins. The known supplemental selenium and not challenged with the
selenoproteins have numerous functions, but many bacteria (Table 5). Mortality of E. coli-challenged
of the selenoproteins still have unknown functions Sel-Plex®-fed broilers was 59% less than mortality
(Table 3). Furthermore, it is not known if all animals in E. coli-challenged broilers given no supplemental
possess equivalent proteins. An excellent review of selenium (Table 5). Additionally, the GSH/GSSG
the functions of selenoproteins in farm animals was ratios in broilers given Sel-Plex® were less than those
presented by Jacques (2001) who pointed out that that were not given supplemental selenium (Table
selenium must exert its greatest influences through 6). This suggested that the GSH/GSH-Px system was
selenoproteins. working more efficiently in broilers fed Sel-Plex®.
In poultry there has been limited research to This conclusion was supported by the observation
ascertain the presence and function(s) of the various that there was less heat shock protein (hsp) 70 in the
selenoproteins. It appears from the few publications intestines of Sel-Plex®-fed birds compared with those
on avian selenoproteins that their functions are given no supplemental selenium (Figure 1).
generally similar to the corresponding protein(s) in Sulfur-containing amino acids (cysteine and
mammals (Petovich and Podorozhnaia, 1981; Ilian methionine) are susceptible to oxidation. The
and Whanger, 1989; Snityns’kyi and Antoniak, 1994). sulfhydryl groups of cysteine residues of proteins are
Of the selenoproteins studied in chickens, it has been normally maintained in a reduced state or they can
reported that there are two deiodinases: Type I that be oxidized by thioredoxins in the cytoplasm
functions in the liver to convert thyroxin (T4) to tri- (Okamoto et al., 1999). During oxidative stress,
iodothyronine (T3), and Type III that functions to thiol-disulfide exchange occurs between cysteine
convert T3 to rT3 and T3 to T2 (May, 1989; Van der residues in protein and oxidized glutathione (GSSG).
Geyten et al., 1997). There are numerous reports on This process serves as a redox-dependent regulator
GSH-Px activity(ies) in chickens, and those reports of various protein functions (Fratelli et al., 2002).
suggest that the GSH-Px enzymes function similarly The oxidized sulfhydryl in methionine can be reduced
to those found in mammals because of their specificity by the methionine sulfoxide reductases (Grimaud et
for reduction of hydrogen peroxide and lipid peroxides al., 2001) and might serve in an antioxidative capacity
(Stadtman, 1980; Pablos et al., 1995a,b; 1998). (Levine et al., 1996; Stadtman et al., 2002). These
Recent evidence has shown that chickens express mechanisms are interrelated because the heat shock
phospholipid-hydroperoxide GSH-Px (Kong et al., proteins elevate reduced glutathione levels by
2003), supporting observations of its presence in promoting an increase in glucose-6-phospho-
several chicken and turkey tissues (Surai et al., 1998a, dehydrogense activity (Preville et al., 1999). The
b). The chicken is different from most mammals heat shock proteins are normally induced during
Table 3. Selenoproteins and their role in the maintenance of homeostasis in animals.

Selenoproteins Function Reference
Glutathione peroxidases (GSH-Px)
GSH-Px-1 (cytosolic) Reduces reactive molecules and free radicals; Rotruck et al., 1973; Flohé et al., 1973
complements action of vitamin E
GSH-Px-2 (gastrointestinal) Antioxidant; reduces ingested lipid hydroperoxides Chu et al., 1993
GSH-Px-3 (plasma) Unknown; selenium carrier Takahashi et al., 1987
Phospholipid hydroperoxide Directly reduces phospholipids and cholesterol Ursini et al., 1985
GSH-Px (spermatozoa and testis) hydroperoxides
GSH-Px- sperm nucleus Protamine thiol peroxidase; responsible for disulfide Behne et al., 1988, 1997
cross-linking; necessary for sperm maturation and
male fertility
GSH-Px-6 Unknown; homologue of GPx-1 Kryukov et al., 2003
Thioredoxin reductases (TrxR)

Cytosolic TrxR (TrxR1) Reduces protein thiols and thioredoxin; provides Tamura and Stadtman, 1996
reducing equivalents to several redox-dependent systems
Testicular TrxR (TrxR2) Reduces protein thiols and thioredoxin; provides Gasdaska et al., 1999; Lee et al.,
reducing equivalents to several redox-dependent systems 1999; Miranda-Vizuete et al., 1999;
Watabe et al., 1999
Mitochondrial TrxR (TrxR3) Reduces protein thiols and thioredoxin; provides Sun et al., 1999
reducing equivalents to several redox-dependent systems
Iodothyronine deiodinases (ID)

ID Type-I Converts thyroxin (T4) to tri-iodothyronine (T3) Behne et al., 1988, 1990; Arthur et al.,
1990; Berry et al., 1991a
ID Type-II Converts T4 to T3 and rT3 to T2 Croteau et al., 1996; Salvatore et al.,
1996
ID Type-III Antioxidant in brain; converts T3 to rT3 and rT3 to T2; Kaplan, 1986; Croteau et al., 1995;
protects developing brain from excess T3 Mortimer et al., 1996
Other selenoproteins
Sel H Unknown Kryukov et al., 2003
Sel I Unknown Kryukov et al., 2003
Sel K Unknown Kryukov et al., 2003
Sel M Unknown Kryukov et al., 2003
Sel N Unknown Kryukov et al., 2003; Lescure et
al., 1999
Sel O Unknown Kryukov et al., 2003
Sel P Selenium carrier; antioxidant Herman, 1977; Motsenbocker and
Tappel, 1982
Sel R Methionine sulfoxide reductase; antioxidant Kryukov et al., 2003; Lescure et
activities and aging al., 1999; Moskovitz et al., 2001
Sel T Unknown Kryukov et al., 2003; Lescure et
al., 1999
Sel U (SEC in chickens and fish; Cys-homologue Unknown Catellano et al., 2004
in mammals)
Sel V Unknown; homologue of Sel W Kryukov et al., 2003
Sel W Unknown; redox activity in muscles and Vendeland et al., 1993; Allan et
other tissues al., 1999
Sel X Unknown Kryukov et al., 2003; Lescure et
al., 1999
15 kDa Selenoprotein Unknown; found in T cells and prostate Gladyshev et al., 1998
18 kDa Selenoprotein (mitochondrial) Unknown Behne et al., 1988; Kyriakopoulos
et al., 1996
Selenophosphate synthetase-2 Biosynthesis of selenocysteine used in Low et al., 1995
selenoprotein synthesis
Table 4. Influence of selenium source on blood and liver total glutathione (TGSH), reduced glutathione (GSH), oxidized GSH (GSSG), GSH/
GSSG, glutathione peroxidase (GSH-Px), glutathione reductase (GR), and hsp70 in chickens before and after mild heat distress.
Variable Treatment Blood (before heat) Blood (after heat) Liver (before heat) Liver (after heat)
TGSH, µM/g Hb Control 15.5 a 17.2a 2786 a 1997 a

or µM/g tissue Selenite 14.4ab 17.0a 2320 a 2120 a
Sel-Plex® 13.4 b 15.9a 2430 a 2297 a
SEM 0.65 0.65 254 254
GSH, µM/g Hb Control 12.1 a 13.0a 2695 a 1951 a
or µM/g tissue Selenite 11.3ab 13.2a 2269 a 2082 a
Sel-Plex® 10.7 b 12.2a 2346 a 2185 a
SEM 0.46 0.46 249 249
GSSG, µM/g Hb Control 1.70 a 2.10a 45.8a 22.8a
or µM/g tissue Selenite 1.56 a 1.91a 25.5a 19.2a
Sel-Plex® 1.35 a 1.82a 41.8a 56.4a
SEM 0.15 0.15 14.7 10.7
GSH/GSSG Control 7.13 a 6.19a 58.84 a 85.57 a
Selenite 7.24 a 6.92a 88.98 a 108.44 a
Sel-Plex® 7.52 a 6.71a 56.12 a 38.74 b
SEM 0.28 0.37 16.27 21.31
GSHpx, mU/mg Hb Control 9.31 c 19.80 c 2198 b 1632 c
or mU/mg protein Selenite 58.76b 91.17 b 3602 a 2936 b
Sel-Plex® 139.15 a 190.36 a 3836 a 3432 a
SEM 7.86 7.86 390 190
GR, mU/mg Hb Control 2.33 a 2.78a 187.1 a 163.6 a
or mU/mg protein Selenite 2.23 a 2.33a 169.6 a 159.0 a
Sel-Plex® 2.43 a 2.46a 173.9 a 176.5 a
SEM 0.28 0.28 9.1 9.1
hsp70 Control 4.28a 5.35a
(ng/mg total protein) Selenite 4.25a 4.37ab
Sel-Plex® 4.38a 3.95b
SEM 0.43 0.43
a,b,c
In a column, means with unlike superscripts within a variable differ significantly (P≤0.05).
Table 5. Performance of 42 days old male broiler chickens given a (Edens, 2001). If the hypothesis presented here is
dietary supplement of Sel-Plex® and challenged with an entero- correct, the selenium in selenomethionine and
pathogenic E. coli.
selenocysteine in proteins might be less reactive with
Treatments Body weight FCR Mortality oxidized GSSG, and the GSSG would be reduced
(g) (g/g) (%) more readily by glutathione reductase as postulated
by Mahmoud and Edens (2003). As a result, when
No Selenium, no E. coli 2047 ± 86 bc 1.95b 16.7b (10/60) 1
Sel-Plex®, no E. coli 2283 ± 67 a 1.82c 5.0c (3/60) organic selenium is fed to broilers, less heat shock
No Selenium + E. coli 1896 ± 95 c 2.09a 36.7 a (22/60) protein would be induced because the cell cytoplasm
Sel-Plex® + E. coli 2098 ± 71 b 1.93b 15.0b (9/60) would be maintained in a more reduced status even
a,b,c
in the face of stressors that promote oxidative distress
In a column, means with unlike superscripts differ (P≤0.05).
1
Numbers in parentheses represents total mortality per treatment. (Table 4).
The data from these two different experiments
oxidative stress and protect sensitive sites on proteins. suggest that in three tissues there might be three or
In the results of two studies presented here, we four different GSH-Px, specifically the classical
concluded that broilers given Sel-Plex® and exposed GSH-Px-1, GSH-Px-2, GSH-Px-3 and monomeric
to either heat stress (Table 4) or enteric an E. coli GSH-Px. Although, the data cannot predict which
challenge (Figure 1) performed better because they isoform was active/not active, it was apparent that
had lower concentrations of liver hsp70 than broilers the GSH/GSH-Px system in the chicken is highly
given sodium selenite or no supplemental selenium responsive to selenium both as sodium selenite and
as organic selenomethionine in yeast protein.
Table 6. Influence of Sel-Plex® on intestinal total glutathione (µM/g), oxidized glutathione (µM/g), reduced glutathione (µM/g), and the
reduced:oxidized glutathione ratios in broiler chickens given a dietary supplement of organic selenium and challenged with an enteropatho-
genic E. coli.
Treatments Total glutathione Oxidized (GSSG) Reduced (GSH) Ratio R:O
No Selenium, no E. coli 18954 ± 1904 ab 1601 ± 196 c 13349 ± 1835 b 8.32 ± 1.05 a
No Selenium + E. coli 21896 ± 1971 a 3047 ± 180 a 19099 ± 1688 a 7.30 ± 1.44 ab
Sel-Plex®, no E. coli 17045 ± 1843 b 2238 ± 174b 13322 ± 1627 b 5.95 ± 1.01 b
Sel-Plex® + E. coli 22500 ± 1971 a 3313 ± 188 a 18911 ± 1757 a 5.71 ± 1.09 b
a,b,c
In a column, means with unlike superscripts differ significantly (P≤0.05).
0.014
0.012
0.010
OD/µg total protein
0.008
0.006
0.004
0.002
0.000
No EC, No Se No EC+Sel-Plex® EC, No Se EC + Sel-Plex®
Treatment groups
Figure 1. Intestinal heat shock protein 70 response to enteropathogenic E. coli (EC) infection in broiler chickens with and without
supplemental Sel-Plex® .
Without a highly functional selenium-dependent the potential to affect the efficiency of conversion of
antioxidant system, the modern broiler chicken would food into energy and metabolic heat production. The
not be as productive. As an illustration, Edens (2001) mitochondria, the cell’s power generators, are
presented performance data of broilers grown to 42 affected directly by T3. Rapid protein synthesis,
days of age. In that experiment Sel-Plex®-fed broilers mitochondrial gene transcription, and synthesis of
had body weight improvement of 20 g over selenite proteins from genetic information are affected by
supplemented broilers and a 50 g improvement over T3. These processes cause turnover of body proteins,
broilers with no selenium supplementation. Feed increase free fatty acids for energy metabolism, and
conversion in Sel-Plex®-fed broilers was improved 3 increase oxygen usage by cells in the body.
points over selenite-fed birds and 9 points over birds Additionally, the cardiovascular system is stimulated
with no supplemental selenium. to a higher level of activity to meet the tissue demands
for increased oxygen caused by higher levels of T3.
Body temperature regulation is also partly under the
Monodeiodinase and triiodothyronine: control of circulating T3 that influences mitochondrial
influences on diverse functions and activity in muscles. The circulating T3 level is highly
responsive to caloric intake and external temperature
poultry performance and is necessary for cold adaptation. If caloric intake
The thyroid hormone, triiodothyronine (T3), has many increases or external temperature decreases, there is
important functions in all animals. In poultry, T3 has an increase in circulating T 3. Conversely, with
starvation T3 decreases along with increased thyroxin Edens (2001) reported that body weight of broilers
(T4). The development and growth of animals is fed organic selenium in Sel-Plex® was improved (70
directly affected by T3 binding to thyroid receptors g/bird and 20 g/bird, respectively) over that of broilers
(TR) that bind to responsive elements on the nuclear fed no supplemental selenium and those fed sodium
DNA triggering gene transcription. This T3 function selenite. Feed conversions of broilers fed organic
in all animals is extremely important in the selenium in Sel-Plex® was also improved (9 points
developing embryo for growth and hatching and 3 points, respectively) over that of broilers fed
efficiency and in the post-hatch chicken for growth. no supplemental selenium and those fed sodium
Recently, a report was given that sodium selenite selenite. Part of the improvement in performance was
actually inhibited the development of the T3-TR bond, due to the status of the metabolically active thyroid
but organic selenium did not inhibit that relationship hormone in the broilers fed Sel-Plex® (Table 7). These
(Brtko et al., 1997). observations were supported by the report of Jianhua
Involvement of thyroid hormones in feathering has et al. (2000), who examined hepatic 5´-deiodinase
long been recognized (Radi and Warren, 1938; Boone activity and thyroid hormone dynamics in broilers
et al., 1950). Feeding thyroprotein increases feathering fed selenium deficient or selenium (selenite) adequate
rate in slow feathering birds. In thyroprotein there is a diets. They concluded that selenium deficiency
predominance of thyroxin (T 4), but in poultry, depressed growth of broilers by inhibiting 5´-
triiodothyronine (T 3) is the most active thyroid deiodinase activity, which in turn decreased the plasma
hormone. It is known that T3 is intimately involved concentration of T3.
in feather development. A small quantity of T3 is The role of selenium in the immune system of
produced in the thyroid, but the greatest quantity is chickens has received little interest to date. We have
converted from T4 to T3 in the liver by the selenium- noted that fast growing, high yielding broilers fed
dependent type I 5′-deiodinase enzyme (Berry et al., selenite at 1.2 ppm Se showed early signs of selenium
1991a) that predominates in the chicken (May, 1989). toxicity by exhibiting decreased body weight,
Experiments have been conducted to compare increased liver weight, and a compromised immune
selenium from sodium selenite and from Sel-Plex® system as indicated by decreased thymus and bursa
on feathering in broiler chickens (Edens, 1996; 2001). of Fabricius weights and abnormally high delayed
It was determined that organic selenium induced type hypersensitivity reactions to phytohemagglutinin
more rapid feathering in auto-sexing, slow feathering antigen (PHA-P), but broilers fed Sel-Plex® at 1.2
male chickens and in their normal feathering sisters ppm were not affected (Gowdy and Edens, 2003).
(Edens, 2001). An evaluation of thyroid hormone Marsh et al. (1986) fed low levels of sodium selenite
levels in broiler chickens was made in an attempt to to chickens and observed increased growth of
relate thyroid hormones to feathering (Edens, 2001). lymphoid organs including an increased thymus
When organic selenium in Sel-Plex ® was fed to weight. Gowdy and Edens (2003) found that feeding
broilers, the circulating percentage concentrations of sodium selenite (0.3 or 0.6 ppm) to control chicks
T4 were lower and T3 concentrations were higher than also caused increased thymus weight compared to
in broilers given sodium selenite (Table 7). These thymus weight from chicks fed either no selenium
observations suggest that organic selenium is more or organic selenium, but with infection, only organic
important than inorganic selenium in the early selenium (0.3 or 0.6 ppm) maintained thymus weight
feathering of chickens and possibly other birds. comparable to weights of non-infected chicks. Gowdy
and Edens (2003) also reported that the chicken T-
Table 7. Mean serum thyroxin (T4, ng/mL), triiodothyronine (T3, cell-mediated wing web response to PHA-P in
ng/mL), and percentage levels in Sel-Plex®-fed, Sel-Plex® + selenite- Sel-Plex®-fed broilers was less than the response in
fed and no supplemental selenium compared with the control
(selenite) over a six week growing period. sodium selenite-fed birds. Presumably, the lesser
delayed type hypersensitivity response associated with
Treatments T4, T4 T3 T3
(ng/mL) (%) (ng/mL) (%) Sel-Plex ® feeding was associated with increased
GSH-Px activity in lymphocytes and granulocytes
No Se 6.05 a 144.3 0.47 c 72.0
Selenite 4.29 b 100.0 0.64 b 100.0 that migrate to the site of antigen stimulation (Brown
Sel-Plex® 3.99 b 88.1 0.77 a 120.0 et al., 2000).
Sel-Plex® + selenite 4.08 b 96.8 0.76 a 118.8 Arthur et al. (2003) reported that selenium is
a,b,c
In a column, means with unlike superscripts differ (P≤0.05). essential in the activation of all elements of the
immune system. The thymus seems to have a special
affinity for selenium because it is the location for immunoactive cells decreases the nutrients that
maturation of T cells that ultimately will control most normally would be destined for protein production
aspects of the immune system. The thymus, at least associated with growth. In animals, with an immune
in mammals, also contains selenium-dependent Type system conditioned to react quickly to an antigen
II deiodinase enzyme, which is used to locally convert challenge and clear that antigen more quickly,
T4 to T3 and rT3 to T2. Thus, it is highly probable performance can actually be enhanced due to less
that any impairment of this enzyme could have effects time utilized in combating the numerous challenges
on T cells and the immune system in diverse locations poultry face every day in their specific environments.
throughout the body of an animal. The thymus These activities might be enhanced in animals given
appears to be exquisitely sensitive to selenium as organic selenium in Sel-Plex® as compared with
shown by the results of Gowdy and Edens (2003); animals fed inorganic sodium selenite. Evidence
and it appears that organic selenium is better tolerated presented by Gowdy and Edens (2003) seem to
than inorganic selenium in the thymus and possibly support this hypothesis.
mature T cells. It is apparent that selenium-dependent 5´-deiodinase
Leng et al. (2003) compared the influence of either activity in poultry has a very important role to play.
sodium selenite or organic selenium sources on On the most fundamental basis, embryonic growth,
immunity in layer chickens. They determined that livability and hatchability are dependent on
organic selenium increased levels of tissue selenium availability of selenium from the dam, and provision
more than 2-fold higher than with selenium derived of organic selenium to the dam improves all of these
from selenite. More importantly, Leng et al. (2003) parameters for the embryo (Edens, 2002).
were the first to show that the organic selenium caused Hatchability of chicken and turkey embryos is under
an increase in CD3+, CD4+, and CD8+ surface the influence of thyroid hormones (Christensen and
markers on T cells located in several lymphoid Biellier, 1982; Christensen, 1985). Christensen et al.
structures in young chickens. The CD3+ marker is (2002) determined that delayed conversion of T4 to
involved in signal transduction and is found in all T T3 was associated with increased embryonic mortality
cells. The CD4+ marker is related to helper T cell- and delayed hatchability. However, if T3 is elevated
mediated expansion of the humoral immune response prior to certain critical periods, embryonic livability
ascribed solely to B cells from the bursa of Fabricius, and hatchability can also be decreased (Christensen
and the CD8+ T cell marker is associated with et al., 2002). Van der Geyten et al. (1997) have
cytotoxicity and cell killing functions. Leng et al. reported that deiodinase 1 (D1) and deiodinase 3 (D3)
(2003) concluded that organic selenium improved in chicken embryos develop in parallel to the plasma
the status of the avian immune system by increasing T3 profiles. D1 increased from day 14 of incubation
the rate and ability of immunocompetent cells to to hatching. D3 increased from day 14 of incubation
respond to antigen challenge. They concluded that until day 17 of incubation then decreased. These
chickens fed organic selenium had more protection enzymatic activities would indicate that there is
against potential pathogens than did chickens fed maximized plasma T3 concentrations at the time of
selenite. Chang et al. (1994) studied the influence of internal pipping and conversion to lung breathing by
sodium selenite and vitamin E on T and B cell the embryo. More recently, an unpublished report
markers and lymphocyte proliferation. Lymphocyte from the poultry industry indicated that hatchability
proliferation was impaired by vitamin E and selenium of turkey eggs from Sel-Plex® -fed hens had a higher
deficiency, but the CD4+, CD8+ T cell populations hatch rate if they had to be stored for as long as 10 to
increased slightly with vitamin E and selenium 14 days.
deficiency. As pointed out by Edens (1996; 2001), broiler
The relationship between the immune system and performance is improved with the use of organic
performance in poultry species is complex. It is selenium. Much of the explanation for the improved
generally believed that a heightened status of the active performance has focused on improved antioxidant
immune system is correlated with decreased status in chicks and poults from organic selenium-
performance. In fact, that situation can become fed hens. Furthermore, post-slaughter yield is
established because an active immune system has improved with the use of organic selenium, and water
higher energetic demands and nutrients are retention as indicated by less drip loss is improved.
recompartmentalized to provide the energy required Because it appears that T3 is very intimately involved
by the immunoactive cells. Increased demand from in these responses, one must conclude that selenium
as organic selenium has a special role to play in the normal tissues or cell lines (Gladyshev et al., 1998).
regulation of poultry selenium-dependent Thioredoxin reductase functions to stabilize
monodeiodinases that convert T4 to T3 and rT3 to T2. disulfide bonds (-S-S-), free sulfhydryl groups (-SH),
and to reduce thioredoxin. In the extracellular
environment, TrxR interacts with disulfide bonds on
Thioredoxin reductase in chickens cell surfaces and extracellular proteins. In the cytosol
of the cell, TrxR primarily stabilizes the sulfhydryl
Thioredoxin reductase (TrxR) belongs to a groups and reduces thioredoxin. Thioredoxin is
superfamily of flavoenzyme disulfide oxidoreductases required inside the cell to provide reducing
including glutathione reductase, mercuric ion equivalents for many different substrates, primarily
reductase, dihydrolipoamide reductase, and alkyl proteins that are vital to the survival of the cell. When
hydroperoxide reductase (Williams, 1992). This thioredoxin undergoes oxidation, it is able to transfer
selenium dependent enzyme has been reported to show reducing power to cellular proteins through TrxR,
increased activity after selenium supplementation to and TrxR, using electrons from NADPH, then
animals as compared with enzyme activity in selenium reduces thioredoxin. All three mammalian TrxRs are
deficient animals (Hill et al., 1997; Berggren et al., selenium-dependent flavoproteins (Tamura and
1999). However, Ganther and Ip (2001) have Stadtman, 1996).
reported that monomethylated selenium in vivo did Thioredoxin, the primary substrate for thioredoxin
not increase TrxR activity and that high reductase, is expressed differentially in chickens as a
concentrations of selenium in a cell-free system product of a single copy gene and is similar to
inhibited TrxR. Wu et al. (2003) reported that there thioredoxins in other species (Jones and Luk, 1988).
was an inverse relationship between dietary selenium Thioredoxin is a high capacity electron donor for
and the activity and expression of TrxR in rat aorta. reductive enzymes that include ribonucleotide
Wu et al. (2003) suggested that the mechanisms reductase, thioredoxin peroxidase, and through thiol/
regulating transcription of GSH-Px and TrxR in the disulfide exchange it reduces cysteine residues in
aorta are different. Wu and colleagues suggested that transcription factors to increase their binding to DNA
GSH-Px expression and activity are directly related thereby influencing gene transcription (Mustacich and
to selenium intake, but TrxR activity is mediated Powis, 2000; Powis and Montfort, 2001). Thioredoxin
mostly by generation of ROM. In long-term selenium also functions as a cell growth factor and inhibits
deficiency studies by Wu et al. (2003), TrxR activity apoptosis. Arner and Holmgren (2000) report that
actually increased, selenium repletion of the rats thioredoxin reduces hydroperoxides, ascorbate and
caused a decrease in TrxR activity, and selenium selenite. Ascorbate is the biochemical link between
adequate animals had the lowest activity of TrxR. vitamin E and selenium because it has been shown to
The decreased activity of TrxR in the aorta of recycle tocopheroxyl to tocopherol in vitro (Burk and
selenium-repleted animals might be a tissue specific Hill, 1999). The thioredoxin-thioredoxin reductase
response, because in other organs/tissues selenium system, which maintains free sulfhydryls, and the
deficiency caused a decrease in TrxR and GSH-Px GSH/GSH-Px system, which is a primary antioxidant
activities (Berggren et al., 1999). Berggren et al. system, work together to regulate a low intracellular
(1999) stated that TrxR behaved in a manner very redox potential (Arner and Holmgren, 2000).
different from other selenoenzymes in that it The TrxR work in chickens has just begun, and
increased its activity with excess sodium selenite and preliminary observations suggest that chicken TrxR
that the increased activity was transitory eventually may be different from mammalian TrxR (Gowdy and
returning to lower activities even with continued high Edens, unpublished). Some of our preliminary
dosing of selenium. Therefore, these observations observations on TrxR are presented in Tables 8-10,
might indicate that TrxR is induced as a safety representing the first observations on tissue and
precaution when its selenocysteine is affected by cellular distributions in chicken and the influence of
severe oxidative stress (Gladyshev et al., 1999). In different selenium sources on its activity in various
mammalian fast growing transformed tumor cells tissues.
with high generation of ROM, thioredoxin and TrxR Organ distribution and activity of TrxR in 3-week
activities are much higher than in normal cells (Liu old broiler cockerels from our current studies are
and Stadtman, 1997; Gladyshev et al., 1998; Ganther, presented in Table 8. Similar to the observations made
1999). There is an inverse relationship between TrxR by Berggren et al. (1999) with the rat, we observed
and GSH-Px activity in tumor cells as compared with that broiler chickens which were not provided
supplemental selenium in their diets had significantly was determined, the matrix TrxR activity was higher
lower TrxR activities than broilers given either than that in the mitochondrial pellet, similar to the
sodium selenite or Sel-Plex®. A second observation observation made in this investigation with chickens
in this distribution study was that the selenium source (Table 9). The post-nuclear and post-mitochondrial
was important in regulating TrxR activity. With few supernatant TrxR activities were comparable to the
exceptions, TrxR activity was higher in Sel-Plex®- cytosolic activity because those post-supernatants are
fed cockerels than in sodium selenite-fed or comprised of the cytosol. The observations with
combination (selenite + Sel-Plex ®)-fed birds. In subcellular distribution of TrxR in chickens are
comparison to mammals, chickens have low TrxR similar to observations made in rat (Rozell et al.,
activities (Smith and Levander, 2001), and the low 1985;1988) and human tissue (Ejima et al., 1999;
activities found in these chickens reflect the fact that Chen et al., 2002). Rozell et al. (1985;1988)
TrxR levels in normal tissues are very low (Liu and concluded that the distribution of thioredoxin and
Stadtman, 1997). TrxR to subcellular structures such as the endoplasmic
reticulum, secretory granules, plasma membrane, and
Table 8. Thioredoxin reductase activity in chicken tissues from 3- at the subplasma membrane was consistent with the
week-old male broilers fed supplemental levels of selenite, Sel- functions in protein processing, secretion, and
Plex®, a combination of 0.15 ppm selenite + 0.15 ppm Sel-Plex®,
or no supplemental selenium 1. formation of nascent protein disulfides. Chen et al.
(2002) found high concentrations of TrxR in the
Tissue Control Selenite Sel-Plex® Sel-Plex® + mitochondria, lysosome, microsome and cytosol in
(0 Se) (0.3 ppm) (0.3 ppm) selenite human liver, and Ejima et al. (1999) found high TrxR
(0.3 ppm)
activity (90%) in the cytosol of the placenta cells
Liver 0.023 b 0.084 a 0.071 a 0.053ab and about 10% of the total cellular activity in
Lung 0.047 b 0.082 a 0.085 a 0.034b mitochondria. An important deduction from these
Heart 0.024 b 0.062 b 0.114 a 0.056b observations is that TrxR plays a significant role in
Kidney 0.061 a 0.087 a 0.079 a 0.051a
Brain 0.060 a 0.076 a 0.149 a 0.129a the nucleus and in the mitochondria. The role of TrxR
Breast muscle 0.028 b 0.069ab 0.083 a 0.066ab in the nucleus should be apparent when it is
Bursa 0.026 b 0.070ab 0.098 a 0.058ab understood that thioredoxin is used to enhance
Thymus 0.038 c 0.092 b 0.140 a 0.076b transcription of DNA (Mustacich and Powis, 2000;
Spleen 0.025 b 0.045ab 0.065 a 0.076a
RBC 0.030 a 0.066 a 0.040 a 0.021a Powis and Montfort, 2001).
Plasma 0.034 a 0.031 a 0.025 a 0.039a
Table 9. Subcellular distribution of chicken thioredoxin reductase
1
TrxR activity (at 412 nm) was determined using the DTNB assay activity (µmol NADPH/min/mg total protein) in liver cells.
that measures the NADPH-dependent reduction of the disulfide
bond in 5,5´-dithiobis(2-nitrobenzoic acid). Calculated results are Cellular distribution µmol/min/mg total protein
based on yield of 2 moles of 2-nitro-5-thiobenzoate per mole of
NADPH consumed. Results are given as the µmol of NADPH Liver homogenate 0.1072
oxidized per minute per mg of total protein. Nuclear pellet 0.1289
a,b,c
In a row, means with unlike superscripts differ significantly Post-nuclear supernatant 0.0865
(P≤0.05). Mitochondrial pellet 0.0650
Post-mitochondrial supernatant 0.1066
The subcellular distribution of TrxR in chicken Mitochondrial lysate 0.1333
Mitochondrial membranes 0.0930
hepatic cells is presented in Table 9. The relatively
high TrxR activity in a liver homogenate (primarily TrxR activity (at 412 nm) was determined using the DTNB assay
cytoplasmic) indicates the importance of the cellular that measures the NADPH-dependent reduction of the disulfide
content of the enzyme. When different fractions of bond in 5,5´-dithiobis(2-nitrobenzoic acid). Results are given as
the µmol of NADPH oxidized per minute per mg of total protein.
the cell were analyzed for TrxR activity, the nuclear
pellet and the mitochondrial lysate had the highest We also wanted to determine the influence of feeding
activities followed by post-mitochondrial supernatant, high levels of sodium selenite and Sel-Plex® on
mitochondrial membranes, post-nuclear supernatant, activity of TrxR in chicken liver homogenates (Table
and the mitochondrial pellet. Rigobello et al. (1998) 10). This was part of a larger study in which the
reported that total TrxR activity of the mitochondrial toxicity of Sel-Plex® was being evaluated in chickens
pellet was higher than the mitochondrial matrix (Gowdy and Edens, 2003). In that study, we
(lysate) in rat liver cells, but when specific activity determined that as little as 1.2 ppm of sodium selenite
was sufficient to allow signs of toxicity to develop cytosolic protein sulfhydryls and disulfide bonds in
in high-yield broiler chickens. At 5 ppm sodium a reduced state. Third, TrxR has antioxidant properties
selenite caused severe growth depression, and at 15 that come into play when the redox potential becomes
ppm all of the chickens fed sodium selenite had more negative due to rapid and large production of
experienced severe growth depression and nearly all ROM in cells. In that capacity, TrxR acts in a manner
had died by two weeks of age. Contrary to those similar to GSH-Px as an antioxidant against ROM.
observations, there was no treatment-associated Without these general functions, animals such as
mortality in the Sel-Plex ®-fed groups and body chickens and other food animal species would suffer
weights were comparable to controls fed 0.3 ppm from oxidative stress and become less productive.
Se. At two weeks of age, the chickens were killed by
carbon dioxide asphyxiation and liver samples were
collected from each bird and analyzed for TrxR (Table Summary
10). It was interesting to observe that chickens given
no supplemental selenium had the lowest liver TrxR • Selenium is essential in all animals.
activity. Feeding either sodium selenite or Sel-Plex® • Organic selenium is the preferred form and has
from 0.3 ppm to 15 ppm induced higher activities of higher bioavailability than inorganic selenium.
TrxR, and Sel-Plex® at 15 ppm Se induced the highest
activity of TrxR. This observation was similar to that • Sodium selenite is a pro-oxidant but seleno-
of Berggren et al. (1999) who also reported selenium methionine is not.
(as selenite) to be capable of inducing in vivo higher • Feed supplements providing organic selenium are
TrxR activity in rats. Thus, chickens and rats respond not the same; exercise caution in making selection.
comparablely with selenium-mediated induction of
TrxR. • Selenium is unique among all trace minerals,
requiring its own mRNA and SECIS.
Table 10. Thioredoxin reductase activity (µmol NADPH/min/mg
total protein) from chicken liver homogenates from 3 week old
• Selenomethionine in body protein provides
male broilers fed high levels of selenite or Sel-Plex® 1. reversible storage of selenium in tissues and
organs.
Treatment µmol/min/mg total protein
• Selenomethionine is integrated into body protein,
Control (0 ppm Se added) 0.042c but inorganic selenium is loosely associated with
Selenite, 0.3 ppm Se 0.084bc thiols via a labile bond.
Sel-Plex®, 0.3 ppm Se 0.071bc
• Selenium must function through specific seleno-
Selenite, 5 ppm Se 0.081bc
Sel-Plex®, 5 ppm Se 0.098ab proteins (25 known) encoded by at least 18 genes.
Selenite, 10 ppm Se 0.090ab • Poultry express selenoproteins, but all avian
Sel-Plex®, 10 ppm Se 0.096ab selenoproteins are not yet described.
Selenite, 15 ppm Se 0.102ab
Sel-Plex®, 15 ppm Se 0.146a • Poultry are known to express selenium-dependent
GSH-Px, iododeiodinase, and TrxR.
1
TrxR activity (at 412 nm) was determined using the DTNB assay
that measures the NADPH-dependent reduction of the disulfide • Performance as indicated by body weight,
bond in 5,5´-dithiobis(2-nitrobenzoic acid). Calculated resulted livability, feed conversion, meat yield, and meat
are based on yield of 2 moles of 2-nitro-5-thiobenzoate per mol quality in poultry is influenced significantly by
of NADPH consumed. Results are given as the µmol of NADPH
oxidized per minute per mg of total protein.
selenoproteins.
a,b,c
Means with unlike superscripts differ significantly (P≤0.05). • Physiological functions as diverse as body
The importance of thioredoxin reductase in living temperature regulation, immunity, resistance to
systems, such as the growing chicken, continues to oxidative stress, reproduction, thyroid function,
be revealed. Currently, it is believed that TrxR growth, development, all endocrine functions,
functions to reduce thioredoxin and maintain a readily digestive processes associated with pancreatic
available supply of the reduced thioredoxin, which function, and more are influenced by selenium
has numerous functions associated with growth and status in poultry.
development. Second, TrxR functions to maintain
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P.R. Ferket 57
Alternatives to antibiotics in poultry production: responses, practical

experience and recommendations
PETER R. FERKET
Department of Poultry Science, College of Agriculture and Life Sciences, North Carolina State
University, Raleigh, North Carolina, USA
During the past 50 years, the livestock and poultry and produce safe meat and egg products. Some of
industries have developed in several areas including these alternatives may include significant changes in
nutrition, genetics, engineering, management, and husbandry practices or the strategic use of enteric
communications to maximizing the efficiency of microflora conditioners, including acidifiers,
growth performance and meat yield. Now these probiotics, enzymes, herbal products, microflora
industries must focus more attention on how animal enhancers, and immuno-modulators. The objective
agriculture affects the environment and food safety. of this paper is to briefly review the use of antibiotic
As in many other industries, the global paradigm is growth promoters as enteric conditioners and discuss
shifting from an emphasis on productive efficiency the potential of non-pharmaceutical alternatives.
to one of public security. Nothing demonstrates this
paradigm shift more clearly than the issues concerning
the use of antibiotic growth promoters. For the past Benefits of feeding antibiotics
four decades, antibiotics have been used in animal
agriculture to improve growth performance and Antibiotic usage in animal feeds has many benefits.
protect animals from the adverse effects of pathogenic It improves food safety by increasing animal health
and non-pathogenic enteric microorganisms. Now, and reducing or eliminating certain pathogens. It
antibiotics have come under increasing scrutiny reduces animal production costs and economic
because of the potential development of antibiotic- benefits are distributed along the food chain,
resistant human pathogenic bacteria after long use including the feed industry, production animal
(Phillips, 1999; Ratcliff, 2000). In response to this agriculture, food processors, retailers, and consumers.
apparent ‘threat’, the European Union banned the Most of the cost savings attributed to antibiotics is
use of subtherapeutic levels of antibiotics to prevent from improved feed conversion, and this response is
disease or promote growth, starting with a ban on highest in fast-growing genetically improved animals
avoparcin in 1997 and a ban on virginiamycin, reared in intensive production systems. Other cost
bacitracin, spiromycin, and tylosin in 1999. savings come from faster growth rate, reduced
Antimicrobials scheduled to be banned by 2006 mortality, greater resistance to disease challenge,
include avilamycin, bambermycin, salinomycin and improved reproductive performance, improved
monensin. In June of 2003, McDonald’s Corp. pigmentation, and better manure and litter quality.
announced that it would prohibit their direct suppliers Rosen (1995) concluded from his review of 12,153
from using antibiotics that are important in human feeding studies that antibiotic growth promoters gave
medicine as growth promoters in food animals after a positive response 72% of the time. The magnitude
2004, and they created a purchasing preference for of responses was dependent upon the type of animal
companies that work to minimize antibiotic use. management, disinfection procedures, age of the farm
Although banning antibiotic growth promoters may buildings, and quality of the feed. Finally, the use of
not be scientifically justified, the tide of public antibiotic growth promoters has a positive impact on
opinion is forcing animal agriculture to develop two important issues facing animal agriculture: animal
alternatives, or at least substantially reduce the amount welfare and environmental stewardship. Animal
of antibiotics used to maintain production efficiency welfare is definitely improved in animals that are
58 Alternatives to antiobiotics in poultry production
healthier due to the disease-suppressing effects of absorption (Visek, 1978) and reduce the metabolic
antibiotics. The improved utilization of dietary demands of the gastrointestinal system. The
nutrients by supplemental antibiotics results in minimization of gastrointestinal bacteria may also
significant reduction in nitrogen, phosphorus, and ease the competition for vital nutrients between the
other nutrients excreted into the environment bird and the microbes (Ferket, 1991). Finally,
(Cromwell, 1999). antibiotics may reduce the adverse effects of
immunological stress on growth performance by
lowering the enteric microbial load. Over-stimulation
Antibiotic modes of action of the host immune system by the resident microflora
could impair the optimum growth and performance
Antibiotics are natural metabolites of fungi that inhibit of the bird (Cook, 2000; Klasing, 1988).
the growth of bacteria. They function by altering
certain properties of bacterial cellular metabolism
resulting in impaired growth or death. Some The antibiotic resistance debate
antibiotics interfere with the building and maintenance
of the cell wall, while others interrupt proper protein During the last 10 years, the use of growth promoting
translation at the ribosomal level. Because of their antibiotics has been criticized for their possible role
elevated rate of growth and proliferation, bacteria in the occurrence of antibiotic-resistant microbes.
are vulnerable to antibiotics that target active cellular Numerous reports have been issued concerning the
metabolism. Limiting the growth and proliferation effects of agriculture-related antibiotics on the
of certain bacteria and inhibiting the production of emergence of antibiotic resistance in human pathogens
various toxins restricts the influence that the microbe (SCAN Report, 1999; DANMAP, 2000). Although a
has upon the host organism. This enables the host to complete ban on the use of subtherapeutic doses of
grow and perform better than if grown under normal antibiotics in animal feed has not yet been enforced
challenge conditions. in many countries, this day may eventually come.
The term ‘Growth Promoter’ has been used for years There is some evidence that the use of antibiotic
to describe the use of subtherapeutic levels of growth promoters in animal and poultry feeds is
antibiotics to improve growth performance. It is an associated with bacterial resistance in human disease
inappropriate term to describe this use of antibiotics therapy. Rapid selection for resistant bacteria when
because they do not promote growth as do anabolic subtherapeutic levels of antibiotics are fed occurs
hormones, such as growth hormone or estrogen-like because of the plethora of bacteria in the gut of
compounds. This may be why the general public animals, the high mutation rates among these bacteria,
confuses this term with the use of anabolic hormones. and the frequent transfer of genes including resistance
The poultry industry does not use anabolic hormones genes. Mathew et al. (2002) demonstrated that
as do the swine and cattle industries. Instead of calling selection for resistant bacteria can occur in as little
them ‘Growth Promoters’, they should be called as two days following administration of a feed-based
‘Growth Permitters’ because they allow the animal antibiotic. Wide use of antibiotic growth promoters
to express its genetic potential for growth without in poultry is one reason the public is placing some
compromise. blame for antibiotic resistance of potential pathogens
Antibiotics limit the growth of detrimental on the poultry industry. Gustafson and Bowen (1997)
microbes, such as Clostridium perfringens (Truscott reported that antibiotic resistance of indigenous E.
and Al-Sheikhly, 1977). They also limit the growth coli of poultry has remained at a relatively high level
and colonization of numerous non-pathogenic species since the 1950s. However, Lou et al. (1995) reported
of bacteria in the gut, including lactobacilli, that removing antibiotics from a swine herd for now
bifidobacteria, bacteroides, and enterococci (Tannock, over 30 years has not eliminated antibiotic resistance.
1997). Antibiotics reduce the production of So, the question that needs to be answered is: Can a
antagonistic microbial metabolites, such as ammonia ban on the use of AGPs reverse the trend in increasing
(Zimber and Visek, 1972), which adversely affect antibiotic resistance of human pathogens? This
the physiology of the host animal. Subtherapeutic question can be answered from recent experiences
levels of antibiotics in the diet also reduce weight after the European ban on AGPs.
and length of the intestines (Visek, 1978; Postma et Following the ban of all food animal growth
al., 1999). A thinner intestinal epithelium in promoting antibiotics by Sweden in 1986, the
antibiotic-fed animals may enhance nutrient
P.R. Ferket 59
European Union banned avoparcin in 1997, and load in the gut, resulting in a reduction in energy
bacitracin, spiramycin, tylosin, and virginiamycin in and protein required to maintain and nourish the
1999. As a consequence of public pressure following intestinal tissues. Because energy required to maintain
the ban in 1999, the use of the two remaining the gut accounts for about 25% of the total basal
antibiotics, bambermycin and avilamycin is scheduled metabolic needs of an animal (Croom et al., 2000),
to be banned in 2006. This ban is logical if the any reduction in gut tissue mass can have a significant
antibiotics banned from prophylactic usage were impact on the amount of energy available for growth
commonly used in human medicine, but that is not and caloric conversion efficiency. The reduced
the case. Only bacitracin is routinely used in human microbial load in the gut by subtherapeutic levels of
medicine. In order to protect their position in the antibiotics also reduces immunological stress,
European market, the Danish government instituted resulting in more nutrients partitioned toward growth
a voluntary ban on the use of AGPs along with a and production rather than toward mechanisms of
penalty tax for use in 1998. In effect, Denmark has disease resistance. In contrast, most alternative
become an ideal laboratory to test the consequences compounds do not reduce overall microbial loads in
of a total European Ban on AGPs. By 2000, the the gut and thus will not promote growth by a
complete ban on the use of AGPs was in effect in mechanism similar to antibiotics. Instead, they alter
Denmark, but then enteric disease problems and the gut microflora profile by limiting the colonization
mortality rates began to mount and the therapeutic of unfavorable bacteria while promoting the
use of antibiotics began to rise sharply. By 2001, the fermentation of more favorable species. Consequently,
total consumption of therapeutic antibiotics almost alternatives to antibiotics promote gut health by several
reached the same amount as the total consumption of possible mechanisms including: altering gut pH,
AGPs before the ban was instituted. In effect, AGPs maintaining protective gut mucins, selection for
(avilamycin, virginiamycin, bacitracin, and tylocin) beneficial intestinal organisms or against pathogens,
that are not typically used to treat human disease were enhancing fermentation acids, enhancing nutrient
replaced by therapeutic antibiotics (ampicillin, uptake, and increasing the humoral immune response.
erythromycin, streptomycin, tetracycline, etc) that Strategic use of these alternative compounds will help
are used to treat human disease pathogens (Hayes optimize growth provided they are used in a manner
and Jensen, 2003). For example, tetracycline use in that complements their modes of action.
Denmark increased from 12,100 kg in 1998 to 27,000
kg in 2001. Now Denmark has mounting tetracycline
resistance in human pathogens, such as Salmonella SANITATION AND PATHOGEN LOAD
typhimurium and Campylobactor jejuni (DANMAP, REDUCTION
2001). Isn’t it ironic that the policy against the use
of AGPs actually resulted in an increase in resistance There is considerable evidence that subtherapeutic
to antibiotics that the public is most concerned about? antibiotics or alternative compounds are most
A simple ban on AGPs will not solve the antibiotic effective when fed to animals raised in unsanitary
resistance problem, but may lead to greater risks to environmental conditions. Good barn sanitation, pest
human and animal health (Casewell et al., 2003). control, biosecurity practices, and litter or manure
Therefore we need to strategically use different feed management are necessary to reduce pathogen load
additives and management practices that will and exposure and minimize the need for antimicrobial
minimize the use of both AGPs and therapeutic therapy. Water must be clean and drinkers must be
antibiotics. properly maintained to minimize spillage and prevent
a bloom of pathogens in the litter and environment
of the animals. Implementation of a good sanitation
General strategies to control gut health program is usually much less costly than any disease
treatment.
without antibiotics
Effective use of feed additives to manage gut health
is dependent upon some degree of understanding of ENHANCE PATHOGEN COLONIZATION
their mechanisms of action. Clearly, the modes of RESISTANCE
action of growth promoting antibiotics and their Colonization of enteric pathogens is dependent upon
alternatives can differ considerably. Subtherapeutic the degree of resistance afforded by the stability of
antibiotics work in part by decreasing the microbial
the resident microflora and the integrity of the There is a complex balance between the gut ecosystem
intestinal mucin barrier in the animal. Older animals and intestinal mucins, and this balance can be altered
are much less susceptible to the colonization of enteric by enteric health conditions and the diet. Although
pathogens than young animals because they have a intestinal mucins and glycoproteins have a protective
more stable and diverse gut microflora that function, they also serve as a nutritional substrate
competitively excludes pathogen colonization. In for some bacteria that thrive in a galactose-rich
contrast, the ability of pathogens to colonize in the environment, such as bifidobacteria (Roy et al.,
gut increases after antibiotic administration because 1991). In pigs, Pestova et al. (2000) observed a
of a loss of resident microflora. The stability of significant decrease in intestinal mucins following
resident microflora can be enhanced by the weaning, and this was partially prevented by the
administration of competitive exclusion cultures inclusion of galactose in the post-weaning diet.
(probiotics) or feeding prebiotic compounds that feed Apparently, the lack of galactose in the post-weaning
the beneficial microflora. Hollister et al. (1999) high starch diet increased the scavenging of galactosyl
reduced salmonella colonization in chicks by feeding units in mucins by some microflora, thus promoting
a live cecal culture from salmonella-free poultry. the degradation of the protective mucin barrier.
Fedorka-Cray et al. (1999) have shown similar Dietary inclusion of compounds that feed beneficial
response to microbial cultures in young swine. Gram- bacteria, such as bifidobacteria, should alleviate their
positive bacteria, including Lactobacillus, attack on the protective mucins. Such compounds
Enterococcus, Pediococcus, Bacillus, and include oligosaccharides or enzymes that liberate
bifidobacteria, and fungi of the Saccharomyces (yeast) galactose from galactosyl polymers, such as galacto-
genus are often fed after antibiotic therapy as a means mannans. More research must be done in this area
of re-introducing a beneficial flora to the gut of of interest.
affected animals. Beneficial bacteria inhibit the
colonization of pathogens by producing volatile fatty
acids that reduce the pH of the brush-border IMMUNE RESPONSE AUGMENTATION
microenvironment or they can block the attachment
of pathogens. Organic acids have strong antibacterial The immune system is the primary defense
effects, especially to Gram-negative pathogens. mechanism of the animal against infectious disease.
Blomberg et al. (1993a) also demonstrated that Augmentation of humoral and cell-mediated
undefined compounds in a culture of lactobacilli immunity will increase an animal’s ability to resist
inhibit the attachment to intestinal components of pigs disease. Although there is a small nutrient cost in
by pathogenic K88 E. coli. They suggested that the production of immunoglobulins, good antibody
compounds produced by the lactobacilli or the titer levels indicate a far more efficient capacity to
lactobacilli themselves bound to the receptor of K88 resist disease by humoral immune responses than an
E. coli in pig intestine, thereby preventing the active inflammatory response (Humphrey et al.,
colonization by the E. coli. 2002). A pro-inflammatory innate immune response
Mucins and glycoproteins associated with the is associated with the mobilization of nutrients away
intestinal brush border serve as a very important from growth and suppression of feed intake. Thus,
barrier protecting the delicate absorptive surface from dietary immunomodulators or vaccines that enhance
the abrasive action of feedstuffs, bacteria colonization, humoral immunity and minimize immunological
and toxins. Mucin, produced by goblet cells, is stress will affect growth performance most positively.
secreted in response to the degree of insult on the Although there is now a considerable amount of
absorptive surface of the gut. Glycoproteins of gut knowledge about systemic immunity, knowledge
mucins specifically bind pathogens and reduce their about gut-associated immunity is still primitive. The
colonization by serving as alternative binding sites to gut is a major interface where the immune system
receptors on host enterocytes. For example, pathogenc can sample the potential disease antigens in the
E. coli K88 adhesins were found to bind to ileal mucus animal’s environment and mount a defensive strategy
from pigs, and Blomberg et al. (1993b) concluded to resist disease. Therefore, the resident microflora
that the intestinal mucus might intercept these will have a marked effect on the amount and profile
pathogens before they can attach to intestinal tissues of immune factors, such as immunoblobulins.
and cause disease. Dietary factors that result in Perdigon et al. (1991) observed that specific
increased mucus secretion may thus indirectly enhance lactobacilli fed to mice resulted in enhanced
an animal’s ability to resist pathogen colonization. protection against S. typhimurium and E. coli by
P.R. Ferket 61
increasing IgA production. IgA, predominantly found when diets contain enzymes and antibiotics together
in the mucus secretions in the respiratory tract and (Bedford, 2000; Elwinger and Teglof, 1991; Danicke
gut, function to attenuate antigens and present them et al., 1999). In a comprehensive literature review,
to lymphocytes for degradation and stimulation of Rosen (2001) concluded that the effect of enzymes
the production of specific antibodies. Dietary was nearly equivalent to the effects of antibiotics on
supplementation of mannan-oligosaccharide (Bio- gain and FCR, and that in combination there was
Mos®) has also been shown to enhance IgA titers in further improvement, but less than the sum of the
the plasma of poultry (Savage et al., 1996) and sow’s two. Enzymes are perhaps the most extensively
milk (O’Quinn et al., 2001). reviewed products that seem to be capable of limiting
An alternative to feeding dietary factors that the performance losses associated with removal of
stimulate gut-associated humoral immunity may be antibiotic growth promoters.
feeding specific antibodies that neutralize pathogenic Because supplemental enzymes mediate their
organisms. To produce the specific antibodies, laying beneficial effects primarily by enhancing feed
hens are exposed to particular antigens to stimulate digestibility and nutrient availability to the host, it
the production of immunoglobulins, which are must be assumed that they also influence the gut
deposited in the egg. These immunoglobulins are then microbial ecosystem. The use of enzymes has been
harvested from the eggs and fed to susceptible young shown to alter the gut microflora populations in the
animals. There may be some limitations to this small intestine and caeca (Choct et al., 1996; Hock
technology, since these immunoproteins are sensitive et al., 1997; Bedford, 2000) and reduce mortality
to heat treatment during feed processing and the rates (Rosen, 2001). Such benefits are brought about
digestive process of the animal. by a more rapid digestion and absorption of starch,
protein and fat from the small intestine, which
effectively limits available substrate for the resident
Nutritional strategies and feed additives flora. In general, the improvement in nutrient
digestibility achieved for the host by the use of an
DIET DIGESTIBILITY AND ENZYME appropriate enzyme is much smaller than the
SUPPLEMENTATION concomitant loss of substrate experienced by
microflora resident in the large intestinal. This starch
Gut health and enteric disease resistance is often and protein removal effect is coupled with the
dependent upon the digestibility of feed components production of exogenous enzyme for fiber-derived
and feed formulation. Poorly digested protein meals oligomers, which serve as substrate for specific
due to improper heat processing causes the populations of bacteria that seem to benefit the host
proliferation of putrifying bacteria in the hindgut, (Bedford, 2000).
which increases toxic metabolites (ammonia and
biogenic amines) that compromise gut health. In
agreement, antibiotics are most effective in birds fed ACIDIFIERS AND ORGANIC ACIDS
diets containing high levels of indigestible protein
(Smulders et al., 2000). Similarly, poultry fed diets Clostridia and pathogenic coliform bacteria often
containing high levels of poorly digested non-starch associated with enteric disease do not grow well in
polysaccharides (NSP) from wheat, barley or rye are media of low pH, so any means to reduce gut pH
more susceptible to enteric disease, such as necrotic should improve an animal’s resistance to enteric
enteritis (Riddell and Kong, 1992; Kaldhusdal and disease. Because organic acids have strong
Skjerve, 1996). Langhout (1999) observed that bacteriostatic effects, they have been used as
dietary NSP significantly increases gut populations salmonella-control agents in feed and water supplies
of pathogenic bacteria at the expense of beneficial for livestock and poultry. Organic acid blends have
bacteria. However, the digestibility of wheat, barley, also been used as acidifiers in baby pig diets to reduce
rye, triticale and even corn-based diets can be enteric disease, but the benefit for poultry seems to
significantly improved through use of exogenous be less conclusive. Dietary acidifiers may work better
enzymes including xylanases, phytases and ß- in baby pig diets because they have more limited
glucanases. The response to dietary enzyme hydrochloric acid production than chicks. Moreover,
supplementation is greater when antibiotics are not dietary organic acids are easily neutralized in the
used than when they are, but the performance duodenum unless they are delivered to the ileum and
responses do not approach the level that is observed below by adsorbent vehicles.
HERBS, SPICES, AND ESSENTIAL OILS the end of the chain (Yun, 1996). A sucrose unit
attached to one additional fructose residue is
Herbs, spices, and plant extracts have been used to commonly referred to as 1-kestose. Nystose contains
make human foods more appetizing for centuries, two additional fructose units, and three additional
and many of them are recognized for their health fructose units is designated as 1F- ß-fructofuranosyl
benefits. Some of these compounds stimulate appetite (Hidaka and Hirayama, 1991). Fructooligo-
(e.g. menthol from peppermint), provide antioxidant saccharides are found in numerous plants such as the
protection (e.g. cinnamaldehyde from cinnamon), or onion, Jerusalem artichoke, garlic, banana, chicory,
suppress microbial growth (carvacrol from oregano). asparagus, and wheat.
These plant-based antimicrobial compounds, which Fructooligosaccharides influence enteric microflora
function in a fundamentally similar way to antibiotic by ‘feeding the good bacteria’, which competitively
compounds produced by fungi, could be used to excludes the colonization of pathogens. Dietary
replace some antibiotic growth promoters. To be most supplementation of FOS provides selective
effective as growth promoters, these herbal enrichment of lactobacilli (Mitsuoka et al., 1987) and
antimicrobial compounds must be supplemented to bifidobacteria (Hidaka et al., 1991). Patterson et al.
the feed in a more concentrated form than found in (1997) found that cecal bifidobacteria concentrations
their natural source. As with antibiotics, continued were increased 24-fold and lactobacilli populations
use of these plant-based antimicrobials may result in increased 7-fold in young broilers fed the FOS-
the development of resistance in some pathogenic enriched diets. Fructooligosaccharides are well
bacteria. However, more research is necessary to utilized by the majority of bifidobacteria strains (B.
confirm this risk. longum, brevis, and infantis) with the exception of
Essential oils from oregano are showing the greatest B. bifidum (Hidaka and Hirayama, 1991). The
potential as an alternative to antibiotic growth bacteroides group also showed a tendency to utilize
promoters. Oregano contains phenolic compounds, FOS as a growth source, while L. fermentum, E. coli,
such as carvacrol, that have antimicrobial activity and C. perfringens failed to utilize FOS as a
(Akagul and Kivanc, 1988). Like antibiotics, oregano fermentative carbohydrate source. Bifidobacteria
essential oils modify the gut microflora and reduce readily ferment FOS because of the innate secretion
microbial load by suppressing bacteria proliferation. of a ß-fructoside enzyme. Bifidobacteria may inhibit
There are some claims that oregano oil can replace other microbes because of their acidic surroundings
anticoccidial compounds, not because they inactivate from the high production of VFAs or the secretion
coccidia, but because they increase the turnover of of bacteriocin-like peptides. The improvement in gut
the gut lining and prevent coccidial attack by health conditions by dietary FOS supplementation
maintaining a more healthy population of gut cells often results in improved growth performance.
(Bruerton, 2002). This mode of action would Ammerman et al. (1988) demonstrated that the
increase the animal’s maintenance energy requirement addition of either 0.25% or 0.50% dietary FOS
because enterocyte turnover is a major proportion of improved feed efficiency from 1 to 46 days of age
the basal metabolic rate. and reduced mortality when fed at the higher level
(0.50%). FOS-treated birds also had less air sac lesions
at day 46.
OLIGOSACCHARIDES
Mannan oligosaccharide (MOS)
Oligosaccharides are promising alternatives to
antibiotic growth promoters because they facilitate Unlike FOS, MOS is not used as a substrate in
and support the symbiotic relationship between host microbial fermentation, but it still exerts a significant
and microflora. Fructooligosaccharide (FOS) and growth-promoting effect by enhancing the animal’s
mannan oligosaccharide (MOS) are two classes of resistance to enteric pathogens. Bio-Mos® (Alltech
oligosaccharides that are beneficial to enteric health, Inc., Nicholasville, KY) is the commercial source of
but they do so by different means. MOS that has been used in most of the published
research literature. Based on the scientific literature,
Fructooligosaccharides (FOS) Bio-Mos® enhances resistance to enteric disease and
Fructooligosaccharide compounds are inulin-type promotes growth by the following means: 1) inhibits
oligosaccharides of D-fructose attached by ß(2-1) colonization of enteric pathogens by blocking
linkages that are attached to a D-glucosyl residue at bacterial adhesion to gut lining; 2) enhances
P.R. Ferket 63
immunity; 3) modifies microflora fermentation to humoral immune response is nutritionally a more

favor nutrient availability for the host; 4) enhances efficient means to resist disease than an active
the brush border mucin barrier; 5) reduces enterocyte inflammatory response (Humphrey et al., 2002).
turnover rate; and 6) enhances the integrity of the Savage et al. (1996) reported an increase in plasma
gut lining. IgG and bile IgA in poults fed diets supplemented
with 0.11% Bio-Mos®. An increase in antibody
Inhibition of pathogen colonization response to MOS is expected because of the ability
of the immune system to react to foreign antigenic
Mannan oligosaccharides, derived from mannans on material of microbial origin. Portions of the cell wall
yeast cell surfaces, act as high affinity ligands, structure of the yeast organism Saccharomyces
offering a competitive binding site for a certain class contained in MOS has been shown to elicit powerful
of bacteria (Ofek et al., 1977). Gram-negative antigenic properties (Ballou, 1970). However, MOS
pathogens with the mannose-specific Type-1 fimbriae may also enhance humoral immunity against specific
attach to the MOS instead of attaching to intestinal pathogens by preventing their colonization leading
epithelial cells and they move through the gut without to disease, yet allowing them to be presented to
colonization. Dietary MOS in the intestinal tract immune cells as attenuated antigens. Indeed as MOS
removes pathogenic bacteria that could attach to the facilitates the secretion of IgA into the gut mucosa
intestinal epithelium (Newman, 1994). Mannose was layer, pathogenic agents become more labile to the
shown by Oyofo et al. (1989a) to inhibit the in vitro phagocytic action of gut-associated lympocytes.
attachment of S. typhimurium to intestinal cells of All animals reared under commercial field
the day-old chicken. Then Oyofo et al. (1989b) conditions are subjected to immunological stress,
provided evidence that dietary D-mannose was depending on the pathogen load in the environment
successful at inhibiting the intestinal colonization of and the vaccination program. The release of cytokines
S. typhimurium in broilers. The ability of MOS to associated with inflammation and the innate immune
interfere with the attachment of pathogenic bacteria response results in fever (which reduces appetite),
in the gut raises the possibility that it could also inhibit causes the mobilization of body reserves (glucose,
the binding between bacteria that is required for amino acids, and minerals) away from liver, muscle
plasmid transfer via conjugation. This kind of and bone, suppresses nutrient absorption in the gut,
inhibition of plasmid transfer in the digestive tract and increases body fluid losses as diuresis and diarrhea.
of mice colonized with human microflora has been The positive growth performance effects observed
described using lactose (Duval-Iflah, 2001). Lou among animals in studies with Bio-Mos® may be
(1995) demonstrated that dietary Bio-Mos ® partly due to its effect on acute immunological stress.
supplementation decreased the proportion of specific Although MOS may enhance humoral immunity,
groups of Gram-negative antibiotic resistant fecal there is some evidence that it may suppress the pro-
bacteria in swine. inflammatory immune response that is detrimental
In an effort to confirm that MOS inhibits pathogen to growth and production. To test this hypothesis,
colonization, Spring et al. (2000) screened different Ferket (2002) induced an acute immune stress in 14-
bacterial strains for their ability to agglutinate mannan day old turkey poults by intraperitoneal injection of
oligosaccharides in yeast cell preparations. Five of LPS from S. typhimurium strain SL 684. The poults
seven strains of E. coli and 7 of 10 strains of S. were fed either 1 kg Bio-Mos®/ton, 20 g virginiamycin/
typhimurium and S. enteritidis were agglutinated by ton, or control diet from day one of age. Cloacal
Bio-Mos® and Sac. cerevisiae cells. However, strains temperatures were measured 8 hrs after the LPS
of S. choleraesuis, S. pullorum, and Campylobacter injection, and then body, liver, spleen, bursa of
were not agglutinated. Although Bio-Mos® does not Fabricius, and intestinal tract weights were recorded.
bind clostridia, it does reduce clostridial numbers in In contrast to the control and the antibiotic-fed birds,
some trials, possibly by enhancing the mucin barrier the Bio-Mos®-fed birds showed no fever response
or stimulating gut associated immunity. 8 hrs post-injection, even though liver and intestine
weights were increased. In other words, the Bio-
Enhancement of immune function Mos®-fed birds retained normal body temperature
Mannan oligosaccharide has been shown to have a after exposure to a pro-inflammatory antigen, while
positive influence on humoral immunity and the controls and virginiamycin-fed birds expressed
immunoglobulin status. As mentioned above, a good elevated body temperature. Under commercial
conditions where birds are subjected to chronic by Bio-Mos®, a decrease in crypt depth approached
immunological stress, Bio-Mos® may help reduce the significance and villi height:crypt depth ratio was
pro-inflammatory response and associated depression significantly greater than the control or virginiamycin
in feed intake and growth. treatments. Iji et al. (2001) also observed an increase
in jejunal villi height:crypt depth ratio by Bio-Mos®
Effects on gut microflora fermentation and supplementation in broilers, but this was due to a
dietary energy utilization significant increase in villi height rather than crypt
depth. These researchers also observed that Bio-Mos®
Even though the ceca are the primary site of gut significantly increased protein/DNA of jejunal
microflora fermentation, microbial fermentation in mucosa, and increased the brush border enzymes
the jejunum has a greater influence on digestion and maltase, leucine aminopepidase and alkaline
nutrient absorption. Measurement of volatile fatty phosphatase. Turkeys receiving Bio-Mos® in our
acid (VFA) content and pH of the jejunum digesta is experiment also exhibited a thinner muscularis layer
one way to evaluate the influence of feed additives and increased the number of goblet cells per mm of
on microbial fermentation. In a study with turkeys, villus height as compared to control birds.
Ferket (2002) observed dietary supplementation of The mucus gel layer coating the surface of the
Bio-Mos® and antibiotics reduced total VFA content intestinal epithelium is the first major barrier to enteric
of jejunum digesta by about 40%. Most of this effect infection. Hence, the production of mucus, as
was attributed to a reduction in propionic acid, which indicated by the number of goblet cells, is an
is the major fermentation product of microflora that important feature in the protective scheme against
use starches and sugars as their primary substrates. pathogens. Feeding Bio-Mos® resulted in an increased
Therefore, Bio-Mos® may improve dietary energy proliferation of goblet cells into the surface of the
availability by reducing the microflora-host villus membrane. The innate immune system
competition for available starches and sugars. Indeed, recognizes key molecular structures of invading
apparent metabolizable energy of the diet was bacteria, including lipopolysacchharides,
increased by about 3% when Bio-Mos ® or peptidoglycans, and possibly the mannose structures
virginiamycin was supplemented to the diet. Another in the cell walls of yeasts. Oligosaccharides containing
benefit to dietary inclusion of Bio-Mos ® was a mannose have been shown to affect the immune
decrease in jejunum digesta pH and ammonia system by stimulating liver secretion of mannose-
concentration in comparison to the antibiotic-fed binding protein. This protein, in turn, can bind to
birds. Lower gut pH suppresses the proliferation of bacteria and trigger the complement cascade of the
putrifying bacteria that excrete ammonia as their host immune system (Newman, 1994). Intestinal
fermentation by-product, and ammonia has a microbes might influence goblet cell dynamics by
detrimental effect on the integrity of gut tissues. releasing bioactive compounds or indirect activation
of the immune system (Bienenstock and Befus, 1980).
Effects on gut tissue integrity and health
The beneficial effects of MOS on the gut microflora,
nutrient utilization, and growth performance may be Conclusion
associated brush border morphology and how it
In response to consumer demands and government
influences enteric disease resistance. To test this
regulations, today’s intensive animal agriculture
hypothesis, Ferket (2002) conducted an experiment
industry must adapt to producing animals in a world
to ascertain effects of Bio-Mos® and virginiamycin
without antibiotic growth promoters. This paper
on jejunum villi morphology. Commercial Hybrid®
presented several alternatives to antibiotics to manage
poults were fed a corn-soy control diet or diets
gut health. Although no single alternative may be as
supplemented with 1 kg Bio-Mos®/tonne or 20 g
effective as antibiotics, a combination of strategies
virginiamycin/tonne starting at 1 day of age. At 14
and feed additives can be used to achieve good gut
days of age, 8 birds per treatment pen were sampled
health and growth performance. The key to selecting
for morphometric measurements including villus
the most cost effective approach will depend upon
height, crypt depth, muscularis thickness, and goblet
the production requirements of each company, and
cell number.
the type of production challenges they face.
Bio-Mos ® had the greatest effect on villi
morphology. Although villus height was unaffected
P.R. Ferket 65
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G.G. Mateos et al. 69
Facing the realities of poultry health and performance without antibiotics in

Europe
G.G. MATEOS1, J.M. GONZÁLEZ-ALVARADO2 AND R. LÁZARO1
1
Departamento de Producción Animal, Universidad Politécnica de Madrid, Madrid, Spain
2
Departamento de Agrobiología. Universidad Autónoma de Tlaxcala, Tlaxcala, México
Introduction
Maintaining the structure of the digestive tract in good manipulation of the composition and nutrient content
health is critical for successful rearing of broilers. of the diet might help to improve GIT health. Options
Dietary factors disrupting mucosal integrity or to minimize enteric diseases associated with
motility of the gastrointestinal tract (GIT) might microflora changes include the use of highly
induce enteric disorders, wet litter problems, poor digestible feeds to improve the structure of the gut,
pigmentation and inefficient growth. Enteric adequate processing of raw materials and diets, and
disorders have been managed through dietary changes the use of exogenous enzymes, organic acids, yeasts,
including the use of in-feed growth promoters and and other additives (Lilburn, 1998; Mateos et al.,
animal proteins. Addition of certain antibiotics to feed 2002). However, data supporting the effectiveness
at low levels is a common practice in poultry of these techniques are equivocal, and changes in flock
production and has been shown to improve weight management, early detection of symptoms of
gain and feed efficiency in the range of 1 to 5% diseases, and careful design of the feeding program
(Thomke and Elwinger, 1998a). The reasons for the are required to reduce the incidence of enteric
improvement are not well understood but some problems.
intestinal organisms such as clostridia and other gram-
positive germs are inhibited by these antibacterial
agents. In fact, the restrictions imposed on the use of Post-hatch nutrition, feed management
in-feed antibiotics and proteins of animal origin in and chick productivity
many European countries have increased the incidence
of clostridiosis and other enteric diseases; and the Growth of broilers during the first days post-hatch is
cost of broiler meat production has increased by of paramount importance for ultimate performance
around 0.01 €/kg (Mateos et al., 2001a; Van der Eijk, of poultry reared for meat. During the first week of
2002). However, consumer concerns about recycling life allometric growth is maximal and the chick
animal proteins and cross-resistance related to the multiplies initial body weight 4 to 5-fold. In commercial
use of additives requires new methods to protect operations a large proportion of chicks remain without
enteric health and improve broiler performance. feed for more than 36 hrs after removal from the
Many poultry integrators in Europe are producing incubator as placement on the farm is frequently
feeds without growth promoters, based exclusively delayed because of hatchery processing and
on vegetable feedstuffs. Under these circumstances, transportation. Furthermore, the yolk sac at hatch
problems related to necrotic enteritis (NE), feed contains less than 1 g of triglycerides and is almost
passage, and overgrowth of intestinal microflora are absent by the third to fourth day of life (Murakami
frequently reported. Numerous natural products, et al., 1988; Bigot et al., 2003). Therefore, residual
including organic acids, probiotics, prebiotics, plant triglycerides in the yolk sac are not a good reservoir
extracts, and immune stimulants have been proposed of nutrients and early access to feed is critical for the
for the control of pathogens in the GIT (Flickinger, newborn chick (Lilburn, 1998). Early access to feed
2003; Hooge, 2003; Chaveeraach et al., 2004). Also, and water stimulates the growth of the GIT and its
70 Facing the realities of poultry health and performance without antibiotics in Europe
absorptive capacity and improves gut integrity and Aranibar (2001) and Aranibar et al. (2001) observed
subsequent performance (Moran, 1990; Noy and that changes in the nutrient content (crude protein,
Sklan, 1999; Corless and Sell, 1999). The GIT adapts lysine and ME concentration) of the diet or in the
to the nature of the digestive contents, a response major energy source used (fats with different fatty
that is modulated by the health status of the gut. In acid profile, starch, and sucrose) or delays in the
case of disturbances, physiological responses take access to feed for up to 48 hrs affected many digestive
place with overgrowth of pathogenic bacteria and a and productive parameters in chicks from 0 to 7 days
reduction of the appetite. In practical conditions birds of age but that the differences tended to disappear
that eat more during the first week of life achieve with age. These results agree with Noy and Sklan
the best final weights and feed conversion at slaughter (2002), who used a series of diets during the first
(Martins, 2003). week of age in which the level of fat, protein, and
The nutrient requirements of baby chicks and poults cellulose varied widely and found differences in
after hatching are not precisely known. Lilburn broiler performance at day 7 but not at day 18. In
(1998) proposes feeding highly digestible protein general, losses in performance incurred by chicks held
ingredients in combination with corn for the first 10 without feed for 36 to 48 hrs are compensated by
days of life to meet the energy and protein needs of increasing the length of time required to reach market
young birds. Batal and Parsons (2002a) observed that weight by a similar period of time. Therefore, at
the ME(n) value for chicks of corn diets based on farm level, where age of the chicks is measured as
soybean or rapeseed meal was very low at 2 and 4 days post-feeding and not post-hatching, only small
days of age, but increased afterwards. However, the differences in final performance will be found
ME(n) of a dextrose-casein diet was high at 2 days associated with a moderate delay of access to feed.
and no further improvements were observed with age
(Table 1). Sulistiyanto et al. (1999) also indicated
that casein was better utilized than fish meal and Digestibility of nutrients
soybean meal for chicks less than 10 days of age.
They also found that energy from corn was utilized The National Research Council (1994) assumes that
better than energy from wheat and sorghum but not digestibility of nutrients is independent of age, an
better than energy from different fat sources. On the assumption that is no longer accepted (Batal and
other hand, Noy and Sklan (2002) observed that baby Parsons, 2002a; Mateos et al., 2002). Digestion and
chicks do not respond to high-fat starter diets; and absorption of nutrients early in life depends primarily
Sklan (2003) indicated that lipoprotein production on pancreatic enzyme activity (Nitsan et al., 1991a,
might limit fat use in very young broilers. b), but the pancreas is immature at hatch. As a
consequence, dietary nutrients are poorly utilized
Table 1. Influence of age on the ME(n):GE ratio of diets for chicks during the first 10 days post-hatching. Gracia et al.
(%).1,2 (2003a) have reported that in the broiler chick, the
Age, Corn- Corn- Corn- Dextrose- Pooled maximal weight (g organ/g of BW) of the
(days) soybean canola synthetic casein SEM proventriculus, gizzard, pancreas, liver, and small
meal meal amino acids
intestine is observed at 4.1, 3.9, 8.1, 4.6, and 7.9
0-2 66 cy 63 bz 84 cx 88 bw 0.8 days of age, respectively (Table 2), data that compare
3-4 68 cy 64 bz 84 cx 88 bw 0.6
7 70bx 65 by 87 bw 88 bw 0.4
well with information from Sell (1996). Batal and
14 73 ax 68 ay 89 aw 88 bw 0.2 Parsons (2002a) found that the ME(n) and the
21 73 ax 69 ay 89 aw 89 aw 0.4 apparent digestibility of starch, fat, and selected amino
Pooled 0.6 0.7 1.0 0.5 acids of a corn-soybean meal diet was low at 2 days
SEM
and reached a plateau at 14 days of age (Table 3).
1
Batal and Parsons (2002a). Lilburn (1998) indicated that overall digestibility of
2
Soy oil was used as the main fat source of the diets.
a-c
Means within a column differ significantly (P<0.05).
lipids in chicks for the first 3 to 5 days of age varies
w-z
Means within a row differ significantly (P<0.05). from 69% to 80% with the higher values
corresponding to unsaturated fats. Therefore, proper
Data from different institutions indicate that dietary lipid sources can be used successfully in prestarter
composition of the prestarter feed and delay of access diets for poultry.
to feed influence chick productivity, but that the Starch digestibility is critical for understanding
effects tend to disappear with age. For example, energy utilization because the starch content of a
Table 2. Changes with age in relative weights of digestive organs is hydrolyzed more rapidly than starch in large
of the chick (% BW). granules. Also, starches with high amylose content
Age, days Day of are less susceptible to amylase attack than starches
maximal with low amylose content. Therefore, rice might be
relative a candidate for prestarter diets for chicks because its
growth starch is very accessible and mostly of type A (easily
0 4 8 15 21 SEM A1 B2 digested compact starch with no free space left for
(n= 20) water), granule size is very small, the amylose content
Proventriculus 0.87 1.46 1.19 0.98 0.75 0.04 4.1 3 to 5 is lowest among cereals, and the grain has very low
Gizzard 5.28 5.75 4.34 3.37 2.72 0.14 3.9 3 to 4 content of ß-glucans and xylans. A recent study
Pancreas 0.15 0.57 0.59 0.49 0.40 0.023 8.1 8 to 9 conducted in our laboratory (González-Alvarado et
Liver 2.55 4.36 4.22 3.74 3.17 0.144 4.6 6 to 8 al., unpublished) has confirmed the potential of rice
Small intestine 2.74 6.09 6.87 4.80 4.33 0.19 7.9 5 to 7
as an energy source in prestarter feeds for broilers.
1
Gracia et al. (2003a). In two different trials we studied the influence of the
2
Sell (1996).
P<0.001 with age (0, 4, 8, 15, 21 days). main cereal of the diet (60% corn vs 60% rice),
processing of the cereal portion of the diet (raw vs
Table 3. Influence of age on apparent fecal digestibility of cooked and rolled) and the inclusion of insoluble fiber
nutrients in New Hampshire x Columbian male chicks.1,2 sources (none vs 3% soy hulls vs 3% oat hulls) for
broilers from 1 to 21 days of age. Rice feeding
Age ME(n) Apparent fecal digestibility (%)
consistently improved feed conversion in both trials,
(days)(kcal/kg DM) Starch Fat Lys Met
but no differences between cereals were observed for
0-2 2,970d 93 c 61 b 78 d 80 c feed intake or daily gain (Table 5). The data indicate
3-4 3,085c 93 c 58 b 81 c 82 c that rice is well utilized by the chick and that its
7 3,185b 97 b 59 b 85 b 87 b metabolizable energy content is approximately 3 to
14 3,429a 99 a 74 a 89 a 92 a
21 3,426a 99 a 73 a 89 a 92 a
5% higher than that of corn.
SEM 26 0.4 1.3 0.7 0.9
Table 4. Influence of age on starch and ether extract fecal
1
Batal and Parsons (2002a). digestibility in broilers (%).
2
Diets with 5.5% added soy oil.
a-d
Age Starch Ether extract
Means within a column with no common superscript differ
1 2
significantly (P<0.05). (days) A B A B
4 95.0 95.0 63.4 60.1
typical poultry diet ranges from 35 to 40%. It is 8 95.8 - 56.9 52.4
believed that α-amylase is produced in excess of 15 96.2 96.7 77.0 74.7
21 97.2 97.2 78.9 78.1
requirements (Moran, 1985; 1992), but several SEM (n =10) 0.15 0.09 1.20 1.44
reports indicate that starch digestion at the end of the 1
Mateos et al. (2002). Corn-soybean meal diet with 2.7% lard.
ileum of young birds is incomplete. Rogel et al. 2
Gracia et al. (2003a). Barley-soybean meal diet with 6% lard.
(1987) observed that in meal diets, fecal digestibility Starch: linear, P<0.001.
of wheat starch was 77.2% at 3 weeks and 97.8% at Ether extract: linear (P≤0.01) and quadratic; P<0.01.
6 weeks of age. Weurding et al. (2001) found that
total tract digestibility of starch varied from 98.9% Table 5. Influence of type of cereal in the diet on feed conversion
(g of feed/g of gain) of broilers from 1 to 21 d of age1.
for tapioca pellets to 31.7% for raw potato starch,
with intermediate values for cereals (93.8 to 98.3%) Trial 1 Trial 2
and legume grains (74.5 to 81.5%). Mateos et al. Age Corn Rice SEM Corn Rice SEM
(days) (n = 36) (n = 18)
(2002) and Gracia et al. (2003a) observed that total
tract digestibility of starch and ether extract in broilers 0 to 4 1.34 1.32 0.013 1.21 1.19 0.016
4 to 8 1.23a 1.16b 0.010 1.26 a 1.21b 0.017
increased with age in both corn- and barley-soybean
8 to 14 1.33 a 1.28b 0.013 1.31 a 1.24b 0.011
meal diets (Table 4). Similar results have been 14 to 21 1.45 1.41 0.014 1.42 1.38 0.019
reported by Yuste et al. (1991), Batal and Parsons 0 to 21 1.38 a 1.34b 0.008 1.35 a 1.30b 0.012
(2002a, b) and Gracia et al. (2003b). Enzyme 1
González-Alvarado et al. (unpublished data). Diets based on
accessibility to starch is determined by the viscosity 60% of raw or cooked cereal.
a-b
of gut contents and the nature and structure of the Means within a column (for each trial) with no common
superscript differ significantly (P<0.05).
starch granules. In general, starch in small granules
Heat processing of ingredients and diets Table 6. Influence of barley processing and enzyme supplementa-
tion on performance of broilers.1
Heat is usually applied to pellet poultry feeds and 0-7 days 0-42 days
also to inactivate thermolabile antinutritional factors ADG (g) FC (g/g) ADG (g) FC (g/g)
contained in some raw materials such as soybeans. Enzymes 2
Recently, expanded feeds (110ºC to 120 ºC for 5 sec) 0 14.6 1.20 53.7 1.71
have been introduced into the market because of 500 ppm 15.5 1.15 57.4 1.65
beneficial effects on feed hygiene, nutrient Heat processing
digestibility, and broiler productivity (Fancher et al., Raw 14.3 1.18 55.5 1.69
1996; Mateos and Lázaro, 2001). The information Heated2 15.5 1.18 55.6 1.68
available on the influence of heat processing on SEM (n = 12) 0.657 0.037 1.19 0.027
digestive physiology and poultry performance is Probabilities
Enzymes 0.05 0.01 0.001 0.001
scarce and contradictory (García et al., 1998; Mateos Heat processing3 0.001 NS NS NS
et al., 2002). Heat processing disrupts feed structure, 1
García et al. (1998).
facilitating the access to nutrients by digestive 2
Xylanase and ß-glucanase complex from Aspergillus niger.
enzymes. However, heat processing also shifts the 3
Average value for micronized and expanded barley.
site of starch digestion, facilitates Maillard reactions,
and solubilizes part of the starch and of the NSP,
increasing digesta viscosity. Plavnik and Sklan (1995) Table 7. Influence of heat processing, enzyme supplementation,
have found that dry extrusion or expansion of a corn and age on fecal starch digestibility of corn diets for broilers (%).1
diet improved the ME(n) by 1.5 to 3%, primarily Broiler age (days)
due to an improvement in fatty acid digestibility but 4 8 15 21 Average
no effect was found by Vukic Vranjes et al. (1994). Heat processing
Commercial information indicates that heat Raw 94.2b 94.9b 95.7b 97.1 95.5
processing of barley and wheat diets increases the Cooked 2 95.8a 96.7a 96.7a 97.3 96.6
incidence of wet litter and that the judicious use of Enzymes3
appropriate enzymes reduces the condition (Nissinen 0 94.8 95.9 96.2 97.3 96.1
et al., 1993), data which are corroborated by our 500 ppm 95.2 95.6 96.2 97.1 96.0
Age (n = 20) 95.0 95.8 96.2 97.2
own results (Lázaro et al., 2003a, b, 2004). García SEM (n = 10) 0.16 0.13 0.19 0.12 0.17
et al. (1998) studied the influence of heat processing 1
Mateos et al. (2002).
of barley (cooked at 99ºC for 50 min) and exogenous 2
99ºC for 50 min followed by rolling.
enzymes (ß-glucanase and xylanase) on broiler 3
Protease, xylanase, and α-amylase complex.
performance at 42 days (Table 6). Heat processing a,b
Means in a column differ significantly (P<0.05).
of barley improved daily gains at 7 days but the effects Age effect (P<0.01).
disappeared thereafter. Enzymes improved
performance at all ages but no interaction of
Table 8. Influence of heat processing of the cereal portion of the
processing and enzymes was detected for any trait. diet on broiler performance from 0 to 21 d of age.1
Similar results have been obtained by Mateos et al.
(2002), working with raw and cooked corn (Amandus Cereal Process2 Age of broilers (days)
Kahl, Reinbeck, Germany) diets (Table 7). In a recent 0 to 4 0 to 21
trial, González-Alvarado et al. (unpublished) have ADG3 FC4 ADG FC
studied the influence of including heat processed corn Corn Raw 14.5a 1.31ab 31.2 1.37ab
or rice in the diet on performance of broiler chicks. Corn HP2 13.5b 1.36a 32.0 1.40a
The diets contained 60% cereal either raw or cooked Rice Raw 13.9b 1.34ab 32.4 1.35b
(90ºC for 50 min and then rolled). Heat processing Rice HP 13.9b 1.30b 31.9 1.32b
SEM (n = 18) 0.125 0.019 0.53 0.011
had little effect on productivity at any age but Main effects
improved feed conversion in diets based on rice, Cereal NS NS NS 0.001
although not in diets based on corn (Table 8). Heat processing 0.001 NS NS NS
1
González-Alvarado et al. (unpublished data).
Particle size, feed form, and whole grains 2
Cooked (90ºC; 50 min) and rolled.
3
Average daily gain, g.
The benefits of feed processing have long been 4
Feed intake per g of body weight gain.
recognized by the feed compound industry. Physical Significant interaction: cereal x HP for ADG (P<0.001) and FC
(P<0.05) from 0 to 4 days and for FC (P<0.05) from 0 to 21 days.
structure of the feed (wet feeding, particle size, feed is the excessive hardness of the pellets, which reduces
form, and inclusion of whole grains) influences GIT feed intake and increase wastage by the bird.
structure, composition of the microflora, nutrient Feeding whole grains to poultry has been a common
digestibility, and feed intake. Fine grinding of practice in Europe for the last 50 years. In fact, some
feedstuffs is a common practice because small particle poultry integrators are diluting broiler rations with
size improves nutrient digestibility and facilitates the up to 25% whole wheat to reduce feed cost of poultry
process of pelleting (Douglas et al., 1990; Lott et diets. Plavnik et al. (2002) observed that the inclusion
al., 1992; Kilburn and Edwards, 2001). However, of 20% whole wheat in diets for broilers improved
finely ground cereals might be detrimental for body weights (2,494 vs 2,431 g; P<0.05) and feed
mucosal cell growth and motility of the GIT. Fine conversion (1.82 vs 1.93 g/g; P<0.05) at 7 weeks
particles produce atrophy of the gizzard, a major and increased gizzard weight (16.5 vs 15.0 g/kg BW;
regulator of intestinal motility (Nir et al., 1994; Jones P<0.05). Svihus et al. (1997) observed that duodenal
and Taylor, 2001). Nir et al. (1994) theorized that digesta from chickens fed whole grain had similar
large particles enhance GIT motility and stimulate particle size to digesta from chickens fed ground grain.
peristalsis and digesta backflow whereas finely In fact, Svihus et al. (2002) observed that
ground particles reduce the reflux of the digestive replacement of ground wheat with whole wheat
contents, resulting in more nutrients passing increased ileal starch digestibility (93% to 99%)
undigested to the hindgut. In a recent study, Kilburn indicating that whole grain feeding may improve bird
and Edwards (2004) have found that coarse soybean performance by stimulating gizzard development and
meal increases bone ash of broiler chicks, probably enhancing enzyme production.
through an improvement in mineral utilization, and
also improves growth and feed efficiency when used
in semipurified diets. Crude fiber, nonstarch polysaccharides,
In commercial practice most diets for broilers and and enzymes
turkeys undergo some type of processing. It is widely
accepted that pelleting enhances feed value by making An excess of fiber in feeds might impair nutrient
more nutrients available for growth. Two areas of digestibility and feed efficiency. Dietary fiber often
interest in this respect are the influence of pellet increases endogenous losses leading to a decrease in
quality on feed intake by young poults and chicks ileal digestibility of starch, protein, and lipids
and on performance of growing and finishing birds. (Souffrant, 2001). As a consequence, current practical
Quality and characteristics of the pellets define the diets for prestarter feeds are based on low fiber
ingestion of prestarter feeds by chicks and poults ingredients such as corn, wheat, and high protein
(Picard et al., 2000). In fact, the industry has started soybean meal. However, dietary fiber is a
to manufacture micropellets or small crumbles of heterogenous class of components differing in
uniform size to maximize intake and improve structure and physiological properties. In general
performance at early ages (Martins, 2003). It is widely terms, soluble fiber increases intestinal transit time,
accepted that pellet quality of corn-based diets for delays gastric emptying, increases pancreatic
broilers should provide more than 60% to 65% intact secretion, and slows absorption, whereas insoluble
pellets at feeder level to maximize bird performance. fiber decreases transit time and enhances water-
However, there is a clear trend toward increasing the holding capacity (Montagne et al., 2003). Recent
energy concentration of diets for broilers and turkeys data indicate that adequate type and quantity of fiber
to maximize growth, which often results in the could reduce digestive disturbances and improve the
inclusion of higher levels of supplemental fat. adaptation of the GIT of monogastric animals to
Usually, an increase in the dietary fat level results in current production systems (Graham and Åman, 1991;
a reduction in pellet quality. Consequently, efforts to Hetland and Svihus, 2001; Mateos et al., 2001b;
increase the energy intake of birds through fat Hetland et al., 2003; Sklan et al., 2003). For
supplementation may be partially offset by a example, proventricular hypertrophy and poor gizzard
reduction in pellet quality. Therefore, any addition development has been linked to the use of low fiber
of fat to the diet of broilers to improve performance diets (Riddell, 1976). Montagne et al. (2003) indicate
must not compromise pellet quality. On the other that, depending on nature and physiological factors,
hand, a problem frequently found in commercial dietary fiber may improve gut health, or alternatively
operations when wheat is the main cereal of the diet enhance gut perturbation and subsequent diarrhoea
in young animals. Therefore, more studies are needed Table 10. Effects of adding fiber sources to a low fiber diet on
to find the maximum and minimum levels of dietary broiler performance at 21 days.1
fiber to be included in diets for poultry, especially at Fiber source ADG (g) FI (g/day) FC (g/g)
young ages. In fact, the British Society of Animal
Science has recently proposed a minimum of crude None 30.7 42.9 1.39
fiber and of neutral detergent fiber in diets for young Oat hulls, 3% 33.1 43.5 1.31
Soy hulls, 3% 33.4 44.8 1.34
pigs (BSAS, 2003) but no studies have been SEM (n = 6) 0.92 1.32 0.019
conducted with young birds. We have studied the P2 0.05 NS 0.001
influence of including different sources of insoluble
1
dietary fiber in diets for broiler chicks (González- Gonzalez-Alvarado et al. (unpublished data).
2
Significance of contrast: none vs hull inclusion.
Alvarado et al., unpublished). Two control diets
consisting of 60% corn or rice (either raw or cooked) Enzymes reduce feed cost when viscous cereals are
and soy protein concentrate were formulated. The the major source of energy. Non-starch
experimental diets included 3% of either soy hulls polysaccharides, specifically the soluble fraction, have
or oat hulls at expense of an inert material. The a negative impact on digestion and absorption of
inclusion of the fiber sources consistently improved nutrients in poultry. The mechanisms by which NSP
broiler performance at 21 days of age (Table 9). In a reduce broiler performance are not well understood.
second test, diets low in fiber (1.5% crude fiber and Soluble NSP increase digesta viscosity and reduce
3.6% NDF) based exclusively on rice and soybean the accessibility of enzymes to starch, protein, and
protein concentrate were formulated. The test diets lipids of the diet. Added enzymes improve bird
consisted of adding 3% oat hulls (a source of lignified performance by increasing nutrient digestibility and
insoluble fiber) or soy hulls (a source of non-lignified feed intake, and balancing intestinal fermentation.
insoluble fiber) at expense of an inert material. The Lázaro et al. (2003a, b) have indicated that for laying
inclusion of the two sources of fiber to the rice-soy hens the beneficial effects of supplementary enzymes
protein concentrate diet improved feed conversion at are mostly due to improved nutrient digestibility,
early stages of growth and improved average daily whereas in broilers an increase in feed intake is also
gain at 21 days of age (Table 10). The inclusion of important. Fat is usually the nutrient whose
either soy hulls or oat hulls to the low fiber diets digestibility is most benefited by added enzymes,
increased relative gizzard weight (% BW) (1.786c vs although improvements of up to 8% for starch and
2.407a and 1.868b for control, oat hulls and soy hulls- of 19% for nitrogen digestibility have been reported
including diets; P<0.001) and relative total digestive in barley diets (Hesselman and Åman, 1986).
weight (% BW) (10.23 b vs 10.76 a vs 10.76 ab%; The available information indicates that enzymes
P<0.01). Also, soy hulls but not oat hulls increased improve nutrient digestibility and poultry
intestinal viscosity at 21 days of age (3.38 vs 3.36 vs performance and that the beneficial effects are more
3.76 cP for control, oat hulls, and soy hulls diets, pronounced in broilers fed viscous grain diets than
respectively; P<0.05). in broilers fed corn-based diets. Also, enzymes are
more effective when heat processed ingredients or
Table 9. Influence of fiber inclusion in the diet on performance of
broilers.1 diets such as expanded feeds are used. Information
on oligosaccharidases is scarce; and research effort
Fiber source ADG (g) FI (g/day) FC (g/g)
to increase its use in diets based on soybeans protein
0-4 days meals and legumes is warranted.
None 13.8 18.7 1.36
Oat hulls, 3% 14.1 18.4 1.31
Soy hulls, 3% 14.0 18.5 1.32
SEM (n = 24) 0.109 0.23 0.016 Nutrition, immunity and enteric
P2 NS NS 0.05
disorders
0-21 days
None 30.7b 42.7 1.39a
Oat hulls, 3% 32.2ab 43.1 1.34b
Birds at hatch have an immature immune system and
Soy hulls, 3% 32.7a 44.2 1.35b are susceptible to enteric disorders associated with
SEM (n = 24) 0.046 0.66 0.010 exposure to pathogens (Bar-Shira et al., 2003).
P2 0.01 NS 0.001 Changes in GIT conditions due to disease have a
1
Gonzalez-Alvarado et al. (unpublished data).
significant impact on the efficiency and requirements
2
Significance of contrast: none vs hull inclusion.
for nutrients in the chick, because bacterial challenges high quality fish meal and of synthetic amino acids,
redirect nutrients from growth toward host defense and a reduction of the protein content of the diet is
(Obled, 2002). Potential limiting amino acids for recommended (Mateos et al., 2001).
immune protein synthesis are unknown, but lysine is In general, broilers fed wheat or other high NSP
probably not limiting (Klasing and Leshchinsky, grains are more susceptible to NE than broilers fed
2000). Rowlands and Gardiner (1998) indicate that corn (Ridell and Kong, 1992; Kaldhusdal and
in humans, certain amino acids (glutamine, arginine, Skjerve, 1996). However, the addition of pentosanases
and ornithine), fatty acids (short chain and n-3 fatty to wheat diets did not affect mortality due to NE,
acids), and nucleotides (DNA) might enhance which indicates that other factors are responsible for
intestinal integrity and support immune function. the increase in clostridia counts observed in the GIT
There is evidence in humans and pigs that luminal of birds fed wheat. Further studies are needed to
glutamine benefits mucosal permeability, is used for investigate the influence of different dietary fiber
gluconeogenesis, and is a major fuel and building sources on GIT motility, intestinal digesta
block for synthesis of nucleotides in rapidly movements, and the occurrence of enteric diseases.
proliferating cells, such as those of the immune system It has been proposed that the control of
and intestinal mucosa (Wu, 1998; Obled, 2002). antinutritional factors present in the diet, including
Whether an exogenous supply of these nutrients will mycotoxins, lectins and trypsin inhibitors might help
enhance the mucosal barrier and support immune to reduce enteric disorders. The use of soybean meal
function in the bird is controversial at the present and soy products has increased in Europe because of
time. restrictions imposed on the utilization of animal
Necrotic enteritis is an acute, infectious, non- proteins and more than 40% of soy products are
contagious disease caused by the overgrowth of frequently used in turkey diets. Yet, soybeans are
Clostridium perfringens that affects the lining of the processed the same way today as they were 60 years
digestive tract in chicks from 2 weeks to 6 months of ago and no methods have been implemented to remove
age. Factors that precipitate outbreaks of the disease the oligosaccharides present in the meal. In addition,
include management stress, subclinical coccidiosis and full-fat soybeans are being included at high levels in
abrupt changes in dietary formulation. Historically, all-vegetable diets, with inadequate control of
in-feed antibiotics have been used for the treatment antinutritional factors in many cases. Many
and prevention of NE and there is strong evidence nutritionists accept concentrations of 6 to 8 mg/kg
that banning antibiotics has contributed to an increase of trypsin inhibitor in treated soybeans. However,
in incidence of the disease. Clarke and Wiseman (2001) have indicated that this
Diet has a marked effect on the development of level should be reduced to less than 4 mg/kg in young
the microflora in the alimentary tract of the chick. animals. Van der Klis and Jansman (2002) have
Two major dietary factors that predispose flocks to estimated that the presence of 5.7 mg of trypsin
NE are the use of cereal grains that increase the inhibitor per kg of diet in piglets increases the
viscosity of digesta and the high levels of crude protein percentage of energy required for maintenance from
in the diet (Drew et al., 2004). Kaldhusdal and 5.5 to 13.1% (Table 11).
Skjerve (1996) observed that the inclusion of corn
in wheat or barley diets for broilers contributed to Table 11. Estimated energy costs for synthesizing endogenous
the prevention of the disease. In addition, dietary protein with graded levels of trypsin inhibitor activity in the pig.1
lactose reduced intestinal counts of clostridia while TIA, Endogenous Endogenous Energy Energy
sucrose, glucose, and fructose were associated with mg/kg 2 N excretion3 N synthesis4 expended5 required, %6
an increase (Riddell and Kong, 1992). Bedford
(2000) hypothesized that the proliferation of C. 0 9.7 38.6 174 5.5
1.9 16.3 65.2 293 9.3
perfringens is facilitated by the presence of large 5.7 23.0 91.9 414 13.1
quantities of dietary protein in the ceca. If this is the
1
case, viscous diets will increase the quantities of Van der Klis and Jansman (2002).
2
nitrogen that escape digestion resulting in a more Trypsin inhibitor activity.
3
Excreted ileal endogenous N (g/d).
frequent occurrence of NE (Al Sheikhly and Al Saieg, 4
Total synthesised endogenous N (g/d).
1980; Riddell and Kong, 1992). When the incidence 5
Energy costs of endogenous protein synthesis (KJ/d).
6
of enteric disorders is high, an increase in the use of Percentage of maintenance requirements.
Conclusions GALT during the immediate post-hatch period.

Dev. & Comp. Immunol. 27:147-157.
Enteric diseases are complex and affected by many Bedford, M. 2000. Removal of antibiotic growth
factors, such as subclinical coccidiosis, stresses, lack promoters from poultry diets: implications and
of hygiene, and immunodepression, but dietary strategies to minimise subsequent problems. World’s
changes that improve gut health and stimulate gizzard Poult. Sci. J. 56:347-365.
development and motility might help to overcome Bigot, K., S. Mignon-Grasteau, M. Picard, and S.
digestive upsets. Coarse grinding, mash feeds, low Tesseraud. 2003. Effects of delayed feed intake on
wheat and protein diets, enzyme supplementation,
body, intestine, and muscle development in neonate
inclusion of whole grains and a minimum amount of
broilers. Poult. Sci. 82:781-788.
a convenient fiber source are some of the solutions
BSAS. 2003. Nutrient requirement standards for pigs.
proposed in this respect. In addition, the inclusion of
essential fatty acids, emulsifiers, organic acids British Society of Animal Science. Penicuik,
probiotics, and prebiotics, as well as immune Midlothian, UK.
enhancers, is also recommended. Most of these Chaveerach, P., D.A. Keuzenkamp, L.J.A. Lipman,
techniques are not sufficiently refined at present and and F. Van Knapen. 2004. Effect of organic acids
further research is needed. Adequate management in in drinking water for young broilers, on
terms of preventive vaccination programs, reduction Campylobacter infection, volatile fatty acid
of stresses, and improvement of hygienic conditions, production, gut microflora and histological cell
together with dietary changes minimize the incidence changes. Poult. Sci. 83:330-334.
of enteric disorders in antibiotic-free fed flocks. Clarke, E., and J. Wiseman. 2001. Comparison of
Removal of in-feed antibiotics from feeds is likely nutritional value of full fat soyabean meals for
to increase production costs moderately, mostly broiler chicks in the UE. Br. Poult. Sci. 41:688-
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NE, but efficient production is still feasible without Corless, A.B., and J.L. Sell. 1999. The effects of
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and functional development of the digestive system
of young turkeys. Poult. Sci. 78:1158-1169.
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R.A. Renema 81
Reproductive responses to Sel-Plex® organic selenium in male and female

broiler breeders: impact on production traits and hatchability
ROBERT A. RENEMA
Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton,
Alberta, Canada
Growth selection and reproduction in

broiler breeders
We expect a lot from modern broiler breeders. These this increased growth efficiency is further complicated
highly growth-selected birds must pass the genetics by the development of ‘yield’ varieties, carrying
for fast and efficient growth to as many offspring as increased amounts of breast muscle, often on a
possible. While crossbreeding and selection programs smaller carcass frame. As selection for broiler breeder
have resulted in annual improvements in broiler egg production is not as heritable or profitable as
growth, breast muscle yield, feed efficiency and selection for growth traits, there are continued
disease resistance, there is a negative relationship increases in the growth potential while egg production
between body weight and egg production in broiler is not emphasized. As a result, Whitehead (2000)
breeders (Robinson et al., 1993). While specialized indicates that geneticists continually compound the
genetic selection has meant that egg production is problem by breeding a bird that, if allowed to exist
not remarkably different from what it was a few years in its freely expressed adult state, is completely unfit
ago, hatching egg producers have had to work hard for life.
at fine-tuning strain specific procedures for nutrient Understanding the ovarian function of the chicken
allocation and photoperiod management. and its interaction with nutritional status, age, and
Achieving success with broiler breeder management strain is likely the most important issue affecting
is like hitting a moving target. Modern broiler stocks poultry breeding companies today. The process
have been reported to grow at 4.6 times the rate of a involves the conversion of genetic, environmental,
1957 random-bred strain due to increased genetic and nutritional cues into a cascade of signals from
potential (Havenstein et al., 2003a). The 6-fold the neuroendocrine system. These signals must be
improvement in carcass yield of 2001 stocks fed a integrated and responded to by the organs and tissues
2001 diet compared to 1957 stocks fed a 1957 diet is primarily involved in reproduction, which will in turn
85-90% due to genetics, and 10-15% due to nutritional produce more signals for both local and distant
changes (Havenstein et al., 2003b). While broiler 42- activities. The resulting eggs produced are the net
day body weight is increasing each year, the 42-day result of the bird’s attempt to coordinate the demands
target body weight for male and female broiler its body and environment have placed on it.
breeders has remained the same, or even decreased The ability of an embryo to survive the incubation
(Rustad and Robinson, 2002). In 1979, Hubbard male process relies on a balance between hatchery
and female breeders were approximately 50% of the management and breeder management. Specific feed
42-day broiler weight. In 2001, this percentage had ingredients, bird age, and flock management
decreased to 36.1 for males and 30.3 for females. In decisions can directly affect semen quality, the oviduct
essence, the degree of feed restriction has continued environment, and the egg environment. These factors
to increase while there is increased competition for a combine to influence the potential of the egg to be
reduced feed allocation. Complications in managing fertile and ultimately to hatch.
82 Reproductive responses to Sel-Plex® organic selenium in broiler breeders
Reproduction in the broiler breeder

The reproductive system of the laying hen is The female oviduct environment can be hostile to
comprised of many organs. The list includes the sperm despite their existence in the SST within the
hypothalamus, the anterior pituitary, the ovary, the oviduct wall. Duration of fertility (measured by
oviduct, the liver and the skeletal system. Small monitoring fertility in consecutive eggs) can be
follicle steroidogenesis, particularly estrogen reduced under conditions of overfeeding (Katanbaf
production, is responsible for transforming a pullet et al., 1989b; Goerzen et al., 1996). It is known that
into a hen. As plasma levels of estrogens increase, fewer sperm survive in some bird strains and when
externally visible features include reddening and excess feed is used (Renema et al., 2001), but it is
enlargement of the comb and wattles, a prenuptial not clear how the surviving sperm are affected, and
feather molt (feather drop) and a widening of the if the remaining sperm are of similar quality to the
pubic bones to permit egg passage. Internally, ones originally placed.
estrogen stimulates liver production of egg yolk lipids
with a significant change in the color and size of the
liver. Finally, the oviduct enlarges and becomes a Factors affecting hatching egg quality
secretory organ for deposition of albumen.
The male focuses on quantity rather than quality There are many factors that can affect the potential
when it comes to sperm production. The hen then of the embryo to survive incubation and generate a
must screen out unsuitable sperm in order to guarantee quality chick. Some of these factors are out of our
production of high quality chicks. Mature sperm spend control, such as hen age, and others can be manipulated
the majority of their time in the oviduct. Following through management decisions, such as egg size and
mating, the hen will store sperm in the highly hatchery environment. It can be difficult to formulate
specialized microenvironments of the sperm storage diets to optimize egg production, fertility, and
tubules (SST) are located in the vaginal region of hatchability as little is known about the nutritional
the oviduct. Only about 1 to 2% of the originally requirements of the embryo (Leeson and Summers,
inseminated sperm enter the SST (Bakst et al., 1994), 1991). Dietary vitamin levels are increased in the
where they exist in a near-dormant state. The survival diet with the hope they will also be increased in the
of the sperm to the point of insemination depends on egg. Yet there can be adverse reactions with this type
a combination of sperm quality and the ability of the of approach, as some vitamins have antagonistic
hen to provide a safe environment for the sperm. relationships with others. Furthermore, there can be
The hormone messages being relayed between the stability issues for long-term storage, as well as for
ovary and the hypothalamic and pituitary control feed processing procedures. With current genetic
centers are altered by feed intake. Besides affecting stocks, if the chick hatches in a weakened state due
follicle formation, and reproductive control, feeding to a vitamin or mineral deficiency, it is more likely
level can alter the viability of the embryo through to succumb to disease now than with previous stocks.
changes to the egg and to the early maturation process. Growth-selected stocks have low immuno-
Excess nutrients are diverted into liver lipids, excess responsiveness (Siegel et al., 1984) due to either
ovarian follicle development, and as abdominal fatpad inadvertent or intentional negative selection pressure
(Etches, 1996). It can be a vicious cycle, with obesity combined with growth efficiency selection. The
continuing to worsen as the rate of egg production developing embryo is especially sensitive to vitamin
remains low and/or goes into early decline due to deficiency, which will result in death, malformation
excess feed intake. The ovaries of growth-selected or some other atypical response (Leeson and
strains are particularly sensitive to overfeeding during Summers, 2001).
the sexual maturation process (Renema et al., 2003). During embryo development, oxidative metabolism
Body weight in broiler breeder hens has been increases substantially over the incubation period and
reported to be negatively correlated with duration of especially in the last few days before hatch (Freeman
fertility and fertile egg production (Bilgili and and Vince, 1974). This normal respiration related to
Renden, 1985). Ultimately, reduced chick production embryo growth results in the production of free
in overfed broiler breeders is the culmination of poor radicals, which can cause tissue damage through lipid
egg production combined with reduced fertility, hatch peroxidation, with polyunsaturated fatty acids being
of fertile, and embryonic viability (Yu et al., 1992). especially vulnerable (Surai, 1999). The chick has
R.A. Renema 83
developed effective antioxidant pathways to prevent glutathione peroxidase (GSH-Px) as well as a

damage. The primary defense mechanism is a group component of many other selenoenzymes. Oxygen
of three enzymes (superoxide dismutase, glutathione metabolism produces free radicals, which have
peroxidase, and catalase), which convert free radicals potentially toxic effects on all biological molecules
produced by cellular respiration into less harmful (Surai, 2000). Glutathione peroxidase aids in the
alcohols (Ursiny et al., 1997). A second level of removal of oxidative compounds in the form of
defense are the natural antioxidants – vitamin E, hydrogen peroxide and hydroperoxides from the cell
carotenoids, ascorbic acid, and glutathione, which (Burk, 1989). Buildup of these substances can impair
protect the developing chick (Surai, 1999). During cell membrane structure and function, and once the
the last week of incubation, fat-soluble antioxidants membrane is damaged decreased productivity and
are moved into the liver and yolk sac membrane. reproductive performance can result (Surai, 2000).
The major fat soluble antioxidant, vitamin E, moves Selenoamino acids have been shown to have higher
from the yolk to the embryo tissue at this time (Gaal bioavailability than traditional inorganic sources
et al., 1995). Ascorbic acid (vitamin C) is the major commonly used for dietary supplementation. They
water-soluble antioxidant, and is produced in the yolk are actively absorbed in the intestine compared to
sac membrane before transport to tissues like the brain the passive absorption of inorganic selenium (Surai,
(Surai, 1999). This helps protect membrane lipids 2002). Furthermore, selenomethionine and
during the large metabolic effort of hatching. The selenocysteine can be incorporated non-specifically
third level of antioxidant defense is the generation into structural proteins (particularly muscle tissue)
of enzymes that rebuild damaged membranes (Surai during protein synthesis. Selenomethionine can be
and Sparks, 2001). substituted for methionine during protein synthesis
due to its similar structure (methionine contains a
sulphur atom instead of a selenium atom). This
ROLE OF DIETARY SELENIUM critical difference between selenium sources allows
the organic selenium compounds in Sel-Plex® to
Selenium is normally provided in the diet in the form contribute to a selenium reserve to be available for
of inorganic sodium selenite. An organic form can prevention of lipid peroxidation (through GSH-Px)
be provided (Sel-Plex®), which is selenium yeast. during stress conditions (Surai, 2002). In the broiler
Yeast, like plants, form selenoamino acids and other breeder, it also enables enhanced transfer of selenium
organic selenocompounds that exist in very reduced from the hen to the embryo (Edens, 2002). Increased
form in comparison to the highly oxidized inorganic antioxidant uptake in the hen due to the maternal
selenium forms (selenite and selenate). Organic diet is linked to increased antioxidant concentrations
minerals are transported intact and retained better in in the developing chick (Surai et al., 1999).
target tissues or organs. Higher selenium in eggs The need for defense against oxidative damage is
reflects increased antioxidative properties of the egg clear in the male, where antioxidant enzymes play a
during storage, therefore preserving the egg for key role in maintaining the sperm cells (Surai et al.,
incubation and potentially increasing hatchability. 1998). Sperm cells contain large amounts of poly-
Cantor (1997) and Paton et al. (2000b) found that unsaturated fatty acids, which allow them to maintain
eggs from Sel-Plex®-fed chickens were significantly flexibility relating to motility (Surai, 2002). However,
higher in selenium than eggs from sodium selenite- this means they are also a target for lipid peroxidation.
fed chickens. Organic selenium has a vitamin Cellular integrity is maintained by GSH-Px, other
E-sparing action through its involvement in vitamin selenoenzymes and vitamin E, which protect the cell
E retention in the plasma and through involvement membranes from oxidative damage (Flohe and
with the primary enzymatic defense system of the Zimmermann, 1970).
embryo against lipid peroxidation. In fact, Some recent research has demonstrated that the
supplementing organic selenium to breeder diets has inclusion of selenium in poultry diets enhances sperm
been shown to increase levels of other antioxidants numbers, and using an organic source (Sel-Plex®)
(vitamin A, E and carotenoids) in the egg (Surai and reduces production of defective sperm, thereby having
Sparks, 2001). a positive effect on the fertilizing potential of the
The protective effects of organic selenium are male (Edens, 2002). Little information is available
especially apparent during the highly oxidative state regarding the effect of dietary selenium source on the
of late incubation and the first few days after hatch. reproductive efficiency of laying hens.
Selenium is an integral part of the antioxidant enzyme
How does dietary selenium source affect The sperm penetration assay of Bramwell et al.
the hen’s contribution to fertility? (1995) was used to quantify the perivitelline layer
sperm holes. An approximately 1 cm2 section of the
(Study 1) perivitelline layer above the germinal disc was cut
Egg production and fertility decline with age. The free, cleaned, mounted to a microscope slide, fixed,
decrease in hatchability and fertility associated with and stained with Schiff’s Acid reagent to generate a
an increase in age might be due to the older hen’s contrast with the sperm holes. The holes were counted
inability to hold sperm in the SST (Fasenko et al., at 100X magnification. The raw numbers and change
1992). Furthermore, the sperm do not retain their in numbers over time were used as a representation
viability as long in the SST of older hens, and are of quantifiable fertilization potential.
released in larger numbers from the SST (Bramwell
et al., 1995). Quantification of fertility is determined
by the occurrence rate of perivitelline holes caused OBSERVATIONS
by the sperm. In past research, killing the hen to obtain Egg traits
the newly ovulated ovum was the only way to
determine sperm hole quantities. Bramwell et al. The use of Sel-Plex® rather than sodium selenite as
(1995) adapted the technique to use eggs for the the dietary selenium source has previously been
determination of perivitelline sperm hole numbers. shown to increase shell breaking strength after 42
This study examined the effects of selenium days in laying hens at 80 wk of age (Paton et al.,
supplementation form and level on female 2000a). Our study found no significant differences
reproductive performance and egg traits. Its intent in egg traits between the various treatments after 30
was to determine the effects of organic (Sel-Plex®) days on the diets. However, the Sel-Plex® treatment
and inorganic selenium supplementation in the laying resulted in numerically the greatest positive change
hen diet on fertilization potential and egg traits. in shell quality during the 30 day period of this trial.
Research with younger birds (26 wk of age) has
indicated no difference in shell quality with the use
METHODS of Sel-Plex® although the comparisons were made
after only 28 days on the diet (Paton et al., 2000a).
We housed 75 hens in individual laying cages at 61 After 9 wks on the diets, shell weights were higher
weeks of age. Three dietary treatments were imposed, in the Sel-Plex® group than in the controls, while
varying in selenium source and level. Twenty-five shell weights in the group given inorganic Se was
hens were fed a control diet, 25 were fed a diet enriched intermediate (Table 1). Egg specific gravity, a
with inorganic selenium in the form of sodium selenite measure of shell quality, was greater in the Sel-Plex®
and 25 were fed a diet enriched with organic selenium treatment than in either the control or selenite groups.
in the form of the Alltech product, Sel-Plex®. All diets While higher dietary selenium levels preserved shell
contain 19% CP and 2875 kcal ME/kg. The control quality to some degree, the organic selenium in Sel-
diet had a selenium inclusion rate of 0.1 mg Se/kg, Plex® proved to have a more substantial effect on
whereas both enriched diets contained a total of 0.3 the preservation of shell quality characteristics.
mg Se/kg with an added 0.2 ppm Se coming from the
organic or inorganic selenium source. The diets were Table 1. Effect of selenium source and level on shell weight and
fed for a 3-week period prior to insemination to ensure egg specific gravity after 9 weeks.
tissue saturation of the new dietary selenium forms Diet Shell weight (g) Egg specific gravity
and concentrations. Control 5.43b 1.0740 b
Following the 3-week acclimation period, all hens Sel-Plex® 5.68a 1.0762 a
were artificially inseminated with 50 µL of neat, Inorganic Se 5.53ab 1.0745 b
pooled semen collected from a group of 22 broiler a,b
breeder males (116 million sperm/dose). Eggs were (P<0.05).
collected from 2 to 7 days after insemination for the
quantification of perivitelline layer sperm holes. Eggs Perivitelline sperm hole assay
traits (weight, specific gravity, yolk and dry shell
The Sel-Plex® treatment group had the highest mean
weight) were measured at 30 and 60 days from the
number of sperm holes in the 2 to 4 days after
start of dietary treatments.
insemination study period, while controls had the
R.A. Renema 85
lowest number (Table 2). Although neither the Sel- The form and quantity of dietary selenium appear to
Plex® nor the control treatment were statistically impact the oviduct environment of the hen. Fewer sperm
different from the inorganic Se treatment, they were are able to survive under low dietary selenium conditions
statistically different from each other. A similar (control) compared to conditions provided by
relationship among the dietary treatments occurred supplementation with an organic selenium source (Sel-
for the 5 to 7 day period, and for a comparison over Plex®). Conditions in the SST may be central to the
the entire 2 to 7 day period. The perivitelline sperm differences noted in the number of sperm being able to
hole numbers declined at a similar rate among dietary reach the site of fertilization. Through a combination
treatment groups between the 2 to 4 day and the 5 to of a more stable, antioxidant-free environment with a
7 day study periods. As the control group started with potentially slowed sperm metabolism, more sperm may
a lower number of sperm holes than either of the be able to survive storage. Selenium source appears to
other two treatment groups, their final numbers at 7 influence the hen’s contribution to the fertility of the
days were very low. The Sel-Plex® and inorganic Se breeder flock.
treatments both retained higher fertility potential
throughout the sampling period allowing for a longer
period of time between artificial inseminations. While Effects of dietary selenium source on the
not statistically different, the average number of fertility and hatchability of broiler
sperm holes at the site of fertilization in the Sel-
Plex® group was higher than that of the inorganic Se
breeders (Study 2)
treatment. This indicates that the greater bioavailability Supplying selenium to broiler breeders in the organic
of selenium in Sel-Plex® compared to inorganic Se may selenoamino acid form may have an important impact
be advantageous for the fertility of the female based on on poultry reproduction at the level of sperm
changes to the oviduct environment. Selenium seems to formation, sperm storage, and in the hatching egg
play an important role in the maintenance of fertility in through increased protection from oxidative damage.
older laying hens. This is most likely due to the selenium- This experiment was designed to provide information
dependent GSH-Px improving the environment of the on the role of dietary selenium form on both female
SST (Surai, 2000). The SST need to maintain a stable and male fertility. While there is evidence on a flock
environment and the elimination or reduction of free basis that selenium source affects broiler breeder
radicals within the tubules is essential. female fertility, it is not as clear how these benefits
are being expressed. Previous work suggests the use
of an organic selenium source (Sel-Plex®) can lead
SUMMARY: STUDY 1 to improved egg production, shell quality, sperm
Selenium supplementation is beneficial to increasing viability, and embryo survival. Egg shell quality may
and maintaining fertility and shell quality in older be enhanced through an altered efficiency of calcium
hens. Supplementation with the organic selenium in metabolism, and sperm quality may be enhanced
Sel-Plex® may have a greater impact on reproductive through protective antioxidant effects in the male and
ability than inorganic sodium selenite. Factors such in the female oviduct. This study assessed some of
as age and length of exposure to the diet also play a these production and fertility traits in broiler breeders
role in the results of both this and past studies. maintained individually.
Table 2. The effect of selenium supplementation and source in the laying hen diet on perivitelline sperm holes.
Number of perivitelline sperm holes Change in perivitelline sperm hole numbers
between 2-4 & 5-7 days
Diet 2-4 days1 5-7 days Total (2-7 days) Absolute change Percentage change
(P≤0.004) (P≤0.045) (P≤0.006) (No.) (%)
Control 19.9 b 4.7b 12.3b 17.1b 73.4
Sel-Plex® 55.8 a 10.3a 32.5a 44.4a 75.1
Inorganic Se 40.7ab 9.5ab 25.1ab 31.1ab 68.5
a,b
Means within a column with no common superscript differ (P<0.05).
1
Number of days after insemination.
The objective of this trial was to characterize between Day 2 and 5 after insemination compared to
specific effects of dietary selenium source on fertility a 46% and 48% drop within non-supplemented and
and embryo viability aspects in commercial broiler selenite-fed birds, respectively.
breeder stocks. A female diet with no added selenium The ability to maintain a viable sperm population
was used to identify the impact of dietary selenium. for as long as possible reduces necessary frequency
Inorganic and organic dietary selenium sources were of insemination. While selenium appears essential to
compared to demonstrate the impact of differences allow the sperm into the oviduct, organic selenium
in selenium accessibility and tissue storage on in Sel-Plex® may have an advantage over inorganic
reproductive traits and embryo survival. Higher rates selenium in keeping the sperm population stable and
of production, fertility, and ultimately chick quality, alive. This is especially important as the hens age
would decrease the number of birds required to and have a reduced sperm storage capacity at the
maintain current rates of production, as well as the uterovaginal junction (Goerzen et al., 1996).
overall cost of production.
Table 3. The effect of dietary selenium level and source on
perivitelline sperm holes of eggs from broiler breeder hens.
METHODS Number of perivitelline sperm holes
2 days1 3 days 5 days
Ross 508 pullets were reared in a light tight facility
following the breeder BW profile (Aviagen Inc). No added Se 83b 46 14 b
From photostimulation (22 wks of age) pullets were Selenite2 150a 58 31ab
Sel-Plex®2 119ab 61 60 a
fed a selenium-free laying ration (No added Se), a
standard ration containing sodium selenite (0.3 mg a,b
Se/kg), or a ration containing selenium yeast (0.3 (P<0.05).
1
mg Se/kg from Sel-Plex®). Thirty hens per treatment 2
Number of days after insemination.
0.3 ppm Se
were inseminated weekly (from 30 wks) using pooled
semen from males fed a standard, sodium selenite
The males on the Sel-Plex® diet produced greater
diet or a diet containing the same amount of Se from
semen volume early in production, with an average
Sel-Plex®. Individual egg production to 58 wk, egg
of 0.36 ml/bird compared to 0.19 ml/bird in males
weight, egg specific gravity, and BW were recorded.
on the selenite diet (36 weeks of age). At 56 weeks
At 35 and 57 wk of age, eggs from 2 to 5 days after
of age, this difference was no longer significant, but
insemination were subjected to the perivitelline sperm
remained at nearly the same magnitude. The
penetration assay to measure the number of sperm
comparison was complicated at the later ages due to
penetrations near the germinal disk. Eggs were
several small males dropping out of semen production
incubated weekly and the hatch residue broken out
part way through the trial (selenite treatment). Testes
to determine fertility, hatchability, and embryonic
of all birds are currently being examined for the
mortality.
presence of functionally active sperm producing cells.
Egg production and egg quality traits

OBSERVATIONS
Birds on the Sel-Plex® diet entered egg production
Sperm management
slightly behind the other feeding treatments (non-
Perivitelline sperm hole numbers of Sel-Plex® and significant difference), but caught up within a few
selenite treatment eggs were similar. Both treatments weeks. Early egg production to 29 wk of age was
had more sperm holes than eggs from unsupplemented not different (Table 4). In fact, the rate of lay was
hens by a factor of 2 to 3 (Table 3). Sel-Plex ® similar through most of the production period.
supplementation improved maintenance of sperm However, during the late lay period (49-58 weeks)
numbers between the day 2 and the day 5 sampling. the hen-housed rate of lay was 68% in Sel-Plex® birds
By day 5, Sel-Plex® eggs still had an average of 60 compared to 61% and 60% in the selenite and non-
perivitelline sperm holes compared to 14 in control supplemented treatments, respectively. The Sel-Plex®
eggs, while selenite treatment eggs were intermediate birds produced an extra 5 eggs/bird during this
(31 holes). These values represented a decline of 31% period, on average. This is an important time to be
in apparent viable sperm population in Sel-Plex® birds producing more eggs, as egg size is higher than in
R.A. Renema 87
Table 4. The effect of dietary selenium level and source on egg production traits of broiler breeders.
Hen-housed egg production Total egg Settable egg Unsettable egg

24-28 wk 29-38 wk 39-48 wk 49-58 wk production production production1
(%) (%) (%) (%) (No.) (No.) (% of total)
No added Se 48.9 88.9 75.6 60.2b 174.5 168.5 3.49

Selenite2 49.7 88.1 73.1 60.1b 172.7 168.6 2.37
Sel-Plex®2 46.4 87.7 75.5 67.7 a 177.7 174.6 1.90
a,b
1
Includes double-yolked, soft-shelled, membranous, and abnormally-shelled eggs.
2
0.3 ppm Se
young breeders, which results in a larger chick size thickness in the Sel-Plex® treatment (not tested). If this
and ultimately a greater broiler weight. Edens (2002) were different, there could be implications for incubation
also indicated that the egg production of Sel-Plex®- success and for defense from contamination in the barn.
fed hens initially lagged behind, but caught up and Hen body weight followed a similar pattern
even surpassed that of the selenite-fed hens after 5 throughout the production period. However, the non-
wk. supplemented hens grew heavier than the other
Ultimately the settable egg production in the dietary treatment hens by 42 weeks of age. This difference
groups was 168.5 (non-supplemented), 168.6 carried through to 58 weeks of age. This comparison
(selenite), and 174.6 (Sel-Plex®) eggs/bird (Table 4). is somewhat artificial, as the body weight profile of
Overall, unsettable egg production ranged from the non-supplemented group was inflated by hens
3.49% in non-supplemented hens to 1.9% in Sel- that dropped out of lay at a fairly young age. Nutrients
Plex ® hens and was not significantly different. they were no longer allocating to egg production went
However, during the late lay period (49-58 wk), the into growth instead. By the end of the trial, 100% of
Sel-Plex® hens produced significantly fewer unsettable the Sel-Plex® hens were still in active production
eggs (0.9%) than non-supplemented hens (3.3%), while only 87% and 90% of the non-supplemented
while selenite hens were intermediate (1.7%). Egg and selenite treatment hens remained, respectively.
weight and shell quality of settable eggs was assessed Lack of production was due to either birds ceasing
throughout the trial and was unaffected. This means production, or to hen mortality (mortality limited to
that if the hen laid a good egg, it also had a good non-supplemented treatment). Reduced mortality has
shell. However, diet affected how many eggs were been linked to selenium supplementation (Arnold et
produced with good shells, as shell defects were the al., 1974), particularly under stress conditions such
primary egg quality problem in unsettable eggs. as an immune challenge (Edens, 2001). However,
Feeding Sel-Plex® organic selenium to laying hens this does not explain the increased proportion of birds
at 80 wk of age has previously been shown to improve still in active lay at 58 weeks of age. Reproduction
shell breaking strength (Paton et al., 2000a). in the broiler breeder can be a fragile state and is
Interestingly, dietary selenium affected the change often the first thing to go when there is stress, or
in shell weight as the hens aged. Between 36 and 56 nutrients are insufficient. The fact that all Sel-Plex®
wk of age, shell weight increased by 0.55, 0.80, and birds were still in production may relate to an
0.76 g in eggs of the non-supplemented, selenite, improved efficiency of nutrient uptake. Presumably
and Sel-Plex®-fed hens, respectively. During this time the gut benefits from the improved protection from
egg size also increased, meaning that the shell was cell membrane damage afforded by the organic
being stretched over more egg, and therefore making selenium. During the late production period, these
up a smaller percentage of total egg weight. The birds were producing more eggs than hens of the
proportion of shell weight dropped by 0.84% of egg other treatments (Table 4) with no effect on their
weight in non-supplemented hens, 0.80% in selenite- body weight relative to that of hens on the other
fed hens, and 0.57% in Sel-Plex® hens between 36 treatments.
and 56 wk of age. The Sel-Plex ® hens were
significantly less affected by age-related declines in Fertility, hatchability, and embryonic mortality
the proportion of egg shell than the non-supplemented
hens. While egg specific gravity was not significantly Prior to 34 weeks, hatchability averaged 88% in Sel-
affected, this may be an indicator of increased shell Plex® treatment eggs compared to 80% in selenite-fed
birds and 77% in non-supplemented birds, and was differences, there appears to be a potentially
similar in all treatments after peak production. protective effect of Sel-Plex® compared to inorganic
Overall, fertility, hatchability, and hatch-of-fertile selenium in the diet.
eggs demonstrated the beneficial nature of dietary Examining this relationship more closely revealed
selenium, but did not differentiate between selenium an interesting trend over time. Late embryonic
sources (Table 5). Fertility, for example, was 86.9% mortality of all treatments was similar at the start of
in non-supplemented hens compared to 90.1% in hens the trial, when all birds were still on a fairly high
on selenite and Sel-Plex® diets. Not including selenium plane of nutrition. As the hens aged, late embryonic
in the diet did not seriously harm hatchability, which is mortality stayed almost constant in non-supplemented
in contrast with work by Latshaw and Osman (1974) and selenite hens, while it decreased in Sel-Plex® hens
demonstrating a drop in hatchability to 18% in selenium- (Figure 1). As feed allocations were reduced with
deficient hens. The current study may have provided age, the micronutrients would have been in shorter
more naturally occurring selenium in the other feed supply. The improved efficiency of selenium uptake
ingredients and the non-supplemented dietary treatment in the Sel-Plex® diet may not have made a substantial
was not imposed until photostimulation (22 weeks difference on hatchability until a nutrient challenge
of age). was faced by the flock. This fits with observations
Embryonic mortality can be a telling identifier of that Sel-Plex® can demonstrate benefits in stressful
specific dietary or genetic effects. Problems with early situations. Heat stress and long-term egg storage are
embryonic mortality (1-14 days of incubation) can examples of stress situations where Sel-Plex® has been
point to nutrient deficiencies. In this study, 5.33% shown to help. Surai and Dvorska (2001) indicate
of non-supplemented embryos died during this period that there are numerous on-farm stress conditions that
compared to 3.72% (selenite) and 3.52% (Sel-Plex®) could be alleviated in part by organic selenium
in the selenium-supplemented hens (Table 5). While supplementation.
selenium source did not make a difference here, Ultimately what determines the success of a broiler
clearly selenium supplementation was shown to be breeder management program is chick production.
important. A beneficial effect of organic selenium In this trial, chick production was calculated from
was expected for the late incubation and hatch period, the hatchability of settable eggs. The unsupplemented
as this is the time of the greatest oxidative load for hens produced an average of 131.3 chicks/hen-housed
the embryo, and when the protective antioxidant by 58 weeks of age, while selenite hens produced
effects of the Sel-Plex ® may be most apparent. 139.1 chicks/hen, and Sel-Plex® hens produced 145.3
Variability among birds reduced the significance of chicks/hen (Table 5). Between the selenite and Sel-
this comparison, however, and late embryonic Plex® selenium source diets, the numerical differences
mortality, dead-in-shells, and hatchery culls totaled in settable eggs, embryonic mortality, hatchability,
3.66%, 3.85%, and 3.14% of eggs set for the non- and hatch of fertile culminated in a difference of 5.8
supplemented, selenite, and Sel-Plex® treatments, chicks in favor of the Sel-Plex® hens. This difference
respectively (Table 5). Based on these numerical increased to 14.1 chicks when compared to the non-
supplemented hens.
Table 5. The effect of dietary selenium level and source on fertility, hatchability, embryo mortality and chick production traits of broiler
breeder females.
Embryo mortality and culls Hatch of Chick

Infertile Day1-14 Day 15-hatch1 Fertility Hatchability fertile2 production3
(%) (%) (%) (%) (%) (%) (No.)
No added Se 13.06 a 5.33a 3.66 86.9b 77.9 88.6 b 131.3b

Selenite4 9.91b 3.72b 3.85 90.1a 82.5 91.5 a 139.1ab
Sel-Plex®4 9.87b 3.52b 3.14 90.1a 83.5 92.5 a 145.3a
a,b
1
Includes embryo mortality to hatch, dead-in-shell, and hatchery culls.
2
Hatchability calculated only from fertile eggs set (infertile eggs excluded).
3
Chick production = hatchability X settable eggs.
4
0.3 ppm Se.
R.A. Renema 89
6 No added Se Inorganic Se Sel-Plex®
4
Mortality (%)
0
30 35 40 45 50 55 60
Age (weeks)
Figure 1. Linear representation of trends in embryonic mortality between 15 days of incubation and hatch (including dead-in-shell
and hatchery culls) of broiler breeder hens fed diets containing no supplemental Se, 0.3 ppm inorganic Se, or 0.3 ppm Sel-Plex®.
SUMMARY: STUDY 2 of the minor ingredients make it into the egg and
ultimately the embryo. Specialized feed ingredients
Reproductive traits were improved with the inclusion are available that behave differently than traditional
of dietary selenium, while Sel-Plex® supplementation ingredients and can enhance egg and chick quality
also improved sperm survival in the oviduct, as well under the right conditions. Together these factors can
as settable egg production late in lay through increased be used to enhance embryo survival.
egg production and reduced shell defects. Ultimately,
chick production was improved in the Sel-Plex®
treatment through more successful settable egg References
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measurements. Selenium is essential in the diet for a Tissue selenium content and serum tocopherols as
successful reproductive effort. Additional benefits of influenced by dietary type, selenium and vitamin
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appears to influence the hen’s contribution to the Bakst, M.R., G. Wishart and J.P. Brillard. 1994.
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production requires an understanding of reproductive Quantitative determination of spermatozoa
physiology, nutrition, and their interaction. Besides penetration of the perivitelline layer of the hen’s
a thorough knowledge of everyday management, ovum as assessed on oviposited eggs. Poult. Sci.
there must also be an awareness of feed ingredients 74:1875-1883.
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environmental effects. Whereas the basic composition element metabolism and function: newer roles of
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physiological stability and performance of broiler Chicken. University Books, Guelph.
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(K.A. Jacques and T.P. Lyons, eds). Nottingham level of inclusion and length of storage on internal
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G.D. Rosen 93
Optimizing the replacement of pronutrient antibiotics in poultry nutrition

GORDON D. ROSEN
Pronutrient Services Ltd., Wimbledon, London, United Kingdom
Introduction
Following recent reviews on the setting and the medicine and farm animal nutrition. In the early
meeting of standards for the efficient replacement of 1950s, opponents of antibiotic routines in poultry
pronutrient antibiotics in pig and poultry nutrition feeds initiated resistance research. Concerns were
(Rosen, 2003b; 2003c), this review concentrates heightened by the Japanese discovery in 1959-1960
attention on ways and means of improving and of infectious or transferable (as against natural)
optimizing the use of pronutrient antibiotics in broiler antibiotic resistance involving natural selection,
and turkey feeds, with particular reference to the mutation and DNA fragment transfer. The Swann
multiplicity of proffered candidates and to the Committee (1969) reported on the use of antibiotics
multiplexity and interactivity of influential genetic, in animal husbandry and veterinary medicine and
environmental, managemental and dietary variables. initiated legislative restrictions banning the use of
penicillin and tetracyclines without veterinary
prescription. The European Economic Community
Why replace pronutrient antibiotics? followed this lead. The US Food and Drug
Administration did not. In 1998, the European Union
Contentiously but concretely, the search for and cancelled its approvals of six feed antibiotics, with
validation of antibiotic replacements stems from the intent to ban the remainder not later than 31
attitudes and demands of consumers and their retail December, 2005.
suppliers and from legislation based on precautionary
principles. Soundly-based estimates of the effects on
production costs and on retail prices are sparse. But Which are the candidates?
the number of products offered as alternatives and
the volume of literature thereon have risen steeply Table 1 contains a short list of the seven main
since 1999. Many hundreds, possibly thousands, of categories of antibiotic replacement candidates.
products are on offer. One can envisage several years’ Whatever their nature or chemical composition, and
work ahead to sort the wheat from the chaff. no matter how numerous and multifarious are their
Irrespective of questions of right or wrong in the known or hypothesized modes of action, the common
proscription of prescription-free usage of antimicrobials thread in the present context resides in their abilities
in food production, the need to deploy replacements is to enhance poultry performance at least as efficiently
expanding rapidly, creating a formidable task for as the replaced pronutrient antibiotics in terms of
scientists and producers in their search for fully-effective feed conversion efficiency, mortality, liveweight,
alternatives. The potential value of feed antibiotics was animal product yield and environmental depollution,
first demonstrated in the US by Moore et al. (1946) measured integrally as net return on investment. The
before commercialization in 1949. Potential problems plethora of proffered candidates is evidenced, by way
caused by bacterial resistance phenomena were early of example, in the 2000-01 Direct-Fed Microbial,
sources of concern in human medicine, veterinary Enzyme and Forage Additives Compendium (Miller
94 Optimizing the replacement of pronutrient antibiotics in poultry nutrition
Publishing Co.), by its lists of 222 candidate products, unsatisfactory because it fails to convey the nutritional
90 enzymes, 72 microbials, 40 yeasts, 13 moulds and benefits of additives, which can be tantamount to
acids and seven oligosaccharides for use in poultry those of nutrients.
production alone. The role of a pronutrient is portrayed in the schema
in Figure 1. This shows the functional relationship
Table 1. Major antibiotic replacements. of a pronutrient in nutrition relative to that of an
antinutrient.
Acids
Anticoccidials
Botanicals Better performance
Enzymes
Nutrients
Microbials
Oligosaccharides
Pronutrient
Nutrients are also included because supplementary

amino acids, minerals, purified energy sources and NUTRIENT MIX
vitamins overlap performance-wise (Rosen, 1997) and
may well overlap with pronutrients in nutritional
improvements, irrespective of differences in modes Antinutrient
of action.
Worse performance
Is nomenclature satisfactory?
More meaningful, standardized, transparent Figure 1. Effects of pronutrients and antinutrients in diets.
terminology in this field would benefit scientists,
legislators and, above all, consumers. Feed additive Pronutrients function via a hundred or more different
antibiotics have been variously named as growth modes of action in extending the value of the limiting
promoters, growth permitters, performance nutrient in a diet. There is thus an overlap between
promoters, performance enhancers, production aids, the provision of a pronutrient and a ‘topping-up’ of
feed economizers, nutrient balancers, efficiency a limiting nutrient. Hence, nutrients are, as
enhancers, digestive enhancers and nutrition aforementioned, included as a Table 1 category for
improvers, which descriptors have often been regarded consideration herein. The choice of a pronutrient or
in several senses as inaccurate, incomplete or a nutrient in replacing feed antibiotics is simply a
inappropriate. Notwithstanding long-term, near- question of relative cost-effectiveness.
universal usage, the word ‘additive’ is nonetheless The terms ‘probiotic’ and ‘prebiotic’ are both
ill-conceived, being insufficiently descriptive. In plagiarisms, appropriated by scientists and suppliers.
practice the term ‘additive’ can generate auras of The US Food and Drug Administration and the
minority, subsidiarity, afterthought, optional extra European Union Commission, as regulatory
and contaminant. It may be suitable for fuels, but authorities, both refused them as vagaries, opting
not for food or feed; but it is doubtful whether respectively for direct-fed ‘microbial’ and
legislators would ever countenance a change. ‘microorganism’. The term probiotic was, as a matter
The term ‘pronutrient’ has been introduced to of fact, first coined by Winter (1955) in his research
replace additive. A pronutrient is defined as a on botanical antibiotics found in cruciferous plants,
substance that improves the value of nutrients. As when he stated that ‘we can call these substances
anticipated, a survey of 100 consumers has confirmed probiotics; they are antibiotic against pathogenic
it as unknown to them, but 85 interpreted it as microbes and they are therefore probiotic for the
‘something good for nutrition’. Therefore it is infected organism’. Gibson and Roberfroid (1994)
suggested that pronutrient could usefully replace the took the ‘prebiotic concept’ from its original and
term “nonnutrient additive” used by the National literally-correct meaning and long-standing usage to
Research Council to distinguish an additive from a define a chemical involved in the origin of life.
nutrient. However, “nonnutrient additive” is Prebiotic is incorrect as an appellation for a
G.D. Rosen 95
biosynthesized molecule, which is biofunctional, on the current status and future needs of replacements
which nourishes a live microorganism and which based on the view of 50 suppliers, users, consultants,
inhabits a live host, in no sense whatsoever, before life. educators, communicators and academics revealed a
The adoption of simple, realistic duplex descriptors large number of problems met in comparing candidate
to simultaneously impart nature and function could efficacies (Rosen, 2003c). Of the 92 nominated, the
be a useful forward step in this field. For example, main problem areas are variation in response, use of
we could specify (a) pronutrient cellulase, formate, uncontrolled tests, inadequate test designs, missing
B. cereus, capsicum, etc.; (b) prophylactic narasin, feed compositions, invalid ‘field’ tests and unjustified
nifursol, P. acidilactici, mushroom polysaccharide, dosage recommendations.
etc., (c) therapeutic tylosin phosphate, lincomycin Serious shortcomings in commonly-used averaging
hydrochloride, penicillin, trimethoprim, etc. and (d) procedures are illustrated in Table 2. For example,
pro-environmental phytase, carbohydrase, protease, the comparison of the effects on feed conversion ratio
etc. Furthermore, it would be advantageous to (FCReff) of enzymes and antibiotics, as such or in
discontinue reference to ‘antibiotic alternatives’ or percentage terms, cannot be meaningful due to their
‘antibiotic substitutes’, thereby avoiding a rekindling 11-day mean duration difference. It is known that
of consumers’ scientifically-unproven, anti-antibiotic FCReff diminishes through starter to finisher phases.
prejudices. The same applies to time span (year) differences,
averaging 1972 and 1987. The coefficients of
variation in response of 129-1,449% constitute a
How are antibiotic replacements warning against comparisons based on small test
compared? numbers.
For some researchers, direct comparisons within
All available properly-controlled test data need to be tests are fundamental, but they are, more often than
taken into account. The use of a handful of tests can not, too expensive. They require much larger, more
illustrate the potential of a product as a starting point, costly experiments to manifest statistically significant
but five tests cannot take account of the wide range differences between pairs of candidates, which
of genetic, environmental, managemental and dietary differences are normally much smaller than those
factors affecting response in praxis. A recent survey between candidates and negative controls.
Table 2. Superficial comparison of the effects of antibiotics, enzymes and microbials (FDIeff, LWGeff, FCReff and MORTeff) on control feed
intake (FDIC), control liveweight gain (LWGC), control feed conversion ratio (FCRC) and control mortality (MORTC) with their coeffi-
cients of variation (CV) in broiler nutrition.
Parameter Antibiotics Enzymes Microbials

n 5,159/1,5211 2,557/4391 234/571
FDIC, g 2,478 2,106 2,636
FDIeff, g 15.0 (0.6%) 32.4 (1.5%) 6.0 (0.2%)
CV, % 970 451 1,449
LWGC, g 1,075 1,043 1,331
LWGeff, g 39.8 (3.7%) 54.3 (5.2%) 25.3 (1.9%)
CV, % 129 147 192
FCRC 2.16 1.99 1.87
FCReff -0.073 (-3.4%) -0.105 (-5.3%) -0.030 (-1.6%)
CV, % 164 185 195
MORTC, % 5.77 6.53 3.76
MORTeff, % -1.40 (-24%) -1.71 (-26%) -0.40 (-11%)
CV, % 535 377 360
DUR*, days 41.0 30.3 35.8
YEAR – 1900 71.6 87.0 86.6
Improvement frequency2, % 74 75 70
1
Mortality.
2
Percentage of tests with improved FCReff and LWGeff.
*DUR = duration of test.
Comparative tests versus a positive control alone are squares, minimum standard deviations about
contraindicated because they provide no evidence of regression and significant partial regression
a beneficial response to either candidate. Table 2 also coefficients for all independent variables. After
starkly indicates a crucial gap in the large majority of exclusion of aberrant (>3 x SD) responses, the
tests, which fail to report mortality, viz. 71%, 83% emergent models are used to estimate nutritional
and 76%, respectively for antibiotics, enzymes and responses and 95% confidence limits for any required
microbials. Interestingly, the response improvement set of values of the component independent variables.
frequencies for these three candidate categories are Differences in these predicted responses are tested
virtually equal in the range 70-75%. Frequencies of for statistical significance. Nutritional response
beneficial response and magnitudes of variation estimates are then used to compute and compare net
therein merit greater attention than hitherto. profits to target liveweight and/or target duration.
These procedures are then used in feed formulation
to quantify specific requirements for pronutrients
How should we determine efficacy? or nutrient counterparts for target production. Thus
one can assess whether a nutrient, e.g. L-methionine,
In the light of the aforementioned shortcomings of would be a better choice than a pronutrient, e.g.
few tests and superficial averaging, the answer to this endoxylanase or Bacillus subtilis. In other words,
question resides in the formulation of optimal should one top up a limiting nutrient per se or increase
multifactorial empirical algebraic models for the amount of the dietary limiting nutrient?
nutritional effects of each candidate. Such models are
used to calculate a requirement for any given set of
circumstances and conditions, in order to compare an Which are key variables?
antibiotic and a candidate or to compare two or more
candidates. Essentially, all published data are accepted. The 48 factors listed in Table 3 exemplify the range
Manifestly-uncontrolled tests are excluded. Computer of variables potentially relevant in the elaboration
filing of all relevant data for the hundred or more of working models. There are also subsets of these
potentially-important variables includes routines for factors to be considered. Routinely dose is considered
elimination of errors in the original reports or in data as linear, quadratic, logarithmic or exponential. For
abstraction and repeats. Performance must be broilers there are four sex types: male, female, mixed
measured from start to finish. If second or later phase (50/50) and as-hatched. Ten to 20 antibiotics and
data are required they are obtained, e.g., by five to 10 anticoccidials may be relevant, alone or
subtracting 0-21 day from 0-42 day values. Standard in admixtures. Different disease challenges can be
statistical packages, e.g. Nie et al. (1975) and multiple identified. The data emanate from more than 100
regression methodology, e.g. Draper and Smith (1981) countries, though 10 or so usually furnish most of
are used to determine best-fit models. the test data. A few individual brands have numbers
Best-fit algebraic models have statistically significant worthy of test. Types of oils and fats, animal protein,
regressions, maximum multiple correlation coefficient and vegetable proteins, including admixtures, total
Table 3. Independent genetic, environmental, management, dietary and nutrient variables used in the elaboration of multifactorial
nutritional models.
Control performance Feed process Maize2 Gross energy3
Duration Antibiotic Sorghum Net energy3
Year of test Anticoccidial Wheat Crude protein
Dose Antihistomonial Barley Crude fat
Initial age Metabolic test Oats Crude fibre
Not day-old Diet marker Rye Calcium
Sex Part-purified diet Animal fat Phosphorus
Phased dose Disease challenge Vegetable oil Lysine
Factor 2 dose1 Favson Animal protein Methionine
Selected birds Institute test Vegetable protein Methionine + cystine
Housing Country Wheat offal Threonine
Stocking density Brand Rice bran Tryptophan
1
Second antibiotic/enzyme/acid/microbial/other pronutrient/nutrient
2
Dietary concentrations (columns 3 and 4)
3
Digestible or metabolizable energy are alternatives.
G.D. Rosen 97
more than 200. For example, in Brozyme (Rosen, 20 significant independent variables account for 64-
2000; 2002) there are, including mixtures of each 72% of the sizeable variations in nutritional responses
type, 71 animal proteins, 15 vegetable proteins, 81 with time and place (Rosen, 2003a). Further progress,
oils and fats and 25 carbohydrate sources. however, is unlikely until editors of peer-reviewed
Based on experience to date, level of negative journals take a lead, for others to follow, in ensuring
control performance, duration of feeding and year routine publication of fundamental variables such as
of test are basic, accounting for 10-50% of variation temperature, altitude, lighting pattern, disease status
in effects. Contrastingly, some highly statistically and mortalities.
significant factors can account for 1% or less. As examples, Figure 2 contains basal models for a
Interactions as product terms are normally relatively group of five important broiler antibiotics in order
small contributors. In models containing 10 or more to illustrate the magnitudes of the effects of key
significant variables, dosage tends to be less variables, with algebraic signs in accordance with
important, often accounting for 7.5% or less of total nutrition science and practice.
variation. These models contain 19 significant independent
Some independent variables are vital for the variables. For LWGeff and FCReff, negative
assessment of praxis values, compared with research coefficients quantify inferior and superior respective
conditions, namely, feed form, use of not-day-olds, response contributions, with increase in LWGC and
males, cages, part-purified diets and specific disease FCRC. Their positive DURs afford better and worse
challenges. effects respectively with age increase. Diagnosed or
If the treated value is used as the dependent variable endemic disease enhances LWGeff and FCReff. The
in modelling instead of the effect, multiple partial regression coefficients of LWGC and FCRC
correlation coefficient squares (R2) are grossly inflated mean that each 100 g better control performance
by the high correlation of control values and effects, would reduce LWGeff by 1.1 g and that each 10 point
e.g. for virginiamycin models (n=306), LWGeff and lower value for FCRC would reduce FCReff by
FCReff R 2 values of 0.14 and 0.44 respectively, 1.6 points.
become 0.97 for LWG and 0.99 for FCR as treated
dependent variables. Such inflation of R2 is misleading
in variation accountancy and for some it harbours a How many tests are needed for a working
delusion that R2 less than 0.4-0.6 is unworthy. model?
Incomplete test reports limit the accountancy of
variation. Recent data on phytase responses in broilers Hitherto, the notional n = c. 50 controlled tests has
offer a useful perspective in which models containing been thought of as a minimum required to produce a
FDIeff = -79.5 -0.0380FDIC + 2.98DUR +1.02EXDAT -24.3COC

R2 .052 se 38.7 0.006 0.507 0.465 7.07
SD 126 p 0.029 0.000 0.000 0.028 0.001
n 1709
LWGeff = - 72.4 - 0.0114LWGC + 1.02DUR + 0.871EXDAT +19.5log(AB+1) -16.8COC +76.1VET

R2 0.171 se 15.0 0.004 0.153 0.183 3.22 2.70 5.56
SD 47.4 p 0.000 0.009 0.000 0.000 0.000 0.0000 0.000
n 1709
FCReff = 0.301 - 0.161FCRC + 0.00306DUR - 0.00159EXDAT - 0.0286log(AB+1) - 0.116VET

R2 0.287 se 0.029 0.007 0.000 0.000 0.007 0.012
SD 0.100 p 0.000 0.000 0.000 0.000 0.000 0.000
n 1709
MORTeff = - 1.13 - 0.647MORTC + 0.0363DUR + 1.08log(AB+1) - 2.12VET

R2 0.685 se 0.805 0.021 0.012 0.336 0.475
SD 3.12 p 0.000 0.000 0.003 0.000 0.000
n 708
Figure 2. Antibiotic models relating feed intake, liveweight gain, feed conversion ratio and mortality effects to level of control
performance, duration, date, dosage, anticoccidial use and disease status.
useful model to quantify the magnitudes of nutritional Using zinc bacitracin models based on 1,164 tests
responses in feed formulation. A recent project and first-generation phytase models (n=296), Table
addressed the question of a minimum platform via 4 shows that zinc bacitracin at 80 ppm affords a five
random fragmentation of a large 1,709/mortality 708 point conversion improvement compared with the iso-
test resource of five of the most important feed cost level of phytase with no effect. In addition, a
antibiotics (i.e. two bacitracins, two tetracyclines and huge increase in phytase dosage (x 20) gives a three
virginiamycin) into smaller subsets down to 34-43 point conversion improvement, for which 42 ppm of
for the elaboration of progressively smaller-based a zinc bacitracin would suffice.
models (Rosen, 2004). The parent models are those
in Figure 2. The analysis of a total of 704 fragmented Table 4. Iso-input cost and iso-feed conversion effect comparisons
resource models revealed that (a) no model at all of zinc bacitracin (n=1164) and first-generation phytases (n=296)
for as-hatched 56 day-old broilers of LWGC=3,266g and
was afforded in 23% of the random fragments; (b) FCRC=2.079.
progressive subset fractionation of the data set sharply
Pronutrient Dosage Basis LWGeff FCReff
reduced the number of significant variables from 79%
in the parent models to 17% in the smallest, which Zinc bacitracin (ppm) 8 0 iso-cost 59 -0.054
42 iso-FCReff 47 -0.032
averaged 1.2 significant independent variables; (c)
the chance of three or more significant variables in Phytase (u/kg) 500 iso-cost -1 0.001
10,000 iso-FCReff 159 -0.032
subset models was one in eight; and (d) the smallest
sets, averaging n = 38/mortality 15, did in toto reveal
all the significant variables, albeit very patchily, Secondly, Table 5 provides a model-based perspective
found in the parent models. on the results of a test from 7 to 28 days of age on
female broiler chicks, which compared a Chinese
herbal preparation, virginiamycin and a negative
How are requirement models used? control (Guo et al., 2000). The herbal at 938 ppm
afforded an FCReff four points inferior to
Requirements are needed for each and every set of virginiamycin at 20 ppm, even though the latter
circumstances for a defined target objective, for manifested, in this test, a response three points below
nutrients per se or for pronutrients. In other terms, its predicted result.
what response might we expect within what
confidence limits for a given dosage of supplementary Table 5. Application of LWGeff and FCReff models to assess the
results of a comparative test on a Chinese herbal formulation vs
methionine compared with an optimal dosage of virginiamycin for LWGC=1,098 g and FCRC=1.554 on 7-28 day-
bacitracin methylene disalicylate, or 6-phytase or a old female birds.
mannan oligosaccharide, etc.? The conjoint (overlap)
Product Dosage Basis LWGeff FCReff
consideration of the value of a limiting nutrient (ppm) (g)
supplement or the improved efficiency of a limited
Chinese herbal 938 tested +15 +0.012
nutrient supply with a pronutrient is of particular Virginiamycin 20 tested -6 -0.027
interest for the comparison of multifunctional 20 predicted* +3 -0.056
substances. This approach could well resolve the *306-test requirement models
ongoing, decades-old dispute on the relativities of
DL-methionine (DLM) and methionine hydroxy- The import and value of a negative control is further
analogues (HMTBA and CaHMTB). The use of evidenced in a 39-day broiler test by Messikommer
requirement models based on all-available, unselected on 50 ppm zinc bacitracin versus a rhubarb
control test data for DLM, HMTBA and CaHMTB supplement (Wenk, 2000). The predicted zinc
with dose expressed simply as product weight (not bacitracin response of -0.074 ± 0.026 illustrates that
molar or other equivalencies in malnutrition tests) the observed value of -0.049 is as expected; and that
should be decisive. Such methodology is imperative the botanical has potential value at 2,500 ppm
because DLM is a mixture of a nutrient and a (-0.038), but not at 5,000 ppm (+0.018).
prenutrient and HMTBA and CaHMTB are bifunctional Thirdly, truncated comparative tests without a
as prenutrient and pronutrient. negative control are preferred by some researchers,
The mode of application and value of multifactorial even though they are unable to confirm efficacy for
models for antibiotics and their replacements can next either test product. An assessment of the results of a
be illustrated in four examples as follows. positive control only test can be made using a model
G.D. Rosen 99
for the test antibiotic. Syrvidis et al. (2003) reported requirement models for Bio-Mos®, quantifying the
a test on Digestarom 1317 Poultry Premium (a so- influences of dosage, level of bird performance,
called phytobiotic) versus Flavomycin-80, but failed presence or absence of other pronutrients, especially
to include test dosages. Hence, a notional negative anticoccidials, bird sex, feed form and limiting
control performance of LWGC = 2,125 g and FCRC nutrients.
= 1.970 was calculated from flavomycin models By virtue of extensive 15-year test programmes,
(n=394) for a presumed dosage of 2 ppm. The exogenous enzymes would appear at present to be
computed negative control values reveal very large the best characterized replacement category. The data
Digestarom responses of LWGeff = 326 g (15.3%) available for organic acids in pigs, microbials and
and FCReff = -0.220 (-11.2%). At today’s production oligosaccharides in poultry and pigs may already
standards, however, a feed conversion as-hatched for suffice for the elaboration of initial working models.
a 40-day 2,125 LWGC would be about 1.730, i.e., But all available data should be used to avoid possible
12.2% less than the computed FCRC of 1.97 at 42 selection bias, as in the organic acid studies of Partanen
days old in this trial. Such a result against a positive and Mroz (1999) and Partanen (2001).
control alone should be treated with reserve. The
availability of further tests against negative controls
would afford a better view on the potential of How should we test admixtures?
Digestarom. The same applies to any product if it
claims value from one or more tests solely against a Problems in the use of admixtures of antibiotic
positive control(s). replacements arise from shortcomings in
Fourthly, even prior to the availability of working nomenclature and posology (dosage science).
models for individual product brands of replacements, ‘Additivity’ and ‘synergism’ are often misused,
test data collections of the latter can be reviewed as usually for sub-additivity. The possibility of
to their potential value as antibiotic replacements. antagonism should always be borne in mind. For
Bio-Mos® mannan oligosaccharide collections of 34 purposes of definition, the possible effects of
tests for broilers (Hooge, 2003a) and 27 tests for admixture are classified herein as sub-additive,
turkeys (Hooge, 2003b) are used to provide an additive, synergistic, ineffective or antagonistic, as
example. Table 6 compares mean liveweight gain and defined for the admixture of 2+3 of A and B
feed conversion responses in broilers and turkeys with providing 4, 5, 6, 2 or 3 and 1 unit(s) of response
corresponding predictions for equivalent respectively. A 2 x 2 factorial test is a good starting
circumstances, using comprehensive multifactorial point, shown in Table 7, which also includes other
models for the antibiotic products virginiamycin iso-cost admixtures for A or B alone or for higher
(n=306) in broilers and zinc bacitracin (n=226) in single dosages of A or B. The acid test for maximal
turkeys. results uses A+B each at its economic optimum.
Table 6. Comparison of mean LWGeff and FCReff in 34 mannan

oligosaccharide (Bio-Mos®) broiler tests with predicted responses Table 7. Scope of admixture tests.
for virginiamycin for LWGC=2,149 g, FCRC=1.879 at 42.2 days-
old and in 27 turkey tests with predicted responses for zinc Treatment Feed Investment
bacitracin for LWGC=5,643 g, FCRC=1.981 at 68.7 days. concentration cost
Species/ Dosage Basis LWGeff FCReff (ppm) (MU*)
product (g)
Basal feed 0 0
Broilers Replacement A a 4
Bio-Mos® 1.04 g/kg 34 test mean +40 -0.042 Replacement B b 8
Virginiamycin 20 ppm 305 test model +21 -0.040 Replacement A + Replacement B a+b 12
Turkey Replacement A 3a 12
Bio-Mos® 1.19 g/kg 27 test mean +127 -0.032 Replacement B 1.5b 12
Zn bacitracin 50 ppm 236 test model +148 -0.041 Replacement A + Replacement B 0.33a + 0.33b 4
Replacement A + Replacement B 0.67a + 0.67b 8
The Table 6 data are encouraging for Bio-Mos® as a *MU money units
replacement when used at a dosage of 1 g/kg in broiler
and turkey feed. More searching comparisons, Nutritional models can provide a useful guide towards
however, must now await the availability of maximum admixture efficiency by pre-determination
of the economic optimal doses of Replacements A
and B. These admixture guidelines are also pertinent References

in situations where pronutrient antibiotics can still
be utilized in admixture with nutrients or pronutrient Draper, N.R. and H. Smith. 1981. Applied Regression
supplements. Analysis, 2nd Edition. John Wylie and Sons, Inc.,
New York.
Gibson, G.R. and M.B. Roberfroid. 1994. Dietary
Quo vadimus? modulation of the human colonic microbiota:
introducing the concept of prebiotics. J. Nutr.
The thesis presented herein essentially advocates that 125:1401-1412.
we should optimize the choice and dosage of nutrient Guo, F., R.B. Kwakkel and M.W.A. Verstegen. 2000.
or pronutrient antibiotic replacements by taking The use of Chinese herbs as alternatives for growth
cognizance of all available data expressed in promoters in broiler diets. In: Abstracts and
predictive, empirically-based, multifactorial multiple
Proceedings, XXI World’s Poultry Congress,
regression requirement models for feed, gain,
Montreal, Canada, Aug. 20-24. CD-Rom, pp. 2.
conversion and mortality effects, at least. Such models
Hooge, D.M. 2003a. Broiler chicken performance
should be updated annually to take account of the
accelerating current flow of scientifically-controlled may improve with MOS. Feedstuffs, January 6,
feeding tests, as seen for example, in the increase of pp. 11-13.
exogenous enzyme publications from 1,422 up to the Hooge, D.M. 2003b. Dietary MOS may have
year 2000 rising to its latest content in the Brozyme application in turkey diets. Feedstuffs 75(18):11-
resource (Rosen, 2002) of 2,175. It is intended also 13. p.42.
to extend these studies to table egg and breeder hens Moore, P.R., A. Evenson, T.D. Luckey, E. McCoy,
and at least ducks among the minor species. C.A. Elvehjem and E.B. Hart. 1946. Use of
Following the lead of this Symposium in ‘re- sulphasuccidine, streptothricin and streptomycin in
imagining the feed industry’, should we not also prune nutrition studies with the chick. J. Biol. Chem.
and improve its verbiage to better its science and 165:437-441.
raise its transparency to consumers, setting a good Nie, N.H., C. Hadlai-Hull, J.G. Jenkins, K.
example for all members of the food chain? Steinbrenner and B.H. Bent. 1975. In: Statistical
In conclusion, it may be apposite, in line with the Package for the Social Sciences, 2 nd Edition.
question and answer format of this review, to McGraw Hill Book Company, New York.
conclude with a Seven Question Test with which the Partanen, K. 2001. Organic acids - their efficacy and
user of an antibiotic replacement can assess the modes of action. In: Gut Environment of Pigs (A.
potential value of a supplier’s Product X. Piva, K.E. Bach Knudsen and J.E. Lindberg, eds).
Nottingham University Press, UK, pp. 79-96.
1) How many properly-controlled feeding tests do Partanen, K.H. and Z. Mroz. 1999. Organic acids
you have on the efficacy of Product X? for performance enhancement in pig diets. Nutr.
Res. Rev. 12:117-145.
2) How many of these have no negative controls?
Rosen, G.D. 1997. In: Proceedings of the 16 th
3) Can you supply a bibliography for 1) and 2)? Scientific Day, Southern African Branch of the
4) How many times out of ten does Product X World’s Poultry Science Association (D.G.
improve liveweight gain and feed conversion? Poggenpoel, ed). October 16, 1997. pp. 60-79.
Rosen, G.D. 2000. Enzymes for broilers: a
5) What are the coefficients of variation in the gain multifactorial assessment. Feed Int’l. 21(12):
and conversion responses? 14-18.
6) What dosage of Product X will maximize return Rosen, G.D. 2002. Exogenous enzymes as
on my investment and why? pronutrients in broiler diets. In: Recent Advances
in Animal Nutrition 2002. (P.C. Garnsworthy and
7) Can you supply me with a model to predict
J. Wiseman, eds). Nottingham University Press,
responses to Product X under my conditions?
UK. 89-103.
Receipt of answers of 1) 30; 2) five; 3) yes; 4) seven; Rosen, G.D. 2003a. Effects of genetic, managemental
5) 100-200%; and 6) “x ppm because . . .” should be and dietary factors on the efficacy of exogenous
encouraging. A ‘yes’ to 7) would be even better.
G.D. Rosen 101
microbial phytase in broiler nutrition. Brit. Poultry Syrvidis, V., R. Bobiniene, V. Priudokiene and D.
Sci. 44S1:S25-S26. Vencius. 2003. Phytobiotics add value to broiler
Rosen, G.D. 2003b. Pronutrient antibiotic feed. World Poultry 19(1):16-17.
replacement standards discussed. Feedstuffs Wenk, C. 2000. Herbs, spices and botanicals; ‘Old
75(30):11-13. p.16. fashioned’ or the new feed additives for tomorrow’s
Rosen, G.D. 2003c. Setting and meeting standards feed formulations? In: Biotechnology in the Feed
for the replacement of pronutrient antibiotics in Industry, Proceedings of Alltech’s 16th Annual
poultry. Proceedings of the 30th Annual Carolina Symposium (K.A. Jacques and T.P. Lyons, eds).
Poultry Nutrition Conference, Carolina Feed Nottingham University Press, UK, pp. 79-96.
Industry Association, Research Triangle Park, Winter, A.G. 1955. The significance for therapy and
October 30. pp. 69-79. diet of antibiotic substances from flowering plants
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are required to elaborate working models for the horseradish). Die Medizinische Nr. 2:73-80.
prediction of nutritional effects of pronutrients in
praxis? Brit. Poultry Sci. In press.
Swann Committee. 1969. Joint Committee on the use
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Stationary Office, London. p. 83.
T.K. Smith et al. 103
Comparative aspects of Fusarium mycotoxicoses in broiler chickens, laying

hens and turkeys and the efficacy of a polymeric glucomannan mycotoxin
adsorbent: Mycosorb®
TREVOR K. SMITH, SHANKAR R. CHOWDHURY AND H.V.L.N. SWAMY
Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada
Introduction
Fusarium fungi thrive in temperate climates around contaminated feeds, particularly with respect to
the world and Fusarium mycotoxins are the reduced feed intake (Smith et al., 1997).
mycotoxins most commonly found in feed grains and It has been reported that the feeding of 100 ppm
forages (Wood, 1992). There are numerous zearalenone had no effect on layer performance,
pathologies characteristic of Fusarium mycotoxicoses fertility or hatchability (Marks and Bacon, 1976).
and this is due to the several chemically distinct groups The feeding of DON at 5 ppm (Hamilton et al., 1985)
of Fusarium mycotoxins, which have very different or 18 ppm (Kubena et al., 1987) did not affect layer
effects on animal metabolism and behavior. Swine performance. A combination of DON (2-3 ppm) and
are usually considered to be the most sensitive species zearalenone (0.4–0.5) also resulted in no adverse
to feed-borne mycotoxins both with respect to feed effects on layer performance (Kesharvarz, 1993).
refusal and to reproductive failure. Ruminant animals Layer performance was adversely affected, however,
are thought to be the most resistant because of the when a corn-based diet was fed containing 17.6 ppm
detoxifying effect of rumen microorganisms. Poultry DON and 1.6 ppm zearalenone (Danicke et al.,
are generally considered to be less sensitive than swine 2002). Adverse effects on layer performance are often
and are often the recipients of contaminated grains greater under field conditions (Keshavarz, 1993). It
diverted from swine feeds. is often difficult, however, to explain why this occurs
The Fusarium mycotoxin most often detected in (Williams et al., 1992).
Canadian-grown grains is the trichothecene There is less information available regarding the
deoxynivalenol (DON, vomitoxin) (Scott, 1997). feeding of contaminated grains to turkeys. Hamilton
Trichothecene toxicosis is characterized by reduced et al. (1985) indicated that turkey poults can tolerate
appetite, lesions of the intestinal tract and diets containing at least 5 ppm DON. Manley et al.
immunosuppression. Another frequently detected (1988) described feed refusal and high mortality in a
compound is zearalenone, an estrogenic mycotoxin commercial turkey flock fed diets containing 81 ppb
that impairs mammalian reproduction. DON + 2.2 ppm salinomycin. The feeding of 4.4
It has been reported that broiler chicks can tolerate ppm DON + 22 ppm salinomycin had no effect on
up to 15 ppm DON from naturally-contaminated feed consumption or viability. Morris et al. (1999)
wheat and oats without adverse effects on observed that the feeding of 20 ppm DON had no
performance (Hulan and Proudfoot, 1982; Kubena adverse effects on poults and no toxicological
et al., 1987). Others, however, observed reduced interaction was observed between DON and
performance and changes in immune function, moniliformin.
hematology and serum chemistry in broiler chicks The discrepancy between the relative tolerance of
fed 16 to 18 ppm DON from naturally contaminated poultry to Fusarium mycotoxins in literature reports
sources (Huff et al., 1986; Kubena et al., 1988; 1989; and the seeming susceptibility of poultry under
Harvey et al., 1991). It is clear however, that broiler commercial conditions may be due to several factors.
chickens are far less sensitive than swine to DON- Many of the experiments conducted in literature
104 Comparative aspects of Fusarium mycotoxicoses in poultry
reports were for fairly short periods. This is often and tissue samples were taken for hematology and
necessary to conserve valuable stocks of purified serum chemistry measurements.
mycotoxins or fungal culture materials. The use of
purified mycotoxins, fungal culture materials or Laying hens
artificially inoculated corn can also bias findings
because the toxicological synergism arising from the One hundred and forty-four, 45-week-old laying hens
feeding of combinations of mycotoxins does not take were fed for 12 weeks diets including: 1) control, 2)
place. contaminated grains, and 3) contaminated grains +
Polymeric mycotoxin adsorbents prevent 0.2% Mycosorb®. Parameters measured included feed
mycotoxicoses by adsorbing mycotoxins in the consumption, egg production, egg shell measure-
intestinal lumen and preventing transfer through the ments, egg quality measurements, relative organ
blood to target tissues (Ramos et al., 1996). The weights and plasma chemistry.
current studies were conducted to determine the
efficacy of Mycosorb ®, a glucomannan polymer Turkeys
extracted from the cell wall of yeast, in preventing
Two hundred and twenty-five day-old male turkey
the adverse effects of blends of grains naturally-
poults were fed corn, wheat and soybean meal-based
contaminated with Fusarium mycotoxins on poultry.
starter (0-3 weeks), grower (3-6 weeks), developer
(6 – 9 weeks) and finisher (10 – 12 weeks) diets in a
12-week experiment. Diets included: 1) control, 2)
Materials and methods contaminated grains, and 3) contaminated grains +
EXPERIMENTAL FEEDSTUFFS
0.2% Mycosorb®. Parameters measured included
weight gain, feed consumption, relative organ
Mycotoxins in the current experiments were provided weights and plasma chemistry.
by a blend of naturally contaminated corn and wheat
purchased from producers in Southwestern Ontario.
The complete diets were analyzed for DON, 3-acetyl- Results
DON, 15-acetyl-DON, nivalenol, T-2 toxin, iso T-2
toxin, acetyl-T-2 toxin, HT-2 toxin, T-2 triol, T-2 MYCOTOXIN ANALYSES
tetraol, fusarenon-X, diacetoxyscirpenol, scirpentriol,
Of the twenty different mycotoxins analyzed for, only
15-acetoxyscirpentriol, neosolaniol, zearalenone,
DON, 15-acetyl DON, zearalenone and fusaric acid
zearalenol, aflatoxin and fumonisin by gas
were found in detectable quantities in all experimental
chromatography and mass spectrometry at the
diets. DON was found to be approximately 0.5 ppm,
Veterinary Diagnostic Laboratory, North Dakota State
5.0 ppm, 9.0 ppm and 10.0 ppm for the four
University, Fargo, North Dakota (Raymond et al.,
experimental broiler diets. The concentrations of 15-
2003). Fusaric acid was determined by the high
acetyl DON were about 0.5 ppm, zearalenone was
performance liquid chromatographic method of
about 0.5 ppm and fusaric acid was about 17 ppm.
Matsui and Watanabe (1988) as modified by Smith
Diets fed to laying hens which contained
and Sousadias (1993) and confirmed by Porter et al.
contaminated grains had an average of 12.0 ppm
(1995).
DON with the other three mycotoxins in the same
ratios as were seen in the broiler trials. In the turkey
trial, diets containing contaminated grains averaged
EXPERIMENTAL DESIGN
6.5 ppm, 7.6 ppm, 10.6 ppm and 13.3 ppm DON for
Broilers the starter, grower, developer and finisher phases with
the other three mycotoxins present in the same
Broiler chicks were fed starter (0 – 3 weeks), grower proportions as were seen for the broiler and layer
(3 – 6 weeks) and finisher (6 – 8 weeks) diets in two experiments.
56-day experiments (Swamy et al., 2002; 2004a).
Diets included: 1) control, 2) low level of
contaminated grains, 3) high level of contaminated BROILERS
grains, and 4) high level of contaminated grains +
0.2% Mycosorb®. Growth rates and feed consumption There was a significant linear decrease in growth rate
were monitored weekly. At the end of the study, blood and feed consumption in the grower period when
increasing levels of contaminated grains were fed to and also prevented a decrease in feed efficiency. At
rapidly growing broilers (Swamy et al., 2004a; Table the end of the experiment, it was observed that hens
1). No significant effect of diet was seen in the starter fed contaminated grains had an increased relative
or finisher periods. When broilers were growing kidney weight compared to controls; this effect was
more slowly, growth depression was observed in the prevented by the feeding of Mycosorb®.
finisher phase (Swamy et al., 2002). At the end of Egg production and egg mass decreased (P<0.05)
the finisher phase in the earlier study, it was observed compared to controls in the first two months when
that the feeding of contaminated grains elevated red contaminated grains were fed. The feeding of
blood cell count and blood concentrations of hemoglobin Mycosorb® prevented this in the first month. The most
and uric acid (Table 2). Biliary concentrations of obvious effect of feeding contaminated grains on
immunoglobulin A were reduced while breast meat plasma chemistry was on uric acid concentrations
redness increased. (Table 4). In each month, plasma uric acid
The feeding of 0.2% Mycosorb® prevented all of concentrations were significantly increased with the
the above dietary effects. feeding of contaminated grains. In each case, this
increase was prevented by the feeding of Mycosorb®.
LAYING HENS
TURKEYS
The feeding of contaminated grains decreased feed
consumption compared to controls in the first month The feeding of contaminated grains reduced growth
(P<0.05) (Table 3). Feed intake increased, however, rates in the starter, developer and finisher phases and
in the second and third months. The feeding of overall (Table 5). The feeding of Mycosorb ®
Mycosorb® prevented this increase in the third month prevented these effects. The most obvious effect of
Table 1. Effect of feeding blends of grains naturally-contaminated with Fusarium mycotoxins on weight gain and feed consumption of broiler
chickens.1
Diet Feed consumption (g/bird)2 Weight gain (g/bird)3
0 - 21 days 21 - 42 days 42 - 56 days 0 - 21 days 21 - 42 days 42 - 56 days
Control 908 2797 2544 435 1678 1303
Low mycotoxins 841 2565 2437 376 1522 1274
High mycotoxins 923 2392 2456 386 1479 1319
High mycotoxins + 0.2% Mycosorb® 968 2472 2532 392 1538 1348
SEM 37 60 27 6 16 13
Linear effect NS4 0.05 NS NS 0.04 NS
1
From Swamy et al., 2004a.
2
Values are least square means; n = 3.
3
Values are least square means; n = 90.
4
Not significant (P>0.05).
Table 2. Effect of feeding blends of grains naturally-contaminated with Fusarium mycotoxins on hematology, serum chemistry and breast
meat coloration of broiler chickens.1
Diet RBC2 Hb 3 Uric acid4 Redness5 Biliary IgA6
Control 2.66 95.0 259 0.45 7.54
Low mycotoxins 2.84 101.1 286 0.67 7.28
High mycotoxins 2.83 99.2 357 0.80 4.99
High mycotoxins + 0.2% Mycosorb® 2.54 91.2 281 0.21 6.54
SEM 0.04 1.37 10.9 0.07 0.29
Linear effect 0.01 0.01 0.009 0.01
1
From Swamy et al., 2002.
2
Red blood corpuscle counts (1012/L); n = 12.
3
Hemoglobin concentration (ppm); n = 12.
4
Uric acid concentration (µmoles /L); n = 12.
5
Unitless scale, 0 = green, 1 = red; n = 15.
6
mm precipitate; n = 15.
Table 3. Effect of feeding blends of grains naturally-contaminated with Fusarium mycotoxins on feed consumption and feed efficiency of
laying hens.
Diet Feed consumption1 (g/hd/day) Feed efficiency2 (feed/egg mass)
Wk 0 - 4 Wk 4 - 8 Wk 8 - 12 Wk 0 - 4 Wk 4 - 8 Wk 8 - 12
Control 119 120 117 1.88 1.92 1.90
Mycotoxins 106 127 132 1.94 2.29 2.23
Mycotoxins + 0.2% Mycosorb® 114 124 121 1.90 2.10 1.94
Pooled SD 9 9 7 0.18 0.27 0.17
Control vs mycotoxins 0.008 0.04 0.0001 NS3 0.001 0.001
Mycotoxins vs Mycosorb® NS NS 0.0006 NS NS 0.003
1
n = 12.
2
n = 12.
3
Table 4. Effect of feeding blends of grains naturally-contaminated with Fusarium mycotoxins on organ weights and plasma uric acid
concentrations of laying hens.
Diet Organ weights (g) Uric acid (µmol/L)
Liver Spleen Kidney Wk 4 Wk 8 Wk 12
Control 44.51 2.2 5.9 376 392 390
Mycotoxins 44.2 2.4 7.6 1009 1154 1030
Mycotoxins + 0.2% Mycosorb® 46.2 2.2 6.6 500 539 487
Pooled SD 7.58 0.66 0.98 188 156 159
Control vs mycotoxins NS2 NS 0.002 0.001 0.001 0.001
Mycotoxins vs Mycosorb® NS NS 0.02 0.001 0.001 0.001
1
n = 12.
2
Table 5. Effect of feeding blends of grains naturally-contaminated with Fusarium mycotoxins on growth of turkeys (g/bird/week).
Diet Starter Grower Developer Finisher Overall
Control 217 378 748 723 517
Mycotoxins 196 492 657 637 496
Mycotoxins + 0.2% Mycosorb® 213 548 756 852 592
Control vs mycotoxins 0.01 0.01 0.01 0.05 0.05
Mycotoxins vs Mycosorb® 0.05 0.05 0.05 0.01 0.01
diet on plasma chemistry was on plasma uric acid the experiment, turkeys fed contaminated grains +
concentrations (Table 6). After 4 and 8 weeks of feeding Mycosorb® had significantly smaller spleens and kidneys
contaminated grains, plasma uric acid concentrations and significantly larger bursas than birds fed
were significantly reduced. This effect was not seen, unsupplemented contaminated grains.
however, when birds received Mycosorb®. At the end of
Table 6. Effect of feeding blends of grains naturally-contaminated with Fusarium mycotoxins on organ weights and plasma uric acid
concentrations of turkeys.
Diet Organ weights (g) Uric acid (µmol / L)
Bursa Spleen Kidney Wk 4 Wk 8 Wk 12
Control 5.31 5.6 16.1 437 339 276
Mycotoxins 5.5 6.1 17.7 299 210 305
Mycotoxins + 0.2% Mycosorb® 6.5 5.1 15.8 321 197 235
SEM 0.30 0.33 0.71 27 15 43
Control vs mycotoxins NS2 NS NS 0.001 0.001 NS
Mycotoxins vs Mycosorb® 0.03 0.05 0.05 NS NS NS
1
n = 12.
2
Discussion LAYING HENS
Broilers, layers and turkeys are all sensitive to The effect of feeding contaminated grains on feed
Fusarium mycotoxicoses. The adverse effects of the intake of laying hens is in contrast to that seen in
diets fed in the current studies are greater than broilers. It would seem that after an initial reduction
literature reports based on the DON content. This is in feed intake and egg production, layers increased
likely because of the relatively short duration of feed intake perhaps in an attempt to boost egg
previously reported trials. The blending of different production. The result, however was a very dramatic
naturally contaminated grains, moreover, also results deterioration in feed efficiency (feed consumed/egg
in a more complex mixture of mycotoxins thereby mass). This may be due, in part, to increased hepatic
increasing the chances of toxicological synergy amino acid oxidation due to the trichothecene-induced
between mycotoxins. reduction in protein synthesis. It is notable that the
mycotoxin-induced elevation in blood uric acid
concentrations is similar to, but greater in magnitude
BROILERS than, the response seen in broilers. The increased
kidney weight seen when contaminated grains were
The observation that feeding contaminated grains to fed is likely due to the increased metabolic burden
broilers reduced growth only in the grower and of excretion arising from increased uric acid
finisher phases supports the concept that broilers do synthesis.
not exhibit feed refusal in a manner similar to swine
fed Fusarium mycotoxin contaminated diets (Smith
et al., 1997). The reason for this species difference TURKEYS
has been shown to be differences in the effects on
brain neurochemistry (Swamy et al., 2004b). The Feeding contaminated grains significantly reduced
feeding of contaminated grains to pigs elevated brain weight gain of turkey poults as early as the second
serotonin concentrations. In broilers, such treatments week of feeding. This is a more rapid effect than
elevated both serotonin and catecholamines thereby was seen in broilers but little effect of diet was seen
canceling the effect of serotonin on appetite on feed consumption. It would appear that turkeys
suppression. It is likely that mycotoxin-induced are more sensitive to this mycotoxin challenge than
growth suppression in broilers was due to gradual broilers. The significant depression in blood uric acid
alterations in metabolism that occurred with extended concentrations after 4 and 8 weeks of feeding is in
feeding of contaminated grains. marked contrast to the responses seen in broilers and
The mycotoxin-induced elevation in red blood cell laying hens. The metabolic reason for this remains
count and hemoglobin is similar to the changes seen to be determined as other indices of blood chemistry
in ascites. It is possible that the hypotensive effect of were largely unaffected by diet.
fusaric acid may be reducing blood flow to the lungs
resulting in an increased need for oxygen trapping
capacity of blood. Elevations in blood uric acid EFFECTS OF MYCOSORB®
concentrations were likely due to the inhibition of
Mycosorb® proved to be a very effective preventative
protein synthesis caused by trichothecenes such as
treatment for Fusarium mycotoxicoses in broilers,
DON and 15-acetyl DON. This would result in
layers and turkeys. The mode of action of Mycosorb®
increased hepatic oxidation of amino acids and
is to prevent intestinal uptake of mycotoxins and
increased uric acid excretion. Red discoloration of
subsequent transfer of mycotoxins to sensitive target
breast meat has been reported in turkeys fed Fusarium
tissues such as liver, kidney, brain and reproductive
culture filtrates (Wu et al., 1994). The discoloration
tract. It is clear from the efficacy of Mycosorb® in
seen in the current study was likely due to increased
these trials that it is capable of adsorbing a
red blood cell count and hemoglobin concentrations
combination of Fusarium mycotoxins and minimizing
as well as to edema arising from the hypotensive
the potential for toxicological synergism.
effects of fusaric acid. The reduced biliary
immunoglobulin A concentrations may have arisen
from trichothecene-induced inhibition of protein
synthesis.
References
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H. Valenta and G. Flachowsky. 2002. Effects of and G.E. Rottinghaus. 1989. Individual and
addition of a detoxifying agent to laying hen diets combined toxicity of deoxynivalenol and T-2 toxin
containing uncontaminated or Fusarium toxin- in broiler chicks. Poult. Sci. 68:622.
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Harvey, R.B., L.F. Kubena, W.E. Huff, M.H. and G.E. Rottinghaus. 1999. The individual and
Elissalde and T.D. Phillips. 1991. Hematologic and combined effects of feeding moniliformin, supplied
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deoxynivalenol: Effects on performance of poultry. 2003. Effects of feeding a blend of grains naturally
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Buckley, T.D. Phillips, G.E. Rottinghaus and H.H. and the efficacy of a polymeric glucomannan
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culture material and T-2 toxin or deoxynivalenol grains and grain-based foods for trichothecenes and
in broiler chicks. Poult. Sci. 76:1239. zearalenone. Food Addit. Contam. 14:333.
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and T.D. Phillips. 1987. Effects of feeding content of swine feeds. J. Agr. Food Chem.
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old through egg production. Poult. Sci. 66:1612. Effect of feeding blends of Fusarium mycotoxin-
Kubena, L.F., W.E. Huff, R.B. Harvey, D.E. Corrier, contaminated grains containing deoxynivalenol and
T.D. Phillips and C.R. Creger. 1988. Influence of fusaric acid on growth and feed consumption of
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Boermans and A.E.Sefton. 2002. Effects of feeding on brain regional neurochemistry of starter pigs
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Fusarium mycotoxins on production and Williams, K.C., B.J. Blaney, R.A. Young and R.T.
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Swamy, H.V.L.N., T.K. Smith, N.A. Karrow and kernels naturally infected predominantly with
H.J. Boermans. 2004a. Effects of feeding blends Fusarium moniliforme and Diplodia maydis. II.
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Pig science
PIG SCIENCE
W.H. Close and K. Turnley 113
Creating technical and educational forums that help pig producers meet
performance and economic goals: the Premier Pig Program™
WILLIAM H. CLOSE1 AND KIM TURNLEY2
1
Close Consultancy, Wokingham, Berkshire, UK
2
Alltech Inc., Melbourne, Victoria, Australia
Introduction
Regardless of where pigs are produced the objectives If pigs are sold at 120 kg body weight, then the target
should be the same: to optimise the quantity of pig should be 1.25 tonne of carcass lean per sow per
meat produced per sow per year or per lifetime, at year, or 2.50 tonne carcass lean per lifetime. The
minimal cost. For the modern sow and pig genotypes, performance of the animal at all stages of production,
with pigs being sold at 100 kg liveweight, the that is the sow, weaner and grow-finish pig, contribute
objective should be to produce at least 1 tonne of to these target levels of performance, as indicated in
carcass lean per sow per year. This is achieved as in Figure 1.
Table 1.
Table 1. Targets for producing 1 tonne of carcass lean per sow per year with pigs sold at 100 kg
liveweight.
100 kg liveweight at slaughter → 75 kg carcass weight

60% lean in carcass → 45 kg carcass lean
23 pigs sold/sow/year → 1.03 tonne carcass lean per year
5 litters/sow/lifetime → 2.06 tonne carcass lean per lifetime
Pig meat production per sow
Litter size Weaning weight Growth rate

Non-productive days Mortality Feed efficiency
Litters/sow/year Growth rate Lean and fat deposition
Management/husbandry Feed efficiency Health status
Health status Management/husbandry
Management/husbandry
Sow Weaner Grow - Finisher
Figure 1. Primary factors affecting pig performance.

114 The Premier Pig Program™
Indeed, it is important to establish target levels of A major feature of the program has been the
performance for each stage of production, so that publication of a manual, which details the appropriate
producers can benchmark the performance on their nutrition and management of the pig at all stages of
own farm relative to industry standards. Suggested production. Information is provided on nutritional
indices for the breeding, weaner and grow-finish needs and on those factors that influence them.
animal are listed in Table 2. Differences in genetic potential, health status, housing
If performance is below expectation, then it is and environmental conditions all influence animal
important to identify those factors that limit performance and these must be considered, as well
performance and to take appropriate actions to avoid as the requirements of the market. Fortunately, our
loss of productivity. This is the aim of the Premier knowledge of the biology and nutritional physiology
Pig Program™. of the pig has advanced sufficiently in recent years
to allow these to be considered in the development
of any feeding and management strategies to meet
Objectives of the Premier Pig Program™ individual needs and animal circumstances.
The manual also suggests target levels of
The Premier Pig Program™ has been developed to performance so that producers can benchmark their
provide independent technical information and farm’s performance against industry standards. It
support appropriate to all sectors of the pig industry discusses what intervention strategies and actions can
worldwide. Although many factors influence be taken on-farm if performance falls below
financial performance, the key factor influencing the expectation. In this way both production and
efficiency and cost of production is nutrition, with economic efficiency can be improved.
the cost of feed representing 60-70% of total The Premier Pig Program™ and the manual cover
production costs in many countries. Understanding the following areas:
the nutritional needs of the pig at all stages of growth
and providing the correct diets and nutritional • Nutrients in the diet
management is fundamental to efficient and profitable
production. • The breeding pig: sow and boar
The major objectives of the Premier Pig Program™ • The weaner piglet
are therefore:
• The grower/finisher pig
• To provide user-friendly technical information • Meat quality
and support to all sectors of the industry
• Environmental issues
• To suggest target objectives for modern pig
production
Nutrients in the diet
• To compare actual and target levels of
productivity and identify areas of concern The objective is to provide a reference base to explain
the major roles of energy, protein, amino acids,
• To propose actions and solutions that can be taken minerals and vitamins for the normal functioning of
on-farm to enhance performance the animal’s metabolism and for the maintenance of
• To provide technology to allow long-term good levels of reproduction and growth, as well as
sustainable production its health and well being. Sources of nutrients are
also discussed.
• To help in the application of new concepts and
technologies
Table 2. Performance targets for sows, piglets and grow-finish pigs.
Sow: 24-25 piglets weaned per year

Weaned piglet: 30 kg body weight at 10 weeks of age; Feed : gain <1.5 : 1
Grow-finish pig: 100 kg body weight at 21 weeks of age; Feed : gain <2.5 : 1
120 kg body weight at 23 weeks of age; Feed : gain <2.7 : 1

The breeding pig: sow and boar

The nutritional and management needs of the A major factor limiting the number of piglets
replacement gilt and the gestating and lactating sow produced per sow per year is the number of litters
are discussed, including those factors that influence per sow per year, which is a reflection of the number
them. This allows practical feeding and management of empty or non-productive days. These are expensive
strategies to be developed to optimise and to sustain in terms of lost productivity; and some idea of the
long-term sow productivity. economic loss for different sow herd sizes is provided
Target levels of performance for the sow are in Table 4. Thus, another important objective of the
suggested, as well as the threshold or intervention Premier Pig Program™ is to provide procedures that
levels when action needs to be taken to avoid loss of allow calculation of the cost of lost productivity, such
productivity (Table 3). The nutritional, management as that associated with the number of empty or non-
and healthcare strategies needed to meet these target productive days.
levels of productivity are discussed and the economic The nutritional and management needs of the boar
costs of lost productivity highlighted. are different from those of the sow and these are
discussed. Strategies need to be developed to optimise
Table 3. Key factors influencing sow productivity. semen quality, since this influences fertilisation rate
Parameter Target Intervention and hence, potential litter size. A summary of the
Culling rate, % 35 >42 major factors that influence sow reproductive
Sow parity at culling 6-7 <3, >8 performance are given in Table 5.
Average parity 5 <3, >8
Sow mortality, % <5 >5
Farrowing rate, % 90 <83
Litters/sow/year 2.4 <2.2 The weaner piglet
Wean-mating interval, days 5 >7
Sows mated within 7 days of weaning, % 9 0 <85 Weaning is a major challenge to the young piglet
Empty days/sow/year 12 >20 and represents a critical period in its life. In nature,
Total piglets born/litter 12.0 <11.0
Piglets born alive 11.3 <10.5
weaning is a gradual process that is completed at some
Mean piglet birth weight, kg 1.4 <1.1 10-12 weeks of age. In commercial practice, weaning
Pre-weaning mortality, % <10.0 >13.0 normally takes place at between 14 and 28 days of
Piglets weaned/litter 10.2 <9.5 age, or sometimes later, and is an abrupt process that
Piglets weaned/sow/year 24.5 <21
Pigs sold/sow/year 23.0 <19
has considerable consequences for the piglet,
Piglet weaning weight, kg * (day 23) 7.0 <6.0 involving substantial changes in its metabolic,
Litter weaning weight, kg * (day 23) 70.0 <60.0 physiological, endocrine and immune processes and
Feed/sow/year, tonne 1.20 <1.00, >1.5 greatly affecting its subsequent growth and health
Sow feed/piglet weaned, kg 50 >55
status.
* These are dependent on the age at weaning
Table 4. Example calculation of the economic loss associated with empty or non-productive days.
No. of empty days/parity* 0 10 20 30 40
Litters/sow/year 2.45 2.30 2.16 2.04 1.93
Piglets reared/sow/year1 24.5 23.0 21.6 20.4 19.3
Reduction in piglets weaned - 1.5 2.9 4.1 5.2
Value of piglets2, € - 45 87 123 156
Empty days/sow/year - 23 43 61 77
Penalty/empty day, € - 2.0 2.0 2.0 2.0
Potential losses by sow herd size:
250 sows 11,250 39,000
500 sows 22,500 78,000
1,000 sows 45,000 156,000
*Assumes a parity of 149 days: 114 days gestation + 28 days lactation + 7 days wean-mating
1
Assumes 10 piglets reared/litter
2
Value of piglet = €30
+
Values may change depending on country
Table 5. Suggested feeding and management strategies to optimize The Premier Pig Program™ therefore discusses:
sow productivity.
• The metabolic, endocrine and physiological

1 Select gilts as early as possible; important for acclimatisation changes that occur at weaning and how these may
2 Feed special gilt rearing diet: flush-feed before mating (high be manipulated for the benefit of the piglet
ovulation rate)
• Growth potential and target levels of performance
3 1st mating at: 220 - 230 days of age
130 - 140 kg body weight • How the nutritional needs can best be met and
16 - 20 mm P2 (condition score: 3.0) feed and water management
2nd or 3rd oestrus period
• Ways to enhance appetite post weaning
4 Reduce feed intake in gilts for 3 weeks post-mating (high
embryo survival) • Environmental and housing requirements
5 Then feed to body condition: target condition score at • How to ensure a high immune and health status
farrowing 3.5 (scale 1-5)
• The importance of good management, husbandry
6 If temperature <20°C, increase feed allowance by 4% per and healthcare practices.
1°C below 20°C
7 Increase feed intake over last 4 weeks of gestation to optimise

birthweight and mammary development If the effects at weaning are minimised, then it is
possible to produce a 30 kg piglet at 10 weeks of
8 Reduce feed 1-2 days before farrowing to facilitate
farrowing process
age. The target levels of performance at the different
stages post-weaning needed to achieve this are
9 After farrowing: gradually increase daily intake over first suggested in Table 6.
week; then feed to appetite
10 Good appetite during lactation is critical for high piglet Table 6. Suggested target levels of performance for the piglet
weaning weight post-weaning.
11 Mean requirements during lactation 100 MJ DE (23 Mcal Age Body Feed Growth
ME) and 60 g lysine/day (days) weight intake rate Feed : gain Mortality
(kg) (g/day) (g/day) (g/g) (%)
12 Minimise loss of body weight and condition. Target
condition score at weaning 2.5 (scale 1-5) 21-35 7 - 10.5 250 250 1.0 <3
13 Piglet weaning weight: 7 kg at 23 days. Each 1 kg litter 35-49 10.5 - 17 575 450 1.3 <1.5
growth rate requires 4 l of milk 49-70 17 - 30 900 600 1.5 <1
Overall 7 - 30 620 460 1.35 <2
14 Feed separate gestation and lactation diets:
- Gestation: 13.0 MJ DE (3.0 Mcal ME) and 6 g lysine/kg

- Lactation: 13.5 - 14.5 MJ DE (3.1 - 3.3 Mcal ME) and
The importance of weaning weight and performance
8 - 11 g lysine/kg in the post-weaning period, and their effects on
subsequent growth rate and performance, are
15 Lactating sows need 30-50 l water/day; nipple flow rate
>2.0 l/min
recognised and ways on how to achieve this are
suggested in Table 7.
16 Reduce demand on sow provide supplementary feeding to
piglets, cross-foster or split-wean
17 Reduce empty or non-productive days: each day costs The grower/finisher pig
~ 2.0 €
18 Organic minerals boost reproductive performance. Role The modern pig has a high potential for growth and
of: Se, Cr, Fe and SowPakTM protein or lean gain. Indeed, growth rates in excess
19 Ensure good sow health and welfare. Role of Bio-Mos®;
of 1.2 kg/day and protein gains greater than 200 g/
also good hygiene day have been achieved in the grow-finish period
under ideal conditions. Although these rates are
20 Don’t forget the boar! Semen quality, high fertilisation rate,
high litter size
seldom achieved in practice, the potential for growth
is higher than that currently achieved on many farms
and target levels are suggested in Table 8. If these
levels of growth are not achieved, then extra feed is
Table 7. Suggestions for achieving good piglet performance post- required and the time taken to reach slaughter weight
weaning. is increased. This increases the cost of production as
shown in Table 9.Ways to achieve good performance
1 Maximise weaning weight: target 7 kg at 23 days (appro-
priate sow feeding) are presented in the Premier Pig Program™. A
summary of the major factors that influence the
2 Provide supplementary feeding when sow milk supply is
inadequate or litter size is >10
performance of the grower-finisher pig, and which
are discussed in the Premier Pig Program™, is
3 Pen according to weaning weight and size presented in Table 10.
4 Special care for smallest piglets (or delay weaning)
Table 8. Suggested targets for the grower-finisher pig under good
5 High feed intake post weaning is critical
commercial conditions.*
6 Growth rate must be at least 200 g/day to maintain body fat
reserves Age Body weight Feed intake Growth rate Feed : gain
(days) (kg) (kg/d) (kg/d) (kg/kg)
7 Feeding high quality diets of correct nutrient specification
is essential 56-84 20-40 1.4 0.70 2.0
8 Phase diets to piglets needs and digestive competence 84-108 40-60 1.9 0.83 2.3
108-129 60-80 2.4 0.95 2.5
9 Feed a little and often just after weaning, with sufficient 129-149 80-100 2.8 1.00 2.8
trough space (50 mm/pig) 149-170 100-120 3.0 0.93 3.2
10 Feeder hygiene is critical: clean at least twice per day Overall 20-120 2.2 0.87 2.5
immediately post-weaning
*Assumes a good rate of protein or lean gain, with a P2 value of
11 Wet (gruel) feeding for first few days is beneficial 10-12 at 100 kg body weight and 12-14 mm P2 at 120 kg body
weight.
12 Adequate number of drinkers (10 piglets per bowl): water
flow rate 1.0 l/minute
13 Temperature: initially 28°C; then reduce by 2°C each week
Meat quality
until 20°C. If housed in shelters, provide ample straw
bedding material and creep area Factors influencing both carcass and meat quality are
discussed, including the components of meat quality.
14 Maintain good air quality: do not reduce ventilation to
maintain temperature
The nutritional and managemental procedures that
can be employed to ensure a more consistent product
15 Avoid draughts and wet floors
of good meat eating quality with desirable flavour
16 Do not overstock: each piglet needs 0.20 m2 in conventional and good keeping and hygiene quality are highlighted.
housing, or 50% more if in alternative bedding systems
17 Lighting: leaving lights on for the first few days may help
18 Promote good health and immunity: treat sick animals Environmental issues
promptly
19 Good hygiene, cleanliness and disinfection between batches
Interest in the environment has intensified in recent
is essential years and there is much discussion on how to reduce
environmental pollution from whatever source. With
20 There must be a high degree of stockmanship
regard to pig production, the major concerns are the
Table 9. The predicted cost of reduced growth rate (20-100 kg body weight).
Feed intake Growth rate Days Feed Extra feed* Value1 Extra overhead Total
(kg/d) (g/d) (kg)
2.00 700 114.3 228.6 44.6 8.9 3.4 12.3

2.05 750 106.7 218.7 34.7 6.9 2.7 9.6
2.10 800 100.0 210.0 26.0 5.2 2.0 7.2
2.15 850 94.1 202.3 18.3 3.7 1.4 5.1
2.20 900 88.9 195.6 11.6 2.3 0.9 3.2
2.25 950 84.2 189.5 5.5 1.1 0.4 1.5
2.30 1000 80.0 184.0 - - - -
*Comparisons are made with a pig growing at 1000 g/day and having a mean feed intake of 2.3 kg/day
*Cost of feed € 200/ton. Each extra day costed at € 0.1/day
1
Values may change depending on country
Table 10. Suggestions for optimizing performance of the grow-finish pig.
Target Suggestion for achieving target
1 What is the growth rate of your pigs? Monitor performance regularly

2 Target growth rate (20-100 kg bodyweight) 850 g/day and FCR of 2.5:1
3 Poor growth costs money. Each 50 g/day reduction in growth (20-100 kg) costs € 2.0/pig
4 Optimise lean tissue growth. Higher growth rate, better FCR, reduced P2 = higher carcass
value
1
5 Phase feed according to the needs of the pig. 20 kg: 14.5 MJ DE and 11.0 g Lys/kg (0.76 g/MJ)
2
100 kg: 12.5 MJ DE and 7.0 g Lys/kg (0.55 g/MJ)
6 Overall herd FCR target: 2.80 kg/kg live weight; 3.50 kg/kg How do your values compare?
carcass weight.
7 Good quality diets are essential. Minimal changes of ingredients and low antinutritional factors
(ANFs)
8 Avoid excessive feed wastage. Wastage is expensive and should not be >5%
9 Good bin hygiene is essential to reduce moulds and mycotoxins. Consider mycotoxin adsorbents
10 Wet feeding or wet and dry feeders Better FCR values and growth rates than dry feeding
11 Water is essential: Requirement is 0.1 l/kg body weight: Consumption is 2-3 times higher in hot conditions (30°C)
20 kg = 2 l/day; 100 kg = 10 l/day
12 Adequate number of drinkers 10 pigs/drinker, water flow rate 1.5-2.0 l/min
13 Environmental needs Are pigs huddling? = too cold!
Are they panting, dirty and lying dispersed throughout the
pen? = too hot
14 Do not overstock Pigs need 0.3 (25 kg) to 0.7 (100 kg) m2/pig in conventional
systems; and more in alternative systems
15 Maintain good air quality and ventilation rate. Avoid dust and noxious (smelly) gases
16 Poor health is the greatest barrier to good growth: promote good Reduce stress, treat sick animals promptly
health and immunity.
17 Good hygiene Cleaning and disinfection between batches is essential. All-in-
all-out
18 Do not stress animals when moving. Good handling and transportation will enhance carcass and
meat quality.
19 High degree of stockmanship is required at all times. Positive interaction with animals
20 Staff morale and skills Provide good facilities for staff and ensure proper staff training.
1
20 kg: 3.35 Mcal ME and 11.0 g Lys/kg (3.3 g/Mcal)
2
100 kg: 2.85 Mcal ME and 7.0 g Lys/kg (2.5 g/Mcal)
excretion of excessive nitrogen and phosphorus and workshops dealing with the breeder, weaner and
the emission of noxious gases, such as NH3, CO, CO2 grower/finisher pig. Approximately 1,000 people
and H 2S. Ways to minimise these pollutants are attended these workshops, representing 90% of the
discussed. Australian and New Zealand pig population. The
positive response from and uptake by the industry
was such that the program has now been launched in
Application of the Premier Pig Asia and Europe with considerable success.
Program™ The Premier Pig Program™ has been designed to
continuously evolve. As new research results and
The program was launched in New Zealand and technical data become available, updates to the
Australia during 2002 and 2003 as a series of three manual will be prepared and distributed. In this way,
the manual will be kept up-to-date and relevant. The

overall objective is to provide practical information
in order to ensure long-term efficiency and
profitability of pig production.
Acknowledgement
It is a pleasure to thank Dr. Bruce Mullan, Department
of Agriculture, Western Australia, Mr. Tony Edwards,
A.C.E. Livestock Consulting Pty. Australia and Dr.
Kate Jacques, Alltech Inc. Lexington, KY, USA, for
their contributions to the development and application
of this program.
J. Hedges 121
Successful feed companies in the future

JIM HEDGES
Hubbard Feeds Inc., Mankato, Minnesota, USA
Like everything else, the feed industry has changed. manufacturer is a conglomeration of what used to be
I cannot think of a lot that has not changed. Most of three quite large individual feed companies, namely
our people use notebook computers. The original ones Land O’ Lakes, Farmland Feed and Purina Mills. How
were called portable computers and we joked that did this come to pass? In part it is due to traditional
they should have been called ‘luggable’. The early feed companies failing to supply what the customer
models were about the size of a small to medium- wanted. My theory is that if the major feed companies
sized suitcase and were certainly not light weight. had produced and marketed premixes when the
Wal-Mart has taken the place of Woolworth and K- market demanded the product, it would have been
Mart. They offer it all with one stop, from food to very difficult for the strictly premix companies to have
motor oil to clothes. They evolved with the times; developed and grown. The big traditional feed
and sell good products at very reasonable prices. Wal- companies had the sales force, technical expertise and
Mart has excellent marketing and they recognize dealer network to fill the market need, but were afraid
trends and change with the times and customer needs they would have several mills sitting idle. The moral
and wants. of the story is you cannot make people buy what you
The feed company that succeeds must be as want to sell. At Wayne Feed when we finally
innovative and market-driven as the computer introduced premix in the early 1980s, we actually
industry and a Wal-Mart Superstore. I have always increased market share by not only gaining customers
tried to pattern our work after the electronic industry. with the premix, but by selling more concentrate as
There are similarities, but the electronics companies well. Without a premix, it was easy for the producer
have some advantages. A VCR 20 years ago retailed (potential customer) to dismiss a salesman. Once we
for approximately $780, while today the price is about had a premix to offer, many producers, when offered
$80 and quality is vastly improved. Research in food the option of either premix or concentrate from the
animal nutrition has always been driven toward same company changed back to a concentrate-feeding
maintaining performance while lowering costs. program since many of the producers in the early
Unlike electronic companies, we cannot control the 1980s really were not big enough to conventionally
commodity market and therefore have less control use premix and soybean meal.
over input costs, but we have made great strides. Two One very important point concerning the list of top
examples of ways we have gained control over feed feed manufacturers is that the ranking is based on
ingredients costs is with the enzyme phytase and production capacity, not actual production. In this
through the use of synthetic amino acids. ranking, the North American Animal Nutrition
The January 1903 Feed Management magazine Companies (most of you know them as Akey) is
article on the Top Feed Manufacturers is quite ranked number 52. I dare say there are very few feed
enlightening (Table 1). In 2001 the magazine companies that feed more animals than this one. This
expanded the survey to include leading integrated suggests that bricks and mortar are not necessarily
poultry and livestock producers. In the 2002 edition needed to sell and service a significant portion of the
of the article, only four of the top 10 feed livestock industry.
manufacturers are actually feed companies. The top The 2003-04 Feedstuffs Reference Issue and Buyers
122 Successful feed companies in the future
Table 1. Leading commercial and integrated feed manufacturers1.
Company name & Production Feed States Complete Pelleted Bulk Dealer-sold
headquarters locations capacity mills served (%) (%) (%) (%)
(thousand tons/yr)
Land O’Lakes Farmland 12,881 131 48 64 54 71 52

Feed/Purina Mills Arden
Hills, MN
Tyson Foods 12,000 40 100 78 100 0
Rogers, AR
Cargill, Inc. 9,500 79 49 55 65 65 60
Minneapolis, MN
Smithfield Foods, Inc., 4,500 100 85 100 0
Smithfield, VA
ADM Alliance Nutrition, 3,200 45 42 50 57 67 59
Inc. Quincy, IL
Pilgrim’s Pride Corp. 3,190 100 100 100 0
Pittsburg, TX
Perdue Farms, Inc. 3,016 12 100 100 100 0
Salisbury, MD
Gold Kist, Inc. Atlanta, GA 3,000 12 5 100 91 100 0
J.D.Heiskell & Co. 2,800 6 65 5 95 5
Tulare, CA
ConAgra Poultry Co. 2,180 100 100 100 0
Duluth, GA
1
Lobo, 2003
Guide lists the top 10 feed manufacturers and is more nothing to do with sales success. Actually in today’s
pertinent for this paper (Table 2). Again, the ranking market it could be argued that milling capacity could
is based on manufacturing capacity, not sales. Ridley be a negative for a feed company. It is extremely
Inc. is ranked No. 9, but if this ranking was based on difficult to be cost competitive and profitable while
animals fed, there would be very few feed companies maintaining a lot of mills operating at 25 to 50%
ahead of Ridley. capacity.
As a normal rule, feed manufacturers cannot make
Table 2. Top US feed companies, based on manufacturing capacity, feed as cheaply as a large livestock producer. A
mid-20031. livestock producer with a modern mill can make
Company Annual manufacturing significantly more tons of feed per hour than a feed
capacity, million tons manufacturer. The livestock producer may have only
1. Land O’Lakes Farmland Feed LLC 12.5 10-20 base formulas of meal feed made in 3-5 ton
2. Cargill Animal Nutrition 8.0 batches in bulk while the commercial feed company
3. ADM Alliance Nutrition 3.2
4. J.D. Heiskell & Co. 2.8 mill may have 200 formulas. Also, most feed
5. Westway Feed Products 2.0 companies are multi-species, which increases the
6. Kent Feeds 2.0 complexity of production. Being multi-species not
7. Southern States Co-op 1.7 only affects the number of formulas, but can
8. PM Ag Products 1.7
9. Ridley Inc.2 1.6 significantly affect sequencing and even the raw
10. Agway 1.0 material inventory. For several years now Ridley has
1
Feedstuffs 2003-2004 Reference Issue & Buyers Guide had a policy to not inventory any ruminant meat and
2
Includes Canadian feed tonnage bone meal or even plasma of ruminant origin. With
these issues on ingredients, products, labor, taxes etc.,
Premix companies hardly even make it into these the large commercial feed mill cannot compete on a
rankings, but again if this were based on animals fed, price per ton basis with single species or a simple
several of these companies would be near the top. meal-producing mill.
These rankings are interesting, but capacity has Given these constraints, how can feed companies
J. Hedges 123
grow and be successful? Most feed companies are made considerably less painful with effective training
trying to grow by acquisition. Definitely this can and product positioning. When Ridley bought the
accomplish the goal, but is not without risk. When a Wayne Feed brand we did not force the Wayne dealers
company that has been a competitor for years is to convert to the Hubbard brand immediately. We
purchased, differing company cultures usually clash. updated the products and said that as long as they
Hubbard has gained significant new business from sold the existing brands in good volume to keep on
the gridlock that can occur with these acquisition selling them. As dealers undergo more and more
patterns in the marketplace. Slow decision-making training sessions and have updated products to offer,
and poor service while two companies decide who is they will change to new products without excess
in charge has enabled us to obtain huge volumes of trauma. They must be confident the changes benefit
business. One customer asked for a loan and he never the customer, not just the parent company.
received a response. He told me he could understand While product brands are important to dealers and
a ‘no’, but no response at all after being a customer small to medium size producers, they are not the main
for 7-8 years drove him away. In another instance we issues with large producers. For all your customers,
gained 2000 tons of new business per month when the products must meet a need either through
one company bought another. Management, in many technology advances or by providing something the
cases, does not seem to realize that change cannot be customer cannot make himself. Unless your goal is
forced on people. If a customer, either a feed dealer toll milling, complete grow-finish or sow diets are
or direct account, is forced to change their way of not a growth option for most of today’s feed
doing business, why not change completely to another companies. For this segment of the market, it is best
company? These customers (dealers) perceived their to supply some type of premix and possibly
original brand being de-emphasized and felt ingredients. To prevent this from being a complete
steamrolled into the new brand. On top of this were bid situation, you need excellent technical support
poor service and poor communication. The result was and formulation help. The diet density and feed
a change to a company that wanted their business budgets are a part of what the successful feed
and supported them. You can buy a company, its mills, company must also supply. For the megaproducers,
trucks and offices, but its dealers and direct customers products are somewhat different for each customer
do not automatically follow. Companies spend a depending on the customer’s feed milling capabilities.
tremendous amount of time and money developing a We have customers who require a 10 lb/ton vitamin/
company culture and image. It is neither easier nor a trace mineral premix sent to some of their toll mills
good idea to discard it without recognizing its value and a 60 lb/ton base mix for other mills. We have
in the marketplace. When the old Hubbard bought other customers with sophisticated mills that we
companies they made the transitions relatively supply with a 2 lb/ton vitamin/trace mineral premix.
successfully, keeping the original brands until time If a livestock producer has a $10 million feed mill,
and market changes deemed it good management he will not be buying a 60 lb/ton base mix. There is
practice to bring the brands into the parent company. room for both segments; you do what the customer
It is critical to understand that we can buy an needs and respect his milling capabilities.
employee’s time; or a business; but simply ‘buying’ One customer need that a feed company can meet
is not effective. Only voluntary actions, those which is in supplying feed milling technology. Most
cannot be bought, are ultimately effective. Trust, for producers need help in this area as well as in
example, must be earned. You earn trust by monitoring ingredient quality. They also need help
consistently doing what is right for the long term, in monitoring finished feed quality. There are a lot of
not just for the next quarter’s results. Customers need large dealers or co-op mills that have tremendous
time to adjust to the new company, because they make grain handling capability and milling capacity, but
decisions for their own reasons, not ours. You grow do not have the nutrition technology that progressive
the business by infusing capital and technically pork producers need. This represents another market
improving the products. Making more than one brand segment for the traditional feed company, but it differs
of feed at the same plant admittedly causes some in that these are not the same products the company
difficulties, but the dealers will be somewhat more supplied years ago. In these situations we supply the
tolerant as long as service is acceptable and feed vitamin/trace mineral premix or base mix and do the
quality is good. Brand names are important to dealers; finished diet formulation while the toll millers do the
and if you are to change the brand name, the dealer grinding, mixing and delivery.
needs time to accept this change. The change can be The starter feed area is definitely an area where there
is opportunity for differentiation from the adjustments needed to ensure that pigs receive 20 g
competition. The early-stage complex diets are of lysine per day. This is an old concept, but many
difficult to manufacture. In addition, most swine people do not understand it to this day. The data in
producers do not have storage capacity for all the Figure 1 show how adjusting the diet can alleviate
ingredients needed for these feeds, and certainly do part of the performance depression that can occur in
not possess the technology to make them. restricted feed intake situations. The optimum lysine
level for these finishing hogs when fed ad libitum is
0.61%. When the hogs are restricted to 85% of ad
Research libitum intake, the optimum lysine level is 0.85% of
the diet. A reduction to 85% of ad libitum is not an
From a technical perspective, this is a key to a academic exercise. Our large customers running mills
successful future. In the past, feeds and feed 20 hrs a day do not have to run that hard in July and
ingredients could be sold based on testimonials, but August, when feed intake falls due to heat stress. Not
this approach no longer suffices. Statistically valid all of the reduction in performance due to heat stress
trials must be conducted in facilities and with genetics can be overcome, but it can be alleviated to some
similar to those of your customers. We made mistakes extent by diet manipulation.
years ago with facilities that were ‘ideal’ for research,
but the results did not apply to the field. The data in Table 5. Effect of feed intake on daily lysine intake in finishing hogs.
Tables 3 and 4 show this very well. Notice the huge
Daily feed Lysine/day Lysine needed Approximate
difference in feed intake between the two groups. Pigs intake (lb) (g) to supply 20 g/day crude protein
housed in the commercial unit stocked at 45 pigs/ (%) (%)
pen consumed 5.1 lbs/day compared to 6.2 lbs/day
for the research facility pigs stocked at 10 pigs per 7.0 20.6 0.65 14.0
6.5 19.2 0.70 14.5
pen. Feed conversion is not greatly different, but the 6.0 17.7 0.75 15.5
data for the research-stocked pigs indicated these pigs 5.5 16.2 0.83 16.5
needed only 0.49% available lysine (the lowest level 5.0 14.8 0.91 17.5
fed) and the commercial-stocked pigs need 0.65%
Hedges (1994), unpublished
available lysine! Our research facilities once had a
limited number of pigs per pen. The performance of
The reference data in Table 6 are accurate under the
the pigs in these facilities was great, yet the salesmen
conditions in which the pigs were fed, but the diet
and dealers complained the commercial hogs stalled
densities recommended would probably not give
out and did poorly in many cases. The diets were
optimum performance in a large commercial unit. The
correct, but only under the circumstances in which
swine producer does not have the luxury of removing
they were fed.
the pigs that are too big or too little; the producers
Table 3. Research pens, 10 pigs/pen.
feed all the pigs.
Early in my career the salesmen accused me of
Avail. lys, % 0.49 0.65 0.77 0.88
Tot. lys, % 0.60 0.77 0.90 1.04
doing ‘Ivory Tower’ research. Actually, they were
St wt, lbs 136.7 140.6 146.1 141.7 correct. There has always been a bit of disconnect
End wt, lbs 236.8 241.6 247.1 240.7 between university recommendations and the feed
ADG, lbs 2.32 2.35 2.34 2.30 industry. There is no need to have a big debate over
ADFI, lbs 6.26 6.41 6.28 6.10
F/G 2.68 2.73 2.70 2.66
this. I am not trying to criticize the excellent scientists
P2 0.77 0.76 0.78 0.77 that did this work, I just do not think you should rely
Campbell (1995)
on university tables for all nutrient recommendations.
The point is, if you are to have industry-leading diets,
Table 4. Commercial pens, 45 pigs/pen. you must develop them yourself. From my
Avail. lys, % 0.49 0.65 0.77 0.88 perspective, the role of the university is to do the basic
St wt, lbs 119.2 119.7 120.6 119.2 work. I have formulated swine diets for 25 years using
End wt, lbs 191.8 197.5 196.6 195.5 digestible amino acid values and I did not generate
ADG, lbs 1.81 1.94 1.90 1.91
ADFI, lbs 5.14 5.11 5.13 5.13
any of the values I used. All the digestible amino acid
F/G 2.83 2.63 2.70 2.69 data was generated by university research; which we
P2 0.53 0.49 0.50 0.50 applied and made practical. We use the enzyme
Campbell (1995)
Table 5 illustrates the importance of feed intake
J. Hedges 125
2.4
Ad libitum feeding (100%)
2.2
2.0
0.61%
ADG (lb/day)
1.8
Restricted feed (85%)

1.6
1.4 0.85%
1.2
0.45 0.60 0.75 0.90 1.05
Dietary lysine (%)
Figure 1. Ad libitum vs restricted feeding: effect on required dietary lysine concentrations (adapted from Coma et al., 1995).
Table 6. Suggested dietary amino acid and protein allowances for swine fed corn-soybean meal diets at various bodyweights1.
Barrows and gilts Barrows Gilts Sows

Item 20-45 45-80 80-120 120-170 170-220 220-280 120-170 170-220 220-280 Gestation2 Lactation3
Amino acids, % of diet4

Lysine 1.20 1.00 0.90 0.75 0.68 0.58 0.84 0.73 0.62 0.55 0.92
Met+Cys5 0.72 0.62 0.56 0.48 0.43 0.37 0.53 0.47 0.39 0.30 0.44
Tryptophan 0.21 0.18 0.16 0.15 0.13 0.11 0.16 0.14 0.11 0.11 0.16
Threonine 0.78 0.67 0.60 0.52 0.48 0.40 0.58 0.51 0.43 0.45 0.58
Arginine6 0.50 0.36 0.33 0.14 0.13 0.10 0.15 0.14 0.11 0.00 0.50
Histidine 0.39 0.32 0.29 0.24 0.21 0.19 0.27 0.23 0.20 0.17 0.35
Isoleucine 0.72 0.60 0.54 0.45 0.40 0.35 0.50 0.44 0.37 0.31 0.50
Valine 0.81 0.68 0.61 0.51 0.47 0.39 0.57 0.50 0.42 0.36 0.76
Leucine 1.20 1.00 0.90 0.75 0.68 0.58 0.84 0.73 0.62 0.45 0.96
Phe+ Tyr7 1.14 0.95 0.86 0.71 0.65 0.55 0.80 0.70 0.59 0.50 1.00
Protein8 20 18 16 14 13 12 16 14 13 12 17
1
Bodyweights are listed in pounds. The suggested protein and amino acid allowances assume that corn-soybean meal diets are fed, and that
the diets contain 1,560 and 1,500 kcal/lb DE and ME, respectively. Ideal ratios (relative to lysine) were used in calculating amino acid
requirements for market pigs considered to be of high-lean genetics.
2
Developing boars should receive 0.90 and 0.75% lysine during the grower and finisher phases, respectively. Specific requirements for the
mature boar have not been established. A diet that is adequate for the gestating gilt should be adequate for the mature boar.
3
A minimum of 28 days lactation is assumed.
4
These allowances were determined with fortified corn-soybean meal diets. Substitution of other grains for corn or other protein sources for
soybean meal should be made on a digestible amino acid basis.
5
Cystine can supply up to 50% of the requirement for sulfur amino acids. Thus, the values given here represent a Met + Cys allowance.
6
Dietary arginine is not essential for gestation.
7
Tyrosine can satisfy 50% of the need for total aromatic amino acids (phenylalanine + tyrosine).
8
These levels of crude protein in a corn-soybean meal diet will generally meet the amino acid requirments, although some supplemental
lysine may be needed.
Source: Baker et al., 2003.
phytase, the efficacy data on which was generated feed formulations. The university diet is much simpler
by the universities in their facilities with graduate and represents the optimum formulation for a
students available. The feed industry nutritionist university nursery with 10 pigs/pen. The other feed
should be able to take this basic work and figure out companys’ diets were probably also developed in
how it can be applied in the commercial world. The small pen nurseries. These trials are more for in-house
vast majority of our research is conducted in barns with benchmarking of performance versus the competition
1000 or more pigs, in industry standard pen sizes. This than for selling. They are not great sales tools;
allows the pigs to experience the same space, social and producers’ interest might be aroused, but normally
ventilation factors they would encounter in normal they do their own tests. In another example Table 8
commercial units. These facilities are all commercial shows what complex diets do for uniformity of gain.
facilities with modifications for weighing both feed and Very healthy pigs with minimal stress can grow quite
pigs accurately. well with less complex diets, except this is not the
We have a 1000-head capacity research nursery that normal situation in the commercial world.
is a contract barn for a large producer. This barn has The grow-finish research barns are actually
pen scales, six feed tanks instead of one and a Mosdal commercial 1000 or 1200-head barns modified with
feed cart with built in scales. The pigs are weaned at scales for weighing the hogs and equipment to
approximately 20 days of age and transported 12 to measure feed intake. Depending on the facility, the
15 hrs to this nursery. When the pigs arrive they are hogs are housed 20 to 32 pigs/pen with normally 7.5
weighed and allotted to treatments, without an ft2/hog. All dietary treatments are replicated 6 to 8
adjustment period. The diets are formulated to meet times. A couple of these barns have dual water lines
the nutritional needs of pigs housed at 22-25 pigs for studying water treatments as well. In total, Ridley
per pen. The data in Table 7 show the performance has two 1000-head conventional nurseries for
difference that can be obtained with different starter research and three wean-to-finish barns of 1000 head
Table 7. Starter diet comparison report sheet from a contract nursery.

HFI New HFI Brand A Brand B Brand C University
Start wt 11.01 11.01 11.06 11.08 11.03 11.03
Day 4
ADG, lbs 0.71 0.71 0.7 0.55 0.63 0.61
F/G 0.62 0.62 0.63 0.62 0.64 0.68
ADFI, lbs 0.43 0.43 0.43 0.33 0.39 0.39
Day 4 wt., lbs 13.86 13.86 13.85 13.29 13.55 13.46
Day 4-11
ADG lbs 0.63 0.63 0.37 0.55 0.62 0.49
F/G 1.21 1.21 1.54 1.19 1.20 1.32
ADFI lbs 0.76 0.76 0.55 0.65 0.74 0.64
Day 11 wt., lbs 18.29 18.29 16.45 17.16 17.87 16.91
Day 11-25
ADG lbs 0.98 1.08 1.02 1.08 0.96 0.82
F/G 1.55 1.37 1.39 1.37 1.48 1.54
ADFI lbs 1.52 1.48 1.41 1.47 1.42 1.26
Day 25 wt., lbs 32.06 33.35 30.67 32.26 31.34 28.42
Day 0-39
ADG lbs 1.06 1.09 1.02 1.03 1.02 0.94
F/G 1.52 1.48 1.44 1.37 1.5 1.48
ADFI lbs 1.61 1.61 1.47 1.41 1.52 1.39
Day 39 wt., lbs 52.16 53.49 50.88 51.08 50.67 47.7
Feed budget
Nursery 1 1 1 0.5 1 1 1
Nursery 2 3 3 1.5 3 3 3
Nursery 3 12 12 12 12 12 12
Nursery 4 50 50 50 50 50 50
Nursery 5 0 0 0 0 0 0
J. Hedges 127
each. Two are for demonstration work and one is Dealers have a business history and a brand they have
equipped for multiple treatments and multiple promoted, and tend to have a higher tolerance of
replications. ‘average’ service than a direct account. In many cases
Ridley also has three conventional 1000 head grow- a direct account communicates personally with the
finish buildings and two 1200-head barns. All of these order staff and there is no buffer. Your order staff must
are equipped for multiple treatment, multiple be trained to deal with direct customers in a
replication trials. Ridley conducts 75-80 trials per knowledgeable, courteous and efficient manner.
year. These normally are nutrition trials, but we also Ridley has been adjusting to this change, although
conduct trials involving management issues. there is still room for improvement, as at every
company. It seems obvious, but orders must be ready
when the customer expects them. Pellets must be
Customer service excellent and not 10% fines. With many big systems,
the pellets are handled several times. If the customer
Exceptional customer service is essential for a is large enough to have his own pellet mill, you had
successful company. Most traditional feed companies better deliver significantly better pellet quality than
have always been only one step away from the end he can make. The successful feed company must have
user. Selling direct is different. Dealers buffer the excellent production people and truck drivers.
company from the end user. Animals eat 24/7. I know As a technical salesman it is much easier to sell
from personal experience that plants and order desk feed from a plant you know will make the feed
personnel do not always understand urgency. In correctly. It is one thing to design or formulate a feed
fairness to the plant personnel, some of our plants on paper, but it must be mixed properly. A mistake
are nearly at maximum capacity and it can be quite a with a 5 lb/ton premix is a big mistake, because it
juggling act to get all the feed out on time. The time will be multiplied many, many times. Proper quality
frame is shortened without the dealer’s inventory assurance is critical; particularly when the customer
serving as a buffer between you and the end user. must perceive that your service is better than the
Table 8. Determining the differences in nursery feeding programs (Location: Contract nursery).
Average weaning FC1 Univ. HFI FC2 FC3 FC4 FC5
weight (lbs) Spec.
Pens 9 9 9 9 9 9 9
Total no. of pigs 92 92 92 92 92 92 92
Starting weight, lbs 11.03 11.03 11.01 11.08 11.08 11.09 11.06
Pigs dead or <40 lbs at 39 days 9 15 5 10 3 17 8
Rep 1a 8.97 45 67 22 33 01 45 33
Rep 2 8.80 13 26 01 13 01 37 13
Rep 3 8.62 12 25 12 25 12 37 01
Rep 4 11.49 27 01 12 12 12 12 12
Rep 5 11.49 01 14 01 14 01 14 14
Rep 6 11.49 13 37 01 13 01 25 25
Rep 7 12.66 01 01 01 17 01 01 01
Rep 8 13.80 01 01 01 01 01 01 01
Rep 9 12.17 01 14 14 01 14 37 01
1
Number of times ranked 1b 4 3 5 2 6 2 4
2
Number of times ranked 2 1 0 3 1 2 1 1
3
4
5
6
7
Total Sum (#*Rank) 24 36 15 29 14 39 21
FC = Feed company
a
number of pigs that died in a given rep and treatment
b
weighted average, the number of times a diet was ranked 1,2, etc. in ADG. HFI five times had best gain, three times second best, one time third, and
one time fourth: 5x1+3x2+1x4=15; the lower the number the better.
competitor’s. Researchers Keiningham and Vaura and correcting problems. The feed industry used to
(2001) discuss customer satisfaction in terms of give away a lot of caps and jackets; and while this is
delighted customers rather than just merely satisfied not a big deal with large customers, a shooting outing
customers. A review of this book suggests that or fishing trip can be a great rapport builder. People
anywhere from 60 to 85% of customers who switched buy from people. It is still a people business whether
firms would have been classified as satisfied by the customer has 100 sows or 100,000 sows.
generally accepted market research measurement Management’s role is to be a leader, a point that
tools. The successful organization needs to seems obvious but is often missed. You do not have
dramatically increase positive customer experience, to like the feed division general manager, but it is
virtually eliminate the negatives and drive customers important people respect him. The head person needs
to new levels of repeat purchasing, loyalty and sheer to receive and respect feedback from the people doing
delight. I have often said if we could make excellent the selling. He also needs to get out and work with
pellets, deliver them on time, put them in the correct enough customers to know the market needs. The big
bin every time, we could not make all the feed we companies that have failed basically ignored the
could sell. You do not lose customers, you drive them market and tried to keep on doing what had made
off. When was the last time you complained about them successful. The successful company will have
great service? focused, energetic and creative people who can sell
without two or three layers of people to tell them
what to do. Feed companies used to have huge
Management bureaucracies, but they can no longer afford them. In
today’s business environment the big don’t eat the
In the almost thirty years I have been in the feed small; the fast eat the slow.
industry I have worked for about 10 feed division The ideal feed company division head would have
managers. An excellent head of a feed division grows the following characteristics:
the business, a mediocre one does not hurt the
business and a bad one destroys the business. The Be a leader. People are good followers if they
absolute key element needed for a successful business have someone they respect to follow. My brother
is people. Without excellent people you have a has a saying “you can lead me a long way but
mediocre or failing company. you are not going to push me very far”. In my
The mistake I have seen most often is too much career I have had leaders that tried to motivate
management. The really good salesmen are independent by intimidation and it did not work well. To be a
thinkers and do not function well in a rigid structure. good leader you have to listen, support and
In today’s business climate with declining margins, encourage your people. Get them the tools they
the successful company must have mainly Indians need and stay out of the way. Too many general
and very few Chiefs. If you are not selling or managers overrate themselves; they cannot make
supporting customers, there is no need for you. the company successful by themselves, without
Anyone that has contact in any way with customers good people they will fail. Poor leaders cannot
needs to have good people skills. Customers have attract good people.
enough options to easily avoid buying from people
they perceive as difficult. Very little in the company Listen to your people. I have a fair reputation of
is necessary until something is sold. When I hire a being a good product developer, but it has not
PhD for my group I ask them if they can sell. If they been hard. The customer will tell you what he
cannot sell, I do not hire them. The PhD nutritionist needs, you must listen and act. The employees
is a technical salesman in today’s business are management’s customers. It is so obvious that
environment. We have accounts where the only person I guess it is hard for management to see at times,
involved is the technical person and the order desk but all they need to do is listen to the people that
personnel at the plant. This is partly because the are in front of the customers and actually making
traditional sales force does not have the confidence sales. Having said this, one of the mistakes feed
to call on anyone with 20,000 sows or more. A good companies make is giving salesmen credit and
account manager can be helpful. They can play a key commission for a huge sale they did not make
role in customer support, helping with communi- simply because it is geographically in their
cating and listening to the customers needs, concerns territory. Just because the animals are in a
J. Hedges 129
salesman’s territory does not mean he should get on people to sell. A lot of salesmen drive by larger
credit, but many times sales managers want to operations because they are not confident enough to
protect their salesmen. We cannot afford this call on them. This cannot be tolerated if you plan to
anymore. Territory selling is changing to succeed. Excellent research, product quality (both the
customer selling, and in some cases, team selling. formula and the production of it), superior service,
and acceptable pricing are all tablestakes. Some
Trust your people. Management must accept that
company is going to supply the nutrition to the
they have employees that want to grow the
livestock industry; and I think it will be a feed
business just as much as they do; and
company. I do not think that ingredient companies,
management should listen and act when these
amino acid, vitamin or dical suppliers, etc. have the
employees request people or equipment to grow
technical expertise or service capability to meet
the business.
livestock producer needs.
Be customer friendly. If in doubt, do what is right It will be quite interesting 15 years from now to
for the customer and then work hard to make it see how the feed industry looks. One area of concern
right for the company. is where are we going to get trained technical people?
Be able to meet with customers and earn their The old power house universities that produced a lot
respect. of good research and trained students in the past have
dwindled to a very few. If we can get good people, I
Be innovative, be able to think outside the box. It think most of the good applied research of the future
seems most feed companies think the only way will be through alliances between the successful feed
to grow is by acquiring another feed company. company and their customers. The large customer and
What is wrong with staffing up and attacking the the feed company need to form such alliances. Our
market with excellent people? Very few feed customers are not just those we sell to and profit from.
companies are operating their mills at full They are partners; and need to be thought of as such.
capacity. If you cannot grow your existing It is far too difficult to earn the trust and respect of an
business, what makes you think you can operate account on any other basis. The feed company’s
an even larger business? objective is to help make the customer profitable. If
Do not be afraid to invest capital to grow the the customer fails, the feed company fails also.
business. Possibly dedicated feed mills for each Most of this may just seem common sense; but
species group is the way to go. With a special knowing and doing are two different things. The feed
purpose mill things can be accomplished from a business is really quite simple; and maybe that is the
production standpoint that are not possible in a problem.
full line plant.
References
Conclusions
Anon, 2003. Feed marketing and distribution.
I think the feed company that really succeeds in the Feedstuffs, September 17, 2003, p. 4.
future will have to quit thinking like this is still 1985. Baker, O.H., R.A. Easter, G.R. Hollis and M. Ellis.
The feed business has changed, but I am not 2003. Swine health & nutrition, dietary nutrient
convinced the management at most feed companies allowances for swine, Feedstuffs, September 17,
realize this - or if they have, they do not know what 2003, pp. 24-29.
to do about it. You will need specialists, not
Campbell, R. 1995. New technologies for the pig
generalists. I have never understood the logic of trying
industry. Jefo Nutrition Inc. Pre-Conference
to get salesmen to be good at all species. Actually,
Symposium, May 18. pp. 18-31.
they are not and they gravitate to the species they are
most comfortable with and the one they think they Coma, J. Carrion and D.R. Zimmerman. 1995. Effect
can be the most successful at. The operations are of feed intake on the lysine requirement of pigs at
bigger, fewer salesmen may be needed but these two stages of growth. Iowa State University, 1994
individuals need to be of higher quality and be more Swine Research Report, January 1995, pp. 49-52.
highly educated. Keiningham, T.L. and T. Vaura. 2001. The Customer
One thing that has not changed is you have to call Delight Principle: Exceeding Customer’s
Expectations for Bottom-Line Success. McGraw-

Hill Trade.
Lobo, P. 2003. Top feed manufacturers improved
insight, Feed Management, January, Vol. 4, Number
1, p. 6.
D. Mahan 131
The role of selenium and Sel-Plex® in sow reproduction

DON MAHAN
Department of Animal Sciences, The Ohio State University, Columbus, Ohio USA
Introduction
The demonstration of selenium (Se) deficiencies in Although this dietary level has not been recognized
the field was first noted where occurrences of sudden as the dietary requirement for all swine production
deaths, white muscle degeneration, liver necrosis, phases (NRC, 1998), it is the level commonly
mulberry heart, gut-edema, uncontrollable diarrhea, incorporated into most swine feeds by many feed
and increased fluid in the pericardial sac were companies. Over the past two decades, the continued
predominant observations in weaned pigs. The occurrence of vitamin E/Se deficiency in sows and
condition was not as prevalent with reproducing gilts pigs has not been completely abated by adding higher
or sows, for during this period of the swine industry dietary levels of these nutrients, particularly vitamin
most sows were housed outdoors on concrete pads or E, and thus different forms and levels of vitamin E
in pasture lots. As complete confinement for and Se have been more actively pursued.
reproducing animals became more common,
increased occurrences of the deficiency in the adult
animal began to emerge. Hyposelenosis in the female Selenium deficiency in the sow
was generally accompanied by prolonged farrowing
times, reduced fertility, increased number of Feeding a semi-purified diet deficient in vitamin E
stillbirths, poor milk letdown during the initial days and Se resulted in a lower litter size and a low number
postpartum, higher incidence of MMA (mastitis, of sows farrowing (Mahan et al., 1974). Sows fed
metritis, agalactia), lower litter sizes, and greater the diet and their piglets were extremely weak at
losses of sows from the herd. Although other factors parturition. None of the sows fed the semi-purified
can also contribute to these clinical signs, current vitamin E/Se deficient diet completed a second
research has confirmed that these observations are parity, but three died during their second pregnancy.
related to Se deficiency. Wilkinson et al. (1977) and Within their reproductive tracts there were both
Mutetikka and Mahan (1993) have confirmed that normal and abnormal fetuses, suggesting that tissue
sows housed on pasture have higher sow serum Se atrophy and oxidative damage had occurred. Sows
and α-tocopherol concentrations than a similar group fed the deficient diet also had elevated serum
of gilts fed in confinement. glutamic oxaloacetic acid transaminase (SGOT)
With the discovery by Rotruck et al. (1973) that enzyme activities by 30 days of gestation, values that
Se was part of the enzyme glutathione peroxidase increased to yet higher levels as gestation progressed.
(GSH-Px), and the recognition that supplemental This enzyme is indicative of cellular damage of sow
dietary Se prevented the occurrence of the or fetal tissue. Consequently, these results suggest
deficiencies under field and research conditions, that diets deficient in Se will deplete sows of body
inorganic Se (sodium selenite or sodium selenate) Se and vitamin E reserves, will result in lower litter
was approved by FDA (1974). Later research using sizes, reduced sow parturition performance, and
selenite as the Se source demonstrated that the weaned increased fetal atrophy and death.
pig required 0.30 ppm Se in the diet. This supplemental Practical diets fed to reproducing sows generally
level was subsequently approved by FDA (1987). have not shown evidence of reproductive failure as
132 The role of selenium and Sel-Plex® in sow reproduction
readily as when the semi-purified diet was fed. A the accumulation of oxidative end-products in
non Se-fortified corn-soybean meal diet fed to gilts conceptus products. Because the amount of Se
and sows resulted in a lower litter size by parity 2, transferred to the fetus and GSH-Px production is
whereas sows fed the same diet fortified with Se evidently relatively low in the developing fetus, the
(selenite) at 0.10 ppm had a larger litter size at parity Se status of the female could be critical to fetal
2 (Mahan et al., 1974). Feeding low Se grains or survival. The transfer of Se to the fetus is not only
diets low in vitamin E and Se, particularly grains of dependent upon the Se status of the female, but also
a high moisture content, has shown lowered upon the source and level of dietary Se provided to
reproductive performance and higher incidence of the dam during gestation.
MMA (Whitehair and Miller, 1985). Most of the
published sow data has thus shown a greater effect
of supplemental Se in later reproductive parities, with Postnatal effects
beneficial responses to supplemental Se more in older
than in younger sows. This supports the concept that When the female is deficient in vitamin E and Se,
sow body depletion occurs with continued the neonatal pig may experience various deficiency
reproductive demands, and that the deficiency onset signs including iron (Fe) toxicosis. Iron toxicity in
is exacerbated with age (Nielsen et al., 1979; Chavez, piglets is a condition where Fe administration to the
1985). Sow body composition research (Mahan and pig becomes ‘free’ in the circulatory system and the
Newton, 1995) has demonstrated that sows with released free Fe exacerbates oxidative reactions thus
heavier litter weaning weights had lower body tissue damaging tissue membranes, ultimately causing the
Se concentrations or a greater loss of several trace death of the pig. Loudenslager et al. (1986)
minerals than sows of a lower productivity. demonstrated that neonatal pigs from sows fed diets
low in Se and vitamin E had a poorer antioxidant
status than those pigs from sows fed diets fortified
Effect on the fetus with adequate levels of vitamin E and Se. Research
with the young chick has also demonstrated that
When the dam’s diet is low in Se, fetal liver Se has chicks fed a Se-deficient diet developed a necrosis in
been shown to decline during the gestation period the muscle tissue suggesting that muscle atrophy may
(Piatkowski et al., 1979). Recent research by occur in the Se-deficient neonate (Bartholomew et
Hostetler and Kincaid (2004) has additionally al., 1998).
demonstrated that oxidative damage occurs in both Because colostrum is high in vitamin E and Se
the sows and fetal liver tissue when a low Se diet is concentrations, it is a major source of these nutrients
fed to the pregnant animal. This was indicated with for the neonatal pig. Consequently, sows with a higher
higher concentrations of the lipid peroxide tissue status of these nutrients would be expected to
malondialdehyde (MDA) and H2O2 in fetal livers, but transfer more through the mammary secretions,
GSH-Px activity of fetal liver was not affected. Their whereupon the neonate would be better able to cope
results also demonstrated that liver GSH-Px activity with the oxidative effects postnatally. Milk is also a
was substantially higher in adult female pigs than in good source of Se and vitamin E, but its concentration
their fetuses. When the Se deficient diet was fed, the is somewhat lower compared with colostrum.
oxidative end products accumulated in the fetus. The Consequently, the weaned pig’s vitamin E and Se
pig fetus has a relatively low production of GSH- status will be largely affected by the sow’s body tissue
Px, which is apparently inadequate to remove the status, what the sow is fed and the amount and form
additional influx of oxidative end products from the transferred through the placental and mammary tissue
dam, thus resulting in an increased concentration in to her milk supply. Mahan (1991; 1994) demonstrated
the fetus. The fetus may therefore be unable to that milk Se and vitamin E concentrations normally
synthesize increased amounts of the GSH-Px enzyme decline with parity, suggesting that the progeny of
to prevent the buildup of these oxidative by-products older sows would be more prone to the deficiency
(i.e., MDA, and H2O2 ). Although supplemental Se onset postweaning than the progeny of younger sows.
has been shown to reduce the production of the lipid The importance of the pig’s Se status at weaning is
peroxide MDA (Sarada et al., 2002) the fetus is demonstrated by the report of Mahan et al. (1974).
apparently unable to do so. Therefore, the sow’s Se In that experiment, pigs were obtained from sows
status may be critical to fetal survival by preventing that were fed either a non-Se-fortified diet or one
D. Mahan 133
that contained 0.10 ppm Se (selenite), but both pig both Se sources, and 3) the organic Se form has been
groups upon weaning were fed a semi-purified diet shown to increase colostrum and milk Se substantially
without supplemental Se. This experiment thus above that of inorganic Se (Mahan and Kim, 1996;
evaluated the post-weaning effects of body tissue Se Mahan, 2000).
reserves accumulated during nursing. The results Because Se yeast has now been approved in the US
presented in Table 1 demonstrated that by 28 days as a dietary Se source for swine (FDA, 2002) as well
post-weaning, the majority of pigs from sows fed as in many other countries, its long-term effects on
the non-Se-fortified diet had several classical the sow and progeny were evaluated (Mahan, 2004).
deficiency signs, while none were present in the pigs The results of that study demonstrated that the
from sows fed supplemental Se. Because of the high numbers of total and live pigs born were lower when
death loss of pigs from the non-Se-fortified sow diet a non-Se fortified basal diet was fed, but generally
at 28 days, they were discontinued; but the other there was no significant difference between the two
treatment group continued on the non Se-fortified dietary Se sources or the two Se levels evaluated
postweaning diet. By 56 days these sow Se- (Figure 1). The number of stillbirths was greater
supplemented pigs also had clinical signs that the (P<0.05) when inorganic Se was fed (Figure 2).
other treatment pig group had demonstrated at 28 Neonatal pigs having a moderate or severe splay-
days. These results indicate the importance of the Se legged condition also appeared to be greater when
status of pigs at weaning in preventing the deficiency the inorganic Se source was fed as compared with
onset. It would appear that feeding the sow diets that organic Se diets, but the responses were not
would meet the Se and vitamin E requirement would significant (Figure 3).
provide an initial body reserve of these nutrients in Serum GSH-Px activity seemed to plateau (P<0.05)
the neonatal and weaned pig, and that these reserves at the 0.15 ppm Se level within each parity (Figure
could be used during periods of Se need. 4). Sows fed the basal diet had consistently lower
GSH-Px activities within each parity as compared
Table 1. Selenium carryover from the sow to the progeny1. with the two Se sources but the values continued to
decline with advancing parity when the basal non-
Sow diet Se-fortified diet was fed. Although sow serum
Basal Basal + GSH-Px activity also declined by parity in all
Itema No added Se 0.10 ppm Se
treatment groups, particularly in parity 3 and 4, the
28-days post-weaning decline was greater when the non-Se-fortified diet
No. pigs examined 6 6 was fed.
White skeletal muscle, % 67 0 There was a consistent decline in serum Se after
Liver necrosis, % 100 0
70 days post-coitum regardless of Se source or Se
Enlarged heart, % 50 0
Gastric ulcers, % 83 0 level fed, suggesting that Se was being transferred to
the conceptus products during this phase of
56-days post-weaningb
No. pigs examined 01 5 reproduction, resulting in the lower circulatory level.
White skeletal muscle, % - 40 The same trends are shown for sow GSH-Px activity
Liver necrosis, % - 100 where GSH-Px activity was lower at 110 days post-
Enlarged heart, % - 40
coitum than at day 70, whereupon the values are
Gastric ulcers, % - 100
correspondingly increased by weaning. The latter is
a
Pigs were fed a semi-purified non-Se fortified diet post-weaning undoubtedly reflective of the increased sow feed and
b
Pigs were removed from the trial due to sudden deaths. Se intake, except for sows fed the non-Se-fortified
1
Mahan et al., 1974
diet (Figure 5).
Although the Se concentration in colostrum and
Effect of inorganic or organic selenium milk increased more with the feeding of organic Se
on sow reproduction compared with inorganic Se, there was a decline in
milk Se as parity progressed but only when the
Feeding organic Se from an enriched Se yeast source inorganic Se or the basal non-Se-fortified diets were
(Sel-Plex®) to reproducing sows as compared with fed, not when the organic Se diet was provided (Figure
sodium selenite has previously been shown to: 1) 6). These results suggest that the Sel-Plex® organic
enhance the Se status of both the sow and progeny, Se source provided a consistent and relatively constant
2) has resulted in equivalent GSH-Px activities for supply of Se during each lactation, and that it
14
Total Live
12
Pigs per litter (no.) 10
0
0.00 0.15 0.30 0.15 0.30 0.30
Basal Sel-Plex® Inorganic Se Combined
Figure 1. Effect of sow dietary Se source (inorganic selenite or Sel-Plex® organic Se) and level (ppm) on total and live pigs born
per litter over the 4-parity period (SEM [total and live]= 0.60, P<0.05). The combination treatment (0.30 ppm Se) contains 0.15
ppm Se from both organic and inorganic Se sources.
1.0
0.8
Stillborn pigs per litter (no.)
0.6
0.4
0.2
0.0
0.00 0.15 0.30 0.15 0.30 0.30
Figure 2. Effect of sow dietary Se source (inorganic selenite or Sel-Plex® organic Se) and level (ppm) on the average number of
stillbirths per litter over the 4-parity period (SEM = 0.05, P<0.05). The combination treatment (0.30 ppm Se) contains 0.15 ppm
Se from both organic and inorganic Se sources.
appeared to be readily incorporated into milk proteins. at weaning as compared with pigs from sows fed Se-
This also suggests that an increasing amount of Se fortified diets (Figure 7). Pigs from sows fed the
would be consumed by the nursing pigs during each non-Se-fortified diet had a higher serum Se in parity
parity when sows are fed organic Se, thus enhancing 1 but the concentrations declined with advancing
the pig’s Se status at weaning. parity. This suggests that sow depletion of Se was a
Consequently, pigs that nursed sows fed the non- contributing factor in milk Se concentration over
Se-fortified diet had lower serum Se concentrations time. Weanling pig serum Se was greater (P<0.01)
D. Mahan 135
40
30
Splay leg pigs (% of total)
20
10
0
0.00 0.15 0.30 0.15 0.30 0.30
Figure 3. Effect of sow dietary Se source (inorganic selenite or Sel-Plex® organic Se) and level (ppm) on the percentage of pigs
showing evidence of the splay-legged condition at birth. Pigs were individually scored at birth (1= normal, 2 = slight, 3, =
severe). The above is a combined total of scores 2 and 3 (SEM = 0.55, P>0.15). The combination treatment (0.30 ppm Se)
contains 0.15 ppm Se from both organic and inorganic Se sources.
1.2 Parity 1 Parity 2 Parity 3 Parity 4

Sow serum GSH-Px activity (units/mL)
1.0
0.8
0.6
0.4
0.2
0.0
0.00 0.15 0.30 0.15 0.30 0.30
®
Basal Sel-Plex Inorganic Se Combined
Figure 4. Effect of parity, dietary Se source (inorganic selenite or Sel-Plex® organic Se), and level (ppm) on sow serum GSH-Px
activity at 110 days post-coitum (SEM = 0.078; Parity (P<0.01)). The combination treatment (0.30 ppm Se) contains 0.15 ppm Se
from both organic and inorganic Se sources.
when sows were fed Sel-Plex ® as compared to Se-fortified diet than when sows were fed the Se-
inorganic Se, responses consistent with previous fortified diets (Figure 8). Pig serum GSH-Px activity
reports (Mahan, 2000). was increased (P<0.05) as sow dietary Se level
Pig serum GSH-Px activity at weaning was also increased to 0.30 ppm Se from both dietary Se
lower (P<0.01) when sows were fed the basal non- sources. These serum GSH-Px activities did not differ
1.2
70 day 110 day Weaning
Sow GSH-Px activity (units/mL)

1.0
0.8
0.6
0.4
0.00 0.15 0.30 0.15 0.30 0.30
Figure 5. Effect of Se source (inorganic selenite or Sel-Plex® organic Se), and level (ppm) on sow serum GSH-Px activity at
various stages of gestation and at weaning (SEM = 0.03, interaction of Se source x level x production phase P<0.01). The
combination treatment (0.30 ppm Se) contains 0.15 ppm Se from both organic and inorganic Se sources.
Parity 1 Parity 2 Parity 3 Parity 4

0.12
0.09
Sow milk Se (mg/L)
0.06
0.03
0.00
0.00 0.15 0.30 0.15 0.30 0.30
Figure 6. Effect of parity, dietary Se source (inorganic selenite or Sel-Plex® organic Se), and level (ppm) on milk Se concentra-
tions at weaning (SEM = 0.003; Parity (P<0.01), interaction of parity by Se treatment (P<0.01)). The combination treatment (0.30
ppm Se) contains 0.15 ppm Se from both organic and inorganic Se sources.
between the two Se sources nor was the interaction responses consistent with previous reports (Mahan
between the factors significant. There was an apparent and Kim, 1996). Because of interest in using hair as
decline in pig serum GSH-Px activity as parity an analytical tool for evaluating sow Se status, hair
progressed in all treatment groups, the reason for Se content was analyzed at the time of weaning for
which is unclear. each treatment group. Hair Se content was greater
Sow tissue Se concentrations were greater at the when sows were fed increasing levels of organic
end of the 4-parity period when organic Se was fed, (P<0.01) or inorganic Se (P<0.05), but the Se
D. Mahan 137
0.10
Pig serum Se (mg/L) 0.08
0.06
0.04
0.02
0.00
0.00 0.15 0.30 0.15 0.30 0.30
Figure 7. Effect of parity, sow dietary Se source (inorganic selenite or Sel-Plex® organic Se), and level (ppm) on serum pig serum
Se concentrations at weaning (SEM = 0.002; Parity (P<0.01); Interaction of parity by Se treatment (P<0.01)). The combination
treatment (0.30 ppm Se) contains 0.15 ppm Se from both organic and inorganic Se sources.
0.60
Parity 1 Parity 2 Parity 3 Parity 4
0.50
Pig serum GSH-Px (units/mL)
0.40
0.30
0.20
0.10
0.00
0.00 0.15 0.30 0.15 0.30 0.30
Figure 8. Effect of parity, sow dietary Se source (inorganic selenite or Sel-Plex® organic Se), and level (ppm) on pig serum GSH-
Px activity at weaning (SEM =.035, Parity (P<0.01)). The combination (0.30 ppm Se) contains 0.15 ppm Se from both organic
and inorganic Se sources.
concentration was substantially greater when the whereas the other treatment groups showed a small
organic Se source was fed (Figure 9). When the but consistent increase by parity but this response
combination of Se sources were fed, sow hair Se was not significant. The sulfur (S) in the amino acid
concentrations were generally similar to the group cysteine in swine hair is thought to be greater (i.e.
fed 0.15 ppm organic Se. Hair Se concentration when >13%) than in other tissues (Mahan and Shields,
sows were fed 0.30 ppm Se from Sel-Plex® seemed 1998). Because of the substitution of S with Se in
to be relatively constant over the 4-parity period, these amino acids (i.e., methionine or seleno-
1.0 Parity 1 Parity 2 Parity 3 Parity 4
0.8
Sow hair Se (mg/kg)
0.6
0.4
0.2
0.0
0.00 0.15 0.30 0.15 0.30 0.30
Figure 9. Effect of parity, dietary Se source (inorganic selenite or Sel-Plex® organic Se), and level (ppm) on sow hair Se concen-
trations at weaning (SEM = 0.028; Parity (P<0.01)). The combination treatment (0.30 ppm Se) contains both organic and
inorganic Se sources.
methionine) the resulting hair Se had a greater relative production, but because it is not as well retained by
Se concentration when selenomethionine was in a the pig and less is transferred to the fetus and milk
higher concentration in the diet. Because these amino products in the adult female, the organic Se form
acids can substitute for each other in the tissues, the might be superior over prolonged reproductive
results suggest that selenomethionine or periods. Although inorganic Se is effective in
selenocysteine would be a major contributor to hair producing the selenoenzymes necessary for
Se when present in the animal’s diet and thus in its antioxidant protection, organic Se seems to be equally
circulatory system. Se was probably present in a effective. The use of organic Se is beginning to show
higher relative concentration when the organic Se advantages as a source of Se for the sow over that of
source was fed at the 0.30 ppm Se level than when inorganic Se. Feeding organic or inorganic Se at 0.15
inorganic Se was the major Se source or when the ppm appears to optimize reproductive performance,
lower (i.e., 0.15 ppm) organic Se level was fed. The but the higher level (i.e., 0.30 ppm Se) of the organic
present experiment suggests that the Se content of Se source will more greatly increase the transfer of
sow hair appears to largely reflect the dietary source Se to the fetus and milk products than the lower 0.15
and level of organic Se that was fed to the sow, and ppm Se level or the inorganic Se source.
does not reflect the sow’s Se status.
References
What does all of this mean ?
Bartholomew, A., D. Latshaw and D. Swayne. 1998.
The results of several experiments show that the sow Changes in blood chemistry, hematology and
may experience hyposelenosis, particularly if an histology caused by a selenium/vitamin E deficiency
inadequately fortified diet is fed. A hyposelenosis and recovery in chicks. Biol. Tr. Elem. Res. 62:7-
condition can affect her reproductive capability, and 16.
it can influence her body’s Se status and Se status of Chavez, E.R. 1985. Nutritional significance of
her progeny. Sow tissue can become depleted of Se selenium supplementation in a semi-purified diet
over time, and sows of higher productivities and fed during gestation and lactation to first-litter gilts
greater age are more prone to the deficiency and their piglets. Can. J. Anim. Sci. 65:497-506.
occurrence. Inorganic Se (selenite) was critical in FDA. 1974. Food additives permitted in feed and
the initial discovery that Se had a vital role in swine
D. Mahan 139
drinking water of animals: selenium. Final rule. from birth to 145 kilograms of body weight, and
Fed. Regis. 39:1355. comparison to other studies. J. Anim. Sci. 76:513-
FDA. 1987. Food additives permitted in feed and 521.
drinking water of animals: selenium. Fed. Reg. Mahan, D.C., L.H. Penhale, J.H. Cline, A.L. Moxon,
52:21001. A.W. Fetter and J.T. Yarrington. 1974. Efficacy
FDA. 2002. Food additives permitted in feed and of supplemental selenium to reproductive diets on
drinking water of animals: selenium yeast. Fed. sow and progeny performance. J. Anim. Sci.
Reg. 67:46850. 39:536-543.
Hostetler, C.E. and R.L. Kincaid. 2004. Maternal Mutetikka, D.B. and D.C. Mahan. 1993. Effect of
selenium deficiency increases hydrogen peroxide pasture, confinement and diet fortification of
and total lipid peroxides in porcine fetal liver. Biol. vitamin E and selenium on reproducing gilts and
Tr. Elem. Res. 97:43-56. their progeny. J. Anim. Sci. 71:3211-3218.
Loudenslager, M.J., P.K. Ku, P.A. Whetter, D.E. National Research Council (NRC). 1998. Nutrient
Ullrey, C.K.Whitehair, H.D. Stowe and E.R. Requirements of Swine, 9th rev. ed., National
Miller. 1986. Importance of diet of dam and Academy Press, Washington, D.C.
colostrum to the biological antioxidant status and Nielsen, H.E., V. Danielsen, M.G. Simese, G. Gissel-
parenteral iron tolerance of the pig. J. Anim. Sci. Nielsen, W. Hjarde, T. Leth and A. Basse. 1979.
63:1905-1914. Selenium and vitamin E deficiency in pigs. I.
Mahan, D.C. 2004. Long-term effects of dietary Influence on growth and reproduction. Acta. Vet.
organic and inorganic selenium sources and levels Scand. 20:276-288.
on reproducing sows and their progeny. J. Anim. Piatkowski, T.L., D.C. Mahan, A.H. Cantor, A.L.
Sci. (accepted). Moxon, J.H. Cline and A.P. Grifo, Jr. 1979.
Mahan, D.C. 1991. Assessment of the influence of Selenium and vitamin E in semi-purified diets for
dietary vitamin E on sows and offspring in three gravid and nongravid gilts. J. Anim. Sci. 48:1357-
parities: reproductive performance, tissue 1365.
tocopherol, and effects on progeny. J. Anim. Sci. Rotruck, J.T., A.L. Pope, H.E. Ganther, A.B.
69:2904-2917. Swanson, D.G. Hafeman and W.G. Hoekstra. 1973.
Mahan, D.C. 1994. Effects of dietary vitamin E on Selenium: biochemical role as a component of
sow reproductive performance over a five parity glutathione peroxidase. Science 179:588-590.
period. J. Anim. Sci. 72:2870-2879. Sarada, S.K., M. Sairam, P. Dipti, B. Anju, T.
Mahan, D.C. 2000. Effect of organic and inorganic Pauline, A.K. Kain, S.K. Sharma, S. Bagawat, G.
selenium sources and levels on sow colostrum and Ilavazhagan and D. Kumar. 2002. Role of selenium
milk selenium content. J. Anim. Sci. 78:100-105. in reducing hypoxia-induced oxidative stress: an
Mahan, D.C. and Y.Y. Kim. 1996. Effect of inorganic in vivo study. Biomed. Pharmacother 56:173-178.
or organic selenium at two dietary levels on Whitehair, C.K. and E.R. Miller. 1985. Vitamin E
reproductive performance and tissue selenium and selenium in swine production. In: Selenium
concentrations in first parity gilts and their progeny. Responsive Diseases in Food Animals. Western
J. Anim. Sci. 74:2711-2718. States Vet. Conf. pg 11.
Mahan, D.C. and E.A. Newton. 1995. Effect of initial Wilkinson, J.E., M.C. Bell, J.A. Bacon and F.B.
breeding weight on macro- and micro-mineral Masincupp. 1977. Effects of supplemental selenium
composition over a three parity period using a high- in swine. I. Gestation and lactation. J. Anim. Sci.
producing sow genotype. J. Anim. Sci. 73:151-158. 44:224-228.
Mahan, D.C. and R.G. Shields, Jr. 1998. Essential
and nonessential amino acid composition of pigs
P. Penny 141
Adding value to pork for producers and consumers: enhancing omega-3

DHA and selenium content of meat
PAUL PENNY
JSR Genetics Ltd, Southburn, Driffield, United Kingdom
Introduction
Pork is possibly the most versatile raw meat product market, it should be seen as a warning and acted upon
compared to chicken, beef, turkey and lamb. By accordingly. To continually improve the image of
utilising sophisticated processing techniques it can pig meat and hence consumption it is necessary not
be easily transformed and presented across a highly only for the retailers but the producers and processors
varied range of quality products such as fresh pork, to portray the correct image to the consumer. There
spare ribs, specialist hams, bacon, sausages and is an ever-increasing demand by the public for varied
salamis. Developing and marketing new pork products diets containing even safer and healthier foods.
is becoming an increasingly competitive activity due Providing the consumer with new and exciting
to the globalisation of the pig industry and the products that can deliver consistent quality and
homogenisation of the markets. Consumers are complement an ever-growing need for a healthy diet
becoming more particular regarding what they buy; and lifestyle should be fully embraced. The successful
they are not only concerned with the welfare of the company of the future must create wealth within a
animal but the health of their own diets and what very challenging environment, requiring the ability
nutrients the presented product can offer over and to deal with confusion, unanticipated market
above alternative protein sources. movements and rapid change. It is therefore extremely
Meat consumption in the EU and USA (per capita important to develop an entire pork chain that is
consumption) is 44 kg and 30 kg, respectively, with knowledge-based. Networking is required to provide
pig meat consumption being higher than all the other synergistic benefits through exchange, association and
meats. The present and future forecast projection for mutuality enabling greater achievements than the
the EU is 45 kg/hd (Table 1), although this is individual working alone.
extremely static when viewed over a 10-year period The need for identifying a positive way to promote
(Pig Progress, 2001). and re-position pork as a healthy, tasty and nutritious
For pork to continue to compete successfully in product is of paramount importance and was the key
the international and national meat protein market, driving force behind the development of Vitapork™.
it must demonstrate an unquestionable stance on This new product delivers substantial amounts of
safety, quality, convenience, healthiness and price. essential omega-3 DHA polyunsaturated fatty acids
Recent data compiled by a leading retailer in the UK together with major antioxidants (selenium and
(Figure 1) clearly showed that pork was seriously vitamin E). It is believed that Vitapork™ has all the
failing to attract the contemporary consumer (Smith, necessary characteristics and final product benefits
2003). Although this may be specific to the UK to fully complement the consumer’s growing appetite
for healthy nutritious food.
Table 1. Per capita pig meat consumption projections in the EU, 1999-2008.
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008
Consumption, kg/head 44.4 43.4 44.2 44.7 44.9 44.7 45.2 45.8 45.8 46.1
142 Adding value to pork for producers and consumers
50
40
Pork is failing to appeal to

Sales by lifestyle category
30
'healthy' and 'convenience'-
20 oriented consumers.
10
-10
-20
-30 Traditional Price Mainstream Finer H althy Conv ni nce

Co
sensitive foods
Figure 1. Pork customer profile (Smith, 2003).
Consumer behaviour products containing high levels of omega-3 DHA fats

such as eggs, offal and oily fish have lost their place
Meat and meat products are important components in the modern diet. Leading nutritional experts and
in the diets of developed countries and their organisations like the Foods Standards Agency within
consumption is affected by various factors such as the UK are now actively promoting the fact that
preparation time, cooking convenience and overall consumers should make every effort to increase their
enjoyment. There is also a shift in the type of meat daily intake of omega-3 polyunsaturates.
product consumed from fresh to processed products.
Consumer needs are constantly changing; and this
will be enhanced in the future through increasing
Omega-3 fatty acids
disposable income. The consumer has a vast choice
of products and must balance lifestyle activities Fatty acids are the major building blocks of all lipids.
against the need to consume food. An understanding Their division into two groups, non-essential, i.e.
will be required of how product presentation must need not be supplied in the diet, and essential, i.e.
respond to these trends. In the next 10-20 years a must be supplied in the diet, has been known since
higher proportion of the population will be over 45 the early 1930s. Fatty acids not only serve as structural
years of age, which means that this group of components of all cells but also take part in and are
consumers will have a larger influence on pork of paramount importance to cellular metabolism.
consumption than the younger generation. The This is particularly so for the essential fatty acids;
traditions and habits of the consumer will gradually and ever since their discovery an ever increasing
change largely due to the consumers’ lifestyles. Sitting number of roles for them have been found ranging
around a table as a family will occur less frequently from basal metabolism to the maintenance of health
and therefore eating out, which involves quick, on- and well-being (BNF, 1992) (Table 2).
the-go consumption of convenience products will The essential fatty acids can be divided into the n-
become a prominent feature of food consumption. 6 group based on linoleic acid (LA) and its longer
Over the last 50 years there has been a significant chain, more unsaturated derivatives, and the n-3
decline in the consumption of omega-3 (n-3) group, which is based on α-linolenic acid (LNA)
polyunsaturated fats. The main reason for this and its derivatives. Until relatively recently, the
reduction of omega-3 DHA consumption is the balance of nutritional interest was heavily weighted
dramatic change in eating habits and more particularly in favour of the n-6 polyunsaturates and their role in
a change in the type of foods consumed. Those food health promotion; the imbalance with the n-3 group
P. Penny 143
Table 2. Summary of descriptions and roles of omega-3 polyunsaturated fatty acids.
• The omega-3 and omega-6 group of fatty acids are essential nutrients.
• α-linolenic acid is the first fatty acid in the omega-3 group and is the precursor to synthesise DHA, however efficiency of DHA
formation is only 5%.
• DHA is a long-chained PUFA and is the most important omega-3 fatty acid.
• DHA is a major constituent of the brain and retina; and is needed for development and maintenance of brain structure and function.
• DHA is an important component of the retina, particularly for infants and the elderly, but also throughout life to help preserve
vision.
• Pregnant women, newborn infants, children up to 18 years old and the elderly are key beneficiaries of added dietary DHA.
• Due to a significant change in eating habits, the average intake of DHA in the EU does not exceed 75 mg per day.
• The recommendation is to consume 200-400 mg DHA daily.
being of an order of magnitude and primarily due to (LCPUFA), eicosapentaenoic (EPA, C22:5) and
the consumption of cooking oils and margarines. The docosahexaenoic acid (DHA, C22:6), in terms of
extent of n-6 polyunsaturate consumption was such human health and well-being. The reason for this
that adverse effects on health were a distinct lack of improvement relates to the complicated
possibility. Furthermore a comprehensive biochemical process of desaturation and chain
involvement for the n-3 polyunsaturates in a wide elongation, which is required to convert LNA into
range of health issues and disease prevention was EPA and DHA. Efficiency of conversion from LNA
becoming increasingly clear (BNF, 1992). As a result, to EPA and DHA within the pig is no more than 5%,
a far more balanced nutritional strategy in the about the same as in the human (Emken et al., 1994).
provision of the two groups of polyunsaturates is now Therefore, the only way of significantly increasing
recognised as essential. With the recognition that the the key LCPUFA, EPA and DHA, in the carcass is
groups of polyunsaturates each have their own by incorporating them directly into the diet.
distinctive metabolic roles and involvements in health
promotion, it is also recommended that emphasis on
the polyunsaturated:saturated fatty acid (P:S) ratio Antioxidant requirements: selenium and
as a measure of dietary acceptability be reduced and vitamin E
that the importance of the separate n-6 and n-3 fatty
acids be recognised through an appropriate total n- Unsaturated fatty acids are particularly susceptible
6:total n-3 ratio or even an LA:LNA ratio (BNF, to oxidation; which is substantially increased in highly
1992; Bruckner, 1992). Furthermore, consumption unsaturated molecules such as DHA through a cascade
of n-3 acids should be increased with an aim to of oxidative events. Oxidation is brought about by
achieving an n-6:n-3 ratio of 6:1. The major fatty the action of oxygen free radicals, which naturally
acid of the omega-3 series, which is seen as being occur and potentially accumulate in living and post-
the most important for the body, is docosahexaenoic mortem cells, on the highly unsaturated lipids in the
acid (DHA). cell membrane and contents (Burton, 1994). The
Many investigations have looked at the effect of breakdown can be further enhanced by post-mortem
increasing the levels of polyunsaturated fatty acids handling that facilitates interaction between pro-
in pig feed, which has been noted to increase carcass oxidant factors and the unsaturated lipids. The
fat content. Feeding linseed oil has been the primary accumulated presence of the oxidised lipid
route taken to achieve this natural modification of metabolites not only poses a potential cytotoxic threat
the fat and muscle tissue. Addition of linseed oil can but also reduces product acceptability due to off-
successfully increase carcass level of LNA (C18:3), flavours, rancidity and malodorous compounds.
the first fatty acid in the omega–3 family. However, However, cells have an array of natural defense
this method of dietary modification has minimal mechanisms against free radical formation and lipid
effect on the most important and highly beneficial oxidative damage (Frust, 1996). These consist of
omega-3 long chain polyunsaturated fatty acids concerted enzyme systems that eliminate free radicals
and a selection of naturally occurring vitamins and substantially intensifies the susceptibility of the
synthesized products, both fat and water soluble, to product to free radical attack. To fully ensure optimal
deter the cascade of events that leads to radical benefits, animals must be fed significant amounts of
accumulation. Important amongst the former are the vitamin E above those normally found in standard
enzymes superoxide dismutase, catalase and production diets. An achievable target for maximising
glutathione peroxidase; and amongst the latter superior product quality would be 5-6 mg vitamin E
vitamins A, C and E, ubiquinones and a range of (as α-tocopherol) per kilogram of muscle tissue. It
natural plant metabolites. The whole sequence of is very difficult to obtain the same effects by treating
events is further affected by a selection of pro-and meat products during processing.
antioxidant multivalent metal ions e.g. iron, copper,
selenium, zinc. With an increasing interest in the
promotion of polyunsaturated components in animal Vitapork™ ‘Choose the healthy option’
products for human consumption, the lipid antioxidant
nutrients, in particular selenium and α-tocopherol Vitapork™ is an innovative approach to enhancing
(vitamin E), are of major interest for protection against the ‘healthiness’ and nutritional value of pork. It is a
tissue oxidative degradation (MacPherson, 1994). ‘brand’ of new lean and healthy meat that can be
The major benefit of selenium is as an antioxidant utilised across a range of final product formats.
enzyme cofactor, preventing damage to cells by The object of the Vitapork™ program was to
oxidation. As part of the glutathione peroxidase develop a new approach toward enhancing the
molecule and other antioxidant selenoenzymes, healthiness of pork. The strategy was to increase the
selenium status plays a major role in the body’s most important LCPUFA, EPA and DHA, and obtain
antioxidant defence. The availability and usefulness the recommended n-6:n-3 ratio without
of selenium from the diet is dependent on the form compromising the physical and organoleptic
in which it is occurs. This can be either natural properties of the carcass. Accepted understanding to
selenoamino acids, which are found in plants or date has suggested that increasing the dietary
inorganic sources like sodium selenite that are usually concentration of linoleic acid (C18:2) n-6, and
present in standard trace mineral premixes. particularly the carcass concentration, e.g. above 15%
Selenomethionine, the primary form present within of the total fat, leads to a substantial softening of the
Sel-Plex® provides added advantages over the sodium depot fat, thereby producing carcasses with highly
selenite. It enables both increased availability and unacceptable processing properties which fail to
tissue selenium reserves that can be used quickly and satisfy consumer requirements (Whittington et al.,
effectively during increased demand. The selenium 1986). However it is now possible to overcome this
accumulated in body proteinaceous tissues when negative outcome by utilising a combination of oil
selenoamino acids are included in the diet provides products in the diet. When elevating the PUFA level,
an opportunity for consumers of such animal products antioxidant protection becomes critical in order to
to benefit through increased intake of selenium. provide the necessary assurance against lipid oxidation.
Vitamin E is the main fat-soluble antioxidant in To fulfil this need a combined selenium (Sel-Plex®)
cell membranes. It is stored in fatty tissue, liver and and vitamin E supplement was paramount.
muscle. It can neutralise free radicals and help stop The extensive amount of literature highlighting the
them from reacting further. In this way it acts like a health benefits of consuming these specific LCPUFA
protective shield around each cell, reducing tissue and the additional benefits of consuming elevated
damage. This beneficial action of being able to slow concentrations of selenium and vitamin E was one
down oxidation clearly identifies vitamin E as an of the main reasons for developing and producing
important protectant for improving the quality of the Vitapork™ product. The need for identifying a
fresh, processed and frozen meat products. positive way to promote and re-position pork as a
Vitamin E can stabilise the colour of red meat, but healthy, nutritious product in terms of PUFA levels
most importantly it stops fat turning rancid and helps without compromising physical properties and
alleviate off tastes and odours. The role and function organoleptic acceptability was also a significant
of vitamin E become absolutely paramount when driving force.
modifications are made to the fatty acid composition A series of controlled experiments and extensive
of the fat and lean tissue. Increasing the field studies were undertaken to investigate various
polyunsaturate content of the diet and hence the diet compositions and feeding time periods (Table
number of double bonds in the fat and meat 3). In association were extensive carcass assessments
P. Penny 145
(Table 4). The total PUFA level in the fat tissue acceptable carcass in terms of processing and final
increased from 18% to 34% in the Vitapork™ product, product formats. The diet formulation utilises key
which would be equivalent to the profile of good active ingredients, such as high-DHA tuna oil, soya
quality table margarine. oil, organic selenium (Sel-Plex®) and vitamin E.
The improvements from implementing the 5 weeks
Table 3. Percentages of DHA in lean meat and fat following pre-slaughter feeding protocol are due to an increase
dietary supplementation for 2-6 weeks. of 300% in DHA and 68% for LA compared to
Weeks of supplementation Lean meat Fat standard product (Figures 2 and 3). The ability to
2 0.61 0.25 significantly increase both carcass EPA and DHA and
4 0.80 0.40 more specifically LA, whilst at the same time
Vitapork™ - 5 0.98 0.63 maintaining overall carcass acceptability, goes against
6 1.01 0.65
previous knowledge regarding fatty acid enhancement
Table 4. Carcass enhancement following a 5-week supplementa- of the carcass. The final product also has the added
tion period. benefit of being fully traceable and checked for
Standard Vitapork™ authenticity, meaning that the carcass can be checked
PUFA, % of fat 18 34
and easily identified by undertaking a simple
n-6:n-3 9.0 7.0 laboratory analysis.
P:S 0.5 1.0 This exciting opportunity of carcass enhancement
has the ability to make pork the first choice option
The Vitapork™ technology involves a diet containing when purchasing meat. Vitapork™ truly delivers
very specific LCPUFA (EPA and DHA) in multi-functional meat, which provides the health
combination with a significant level of LA. To conscious consumer with a high quality product
demonstrate the robustness of the Vitapork™ feeding containing enhanced levels of omega-3 DHA,
protocol, three separate commercial units each having selenium and vitamin E (Table 6).
different housing facilities were used during the
Table 6. Characteristics of VitaporkTM.
finishing period. In addition, three genotypes were
investigated to show that the technology can be • Vitapork™ can provide 50 mg of DHA per 100 g of pork
broadly utilized. All animals were weighed at the
• Vitapork™ is significantly differentiated from standard pork
beginning and end of the supplemental period
(Table 5). • Vitapork™ delivers clear and identifiable benefits
consistently and provides a novel premium meat product.
Table 5. Growth and carcass response following the five-week • Vitapork™ complements consumer needs for quality,
supplemental period. healthy and convenient products.
Control VitaporkTM
Genotype A - n=16
Start weight, kg 61.5 62.5
End weight, kg 88.5 90.0 Summary
Daily gain, g/day 771 785
P2, mm 9.3 9.7 The secret to producing good quality food is to
Genotype B - n=80 identify the consumer’s quality criteria and act
Start weight, kg 64.3 64.8 appropriately to meet them. Pig meat will need to be
End weight, kg 93.5 92.9 marketed to the consumer based on production using
Daily gain, g/day 834 822
P2, mm 10.4 10.3
natural ingredients, and it will be highly beneficial
if products can be traced from production through to
Genotype C - n=40
Start weight, kg 64.0 64.1 manufacturing.
End weight, kg 98.9 98.4 The consumer must also be able to recognise these
Daily gain, g/day 992 980 branded meat products on the shelf and feel
P2, mm 11.0 11.2 comfortable with the endorsement. Regaining the
required image and restoring consumer trust requires
The carcass produced by the Vitapork™ diet provided a co-ordinated effort and a vision shared by all
both an enhanced LCPUFA content and improved n- participants in the food chain including farm
6:n-3 ratio, but equally importantly provided a very suppliers, service providers, farmers, meat processors,
60
+300%
50
DHA (mg per 100 g)

40
30
20
10
0
Control Vitapork™
Figure 2. Effect of the Vitapork™ feeding protocol on DHA content of pork (100 g portion: 92.5 g lean and 7.5 g fat).
2000
+ 68%
1800
C18:2n-6 (mg per 100 g)
1600
1400
1200
1000
800
Control Vitapork™
Figure 3. Effect of the Vitapork™ feeding protocol on linoleic acid content of pork (100 g portion: 92.5 g lean and 7.5 g fat).
retailers and government. Information needs to flow Bruckner, G. 1992. In: Fatty Acids in Foods and their
along the chain in both directions, which can boost Health Implications. (ed. C.K. Chow), Marcel
efficiency and allow all to benefit from the added Dekker, New York, 631-646.
value achieved. Burton, G.W. 1994. Vitamin E: molecular and
biological function. Proc. Nutr. Soc. 53:251-262.
References Emken, E.A., R.O. Aldof, R.M. Gulley. 1994.
Dietary linoleic acid influences desaturation and
British Nutrition Foundation. 1992. Unsaturated acylation of deuterium-labelled linoleic and
Fatty Acids: Nutritional and Physiological linolenic acids in young adult males. Biochim.
Significance. Chapman and Hall, London. Biophys. Acta 1213:277-288.
P. Penny 147
Frust, P. 1996. The role of antioxidants in nutritional Smith, C. 2003. The challenges of selling pork and
support. Proc. Nutr. Soc. 55:945-961. pork products in a sophisticated market. World Pork
MacPherson, A. 1994. Selenium, vitamin E and Congress, Birmingham, UK.
biological oxidation. In: Recent Advances in Animal Whittington, F.M., N.J. Prescott, J.D. Wood, M.
Nutrition. (P.C. Garnsworthy and D.J.A. Cole, eds) Enser. 1986. The effect of dietary linoleic acid on
Nottingham University Press, UK. 3-30. the firmness of backfat in pigs of 85 kg live weight.
Pig Progress. 2001. The EU-15 and its enlargement. J. Sci. Food Agric. 37:753-761.
Elsevier International Business Information, The
Netherlands. Vol. 17(9): 32-35.
K.J. Stalder et al. 149
Reducing the environmental impact of swine production through nutritional

means
K.J. STALDER1, W.J. POWERS1, J.L. BURKETT1, AND J.L. PIERCE2
1
Department of Animal Science, Iowa State University, Ames, Iowa, USA
2
North American Biosciences Center, Alltech, Inc., Nicholasville, Kentucky, USA
Introduction
Livestock production is becoming more concentrated Table 1. Pig density for selected countries1.
in many parts of the world and pork production is no Country or region Pig inventory Pigs per Pigs per sq
exception. This is particularly evident when acre (hectare) mile
examining a recent Canadian report (Saskatchewan Europe
Agriculture Food and Rural Revitalization, Statistics Germany 25,958,000 0.89 (2.20) 569.1
Spain 23,858,000 0.74 (1.83) 474.6
Canada, 2003) showing the pig densities per square France 15,290,000 0.34 (0.83) 214.7
mile in different countries of the world (Table 1). Netherlands 13,000,000 5.81 (14.36) 3,720.6
More and more people residing in rural areas are not Denmark 12,990,000 2.29 (5.67) 1,467.9
accustomed to practices associated with crop and Belgium 6,851,000 3.29 (8.14) 2,107.4
United Kingdom 5,588,000 0.40 (0.99) 256.1
livestock production. Certainly, producers want to
remain profitable in order to continue in the pork Asia
Japan 9,612,000 0.88 (2.16) 560.1
business in the future. However, most if not all pork China 464,695,000 1.31 (3.24) 838.0
producers also maintain the goal of producing pork United States
in a socially acceptable and environmentally sound Iowa 15,000,000 0.56 (1.38) 357.9
manner (Coffey, 1999). The actual numbers of pigs North Carolina 9,600,000 1.71 (4.23) 1,095.2
produced has not changed drastically since 1900 Minnesota 6,100,000 0.28 (0.70) 181.7
Illinois 3,950,000 0.16 (0.40) 104.6
(Table 2). In fact, the environmental impact of pork Indiana 3,100,000 0.23 (0.57) 148.4
production is likely less today than 100 years ago
Canada
when examined on a per pig basis. However, the same Quebec 4,280,000 0.93 (2.31) 597.7
number of pigs is being produced on fewer farms, Ontario 3,700,000 0.41 (1.01) 261.0
which increases the environmental risk in a Manitoba 2,750,000 0.22 (0.55) 143.3
concentrated area (Table 2). In 2000, 51% of the Alberta 2,100,000 0.08 (0.19) 49.6
Saskatchewan 1,211,000 0.03 (0.07) 16.9
pigs marketed were coming from operations that
1
marketed more than 50,000 pigs annually (National Adapted From Saskatchewan Agriculture Food and Rural
Revitalization, Statistics Canada.
Pork Board, 2002).
The general public is increasingly concerned with Table 2. Inventory of pigs, number of operations and lean meat
the environmental circumstances under which food produced per pig in the US from 1900 to 2000.1
is produced. Public concerns center around soil Year Number of Number of Pigs per Average retail
(accumulation and runoff of minerals from land hogs operations operation meat yield per
where manure is applied), water (surface and ground carcass
water), and air quality environmental issues. Federal, 1900 62,879,000 4,335,989 14.5
state, and local laws have and will continue to impose 1920 59,350,000 4,852,430 12.2
1940 34,040,000 3,767,875 9.0
regulations designed to address the environmental 1960 59,030,000 1,848,784 31.9 124
concerns associated with crop and livestock 1980 67,353,000 670,350 100.5 133
production. Jongbloed and Lenis (1998) reported that 2000 59,073,000 86,360 684.0 151
many countries have limited the use of livestock 1
Coffey et al., 1999 and National Pork Board Pork Facts,
2002/2003.
150 Reducing the environmental impact of swine production
manure or the number of animals per hectare of land. 3) Avoid over-formulating diets.
Livestock producers must implement these
4) Feed for optimal growth rather than maximum.
regulations in times when margin or profit per animal
sold continues to decline (National Pork Board, 5) Use of crystalline amino acids and high quality
2002). The combination of laws, neighbor relations, protein sources.
and economic concerns along with the concentration
6) Improve mineral availability and/or use sources
of pork and other livestock production brings about
with high bioavailability.
challenges for producers. The objective of this paper
is to review the nutritional methods to reduce the 7) Feed to the pigs exact needs by stage of growth
environmental impact of swine production. and gender.
8) Reduce feed wastage.
Feeding practices and excretions

It is clear that not all dietary nutrients ingested are
Food animals convert feed nutrients into various animal absorbed, utilized, and retained by the pig. Kornegay
products including meat, milk, eggs, wool, hides, draft and Harper (1997) outlined the percentages of various
power, feces, urine, and gases (fermentation and nutrients digested and retained by grow–finish pigs
respiration). The amounts of nutrients required depend (Table 3). The percentage of nutrient digested ranged
on the animal’s genetic capacity for each of these from 5% to 88% and the percentage of nutrient
products under a variety of environmental conditions. retained ranged from 10% to 70%. This clearly points
Today, producers are able to control what the pig out that it is not possible for 100% utilization of
eats because modern swine systems utilize nutrients by pigs or other livestock. However, several
confinement facilities in a very large proportion of factors can influence the quantity of each nutrient
the industry. Relatively few animals are raised in retained or excreted (Kornegay and Verstegen, 2001).
outdoor systems where pigs can forage for food other These factors include age, class, and nutritional status
than the diet provided by the producer. As a result, of the pig; source, quality, amount, and proportion
manure concentrations of elemental nutrients in swine of nutrients provided in the diet; processing methods;
are direct functions of nutrient inputs in the diet (Van and environmental influences (Kornegay and
Horn and Powers, 2004). Until recently, livestock Verstegen, 2001).
producers were generally concerned only with
maximizing food animal production. However, when Table 3. Nutrient digestion and retention by growing – finishing
swine.1
including livestock production into a whole farm plan,
often maximization of production output does not make Percentage of intake
economic sense. A better approach would be to develop
a farm plan that optimizes livestock production for the Mineral Digested Retained
operation as a whole. Nitrogen 75-88 40-50
There are several ways that diets fed to pigs can be Calcium 40-75 25-70
altered to reduce the nutrients excreted and the odors Phosphorus 20-70 20-60
produced from pork production thereby reducing Magnesium 20-45 15-38
Sodium 35-70 13-25
environmental impact. Those nutrients that are Potassium 60-80 10-20
generally considered to cause the most environmental Zinc 10-40 Similar to digested
concern are phosphorus, potassium, carbon dioxide, Copper 10-25 Similar to digested
methane, ammonia and the nitrogenous compounds Iron 5-35 Similar to digested
Manganese 8-40 Similar to digested
nitrite/nitrate, and nitrous oxide. Kornegay and Harper
(1997) outlined means to accomplish a reduction in 1
Adapted from Kornegay and Harper, 1997
the excretion of the components of manure that pose
the greatest environmental concern: The nutrient requirements of swine are well
documented (NRC, 1998). However, these
1) Improve feed efficiency. requirements are based on research conducted on pigs
2) Know the pig’s nutrient requirements and nutrient that do not have the genetic capacity for lean accretion
composition of feedstuffs. shown in some modern pig lines. Additionally, as
shown in Table 3, many nutrients have a range of
digestibility and retention values that are dependent digestibility and retention values of several minerals.
on feed quality, processing, and other factors. For It is clear that mineral bioavailability can vary
these reasons, many nutritionists and feed suppliers substantially. Awareness of this variation causes many
commonly recommend nutrient levels that include nutritionists to over-fortify minerals in swine diets,
safety margins, resulting in diets that are formulated the excess of which is excreted. Phosphorus, zinc,
to exceed NRC requirements. Nutritionists commonly and copper, specifically, have received attention
over-fortify diets to ensure that the pig has a supply (Moore et al., 2001). Phosphorus builds up in soils
of nutrients that allows the animal to meet its genetic where manure is applied and can pose runoff
capacity for growth under a variety of conditions. problems, which in turn contributes to eutrophication
There are two requirements needed to minimize of surface waters. Land application of minerals has
the amount of nutrients excreted and maximize the become an increasing environmental concern and is
nutrient quantities digested and retained. First, the now regulated in many livestock-producing regions.
pig’s nutritional needs must be accurately known for As previously indicated, phosphorus is one of the
every genetic type, size and gender of pig, under all minerals fed in relatively high amounts due to its
environmental conditions. Second, the bioavailability poor availability and its importance in various
of each nutrient from every feed source must be metabolic functions in the pig. The relatively high
known. Unfortunately, neither is known precisely at inclusion rates are due to a high proportion of the
the present time. phosphorus in feedstuffs commonly fed to pigs being
in an unavailable form (phytate). Cromwell and
Coffey (1991) reported that as much as two thirds of
NITROGEN the phosphorus in corn-soybean meal swine diets is
in phytate form.
Feeding diets that more closely provide the pig with One way to increase phosphorus bioavailability is
the ideal amino acid pattern has been shown to reduce by including the enzyme phytase in swine rations.
the amount of nitrogen excreted. Traditionally, corn– This enzyme releases a portion of the phytate making
soybean meal diets have an excess of some amino it available to the pig, which in turn reduces the need
acids in order that the requirement of the most limiting for the inclusion of inorganic phosphorus in the diet.
amino acid, lysine, is met. However, excess protein Figure 1, adapted from Kornegay et al. (1998) shows
can be reduced substantially through balancing diets the percentage reduction in phosphorus excreted when
using a reduced amount of soybean meal and phytase is supplied in swine diets with reduced
including crystalline amino acids (Carter et al., 1996). inorganic phosphorus inclusion. McMullen and
By including the crystalline amino acids lysine, Karsten (2001) reported that the inclusion of phytase
threonine, and tryptophan, nitrogen excretion was combined with reduced inorganic phosphorus resulted
reduced by nearly 30% (Bridges et al., 1995). in approximately 20% reduction in phosphorus
Similarly, feeding diets that contain protein sources excreted. Additionally, this study also consistently
that are highly digestible reduces the amount of each demonstrated improved feed efficiency of pigs fed
specific nutrient excreted when the ration is balanced diets containing phytase. Pierce et al. (2001)
based upon bioavailability of the most limiting demonstrated that phytase did not improve feed
nutrient. Kerr and Easter (1995) reviewed several efficiency, but did tend to improve rate of gain. Lyons
articles and estimated that for each percentage point and Cole (2000) summarized several studies, showing
reduction in crude protein obtained by balancing the that a 0 to 0.20 percentage decrease in dietary
diet using crystalline amino acids, nitrogen excretion phosphorus combined with the use of phytase reduced
is reduced by approximately 8 percentage points. phosphorus excretion 23 to 53%.
These results have been more recently supported by There are numerous other studies that demonstrate
those of Sutton et al. (1998), Otto et al. (2003), and improved grow–finish swine performance when
Shriver et al. (2003). phytase is added to diets. This can have positive
environmental impact several ways. First, the direct
effect of reduced phosphorus in the manure of pigs
PHOSPHORUS AND OTHER MINERALS fed diets containing phytase and reduced amounts of
Poor availability of minerals can pose a greater added inorganic phosphorus. Second, if feed
environmental problem. Of particular concern are the efficiency is improved, the same number of pigs
heavy metals and other minerals. Table 3 shows the would excrete less nutrients as it would take less feed
45
40
35
Decrease in P excreted (%)
30
25
20
15
10
0
0.179 0.184 0.193 0.2 0.208 0.219 0.233 0.275
Total phosphorus in excreta (%)
Figure 1. Reduced phosphorus excretion from grow-finish pigs fed increasing amounts of supplemental phytase
(adapted from Kornegay et al., 1998).
to produce the same amount of gain. Third, if rate of (Figure 2). Leeson (2003) found that using trace
gain is improved and pigs are sold at the same minerals with greater bioavailability (Bioplex™ trace
weights, the pigs would be on feed for fewer days mineral) did not affect body weight gain and had
thus reducing the amount of feed needed for little effect on feed efficiency of broilers even when
maintenance and reducing the amount of nutrients fed at 20% of the inorganic trace mineral level. The
excreted. Lastly, it has been documented that phytate authors are conducting a similar study with swine
increases the dietary requirement for zinc. If phytase although the lowest level of the Bioplex™ trace
is utilized and the phytate thereby hydrolyzed, then mineral inclusion is not as low as that used in the
the amount of dietary zinc can be reduced, thereby Leeson (2003) broiler study.
reducing the potential environmental impact of excess
dietary zinc.
Trace minerals are becoming an environmental PHASE FEEDING
concern in areas that rely on repeated land application
of manure. Metals accumulate in the soil when fed Another way to reduce the amount of nutrients
in excess to pigs. These minerals pose problems, as excreted is to meet the pig’s requirements for growth
they can adversely affect the growth of aquatic more precisely. This can be accomplished many ways.
organisms or even become toxic to some fish like It is well known that different genetic lines have
blue gill, minnows, and rainbow trout (Davis, 1974). different capacities for lean growth deposition
Additionally, certain metals tend to accumulate in (Schinckel and deLange, 1996; Thompson et al.,
the food chain and pose a toxicity problem to sensitive 1996). Producers should know the genetic capacity
animal species, such as sheep. Use of minerals with of their pigs for growth, lean deposition, milk
high bioavailability and phase feeding to meet production, etc. in order to match diet nutrient
physiological needs can substantially reduce mineral fortification with level of production. Additionally,
amounts excreted and the environmental risk of soil- it is well known that the requirements of nursery and
applied manure. grow–finish swine change as body weight and feed
Pierce et al. (2001) demonstrated that swine fed intake increase. The Iowa State Life Cycle Swine
diets containing organic minerals (Bioplex™) had Nutrition Guide (Holden et al., 1996) demonstrates
similar performance to those fed inorganic sources, how diets can be modified for pigs of different body
but had a 46% decrease in fecal copper concentrations weights. Similarly, it has been shown (Schinckel et
al., 1996) and accepted by the industry that the
400 Males Females

350
300
Fecal Cu (ppm)
250
200
150
100
50
0
Control Cu sulfate Bioplex Cu
Figure 2. Effect of copper source on fecal copper content of growing pigs (Pierce et al., 2001).
nutrient requirements of barrows and gilts are represents an environmental burden. However, there
different and indeed recommendations for feeding is also an environmental impact associated with
them are different (Holden et al., 1996). The most underfeeding when deficiencies result in slower
appropriate way to meet the nutrient requirements growth and/or poorer feed efficiency.
of pigs and minimize the excreted nutrients would
Table 4. Effect of phase feeding on nitrogen excreted in pigs from
be to change the diet on a daily basis. However, this 25 to 105 kg. 1
is not practical and in most cases not more than 4-6
Single feed Two-feedsa Three-feedsb
diet changes are recommended for barrows and gilts (17% CP) (17% then 17%, followed
throughout the grow–finish phase (Holden et al., 15% CP) by 15%,
1996). The majority of US pork producers are followed by
implementing some form of phase feeding and a large 13% CP
portion implement split sex feeding. In this manner, Nitrogen output, g/day 31.9 29.0 26.7
pigs of different genders and live weights can be fed Percentage of two-feed 110 100 92
strategy
diets that more closely meet their needs. This type of
a
feeding method reduces the environmental impact to Crude protein changed at 55 kg
b
Crude protein changed at 50 and 75 kg
a larger degree when compared to a feeding method 1
from Henry and Dourmad, 1993
that utilizes a diet that meets the needs of a younger,
smaller animal and is fed throughout the full grow – It has been demonstrated that improving growth and
finish period. feed efficiency can reduce the environmental impact
Henry and Dourmad (1993) demonstrated that of livestock production. As previously mentioned,
nitrogen output could be reduced when grow-finish genetic lines that have higher lean content typically
pigs were fed two or three diets rather than a single have better feed efficiency compared to lines that
phase feeding regimen (Table 4). Clearly, when have less lean growth capacity. However, there are
feeding based on averages (either live weight or across numerous other factors that can influence the growth
sexes), the compromise implied will affect nutrient and feed efficiency of grow-finish pigs. Certainly,
excretion. First, when feeding by average weight health challenges can reduce growth and feed
whether for one phase or multiple phases, at some efficiency and could contribute to increased
point the pig is overfed while at other times the pig environmental impact of pork production. Further,
is underfed. Obviously, when the pig is overfed the diseases that result in poorer absorption of dietary
unused nutrients are converted to waste, which nutrients would have a negative environmental
impact. Maintaining a healthy herd can beneficially Table 5. Least square means (± SE) of output of nutrients by swine
affect both the pork producer’s bottom line and the genetic groups.1
environment. Other environmental and management Genetic group
factors can affect feed efficiency and, hence, alter Nutrient, g Paternal x Paternal line Maternal line
the environmental burden associated with the farm. maternal line
Such factors include poor ventilation, overcrowding, Nitrogen 962 ± 39 x 971 ± 36 x 860 ± 43y†
mycotoxins in feed, and a host of other factors. Ammonia 400 ± 28 x 442 ± 26 x 428 ± 31 x
Additionally, proper feed processing influences Phosphorus 291 ± 8x 281 ± 8x 218 ± 9 y
Calcium 281 ± 12 x 245 ± 11y† 184 ± 13 z
nutrient availability by reducing anti-nutritional Zinc 8.32 ± 0.33y† 9.03 ± 0.30x 5.97 ± 0.37z
factors (Han et al., 2001). Iron 8.93 ± 0.38x 9.20 ± 0.35x 7.27 ± 0.43y
Copper 0.94 ± 0.03x 0.97 ± 0.03x 0.67 ± 0.04y
Potassium 408 ± 17 x 418 ± 15 x 370 ± 19y†
FEED WASTAGE a
Adjusted for total weight of pigs (n=8) in pen and feed
disappearance. Total output of liquid and solid material for 3
Another obvious way to reduce the environmental days.
x, y, z
Values with different superscripts in the same row differ
impact of pork production is to control feed wastage. significantly (P<0.01).
Van Heugten and Van Kempen (1999) indicated that 1
Adapted from Crocker and Robison (2002).
feed wastage ranges from 2 to 20% depending on
the country and particular study involved. Producers
should pay close attention to feeder adjustment to emissions would be to reduce the amount of nitrogen
ensure that wastage is minimized. Other factors, such excreted (Powers, 2002). This can be accomplished
as feeder design, feeder placement, and maintenance by not over-fortifying crude protein. As previously
can influence feed wastage. mentioned, it is possible to meet the amino acid needs
of the pig more closely by using crystalline amino
acids without over-fortifying the diet with protein
GENETIC INFLUENCES sources such as soybean meal. This will allow the
diet to more closely meet the pig’s amino acid needs
Crocker and Robinson (2002) demonstrated that there rather than over-fortifying the diet with some amino
is a genetic component to nutrient content in swine acids such that other amino acid requirements are
excreta. Table 5, adapted from Crocker and Robison, met. Excretion of excess amino acids requires energy,
shows the genetic differences in various nutrient which could otherwise be used for growth. For each
contents in the excreta of swine. Pigs from the 1% decrease in dietary crude protein, on average a
maternal line excreted less phosphorus, calcium, corresponding ammonia reduction of 10% is observed
copper, zinc, and iron than did the paternal line or (Sutton et al., 1997; Kay and Lee, 1997; Blair et al.,
the F1 cross of the two lines. These differences, on a 1995; Jacob et al., 1994; Aarnink et al., 1993).
daily basis, may be the result of slower growth and However, the diets using several crystalline amino acids
reduced feed intake of the maternal line. However, have tended to be more costly than those diets using
one should examine the total amount of nutrient soybean meal and thus producers resist implementation.
excreted over the entire growing phase. Some lines While reducing dietary crude protein lowers
grow faster and excrete more nutrients per day, but ammonia emissions, it may not help reduce the
excrete less total nutrients throughout the growing offensiveness of swine manure odor. A study by Otto
phase when compared to lines that grow slower, et al. (2003) demonstrated that an ammonia emission
excrete fewer nutrients on a daily basis, but require a reduction was obtained when dietary crude protein
greater number of days to reach the same weight. was reduced from 15% to 9% or 6% in diets that
The Crocker and Robison (2002) study also showed were supplemented with crystalline amino acids. This
that gilts excrete lower levels of all nutrients compared study also reported that odors from manure from pigs
to barrows. fed these diets were more offensive than the manure
from pigs fed the control 15% crude protein diet.
PROTEIN
FIBER AND CARBOHYDRATES
Nutritional strategies exist to reduce odor and its
components. The obvious method to reduce ammonia Sutton et al. (1998) reported that adding low levels
of an oligosaccharide and cellulose reduced the manure are emitted during storage, treatment,
nitrogen content of fresh swine manure, especially transport and disposal (Van Horn and Powers, 2004).
the ammonia nitrogen fraction. This study indicated While the odor-causing compounds emitted are not
that inclusion of these products stimulates at toxic levels downwind of swine facilities, the odors
fermentation in the colon, reducing the amount of can be offensive to those living around swine
ammonia in the fresh manure. An additional benefit operations. The offensiveness of livestock odors can
from the addition of cellulose to the pig’s diet was be dependent on several factors that are generally
that the pH of fresh and stored manure was reduced, subjective. Not all odors smell exactly the same to
which would help control ammonia volatilization. each person nor is the degree of offensiveness the
This study did not evaluate the economics of the diets same from person to person. The odors associated
or cost of gain for the pigs on test. Other tests have with swine production are caused by the products of
used non-starch polysaccharides and found that total anaerobic decomposition of manure (Van Horn and
nitrogen excretion did not differ but route of excretion Powers, 2004). Certainly, diet composition can affect
shifted from urine to fecal-excreted nitrogen (Canh the amount of substrate available for anaerobic
et al., 1997). Fecal-excreted nitrogen is less volatile decomposition. At the present time, the gases most
than urine nitrogen. associated with odor concerns are ammonia and
hydrogen sulfide. They also are the most commonly
regulated. Methane gas is also a concern, but not from
SULFUR an odor standpoint as it is odorless. Methane gas
concerns arise from its contribution to greenhouse
Many of the odors found most offensive have the gases and global warming (Van Horn and Powers,
‘rotten egg’ smell indicative of sulfur containing 2004).
compounds. Shurson et al. (1998) concluded that
reducing the amount of dietary sulfur resulted in a 2
to 40% reduction in odor concentrations. One might Conclusions
also theorize that allowing pigs to consume water
with high sulfur content might also contribute to Livestock production is becoming more concentrated
manure odor unless other dietary sources of sulfur in many parts of the world and pork production is no
(i.e. trace mineral mixes) are adjusted accordingly. exception. The fact that the same number of pigs is
Reduced sulfur compounds are formed under being produced on fewer farms does increase the
anaerobic conditions (Mackie et al., 1998). This type environmental risk. When the environmental risks are
of condition is typically found when manure is stored combined with the general public concerns about the
in deep pits or lagoons and is released when the environment and a larger number of rural households
storage structure is agitated. However, other storage without livestock or experience with animal
systems, like shallow pit recharge and flush, are not production, livestock producers must find ways to
likely to facilitate the production of hydrogen sulfide reduce the environmental impact of their operations.
containing compounds within the building. Van In many cases federal, state, and local laws have
Kempen and van Heugten (2003) hypothesized that imposed regulations designed to reduce the
when these compounds are formed in the anaerobic environmental impact of livestock production and
portion of lagoons, the hydrogen sulfide compounds producers are forced to comply. The environmental
are metabolized by aerobes in the upper layer of a impact of pork production can be reduced through
properly function lagoon. Therefore, strategies are nutritional methods. Many of the nutritional methods
site-specific in nature if they are to be effective. can have the added benefit of reducing costs of
production to pork producers.
Diet and air quality

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161-186.
C.D. Mateo and H.H. Stein 159
Nucleotides and young animal health: can we enhance intestinal tract

development and immune function?
C.D. MATEO AND H.H. STEIN
Department of Animal and Range Sciences, South Dakota State University, Brookings, South
Dakota, USA
Introduction
The development of antibiotic resistance in humans only limited information is available about the role
has led to a growing interest in antibiotic-free animal of nucleotides in the development of the immune
production worldwide (Bager et al., 2000). system and intestinal health. The objective of the
Unconventional management, feeding practices, and current contribution is to give an update on the current
additives that act as alternatives to antibiotics have understanding of the roles and functions of nucleotides
been tested (Turner et al., 2001; Stein, 2002). in young animal feeding.
Research in human nutrition has demonstrated that
the inclusion of nucleotides in parenteral formulas
and infant milk formulas improves intestinal health Nucleotide biochemistry and
and the development of the immune system in infants. nomenclature
Because of their role in maintaining intestinal health,
dietary nucleotides may act as an alternative to Nucleotides are ubiquitous molecules with
antibiotics in the feeding of young animals. If this considerable structural diversity. They are composed
were true it would provide the livestock industry with of a nitrogenous base linked to a pentose sugar to
an alternative when looking to formulate antibiotic- which at least one phosphate group is attached (Figure
free feeds. This would be helpful in particular in 1). The pentose sugar may either be a ribose for a
diets for young animals where in-feed antibiotics are ribonucleic acid (RNA) or a 2´-deoxyribose for a
most efficient. However, very little data relative to deoxyribonucleic acid (DNA). The nitrogenous base
the young animal’s need for nucleotides exist; and can be either a purine or a pyrimidine. Pyrimidine
N
Phosphate group N Phosphate group N

attached to C5 Base attached to C5 Base
-2O P O CH2 N -2O P O CH2 N N

3 3
O O H
Glycosidic Glycosidic
H H H H
H H linkage H H linkage
OH OH OH OH
Pentose sugar Pentose sugar
Pyrimidine nucleotide Purine nucleotide

Figure 1. Structure of a nucleotide.
160 Nucleotides and young animal health: can we enhance intestinal tract development and immune function?
bases are composed of six-membered rings and Sources of nucleotides in food and feed
comprise uridine, cytosine, and thymine (Table 1).
Purine bases have an additional five-membered ring Nucleotides, particularly IMP, are mainly found in
and comprise adenine, guanine, and hypoxanthine. food rich in protein (Carver and Walker, 1995).
The phosphate group may be in a mono-, di-, or tri Generally, feed or food ingredients containing cellular
phosphate form, and is commonly esterified to the elements are potential dietary sources of nucleotides
C-5′ hydroxyl group of the pentose sugar (Rudolph, in the form of nucleoproteins. Organ meats, poultry,
1994). and seafood are good sources of nucleoproteins
When the phosphate group is absent, the compound (Kojima, 1974; Clifford and Story, 1976; Barness,
is known as a nucleoside. Nucleosides are formed by 1994). Yeast protein sources, baker’s or brewer’s yeast
a nitrogenous base attached to a pentose sugar through and yeast extract, are ingredients that have a relatively
a glycosidic linkage between the N-1 nitrogen of a high concentration of nucleotides (Maloney, 1998;
pyrimidine or the N-3 nitrogen of a purine, and the Ingledew, 1999; Tibbets, 2002). Feed ingredients are
C-1 carbon of the pentose sugar (Voet and Voet, not routinely analyzed for nucleotide concentrations,
1995). A chain of nucleotides attached together via a but data are available for a few ingredients (Table
phosphodiester linkage at the 3C and 5C positions of 2). Most commonly used feed ingredients contain
neighboring ribose units are called polynucleotides relatively low amounts of nucleotides.
or nucleic acids. Nucleic acids conjugated to proteins
are called nucleoproteins.
Table 1. Nucleotide nomenclature.

Base: Product: Nucleoside Ribo- Deoxyribo- Diphosphate Triphosphate
nucleotide a nucleotideb nucleotide c nucleotided
Purines:
Adenine Adenosine AMP dAMP ADP/ dADP ATP/ dATP
Guanine Guanosine GMP dGMP GDP/ dGDP GTP/ dGTP
Hypoxanthine Inosine IMP - - -
Pyrimidines:
Cytosine Cytidine CMP dCMP CDP/ dCDP CTP/ dCTP
Uracil Uridine UMP dUMP UDP/ dUDP UTP
Thymine Thymidine dTMP dTDP dTTP
a
AMP = adenosine 5′-monophosphate; GMP = guanosine 5′-monophosphate; IMP = inosine 5′-monophosphate; CMP = cytidine 5′-
monophosphate; UMP = uridine 5′-monophosphate
b
dAMP = deoxyadenosine 5′-monophosphate; dGMP = deoxyguanosine 5′-monophosphate; dCMP = deoxycytidine 5′-monophosphate;
dUMP = deoxyuridine 5′-monophosphate; dTMP = deoxythymidine 5′-monophosphate
c
ADP = adenosine 5′-diphosphate; dADP = deoxyadenosine 5′-diphosphate; GDP = guanosine 5′-diphosphate; dGDP = deoxyguanosine
5′-diphosphate; CDP = cytidine 5′-diphosphate; dCDP = deoxycytidine 5′-diphosphate; UDP = uridine 5′-diphosphate; dUDP =
deoxyuridine 5′-diphosphate; dTDP = deoxythymidine 5′diphosphate
d
ATP = adenosine 5′-triphosphate; dATP = deoxyadenosine 5′-triphosphate; GTP = guanosine 5′-triphosphate; dGTP = deoxyguanosine 5′-
triphosphate; CTP = cytidine 5′-triphosphate; dCTP = deoxycytidine 5′-triphosphate; UTP = uridine 5′-triphosphate; dTTP =
deoxythymidine 5′-triphosphate
Table 2. Nucleotide concentration in some commonly used feed ingredients (as is basis)a.
Nucleotide (mg/g)
Ingredient 5′CMP 5′AMP 5′GMP 5′UMP 5′IMP
Barley 0.002 0.001 0.001 0.000 0.001
Casein 0.001 0.000 0.000 0.000 0.000
Corn 0.003 0.002 0.003 0.000 0.001
Fish meal 0.026 0.011 0.002 0.001 0.035
Naked oats 0.003 0.003 0.003 0.001 0.001
Plasma protein, spray dried 0.002 0.002 0.002 0.000 0.001
Red blood cells, spray dried 0.000 0.044 0.003 0.002 0.006
Soy protein concentrate 0.000 0.001 0.002 0.000 0.001
Soybean meal, 44 % 0.016 0.008 0.003 0.009 0.002
Whey, dried 0.270 0.019 0.000 0.001 0.004
a
Data from Mateo et al. (2004a)
The nucleotide concentration in the milk of lactating Digestion, absorption and metabolism of
mammals is species-specific and the concentration nucleotides
of most nucleotides changes during the lactation
period (Table 3) (Johke, 1963; Gil and Sanchez- Dietary nucleoproteins, nucleic acids, and nucleotides
Medina, 1981; Gil and Sanchez-Medina, 1982; Mateo need to be enzymatically hydrolyzed prior to
et al., 2004). In a recent experiment in our absorption because only nucleosides, bases, and small
laboratory, we demonstrated that the concentration amounts of nucleotides are absorbed. This process
of 5′AMP, 5′CMP, 5′GMP, 5′IMP, and 5′UMP in takes place in the small intestine. Endonucleases,
porcine milk changed during the initial two weeks phosphodiesterases, and nucleoside phosphorylase are
of lactation, but was relatively constant after that the major enzymes involved in this process (Figure
(Mateo et al., 2004b). 2). These enzymes originate from the brush border
Because of the species differences in milk epithelium (Markiewicz, 1983; Morley et al., 1987),
nucleotide concentration, it is possible that the pancreatic juice (Weickman et al., 1981), and bile
nucleotide requirement may also vary among species, (Holdsworth and Coleman, 1975). In contrast to
but at this point there are no data available on the adults, the exact metabolism of nucleic acids ingested
nucleotide requirements of animals. The demand for by infants is unknown (Carver and Walker, 1995).
nucleotides increases during periods of stress and rapid However, an attempt to evaluate the capability of
growth. Therefore, the requirement may be elevated infants to metabolize nucleic acids and nucleotides
during the immediate post-weaning period of livestock was made by Thorell et al. (1996), who suggested
species. Current research in our laboratory is that enzymes for nucleotide catabolism are present
addressing this hypothesis.
Table 3. Concentration of adenosine 5′′-monophosphate (AMP), guanosine 5′′-monophosphate (GMP), inosine 5′′-monophosphate (IMP),
citidine 5′′-monophosphate (CMP), and uridine 5′′-monophosphate (UMP) in milk from different species (µmoles/100ml)ab.
Nucleotide Milk Day of lactation
5-7 8 10-11 14-15 21 28-31
e e
AMP Human 2.24 - - 2.60 - 2.02e
Bovine 3.15d 1.80c - 2.91 d 1.81d -
Caprine 11.00 d 6.30d 12.20 c 2.79 d - 4.07d
Ovine - 15.67 d - 11.87 d - 8.47d
Equine - - 0.50c - - -
Porcine 12.80f - - 6.80 f 4.30f 3.00 f
CMP Human 3.10 e - - 2.64 e - 1.87e
Bovine 3.02d 6.20c - 4.90 d 4.12d -
Caprine 8.07d 5.86d - 2.28 d - 3.55d
Ovine - 23.30 d - 7.17 d - 8.70d
Equine - - 1.50c - - -
Porcine 7.10 f - - 3.50 f 2.30f 2.50 f
GMP Human 0.50 e - - - - 0.32e
Bovine 0.83d - - - -
Caprine - - 1.70c 0.99 d - 0.70c
Ovine - 1.50d - - - -
Equine - - - - - -
Porcine 14.00f - - 10.20f 6.00f 7.10 f
IMP Porcine 2.60 f - - 1.40 f 0.90f 0.40 f
d d
UMP Bovine 2.87 1.30 - - - -
Caprine 12.37 d 12.59 d 5.90c 16.08 d - 12.64d
Ovine - 65.16 d - 20.07 d - 26.08d
Equine - - 7.70c - -
Porcine 263.10 f - - 144.00 f 122.80 f 104.00 f
a
Number of samples analyzed varied between 4 and 12
b
Nucleotide analysis via enzymatic analysis or HPLC.
c
Data from Johke (1963)
d
Data from Gil and Sanchez-Medina (1981)
e
Data from Gil and Sanchez-Medina (1982)
f
Data from Mateo et al. (2004b)
Nucleoproteins
Proteases ENTEROCYTE
Nucleic acids
Pancreatic nucleases /
Phosphodiesterases
Purines and
Nucleotides Pyrimidines
(mono, di, tri)
Pi Intestinal alkaline
phosphatases /nucleotidases
Nucleosides Nucleosides
Pentose sugars Nucleosidases
Purines and Purines and

Pyrimidines Pyrimidines
Figure 2. Digestion and absorption of nucleic acids and their related products (adapted from Quan and Uauy, 1991).
in the fetal small intestine and act on the nucleotide tissue. If these products are not re-utilized for
containing substrates. nucleotide production or are unabsorbed, the purine
The duodenum has the greatest absorptive capacity and pyrimidine bases are catabolized into uric acid
(Bronk and Hastewell, 1987). Differences in the and ß-alanine or ß-aminoisobutyrate (Rudolph, 1994;
efficiency of uptake among nucleosides have been Carver and Walker, 1995; Thorell et al., 1996). In
reported with guanosine being taken up most rapidly mammals except for primates, uric acid is further
(Sanderson and He, 1994). Under physiological catabolized into allantoin via the enzyme uricase.
conditions, nucleotides have a limited capacity to pass Allantoin is then excreted into the urine. In avian
through cell membranes (Sanderson and He, 1994). species and primates, uric acid is excreted via the
This may be due to the absence of a nucleotide urine. The catabolic products of pyrimidine bases
transport system. Nucleotides also have a high (i.e., ß-alanine and ß-aminoisobutyrate) are further
negatively charged phosphate group that hinders metabolized into ammonia, carbon dioxide and
absorption. Therefore, the nucleoside form is the acetyl CoA.
major vehicle for entry of purines and pyrimidines
into the epithelial cells. More than 90% of dietary
and endogenous nucleosides and bases are absorbed Synthesis of nucleotides
into the enterocyte (Salati et al., 1984; Uauy, 1989).
Nucleoside transport into the enterocyte occurs by Humans and animals can synthesize nucleotides de
facilitated diffusion and by specific Na+-dependent novo provided that the precursors are available. This
carrier mediated mechanisms (Bronk and process takes place in the cytosol of hepatocytes
Hastewell, 1987). where all the enzymes for purine and pyrimidine
From the enterocyte, partial metabolic products of synthesis are available. The purine IMP is synthesized
dietary and endogenous nucleotides and nucleosides from α-D-ribose-5-phosphate via a process involving
enter the hepatic portal vein. These molecules are 11 reactions. In the first reaction, α-D-ribose-5-
carried to the hepatocytes for further metabolism. phosphate is phosphorylated at C1 by a phosphate
From the liver, partial metabolic products of dietary group donated by ATP to form 5-phosphoribosyl-1-
and endogenous nucleotides and nucleosides are pyrophosphate (PRPP). In the second reaction, an
released into systemic circulation and enter muscle N-glycosidic bond is formed to synthesize 5-
phospho-ß-D-ribosylamine. Glutamine is the N- Physiological roles of nucleotides

donor in this reaction. Glycine, aspartate, and
tetrahydrofolate derivatives are other precursors The concentration of ribonucleotides is relatively
needed in the synthesis of IMP. Both AMP and GMP constant in all cells, while the concentration of
are subsequently formed from IMP via deoxyribonucleotides varies with the stage of the cell
adenylosuccinate and xanthosine monophoshate, cycle (Barness, 1994). Nucleotides are the building
respectively (Rodwell, 2000). The precursors for blocks for nucleic acids (i.e., DNA and RNA).
pyrimidine synthesis are carbamoyl phosphate and However, nucleotides also have physiological roles
aspartate. The pyrimidine UMP is formed in a process in the body such as being a source of energy (i.e.,
involving six reactions. A dephosphorylation of UMP ATP and GTP), functioning as cofactors in oxidation
yields UDP, which is subsequently turned into CMP and reduction reactions (i.e., FAD, NAD +, and
or TMP. Glutamine and N5N10-methylene-folate are NADP+), serving as physiological regulators (i.e.,
needed in the synthesis of CMP and TMP, respectively cAMP and cGMP), and carrying activated
(Rodwell, 2000). The de novo synthesis of both purine intermediates (i.e., UDP-glucose, CMP-sialic acid,
and pyrimidine nucleotides is a metabolically costly and CDP-choline) and acyl groups (i.e., CoA).
process requiring a significant amount of energy in Rapidly dividing tissues have increased DNA
the form of ATP. replication and RNA synthesis (Cory, 1992). Because
Synthesis of a nucleotide from a nucleoside and an of the role of nucleotides in RNA synthesis,
inorganic phosphate group is accomplished via the regeneration of new tissue after injury or relative
Salvage Pathway. The nucleosides used in the nutrient deficiency is accelerated (Iwasa et al., 1997;
Salvage Pathway may originate from dietary sources Yamauchi et al., 1998). Replication of DNA occurs
because most dietary nucleotides are changed to during the S phase of the cell cycle, and during this
nucleosides prior to absorption. The Salvage Pathway time, the enzyme activity is increased for purine and
may also be used to re-synthesize nucleotides via pyrimidine synthesis and nucleotide interconversion
phosphoribosylation of purines and pyrimidines (Carver and Walker, 1995). Therefore, there is an
formed during the catabolism of nucleotides. This increase in demand for nucleotides during cell division
pathway may spare energy and allow cells (i.e., and growth (Tsujinaka et al., 1999).
leukocytes, erythrocytes, bone marrow cells, Dietary nucleotide supplementation has been
intestinal mucosal cells and lymphocytes), which are associated with both humoral and cellular immunity,
incapable of de novo synthesis, to maintain their but the exact mechanism has not been elucidated.
nucleotide pools (Sanderson and He, 1994). Nucleotide deprivation caused the arrest of T-cells
Purines and pyrimidines are synthesized in equal in the G phase of the cell cycle, preventing a response
amounts on a molar basis. The synthesis is strictly to various immunological signals that occur by
regulated via feedback and allosteric regulations transition to the S phase (Kulkarni et al., 1994).
(Rodwell, 2000). Nucleotide deprivations also caused a decrease in
phagocytic activity, lymphokine production, and/or
inhibited lymphocyte maturation (Paubert-Braquet
Storage of nucleotides et al., 1992). Dietary nucleotides contribute to the
circulating pool of nucleosides available to stimulate
Nucleotide metabolism is characterized by constant leukocyte production (Kulkarni et al., 1994; Carver
synthesis and catabolism. Tracer studies in animals and Walker, 1995). Therefore, there is an elevated
indicate that 2 to 5% of dietary nucleotides are retained need for nucleotides indicated during periods of
in the small intestine, liver, and skeletal muscle tissue immunological challenges.
pools (Saviano and Clifford, 1978). Increased tissue Dietary factors play a role in the antibody response
retention has been reported in young animals (Kobota, to immunization of infants. Infants fed milk formula
1969) and during fasting (Saviano and Clifford, 1978; fortified with nucleotides had better responses to
Gross and Saviano, 1991). This may be a immunization as evidenced by an increase in humoral
manifestation of a physiological requirement. antibody response (Fanslow et al., 1988; Pickering
Nucleotide pools are larger in differentiated (i.e., et al., 1998) and increased cytokine production
cancerous) cells than in undifferentiated (i.e., (Carver et al., 1991). In vivo studies in mice show
nonmalignant) cells (Sanderson and He, 1994). This similar results to nucleotide supplementation
suggests that undifferentiated cells are more dependent (Jyonouchi et al., 1993; Jyonouchi, 1994).
on the dietary supply of nucleotides. Nucleotide-free diets supplemented with single
nucleotides (i.e., AMP, GMP, or UMP), have been mented in calf milk replacers improved intestinal
shown to increase immunoglobulin concentration health as demonstrated by improved feacal scores
(Navarro et al., 1996). Dietary supplementation of compared to the non-supplemented control group
purified nucleotides to milk replacers fed newborn (Oliver et al., 2002). In piglets, nucleotides in
bull calves challenged with lipopolysaccharide (LPS), concentrations similar to those in human milk exert
resulted in a trend toward higher mean IgG levels in a protective effect in the intestinal lumen against an
supplemented compared to the unsupplemented calves inflammatory response to ischemia-reperfusion
(Oliver et al., 2003). Nucleotide supplementation also (Bustamante et al., 1994).
increased lymphocyte stimulation to phytohaemag- Dietary nucleosides enhance the growth and
glutinin and concanavalin-A challenges in weanling maturation of intestinal epithelial cells as evidenced
piglets by 50 and 30%, respectively (Zomborsky- by an increased formation of mucosal protein, DNA,
Kovacs et al., 1998). Similar results were observed taller villi in the small intestine and increased
to challenges by keyhole limpet hemocyanin (KLH) maltase:lactase enzyme ratio (Uauy et al., 1990;
and non-specific T-cell mitogens in piglets fed yeast Carver, 1994). Dietary nucleotides may also stimulate
RNA (Cameron et al., 2001). Pigs with pathogenic differentiation (Sanderson and He, 1994). Parenteral
E. coli infection fed diets supplemented with NuproTM, supplementation of nucleic acids supports mucosal
a yeast extract as a source of nucleotides, have reduced cell proliferation and function as demonstrated by
diarrhea and improved weight gain and feed increased mucosal wet weight, protein and DNA
conversion compared to pigs fed the control diet contents, villous height, but not crypt depth, and
(Maribo, 2003). Results of these studies imply that narrower tight junctions of the jejunal width (Kishibuchi
dietary sources of nucleotides play a role in et al., 1997; Tsujinaka et al., 1999). The development
developing, maintaining, and enhancing the immune of the gastrointestinal tract directly affects the degree
system. of nutrient absorption and ultimately, animal growth.
Dietary nucleotides enhance intestinal absorption Because of the role of nucleotides in maintaining
of iron, affect lipoprotein and long chain intestinal morphology and maturation, dietary
polyunsaturated fatty acid metabolism, have trophic nucleotides may be needed during the immediate post-
effects on the intestinal mucosa and liver, and reduce weaning period to maintain the structure and growth of
the incidence of diarrhea (Cosgrove, 1998; Schlimme the intestinal tract.
et al., 2000). Fecal flora of infants fed a nucleotide-
supplemented commercial milk formula had a
predominance of bifidobacteria (Tanaka and Mutai, Are dietary nucleotides needed in diets
1980), while enterobacteria predominated in the fecal for weanling pigs?
flora of infants fed a commercial formula without
nucleotide supplementation (Uauy, 1994). These The need for nucleotides is elevated during periods
studies suggest that nucleotide supplementation may of rapid growth, during periods of stress, and in
positively influence the microflora in the immuno-compromised animals. In newly weaned
gastrointestinal tract, which leads to a lowering of pigs, all of these factors are present – therefore, it is
gastric pH and hinders the proliferation of pathogenic expected that they would have a high requirement
bacterial species as evidenced by a lower rate of for nucleotides during this period. Because nucleotide
diarrhea (Yu, 1998). synthesis is an energy and glutamine-requiring
Nucleotide supplementation increased bacterial process and because newly weaned pigs are often
resistance of mice inoculated with Candida albicans deficient in both energy and glutamine, it is possible
(Fanslow et al., 1988) and Staphylococcus aureus that pigs are not able to synthesize sufficient quantities
(Kulkarni et al., 1986; Carver, 1994). Intraperitoneal of nucleotides during the immediate post-weaning
administration of nucleotides and nucleosides period. If this is correct, dietary nucleotides must be
decreased bacterial translocation, number of colony supplied. In a typical starter diet for weanling pigs,
forming units, and increased the survival of mice the concentration of 5′CMP is close to the
against methicillin-resistant S. aureus (Yamamoto et concentration found in sow’s milk during the last
al., 1997). Therefore, nucleotides are capable of half of lactation, but the concentrations of 5′AMP,
increasing resistance to a wide range of potentially 5′GMP, 5′IMP, and 5′UMP are much lower than in
pathogenic bacteria. sow’s milk (Table 4). Assuming that the concentrations
Dietary nucleotides from yeast extract supple- of nucleotides in sow’s milk are representative of the
Table 4. Calculated nucleotide concentration of a starter diet for increased number of probiotic bacteria (i.e. L.
weanling pigs a . acidophilus and Bifidobacterium spp.) may also result
Nucleotide (ppm) in improved intestinal morphology and improved
CMP AMP GMP UMP IMP nutrient uptake. This hypothesis is being tested in a
current research project at South Dakota State
Total in starter 58.99 6.46 2.03 1.00 4.33 University.
diet b
Sows milk c 56.00 117.50 185.5 2334.50 23.5
Difference 2.99 -111.04 -183.47 -2333.50 -19.17
Conclusion
a
Data from Mateo et al. (2004a)
b
Diet formulated to contain the following feed ingredients: Corn, Nucleotides are molecules with considerable structural
49.32%; Whey powder, 20%, Soybean meal, 8%; Fish meal,
8%; spray dried protein plasma, 7.5%, vitamins, minerals, oil,
diversity. They are composed of a nitrogenous base
and crystalline amino acids, 7.18%. linked to a pentose sugar to which at least one
c
Data from Mateo et al. (2004b) phosphate group is attached. Feed or food ingredients
containing cellular elements are potential sources of
nucleotides. Nucleotides have many important
requirement of the piglets, it is easily concluded that
physiological, gastrointestinal, and immunological
a starter diet for young pigs is deficient in four of
functions in the body. The exact metabolism of
the five nucleotides. It may, therefore, be beneficial
nucleic acids ingested by young animals is unknown.
to add additional nucleotides to such diets. In a recent
Synthesizing nucleotides de novo is metabolically
experiment in our laboratory, we added nucleosides
costly compared to synthesis via the Salvage Pathway.
to a deficient starter diet. We used nucleosides rather
During periods of rapid growth and development,
than nucleotides because dietary nucleotides need to
disease challenges, injury or stress, dietary nucleotide
be digested to nucleosides before absorption. Blood
supplementation may be beneficial because of the
samples and fecal samples were collected from
role of nucleotides in developing and enhancing
weanling pigs on the day of weaning, on day 7 post-
immunity, maintaining intestinal health, and
weaning and on day 14 post-weaning. Blood samples
preserving energy. However, more research is needed
were analyzed for IgG concentrations, while the
to verify this hypothesis.
microbial flora was quantified in the samples. Results
of the experiment showed that fecal counts of
Clostridium perfringens were reduced in samples
collected from pigs fed a nucleoside-supplemented References
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Dairy and
RUMINANTS
beef cattle
L.O. Abdelhadi 171
Evaluating inoculants for forage crops in Argentine beef and milk grazing
systems: effects on silage quality and system profitability
LEANDRO O. ABDELHADI
El Encuentro Livestock farm, Research & Extension in Ruminant Nutrition, Alltech Argentina, Cnel.
Brandsen, Argentina
Introduction
Beef and milk production in Argentina rely mainly 2003) and Abdelhadi and Santini (2003). An increased
on grasslands and improved pastures; and conserved stocking rate is the only benefit of corn or grain
forages or concentrates are fed to the animals sorghum silage supplementation of pasture.
generally when DM availability or nutrient content Nevertheless, Argentina’s producers are far from
of pastures is insufficient to satisfy animal having the high quality silages needed in order to
requirements. Among conserved forage alternatives, justify increasing silage use in rations or to replace
silage production is increasing because of its many concentrate in diets fed high producing animals. If
advantages over other supplements including: we analyze corn silage, one of the easier crops for
making good silage due to its high water soluble
• Less dependence on weather compared with hay. carbohydrate (WSC): buffering capacity (BC) ratio,
• Nutrient content maintained, similar to field crop. it is clear that digestibility coefficients are lower than
in other countries, from a crop of about 75% in vitro
• Higher DM production at low cost compared with dry matter digestibility (IVDMD) at harvest stage
grains. (Figure 1).
There are three major events for making good
Due to the high quality of pastures and relatively
silage: 1) exclusion of air before sealing the silo
low silage inclusion in Argentine diets (<30%, DM
(generally depends on contract workers); 2) rapid
basis), good animal performance was obtained when
rate and extent of pH drop (depends on the extent of
silages supplemented pastures compared with ad
fermentation); and 3) storage phase (depends on
libitum grazing, as shown by Abdelhadi et al. (2001;
farmer management) (Kung, 2001).
90
80
70
IVDMD, %
60
50
40
Holland Belgium USA France Argentina
Figure 1. Corn silage in vitro DM digestibility (IVDMD) around the world (Data from: Schroeder et al., 2000; INRA, 1989; Roth
and Undersander, 1995; Deinum and Struik, 1985; De Boever et al., 1997).
172 Evaluating inoculants for forage crops in Argentine beef and milk grazing systems
The data in Figure 1 come from samples taken in completely randomized design with a 2 x 2 factorial
the silo, not in the feedbunk, and so are the result of arrangement (two stages of maturity with or without
Events 1 and 2. Because the work of contractors is Sil-AllTM inoculation).
often difficult for the farmer to manage, our research
was directed at controlling the fermentation process.
Together with representatives from Alltech Argentina RESULTS
and Monsanto, we conducted a series of studies to
evaluate utilization of Sil-AllTM inoculant to increase DM recovery and fermentation
quality of ensiled crops and hence animal production A maturity x inoculant interaction was found
and profitability of pasture based systems at the El (P<0.06) for DM recovery. Inoculation increased
Encuentro farm (35°23´S; 58°25´W). (P<0.08) silage DM recovery from 92.4% (control)
to 97.9% in silages harvested at the milk stage of
maturity, while no effect was noted (P<0.25) in silage
Effects of Sil-AllTM inoculation on harvested at the dough stage.
fermentation and nutritive value of grain Parameters associated with silage fermentation are
sorghum (whole head) ensiled at two presented in Table 1. Sil-AllTM inoculation decreased
stages of maturity silage temperature after three days of air exposure
(P<0.01) by 0.7 and 1.4 ºC for milk and late dough
METHODS grain stages of maturity, respectively. No effects of
inoculation were detected for pH (P<0.93) or NH3-N
The same grain sorghum hybrid was seeded on (P<0.53), while air exposure increased pH and
December 3 and 20, 2002 on a commercial dairy decreased NH3-N of silages at either stage of maturity
farm (El Recuerdo). Harvest occurred on March 26, (P<0.01).
2003, therefore we had the same cultivar at two stages
of maturity (milk and dough stages). A Class Jaguar Table 1. Sil-AllTM effects on temperature and silage fermentation
860 harvester equipped with a grain cracker was used; of grain sorghum (whole head) at milk or late dough stages of
and the chop length was 7.9 mm. Within 30 min of maturity1.
chopping, samples of fresh material from each stage Temperature Fermentation
of maturity were frozen and triplicate mini silos (ºC) pH NH3-N (% of CP)
constructed of PVC pipe (10 x 39 cm) were made Sampling time Post air Post- Post air Post- Post air
with or without Sil-AllTM inoculation. Weights of silos exposure ensiling exposure ensiling exposure
both empty and full were recorded, and silos were Milk stage
then stored at ambient temperature of about 15 to Control 19.0 4.13 7.37 5.56 1.23
20ºC. After a three month storage period (from March (0.0) (0.0) (0.2) (0.4) (0.2)
to June, 2003) final weights of the full silos were Sil-AllTM 18.3 4.17 7.61 5.33 1.03
recorded and the silage was hand-mixed thoroughly. (0.6) (0.0) (0.1) (0.3) (0.4)
Late dough stage
Dry matter (DM) recovery was calculated as in Kung Control 20.0 4.71 6.90 4.43 2.26
et al. (2003) and pack density in each mini silo was (0.0) (0.1) (0.8) (0.7) (0.7)
estimated as in Johnson et al. (2002). Approximately Sil-AllTM 18.6 4.82 6.44 4.40 3.53
50% of the mixed material was frozen and the (0.6) (0.1) (1.2) (0.1) (1.8)
remainder was exposed to air for three days after Maturity effect (M) 0.02 0.63 0.26
which samples were frozen. Frozen samples of each Sil-AllTM effect (S) 0.01 0.93 0.53
material pre-ensiling (PRE), post-ensiling (POS) and Sampling time effect
(ST) n.d. 0.01 0.01
post-air exposure (PAE), were oven-dried (60ºC), M x S interaction 0.19 0.47 0.21
ground through a 1 mm screen (Willey Mill, M x S x ST
Philadelphia, PA) and analyzed for DM, in vitro interaction n.d. 0.38 0.33
organic matter (OM) degradability (Villalba, 2001), 1
Treatment means (± standard deviation)
WSC (Bailey, 1958), crude protein (CP) (Horneck
and Miller, 1998) and NDF (van Soest, 1991); plus
Nutrient content and in vitro degradability
pH (Cole Parmer portable pH meter) and ammonia-
N (NH3-N) (Weatherburn, 1967) in POS and PAE Neither DM, CP nor NDF concentration were
samples. Statistical analysis was conducted using the affected by inoculation, while WSC in silage
general linear model in SAS (1989) using a increased with Sil-All TM (P<0.05) (Table 2).
L.O. Abdelhadi 173
Significant maturity effects were detected for nutrient economic point of view. Again, increased maturity
concentrations in pre-ensiled samples (P<0.01), negatively influenced quality of material at ensiling.
which underscores the importance of defining harvest
time for grain sorghum silage. Effects of Sil-AllTM inoculation on
In vitro OM degradability was determined after fermentation and nutritive value of
incubation of PRE, POS and PAE samples with
ruminal fluid (Table 3). No significant effects of Sil-
whole-plant grain sorghum silage
All TM inoculation were noted across the entire METHODS
incubation time (P<0.5), but depending on time of
incubation, increases in degradability from 3 to 5 Grain sorghum (MS2, Morgan hybrid) was direct
points were noted and should be considered from an seeded on December 5, 2002 at the Los Aromos dairy
Table 2. Sil-AllTM effects on nutrient concentration of whole-head grain sorghum silage at milk or late dough stages of maturity1,2,3.
Nutrient content (% of DM)
DM, % WSC CP NDF
Sampling time PRE POS PAE PRE POS PAE PRE POS PAE PRE POS PAE
Milk stage
Control 35.1 32.7 32.6 10.9 8.0 11.3 6.8 7.1 6.9 43.2 42.7 46.9
(0.2) (0.2) (0.3) (2.1) (1.3) (1.0) (0.2) (0.4) (0.4) (4.0) (1.1) (1.4)
Sil-AllTM 35.1 35.3 32.8 10.9 10.4 11.7 6.8 6.8 7.5 43.2 42.0 43.8
(0.2) (1.9) (0.6) (2.1) (1.8) (3.1) (0.2) (0.7) (1.0) (4.0) (0.9) (1.9)
Late dough stage
Control 42.9 40.2 41.0 8.9 13.4 11.6 5.2 5.3 5.4 45.4 39.7 42.1
(2.1) (0.8) (0.6) (0.3) (1.1) (1.0) (0.1) (0.2) (0.7) (1.0) (4.1) (3.2)
Sil-AllTM 42.9 38.8 41.8 8.9 14.4 14.3 5.2 5.1 5.7 45.4 41.7 40.8
(2.1) (0.1) (2.8) (0.3) (1.5) (0.5) (0.1) (0.1) (0.3) (1.0) (1.0) (1.6)
Maturity effect (M) 0.01 0.01 0.01 0.19
Sil-AllTM effect (S) 0.41 0.05 0.62 0.54
Sampling time effect (ST) 0.01 0.01 0.17 0.03
M x S interaction 0.21 0.81 0.83 0.37
M x S x ST interaction 0.09 0.38 0.77 0.79
1
Treatment means (± standard deviation); 2PRE= pre-ensiling, POS= post-ensiling, PAE= after 3 days air exposure
3
DM= dry matter, WSC= water soluble carbohydrates, CP= crude protein, NDF= neutral detergent fiber
Table 3. Sil-AllTM effects on in vitro organic matter degradability of whole-head grain sorghum silage at milk or late dough stages of
maturity1,2.
In vitro organic matter degradability (%)
Hours of incubation 24 48 72
Sampling time PRE POS PAE PRE POS PAE PRE POS PAE
Milk stage
Control 43.7 38.5 30.6 64.2 54.1 52.1 73.7 62.3 62.1
(2.0) (1.9) (2.3) (0.7) (1.35) (1.4) (0.9) (1.1) (2.1)
Sil-AllTM 43.7 36.5 27.0 64.2 55.0 49.4 73.7 64.7 68.3
(2.0) (3.8) (1.4) (0.7) (4.4) (4.1) (0.9) (5.1) (4.6)
Late dough stage 28.0 31.4 27.4 51.1 52.5 48.9 67.3 63.4 63.2
(1.5) (1.1) (2.2) (1.3) (2.3) (2.2) (4.3) (2.5) (0.9)
Control 28.0 31.4 30.1 51.1 52.2 53.9 67.3 63.3 61.7
(1.5) (1.7) (2.8) (1.3) (2.5) (1.9) (4.3) (3.1) (6.4)
Sil-AllTM 0.01 0.01 0.29
Maturity effect (M) 0.57 0.55 0.34
Sil-AllTM effect (S) 0.01 0.01 0.01
Sampling time effect (ST) 0.08 0.17 0.46
T x S interaction 0.20 0.06 0.20
1
farm (35º10´S; 58º14´W). Harvesting occurred on fermentation or following aerobic exposure (Table
March 27, 2003 when grain was at the milk stage. 5). In vitro OM degradability of air-exposed silage
Ensiling, sampling methods and quality was lower than the pre-ensiled samples (P<0.01)
determinations were as in the first experiment. Data (Table 6). Depending on time of incubation, Sil-AllTM
were analyzed as a completely randomized design tended to reduce losses in degradability (by 3.5 points).
and treatment comparisons were obtained by ANOVA
using the PROC GLM procedures of SAS (1988). Table 4. Sil-AllTM effects on temperature and silage fermenta-
tion of whole-plant grain sorghum silage at the milk stage of
maturity1,2.
RESULTS
Silage Fermentation
DM recovery and fermentation Temperature pH NH 3-N
(ºC)
Dry matter recovery was not statistically different, Sampling time PAE POS PAE POS PAE
however the numerical increase from 92.1% (control)
Control 22.3 4.1 5.4 6.9 3.5
to 95.1% with Sil-All TM inoculation could be of (2.8) (0.1) (1.3) (1.2) (1.9)
economic relevance. Temperature after three days
Sil-AllTM 23.6 4.1 4.8 6.7 2.8
exposure to air was unaffected (P<0.34) by (0.5) (0.0) (0.3) (0.6) (1.3)
inoculation, as were pH and NH3-N values (Table 4). Sil-AllTM effect (S) 0.34 0.52 0.61
Sampling time
Nutrient content and in vitro degradability effect (ST) n.d. 0.04 0.01
S x ST interaction n.d. 0.42 0.76
There were no significant effects of inoculation on 1
Treatment means (± standard deviation).
nutrient quality of silage samples taken either after 2
POS= post-ensiling, PAE= after 3 days air exposure
Table 5. Sil-AllTM effects nutrient concentration of whole-plant grain sorghum silage at milk stage of maturity1,2,3.
DM, % WSC CP NDF
Control 29.4 27.5 27.1 10.1 6.9 7.7 6.6 7.1 7.6 48.2 52.2 54.4
(0.6) (0.3) (0.5) (0.6) (0.3) (1.3) (0.6) (0.5) (0.2) (1.7) (0.7) (2.4)
Sil-AllTM 29.4 28.6 27.4 10.1 6.9 8.0 6.6 7.6 8.0 48.2 50.8 52.
(0.6) (1.9) (0.3) (0.6) (0.7) (1.4) (0.6) (0.1) (0.7) (1.7) (1.6) 7 (1.9)
S x ST interaction 0.57 0.89 0.74 0.69
1
2
PRE= pre-ensiling, POS= post-ensiling, PAE= after 3 days air exposure
3
Table 6. Sil-AllTM effects on in vitro organic matter degradability of whole-plant grain sorghum silage at milk stage of maturity1,2.
Item In vitro organic matter degradability (%)
Control 43.3 36.2 33.2 64.1 55.8 51.1 73.6 65.2 60.9
(2.8) (4.4) (2.3) (2.2) (6.2) (5.1) (2.0) (7.3) (5.6)
Sil-AllTM 43.3 40.2 34.3 64.1 59.2 53.7 73.6 68.5 63.3
(2.8) (2.2) (4.5) (2.2) (2.4) (2.8) (2.0) (2.7) (2.5)
Sil-AllTM effect (S) 0.30 0.29 0.36
S x ST interaction 0.57 0.73 0.78
1
2
PRE= pre-ensiling, POS= post-ensiling, PAE= after 3 days air exposure
L.O. Abdelhadi 175
Effects of Sil-AllTM inoculation on Table 7. Sil-AllTM effects on temperature and silage fermentation
of whole-plant corn silage at late dough stage of maturity1,2.
fermentation and nutritive value of
Silage Fermentation
whole-plant corn silage Temperature pH NH3-N
(ºC)
METHODS
Sampling time PAE POS PAE POS PAE
Corn (M369, Morgan hybrid) was seeded on October Control 21.6 4.34 6.32 7.76 5.06
25, 2002 in Los Aromos dairy farm (35º10´S; 58º14´W). (0.5) (0.2) (1.7) (0.4) (1.4)
Ensiling, sampling methods and quality determinations Sil-AllTM 20.0 4.13 5.61 6.96 3.60
were as in the previous experiments, but harvest occurred (0.0) (0.1) (1.4) (0.5) (0.7)
at the late dough stage of maturity on March 27, 2003. Sil-AllTM effect (S) 0.01 0.05 0.50
Statistical analysis was as in the second experiment. Sampling time
effect (ST) n.d. 0.01 0.03
S x ST interaction n.d. 0.52 0.71
RESULTS
1
Treatment means (± standard deviation).
2
DM recovery and fermentation POS= post-ensiling, PAE= after 3 days air exposure
Inoculation with Sil-AllTM increased DM recovery Nutrient content and in vitro degradability
(P<0.05) from 94.5% (control) to 99.6%. The added
5.1 percentage points represents a large amount of A significant increase in DM percentage (P<0.02) and
forage. Sil-AllTM inoculation decreased pH (P<0.05) decreased NDF content (P<0.09) were detected in
measured in samples taken either after silage inoculated corn silage (Table 8). Neither WSC nor CP
fermentation or after aerobic exposure (Table 7). Heating concentrations were affected by inoculation. A trend
was also reduced in the inoculated silage by 1.6ºC. toward increased in vitro OM degradability was detected
Ammonia-N tended to be lower in inoculated silage after 48 (P<0.19) and 72 hrs (P<0.17) of incubation in
but was not statistically different. Sil-AllTM-treated material (Table 9). The added 3.5%
Table 8. Sil-AllTM effects on nutrient concentration of whole-plant corn silage at late dough grain stage of maturity1,2,3.
DM, % WSC CP NDF
Control 37.3 36.2 35.7 9.5 10.7 11.0 8.2 9.6 9.0 47.7 44.6 45.8
(0.4) (0.6) (2.5) (0.7) (0.8) (0.8) (0.0) (0.1) (0.4) (0.6) (1.8) (4.1)
Sil-AllTM 37.3 38.9 38.0 9.5 10.1 11.5 8.2 9.0 9.8 47.7 42.1 42.8
(0.4) (0.2) (1.7) (0.7) (0.8) (0.8) (0.0) (0.3) (0.6) (0.6) (1.0) (2.2)
S x ST interaction 0.21 0.84 0.01 0.45
1
Treatment means (± standard deviation); 2PRE= pre-ensiling, POS= post-ensiling, PAE= after 3 days air exposure;
3
Table 9. Sil-AllTM effects on in vitro organic matter degradability of whole-plant corn silage at late dough stage of maturity1,2.
Item In vitro organic matter degradability (%)
Control 40.7 37.4 34.2 60.5 54.0 53.3 69.6 62.6 62.6
(2.5) (2.8) (7.1) (3.6) (3.4) (6.5) (4.1) (3.6) (5.9)
Sil-All 40.7 39.0 38.2 60.5 57.2 58.6 69.6 66.3 68.8
(2.5) (3.9) (2.5) (3.6) (4.2) (3.8) (4.1) (4.2) (4.6)
Sil-All effect (S) 0.34 0.19 0.17
S x ST interaction 0.69 0.58 0.58
1
digestible material is similar to values reported for grain Conclusions

sorghum silage.
Even though ensiling grain sorghum and maize is
Economic evaluation of Sil-All TM easier due to higher WSC:BC ratio, significant losses
inoculation for Argentine systems have been quantified in Argentine silos. These
experiments were conducted to evaluate the Sil-AllTM
Because silage inclusion in beef and dairy cattle diets in inoculant as a management tool to reduce such losses.
Argentina is only moderate (<30%, DM basis) less Data obtained showed increased DM recovery and in
benefit will be achieved when increasing silage quality vitro OM degradability of ensiled crops when Sil-
than in other feeding systems. The chief benefit of AllTM was used. In addition, a better fermentation
inoculation in the Argentine system is the added DM pattern and lower NDF content was reported
recovery, which along with improved silage quality, depending on crop evaluated. Since silages in
has the potential to increase animal production and hence Argentina supplement grazing cattle, we evaluated
economic return. these increases in terms of quantity and quality in
To calculate profitability of a silage inoculation economic terms, finding a benefit:cost ratio of 6.5:1
program for Argentina, we considered DM recoveries (beef) or 8.8:1 (dairy) USD per hectare of grain
and increases in degradability obtained. These data were sorghum or corn ensiled. These results will give
transformed to extra animal live weight gain (LWG) or farmers the technical and economic information
milk production using the following criteria: needed to implement an inoculation program for
pasture-based systems.
1) 8.8 kg DM of silage = 1 kg LWG/ha and 5.3 kg
digestible DM (DDM) = 1 kg LWG/ha in grazing
beef cattle (Abdelhadi et al., 2001; 2003; Abdelhadi References
and Santini, 2003).
2) 1.45 kg DM of silage = 1 kg milk and 0.87 kg Abdelhadi, L.O., F.J. Santini and G.A. Gagliostro.
DDM = 1 kg milk in grazing dairy cows (Abdelhadi 2001. Whole plant silage as a supplement for
et al., 2001) (using a mean digestibility of about grazing cattle: Effects on productive performance
60% (DM basis) generally obtained in commercial and ruminal environment. Rev. Arg. Prod. Anim.
farms) (Table 10). 21(3-4):147-158.
Abdelhadi, L.O., F.J. Santini and G.A. Gagliostro.
Finally, the cost of inoculation was deducted from extra 2003. Corn silage or high moisture corn
return to obtain the benefit:cost ratio. Benefit:cost ratios supplements for beef heifers grazing temperate
of 6.5:1 (beef) and 8.8:1 (dairy) USD per hectare of
pastures: Effects on performance, ruminal
grain sorghum or corn ensiled were obtained (Table 10).
fermentation and in situ pasture digestion. Anim.
Table 10. Benefit:cost ratio of Sil-AllTM inoculation in ensiled corn Feed Sci. and Tech. (In press).
or sorghum silages for supplementing grazing beef or dairy cattle. Abdelhadi, L.O. and F.J. Santini. 2003. Replacing
Item Beef Dairy corn silage with grain sorghum silage to supplement
Extra DM recovery1, kg/ha 800 800 growing steers grazing high quality pastures:
Extra digestible DM2, kg/ha 700 700 Effects on performance and ruminal fermentation.
DM silage/kg LWG or milk, kg 8.8 1.45
DDM silage/kg LWG or milk, kg 5.3 0.87
Anim. Feed Sci. and Tech. (In press).
Extra LWG or milk potentially obtained, kg 223 1356 Bailey, R.W. 1958. Reactions of pentoses with
Market prices3 per kg of beef or milk, USD 0.69 0.16 anthrone. Bioch. J. 68:669.
Extra $ obtained per ha of ensiled crop 153.9 216.9 De Boever, J.L., B.G. Cottyn, D.L. De Brabander,
Cost of inoculation (USD) for silage yield 24.6 24.6
obtained4 J.M. Vanacker and Ch.V. Boucqué. 1997. Prediction
Benefit:cost ratio 6.2:1 8.8:1 of feeding value of maize silages by chemical
1
Mean production DM/ha for sorghum and corn = 20 tons, parameters, in vitro digestibility and NIRS. Anim.
considering DM recovery of 5% extra by inoculation. Feed Sci. and Tech. 66:211.
2
Mean production DDM/ha for sorghum and corn = 12 tons, Deinum, B. and P.C. Struik. 1985. Improving the
considering an extra of digestible DM of 3.5 points which is
5.83% extra of DDM/ha. nutritive value of forage maize. Proc. 13th Congress
3
Expressed in USD, considering local money exchange of 2.9 of the Maize and Sorghum Section of Eucarpia,
Argentinean pesos = $1. September 9-12, 1985, Wageningen. The
4
Mean of about 60 tonnes of fresh material per hectare.
Netherlands, pp. 77-90.
L.O. Abdelhadi 177
Horneck, A.D. and R.O. Miller. 1998. Determination Roth, G.W. and D. Undersander. 1995. Corn silage
of total nitrogen in plant tissue. In: Handbook of production, management and feeding. American
Reference Methods for Plant Analysis (Y.P. Kalra, Society of Agronomy, Inc., Crop Science Society
ed). Soil and Plant Analysis Council, Inc. CRC of America, Inc. and Soil Science of America, Inc.,
Press, p. 300. p. 42.
Institut Nationale Recherche Agronomique (INRA). SAS. 1989. SAS/STAT User’s Guide: Ver. 6, 4th Ed.,
1989. Ruminant nutrition. Recommended Vol. 2, Cary NC: SAS Institute Inc.
allowances and feed tables (R. Jarrige, ed). Paris: Schroeder, G.F., J.C. Elizalde and J.P. Fay. 2000.
INRA, p. 389. Characterization of the nutritive value of corn silage
Johnson, L.M., J.H. Harrison, D. Davidson, W.C. produced in Buenos Aires province. Rev. Arg. Prod.
Mahanna, K. Shinners and D. Linder. 2002. Corn Anim. 20(3-4):161-177.
silage management: Effects of maturity, inoculation van Soest, P.J., J.B. Robertson and B.A. Lewis. 1991.
and mechanical processing on pack density and Methods for dietary fiber, neutral detergent fiber
aerobic stability. J. Dairy Sci. 85:434-444. and nonstarch polysaccharides in relation to animal
Kung, L., Jr. 2001. Silage fermentation and additives. nutrition. In: Proceedings of the Symposium on
In: Proceedings of Alltech’s 17 th Annual Carbohydrate Methodology, Metabolism, and
Symposium. (K.A. Jacques and T.P. Lyons, eds). Nutritional Implications in Dairy Cattle. J. Dairy
Nottingham University Press, Nottingham, UK, pp. Sci. 74: 3583-3597.
145-159. Villalba, S.E. 2001. Producción de gas in vitro. Uso
Kung, L., Jr., C.C. Taylor, M.P. Lynch and J.M. de la técnica para estimar la degradabilidad de los
Neylon. 2003. The effect of treating alfalfa with alimentos para rumiantes. Trabajo final de
Lactobacillus buchneri 40788 on silage graduación. Facultad de bromatología UNER.
fermentation, aerobic stability and nutritive value Weatherburn, M.W. 1967. Phenol-hypochlorite
for lactating dairy cows. J. Dairy Sci. 86:336-343. reactions for determination of ammonia. Anal.
Chem. 39:971-974.
V. Akay et al. 179
Optigen® 1200: controlled release of non-protein nitrogen in the rumen

VEYSEL AKAY1, JEFF TIKOFSKY1, CORWIN HOLTZ2 AND KARL A. DAWSON1
1
2
Comsen Dairy Consultation, LLC, Dryden, New York, USA
Introduction
Non-protein nitrogen (NPN) includes any compound utilization of urea (Stern et al., 1994). In contrast,
that contains nitrogen (N) but which is not present in greater dietary concentrations of NSC have increased
the polypeptide form of proteins. Inexpensive NPN, the utilization of ruminal ammonia for the synthesis
especially urea, is very useful in ruminant rations of bacterial protein (Hoover and Stokes, 1991). These
because it is hydrolyzed to ammonia in the rumen findings indicate that microbial metabolism in the
and can be incorporated by microbes into amino acids rumen is a complex process requiring an
and bacterial proteins, which are utilized subsequently understanding of both the rate and extent of
by the host animal. As a result, NPN is used in dairy carbohydrate digestion and ammonia supply for
and beef rations as a less expensive alternative to efficient bacterial growth in the rumen.
pre-formed proteins of plant origin such as soybean
meal or cottonseed meal, or of animal origin such as
fish meal or blood meal. Controlled release non-protein nitrogen
A variety of factors must be considered when compounds
utilizing urea. The amount of urea that can be used
is limited because of the rapid hydrolysis of urea to Attempts at synchronizing the ruminal production
ammonia in the rumen. This rapid breakdown to of ammonia with ruminal energy digestion have
ammonia can occur at a much faster rate than focused on the development of controlled release urea
ammonia assimilation by the rumen bacteria, resulting compounds for more than 30 years. Previously
in accumulation and escape of ammonia from the developed controlled release NPN compounds such
rumen (Satter and Roffler, 1975). As a result, urea as isobutylidine monourea (Mathison et al., 1994),
can only be used as a source of nitrogen when there biuret (Löest et al., 2001), Starea (Bartley and Deyoe,
is an adequate supply of readily fermentable 1975), formaldehyde-treated urea (Prokop and
carbohydrate available to synthesize bacterial protein. Klopfenstein, 1977), and linseed oil-coated urea
Because ammonia produced in the rumen is used for (Forero et al., 1980), have not been as advantageous
bacterial growth and bacterial growth is dependent as urea because a substantial part of the NPN in these
on energy availability, it is important that the rate of compounds may leave the rumen without being
ammonia production in the rumen be coordinated converted to ammonia, thus reducing its incorporation
with the rate of carbohydrate fermentation. If the into bacterial protein. They are also less advantageous
rates for ruminal ammonia production and energy because the ammonia formation from these
digestion are not synchronized, ammonia compounds in the rumen, though slower than urea,
concentration in the rumen will increase after feeding is still too fast to improve the utilization of nitrogen
(Newbold and Rust, 1992; Henning et al., 1993). by rumen bacteria (Owens and Zinn, 1988; Henning
However, simply increasing supplemental dietary et al., 1993).
nonstructural carbohydrate (NSC) does not guarantee These data clearly indicate that a new approach to
either an increase in bacterial protein yield or supplying controlled release NPN is needed. Optigen®
efficiency of bacterial protein synthesis and improved 1200 is prilled urea coated with a biodegradable
180 Optigen® 1200: controlled release of non-protein nitrogen
polymer that has a controlled release property. This In situ nitrogen disappearance of Optigen® 1200
material is a highly concentrated nitrogen source that followed a pattern which was more similar to soybean
can enhance rumen function by supplying nitrogen meal than urea (Figure 1). Nitrogen disappearance
to rumen bacteria at a rate that optimizes the of urea followed a linear pattern, while the
conversion of nitrogen into bacterial protein. disappearance of nitrogen from Optigen ® 1200
Optigen® 1200 also increases the nitrogen density of followed a 2 nd order polynomial pattern. This
the protein fraction of the diet, enhances the growth of indicated that for urea there was one rapid nitrogen
fiber utilizing bacteria, and creates more space for the disappearance rate, but for Optigen® 1200, there were
inclusion of digestible fiber and energy in the ration. two different disappearance rates. Optigen® 1200 had
an intermediate disappearance rate during the first
16 hrs of rumen fermentation. This was followed by
Experiments evaluating the efficacy of a slower disappearance rate from 16 to 30 hrs. This
Optigen® 1200 two phase disappearance pattern was similar to the
pattern observed for soybean meal. During the first
IN SITU NITROGEN DISAPPEARANCE 3 hrs of fermentation, Optigen® 1200 had a faster
rate of disappearance than soybean meal, but had a
In situ nitrogen disappearance of Optigen 1200® was slower rate of disappearance between 3 and 16 hrs of
compared with that of urea and soybean meal. In fermentation. These results clearly demonstrate that
these studies, samples of Optigen® 1200, feed grade Optigen® 1200 is a controlled release source of NPN,
urea and soybean meal were placed in polyester bags. since typical NPN compounds like urea have a
Sample size was adjusted so that the sample:bag disappearance rate that is much faster than soybean
surface area ratio was similar for all nitrogen sources. meal.
The bags were suspended in the rumen of a cannulated
animal. There were two bags per time point and three
replications. Time points for bag incubation were 0, BACTERIAL PROTEIN SYNTHESIS AND
0.5, 1, 2, 3, 4, 6, 8, 12, 16, 24 and 30 hrs. When the DISAPPEARANCE OF NUTRIENTS IN
polyester bags were removed from the rumen, they CONTINUOUS CULTURE FERMENTORS
were washed with distilled water until the rinse water
was clear. The bags were dried in a forced air oven Studies with rumen-simulating continuous culture
at 60oC. Samples were composited by time point and fermentors have been conducted to evaluate the effects
then analyzed for nitrogen.
100
Nitrogen disappearance (%)
80
60 Optigen® 1200
Soybean meal
40 Urea
20
0
0 5 10 15 20 25 30
Hours of rumen incubation
Figure 1. In situ nitrogen disappearance of Optigen® 1200, soybean meal and feed grade urea.
V. Akay et al. 181
of Optigen ® 1200 addition on bacterial protein formaldehyde or molasses did help avoid ammonia
synthesis and disappearance of nutrients. Continuous toxicity, they did not affect nutrient disappearance.
culture fermentors were inoculated with 1000 ml of
rumen fluid and 50 g of digesta taken from a
ruminally-cannulated Holstein. Each fermentor was LACTATING DAIRY COWS: EXPERIMENT 1
fed the control diet or Optigen® 1200 diet (0.66%
Optigen® 1200 on a DM basis) at a rate of 75 g DM Two hundred and twenty lactating dairy cows on a
per day in three equal feedings. The fermentors were commercial dairy in western New York were used to
operated for a 5-day adjustment period, followed by compare a control diet with a diet containing
a 3-day sampling period. During the sampling period, Optigen® 1200. Groups were balanced for days in
effluent was collected daily and a portion was freeze- milk, parity, and milk yield at the start of the
dried and ground for the analysis of acid detergent experiment. Both groups were fed the control diet
fiber (ADF), neutral detergent fiber (NDF), total from May 16 through June 15. During the baseline
carbohydrate (CHO) and organic matter (OM). period, daily milk weights for individual cows were
Optigen® 1200 resulted in more bacterial protein recorded and DHIA Dairy One (Ithaca, NY) took
synthesis and faster disappearance of nutrients milk samples every 14 days. The treatment period
compared to the control diet (Figure 2). Bacterial started on June 16 and ended on August 25, during
protein synthesis was 6% higher for Optigen® 1200. which time individual cow daily milk weights were
Optigen® 1200 also increased the disappearance of again recorded and DHIA took milk samples every
ADF, NDF, total CHO and OM in the amounts of 14 days. Individual cow milk samples were analyzed
16.6, 6.8, 4.0 and 8.0%, respectively. Improved for CP, fat and milk urea N.
nutrient disappearance with Optigen ® 1200 was The ingredient composition of the diets is shown
apparently due to an increased number of bacteria in in Table 1. The amount of forage dry matter was
the rumen, which increased digestion of these similar between treatments, both containing 22 lbs.
nutrients. Ruiz et al. (2002) reported higher DM, N The diets contained 41.5% forage and 58.5%
and NDF disappearance when urea was added to the concentrate. When Optigen® 1200 was added to the
diet of lactating dairy cows. However, Owens and diet, the amounts of urea and soybean meal were
Zinn (1988) reported that while controlled release reduced and carbohydrates (wheat midds and ground
NPN compounds, such as Starea, biuret, certain corn) were used to replace these protein supplements.
coating materials and most complexes of urea with Both diets contained 0.44 lbs of buffer per cow as
part of the mineral supplement.
110 Control Optigen® 1200
90
70
%
50
30
Bacterial ADF NDF Total CHO OM
protein disappearance disappearance disappearance disappearance
synthesis
Figure 2. Bacterial protein synthesis and disappearance of nutrients in rumen-simulating continuous culture fermentors fed a
control diet or a diet that contained 0.66% Optigen® 1200 on dry matter basis.
Dietary nitrogen fraction concentrations are in Table treatment, stage of lactation, and treatment by DHIA
2. The diets differed in the amount of rumen test day. Cow was considered as a random effect in
undegradable protein (RUP), starch and rumen this model. Only cows that had four or more DHIA
degradable protein (RDP). The addition of Optigen® test days during the test period were included in the
1200 to the diet resulted in a 5.55% decrease in RUP analysis. For the statistical analysis, data from 206
content, and a 3.06% increase in starch content as cows were analyzed. Multiple test days per cow were
well as a 4.12% increase in RDP content of the diet. treated as repeated measures.
Optigen® 1200 significantly altered milk yield
Table 1. Ingredient composition of experimental diets fed during (Table 3). This treatment supplied more RDP and
Experiment 1 (DM basis). more rumen degradable starch compared to the
Control Optigen® 1200 control diet. It is likely that Optigen® 1200 stimulated
Ingredients (lb DM/day) (lb DM/day) rumen bacterial growth and ruminal digestion of the
Corn silage 15.38 15.37 diet because milk yield increased 8.14 lbs/day in the
Haylage 6.59 6.59 group given Optigen® 1200. This represents a 9.74%
Whole cottonseed 4.40 4.40
Ground corn 13.55 14.00 increase in milk yield. One explanation for the
Cane molasses 1.71 1.71 increase in milk yield could be the enhanced ruminal
Soybean meal 3.85 1.65 starch digestion. Huber and Herrera-Saldana (1994)
Feed grade urea 0.22 0.10 reported a 9% increase in milk yield when dry rolled
Optigen ® 1200 0 0.50
Brewer’s grains 0.97 0.88 sorghum was replaced with steam-flaked sorghum,
Wheat midds 0 1.76 which increased ruminal starch digestion. Hoover
Soybean hulls 0.97 0.88 and Stokes (1991) reported that as the RDP of the
Soy Plus 1.94 1.76 diet was increased from 10% to 14% of DM,
Agway Hi-Lysine Protein supplement 0.50 0.51
Corn gluten meal 0.29 0.26 carbohydrate digestion increased from 60 to 70%.
Tallow 0.44 0.49
Limestone 0.93 0.96 Table 3. Least squares means of milk yield and percentage of milk
Custom min/vit plus buffer 1.26 1.18 fat and protein in Experiment 1.
Total dry matter 53.00 53.00
Items Control SE Optigen® SE P
1200
Table 2. Nutrient composition of diets fed during Experiment 1
(DM basis)1.
Milk yield, lb/d 83.56 2.65 91.70 2.37 < 0.01
Nutrients Control Optigen® 1200 Milk fat, % 3.72 0.08 3.48 0.07 < 0.01
Fat yield, lb/d 3.11 0.09 3.19 0.08 0.48
Crude protein, % 18.20 18.40 Milk protein, % 3.01 0.03 2.95 0.03 0.03
Degradable protein, % DM 11.65 12.15 Protein yield, lb/day 2.47 0.07 2.71 0.06 < 0.01
Soluble protein,% of CP 29.81 30.47
Undegradable protein, % of CP 36.10 34.00
NFC:DIP2 3.24 3.15
Net energy, mcal/lb 0.78 0.78
It is likely that using Optigen® 1200 as a nitrogen
ADF, % 19.64 19.57 source increased rumen efficiency compared to urea
NDF, % 30.39 31.02 and soybean meal. Milk fat and milk protein content
Forage, % of DM 41.50 41.50 were decreased compared to controls. This was
NFC, % 37.69 38.22
Starch, % 28.11 29.00
expected since milk volume was increased by 9.74%.
Calcium, % 1.10 1.10 Yield of milk fat was unaffected. On the Optigen®
Phosphorus, 0.45 0.45 1200 diet, milk protein yield was increased by 8.9%
Potassium, % 1.67 1.38 even though RUP was reduced by 5.55%. This
Magnesium, % 0.30 0.30
Sodium, % 0.44 0.50
suggests that when Optigen® 1200 was used as a
Sulfur, % 0.23 0.23 nitrogen source, ruminal bacterial protein production
1
Estimated values based on forage analysis and diet composition.
was increased.
2
Non-fibrous carbohydrate:degradable intake protein. Based upon animal performance, Optigen® 1200
can be used to enhance rumen function by creating
An injection of bST was given to both groups on the space in the diet to optimize carbohydrate and protein
same day every 14 days. At the start of the experiment, digestion rates. In the present experiment, Optigen®
both groups contained the same percentage of cows 1200 partially replaced soybean meal and urea in
receiving bST injections. DHIA test day data were the diet. Feeding 0.5 lbs of Optigen® 1200 allowed
analyzed using the Proc Mixed Model procedure of for a reduction in soybean meal by 2.2 lbs of dry
SAS (1999). Fixed effects in the SAS model included matter, a reduction in urea by 0.12 lbs of dry matter,
V. Akay et al. 183
and a reduction in Soy Plus® by 0.18 lbs of dry matter. Table 4. Composition of experimental diets fed during Experiment
This created 2.0 lbs of dry matter space in the diet. 2 (DM basis).
This additional dry matter space was filled with wheat Control Optigen® 1200
midds (1.76 lbs of DM) and finely ground corn (0.45 Ingredients (lb DM/day) (lb DM/day)
lbs of DM). These dietary changes resulted in greater Corn silage, 1999 14.35 14.64
milk yield and milk protein yield, but were Haylage, 1st cutting 7.81 7.90
Idaho hay, 1999 2.70 2.70
accompanied by a lower milk fat percentage. Whole cottonseed 4.14 3.72
Both diets were similar in NDF, NSC and starch Ground corn 13.09 14.02
content. Using Optigen® 1200 as a nitrogen source Soybean meal, 48% CP 3.34 1.98
allowed the successful manipulation of rate of Feed grade urea 0.06 0
Optigen® 1200 0 0.35
carbohydrate digestion. Fiber from wheat midds is Corn gluten meal, 60% CP 0.74 0.56
digested more rapidly in the rumen compared to fiber Blood meal 0.26 0.39
from corn (Hoover and Miller, 1991). Wheat midds Agway Amino Plus 2.06 2.00
have a lower ‘rumen fill’ value than corn, barley and MolMix Prime 28 liquid supplement 0.96 0.96
Tallow 0.20 0.30
distiller’s grains (Varga et al., 1984). Replacing Limestone 0.41 0.38
soybean meal and Soy Plus® (high in RUP) with Dicalcium phosphate 0.28 0.33
wheat midds and ground corn changes the rumen fill Megalac 0.49 0.57
values of these diets and the rate of starch and fiber Custom min/vit plus buffer 1.41 1.48
Total dry matter 52.30 52.28
digestion. Thus, the Optigen® 1200 diet would have
a lower rumen fill value and faster rate of digestion
compared to the control diet. When diets with low Control and Optigen® 1200 diets contained similar
rumen fill were compared to diets with high rumen amounts of crude protein and net energy (Table 5).
fill, the diets with low rumen fill resulted in greater The diets differed in the amount of RUP and starch.
NDF and total carbohydrate digestion, and also higher The addition of Optigen® 1200 to the diet resulted in
milk production (Hoover and Miller, 1991). a 4.29% decrease in RUP content and an 2.56%
increase in starch content. The RUP was reduced in
the Optigen® 1200 diet. One hypothesis for this is
LACTATING DAIRY COWS: EXPERIMENT 2 that a controlled release nitrogen supplement like
Optigen® 1200 would enhance ruminal bacterial
In a second trial, 240 lactating dairy cows on a growth and bacterial protein production.
commercial dairy in central New York were used to
evaluated performance response to Optigen® 1200. Table 5. Nutrient composition of diets fed during Experiment 2
(DM basis)1.
Groups were balanced for days in milk, parity and
milk yield at the start of the experiment. Both groups Nutrients Control Optigen® 1200
were fed the control diet for a 15-day baseline period, Corn silage, 1999 14.35 14.64
followed by a treatment period lasting one month. Crude protein, % 18.53 18.56
Individual daily milk weights were recorded and Degradable protein, % DM 11.98 12.13
DHIA Dairy One (Ithaca, NY) took milk samples Soluble protein,% of CP 33.26 33.19
Undegradable protein, % of CP 34.98 33.48
every 14 days. Individual cow milk samples were NFC:DIP ratio2 3.24 3.26
analyzed for CP, fat and milk urea nitrogen by the Net energy, mcal/lb 0.80 0.80
New York DHIA Milk-Testing Laboratory (Ithaca, ADF, %3 19.41 19.24
NY). Daily feed intake by group was measured. Orts NDF, %4 28.50 28.28
Forage, % DM 47.50 48.26
were taken daily from the feed bunk and moisture NFC, % 38.80 39.51
determined using a Koster moisture tester. Starch, % 30.79 31.60
The ingredient composition of the diets is shown Calcium, % 0.95 0.95
in Table 4. Forage supplied 24.86 and 25.24 lbs of Phosphorus, 0.49 0.49
Potassium, % 1.45 1.45
the DM on control and Optigen ® 1200 diets, Magnesium, % 0.33 0.33
respectively. These diets contained 47.5% and 48.26% Sodium, % 0.61 0.61
forage on a DM basis, respectively. When Optigen® Sulfur, % 0.28 0.30
1200 was added to the diet, the amounts of urea and 1
Estimated values based on forage analysis and diet composition.
soybean meal were reduced. Carbohydrates (ground 2
Non-fibrous carbohydrate:degradable intake protein.
corn and corn silage) replaced these protein 3
Acid detergent fiber.
supplements. Both diets contained 0.70 lbs of buffer 4
Neutral detergent fiber.
per cow per day as part of the mineral supplement.
Daily milk yield data were analyzed using Proc Mixed Table 7. Estimated nutrient intake based on dry matter intake and
Model procedure of SAS (1999). Daily observations diet composition in Experiment 2.
were treated as repeated measures. Fixed effects in Items Control Optigen® 1200 Difference
the SAS model included parity, treatment, stage of Dry matter, lb/d 52.74 50.77 -3.88%
lactation, test day, and the interaction of treatment × Forage, lb/d 25.05 24.50 -2.20%
stage of lactation, treatment × test day, and treatment Crude protein, lb/d 9.77 9.42 -3.58%
Undegradable protein, lb/d 3.42 3.15 -7.89%
× parity. Cow was considered as a random effect in NFC, lb/d 20.46 20.06 -1.96%
this model. Daily DM intake by group was analyzed Starch, lb/d 16.24 16.04 -1.23%
using the Proc Mixed Model procedure of SAS
(1999). Fixed effects in this model were treatment addition of forage (0.38 lbs of DM) and ground corn
and date. Daily observations by group were treated (0.93 lbs of DM) filled this additional space. These
as repeated measures. dietary changes did not result in greater milk yield,
Delivering a part of the total dietary nitrogen in but did increase milk fat percentage. The lack of milk
the form of Optigen® 1200 had a significant effect yield response can be explained by the fact that non-
on DM intake but not on milk yield (Table 6). Dry fibrous carbohydrate and starch intake were lower in
matter intake was 1.97 lbs lower in the Optigen® the Optigen® 1200 diet compared to the control diet
1200 group compared to controls. Milk fat (Table 7). The increase in milk fat percentage,
percentage was greater on the Optigen ® 1200 however, cannot be explained by greater forage intake.
treatment, but fat yield was unaffected. Milk protein Forage intake was actually lower for the Optigen®
percentage was greater in the control group while 1200 diet due to the lower dry matter intake (Table
protein yield was unaffected. 7). The increase in fat percentage may be explained
by greater forage digestion, which could have
Table 6. Least square means of milk yield and percentage of milk
fat and protein in Experiment 2.
occurred due to greater ruminal bacterial activity in
the Optigen® 1200 diet. Since the intake of RUP was
SE Optigen ® 1200
Items Control SE P
7.89% less in the group given Optigen® 1200 while
DM intake, lb/d 52.74 0.20 50.77 0.20 < 0.01 milk yield was similar, bacterial protein production
Milk yield, lb/d 88.51 0.84 89.00 0.85 > 0.50
in the rumen must have increased by 7.89%.
Milk fat, % 3.76 0.06 3.93 0.07 0.065
Fat yield, lb/d 3.41 0.04 3.55 0.04 0.268 Enhanced bacterial growth and microbial protein
Milk protein, % 3.02 0.02 2.96 0.03 0.087 production in response to Optigen® 1200 is a likely
Protein yield, lb/d 2.67 0.04 2.63 0.04 > 0.50 explanation for the improvement in efficiency of milk
production (4.2%).
Using Optigen® 1200 as a rumen nitrogen source
increased rumen efficiency. Efficiency of milk
production (lb milk/lb DMI) was greater for cows Conclusion
given Optigen® 1200 (1.75 vs. 1.68). This represents
an improvement in efficiency of 4.2%. Optigen® 1200 has an in situ nitrogen disappearance
Indirect evidence for an improvement in ruminal rate very similar to soybean meal. Bacterial protein
efficiency can be seen when actual intake of nutrients synthesis and disappearance of nutrients in continuous
is calculated based on actual DM intake and diet culture fermentors were greater when Optigen® 1200
composition (Table 7). The cows receiving Optigen® was present in the diet. Optigen® 1200 increases the
1200 consumed 3.58% less protein per day and 7.89% crude protein density of the protein fraction of the
less RUP per day compared to controls. Since both diet because it is a highly concentrated source of
milk yield and milk protein yield were similar, the nitrogen. By increasing diet protein density, space is
use of Optigen® 1200 likely increased the supply of created in the formulation, which allows nutritionists
bacterial protein passing into the small intestine. to manipulate the carbohydrate fraction of the diet.
Optigen ® 1200 was substituted in the diet for These manipulations resulted in greater milk yield
soybean meal, urea and whole cottonseed. Feeding and milk component yield including greater milk fat
0.35 lbs of Optigen® 1200 allowed a reduction in percentage. In both in vivo experiments, the amount
soybean meal by 1.36 lbs of dry matter, a reduction of RUP was reduced in the Optigen® 1200 diets
in urea by 0.06 lbs of dry matter, and a reduction in compared to the control diets without a loss in milk
whole cottonseed by 0.42 lbs of dry matter. This yield. These results indicate that Optigen® 1200 can
created 1.84 lbs of dry matter space in the diet. The be used successfully in dairy cattle diets to create
V. Akay et al. 185
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W. Chalupa et al. 187
Dairy nutrition models: their forms and applications

WILLIAM CHALUPA, RAY BOSTON AND ROBERT MUNSON
School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania, USA
Introduction
For some years it has been evident that dairy cow properties of production and scientific models are
nutrition models are vital to the continued success of presented in Table 2.
the dairy industry. This is especially true as we
recognize the importance, for example, of ruminal Table 1. Model levels1.
microbes and metabolism in body tissues to nutrient Level Description of level
requirements. In addition, our production emphasis
has shifted from only milk volume and fat to include i+1 Collection of organisms (herd, flock, crop)
milk protein percentage and yield. Mathematical i Organism (animal, plant)
i-1 Organs
models of nutrition have been in use for over three i-2 Tissues
decades and have stimulated improvement in feeding Cells
cattle. However, more complete data sets available Organelles
in recent years combined with more precise 1
Adapted From France and Thornley (1984).
mathematical approaches have now allowed us to
improve models of nutrient use tremendously. Such
models will be used more frequently in the future
SCIENTIFIC MODELS
for support of decisions not only on the nutrition of
cattle, but for other aspects including farm economics Scientific models are usually developed upward from
and environmental impact. basic experimental data pertaining to metabolic
processes. Scientific models assume that a living
system can be described in terms of a set of ‘critical’
Dairy nutrition models: forms and roles metabolic transactions encapsulated in organs. The
goal is to translate in vitro experimental data into
Nutritional models vary in complexity according to chemical reactions representing the essential metabolic
objectives. A typical scheme of model levels needed processes. Differential equations of the mass balance
to represent a system is found in Table 1. Information and Michaelis Menten forms are used to describe
about a system must be at least one level below the substrate level changes as the system equilibrates to
system explored with the model. Thus, models a (new) steady state because of nutritional and
describing herds operate at the animal level or below, digestive inputs. Implicit to these models are two
those describing animals require details at the organ basic assumptions: firstly, that in vivo metabolic
level and lower and so on. pathways can be represented using the critical
In practice, models only need details that have transactions modeled from in vitro experimental data,
significant bearing on consequences of changes arising and secondly, that cellular level metabolic processes
from inputs to the system (Production Model) or as can be aggregated to the organ level to effectively
much detail as is necessary to explore the system in model whole animal function. Baldwin, at the
new and different ways (Scientific Model). Salient University of California, and his colleagues (Baldwin
188 Dairy nutrition models: their forms and applications
Table 2. Properties of production and scientific models1.
Feature Production model Scientific model
Purpose Predict response Understand process

Form Response surface equations Differential equations (state equations)
Parameters Polynomial coefficients derived from data fitting Biochemical reaction properties
Aggregation step None; model derived from aggregated experiments Chemical processes aggregated to organ and animal
level functions
Solution process Simple explicit solution of equations Complex systems of differential equations requiring
special software
Outputs Computed indicators of adequacy of inputs and Steady state solutions to transactions in terms of
production cost measures scientifically significant indicators
Character Empirical and static Dynamic and mechanistic
1
Adapted from Boston et al. (2003)
et al., 1987a,b,c) have produced a comprehensive stimulated more precise thinking and
integrated model that describes digestion and experimentation. Better data were incorporated into
metabolism of the dairy cow with dynamic, newer versions of models. Largely because of
mechanistic equations of physiological processes. concepts in these increasingly precise models, rations
for dairy cows usually now contain feed ingredients
that are resistant to ruminal degradation. This
PRODUCTION MODELS increases overall efficiency of dairy cow feeding.
The need for more accurate models to define rumen
Production models are primarily used to portray bacterial and whole animal requirements, to assess
animal responses to different inputs. They are usually feed utilization and to predict production responses
created from collections of response surface models led to the development of the Cornell Net
that are developed from animal or herd level Carbohydrate and Protein System (Fox et al., 1992;
experiments. Thus, these models are developed O’Connor et al., 1993; Russell et al., 1992; Sniffen
downward. They are valid within the domain of data et al., 1992). The CNCPS is a mix of empirical and
underpinning the individual response surfaces and mechanistic approaches that describe feed intake,
are as accurate as the response models themselves. ruminal fermentation of protein and carbohydrate,
A theme for the development, refinement and intestinal digestion and absorption, excretion, heat
deployment of empirical production models is seen production, and utilization of nutrients for
in the development and implementation of the maintenance, growth, lactation and pregnancy. When
National Research Council dairy cow models (NRC, the CNCPS was evaluated with data from individual
1978; 1989; 2001). In 1978, response equations were dairy cows where the appropriate inputs were
used to predict crude protein and energy needs of the measured and changes in energy reserves were
dairy cow. The 1989 model used a system of protein accounted for, 90% of the variation in actual milk
utilization that partitioned dietary protein into rumen production of individual cows was explained with a
degradable (DIP) and rumen undegradable (UIP) 1.3% bias. The model accounted for 76% of the
fractions (NRC, 1985). Growth of microorganisms variation in individual cow milk production with an
in the rumen was driven by energy intake (TDN, 8% underprediction bias when energy was first
NE L). In 2001, the National Research Council limiting and 84% of the variation with a 1.1%
released a new dairy cow model that contains some overprediction bias when protein was first limiting
of the mechanistic approaches in the CNCPS/CPM- (Fox et al., 2004).
Dairy that are described below.
Other empirical production models include the
VEM-DVE/OEB (Dutch; Tamminga et al., 1994), Dairy nutrition software
AFRC (British; AFRC, 1990; 1992), CSIRO
(Australian; CSIRO, 1990) and INRA (French; Dairy nutrition models often do not contain tools for
INRA, 1989) systems. These early production models computer assisted ration formulation. Software
included with the 1989 and 2001 NRC dairy nutrition ‘no feasible solution’. This provided no direction to
models allowed calculation of nutrient requirements obtaining a solution. Newer optimization methods
and evaluation of rations but did not provide for provide direction by listing nutrient constraints that
formulation of rations. are not met.
In both empirical models and in the CNCPS/CPM-
DAIRY, nutrients like crude protein, fat,
AUTO-BALANCING carbohydrates (fiber and non-fiber) and minerals are
constant proportions of the ingredient regardless of
The usual objective of auto-balancing is to produce the amount of feed consumed. Thus, supply of these
an ‘optimal ration’ at the lowest cost. Constraints nutrients is a linear function of intake. In empirical
(minimum and maximum amounts) are set for both dairy cow models, metabolizable protein and energy
nutrients and feed ingredients. Nutritional constraints (metabolizable and net) values also are not affected
describe the requirements of cows to perform specific by intake and thus are constant. Thus linear
or multiple functions (maintenance, growth, lactation, programming can be used for auto-balancing. The
pregnancy). Nutritional constraints include dry matter CNCPS/CPM-DAIRY has a dynamic rumen sub-
intake, energy (metabolizable and net), protein model wherein the passage rate of feeds (determined
(crude, soluble, bypass and metabolizable), mainly by feed intake but also adjusted by ration
carbohydrates (fiber and non-fiber), fat, minerals; forage content and particle size) determines the
and in the case of newer models like the CNCPS/ outflow of nutrients from the rumen system. Thus,
CPM-DAIRY, amino acids and rumen available nutrients like metabolizable protein, metabolizable
nitrogen (peptides and ammonia) are included. Feed energy, amino acid content of metabolizable protein,
ingredients are selected on the basis of the major and rumen available protein (peptides and ammonia)
nutrients that they provide (i.e. fiber from forages, are not constant but vary according to feed
non-fiber carbohydrates from grains, protein from consumption and ration ingredients. These features
oilseed meals). Feed constraints are set based on the of the dynamic digestion models mean that the
availability of purchased ingredients and inventory problem of dairy cow ration optimization is no longer
of ingredients on the farm or contracted for purchase. the province of linear programming and nonlinear
The amount of an ingredient specified is often programming packages are required. Implementing
adjusted by the formulator to take into account a constrained, nonlinear optimization is not without
minimum amount that the formulator feels rations problems. If the nutrition model contains
should contain or the maximum amount that the discontinuous (break-point) functions, continuous
formulator feels can be tolerated by the animal. The mathematical models must be developed to describe
amount of a feed ingredient should not be limited by the discontinuous functions (Boston et al., 2000).
high cost because optimization programs will control Whereas a linear programming problem can be solved
the inclusion of expensive feeds. Thus, the auto- from any starting point, a nonlinear programming
balancing (optimization) task is to find the least cost problem requires a ‘good’ feasible starting point to
combination of feed ingredients within their ‘effectively’ start the solution process. Finally, a linear
minimum and maximum constraints that provide programming optimization problem has just one
nutrients that are within the specified minimum and solution. This is not so for nonlinear optimization.
maximum ranges. When the foregoing is achieved, Ration formulation was one of the first applications
the auto-balancing process has provided a solution of linear programming. Not only could solutions be
to the specifications defined by the formulator. found in seconds, but building on contributions of
Ration formulators often are discouraged when the Dantzig (1995) to operational research, we also were
optimization process does not give a solution as able to derive an array of other very helpful economic
defined above. This simply means that a combination properties (shadow prices) relating to our optimal
of feed ingredients in amounts within the minimum solution. For example, we could discover the cost
and maximum ranges cannot provide nutrients within ranges over which feeds within the optimal ration
the specified ranges. To find a solution, the formulator remained there, as well as which amongst the feeds
should either expand (relax) the feed ingredient and not selected in the optimal ration were candidates
nutrient constraints or include additional ingredients for inclusion if cost decreased.
that are good sources of limiting nutrients. Older Bath and Bennett (1980) at The University of
optimization methods simply indicated that there was California were amongst the earliest to employ linear
programming to formulate rations for maximum scheme was developed by the Systems Programming
income over feed costs. Galligan and coworkers Group at the University of Maryland (Zhou and Tits,
(1986) at the University of Pennsylvania programmed 1997) and employs a forward sequential quadratic
the 1978 NRC dairy nutrition model into Lotus 1-2- programming approach. Version 1, released in 1998,
3 with auto-balancing of rations provided by Einfin. also contained a modification of NRC (1989).
Spartan represented an excellent effort in software Versions 2 and 3 only contain the CNCPS and are in
development by the group at Michigan State beta testing with release anticipated in 2004. Version
University that was based on NRC models and 3 has expanded carbohydrate fractions, a lipid
included auto-balancing (Vandehar et al., 1992). submodel (Moate et al., 2004) and incorporates NRC
CPM-Dairy is a combined effort by researchers at (2001) mineral requirements.
Cornell University, the University of Pennsylvania
and the W.H. Miner Agricultural Research Institute
(Boston et al., 2000). CPM-Dairy contains the COMMERCIALLY AVAILABLE SOFTWARE
CNCPS and is a field efficient program for use with
growing, lactating and dry cows. The optimization Table 3 contains a list of commercially available
software for formulation of dairy cattle rations. NRC,
Table 3. Some commercially available dairy nutrition software.
Software Developers Dairy Nutrition Model1 Website
Feed Ration Balancer Feed Management Systems NRC, User defined http://www.feedsys.com/
CamDairy Cam Software Proprietary model http://epicentre.massey.ac.nz/
Downloads/Software/CD_install.pdf
The Consulting Nutritionist Dalex Computer Systems, Inc. User defined nutrient http://www.dalex.com/
requirements, CNCPS, NRC
CPM-Dairy Cornell U., U.Pennsylvania, CNCPS http://mail.vet.upenn.edu/~ejjancze/
Miner Institute http://mail.vet.upenn.edu/~ejjancze/
cpmbeta3.html
CNCPS Cornell University CNCPS http://www.cncps.cornell.edu/cncps/
main.htm
Dairy Ration System ACS Computer Services NRC http://www.acsdrs.com
Formulate2 Central Valley Nutritional NRC http://www.formulate2.com/
Associates
INRAtion - PrevAlim INRA INRA http://www.cnerta.educagri.fr/unites/
lpa/lpa.htm#inration
Mixit-Win Agricultural Software User defined minimum and http://www.asc-mixit.com/
Consultants, Inc. maximum nutrient amounts
Molly U. California, Davis Molly http://animalscience.ucdavis.edu/
research/molly/default.htm
PCDairy-2 U. California, Davis NRC http://animalscience.ucdavis.edu/
extension/pcdairy.htm
RationPro ProfitSource NRC, User-defined http://www.rationpro.com/
RumNut A.T Chamberlain AFRC, PDI http://www.rumnut.com/proginst.pdf
Shield U. California, Davis Proprietary model http://animalscience.ucdavis.edu/
extension/shield.htm
SigaDairy Siga Farm Software NRC, User-defined http://www.siga.net
Spartan Michigan State U. NRC with modifications http://www.msu.edu/user/ssl/index.htm
Trilogic Trilogic Systems NRC, User-defined http://trilogic-systems.com/

requirements, proprietary amino
acid/ carbohydrate model
N.RC, National Research Council; CNCPS; Cornell Net Carbohydrate and Protein System; INRA, Institut National de la Recherche
Agronomique, MOLLY, a dynamic, mechanistic computer model of a dairy cow; AFRC, Agricultural and Food Research Council;
PDI (INRA), Proteines varies reellenment digestibles dans l’Intestin grele.
INRA and CNCPS dairy nutrition models are used DM) and contained 41.5% non-fiber carbohydrate
in some of the software packages whereas proprietary (NFC). High moisture corn, which has an initial high
or user-defined models are used in others. Linear rate of fermentation in the rumen, contributed a
programming is used for auto-balancing in empirical substantial amount of NFC.
models. In CNCPS, biological values for
Table 4. Field application of CPM-Dairy.
metabolizable energy, metabolizable protein, passage
rate, bacterial yield efficiencies and degradation rate Ration
of available fiber, which depend upon feed intake Pre-CPM-Dairy CPM-Dairy
and the ingredients selected, are first estimated and Ingredients (%DM)
then rations are balanced using linear programming. Alfalfa silage 27.14 15.21
In CPM-Dairy, a nonlinear optimizer is used to auto- Corn silage 22.79 37.16
High moisture corn 24.23 18.95
balance rations according to the CNCPS (Zhou and Soybean hulls 6.92
Titts, 1997). Soybean meal 5.63 3.91
Corn distiller’s grains 5.51
Dry brewer’s grains 3.34
Whole cotton seed 9.29 10.00
Application of dairy nutrition models Fish meal 1.38
Blood meal 0.55
CPM-Dairy is used by veterinarians, nutrition Animal-marine protein blend 3.38
consultants and the feed industry to evaluate and Megalac 0.59
Megalac Plus 0.49
formulate rations for dairy cattle. Salt 0.40 0.22
At New Bolton Center, our Field Investigation Unit Sodium bicarbonate 0.50 0.80
was presented with a case where milk production in Mineral and vitamin mix 0.58 1.03
a 200-cow dairy was only 30 kg/d although cows Total dry matter, kg/d 22.46 22.46
Cost, $/day 3.65 3.82
were milked three times daily and were treated with
bST. Early lactation cows frequently went off feed Carbohydrates
Non fiber1, %DM 41.5 39.0
and had diarrhea. Feces contained undigested corn, Neutral detergent fiber, %DM 30.4 33.3
believed to be high moisture corn. Effective neutral detergent fiber,
There were two high production groups (heifers %DM 21.0 22.9
and high cows) and a low production group. The Protein
heifers and high production cows were housed in a Crude, %DM 18.6 17.8
Undegraded, %CP 33.7 37.2
new free-stall barn with excellent ventilation and cow
Metabolizable, kg/d 2.28 [100%] 2 2.49 [100%]
comfort. Cows were fed three times daily with Bacterial 1.25 [ 55%] 1.34 [ 54%]
frequent ‘push-up’ of feed. Feed bunk space was 1.7 Undegraded 1.03 [ 45%] 1.15 [ 46%]
feet per cow with no headlocks, and cows had good Metabolizable amino acids
water access. Remaining milking cows were housed Methionine, g/d 43 55
in a renovated free-stall barn. They were fed twice Met/MP 1.89 2.19
Lysine, g/d 142 173
daily with frequent ‘push-up’ of feed. Feed bunk space Lys/MP 6.24 6.94
and ventilation were good. Non-lactating cows were
Nutrient limited milk, kg/d
housed on a bedded pack. Metabolizable energy 44.0 43.8
CPM-Dairy was used to evaluate the existing rations Metabolizable protein 34.4 39.1
and to formulate new rations. In Table 4 are details Rulquin Ratio3 32.8 39.0
of pre-CPM-Dairy and CPM-Dairy rations for the 1. Includes sugars, starch, pectin, B-glucans and acids produced
high producing animals (heifers and high cows). during silage fermentation. Silage acids are not fermented
The pre-CPM-Dairy ration was formulated for a further in the rumen and do not provide energy for bacterial
growth.
target of 39 kg/d milk with 3.6% fat and 3.1% crude 2. Values in brackets are percentages of metabolizable protein
protein. According to CPM-Dairy, this ration was 3. Responses to amino acid ratios calculated according to
low in metabolizable protein and would only support equations in Rulquin and Verite (1993) and added to metaboliz-
34.4 kg/d milk. Poor amino acid ratios (Met/ able protein-limited milk.
MP=1.89; Lys/MP=6.24) could reduce milk
production by 1.6 kg/d so that expected milk on the Objectives in formulating the new ration were to 1)
basis of metabolizable protein and balance of amino provide less NFC without compromising total
acids was only 32.8 kg/d. The ration was marginal carbohydrate fermentability, 2) increase eNDF, 3)
in eNDF (21% vs the guideline of 23% of ration correct the deficiency of metabolizable protein, and
(4) improve amino acid balance.
Reducing amounts of high moisture corn (19 vs Two weeks after the ration change, manure still
24% DM) and alfalfa silage (15 vs 27% DM), contained excessive undigested high moisture corn.
increasing the amount of corn silage (37 vs 23% DM) Grinding the high moisture corn through a 0.635 cm
and including soybean hulls (7 vs 0% DM) reduced screen reduced corn in feces and milk production
NFC (39.0 vs 41.5%). Although NFC was lower, total increased another 2 kg/d. Herd milk production now
fermentable carbohydrates were higher (45 vs 43% averaged 36 kg/d. For the next 12 months, average
ration DM) because of the high ruminal fermentation milk production for the total herd was 36 to 38 kg/d.
of NDF in soybean hulls and corn silage. Increasing
ration forage (52 vs 50% DM) and including soybean
hulls increased NDF (33.3 vs 30.4%) and eNDF (22.9 References
vs 21.0%).
Replacing a portion of the soybean meal and all Agricultural and Food Research Council. 1990.
of the corn distiller’s grains and dried brewer’s grains Technical Committee on Responses to Nutrients,
with animal and marine proteins increased the supply Report no. 5. Nutritive requirements of ruminant
of metabolizable protein from rumen undegraded animals: energy. Nutrit. Abstr. and Rev. Series B
protein by 0.12 kg (1.15 vs 1.03 kg/d). Metabolizable 60:729-804.
protein from ruminal bacteria was increased (1.34 vs Agricultural and Food Research Council. 1992.
1.25 kg/d) for two reasons. First, bacterial growth is Technical Committee on Responses to Nutrients,
driven mainly by energy derived from fermentation Report no. 5. Nutritive requirements of ruminant
of carbohydrates. As noted above, the CPM-Dairy animals: protein. Nutrit. Abstr. and Rev. Series B
ration contained more rumen fermentable 62:787-835.
carbohydrates (45 vs 43 % DM, 10.05 vs 9.76 kg/d) Baldwin, R.L., J. France and M. Gill. 1987a.
than the pre-CPM-Dairy ration. Second, bacterial Metabolism of the lactating cow I. Animal elements
growth is decreased when rumen pH decreases. The
of a mechanistic model. J. Dairy Res. 54:77-105.
CPM-Dairy ration contained more eNDF than the
Baldwin, R.L., J.H.M. Thornley and D.E. Beever.
pre-CPM-Dairy ration. This stimulates cud chewing,
1987b. Metabolism of the lactating cow II.
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escape protein and from bacterial protein alleviated Res. 54:107-131.
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CPM-dairy ration was 39.1 kg/d compared to 34.4 Properties of mechanistic models suitable for
kg/d for the pre CPM-dairy ration. evaluation of energetic relationships and factors
Animal and marine proteins and ruminal bacteria involved in the partition of nutrients. J. Dairy Res.
are excellent sources of lysine. Thus, the combination 54:133-145.
of ruminal escape and bacterial protein improved Lys/ Bath, D.L. and L.F. Bennett. 1980. Development of
MP (6.94 vs 6.24). Fish meal, but not blood meal a dairy feeding model for maximizing income
nor rumen bacteria are good sources of methionine. above feed costs with access by remote computer
Met/MP (2.19 vs 1.89) was improved by using terminals. J. Dairy Sci. 63:1397-1389.
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Moate, P.J., W. Chalupa, T.C. Jenkins and R.C. M. Allen, C. Sniffen and R. Black. 1992. Spartan
Boston. 2004. A model to describe ruminal Dairy Ration Evaluator/Balancer. Michigan State
metabolism and intestinal absorption of long chain Univ., East Lansing.
fatty acids. Anim. Feed Sci. Tech. 112:79-105. Zhou, J.L. and A.L. Tits. 1997. User’s Guide for
National Research Council. 1978. Nutrient FFSQP Version 3.5. Electrical Engineering Dept.
Requirements of Dairy Cattle. National Academy and Institute for Systems Res. Univ. Maryland,
Press, Washington, D.C. College Park.
National Research Council. 1985. Nitrogen Usage in
Ruminants. National Academy Press, Washington,
D.C.
S.I. Kehoe and A.J. Heinrichs 195
Gastrointestinal development in dairy calves

S. I. KEHOE AND A. J. HEINRICHS
Dairy and Animal Science Department, The Pennsylvania State University, University Park,
Pennsylvania, USA
Gastric enzymes in the dairy calf

The role of gastric enzymes is important for the 1987; Hill et al., 1970). Pregastric esterase
digestion of the diet of the young ruminant animal hydrolyzes about 20% of all milk fat glyceride
due to its undeveloped rumen and reliance on the linkages, mainly short chain fatty acids, indicating
abomasum and small intestine for nutrient digestion its role in lipolysis (Bondi, 1987; Hill et al., 1970;
and assimilation. The ability of neonates to digest Pitas and Jensen, 1970).
and utilize high concentrations of milk fat, especially The preference of pregastric esterase for glyceride
with low concentrations of intestinal lipases, is due linkages of butyric acid has been reported to result
to a combination of enzymes called pregastric esterase in 59% of butyrate linkages hydrolyzed in vitro as
(Huber et al., 1961). This complex of lipolytic compared with 7% of higher fatty acids (Ramsey
enzymes provides the majority of lipid breakdown and Young, 1961b). Butyric acid is highly soluble in
within the abomasum, similar to salivary α−amylase water and is hydrolyzed faster than longer chained
in monogastrics. Pregastric esterase is composed of fatty acids, passing quickly from the abomasum to
at least six different enzymes secreted from four areas the small intestine. Breakdown of butyric acid and
of the glosso-epiglottic area of the mouth, including other fatty acids is higher in milk fed orally than
the vallate papillae region of the tongue, the glosso- milk that is infused into the abomasum, indicating
epiglottic area, the pharyngeal end of the esophagus pregastric esterase is required (Otterby et al., 1964a;
and the submaxillary salivary gland (Moreau et al., Otterby et al., 1964b; Russell et al., 1980). Other
1988; Ramsey et al., 1956). It is of interest to note less soluble fatty acids are trapped by curd particles
that adult ruminants in general are not capable of and slowly migrate to the small intestine (Otterby et
digesting diets high in fat, yet it is one of the main al., 1964a; Ramsey and Young, 1961a). Although
components in the diet of a neonatal ruminant. pregastric esterase plays a primary role in lipolysis
Although the term pregastric esterase is commonly in the abomasum, calves fed only through infusion
used to describe esterases (defined as enzymes capable of milk into the abomasum, which decreases
of hydrolyzing esters in solution) it also contains stimulation of pregastric esterase secretion, are still
lipases, which have a more specific role in digestion able to digest milk fat most likely by pancreatic lipase
(defined as specialized enzymes that hydrolyze fatty action (Russell et al., 1980).
acids from water insoluble glycerol esters). Pregastric Pregastric esterase may also be found in the digesta
esterase has become the common term for enzymes of the small intestine, which was thought to indicate
of lipolytic or esterolytic nature secreted by a role in intestinal digestion of lipids (Otterby et al.,
mammalian oral tissues (Nelson et al., 1960). 1964b). Later work reported that pregastric esterase
Pregastric esterase is stimulated by nursing in the has a diminished effect once it enters the duodenum
young calf (Huber et al., 1961; Moreau et al., 1988; and no effect at all if pancreatic enzymes are not
Ramsey and Young, 1961a). During clotting of milk present (Gooden, 1973).
that occurs after ingested milk reaches the abomasum A small part of lipolysis in the abomasum was
in young ruminants, pregastric esterase begins the reported to be caused by a gastric lipase secreted
breakdown of lipids within the casein clot (Bondi, directly from abomasal tissues. Toothill et al. (1976)
196 Gastrointestinal development in dairy calves
used abomasal pouch secretions to prevent oral or Pancreatic enzymes

pancreatic contamination and found no lipolytic
digestion, concluding that lipolysis in the abomasum During the first two days of life, high concentrations
is solely due to pregastric esterase. Other researchers of abomasal enzymes clot colostrum and allow
also concluded that gastric lipase is similar to immunoglobulins to pass into the small intestine.
pregastric esterase and must have been mistakenly Pancreatic enzymes are found in low concentrations
identified as a separate enzyme (Nelson et al., 1960; at birth until two days of age in young ruminants,
Otterby et al., 1964b). allowing immunoglobulins to remain intact. It is not
Although most of the enzymes in the alimentary until after two days of age that concentrations begin
tract of the young calf seem underdeveloped, enzymes to increase until around 42 days of age (Guilloteau
that primarily coagulate milk protein are produced et al., 1983; Guilloteau et al., 1984).
in high concentrations. Chymosin, pepsin and Pancreatic amylase is a glycosidic enzyme found
hydrochloric acid coagulate milk, retaining casein in the small intestine, making up 5-6% of total protein
and fat and allowing nutrients to slowly pass into the in human pancreatic secretions (Lowe, 1994).
small intestine (Cruywagen et al., 1990; Guilloteau However, in ruminants, only 2% of total protein in
et al., 1983; Guilloteau et al., 1984). Chymosin, pancreatic secretions is α−amylase indicating a
formerly called rennin, is found in high concentrations decreased intestinal ability to hydrolyze starch (Keller
in the newborn calf and lamb and decreases with age et al., 1958). Starch is not part of the diet of milk-
and at weaning (Cybulski and Andren, 1990; fed ruminants and mature ruminants are able to
Guilloteau et al., 1983; Guilloteau et al., 1984; hydrolyze starch in the rumen. Pancreatic amylase is
Guilloteau et al., 1985). However, calves kept on found at low levels in the newborn and increases with
milk for an extended period of time retain higher age (Guilloteau et al., 1984; Huber et al., 1961; Le
chymosin concentrations than weaned calves Huërou et al., 1992; Morrill et al., 1970).
indicating abomasal enzymes are regulated by Pancreatic fluid also contains two nucleases,
development of the rumen as well as high deoxyribonuclease I (DNase) and ribonuclease
concentrations of lipids and casein in the diet (RNase). Although there is little known about either,
(Cybulski and Andren, 1990; Guilloteau et al., 1985). RNase is required in weaned calves to recover
The bovine abomasum secretes at least three phosphorus from bacterial RNA (Lowe, 1994).
proteases, including pepsin A, pepsin B (also known Unlike other pancreatic enzymes, all peptidases are
as gastricsin or pepsin II), and chymosin, all secreted secreted as zymogens or proenzymes. The main
as proenzymes from the mucous cells of the pyloric activator of pancreatic peptidases, trypsinogen, is also
and fundic glands and requiring a pH of less than 4 secreted as a proenzyme and is cleaved by
to become active (Cybulski and Andren, 1990). Pepsin enteropeptidase to the active form of trypsin. Trypsin
A is found in high concentrations at birth and remains then cleaves other proenzymes, including
constant with increasing age of both calves and lambs chymotrypsinogen, procarboxypeptidase A,
(Guilloteau et al., 1983; Guilloteau et al., 1984; procarboxypeptidase B and procolipase, to their active
Guilloteau et al., 1985), until about 44 days (Huber forms of chymotrypsin, carboxypeptidase A and B
et al., 1961). After weaning, the ratio of pepsin to and colipase (Lowe, 1994). Trypsin amounts secreted
chymosin increases the need to digest protein in solid in pancreatic juice are low in the newborn ruminant
feed rather than casein (Cybulski and Andren, 1990; and increase with age during the first two to four
Guilloteau et al., 1983; Guilloteau et al., 1985). weeks in both the lamb and the calf. Chymotrypsin
Pepsin A and chymosin adequately coagulate milk in is higher in the young animal than trypsin but the
the abomasum of the young calf, and therefore pepsin ratio decreases with age (Guilloteau et al., 1983;
B is not found until weaning, when concentrations Guilloteau et al., 1984; Huber et al., 1961).
of chymosin decrease and the pH of the abomasum Pancreatic lipases, such as colipase and
acquires a broader range due to varied feed (Cybulski phospholipase A2, are low at birth and then increase
and Andren, 1990). The potential for pepsin, trypsin, and remain constant (Gooden, 1973; Guilloteau et
chymotrypsin and amylase to be secreted from the al., 1984; Huber et al., 1961; Le Huërou et al.,
pancreas increases as the amounts of starch and protein 1992). These lipases are highly active at a pH of 8.5
increase in the diet (Garnot et al., 1977; Guilloteau and specifically hydrolyze triglycerides and
et al., 1985). Therefore these generally increase with phospholipids. Guilloteau et al. (1984) reported that
age and increasing dry matter intake of grains in from birth until three weeks of age, the colipase/
the diet. lipase ratio is higher than one indicating pancreatic
activity is entirely expressed in pancreatic juice in drastically decline at weaning. However, feeding a
the intestinal lumen. Although calves one to two diet high in lactose does not alter lactase
weeks of age have a diminished capacity to absorb concentrations (Huber et al., 1961).
lipids if pancreatic enzymes are removed, there still Although digestive enzyme concentrations are
remains lipolytic activity in the intestinal lumen dependent on age of the calf, early researchers
(Gooden, 1973; Gooden and Lascelles, 1973). This reported that digestive enzyme secretions are not
is most likely due to enzymes present in the brush affected by diet of the calf (Guilloteau et al., 1983;
border of intestinal villi. Guilloteau et al., 1984; Huber et al., 1961; Young et
al., 1960). More recent work has found alterations
in secretions of digestive enzymes when different
Brush border enzymes sources of protein are used in milk replacers.
Unprocessed soy protein in milk replacers often alters
There are many peptidases found in the microvilli of intestinal morphology by decreasing absorptive
enterocytes. The main hydrolases found in the brush surface area of intestinal villi as well as concentration
border are aminopeptidase N, aminopeptidase A, and of disaccharidases in the brush border (Pederson and
dipeptidyl peptidase IV (Le Huërou-Luron, 2002). Sissons, 1984). Trypsin and chymotrypsin secretions
All of these peptidases are designed to cleave specific are also inhibited by allergens and anti-trypsin factors
terminal amino acids from proteins (Palmer, 1995). in soy protein when ingested by the young calf
Aminopeptidase N hydrolyzes peptides in a stepwise (Gorrill et al., 1967; Guilloteau et al., 1986; Mir et
manner up to a certain point at which dipeptidyl al., 1991). Soy protein may be included in milk
peptidase IV finishes the hydrolysis (Le Huërou- replacer fed to calves older than 20 days of age in
Luron, 2002). Aminopeptidase A, aminopeptidase N, low concentrations but must be processed to decrease
and alkaline phosphatase are highest in the calf until concentrations of potential allergens as well as
two days of age and then decrease until one week of increase protein digestibility (Mir et al., 1991).
age. They remain constant until weaning, at which Digestibility of milk replacer is affected by other
time they increase (Le Huërou et al., 1992). additions, such as using high concentrations of starch
There are four major disaccharidases found in the as alternative energy sources. Incorporating starch
brush border of the small intestine including maltase- into milk replacer for preruminants is not
glucoamylase, sucrase-isomaltase, lactase, and recommended due to low concentrations of pancreatic
trehalase (Le Huërou-Luron, 2002). Sucrase- amylase present in the intestine. Starch contains 17-
isomaltase is not present in cattle and addition of 30% amylose and 70-83% amylopectin. Amylose
sucrose to the diet of young ruminants causes an consists of glucose units arranged in a linear polymer
increase in scours (Huber et al., 1961; Le Huërou et bound with α(1,4) glycosidic linkages and
al., 1992; Le Huërou-Luron, 2002). amylopectin is bound by α(1,6) branch points along
Maltase-glucoamylase hydrolyzes α(1-4) the α(1,4) linked glucose polymer. These bonds must
glucosidic bonds whereas isomaltase hydrolyzes α(1- be broken before absorption can occur and the low
6) glucosidic bonds (Le Huërou-Luron, 2002). concentrations of pancreatic amylase present do not
Maltase concentrations are low at birth and increase hydrolyze enough bonds to provide adequate energy
between 7 and 119 days after birth. Isomaltase is at for the calf (Morrill et al., 1970).
low concentrations in the newborn and increases after However, once the calf is older and the rumen is
weaning (Le Huërou et al., 1992). Trehalase, one of more developed, starch becomes a primary part of
the four disaccharidases, is able to hydrolyze trehalose the diet. Development of the rumen including
to produce free monomeric glucose for absorption formation and elongation of rumen papillae is critical
(Le Huërou-Luron, 2002). in transforming the monogastric calf to the ruminant
Lactase is the only enzyme capable of ß(1-4) dairy heifer (Church, 1988). More economical diets
glucosidase and ß(1-4) galactosidase activity. It is and reduced labor required for feeding are the driving
able to break down lactose to glucose and galactose forces to demand this change as early as possible.
as well as cellobiose to two monomers of glucose The primary limiting factor for weaning dairy calves
(Le Huërou-Luron, 2002). Lactase activity is highest from milk or milk replacer is having adequate
at birth and decreases until about three weeks of age development of the rumen in order to maintain post-
(Huber et al., 1961) or one week of age (Le Huërou weaning growth at desired rates.
et al., 1992) and then remains constant until levels
Rumen development is stimulated by the presence Holstein bull calves to study effects of calf starter
of microbial produced volatile fatty acids, primarily supplemented with 0, 6, and 12 g/h/d of amylase as
butyric acid (Beharka et al., 1998; McLeod and supplied by AmaizeTM. Calves were assigned to a
Baldwin, 2000). Volatile fatty acids introduced into treatment group on arrival with a total of five calves
the rumen as purified sodium salts, especially sodium per treatment group. Calves were fed milk replacer
butyrate, increase epithelial development (Tamate et (Land O’Lakes 20% all-milk protein, 20% fat
al., 1962). Butyric acid provides energy for the product), at a rate of 10% of arrival body weight
rumen wall for maintenance and development. This twice per day for the duration of the trial. Calves
development includes the formation of papillae and were fed calf starter (18% CP, Purina Mills, Inc.)
thickening of the rumen wall, including increased and water once a day on an ad libitum basis. Grain
capillary development (Weigand et al., 1975). Any was fed in the morning after milk feeding and was
additional butyric acid that is not needed as energy done in such a way that the first 200 g/d of grain
for the rumen is transported into the blood stream as contained the treatment dosage (6 or 12 g/d) of
β-hydroxybutyrate. Around 90% of butyrate AmaizeTM. After calves ate this amount each day they
produced in the rumen is directly absorbed (Bergman, were offered ad libitum grain for the remainder of
1990; Weigand et al., 1975). Rumen wall growth the day. This was done so that calves were receiving
and capillary development are needed to increase the the treatment at the desired rate of daily intake at the
ability of this organ to absorb rumen produced energy earliest possible age. Fecal and health scores were
substrates. Since the microbial end products of starch monitored daily. Calves were euthanized at the end
are propionic and butyric acids, rumen development of 5 weeks (35 days) of age. The reticulorumens were
is most influenced by ingestion of grain and the sampled in nine areas for papillae analysis as shown
digestion of the starch components in the grain. When in Figure 1.
starch is broken down in the rumen, volatile fatty Samples were taken from the rumen from the
acids (VFAs) are produced. (Greenwood et al., 1997; following regions: the caudal portion of the caudal
Krehbiel et al., 1992; Nocek et al., 1984; Weigand ventral blind sac (A), the right and left caudal dorsal
et al., 1975). blind sac (RB and LB), the right and left cranial
In the young pre-ruminant calf, the ability to begin dorsal blind sac (RC and LC), the right and left cranial
lowering the pH helps provide an environment for ventral blind sac (RD and LD), and the right and left
the continuation of a normal rumen bacterial and caudal ventral blind sac (RE and LE). The rumen
protozoan population. Any way to augment the papillae were measured according to length, width,
process of the digestion of starch may allow for faster papillae per cm 2 , and rumen wall thickness
breakdown of starch by bacteria into propionic acid (Lesmeister et al., 2002). All 15 calves arrived
for absorption as energy and butyric acid to stimulate healthy, there was no mortality, and no calves were
development of the rumen. Addition of exogenous treated for clinical disease during the study period.
α-amylase may be one way to increase starch The primary measure of interest of this study was
digestion in the ruminant animal. α-Amylase rumen development. All areas of the rumen were
hydrolyzes α(1,4) glycosidic linkages but is unable sampled and measured as shown in Figure 1. Tables
to hydrolyze α(1,6) bonds in the branch points. 1, 2, and 3 along with their accompanying Figures
Because of this, α-amylase only begins starch 2, 3, and 4, show the rumen papillae length, width,
breakdown and the rest is continued through microbial and density. These measurements are primary aspects
digestion. of the rumen that are related to the tissue surface
Thus, it was of interest to determine whether the area available for volatile fatty acid absorption. Each
addition of two levels of amylase (AmaizeTM, Alltech area grows at a different rate and is quite different in
Inc.) supplied in calf starter could have an effect on size, even in adult ruminants. Therefore the sampling
rumen development by increasing the initial rate of pattern as shown was used to quantify the entire
starch digestion in the developing rumen. reticulorumen.
Papillae length had five measured areas where the
6 g/hd/d treatment was significantly greater (P<0.10)
A study of the effects of added amylase in than either the control (0 g/hd/d) or the 12 g/hd/d
AmaizeTM treatments. In 6 of 9 areas papillae width was
significantly greater (P<0.05) in the 6 g/hd/d group
Recently Gehman et al. (2003) utilized fifteen than the control and in 4 areas was greater than the
Figure 1. Rumen sampling method: A = caudal portion of caudal ventral blind sac, RB and LB = right and left caudal dorsal blind
sac, RC and LC = right and left cranial dorsal blind sac, RD and LD = right and left cranial ventral sac, RE and LE = right and left
caudal ventral blind sac.
Table 1. Least squares means of papillae length (cm) for 9 areas of the rumen in calves fed 0, 6, or 12 g/d of amylase in grain.
AmaizeTM Comparison of means

(g/head per day) P value
Rumen area1 0 6 12 SE 6>0 12 > 0 6 > 12
RB 0.092 0.111 0.101 0.010 NS NS NS
LB 0.104 0.113 0.096 0.007 NS NS 0.09
RC 0.073 0.095 0.088 0.009 0.09 NS NS
LC 0.101 0.116 0.091 0.008 NS NS 0.02
RD 0.192 0.203 0.168 0.020 NS NS NS
LD 0.139 0.141 0.136 0.010 NS NS NS
RE 0.060 0.074 0.046 0.010 NS NS 0.04
LE 0.041 0.046 0.028 0.006 NS NS 0.04
A 0.099 0.111 0.106 0.010 NS NS NS
1
Rumen areas sampled: RB = right caudal dorsal blind sac, LB = left caudal dorsal blind sac, RC = right cranial dorsal blind sac, LC = left
cranial dorsal blind sac, RD = right cranial ventral blind sac, LD = left cranial ventral blind sac, RE = right caudal ventral blind sac, LE = left
caudal ventral blind sac, A = caudal portion of caudal ventral blind sac. Figure 1 shows location of these areas.
Table 2. Least squares means of papillae width (cm) for 9 areas of the rumen in calves fed 0, 6, or 12 g/d of amylase in grain.

Rumen area1 0 6 12 SE 6>0 12 > 0 6 > 12
RB 0.057 0.078 0.068 0.008 0.07 NS NS
LB 0.058 0.083 0.066 0.007 0.01 NS 0.07
RC 0.055 0.079 0.073 0.008 0.04 NS NS
LC 0.067 0.091 0.071 0.008 0.04 NS 0.07
RD 0.081 0.090 0.095 0.013 NS NS NS
LD 0.068 0.087 0.086 0.012 NS NS NS
RE 0.051 0.072 0.052 0.007 0.04 NS 0.05
LE 0.048 0.056 0.035 0.006 NS NS 0.01
A 0.059 0.083 0.075 0.008 0.03 NS NS
1
Table 3. Least squares means of papillae per cm2 for 9 areas of the rumen in calves fed 0, 6, or 12 g/d of amylase in grain.
Rumen area1 0 6 12 SE 6>0 12 > 0 6 > 12
RB 69.81 92.76 83.84 7.71 0.04 NS NS
LB 67.28 79.76 80.57 9.23 NS NS NS
RC 53.80 74.99 66.73 7.42 0.05 NS NS
LC 59.61 70.89 82.31 9.83 NS NS NS
RD 77.37 69.38 86.06 6.61 NS NS 0.07
LD 69.97 72.28 88.55 6.01 NS 0.03 0.06
RE 51.80 75.11 67.09 8.23 0.05 NS NS
LE 48.83 63.90 67.67 6.56 NS 0.05 NS
A 70.03 83.39 90.18 9.17 NS NS NS
1
0 g/hd/d
0.25
6 g/hd/d
12 g/hd/d
0.20
0.15
Length (cm)
a a
ab ab
b a ab b
0.10
b a
ab
0.05 b ab a
b
0.00
RB LB RC LC RD LD RE LE A
Area of rumen
Figure 2. Least squares means of papillae length (cm) for 9 areas of the rumen in calves fed 0, 6, or 12 g/d of AmaizeTM in grain.
0 g/hd/d
0.10
6 g/hd/d a
0.09 12 g/hd/d
a a
0.08 a a
ab
ab b a
ab
Width (cm)
0.07 b b
b b
0.06 b a
b
b b
0.05 ab
0.04
b
0.03
Area of rumen
Figure 3. Least squares means of papillae width (cm) for 9 areas of the rumen in calves fed 0, 6, or 12 g/d of AmaizeTM in grain.
0 g/hd/d
100
a 6 g/hd/d
90 12 g/hd/d a
a
ab
80 ab
a
Papillae per cm2
a
b
b b b
70 ab ab a
ab
60
b
b
50 b
40
Area of rumen
Figure 4. Least squares means of number of papillae per cm2 for 9 areas of the rumen in calves fed 0, 6, or 12 g/d of AmaizeTM in grain.
Figures 2-4. Rumen areas sampled: RB = right caudal dorsal blind sac, LB = left caudal dorsal blind sac, RC = right cranial dorsal
blind sac, LC = left cranial dorsal blind sac, RD = right cranial ventral blind sac, LD = left cranial ventral blind sac, RE = right
caudal ventral blind sac, LE = left caudal ventral blind sac, A = caudal portion of caudal ventral blind sac. Figure 1 shows location
of these areas. Within each rumen area, bars with different letters differ at the P < 0.10 level.
12 g/hd/d group. In no cases was the 12 g/hd/d group as this is quite consistent across all areas of the rumen.
greater than controls. In papillae per cm2, there were Grain intake was similar across all treatment groups
three areas where the 6 gm/hd/d group were larger and was quite typical of calves in summer
(P<0.05) than the controls and two areas where the temperatures and appeared adequate to support
12 g/hd/d were greater than controls along with two weaning of the calves by the end of the experiment.
areas where the 12 g/hd/d group were greater than All animal growth measurements taken including
the 6 g/hd/d group. Rumen wall thickness was not heart girth, body weight, hip width, and withers
affected by treatment. These data show that the height were similar for all treatments. These appeared
amylase affected rumen growth and increased normal for calves of this age and feeding regime with
parameters related to nutrient absorption. The lack moderate levels of milk replacer and grain intake.
of difference in rumen wall thickness is not surprising In summary, the enzyme systems of the neonatal
Table 4. Least squares means of rumen wall thickness (cm) for 9 areas of the rumen in calves fed 0, 6, or 12 g/d of amylase in grain.

Rumen area1 0 6 12 SE 6>0 12 > 0 6 > 12
RB 0.126 0.141 0.134 0.011 NS NS NS
LB 0.126 0.136 0.136 0.008 NS NS NS
RC 0.138 0.150 0.144 0.011 NS NS NS
LC 0.159 0.152 0.158 0.011 NS NS NS
RD 0.176 0.175 0.157 0.011 NS NS NS
LD 0.161 0.190 0.174 0.012 NS NS NS
RE 0.140 0.139 0.131 0.015 NS NS NS
LE 0.139 0.147 0.144 0.008 NS NS NS
A 0.130 0.130 0.130 0.006 NS NS NS
1
dairy calf are quite complex, yet well suited for an Gorrill, A.D.I., J.W. Thomas, W.E. Stewart and J.L.
animal that begins life as a monogastric and is Morrill. 1967. Exocrine pancreatic secretion by
transformed to a ruminant. The development of the calves fed soybean and milk proteins diets. J.
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Guilloteau, P., T. Corring, J.A. Chayvialle, C.
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M.F. Hutjens 205
Meeting the educational needs of dairy clientele in 2020

MICHAEL F. HUTJENS
Changes in dairy production patterns

Ways in which the educational needs of the dairy • Total cow numbers declined less than 1%
clientele in 2020 and beyond are met will change annually. This trend may slow depending on
compared to those used in historical and trends in consumption (demand for dairy
contemporary dairy educational programs. After products).
interacting with dairy clientele (farm managers, agri- • Milk yield per cow increased on average 2.1%
business personnel, veterinarians, educators, and annually, a figure which includes the impact of
consultants) for over 33 years, new ‘teaching’ BST (commercial use began in 1994).
approaches and opportunities are appearing and will Opportunities remain, as the average milk yield
continue to develop in all sectors of the industry. is 18,814 lbs while many herds average over
Looking back to examine changing industry patterns 25,000 lbs and individual cows can exceed 40,000
allows us to look ahead and begin making strategic lbs of milk. Larger herd sizes are associated with
plans to serve this dynamic group. Nutrition and feed increased milk yield per cow.
management examples and applications will be used
in this paper, but these concepts could apply to repro- • The number of dairy herds has dropped by 44%.
duction, milk quality, genetics, manure management, This trend will continue as herd sizes increase to
and other related disciplines. meet the demand for milk by consumers. The
shift to larger herd size will reduce the number
of dairy farm managers while increasing demands
LOOKING BACK: CHANGES IN THE PAST 10 for unique services (feeding, reproduction, health,
YEARS and financial consultants).
The largest shift in US dairy production patterns has • Average herd size doubled in the last 10 years.
been where milk is being produced in the country. This trend will depend on the management style
The western area in 1993 produced 35% of all US of the farm manager or family.
milk and had 31% of the dairy cows. In 2002, the • Milk prices remain constant, leading to tighter
West accounted for 41% of US dairy cows and profits margins and increased emphasis on
produced 45% of all milk, growing in average herd efficiency.
size from 190 cows to 412 cows per herd. Table 1 Table 1. US dairy statistics, 1993 and 2003.
summarizes changes over the last 10 years (1993 to
1993 2003
2003). Summary points are listed below.
Total milk produced, billion lbs 151.0 169.8
Number of cows, million 9.705 9.025
• Total milk yield increased on average 1.2% Number of dairy farms 124,942 70,209
annually. The trend toward more milk may shift, Herd size, cows per farm 78 130
depending on the aging US population, new dairy Milk yield per cow, lbs per year 15,554 18,814
products (e.g. a ‘new’ pizza), health concerns, Milk price (Class 3), $ per 100 lb 11.80 11.40
advertising and marketing of dairy products, and Halladay, 2004
export opportunities.
206 Meeting the needs of dairy cattle clientele in 2020
THE DAIRY INDUSTRY IN 2020 • Marketing risk could be a factor if milk processors
provide incentives for larger milk volumes picked
Milk production will increase while cow numbers up at the farm. The swine industry has future
decline and dairy farm numbers will drop. Larger contracting and shackle space guarantees to ensure
herd sizes (250 to 3000 cows per unit with multiples timely marketing. Will smaller dairy farms have
of these base numbers) will continue. Dairy farms a market?
will produce 1.2 to 1.5 million lbs of milk per full
time employee equivalent. Milk processing companies
Table 3. Percentage of milk production by herd size in the US.
may target semi-load(s) of milk from the farm (600
milking cows per load). Table 2 summarizes Farm size (number of cows) 2000 2010 2020
projected numbers of dairy farms in the United States,
assuming that milk consumption of dairy products 1 to 49 9.5 3.4 0.6
50 to 99 19.4 8.2 2.3
remains constant (LaDue et al., 2004). 100 to 199 17.3 10.1 3.8
200 to 499 18.0 15.3 8.7
Table 2. Projected number of dairy farms in the US. Over 500 cows 35.8 63.0 84.6
Farm size (number of cows) 2000 2010 2020 LaDue et al., 2004
1 to 49 52,920 18,235 2,821

50 to 99 31,360 12,751 3,549
100 to 199 12,865 7,204 2,655 Dairy managers as education clients
200 to 499 5,350 4,292 2,335
Over 500 cows 2,675 3,294 3,361 In the education process to date, the primary focus
Total 105,170 45,777 14,721
has been the dairy farm manager. Dairy farm systems
LaDue et al., 2004 can be divided into different management groups
requiring alternative methods for education delivery.
Large herds (over 500 cows) increased cow numbers
by 4% over the last year. The majority of milk will
be produced on farms milking over 500 cows. FAMILY DAIRY SYSTEMS
Distribution of cows within herds will be a key factor
that could impact sustainability of the operation and Traditional ‘family’ dairy systems will continue to
the need for various types of educational alternatives. be a primary management group. This group will
Table 3 lists the distribution of milk production by have the following characteristics that will dictate
herd size. Factors that could alter trends are outlined educational approaches.
below.
• The labor source will include the owner,
• Economic risk favors larger herd sizes by $0.50 daughter(s) and son(s) in-law, herdsperson and/
per 100 lbs (cwt) of milk produced. Smaller herds or part-time youth.
will need to offset this advantage to remain • The size of the operation will depend on the
competitive. number of family members employed with a
• Health and disease risk increase with herd size as target of 50 to 70 cows per full time employee
more people and higher cattle concentration equivalent.
occur. • Milk production per cow will be high (25% above
• Environmental risk will be a concern as current the national average).
concentrated animal feed operations (CAFO) • Sales of surplus dairy cattle can be a source of
regulations apply only to large farms. In the income. Registered dairy cattle breeders may
future, all farms may be required to meet these remain in this category.
regulations. Smaller herds with fewer cows over
Educational programs that could appeal to this group
which to spread this investment will be forced to
include traditional approaches such as mass media,
make economic decisions. Environmental Quality
educational meetings, tours, and targeted topics
Incentive Program (EQIP) can provide up to
(accelerated calf growth, lameness, milk components,
$450,000 per farm depending on future funding
and crossbreeding).
commitments by the government.
M.F. Hutjens 207
LOW INPUT DAIRY SYSTEMS • Labor will be provided by outside resources,

necessitating labor management skills and
Low input dairy systems are popular in regions that strategies for dealing with language limitations.
have land suited to grazing and a traditional dairy
history. These dairy farms have been referenced as • Economics will be the driving factor as decisions
‘sustainable farms’ with the following characteristics. are based on return on investment (not
convenience or drudgery).
• Family members are the only source of labor. • The ability to assemble semi-loads of milk will
• Herd size will remain stable over time dictated allow market alternatives and premiums.
by labor and land resources. • Profit margins for suppliers will be tight as
• Milk yield may be below the national average volume discounts may be expected by these dairy
(90%). managers.
• The ‘New Zealand system’ dairies are included
in this group (pasture systems, larger number of This group will require unique educational
cows (100 to 150 lactating cows per full time opportunities that may be targeted specifically for
equivalent), and large parlors to reduce milking their farm(s), for example heat detection systems,
time to less than 2 hrs per milking). feed delivery, or benchmarks to monitor economic
success. The educational program may be delivered
• Investment per cow is minimized with little at the farm level allowing employees or managers to
equipment owned, custom planting of crops and balance time availability with specific farm learning
harvesting of forages, and/or off-farm purchase needs. Attending traditional educational programs is
of feeds. not popular; while networking with similar operations
• Off-farm employment may be needed for is perceived as more useful (invitational meetings,
supplemental income and insurance benefits. one-on-one sessions with specialists, and company-
sponsored activities).
The educational needs of this group will be specific
for individual dairy managers and will apply to their
Agri-business as client
particular situation. Tours, success stories in mass
media, and production tips will be useful educational A second group of clients for educational programs
approaches. These individuals will need educational are agri-business ‘educators’ as they serve dairy
efforts to be close to their farms, as travel time or managers. ‘Training the trainer’ multiplies educator
employment will be limiting factors. Weekend impact, resulting in more effective changes on farms,
programs may be needed. and a greater ability to deliver the recommendation
(for example by including the educational concept
in the feed delivered to the farm). Three groups are
INTENSIVE DAIRY FARMS outlined below.
Intensive dairy farms reflect larger dairy units (listed
in Tables 2 and 3). These farms will increase in
VETERINARIANS
number as competition and milk processors favor
these systems. The following characteristics can apply Veterinarians are ‘the’ on-farm experts (defined by
to this dairy model. the dairy manager) that understand herd dynamics
and health changes. If the veterinarian supports a
• Herd size will vary from 250 to 3000 cows with feeding or management change, it will happen on
multiples of each size per farm (for example a her or his client farms. The veterinarian can be an
herd of 12,000 cows may consist of units with effective method to indirectly reach dairy managers.
3000 cows for optimal management by one If one Midwest dairy veterinarian is ‘sold’ on a
management team). concept (for example drenching cows), she or he can
• Milk yield will be high as the latest technology implement this program on 30 to 60 dairy farms.
and health programs will enhance performance Training for veterinarians can include AABP
and quality. (American Association of Bovine Practitioners)
national meetings and programs, state veterinary through a new program initiative called U of I On-
meetings, and local programs. Some states require Line. Administrators felt the future for education was
continuing education (CE) credits be earned by on-line, reaching students across the state and around
veterinarians (another reason why veterinarians are the world. Because dairy feeding classes had been
potential clients). taught across Illinois as traditional classes in the 1990s
(classroom style with face-to-face lectures), a
competitive grant was obtained ($122,200). The
REGIONAL FEED SUPPLIERS Illinois Dairy Certificate program was the only
successful grant in the College of Agriculture and is
Regional feed suppliers can react quicker than national outlined below.
feed suppliers. These suppliers are aware of local
needs, are seen as part of the community (anti- • Five on-line classes were developed
Walmart image), and maintain ties to the community.
These organizations may not have large research and - Principles of Dairy Science (AnSci 200)
marketing departments, and need to develop ways of - Advanced Dairy Management (AnSci 300)
staying current and updated.
- Advanced Dairy Nutrition (AnSci 373)
- Advanced Dairy Reproductive Physiology
CONSULTANTS (AnSci 374)
- Milk Secretion and Mastitis and Quality
Consultants will play a more important role as
(AnSci 375)
increased herd sizes creates the demand for more
specific information and guidelines. Many of these • An on-line laboratory section is available to apply
individuals will have advanced degrees (beyond the principles in the five classes including breed
bachelor degree) and understand the science behind identification, CMT milk test, hand calculated
research studies. These individuals or groups will need and computerized ration formulation, feed
to maintain cutting edge knowledge of their identification, heifer growth, milk progesterone
specialties through non-traditional approaches. testing for pregnancy, and record analysis.
Unique educational programs specific for this group • When the student completes the five classes and
(such as one-on-one discussions) may be popular. lab section, they receive a Dairy Certificate from
the University of Illinois (certified by the
Graduate College at the University of Illinois).
The Illinois distance learning approach This is not a masters’ degree, but indicates
successful completion of the course material and
Illinois Extension continues to change educational exams.
programs as their clientele base changes. With slightly
over 1200 dairy farms (ranging from 27 to 2700 cows
per herd), Illinois Extension delivers research results Individuals not interested in the Dairy Certificate
and recommendations via ten area Illinois Dairy Day Program can enroll in specific classes as needed or
programs, an annual dairy research summary, dairy desired. The enrollment costs vary from $400 to $500
records program using PC DART, and regional depending on whether the student is enrolled for
meetings arranged through the Four State Dairy Team credit (undergraduate or graduate) or audit (visitor)
(WI, MN, IA, and IL). Mass media plays a major status and hours of credit. Each class has the
role delivering information through Hoard’s following characteristics.
Dairyman, Dairy Today, Hoard’s West, Dairy Herd
Management, Midwest Dairy Business, AgriView, • Classes are conducted for 10 to 11 consecutive
AgriNews and other media outlets. Participating in weeks.
national and international programs expands the reach • Each class has a CD that has all lectures recorded,
and focus of Illinois dairy extension programs. All edited, and ready to review when the student has
information is available on the internet at http:// time to listen during the week.
www.traill.uiuc.edu/dairynet/.
Another opportunity occurred in 1998 when the • Each week’s assignment consists of 4 to 8
University of Illinois offered instructional grants modules with 15 minute listening time (single
M.F. Hutjens 209
topic lecture) including 15 to 20 slide sets that and eight consultants. Table 4 summarizes their
can be printed to take notes and list questions ranking and emphasis of the five factors.
while listening.
Table 4. Survey results from the 31 specialists divided by groups
• Once a week, instructor(s) will hold a formal class and ranking.
for 1 hr on the internet. Students can listen to the Item Veterinarians Extension Consultants Total
discussions, type in questions, and review agents
homework. All 1 hr programs are recorded and Forage quality 13 (5) 5(3) 4(3) 33
archived for students that cannot attend or need Cow comfort 9 1 7(2) 24
to review. Transition cow diets 8(5) 2 6 21
Feed bunk management 8(1) 3 4(1) 17
• For students enrolled for academic credit, Feed availability 3(1) 2 2 10
homework is assigned each week (requires 15 to Feed consistency 4 3 2 9
30 minutes to complete depending on the Feed particle length 4 1 1 6
Feed refusal
student’s background) and is sent electronically management 2 0 0 2
to the instructor for grading. A take-home final Personnel mixing
exam completes class assignments. Discussion rations 1 1 1 3
questions and research paper reviews can be used Dry matter intake 4 (3) 0 0 7
Feed palatability 1 0 0 1
by instructors to enhance the learning experience. Moldy feed 1 0 0 1
Bunk silo management 1 0 4 5
Water quality and
The CD can also be purchased as a reference tool to management 2(2) 2(2) 4(1) 13
learn or use. The Advanced Dairy Feeding class Nutrient balance and
(AnSci 373) has trained approximately 350 students form 5 3 2 10
using the internet class while over 1200 CDs have
been sold ($45 for US students; $60 for foreign
students). The CD can be purchased at http:// The number in parentheses indicates the number of
www.fass.org/UofI/. individuals that listed this factor as the number one
Evaluation of the distance learning approach has ranked factor limiting cows for increased milk
been favorable to excellent (Hutjens and Baltz, 2000). production. Each factor was ranked by awarding one
The availability of dairy education on an ‘as needed’ point for the area or factor and an additional one
basis, the flexibility of CD modules, ability to interact point if it was the respondent’s first-ranked factor.
with instructors without being on campus, use of Forage quality was number one in the group. Bunk
expertise from several different areas and universities, management would have been first if all sub-factors
and CD teaching applications have been listed as (listed under bunk management in Table 4) were
advantages of the Illinois system. included as one factor. Respondents were specific
Agri-business has also partnered with the Illinois about numerous feed bunk-related factors. It is also
CD/Internet distant learning approach through support interesting to note the differences in the importance
of program development, distribution of the CDs to and variation of responses by job classification and
their key customers and participation in continuing on-farm responsibilities.
education programs for specific disciplines.
In summary
Future nutritional challenges After reviewing changes in the US dairy industry,
As Holstein herds achieve higher milk yields, clientele needs, and reduced extension commitments,
nutrition needs increase to meet milk and the author’s biases on futuristic dairy educational
reproduction requirements. The University of Illinois programs are outlined below.
Extension conducted a national survey using the
Monsanto Dairy Advisers and Technical Support • The need of for research-based information will
Specialists. Each person was asked to rank in order be remain important in 2020.
five factors that they see limiting cows from reaching • Dairy managers will want more specific and
high yield milk. Thirty-one individuals responded targeted educational information that meets needs
including 17 veterinarians, six extension educators, for their farms and employees.
• One-on-one training and education will be in • Extension educators need to provide futuristic
greater demand (extension administration will not information (food safety, environment quality,
encourage this method of education delivery). consumer concerns, and systems approach) along
with traditional information (feeding, mastitis,
• Regional feed companies will be a factor if they
culling, and other management topics) in a format
can develop a local identity and meet local needs.
the clientele need and support.
• The internet and distance learning approaches will
continue to be a way to reach dairy clientele with
information. References
• Land grant universities will reduce faculty time
for traditional extension programs, require cost Halladay, D. 2004. Ten years later, how much has
recovery, and will emphasize the need for changed? Hoard’s West 6:1:W2.
educational grants. Hutjens, M.F. and J.H. Baltz. 2000. Keeping extension
• The dairy industry (producers and agri-business) programs current in order to meet the needs of a
will need to come forward and support extension dynamic dairy industry. J. Dairy Sci. 83:1412.
efforts (financially and vocally) or lose these LaDue, E., B. Gloy and C. Cuykendall. 2004. Which
educational efforts. farms will produce the nation’s milk in 2020?
Hoard’s Dairyman 49:2:49.
C.J. Richards and H.D. Loveday 211
Redefining selenium nutrition using organic selenium (Sel-Plex®): defining

maximal acceptable tissue residues in beef
C.J. RICHARDS AND H.D. LOVEDAY
Animal Science Department, University of Tennessee, Knoxville, Tennessee, USA
Introduction
Selenium (Se) is an essential nutrient for humans tract (Podoll et al., 1992). This can result in most of
and livestock. Beef cattle have a general requirement the Se from selenite supplementation being excreted
of 0.1 mg/kg of selenium in the diet (NRC, 1996). in feces (Lopez et al., 1969). This is of concern when
Many regions of the United States have soils that are supplementing beef cattle diets, because Se
low in Se. Because forage and feedstuff Se supplementation has been restricted to supplying a
concentrations vary with soil Se concentrations, the maximum of 0.3 mg/kg of Se by the US Food and
Se status of US cattle herds is highly variable. Across Drug Administration (FDA, 1997). With the
the country 18.2% of the cows and heifers have blood supplemental level limitations, it is desirable to have
Se concentrations that are considered low or marginal additional, more available sources of Se to use in
(Dargatz and Ross, 1996). In the southeastern region beef cattle diets.
of the US, 42.4% of the cattle and 35.8% of the Selenium found in feedstuffs is most commonly
herds evaluated were considered to have at least a associated with sulfur amino acids where it replaces
marginally deficient Se status. These deficiencies, the sulfur in methionine and cysteine (Levander,
along with stressors of marketing and transporting, 1983). This is similar to the predominant form found
results in many calves arriving at feedlots with in selenium yeast, which is selenomethionine (Kelly
varying degrees of Se reserves. As one of the major and Power, 1995). Research has shown that selenium
ingredients in feedlot cattle diets, corn contains a from yeast is metabolized differently and has a greater
sufficient Se concentration (average = 0.13 mg Se/ availability than sodium selenite. Selenomethionine
kg diet) to meet finishing cattle requirements. randomly substitutes for methionine in proteins while
However, standard deviation on the 17 samples selenite is incorporated into selenoproteins (Finley,
reported in the Nutrient Requirements of Beef Cattle 2000).
(NRC, 1996) was 0.11 mg/kg. Because commodity Pehrson et al. (1999) and Gunter et al. (2003)
feedstuffs are not commonly analyzed for have shown that feeding Se yeast from Sel-Plex®
microminerals, Se is routinely supplemented to ensure results in greater whole blood concentrations of Se
adequacy. than when sodium selenite is fed. However, data
Since 1974, sodium selenate and selenite have been evaluating the effects of Se yeast supplementation
approved for use as supplements for livestock, with on Se tissue concentrations in finishing cattle are not
the feed industry primarily using the sodium selenite available. Therefore, the objective of this experiment
form. The two forms appear to have similar relative was to determine the effect of feeding a basal diet
biopotencies (Mason and Weaver, 1986; Podoll et balanced to meet the finishing cattle Se requirement
al., 1992), but in the functional ruminant, sodium or the basal diet supplemented with 0.3 mg/kg Se
selenite is known to be readily absorbed into feed from Sel-Plex ® Se yeast on corresponding calf
particles or reduced to insoluble elemental Se or performance, serum Se concentrations and to
selenides in the acidic environment of the digestive determine whether edible animal products (liver and
muscle) exceed allowable Se concentrations.
212 Redefining selenium nutrition using organic selenium (Sel-Plex®)
Materials and methods composition of the Se premixes. Feeding personnel

only knew the color code of Se premix to be included
ANIMALS in each diet. Similarly, sample codes for lab analyses
were labeled with an additional color-coding system
Thirty-five heifers and 32 steers were purchased so personnel conducting the Se assays did not know
through local markets, vaccinated for IBR, BVD, which samples corresponded to which dietary
PI3, and BRSV with Triangle 4 plus type II BVD treatment code.
(Fort Dodge, Overland Park, Kansas) and with a
Clostridial 7-way plus Somnumune from AgriLabs Table 1. Composition of initial grain acclimation diet.
(St. Joseph, MO). Calves were then delivered to The
University of Tennessee Agricultural Experiment Ingredient % (Dry matter basis)
Station in Knoxville, Tennessee. Upon arrival, they
Cracked corn 36.90
were ear-tagged and tattooed in both ears, treated Alfalfa pellets 45.00
for internal and external parasites (EPRINEX ®; Corn gluten pellets 10.00
Merial, Duluth, GA) and treated with Nuflor Molasses 5.00
Salt 0.30
(Schering-Plough Animal Health, Union, NJ). Calves
Fish meal 1.80
were maintained on a cool season pasture, and Vit/mineral premixa 1.00
following a minimum interval of 28 days, all calves
a
were weighed and heifers were verified open by CO-OP All Purpose Cattle Mineral, Tennessee Farmers Co-op,
LaVernge, TN; Ca, 27.00%; P, 4.50%; Salt, 19.20%; Mg, 0.30%;
ultrasonography. Twenty steers and 20 heifers were
K, 0.10%; Co, 24 mg/kg, Cu, 1,000 mg/kg, I, 36 mg/kg; Mn,
selected, blocked by sex and randomly allotted to 3,000 mg/kg; Se, 20 mg/kg; Zn, 3,500 mg/kg; Vit. A, 528,000
one of eight groups of five calves. This provided IU/kg; Vit. D, 99,000 IU/kg; Vit. E, 308 IU/kg.
four replicates per treatment (two replicates per sex).
Groups were randomly assigned to pens. Pens were
in a partially covered, concrete floor-feeding barn Table 2. Composition of finishing diets.
with free choice access to automatic water fountains.
Ingredient % (Dry matter basis)
After calves were assigned to pens, they were fed a
common diet (Table 1) containing 0.20 mg/kg Cracked corn 74.22
supplemental Se from sodium selenite. Grain Alfalfa pellets 10.00
Corn gluten pellets 10.00
acclimation was accomplished by decreasing the levels
Molasses 3.50
of alfalfa pellets from 45% to 35%, 25%, and 15% Urea 0.38
with the difference being made up by cracked corn. Vitamin and mineral supplementa 1.75
Fish meal was included in the acclimation diets Treatment premixb 0.15
allowing calves to become accustom to its a
See Table 3 for composition of vitamin/mineral supplement.
consumption. High Se concentrations in fish meal b
See Table 4 for composition of selenium premixes.
allowed it to be an alternate feedstuff to be included
in the final diet to maintain an adequate basal diet Se Calf weights were recorded two consecutive days
concentration if other feed ingredients varied between immediately prior to initiation of feeding
lots. Fish meal would replace a portion of corn and experimental diets. Calves were weighed every 28
urea if needed. After the acclimation period, pens of days for the duration of the experiment except for
calves were randomly assigned, within sex, to one of the final period, which was 18 days. Final weights
two finishing diet treatments. Treatment finishing were the average of weights taken the final two days.
diets consisted of a control basal diet (Table 2) Calves were fed their respective diets free choice,
balanced to contain 0.20 mg/kg (as-fed) of Se without with daily replenishment. Prior to daily feeding, any
supplemental Se and the basal diet plus 0.30 mg/kg feed present from the previous day’s feeding was
(as-fed) of Se from Sel-Plex® (Alltech, Nicholasville, removed from the feed bunk, weighed, recorded, and
KY). discarded. To verify Se concentrations of the finishing
diets, a 0.45 kg sample was collected from each daily
batch of complete finishing diet. These samples were
EXPERIMENTAL FINISHING PERIOD collected, composited into a weekly sample, and a
0.91 kg subsample retained for Se analysis.
This experiment was conducted as a double-blinded Additionally, a 0.45 kg sample of all major feed
experiment in which only the researcher knew the
ingredients was collected upon delivery to the animal the laboratory the same morning, allowed to coagulate,
facility and Se content determined to verify a centrifuged for 20 min (2,600 x g), serum harvested,
consistent concentration in the basal diet. and frozen (-4°C) for subsequent Se analysis.
The control treatment premix contained only fine
ground corn (Table 4). The Sel-Plex ® treatment
premix consisted of ground corn with 200 mg/kg of HARVEST MEASUREMENTS
Se from Sel-Plex ® . Treatment premixes were
sampled and analyzed for Se content before feeding On day 130, animals were loaded for overnight
to ensure proper inclusion level. Both treatment transport to a commercial harvest facility. Animals
premixes were blended in the treatment diets to were harvested on day 131. Carcasses were identified,
provide 0 or 0.3 mg/kg of Se to yield total dietary hot carcass weights recorded, and liver samples
concentration of 0.2 or 0.5 mg/kg Se. Both finishing collected. Liver samples were collected from each
diets used the same vitamin and mineral supplement, liver lobe (approximately 100 g from each lobe for a
which did not contain supplemental Se, at a rate of total of 300 g). Samples were placed in labeled plastic
1.75% (dry matter basis) of the total diet. Final feeds bags and packed in dry ice for transport to the
were manufactured daily by mixing the major feed laboratory where they were stored frozen (-4°C) until
ingredients, supplement, and premixes in a horizontal analyzed. After a 48 hr chill, standard USDA Yield
paddle mixer (Marion Mixers, Inc.; Marion, IA) and Grade and Quality Grade data were recorded as
then slowly adding liquid molasses. determined by a USDA grader. Adjusted fat thickness
was determined by measuring fat depth ¾ the distance
Table 3. Composition of finishing diet vitamin/mineral supplement around the longissimus muscle at the 12th rib. This
and mineral premix. fat measurement was then subjectively adjusted
according to USDA guidelines to reflect the overall
Ingredient % (Dry matter basis)
carcass fatness. Ribeye area (sq cm) was measured
Vitamin/mineral supplement using a standard USDA grid. Each 12 th rib
Limestone 80.00 longissimus muscle area was measured twice and the
Salt 17.14 average recorded. The percentage of kidney, pelvic
Mineral premix 1.71
Vitamin premixa 1.14
and heart fat (% KPH) was determined to the nearest
0.5%. These yield grade factors were used in the
Mineral premix
Fine ground corn 45.76 following USDA Yield Grade formula: YG = 2.50 +
Molasses 4.00 (2.50 × adjusted fat thickness) + (0.20 × %KPH) +
Magnesium oxide 17.70 (0.0038 × hot carcass weight) – (0.32 × ribeye area).
Zinc sulfate 16.55 Carcass maturity and marbling degree evaluations
Iron oxide 9.00
Manganous oxide 2.60 were used to determine USDA Quality Grade. After
Copper sulfate 3.95 completing the carcass evaluations, loin tissue
Potassium iodide 0.44 samples (500 g) were collected at the 12th rib, placed
a
in plastic bags, identified, and packed in dry ice for
CO-OP Vitimin ADE Micro Mix, Tennessee Farmers Co-op,
LaVergne, TN; Vitamin A, 11,000 IU/kg; Vitamin D, 880 IU/kg, transport to the laboratory where they were stored
and Vitamin E, 5.5 IU/kg. frozen (-4°C) until analyzed.
Table 4. Composition of finishing diet treatment premixes.

SELENIUM DETERMINATION
% (as-fed basis)
Ingredient Control Sel-Plex®
Upon retrieval from the freezer and thawing, a scalpel
Fine ground corn 100.00 80.75 blade was used to remove similar slices (by weight)
Sel-Plex®a 0.00 19.25 from each of the three liver lobe sections of each
a
animal to be combined for a single analytical
Alltech, Nicholasville, KY.
observation. This procedure was repeated two more
times to produce triplicate samples. Each loin sample
Blood was collected from all calves on days 1, 28,
was divided into four approximately equal quadrants
56, 84, 112, and 129. Whole blood was obtained via
and a core sample was obtained from each quadrant
jugular venipuncture and collected into Vacutainers®
using a No. 10 cork borer (150 mm in diameter) so
(Becton Dickinson and Co., Franklin Lakes, NJ).
that a sample from each quadrant was represented in
Samples were immediately placed on ice, taken to
each sample for Se analysis. This procedure was
repeated two more times to produce triplicate samples. 0.13 mg/kg and averaged 0.08 mg/kg Se with values
Sample size from this process was approximately 6 ranging from 0.05 to 0.11 mg/kg Se. Four lots of
g. This was larger than could be digested in available dry corn gluten feed pellets Se concentrations averaged
test tubes, so loin samples were predigested and sub- 0.35 mg/kg and ranged from 0.35 to 0.38 mg/kg Se.
sampled for Se determination. Feed samples were One lot of fish meal contained 2.79 mg/kg Se. Grain
ground to pass a 1 mm screen in a Wiley Mill (Arthur acclimation diets were all supplemented with 0.2 mg/
H. Thomas Company, Philadelphia, PA) prior to kg Se from a commercial vitamin/mineral premix
analysis. (CO-OP All Purpose Cattle Mineral) and received
Selenium concentration of all samples (feed, serum, an additional 0.06 mg/kg from fish meal; however,
meat and liver) was determined using methodology the diet Se concentrations varied due to changing
based upon the fluorometric procedure by Koh and alfalfa pellet:corn ratios. Analysis of feedstuffs used
Benson (1983) as published by the American in the finishing treatment occurred after delivery to
Association of Analytical Chemists (AOAC, 1990). our research facility and before being incorporated
Samples were analyzed for Se in triplicate and a into treatment diets. The control weekly diet Se
coefficient of variation (CV) determined. If triplicates analysis averaged 0.17 mg/kg Se with a standard
of a sample had a CV higher than previously deviation of 0.06 mg/kg and the Sel-Plex ® diet
determined acceptable, samples were rerun. In averaged 0.49 mg/kg Se with a standard deviation of
addition, a standard from the National Institute of 0.04 mg/kg.
Standards and Technology (No. 1577b bovine liver)
of known Se content was assayed with each run of
samples. If the bovine liver standard for a run was PERFORMANCE MEASURES
not within the acceptable range, the run was
reanalyzed. All feed, serum and tissue Se Initial body weights averaged 307 (±3.2 kg; Table
concentrations are reported on an as-is basis. 5) at allotment and averaged 360.8 (±3.5 kg) at the
beginning of finishing diet treatments. Treatment had
no effect on body weight on any of the weigh dates.
STATISTICAL ANALYSIS A tendency (P>0.02) toward heifers being lighter
than steers at the initiation of treatments became
Feed consumption, growth performance, and tissue significant on day 56 due to steers weighing 41.4 kg
Se concentrations were analyzed as a randomized more than heifers and remained significant at day
complete block design using the Mixed Procedure of 84. The sex effect was not significant (P>0.02) for
SAS (Version 8.2, 1999; Carry, NC). The model weights taken on day 112 or the final weights and
included sex, diet treatment and the interaction with there was no treatment by sex interaction for any of
pen as the experimental unit. Differences in means the body weight measurements. Treatment had no
were separated using the LSD procedure of SAS. effect on the average daily gain, feed intake, or
Serum Se concentrations, collected at fixed times gain:feed ratio for any of the four 28-day periods,
throughout the experiment, were analyzed using the the final 18 days, or the total treatment period. There
repeated measure methodology of SAS (1999). For were no significant sex effects or treatment by sex
all response variables measured, results were interactions for average daily gain, feed intake or
considered significant at a confidence level of gain:feed ratio.
P<0.01. Lack of change in growth performance, feed
intake, and feed efficiency determined in this
experiment were expected due to both diets exceeding
Results and discussion the Se requirements of the cattle. The general
recommendation for beef cattle dietary Se is 0.1 mg/
DIET SELENIUM kg (NRC, 1996) and the computer model, adjusted
for conditions of this experiment, predicts a
All feedstuffs were purchased from the local feed requirement of 1.48 mg/day. The average daily
distributor as mill run ingredients without prior consumption of Se throughout the experiment was
screening for Se content. Five lots of alfalfa pellets 2.85 times greater for the Sel-Plex® treatment with
had an average Se concentration of 0.64 mg/kg and Controls consuming 1.71 mg/day and the Sel-Plex®
ranged from 0.47 to 0.94 mg/kg. Twelve lots of corn group consuming 4.88 mg/hd/day of Se. No calves
were all below the NRC (1996) published value of from either treatment showed signs or were treated
Table 5. Weight gains, average daily gains, feed intakes and gain:feed ratios.
Treatment P-value
®
Response variable Control Sel-Plex SEM Sex Treatment Treatment x Sex
Body weight, kg
Allotment 304.0 309.9 3.2 0.02 0.26 0.97
Initiation 358.5 362.2 3.5 0.02 0.51 0.95
Day 28 402.9 401.0 7.8 0.03 0.87 0.37
Day 56 431.9 436.2 6.1 0.01 0.65 0.71
Day 84 477.4 478.2 6.1 0.01 0.94 0.97
Day 112 510.9 510.0 9.0 0.03 0.94 0.98
Final 524.9 523.0 8.2 0.02 0.88 0.81
Average daily gaina, kg
Period 1 1.65 1.39 0.17 0.09 0.34 0.26
Period 2 1.04 1.26 0.20 0.49 0.48 0.39
Period 3 1.63 1.50 0.10 0.30 0.44 0.41
Period 4 1.20 1.14 0.17 0.34 0.81 0.99
Period 5 0.50 0.46 0.15 0.68 0.89 0.69
Overall 1.29 1.23 0.07 0.12 0.63 0.75
As-fed feed intake, kg
Period 1 9.3 9.4 0.2 0.02 0.61 0.73
Period 2 9.2 9.3 0.3 0.07 0.79 0.71
Period 3 10.3 10.3 0.5 0.57 0.97 0.81
Period 4 10.4 10.8 0.5 0.41 0.73 0.99
Period 5 10.0 9.9 0.4 0.21 0.82 0.54
Overall 9.8 9.9 0.3 0.16 0.83 0.94
Gain:feed ratio
Period 1 0.176 0.148 0.017 0.16 0.31 0.29
Period 2 0.113 0.135 0.020 0.75 0.49 0.35
Period 3 0.158 0.145 0.007 0.07 0.26 0.17
Period 4 0.113 0.105 0.011 0.37 0.60 0.95
Period 5 0.050 0.046 0.015 0.77 0.86 0.80
Overall 0.131 0.124 0.004 0.14 0.25 0.46
a
Period 1 = days 1 to 28; Period 2 = days 29 to 56; Period 3 = days 57 to 84;
Period 4 = days 85 to 112; Period 5 = days 113 to 130; Overall = days 1 to 130.
for illness while consuming experimental diets ribeye area (average = 83.25 sq cm), USDA yield
indicating that the addition of 0.3 mg/kg Se from Se grade (average = 3.0) or USDA quality grade (average
yeast did not result in any negative health effects. = Choice 0). No sex effect or treatment by sex
These results agree with research from Hintze et al. interaction was detected for any measure. The lack
(2002) in which steers were fed 55% concentrate diets of changes in carcass measures agrees with the lack
that contained 0.62 or 11.9 mg/kg Se from feedstuffs. of differences determined in performance.
Other results from additional Se in deficient diets
have been variable. When growing heifers, deemed
to be receiving a Se deficient diet (Maas, 1998), were SERUM SELENIUM
dosed with boluses releasing 3 mg of Se from sodium
selenite each day, no performance differences were Concentrations of serum Se were affected by
detected. In contrast, Nunn et al. (1996) determined treatment, time and a treatment × time interaction
that additional Se from Se boluses increased feed (Figure 1). Serum Se was not affected by sex, a sex
efficiency of steers offered a diet of Se deficient hay. × time interaction or a treatment × time × sex
interaction. On day one, there was no difference in
serum Se concentration between the treatments. There
CARCASS MEASURES was a difference between the Control and Sel-Plex®
treatments at all subsequent measurements. Within
No treatment differences were detected in hot carcass the Control treatment, serum Se dropped from day 1
weight (average = 321.2 kg; Table 6), 12th rib fat to 56 (0.091 to 0.078 µg/ml) and then reached a
(average = 1.41 cm), % KPH fat (average = 2.67), plateau (average = 0.080 µg/ml) with no point
Table 6. Carcass weight, 12th rib fat, KPHa fat, ribeye area, yield grade and quality grade.
Treatment P-value
®
Response variable Control Sel-Plex SEM Sex Treatment Treatment x Sex
Hot carcass wt, kg 323.2 319.2 7.0 0.13 0.71 0.92

12th Rib fat, cm 1.47 1.35 0.05 0.02 0.18 0.03
KPH, % 2.68 2.65 0.06 0.05 0.78 0.10
REAb, sq cm 85.0 81.5 3.6 0.56 0.53 0.51
Yield grade 3.0 3.0 0.2 0.29 0.98 0.21
Quality gradec 19.9 20.1 0.3 0.28 0.60 0.28
a
Kidney, pelvic and heart fat.
b
REA = ribeye area.
c
19 = Choice-, 20 = Choice0 and 21 = Choice+.
0.12 *+ * *
* *
0.10
Serum Se concentration (µg/ml)
*+ *
*+ * *
0.08
0.06 Treatment, P<0.01

Sex, P>0.32
Control Time, P<0.01
0.04 Treatment*time, P<0.01
Sel-Plex®
Treatment*sex, P>0.89
0.02 Sex*time, P>0.33
Treatment*sex*time, P>0.21
0.00
1 28 56 84 112 130
*Treatment within time, P>0.01 Days

+Within treatment from previous time
Figure 1. Serum selenium concentrations.
differing from the previous time point for the areas with naturally high Se feedstuffs could be viable
remainder of the experiment. This drop is associated sources of Se for human diets. They obtained steer
with the change in dietary concentrations. Grain calves from areas with moderate and high Se soil
acclimation diets all had 0.2 mg/kg of supplemental concentrations and consequently feedstuffs consumed
Se in addition to varying levels from feedstuffs while by cattle. Those steers were fed diets containing 0.62
the Control treatment had a total concentration of or 11.9 mg/kg Se. Steers from the high Se area had
0.2 mg/kg Se. In the Sel-Plex® treatment, serum Se an initial plasma Se concentration of 0.56 µg/ml and
increased between day 1 and 28 and then reached a steers from the moderate area had concentrations of
plateau at an average concentration of 0.11 µg/ml. 0.12 µg/ml. The serum levels from the moderate area
While this was 1.39 times the concentration in the are comparable to calves at initiation of the present
Control, the design of this experiment did not allow experiment, but levels from the high Se areas are
differentiation between effects caused by increasing greater than any levels measured in the current
dietary Se concentrations in general or specific effects experiment. Neither steers from the high Se area fed
from Se yeast. the 11.9 mg/kg diet or steers from the moderate area
Hintze et al. (2002) conducted an experiment with fed the 0.62 mg/kg diet showed a change in plasma
growing beef cattle diets to determine if cattle from Se concentrations. Steers from the moderate area fed
the 11.9 mg/kg diet had increased plasma Se (Figure 2) but was not affected by sex or a treatment
concentrations within two months to levels similar by sex interaction. The Se content of the loin muscle,
to steers previously exposed to high Se. Steers as collected in this experiment, has been determined
previously exposed to high Se levels and placed on to be representative of several of the edible muscle
the 0.62 mg/kg diet had plasma Se levels drop to tissues including cuts from the round, sirloin,
concentrations that, at the end of the 105-day feeding shoulder clod and ribeye muscles (Hintze et al.,
period, were not different from calves originating 2002).
from the moderate area and receiving the same diet. While there was a 1.9-fold increase in the muscle
The lowest level of Se fed by Hintze et al. (2002) Se concentration between the Control and Sel-Plex®
was slightly higher than the levels fed in our Sel- treatments (Figure 2), both values are still lower than
Plex® treatment and higher than our Control. Calves values obtained from calves in moderate or high Se
fed the 0.62 mg/kg diet in the experiment of Hintze areas of the United States and those fed diets
et al. (2002) had similar plasma Se concentrations to consisting of feedstuffs raised in high Se regions.
serum concentrations in our Sel-Plex® calves (0.13 They were also lower than sheep fed a corn-based
µg/ml vs 0.11 µg/ml, respectively). Both values are diet with the addition 0.3 mg/kg inorganic Se (Podoll
in a range described as normal (~0.10 µg/ml; Maas, et al., 1992) or the upper level determined normal
1998). Awadeh et al. (1998) found similar results by the US Food and Drug Administration (FDA, 1997;
when growing calves were fed diets containing 0.41 0.4 mg/kg). In the experiment by Hintze et al. (2002),
mg/kg (basal) and basal + sodium selenite to 0.73 steers purchased from high Se regions arrived with
mg/kg Se and showed that serum Se increased with muscle biopsy Se concentrations of 2.10 mg/kg, while
dietary Se supplementation (0.12 µg/ml vs 0.29 µg/ calves from moderate regions averaged 0.40 mg/kg.
ml, respectively). Results from the above experiments, After those calves were fed diets containing 11.9 or
along with the changes during the initial points of 0.62 mg/kg of Se, steers from the high region and
the current experiment demonstrate serum Se fed the 11.9 mg/kg diet had ribeye muscle Se
concentrations closely following dietary levels of Se. concentrations of 2.06 mg/kg and the moderate area
with 0.62 mg/kg diet contained 0.35 mg/kg. While
analyzing ground beef from animals raised in various
MEAT SELENIUM regions of the US, Finley et al. (1996) found that Se
concentrations varied from 0.34 mg/kg in samples
Average meat Se concentration was 0.12 mg/kg obtained from cattle raised in eastern North Dakota
higher for the Sel-Plex ® treatment than Control to 0.06 mg/kg for samples from cattle raised in
0.30
Treatment, P<0.01
Meat selenium concentration (mg/kg)
0.25 Sex, P>0.89

Treatment*sex, P>0.83
SEM = 0.009
0.20
0.15
0.10
0.05
0.00
Control Sel-Plex®
Figure 2. Meat selenium concentrations.

southwestern Missouri. These experiments show that Average liver Se concentrations for cattle on the Sel-
there is currently great variability in the Se content Plex® treatment were 0.41 mg/kg higher than that of
of beef reaching the retail market. the Control treatment. Liver concentrations of Se are
Hintze et al. (2002) suggested based on their data generally related to dietary Se concentration, but not
that 2.1 to 2.5 mg/kg could be the maximum amount as highly correlated as other tissues (Hintze et al.,
of Se that can be incorporated into muscle. This would 2001). Podoll et al. (1992) reported that sheep fed a
be consistent with findings that Se present in high corn diet with 0.3 mg/kg Se supplementation
proteinaceous tissue is primarily selenomethionine from selenate or selenite had similar liver
and selenocysteine with the ratio of these forms concentrations (1.79 and 2.10 mg/kg, respectively).
depending on the feedstuffs consumed (Beilstein and Steers in the experiment of Hintze et al. (2002) had
Whanger, 1988). Because methionine and cysteine liver Se concentrations ranging from 0.89 to 5.94
contents of muscle are not affected by Se addition to mg/kg. Both of these experiments consisted of diets
the diet, Se content of muscle could only increase to meeting the 0.3 mg/kg supplemental Se restriction,
a relative level based on the proportions of methionine but have liver concentrations greater than the range
and cysteine containing Se. Therefore, differences of 0.1 to 1.2 mg/kg determined as normal by the
in the incorporation rates of Se into muscle tissues FDA or those observed in this experiment.
due to form (inorganic vs organic) of dietary Se would The sex effect was a result of heifers within each
be expected. Several publications have reported treatment having higher liver Se concentrations than
muscle incorporation comparisons between feeding steers. The authors know of no other data that
of inorganic and organic Se sources and shown that demonstrate an effect of sex on liver Se. While
Se from organic sources is more readily incorporated differences between sexes on the Control diet were
into tissues than inorganic sources of Se (Ullrey et small (0.02 mg/kg), heifers on the Sel-Plex ®
al., 1977; Ammerman et al., 1980; Beilstein and treatment had higher liver Se concentrations (0.19
Whanger, 1988; Ekholm et al., 1991). Despite mg/kg) than did steers on the same treatment, which
incorporation rate differences between dietary forms, led to the treatment × sex interaction. Because calves
Hintze et al. (2002) noted there is high correlation for this experiment were purchased through local
between dietary and meat Se concentrations. market channels and most likely represent many
sources and varied backgrounds, liver differences that
occurred could be a carryover effect from prior
LIVER SELENIUM management. Serum Se concentrations were not
different at the initiation of the experiment, but they
Liver Se concentrations were affected by treatment, are noted to be more representative of Se status over
sex and a treatment × sex interaction (Figure 3). a shorter period than liver tissues (Podoll et al., 1992).
1.00 Control
Treatment, P<0.01 Sel-Plex®
Sex, P>0.01
Liver seleniun concentration (mg/kg)
0.80 Treatment*sex, P>0.01

SEM = 0.001
0.60
0.40
0.20
0.00
Steer Heifer Steer Heifer
Figure 3. Liver selenium concentrations.

Differences in responses of liver tissues to Effect of supplemental selenium for beef cows on
supplementation may be due to an adaptation to the performance and tissue selenium concentrations
prolonged high dietary Se concentrations. Hintze et of cows and suckling calves. J. Anim. Sci. 51:1381-
al. (2002) determined that calves from a moderate 1386.
Se background and fed a high Se diet had higher AOAC. 1990. Official Methods of Analysis (15th
liver Se concentrations than steers from a high Se Ed.). Association of Official Analytical Chemists,
background and fed high Se diets. They proposed Arlington, VA.
that this resulted from differences in the up-regulation Awadeh, F.T., R.L. Kincaid and K.A. Johnson. 1998.
of Se methylation by S-adenosylmethionine, which Effect of level and source of dietary selenium on
is necessary for Se excretion. Another potential concentrations of thyroid hormones and
explanation, demonstrated in the rat liver, may be
immunoglobulins in beef cows and calves. J. Anim
that 83% of Se is associated with GSH-Px compared
Sci. 76:1204-1215.
to relatively low levels of that form in muscle (Behne
Behne, D., and W. Wolters. 1983. Distribution of
and Wolters, 1983).
selenium and glutathione peroxidase in the rat. J.
Nutr. 113:456-461.
Beilstein, M.A., and P.D. Whanger. 1988.
Implications
Glutathione-peroxidase activity and chemical forms
Results of this experiment indicate that supplementing of selenium in tissues of rats given selenite or
0.30 mg/kg of selenium from selenium yeast in a selenomethionine. J. Inorgan. Biochem. 33:31-46.
feedlot diet adequate in selenium does not affect Dargatz, D.A., and P.F. Ross. 1996. Blood selenium
animal performance, but does increase serum and concentration in cows and heifers on 253 cow-calf
tissue selenium concentrations. However, the range operations in 18 states. J. Anim. Sci. 74:2891-2895.
of normal liver concentrations provided by the Food Ekholm, P., P. Varo, P. Aspila, P. Koivistoinen and
and Drug Administration is 0.1 to 1.2 mg/kg of L. Syrjalaovist. 1991. Transport of feed selenium
selenium. Selenium in meat is noted to be one-third to different tissues of bulls. Br. J. Nutr. 66:49-55.
to one-fourth the levels in the liver. This results in FDA. 1997. Food additives permitted in feed and
normal meat Se levels ranging from 0.025 to 0.4 drinking water of animals. Selenium. Fed. Regist.
mg/kg. Calves fed Sel-Plex® selenium yeast at 0.3 62:44892-44894.
mg/kg had average liver selenium concentrations of Finley, J., L. Matthys, T. Shuler and E. Korynta.
0.83 mg/kg and meat samples averaged 0.29 mg/kg. 1996. Seleium content of foods purchased in North
Therefore, selenium residues in edible tissue from Dakota. Nurt. Res. 16:723:728.
feedlot beef cattle supplemented with 0.3 mg/kg Finley, J.W. 2000. Does selenium accumulation in
selenium from selenium yeast do not exceed allowable
meat confer a health benefit to the consumer? Proc.
levels or other published levels.
Am. Soc. Anim. Sci., 1999. Available at: http://
www.asas.org/JAS/symposia/proceedings/0911.pdf.
Gunter, S.A., P.A. Beck and J.M. Phillips. 2003.
Acknowledgements Effects of supplementary selenium source on the
This project was conducted with support from Alltech performance and blood measurements in beef cows
Inc. (Nicholasville, KY). We express our appreciation and their calves. J. Anim. Sci. 81:856-64.
to Howard Blalock, Linda Miller, Del Harris, Malinda Hintze, K.J., G.P. Lardy, M.J. Marchello and J.W.
Burkhart, Eddie Jarboe, and R. Brent Pugh from the Finley. 2001. Areas with high concentrations of
Animal Science Department along with Drs. John selenium in the soil and forage produce beef with
Hodges and Bobby Simpson and their staff from the enhanced concentrations of selenium. J. Agric. Food
University of Tennessee-Knoxville Experiment Chem. 49:1062-1067.
Station for their help in completing this project. Hintze, K.J., G.P. Lardy, M.J. Marchello and J.W.
Finley. 2002. Selenium accumulation in beef:
Effect of dietary selenium and geographical area
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rats of selenium from intrinsically and extrinsically 45:559-565.
labeled isolated soy protein. J. Nutr. 116: 1883-1888.
D. Sauvant 221
Rumen acidosis: modeling ruminant response to yeast culture

D. SAUVANT, S. GIGER-REVERDIN AND P. SCHMIDELY
INAPG Département des Sciences Animales – UMR INRA–INAPG Physiologie de la Nutrition et
Alimentation, Paris, France
Introduction
Ruminal acidosis is a fairly well known digestive yeast supplements published in scientific papers. Two
disorder (Owens et al., 1998). Acute acidosis occurs databases were built from in vitro experimental data
when a ruminant animal ingests quickly a large measured with either mono- or mixed cultures of
quantity of rapidly fermentable carbohydrates. As a rumen microbes. Two in vivo databases were also
consequence, the rumen pH decreases below 5.0 and built. The first contained data on rumen fermentation
lactic acid accumulates in the rumen fluid and in the and digestion measured in vivo on ruminally- and
blood. Death is a common outcome. Before this dual- (rumen and duodenal) cannulated animals. The
dramatic event, there is subclinical acidosis second database contained trials performed on
(5.5<pH<6.2), which is a frequent situation for high lactating dairy cows with simultaneous data of
yielding animals receiving diets deficient in fibre and digestibility.
rich in highly digestible substrates formulated to meet Interpretation of these databases was based on a
high energy requirements. Several drawbacks are statistical meta-analyses (St Pierre, 2001). The
associated with subclinical acidosis. Low pH in the publications were separated into experiments that
rumen over a long period of time inhibits intake and were individually encoded. The basic statistical model
cell wall digestion. This last aspect alters the energy applied to the data was:
value of the diet, particularly of its forage component.
Moreover, the VFA profile in the rumen fluid is Yijk = µ + YEASTi + EXPj + Eijk
altered with a low acetate:propionate ratio and
sometimes a significant accumulation of lactic acid. Where:
One of the outcomes of subclinical acidosis is low Yijk: observations
milk fat content, which can fall below 3%. Several µ: overall mean
other diseases are associated with subclinical acidosis, YEASTi: effect of yeast (yeasts vs control)
as it is a contributing factor in abomasal displacement, EXPj: influence of experiment j
liver abcesses and lameness. Eijk: residual error
The objective of this paper is an overview of the
quantitative relationships that link aspects of rumen All the models were used without weighting the
acidosis and use of yeast supplementation in ruminant observations.
diets, particularly diets fed lactating cows.
Results
Methods
IN VITRO EXPERIMENTS
To reach this target we have considered the problem
Monoculture and co-culture
from the viewpoint of statistical modeling. Databases
were constructed from experiments involving dietary A limited number of publications described
222 Rumen acidosis: modeling ruminant response to yeast culture
experiments where monocultures of rumen bacteria identifying and controlling the specific experimental
were performed with or without Saccharomyces influences. This is particularly true when the number
cerevisiae. An initial goal was to study the impact of of available and (or) complete publications is limited,
yeast supplements in the media on lactate utilisation as is presently the case. A useful way to integrate
by lactate fermenters, either Megasphaera elsdenii these data is to create simple mechanistic models of
or Selenomonas ruminantium (Chaucheyras et al., in vitro fermentations to connect these experiments
1995; Rossi et al., 1995; Callaway and Martin, 1997). and to improve our comprehension of the mode of
There was a clear dose-dependent increase in the action of yeast. As an example, Figure 2 is a diagram
utilisation of lactate, suggesting a role for yeast in of a model suited to test the influence of adding yeast
decreasing rumen acidosis. Unfortunately, as the on the lactate producers and lactate utilizors. With
measured items and the experimental conditions were this model it was possible to achieve fairly good
very different it was difficult to empirically pool the accuracy between simulated and the experimental data
data on lactate metabolism. Simultaneously the of Rossi et al. (1995).
acetate:propionate ratio decreased as is shown in
Figure 1. Mixed rumen mixed cultures
Interesting experiments were also performed with
cocultures of M. elsdenii and Streptococcus bovis, a A database was built from publications dealing with
lactate producer (Chaucheyras et al., 1995). In this the impact of yeast on in vitro mixed cultures in rumen
work, adding yeast boosted the biochemical activity fluid. It contained 21 publications, pooling 49
of the organism. Other trials demonstrated that yeast experiments and 121 treatments. In each experiment
addition to the medium improved cellobiose digestion there was a control compared to one or several
by fibrolytic bacteria, either Fibrococcus succinogenes treatments where a yeast supplement was added to
or Ruminococcus albus (Callaway and Martin, 1997). the culture (n = 89) or to the donor’s rumen fluid (n
Several assumptions were made to explain the mode = 32). There were two in vitro methods, either in
of action of the yeast supplements, including batch (n = 69) or in continuous culture devices (n =
provision of soluble growth factors (amino acids, 52). For each statistical treatment the provided
organic acids, vitamins), or possibly by improving information were the LS means (yeast vs control),
the anaerobic conditions. the numbers of treatments (n) and of experiments
One of the major difficulties in pooling and (Nexp), the value of the residual standard deviation
interpreting databases of mono- and co-cultures is (rsd) and its unit, the probability of the test (P) and
Nisbet and Martin (1991)

1.6 Rossi et al. (1995)
Chaucheyras et al. (1995)
Acetate:propionate ratio
Calaway and Martin, 1997
1.1
0.6
0 1 2 3 4 5
Filtrate of yeast (%v/v)
Figure 1. Influence of adding yeast on the acetate:propionate ratio in vitro in co-culture with M. elsdenii.
D. Sauvant 223
k kcr kmax
Absorbance
Lactate Megasphaera
mmC in M. elsdenii
Initial YL
lactate YEAST
mmol C
in lactate utilizers
mmCVFA
kr = fractional growth rate YL = growth yield of lactate

kmax = max. of kr YEAST = impact of yeast culture
k = Michaelis constant mmC = mmol of carbon
Figure 2. Simple model of the influence of yeast on Megasphaera elsdenii.
the percentage of ‘aberrant treatments’ (a) which There was no effect of treatment on VFA
presented normalised residuals larger than 2. concentrations or production (66.38 vs 65.85, n =
There was an increase in pH in response to yeast 95, Nexp = 36, rsd = 5.0 mM, P = 0.62, a = 5.3%).
supplements (6.35 vs 6.32, n = 94, Nexp = 37, rsd = The acetate:propionate molar ratio was slightly
0.05, P = 0.028, a = 6.4%). Where data were few, decreased, but this effect was not significant (2.99 vs
this impact was partly confirmed as a trend (n = 82 , 3.05, n = 98, Nexp = 38, rsd = 0.20, P = 0.131, a =
P = 0.11) when the analysis was conducted within 5.1%). The proportions of the isoacids were not
the experiment and with the volatile fatty acid (VFA) altered by yeast supplementation (6.33 vs 6.06, n =
concentration used as a covariate. This trend suggested 38, Nexp = 15, rsd = 0.36%, P = 0.31, a = 9.5%).
an eventual favorable effect of yeast on the pH of Also, there appeared to be no influence of yeast
the medium. To go further in the analysis several supplementation on lactic acid concentration in the
sub-bases were built to respond to specific issues. medium (0.646 vs 0.667, n = 32, Nexp = 11, rsd =
When only the data with pH<5.5 were considered 0.105 mM, P = 0.603, a = 3.1%). Similarly, there
(Jouany et al., 1998; Lynch and Martin, 2002), the was no effect when lactic acid concentration was
influence of yeast supplementation was more marked corrected by the VFA concentration, the two items
(pH increase was 0.055 vs 0.024) but less significant being linked.
(5.32 vs 5.26, n = 14, Nexp = 6, rsd = 0.05, P = 0.064, The molecular hydrogen status did not seem to be
a = 7.1%). Pooling the data of Carro et al. (1992), altered by yeast. Effectively CH 4 content or
Zelenak et al. (1994) and Lynch et al. (2002) allowed production (13.6 vs 13.4, n = 58, Nexp = 23, rsd = 1.3
testing where there was any interaction between pH mM CH4, P = 0.576, a = 1.7%) and H2 content or
response and the level of cell wall (CW) in the diet production (0.444 vs 0.440, n = 44, Nexp = 17, rsd =
or feed. The pH actually increased for feeds or diets 0.060 mM H 2, P = 0.842, a = 4.6%) were not
having a higher level of CW, however there was no significantly modified by adding yeast. For CH4 there
effect of yeast on pH and no interaction between CW was a positive correlation with VFA, however there
and yeast. was no effect of yeast when VFA were considered as
a covariate. Globally there was no influence of yeast matter (OMD) digestibility, and in situ
on NH3 content or production (154.4 vs 151.0, n = measurements. There was a lack of basic information
55, Nexp = 21, rsd = 10.6 mg N-NH3/L, P = 0.275, a = on either yeast concentration, or live weight of the
7.3%). However, as the experiments measuring NH3 animals, or dry matter intake, or chemical analysis
levels lower than 100 mg N-NH3/L were selected, of the diet in some of the studies. The yeast effect
there was a significant increase in response to yeast was tested with the basic model including the trial
(72.1 vs 57.8, n = 21, Nexp = 7, rsd = 5.9 mg N-NH3/ and yeast effects.
L, P = 0.002, a = 0%). There were non-significant increases in response
Microbial N production tended to be increased by to yeast supplements in pH (6.341 vs 6.320, n =
adding yeast (837.4 vs 791.0, n = 17, Nexp = 8, rsd = 168, Nexp = 70, rsd = 0.015, P = 0.288, a = 8.3%), in
52.1 mg microbial N/d, P = 0.107, a = 11.7%). Also VFA concentrations (99.1 vs 97.8, n = 156, Nexp =
the efficiency of microbial growth tended to be 64, rsd = 75.8 mM, P = 0.386, a = 8.8%), in the
increased by yeast (21.8 vs 20.4, n = 17, Nexp = 8, rsd acetate/propionate ratio (3.24 vs 3.17, n = 163, Nexp
= 1.48 g microbial N/kg RFOM, P = 0.099, a = = 69, rsd = 0.078, P = 0.110, a = 3.7%), and a non-
11.8%).The number of protozoa in the medium was significant decrease in lactic acid concentration (1.38
unaffected by yeast (4.48 vs 4.53, n = 32, Nexp = 13, vs 1.45, n = 39, Nexp = 15, rsd = 0.417, P = 0.729, a
rsd = 0.16 log10 protozoa/ml, P = 0.494, a = 6.25%). = 10.3%). Except for the pH and the acetate/
In contrast, the total number of viable bacteria was propionate ratio, these data were fairly similar to
significantly increased by yeast (8.67 vs 8.57, n = 26, the in vitro responses.
Nexp = 9, rsd = 0.08 log10 bacteria/ml, P = 0.009, a = Yeast tended to increase organic matter
0%). Moreover, on a larger number of data, the digestibility (71.4 vs 70.8%, n = 66, Nexp = 31, rsd =
number of cellulolytic bacteria was significantly 3.23, P = 0.148, a = 9.0%). There was a similar
increased by yeast supplementation (7.65 vs 7.20, n = trend for increased in situ DM degradability (55.9
32, Nexp = 12, rsd = 0.32 log10 bacteria/ml, P = 0.002, vs 55.0%, n = 75, Nexp = 32, rsd = 7.12, P = 0.145,
a = 6.2%). All these data suggested an increase of a = 5.3%), which was interestingly related with
microbial proliferation by adding yeast in vitro. rumen pH within (w) the experiment (isDMDw =
The apparent degradability of substrate dry matter 10.0 pHw, n = 72, R2 = 26%, rsd = 1.8%, a = 4.0%).
was not significantly increased by yeast addition (58.1 Otherwise it must be stressed that when the 23
vs 57.7, n = 57, Nexp = 22, rsd = 2.7%, P = 0.613, a = treatments having less than 40% dietary concentrate
7.0%). In contrast, on a smaller set of data the were considered, there was a good fit and a significant
degradability of NDF was improved by yeast increase of OMD (64.6 vs 63.2%, n = 23, Nexp = 11,
supplementation (53.3 vs 50.3, n = 32, Nexp = 15, rsd rsd = 1.33, P = 0.033, a = 0%). There was no
= 4.2%, P = 0.062, a = 6.25%). This last aspect is statistical influence of yeast on whole tract
consistent with the above cited results on the number digestibility of NDF (53.5 vs 54.0%, n = 47, Nexp =
of bacteria, particularly the cellulolytic species. 22, rsd = 2.19, P = 0.522, a = 8.5%) and ADF (48.4
vs 47.2%, n = 37, Nexp = 16, rsd = 5.7, P = 0.555, a
= 5.4%). This last aspect did not confirm the in
IN VIVO EXPERIMENTS vitro data.
When the yeast concentration was available in the
Rumen fermentation and digestion paper or could be estimated because the yeast
In order to quantify the effects of yeast on in vivo description was sufficient, we used a model including
ruminal fermentation we pooled into a database the the trial effect and the yeast dose. It was expressed
results of 55 publications corresponding to 78 as log10(CFU/100 kg of live weight) because the
experiments and 186 treatments. This database was database concerned cattle and sheep. Globally, similar
much larger than the in vitro database. Yeast cultures conclusions could be obtained with this second
were from at least eight different commercial approach. Nevertheless, it should be stressed that
preparations. Half of the trials (28 of 56) used Yea- for pH, yeast dose is at the limit of the level of
Sacc1026® or a source of strain 1026. In some papers, significance:
two yeasts were compared. In all the papers, some
rumen parameters were available, at least pH and/or pH = 6.32 + 0.00556 log10 (yeast concentration)
VFAs. Some additional data were noted when (n = 147, Nexp = 64, rsd = 0.032, P = 0.086,
available such as in vivo dry matter (DMD) or organic a = 8.2%)
D. Sauvant 225
This increase in pH value in response to yeast literature suggested that effects can differ according
supplementation is consistent with the in vitro data. to stage of lactation, we coded this as a factor with
In order to test whether some strains are more three levels: EL for experiments starting from calving
effective than others, we also studied two sub- or during pregnancy and ending before 70 DIM
databases concerning the two most studied strains: (peak), ELML for experiments starting before peak
Yea-Sacc1026®, and Diamond V. There was no statistical and ending during mid-lactation, and ML for
effect of the yeast whatever the sub-database experiments starting past peak of lactation.
concerned. In the residual of the negative equation Data recorded for animal response to yeast
linking pH and VFA concentration including a trial supplement were dry matter intake (DMI), raw milk
effect, the yeast effect was statistically significant yield, milk fat content (MFC), milk protein content,
for the whole database (P = 0.024) and for the Yea- and body weight change (BWC). Simultaneously, data
Sacc1026® database (P = 0.034). on NDF, ADF and CP digestibility were recorded.
To look for any optimum yeast dosage, a dose effect The basic model was applied and three others were
has been tested on the 10 experiments where three also used:
levels were given. There was no dose effect on rumen
pH, rumen volatile fatty acids, acetate:propionate ratio Yijk = µ + YEAST + EXP + Eijk
or ammonia concentration.
to test the effect of each type of yeast.
Responses of lactating cows
Yijk = µ + YEAST + stage of lactation + YEAST *
The data base was constituted from 35 publications stage of lactation + Eijk
pooling 40 experiments and 122 treatments (3272
cows) to quantify the effects of yeast culture on dry to test the effect of yeast in interaction with stage of
matter intake (DMI), raw milk yield (RMY), milk lactation (EL, ELML, or ML).
fat content, milk protein content, and body weight
change (BWC) of dairy cows. The choice of the data Yijk = µ + YEAST + %Conc + YEAST * Conc +
was based on the fact that industrial fermentation Eijk,
processes have rapidly changed since the first
publications, consequently only the recent data were
to test the interaction between yeast and %Conc,
considered. The yeast were partitioned among
where %Conc is the percentage of concentrate in the
different products (DiamondV-XP: 16 groups; Yeast+:
diet.
13 groups, Yea-Sacc1026®: 16 groups, others: 21 groups
(Cell-Con, Western 2x225, Levucell), which were
There was a trend (P = 0.08) toward increased raw
pooled because of a low number of groups). Doses
milk yield by 1.3 kg when all types of yeast and all
used largely differed according to the type of
stages of lactation were taken together (Table 1). No
products. Diets were representative of most of the
effects of on milk composition, DMI or BW change
rations used in early- and mid-lactation with high
were observed. ADF digestibility tended to be
concentrate diets for high producing cows. Most diets
increased (P=0.15, +2.8%). This last result was
were fed as TMRs, once or twice a day. Because
consistent with data observed in vitro.
Table 1. Effect yeast on milk performance of dairy cows in experiments (Nexp) realised during early, early-mid, or mid lactation 1.
No. of TRT P< RMSE
experiments Ntreat CTRL Yeast Exp Yeast
Dry matter intake, kg/d 34 99 20.0 ± 0.3 20.2 ± 0.3 <0.01 P=0.42 1.8
Raw milk yield, kg/d 39 112 32.2 ± 0.4a 31.3 ± 0.4 b <0.01 P=0.08 2.5
Milk fat content, g/l 37 104 37.0 ± 0.2 37.1 ± 0.2 <0.001 P=0.75 1.4
Milk protein content, g/l 37 104 31.5 ± 0.1 31.4 ± 0.3 <0.001 P=0.40 0.9
Body weight change, kg/d 34 34 -0.1 ± 0.4 0.1 ± 0.4 P=0.82 P=0.35 1.5
N digestibility, % 6 15 67.4 ± 1.7 69.9 ± 1.6 <0.001 P=0.32 4.4
ADF digestibility, % 6 13 46.6 ± 1.6 49.4 ± 1.5 <0.001 P= 0.15 3.8
1
Data are presented as least square means ± se. RMSE: root mean square error of the model.
When concentrate percentage was included in the that by selecting references, it is quite possible to
analysis, no effect of yeast and no interaction between conclude either a positive or a negative effect of
yeast and percentage of concentrate was significant adding yeast for any given parameter! Therefore, in
(data not shown). The role of some other possible order to obtain more relevant and reliable conclusions
interfering factors were also tested. As for concentrate it was decided to perform a 2-step approach. The
percentage, there was no influence of the dietary NDF first step was to try to be as exhaustive as possible of
or ADF or of the RMY of the control group. In the published results. The second step was to build
contrast, there was an interesting influence of the databases in each area where a sufficient number of
milk fat content of the control group (Figure 3). This experiments were carried out and published. This is
figure shows that there is an increase in milk fat the origin of the four databases mentioned in this
content in response to yeast supplementation when paper.
its value is low, suggesting the presence of subclinical In the current work we had a clear confirmation of
acidosis. the large variability of data among papers. It must
be considered that the present approach is only the
first step of a more detailed and systematic analysis
Discussion of all the specific factors which could explain, at
least partly, the residual variations of the statistical
Several authors have reviewed the influences of yeast models. Among these factors there are the strain and
supplements on rumen digestion and animal the actual level of yeast, comparison between yeast
performance (Ali Haimoud-Lekhal et al., 1999; and fungi which were excluded in this work,
Jouany, 1999; Lescoat et al., 2000; Garza-Cazares et methodological differences (batch vs continuous
al., 2001; Chaucheyras-Durand and Fonty, 2002; culture, adding yeast to the animal vs the fermentor,
Robinson, 2002). However, except for the first cited method of rumen sampling, etc.), dietary
paper, they were largely qualitative or narrative. specifications, type of animal and level of dry matter
Otherwise, several authors have noted that there was intake, type of experimental design, etc. The major
large variability among publications in the results of technical difficulties that we met in this work were
yeast influences on ruminant nutrition. That means fairly classical, the tables of data were largely
5.0
4.0 MFC (S. cerevisae - Control), g4
3.0
Milk fat content (change)
2.0
1.0
MFC (Control), g1
0 .0
25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0
-1 .0
-2 .0
-3 .0
-4 .0
-5 .0 MFC (S. cerevisae - CTL) = 17.8 (± 8.1) – 0.50 (± 0.24) *MFC (CTL)
No. of observations = 59, Nexp - 37, RMSE = 1.32
Figure 3. Influence of control milk fat content (MFC) on MFC change with the addition of S. cerevisae in dairy cows.
D. Sauvant 227
incomplete for our purposes and the experimental of the meta-analysis the threshold value of
methodologies and conditions varied largely from significance is of importance.
one publication to another. Moreover some other The results were not systematically consistent from
specific difficulties were met, the major one being one database to another. Some comments have already
the fact that several of the experiments were available been noted in the text on this aspect. In the in vitro
only in form of abstracts and that most of those were data it was fairly clear that pH was increased,
very incomplete. This situation can induce bias in suggesting a preventative role of yeast toward
the meta-analysis, because non-significant results are acidosis. The results were not so clear in vivo, however
generally not presented in an abstract. For a further they were globally consistent. Moreover the
step, it would be very important to have more interference of either the percentage of concentrate,
complete information on all these studies. Another or the dietary NDF, rapidly degradable carbohydrates,
problem was that another group of the publications cannot be easily tested due to the scarcity of
dealing with digestion was very incomplete with, for experiments targeted for that purpose and due to the
example, poor information on the specifications of lack of dietary specification. On this aspect the in
the diet, or substrate used or on the animal vivo database with dairy cows provided an interesting
characteristics. All these aspects mean that there was observation, which suggested that yeast supplementation
obviously a large range in the ‘quality value’ of the is more efficient in restoring milk fat content when it is
various published experiments dealing with yeast low, which is suggests subclinical acidosis.
response. This suggests that it would be useful to re- Further detailed interpretation is also needed
run the statistical treatment by weighting the regarding VFA and lactic acid production and profiles.
experiments using a ‘quality index’. Another way of On this aspect the results of the in vitro simple culture
weighting the data would be to take into account the could not be confirmed with mixed cultures or in
residual variations of each experiment. For all these vivo. As such, this paper cannot provide useful
reasons, whatever the conclusions that could be now information about the influence of yeast on hydrogen
drawn, it is very important to consider that the all status and redox aspects. On these points a more
the results presented here cannot be considered as detailed interpretation will be necessary.
definitive. Lastly, it is probable that non-positive or Microbial concentrations and activities were
non-significant results had a lower probability of enhanced by yeast in co-culture and in mixed cultures.
being published than positive or significant trials, These data tended to be confirmed by others, such as
which induced a bias in the conclusions. the fibre degradability and digestibility data.
The method of meta-analysis that we have used is However, there is a clear lack of in vivo data to
now considered the most suitable (St Pierre, 2001). confirm these observations on microbial activities.
It is of interest to study the nutritional impact of Experiments with duodenally-canulated animals
rumen defaunation (Eugene et al., 2004) and to could provide more accurate and extensive knowledge
quantify the biases of rumen mechanistic models of the impact of yeast on quantitative metabolism in
(Offner and Sauvant, 2004). This method of the rumen.
statistical modeling has the advantage of splitting A mechanistic modeling approach was only
variation among and within experiments. The latter considered at the level of the co-culture. The first
is therefore controlled and cannot interfere with the results were encouraging; and it will be useful to
former. However, as such, the meta-design and the develop a mechanistic model of the rumen to take
model cannot allow us to easily extract and analyze into account the mode of action of yeast on some
the interactions between the experimental influences basic metabolic process such as lactate and VFA
and the impact of adding yeast. Obviously this last metabolism, microbial growth and activities. In this
issue is a big one in the current context. Another big model it will be necessary to include the major
issue in meta-analyses is the way to treat the aberrant principles of thermodynamics (Heijnen and Van
treatments and (or) experiments. For the present it Dijken, 1992). This aspect, which has been little
was decided to include all the data in the process of explored, could help in understanding any influence
interpretation. However it was decided to indicate of yeast on oxygen/hydrogen balance. As published
the proportion of treatments that could be considered today, the rumen mechanistic models present some
aberrant. Moreover, it was also decided to indicate strong limitations (Offner and Sauvant, 2004) and
the probability level of the test because in the context cannot be used to investigate these aspects. Therefore
such a project would allow significant progress in Pariza and J. Nelson, eds). AOCS Press, USA.
methods of modeling the nutritional impact of yeast Heijnen, J.J. and J.P. van Dijken. 1992. In search of
and other dietary factors in ruminants. a thermodynamic description of biomass yields for
the chemotrophic growth of microorganisms.
Biotechnol. Bioeng. 39:833-858.
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L.W. Whitlow and W.M. Hagler Jr. 231
The top ten most frequently-asked questions about mycotoxins, cattle and
dairy food products
L.W. WHITLOW1 AND W.M. HAGLER, JR.2
1
Department of Animal Science, North Carolina State University, Raleigh, North Carolina, USA
2
Introduction
Mycotoxins are toxic secondary metabolites produced ability of the mycotoxin-producing fungus to cause
by fungi (molds). Secondary metabolites are a plant disease, thus helping to create an environment
chemicals produced by the fungus that are not conducive for growth of the fungus (CAST, 2003).
essential for growth. Mycotoxins are chemically In experiments, Fusarium graminearum and F.
diverse, represent a variety of chemical families, and verticillioides were genetically altered so that they
range in molecular weight from c. 200 to 500. A would not produce trichothecenes or fumonisins,
practical definition of a mycotoxin is a fungal respectively. Results were mixed, demonstrating that
metabolite that causes an undesirable effect in trichothecenes play an important role in wheat head
exposed animals. The undesirable effect or disease blight and corn ear rot caused by F. graminearum
caused by a mycotoxin is a mycotoxicosis (Nelson et (Desjardins and Hohn, 1997; Harris et al., 1999),
al., 1993). Exposure is generally through but that fumonisins are not required for corn ear rot
consumption of contaminated feedstuffs, although caused by F. verticillioides (Desjardins et al., 2002).
dermal contact or inhalation of certain mycotoxins It is also possible that immune suppression in
can also cause undesirable responses. Mycotoxins animals by certain mycotoxins is in fact a mechanism
exhibit broad and variable biological effects in to allow infectivity by the fungus. Some fungi are
animals. Mycotoxins can cause damage to organ infectious pathogenic agents that cause a mycosis
systems, reduce production and reproduction and (fungal infection) that has a detrimental effect on
increase disease by reducing immunity. Some the host animal. Aspergillus fumigatus is thought to
mycotoxins are carcinogens. Some target the liver, be a fairly common mold in both hay (Shadmi et al.,
the kidney, the digestive tract or the reproductive 1974) and silage (Cole et al., 1977). Aspergillus
system. Symptoms are wide ranging including fumigatus has been proposed as the pathogenic agent
decreased feed consumption, poor feed utilization, associated with mycotic hemorrhagic bowel syndrome
weight loss, reduced performance, estrogenic effects, in dairy cattle, which has also been attributed to
vomiting, diarrhea, nervous disorders, tissue necrosis, Clostridial infections and other factors (Puntenney
hemorrhage, tumors, abortions and death. et al., 2003). Such mycoses occur in immuno-
suppressed animals. Dairy cows are immune
suppressed in early lactation. Aspergillus fumigatus
Do molds cause animal problems or do also produces a mycotoxin, gliotoxin, which is an
immune suppressant. It is possible that immune
molds simply produce mycotoxins for that
suppression by gliotoxin is a mechanism that allows
purpose? infectivity by the fungus. Gliotoxin was found in
peritoneal lavages from mice innoculated and infected
There is no confirmed reason for the existence of
with A. fumigatus (Eichner et al., 1988). Gliotoxin
mycotoxins. Most theories suggest that mycotoxins
has also been found in the udder of cows naturally
exist to protect or enhance the existence of the fungus.
infected with A. fumigatus, while other known
Recent speculation is that mycotoxins increase the
mycotoxins produced by this fungus were absent
232 The top ten most frequently-asked questions about mycotoxins, cattle and dairy food products
(Bauer et al., 1989). Interactions with trichothecene species in the world, but only about 69,000 are
mycotoxins may also be a factor in occurrence of a currently identified. Turner (1978) and Turner and
mycosis because reductions in cellular immunity can Aldridge (1983), catalogued 3200 secondary
reduce resistance to a mycosis. Niyo et al. (1988a, metabolites produced by 1600 fungal species, or two
b), showed that rabbits exposed to T-2 toxin had a secondary metabolites per fungal species. Cole and
decrease in phagocytosis of Aspergillus fumigatus Cox (1981) classified 10% of the fungal secondary
conidia by alveolar macrophages and an increase in metabolites as mycotoxins. If there are indeed 1.5
severity of experimental aspergillosis. Richard (1991) million fungal species and if each produces two
has suggested that medical mycologists should consider secondary metabolites and if 10% of secondary
this aspect of infections caused by any toxigenic metabolites are mycotoxins, there are potentially
fungus, especially those that produce immuno- 300,000 mycotoxins. More conservatively, if there
suppressive compounds. Fungal pathogens include are 100,000 fungal species producing 200,000
Aspergillus fumigatus, Candida albicans, Candida secondary metabolites, there may be 20,000
vaginitis and certain species of Fusarium. mycotoxins in nature. Potentially, many more
Fungi are deterioration organisms. Therefore, mycotoxins exist than have been identified.
feedstuffs on which they grow are deteriorated and The potentially large number of unidentified
have an altered nutritional value including decreases mycotoxins and the fact that commercial laboratory
in fat, protein and carbohydrates, which can affect analyses are not available for many of the identified
performance and health (DiConstanzo et al., 1995). mycotoxins suggests that a mycotoxicosis can occur
Cook and Wu (1991) itemized some of the nutritional without any possibility of identifying the mycotoxin,
changes in feeds occurring with mold growth or all of the mycotoxins, that may be interacting to
including a decrease in lysine and thiamin and an produce the mycotoxicosis.
increase in fiber. Some of the interactions of The frequency of the occurrence of mycotoxins and
mycotoxins with nutrients have been reviewed the proportion of feeds that are contaminated indicate
(Schaeffer and Hamilton, 1991). that animal exposure is high. The FAO has suggested
that 25% of the world’s crops are affected (CAST,
1989). Certainly raw agricultural products are more
How many mycotoxins exist – how likely to be contaminated than the human food supply.
frequently are they found? Mycotoxins are present worldwide with some
geographical differences mainly resulting from
Hundreds of mycotoxins have been identified, but climatic differences. There are differences in
other than the major mycotoxins, most have not been mycotoxins by type of feedstuff. Occurrence and
extensively researched and even fewer have good concentrations are variable by year, which is expected
methods of analysis available. The major classes of because of the annual variation in weather conditions
mycotoxins are aflatoxins, zearalenone, tricho- and plant stresses known to affect mycotoxin
thecenes, fumonisins, ochratoxin A and the ergot formation (Coulumbe, 1993). Summaries of surveys
alkaloids. These mycotoxins are the more likely showing the incidence and concentrations of
causes of mycotoxicoses in dairy cattle and other mycotoxins in various feedstuffs have been published
domestic animals because they occur more frequently (CAST, 2003; Wood, 1992; Wood and Trucksess,
and have the potency to cause toxicities. However, 1998). Feed samples submitted by North Carolina
there are many reports of mycotoxicoses that have farmers over a 13 year period (Table 1) indicate that
occurred as a result of those mycotoxins that are mycotoxins in feeds occur commonly at unsuitable
categorized as minor in importance (CAST, 2003; concentrations (Whitlow et al., 1998). It can be
Lacey, 1991). concluded that mycotoxins occur frequently in a
Riley (1998) put forth an argument that only a variety of feedstuffs and are routinely fed to animals.
small proportion of mycotoxins have yet been
identified. One factor that supports this idea is the
high rate of discovery of new mycotoxins. To further What conditions support mold growth
support this idea, Riley (1998) cited the following and mycotoxin formation?
facts, which we have taken the liberty to condense.
Riley (1998) noted that Hawksworth (1991) estimated Perhaps the major mycotoxin-producing fungal
that there may be as many as 1.5 million fungal genera, in terms of research in the United States, are
Table 1. Mycotoxin occurrence by concentration in all feeds submitted for analysis by North Carolina farmers over 13 years.
Aflatoxin Deoxynivalenol Fumonisin T-2 Toxin Zearalenone
Number of samples 3266 5053 822 5136 4563

Low, % of samples below (concentration) 6.4 (<20 ppb) 18.2 (<500 ppb) 32.6 (<5000 ppb) 1.5 (<100 ppb) 7.1 (<300 ppb)
High, % of samples above (concentration) 4.0 (>19 ppb) 28.2 (>499 ppb) 9.4 (>4999 ppb) 6.6 (>99 ppb) 8.3 (>299 ppb)
Aspergillus, Fusarium, and Penicillium. Many species fungicide to the mold organism may cause increased
of these fungi produce mycotoxins in a variety of mycotoxin production (Boyacioglu et al., 1992;
feedstuffs. Claviceps spp. particularly in small grains Gareis and Ceynowa, 1994).
and Epichloe and Neotyphodium in fescue grass all Aspergillus species normally grow at lower water
produce ergot alkaloids. These fungi and their activities and at higher temperatures than do the
mycotoxins are also a concern, but have a more Fusarium species. Therefore, Aspergillus flavus and
specific host-fungal relationship than do Aspergillus, aflatoxin in corn are favored by the heat and drought
Fusarium, and Penicillium fungi. Molds are fungi stress associated with warmer climates. Penicillium
that grow in multicellular colonies, as compared with species grow at relatively low water activities and
yeasts that are single cellular fungi. Molds can grow low temperatures and are widespread in occurrence.
and mycotoxins can be produced pre-harvest or during Penicillium molds are more common in storage than
storage, transport, processing, or feeding. Mold in preharvest, but can grow in the field under very
growth and mycotoxin production are related to wet conditions. Because both Aspergillus and
weather extremes (causing plant stress or excess Penicillium can grow at low water activities, they
hydration of stored feedstuffs), to inadequate storage are considered storage fungi (Christensen et al.,
practices, to low feedstuff quality, and to faulty 1977).
feeding conditions. In general, environmental Growth of A. flavus can occur at 86-87%
conditions - heat, water, and insect damage - cause equilibrium relative humidity (RH) (Davis and
plant stress and predispose plants in the field to Diener, 1983). Field infection of corn with A. flavus
mycotoxin contamination. Because feedstuffs can be (Wicklow, 1983) is expected when temperatures,
contaminated pre-harvest, control of additional mold including nighttime temperatures, are high and there
growth and mycotoxin formation is dependent on is drought stress. Growth conditions in the southern
storage management. After harvest, temperature, US result in routine aflatoxin contamination of crops,
moisture content, and insect activity are the major but aflatoxin can be found in crops grown in other
factors influencing mycotoxin contamination of feed regions in years when weather conditions are
grains and foods (Coulumbe, 1993). conducive. For example, 8% of samples of
Molds grow over a temperature range of 10-40°C midwestern US corn grain from the 1988 drought
(50-104°F), a pH range of 4 to 8, and above 0.7 aw season contained aflatoxin (Russell et al., 1991).
(equilibrium relative humidity expressed as a decimal Corn is susceptible to A. flavus infection via the silks
instead of a percentage). Mold can grow on feeds (Marsh and Payne, 1984) and stress conditions at the
containing more than 12-13% moisture. In wet feeds time of anthesis (pollination) lead to preharvest
such as silage, higher moisture levels help exclude aflatoxin contamination in corn. A. flavus spores as
air and molds grow only if oxygen is available. The inoculum are plentiful at this time. In North Carolina,
conditions most suitable for mold growth may not insect activity appears less important in the events
be the optimum conditions for mycotoxin formation leading to aflatoxin contamination of corn than it
in the laboratory. For example, the Fusarium molds appears to be in Georgia (Payne, 1983). Aflatoxin is
associated with alimentary toxic aleukia have been a greater problem in cottonseed grown in the
reported to grow prolifically at 25-30°C without southwestern US than in the southeastern US
producing much mycotoxin, but at near-freezing (Ashworth et al., 1969). The complex effects of
temperatures large quantities of mycotoxins were relative humidity, temperature, precipitation, and their
produced with minimal mold growth (Joffe, 1986). daily variations may interact to produce conditions
Field applications of fungicides may reduce mold conducive to A. flavus infection and aflatoxin
growth, in turn reducing the production of production in the Southwest (Ashworth et al., 1969).
mycotoxins. However, the stress or shock of the Early harvest and a decrease in late-season irrigation
may reduce contamination (Russell et al., 1976). 1. Find the mycotoxin in suspect substrate from
Experimentally, the use of spores of nontoxigenic A. the toxicosis outbreak.
flavus isolates in southwestern cotton fields has
2. Find in the substrate a fungus that produces the
resulted in greatly reduced aflatoxin levels in
toxin.
cottonseed (Cotty et al., 1994). Improperly stored
cottonseeds are susceptible to mycotoxin 3. Induce the toxicosis in experimental animals by
contamination if mold activity is allowed. ingesting or contacting the toxin.
The Fusarium species are generally considered to
be field fungi and were thought to proliferate before Once the mycotoxicosis is established as a disease
harvest (Christensen et al., 1977). However, Fusarium entity, it is no longer necessary to repeat the process.
species may also grow and produce mycotoxins under Recognition of the mycotoxicosis symptoms and
certain storage conditions. In corn, Fusarium molds mycotoxin presence in feed provide an adequate basis
are associated with ear rot and stalk rot, and in small for diagnosis. If symptoms unique to that mycotoxin
grains, they are associated with diseases such as head are observed, then it is not necessary to determine
blight or commonly referred to as scab (Tuite et al., that the mycotoxin is in the feed.
1974). Fusarium is associated with excessive moisture Mycotoxins result in a progression and diversity
at flowering. In corn, Fusarium diseases are more of symptoms that can be confusing and can make
commonly associated with a cool wet growing season, diagnosis difficult (Hesseltine, 1986; Schiefer, 1990).
with insect damage, warm conditions at silking, and Symptoms from field cases can be different from
wet conditions late in the growing season (Trenholm those observed under controlled experimental
et al., 1988). conditions because in field cases there may be
Of the Fusarium species F. graminearum is a major multiple mycotoxins, variable dosages at irregular
producer of deoxynivalenol (DON) and zearalenone intervals, uncontrolled environments, and various
(ZEN), but other species of Fusarium also produce interacting stress factors. Diagnosis is complicated
DON and ZEN, as well as other mycotoxins by a lack of research, by a lack of feed analyses, by
(Christensen et al., 1988; Marasas et al., 1984). numerous possible mycotoxins, by nonspecific
Conditions exacerbating ZEN accumulation in corn symptoms, and by immunosuppression resulting in
include weather that holds moisture content at 22- opportunistic diseases that produce confounding
25%, or delayed harvest (Abbas et al., 1988). symptoms. Therefore, a definitive diagnosis of a
Zearalenone has been reported to occur in corn, other mycotoxicosis is difficult from general symptoms,
grains, and silage in many areas of the world. specific tissue damage, or even feed analyses.
Weathered soybeans have also been reported to be However, experience with mycotoxin-affected herds
contaminated with ZEN (Hagler et al., 1989). ZEN greatly increases the probability of recognizing a
is also found in wheat, barley, oats, sorghum, sesame mycotoxicosis. A process of elimination of other
seed, hay, and silages. DON occurs in cereal grains factors, coupled with feed analyses and unique
worldwide and can increase in stored grain with kernel symptoms can help identify a mycotoxicosis. Another
moisture contents of 22–25%. Minimum tillage and practice helpful in diagnosis is an observation of
no tillage production are believed to increase the positive responses or alleviation of symptoms after
amount of disease in small grains and corn/wheat the use of products known to be effective in reducing
rotations because of increased inoculum survival on mycotoxin exposure to animals. Examples of such
crop residue (Trenholm et al., 1988). T-2 toxin is products are mold inhibitors and mycotoxin
produced primarily by F. sporotrichioides and F. poae, sequestering agents. Regardless of the difficulty of
but is also produced by other species of Fusarium diagnosis, mycotoxins should be considered as a
(Marasas et al., 1984). T-2 (and DAS) is often found possible cause of production and health problems
in barley, wheat, millet, safflower seed, and in mixed when pertinent symptoms exist and problems are
feeds. not directly attributable to other typical causes
(Schiefer, 1990).
How is a mycotoxicosis diagnosed?
Hamilton (1978) presented an interpretation of the How do mycotoxins affect dairy cows?
application of Koch’s postulates to mycotoxins. The Mycotoxins can increase incidence of disease and
postulates as modified are:
reduce production efficiency. Some of the gross carbohydrate and lipid metabolism (Cheeke and
effects of mycotoxins can include: 1) intake reduction Shull, 1985).
or feed refusal, 2) reduction in nutrient absorption Depending on interactions with other factors,
and metabolism, 3) digestive disorders including aflatoxin concentrations as low as 100 ppb may be
hemorrhage and necrosis, 4) tissue and organ damage, toxic to dairy and beef cattle, however the toxic level
5) gangrene of the extremities, 6) endocrine effects, is generally considered to be between 300 to 700
7) reproductive disorders, embryonic death, ppb. Garrett et al. (1968) showed an effect on weight
abortions, 8) nervous disorders, tremors, unco- gain and intake with diets containing 700 ppb
ordination, 9) suppression of the immune system, aflatoxin, but if increases in liver weights are used as
and 10) death. Symptoms will be dependent on the the criteria for toxicity, then 100 ppb would be
mycotoxins present. In the field, animals experiencing considered toxic to beef cattle. Guthrie (1979)
a mycotoxicosis may exhibit a few or many showed a decline in reproductive efficiency when
symptoms. They may simply be unthrifty, with a lactating dairy cattle in a field situation were
rough or dull hair coat, have an undernourished consuming 120 ppb aflatoxin. When cows were
appearance, impaired reproduction, and(or) a mixed changed to an aflatoxin-free diet, milk production
infectious disease profile. Some of the symptoms increased over 25%. Patterson and Anderson (1982)
observed with a mycotoxicosis may be secondary, and Masri et al. (1969) also suggest that 100 ppb
resulting from an opportunistic disease that is present may reduce milk production.
because of immune suppression caused by the Aflatoxin produced from culture was shown to be
mycotoxin exposure. more toxic to dairy cattle than pure aflatoxin added
Toxicity occurs at the cellular level. Aflatoxin causes to diets (Applebaum et al., 1982). This is thought to
DNA changes, cell deregulation, cellular changes and result from other mycotoxins present in the natural
death. Deoxynivalenol inhibits protein synthesis culture. Under certain conditions, A. flavus also
resulting in disruption of cytokine regulation, altered produces sclerotia, or resting bodies, which contain
cell proliferation and cell death. T-2 toxin inhibits indole alkaloids such as aflatrem (Wicklow, 1983).
protein synthesis with subsequent cell death. Cyclopiazonic acid (CPA), a toxic indole tetramic
Fumonisin alters enzyme activity, which disrupts lipid acid, is also produced by A. flavus (CAST, 1989).
metabolism resulting in cell deregulation and cell The role of these and other toxins produced by A.
death. Zearalenone binds with cytosolic estrogen flavus in aflatoxicoses is not known. Aflatoxin lowers
receptors causing an estrogenic response and altering resistance to diseases and interferes with vaccine-
hormonal control (Riley and Norred, 1996). induced immunity in livestock (Diekman and Green,
1992).
The Food and Drug Administration (FDA) has
AFLATOXINS established nonbinding action levels as informal
guidelines for enforcement of aflatoxin control in
Aflatoxins are a family of extremely toxic, mutagenic, feedstuffs (Table 2, Wood and Trucksess, 1998).
and carcinogenic compounds produced by Aspergillus Blending contaminated ingredients with
flavus and A. parasiticus (Deiner et a1., 1987; uncontaminated ingredients with the purpose of
Kurtzman et al., 1987). Toxigenic A. flavus isolates reducing aflatoxin concentrations is not allowed.
produce aflatoxins B 1, and B 2 and toxigenic A.
parasiticus isolates produce aflatoxins B1, B2, G1, and Table 2. US Food and Drug Administration action levels for total
G2 (Cotty et al., 1994). aflatoxins in food and feed.
Symptoms of acute aflatoxicosis in mammals Food or feedstuff Concentration (ppb)
include inappetance, lethargy, ataxia, rough hair coat, All products, except milk, designated for humans 20
and pale, enlarged fatty livers. Symptoms of chronic Corn for immature animals and dairy cattle 20
aflatoxin exposure include reduced feed efficiency Corn and peanut products for breeding beef cattle,
and milk production, icterus, and decreased appetite swine, and mature poultry 100
Corn and peanut products for finishing swine
(Nibbelink, 1986). Reduced growth rate may be the (>100 lb) 200
only clue for chronic aflatoxicosis and other Corn and peanut products for finishing beef cattle 300
mycotoxicoses (Raisbeck et al., 1991; Pier, 1992). Cottonseed meal (as a feed ingredient) 300
The mechanism by which aflatoxins reduce growth All other feedstuffs 20
Milk 0.5a
rate is probably related to disturbances in protein,
a
Wood and Trucksess, 1998.
ZEARALENONE
Zearalenone and zearalenol are estrogenic metabolites herds, examined by palpation and determined to be
of several species of Fusarium. Chemically, cycling, had lower blood ZEN+M levels than did
zearalenone (ZEN) is a resorcylic acid lactone which cows that were not cycling. The reproductive
does not have actual toxicity. Zearalenone is the cause problems in dairy cattle were associated with ZEN+M
of hyperestrogenism, the estrogenic syndrome, in concentrations of about 400 ppb in the pasture
swine. F. graminearum is the major ZEN-producing samples.
fungus of the Fusarium species that cause corn ear
and stalk rots, but other species of Fusarium produce
ZEN, as well as other mycotoxins (Christensen et TRICHOTHECENES
al., 1988).
Zearalenone is rapidly converted to α− and ß- Trichothecenes are a family of 200-300 related
zearalenol in rumen cultures (Kiessling et al., 1984). compounds that apparently exert their toxicity
α−Zearalenol is c. four-fold more estrogenic in rats through protein synthesis inhibition at the ribosomal
than ZEN, while ß-zearalenol is about equal in level. Several species of Fusarium and related genera
strength to ZEN (Hagler et al., 1979). However, ZEN produce trichothecenes. T-2 toxin, diacetoxyscirpenol
has been considered of less importance to ruminants. (DAS), and DON are commonly found in agricultural
Ruminal conversion of ZEN was found to be about commodities (Desjardins et al., 1993). However,
30% in 48 hrs (Kallela and Vasenius, 1982). A except for DON, it appears that most contamination
controlled study with nonlactating cows fed up to with T-2 toxin and DAS occurs post-harvest. The
500 mg of ZEN (dietary concentrations of about 40 toxic effects of trichothecenes include gastrointestinal
ppm ZEN) showed no obvious effects except that effects such as vomiting, diarrhea, and bowel
corpora lutea were smaller in treated cows (Weaver inflammation. Anemia, leukopenia, skin irritation,
et al., 1986b). In a similar study with heifers feed refusal, and abortion are also common. The
receiving 250 mg of ZEN by gelatin capsule (dietary trichothecenes, as a group, are immuno-suppressive
concentrations of 25-30 ppm ZEN), conception rate (Sharma, 1993).
was depressed about 25%; otherwise, no obvious
effects were noted (Weaver et al., 1986a). Several
case reports have related ZEN to an estrogenic DEOXYNIVALENOL
response in ruminants and sometimes included The impact of DON on dairy cattle is not established,
abortions as a symptom (Kallela and Ettala, 1984; but clinical data show an association between DON
Khamis et al., 1986; Mirocha et al., 1968; Mirocha contamination of diets and poor performance in dairy
et al., 1974; Roine et al., 1971). Other cattle herds, but without establishing a cause and effect
responses may include vaginitis, vaginal secretions, (Whitlow et al., 1994). DON may therefore be a
poor reproductive performance and mammary gland marker for low-quality mycotoxin-contaminated
enlargement of virgin heifers. In a field study feeds in these herds. Other case reports help
(Coppock et al., 1990), diets with about 750 ppb substantiate an association of DON with poor
ZEN and 500 ppb DON resulted in poor consumption, performing dairy herds (Gotlieb, 1997 and Seglar,
depressed milk production, diarrhea, and total 1997). DON has been associated with reduced feed
reproductive failure. New Zealand workers (Towers intake in nonlactating dairy cattle (Trenholm et al.,
et al., 1995a,b; Sprosen and Towers, 1995; Smith et 1985). There was a trend (P<0.16) for a 13% loss in
al., 1995) have successfully estimated intake of ZEN 4% fat corrected milk in a study utilizing 18
and its metabolites (ZEN+M) by measuring urinary midlactation dairy cows (average 19.5 kg milk),
ZEN and its metabolites which include zearalanone, consuming diets shown to contain no common
α- and ß-zearalenol and α- and ß-zearalanol. ZEN+M mycotoxins other than DON which was at levels of
intake predicted from urinary ZEN+M was associated approximately 0, 2.7 and 6.5 ppm in treatment diets
with reproductive disorders in sheep and dairy cattle. (Charmley et al., 1993). Noller et al. (1979) used 54
In sheep, ZEN+M was related to lower conception, lactating dairy cows in a 3 x 3 latin square experiment
reduced ovulation, increased twinning rates and a 10 with 21-day feeding periods. Gibberella zeae (F.
to 20 % decline in fertility of ewes. With dairy cattle, graminearum) infected corn was used to provide
herds with low fertility had higher levels of blood estimated concentrations of 0, 1650 and 3300 ppb
and urinary levels of ZEN+M. Individual cows within
DON and 0, 65 and 130 ppb of ZEN in three with T-2 developed severe depression, hindquarter
experimental diets. While neither intake nor milk ataxia, knuckling of the rear feet, listlessness and
production (22.9 kg/d) were affected, cows that anorexia (Weaver et al., 1980).
received contaminated grain gained significantly less
weight. Conversely, Ingalls (1996) fed lactating cows
diets containing 0, 3.6, 10.9 or 14.6 ppm of DON FUMONISINS
for 21 days, without an apparent effect on feed intake
or milk production (30 kg/d). DiCostanzo et al. This family of mycotoxins is produced by the species
(1995), in a review of several individual studies, of Fusarium in the Liseola section. F. verticilloides
concluded that beef cattle and sheep can tolerate up (formerly F. moniliforme), a species that is almost
to 21 ppm of DON without obvious deleterious ubiquitous in corn, and F. proliferatum are the main
effects. species producing high yields of fumonisins.
The FDA had provided an advisory for DON Fumonisins B1, B2, and B3 (FB1, FB2, and FB3) are
concentrations in wheat and wheat-derived products produced in fungal cultures or found in naturally
(Table 3) (Wood and Trucksess, 1998). contaminated corn samples (Cawood et al., 1991).
Feed infected with F. verticilloides has long been
associated with outbreaks of blind staggers (equine
T-2 TOXIN leucoencephalomalacia, ELEM) in equines (Wilson
et al., 1985). Fumonisin B1 was first isolated in South
T-2 toxin is produced primarily by F. sporotrichioides Africa where F. moniliforme has long been associated
and F. poae, but is also produced by other species of with animal problems (Gelderblom et al., 1988).
Fusarium (Marasas et al., 1984). Data with cattle Fumonisin has been shown to cause
are limited, but the toxicity of T-2 toxin in laboratory leucoencephalomalacia in horses (Marasas et al.,
animals is well-documented (Wannemacher et al., 1988), pulmonary edema in swine (Harrison et al.,
1991). T-2 toxin is a very potent mycotoxin associated 1990) and hepatoxicity in rats (Gelderblom et al.,
with gastroenteritis, intestinal hemorrhages (Petrie 1991). Fumonisins are structurally similar to
et al., 1977; Mirocha et al., 1976) and death (Hsu et sphingosine, a component of sphingolipids.
al., 1972; Kosuri et al., 1970). T-2 toxin fed to cattle Sphingolipids are in high concentrations in myelin
at 0.64 ppm for 20 days resulted in death and bloody and in certain nerve tissues. Fumonisin toxicity is
feces, enteritis, and abomasal and ruminal ulcers (Pier thought to result from disruption of sphingolipid
et al., 1980). Kegl and Vanyi (1991) observed bloody biosynthesis (Riley et al., 1996). A USDA, APHIS
diarrhea, low feed consumption, decreased milk survey of 1995 corn from Missouri, Iowa and Illinois
production and absence of estrus cycles in cows found that 6.9% contained more than 5 ppm
exposed to T-2. Weaver et al. (1980) showed that T- fumonisin B1 (Anon., 1995). Murphy et al. (1993)
2 was associated with feed refusal and gastrointestinal reported fumonisin concentrations in corn for the
lesions in a cow, but did not show a hemorrhagic Iowa, Wisconsin, and Illinois crops. Incidence of
syndrome. Serum immunoglobulins and certain contamination was greater than 60% and
complement proteins were lowered in calves concentrations ranged from 0 to 37.9 ppm. Corn
receiving T-2 toxin (Mann et al., 1983). Gentry et screenings contained c. 10 times the fumonisin content
al. (1984) demonstrated a reduction in white blood of the original corn.
cell and neutrophil counts in calves. A calf intubated
Table 3. US FDA advisory levels for deoxynivalenol in wheat-derived products.1
Product Concentration (ppm)
All finished wheat products, e.g. flour, bran and germ, for human consumption 1
Grains and grain by-products destined for ruminating beef cattle and cattle
in feedlots older than 4 months and for chickens (these ingredients should not exceed 50% of the diet) 10
Grains and grain by-products destined for swine
(these ingredients should not exceed 20% of the diet) 5
Grains and grain by-products for all other animals
(these ingredients should not exceed 40% of the diet) 5
1
Wood and Trucksess, 1998
While FB1 is thought to be much less potent in OCHRATOXIN A

ruminants than monogastrics, work by Kriek et al.
(1981) suggested that fumonisin was toxic to sheep. This mycotoxin is produced by species of Penicillium
Osweiler et al. (1993) fed young steers 15, 31 or and Aspergillus, and is a causative agent of kidney
148 ppm fumonisin in a short term study (31 days). disease in pigs that has been referred to as mycotoxin
There were no significant effects on feed consumption porcine nephropathy, producing symptoms including
or gain; however, there was a trend toward lower diarrhea, increased water consumption, diuresis and
intake and weight gains for those fed 148 ppm. With dehydration (Krogh, 1979). OTA is rapidly degraded
the highest feeding level, there were mild liver lesions in the rumen and thus thought to be of little
in calves, and the group had elevated liver enzymes consequence unless consumed by young pre-ruminant
indicative of liver damage. Lymphocyte blastogenesis calves (Sreemannarayana et al., 1988).
was significantly impaired at the end of the feeding
period in the group having the highest dose.
Dairy cattle (Holsteins and Jerseys) fed diets CITRININ
containing 100 ppm fumonisin for approximately 7 Citrinin can co-occur with OTA, is produced by both
days prior to freshening and for 70 days thereafter Penicillium and Aspergillus, and like OTA targets the
demonstrated lower milk production (6 kg/cow/day), kidney (Kitchen et al., 1977). Symptoms of pruritis,
explained primarily by reduced feed consumption. pyrexia and hemorrhagic syndrome in a dairy herd
Increases in concentrations of serum enzymes were attributed to citrinin (Griffiths and Done, 1991).
suggested mild liver disease (Diaz et al., 2000). Dairy
cattle may be more sensitive to fumonisin than are PATULIN
beef cattle, perhaps because of greater production
stress. Patulin is produced by Penicillium, Aspergillus, and
Fumonisin has been shown to be carcinogenic in Byssochlamys and may be found in silage (Dutton et
rats and mice (NTP, 1999), and has been associated al., 1984; Hacking and Rosser, 1981). Patulin has
with esophageal cancer in humans in China (Chu and been incriminated as a possible toxin in Europe and
Li, 1994) and South Africa (Rheeder et al., 1992). New Zealand (Lacey, 1991).
Therefore, fumonisin contamination has implications
for human health, at least from a regulatory
perspective. The FDA released guidance for PR TOXIN
fumonisin levels in human foods and animal feeds in
late 2001 (Table 4). Produced by Penicillium roquefortii, PR toxin has
Table 4. US FDA guidance for industry on fumonisin levels in human foods and animal feeds.1
Total fumonisins (FB1+FB2+FB3)
(ppm)
Human foods
Product
Degermed dry milled corn products
(e.g., flaking grits, corn grits, corn meal, corn flour with fat content of < 2.25%, dry weight basis) 2
Whole or partially degermed dry milled corn products
(e.g., flaking grits, corn grits, corn meal, corn flour with fat content of > 2.25 %, dry weight basis) 4
Dry milled corn bran 4
Cleaned corn intended for masa production 4
Cleaned corn intended for popcorn 3
Animal feeds
Corn and corn by-products intended for:
Equids and rabbits (no more than 20% of diet)2 5
Swine and catfish (no more than 50% of diet)2 20
Breeding ruminants, breeding poultry and breeding mink and including lactating dairy cattle and hens laying eggs 30
for human consumption (no more than 50% of diet)2
Ruminants >3 months old being raised for slaughter and mink being raised for pelt production 60
(no more than 50% of diet)b
Poultry being raised for slaughter (no more than 50% of diet)2 100
All other species or classes of livestock and pet animals (no more than 50% of diet)2 10
1
Federal Register, 2001.
2
Limits on ingredients are on a dry weight basis
been found in silage (Hacking and Rosser, 1981) and a range of diseases. The majority of human health
was the suspected vector in a case study with risk from mycotoxins is from consumption of
symptoms of abortion and retained placenta (Still et contaminated grains and nuts. While many
al., 1972). Surveys of grass and corn silage in Europe mycotoxins are common contaminants of feedstuffs
have found P. roquefortii in up to 40% of samples and several mycotoxins have been shown to occur in
(Auerbach, 2003). the milk of dairy cattle, concentrations are extremely
low because only a small fraction of the amount
consumed by a cow is transferred to milk in the parent
DICOUMAROL form or as a derivative. Aflatoxin is the only
mycotoxin that has received regulatory action in the
Dicoumarol is produced from natural plant US as a possible contaminant in milk. This is because
compounds when Penicillium or Aspergillus molds aflatoxin transfer from feed to milk is greater than
grow on sweet clover or sweet vernal grass. for other mycotoxins. Also, aflatoxin is carcinogenic,
Dicoumarol interferes with the function of vitamin highly toxic to humans, and because milk is a primary
K, resulting in a hemorrhagic syndrome. Moldy component of the diet of infants. The US FDA
sweet clover poisoning is discussed by Radostits et indicated that aflatoxin is the only mycotoxin that
al. (1980). currently warrants regulation in milk (Wood and
Trucksess, 1998).
ERGOT ALKALOIDS
AFLATOXIN
One of the earliest recognized mycotoxicoses is
ergotism caused by a group of ergot alkaloids. They Milk aflatoxin residues are the result of transformation
are produced by several species of Claviceps, which of the parent compound in the liver and its subsequent
infect the plant and produce toxins in fungal bodies secretion into milk. Aflatoxin B1 results in milk
called sclerotia or ergots. Ergotism primarily causes residues of aflatoxin M1, while aflatoxin B2 results
a nervous or gangrenous condition in animals. in milk residues of aflatoxin M2. Small amounts of
Symptoms are directly related to dietary other derivatives such as aflatoxin M4, Q 1, and
concentrations and include reduced weight gains, aflatoxicol can also be found in milk; however
reduced milk production, and agalactia (Robbins et aflatoxin M1 is the primary residue (Wood, 1991).
al., 1986). Sclerotia concentrations above 0.3% are Van Egmond (1989) concluded that aflatoxin carry-
related to reproductive disorders. Fescue infected with over from feed to milk is approximately 1-2%.
Neotyphodium or Epichloe may contain toxic alkaloids Frobish et al. (1986) found greater aflatoxin transfer
associated with ‘fescue toxicity’ (CAST, 2003). to milk when the toxin was supplied by contaminated
Fescue is a major pasture grass in the US, growing cottonseed meal than when it was supplied by
widely throughout the lower midwest and upper contaminated corn. Percentage transfer of aflatoxin
south. Over half of the fescue is endophyte-infected, to milk was not affected by concentration in the feed
making this a serious problem for cattle and horse or by milk production level of the cow. They
producers. Endophyte-free varieties are available, but concluded that concentration of aflatoxin M1 in milk
they are not as hardy as infected varieties. Fescue was approximately equal to 1.51% of the
infected with a nonpathogenic endophyte may be more concentration of aflatoxin B1 in the diet. Therefore a
field hardy and less toxic. concentration of 33 ppb in the total diet would result
in a 0.5 ppb concentration in milk (3.9 ppb in the
milk dry matter, assuming 12.8% milk solids). Figure
Are dairy products contaminated when 1 shows the extent to which four toxin adsorbents
dairy cattle consume mycotoxins? added to the diet of dairy cows reduced aflatoxin M1
in milk (Diaz et al., 1999).
Moy (1998) reviewed the international efforts to Regulatory pressures and a widespread awareness
evaluate and reduce the human risks of mycotoxins. have helped minimize aflatoxin problems. Surveys
He stated that “human health problems caused by the of aflatoxin B1 concentrations in feedstuffs conducted
consumption of most mycotoxins are complex and during the 1980s resulted in lower levels than for
poorly understood”, but they may be responsible for surveys conducted in the 1970s (Van Egmond, 1989).
70 64.6
58.5
60
50
Reduction (%)
40
31.4
30
20
10 5.4
0
Sodium Calcium Mycosorb® Activated
bentonite bentonite charcoal
Figure 1. Effects of sequestering agents on milk aflatoxin residues (Diaz et al., 1999).
The United States General Accounting Office (GAO, an oral dose of 920 mg and found less than 4 ppb of
1991) concluded that industry, federal and state free and conjugated DON in the milk. DON was
programs are effective in detecting and controlling excreted in milk primarily as DOM-1, but excretion
aflatoxin and that it is doubtful that additional rate is extremely low at 0.0001% of the dose. Côté
programs or limits would reduce the risk of aflatoxin et al. (1986) found no DON, but up to 30 ppb of
in the food supply. The GAO specifically examined DOM-1 in milk of cows fed DON at about 300 mg/day
the state-administered program in the state of Georgia (66 ppm) for five days.
as a part of its report. In 1989, 13% of corn samples
tested by the Georgia Department of Agriculture
exceeded 20 ppb. On farms, 3.9% of tested milk ZEARALENONE
exceeded limits while at the retail level only 0.4% of
milk was in violation. Current surveillance programs Shreeve et al. (1979) fed dairy cows about 1 ppm
in the US aimed at reducing food residues make it zearalenone for 11 weeks without detecting a milk
very unlikely that aflatoxin will be fed at high enough residue. Prelusky et al. (1990) administered up to 6
levels and for sufficient duration to have significant g of zearalenone per cow daily and found a total
production or health effects on dairy herds in those milk residue of up to 16 ppb, which represented about
regions that have an active program. 0.01% of the dose. Hagler et al. (1980) administered
Dairy cattle feeds should contain less than 20 ppb 5 g zearalenone in ground feed to a lactating dairy
aflatoxin to prevent milk residues above 0.5 ppb. cow that was milked twice daily with samples
Concentrations of aflatoxin should be conservatively collected until 120 hr after dosing. Only trace levels
low because of uncertainties in sampling and analysis, of zearalenone were found in the milk obtained at
nonuniform distribution of aflatoxin, and potential 96, 108 and 120 hr after dosing and trace levels of
for more than one source of aflatoxin in the diet. zearalenol were also found in the milk at 108 and
120 hr after dosing. Mirocha et al. (1981) found that
zearalenone and its metabolites reached levels above
DEOXYNIVALENOL 1 ppm in milk representing about 0.7% of the
zearalenone dosage, which was 25 ppm for eight days.
DON is changed to DOM-1 in the rumen with
estimates of 24 hr degradation of about 50% (King
et al., 1984). Deoxynivalenol and metabolites are T-2 TOXIN
rapidly excreted, primarily through urine (Côté et
al., 1986; Prelusky et al., 1984; Prelusky et al., Residues of T-2 and its derivatives have been found
1987). Prelusky et al. (1984) administered DON in in milk, but have a low transfer rate from feed to
milk. After 72 hrs, an orally administered dose of T- the urine and 6% in milk. Of the radioactivity in
2 at 0.42 mg/kg of body weight (approximately 36 milk, only a small amount was in the form of
ppm) was almost completely excreted in the feces ochratoxin A (OTA), representing 0.026% of the
and urine (Yoshizawa et al., 1981; Yoshizawa et al., dosage administered. In a study with lactating cows
1982). Milk residues, which reached a maximum of where ochratoxin A was fed at 317 to 1,125 ppb for
about 35 ppb, suggest that about 0.2% of T-2 and its 11 weeks, neither ochratoxin A nor its metabolite
metabolites are secreted in milk. In the lactating cow ochratoxin α were detected in milk (Shreeve
administered radioactive labeled T-2 toxin, three et al., 1979).
metabolites (3'-hydroxy-T-2 toxin, 3'-hydroxy-HT-
2 toxin and 3'-hydroxy-7-hydroxy-HT-2 toxin)
accounted for 30-40% of the radioactivity in urine, SUMMARY
60-70% of radioactivity in milk and 50-60% of the
radioactivity in blood plasma. Other metabolites Several other mycotoxins, or their derivatives, may
included HT-2 toxin, neosolaniol and 4- be found in extremely small amounts in milk. Other
deacetylneosolaniol. Other investigators (Robinson than aflatoxin, they are not considered likely human
et al., 1979) have measured T-2 up to a peak of 160 health hazards in milk. It is thought that significant
ppb in milk on the fifth day after starting oral residues of these other mycotoxins occur in milk only
intubation with daily doses of 182 mg of T-2 toxin when very high, nonclinical levels are administered
for 15 consecutive days (equivalent to about 9 ppm to cows. Additionally the derivatives are generally
in the diet, assuming a daily consumption of 20 kg). less toxic than the parent compound. Aflatoxin is the
only mycotoxin that has received regulatory action
in the US as a possible contaminant in milk.
FUMONISIN Regulatory efforts have successfully reduced the risk
of aflatoxin in the food supply in the US (GAO,
Fumonisin B1 carryover from feed to milk is thought 1991). Efforts to prevent aflatoxin formation, to
to be negligible (Richard et al., 1996; Scott et al., divert contaminated ingredients away from dairy
1994). Prelusky et al. (1996) reported studies where feeds usage, and to use feed additives that reduce
dairy cattle were administered fumonisin B1 either aflatoxin absorption by the animal, have contributed
orally or intravenously. The oral dosages were to fewer milk contamination problems.
approximately equal to dietary concentrations of 60
to 300 ppm. The intravenous dosages were stated to
be similar to dietary concentrations of 125 to 500 What are the safe levels of mycotoxins for
ppm. No fumonisin B 1 or its metabolites were dairy cattle?
detected in milk (detection limit of 0.5 ppb for
fumonisin B1). Maragos and Richard (1994) analyzed Some of the same factors that make diagnosis difficult
155 milk samples collected in Wisconsin during a also contribute to the difficulty of establishing levels
period when feeds were reported to be severely of safety. These include lack of research, sensitivity
affected by mold. Additionally, 10 samples were differences among animal species, imprecision in
collected in Illinois. Feed samples associated with sampling and analysis, the large number of potential
these milk samples were not collected and thus mycotoxins, and interactions with other mycotoxins
fumonisin B1 concentrations in feed were unknown. and stress factors (Hamilton, 1984; Schaeffer and
Only one of the 165 milk samples tested positive for Hamilton, 1991). Mycotoxin effects are also
fumonisin B1, which was determined to be 1.29 ppb. moderated by factors such as sex, age, duration of
This suggests that fumonisin can occur in milk, but exposure, and stresses of the environment and
is likely to be at very low levels. production. The known dietary factors that interact
with mycotoxins include nutrients such as fat, protein,
fiber, vitamins and minerals (Brucato et al., 1986;
OCHRATOXIN Coffey et al., 1989; Smith et al., 1971).
Naturally contaminated feeds are more toxic than
Goats were administered a single dose of radiolabeled feeds with the same level of a pure mycotoxin
ochratoxin A at 0.5 mg/kg (Nip and Chu, 1979). supplemented into the diet. Jones et al. (1982)
Cumulative excretion of radioactivity over seven days demonstrated that productivity losses in commercial
indicated that 53% was excreted in the feces, 38% in
broiler operations can occur when aflatoxin protozoal than bacterial activity (Kiessling et al.,
concentrations are below those shown by controlled 1984).
research to be of concern in laboratory situations.
Aflatoxin produced from culture was more toxic to
dairy cattle than pure aflatoxin added to diets Which feed mycotoxin source is the
(Applebaum et al., 1982). In swine, Foster et al. greatest problem - grains or silages?
(1986) demonstrated that a diet containing pure added
DON was less toxic than diets with similar Almost any type of feed can be contaminated with
concentrations of DON, which were supplied from mycotoxins. Table 5 compares the incidence and
naturally contaminated feeds. Smith and MacDonald concentrations of mycotoxins in corn grain and corn
(1991) have suggested that fusaric acid, produced by silage over a nine year period. Perhaps the worst case
many species of Fusarium, occurs along with DON scenario for a dairy producer may be to have the on-
to produce more severe symptoms. Lillehoj and farm stored feeds contaminated. Therefore, the
Ceigler (1975) give an example where penicillic acid season’s supply of feed is contaminated and not just
and citrinin were innocuous in laboratory animals the current load that is used over a short time period.
when administered alone but were 100% lethal when This is because toxicity is a function not only of
given in combination. These studies strongly suggest amount but also duration of feeding. When the on-
the presence of other unidentified mycotoxins in farm stored feed is contaminated, the dairy producer
naturally contaminated feeds. It is well documented faces a difficult decision. The contaminated feed
that several mycotoxins may be found in the same could be fed as normal, diluted with purchased feed
feed (Hagler et al., 1984). Abbas et al. (1989) or not used. Increased costs may be incurred from
demonstrated that Fusarium species isolated from additional feed purchases or reduced income may
Minnesota corn produced multiple mycotoxins. result from a loss in animal performance. The
Because animals are fed a blend of feedstuffs and contaminated feed may be either grain or forage;
because molds produce an array of mycotoxins, many however, dairy producers are more likely to store
mycotoxin interactions are possible. forage than grain. Therefore, it is important to discuss
Interactions with other stress factors make mycotoxin contamination of forages.
recommendations difficult. Animals under Many different mycotoxins have been found to
environmental or production stress may show more occur in forages either in the field, or in storage as
pronounced symptoms. It is clearly shown that there is hay or silage (Lacey, 1991). Some mycotoxicoses in
a temperature interaction with fescue toxicity such that cattle resulting from contaminated forages have been
more pronounced symptoms are expressed during heat reviewed (Lacey, 1991; Gotlieb, 1997; Seglar, 1997;
stress (Bacon, 1995). Fumonisin at 100 ppm has been Whitlow, 1993; Whitlow and Hagler, 1997). It is
shown to reduce milk production in dairy cattle (Diaz unclear how much of the mycotoxin contamination
et al., 2000), and in a separate study to not of forages occurs prior to harvest. Fresh feed can be
significantly affect average daily gain in beef cattle contaminated with mycotoxins at harvest; however,
fed 148 ppm (Osweiler et al., 1993). While this mold can grow in harvested forages. The limiting
contrast may reflect a difference in the duration of factor for mold growth in hay is low moisture
feeding, number of animals studied, etc., it may also content. When hay is stored too wet, mold is likely
suggest differences due to greater stress in early to grow, produce heat and cause heat damage. The
lactation dairy cattle as compared with young growing limiting factors for mold growth in silage are a low
beef cattle. pH and high moisture, which limit air infiltration. If
Because of partial degradation in the rumen, silage is stored too dry or insufficiently packed and
mycotoxins are less toxic to cattle than to most other covered, infiltration of air allows for microbial
animals. However mycotoxins are not completely activity which depletes silage acids, allowing pH to
degraded and some of the degradation products rise and molds to grow. Some of the Penicillium molds
remain toxic (Kiessling et al., 1984). Extent of grow at a low pH (Auerbach, 2003). Silage on the
ruminal degradation appears to be variable. It is feeding face must be removed at a rate that prevents
speculated that feeding situations resulting in a faster air infiltration into the silage mass resulting in
rate of ruminal feed passage or a low population of conditions that support mold growth. Practical
protozoa in the rumen may reduce mycotoxin recommendations are to feed 6 to 12 inches daily
degradation in the rumen. Ruminal degradation of from the feeding face to prevent mold.
mycotoxins appears to be more dependent on
Table 5. Occurrence of five mycotoxins in corn silage and corn grain in samples submitted for analysis by producers in North Carolina over a
nine-year period1.
Aflatoxin (>10 ppb) Deoxynivalenol (>50 ppb) Zearalenone (>70 ppb) T-2 toxin (>50 ppb) Fumonisin (>1 ppm)
Corn silage
n Positive X ± sd n Positive X ± sd n Positive X ± sd n Positive X ± sd n Positive

(%)2 (%) (%) (%) (%) X ± sd
461 8 28 ± 19 778 6 6 1991 ± 2878 487 30 525 ± 799 717 7 569 ± 830 6 3 37 N/A
Corn grain
231 9 170 ± 606 362 70 1504 ± 2550 219 11 206 ± 175 353 6 569 ± 690 3 7 60 N/A
1
Whitlow et al., 1998
n = number of samples
2
Percentage of samples positive above given concentrations
It appears that Aspergillus flavus does not grow well in in cattle (Still et al., 1971). OTA in moldy forage
hay or silage, however, aflatoxin concentrations up to 5 has also been implicated in cattle deaths (Vough and
ppm have been reported (Kalac and Woolford, 1982). Glick, 1993).
We have detected low levels of aflatoxin (<100 ppb) The most important pasture-induced toxicosis in
in corn silage and alfalfa. Table 5 shows that the the US is tall fescue toxicosis caused by endophytic
frequency of aflatoxin in corn silage is not different alkaloids (Bacon, 1995). Other forage toxicoses of
from the frequency of aflatoxin in corn grain, but fungal origin include ergotism, perennial ryegrass
the concentrations are lower. The frequency and staggers, slobbers syndrome (a hemorrhagic disease
concentrations of some Fusarium-produced that is associated with dicoumarol produced in fungal
mycotoxins are also compared in Table 5. There is a infected sweet clover and sweet vernal grass), and
trend toward a higher frequency of ZEN in corn silage syndromes of unthriftiness and impaired reproduction
than in corn grain. associated with Fusarium (Cheeke, 1995).
Aspergillus fumigatus is thought to be a fairly
common mold in both hay (Shadmi et al., 1974) and
silage (Cole et al., 1977) and may be a vector in the How can mycotoxin effects be reduced?
development of mycoses in dairy cattle. Silage was
found to contain fumigaclavine A and C and several Pre-harvest control has involved agronomic practices
fumitremorgens (Cole et al., 1977). Animal that minimize mycotoxin accumulation in the field.
symptoms included generalized deterioration typical These include proper irrigation, pesticide application,
of protein deficiency, malnutrition, diarrhea, resistant or adapted hybrids, proper tillage and
irritability, abnormal behavior and occasionally death. fertilization. Unfortunately, breeding for mycotoxin-
The hay was fed to goats and rats and resulted in resistant hybrids has been only partially successful.
retarded growth and histopathological changes in the Munkvold et al. (1999) have shown that compared
livers and kidneys. with nontransgenic corn, Bacillus thurengiensis (Bt)-
Surveys of grass and corn silage in Europe have transgenic corn had less corn borer damage, less F.
found an occurrence of P. roquefortii in as many as verticillioides infection, and lower fumonisin
40% of samples (Auerbach, 2003). PR toxin, contamination. Fungicides have shown little efficacy
produced by Penicillium roquefortii, has been found in controlling pre-harvest aflatoxin contamination in
in silage (Hacking and Rosser, 1981) and was the corn (Duncan et al., 1994).
suspected vector in a case study with symptoms of Post-harvest approaches for management of
abortion and retained placenta (Still et al., 1972). mycotoxin contamination include mycotoxin analysis
Vesely et al. (1981) reported that in cows fed corn of feedstuffs and diversion of contaminated lots;
silage containing PR-toxin there was loss of appetite, ammoniation of corn and cottonseed to destroy
gut inflamation, rumen stasis and abortion. Moldy aflatoxin; dilution; and storage technology (Trail et
alfalfa hay containing Aspergillus ochraceus was al., 1995). Mycotoxin-contaminated grains can be
implicated as producing OTA associated with abortions used for ethanol production, and in some cases
mycotoxin-contaminated grains can be diluted with production losses, impaired immunity and significant
clean feeds (Desjardins et al., 1993). The FDA does economic losses. However, analytical techniques for
not allow dilution of aflatoxin-contaminated feeds, mycotoxins are improving, costs are decreasing and
which is considered adulteration. The best strategy several commercial laboratories are available which
for post-harvest control of mycotoxins is proper provide screens for an array of mycotoxins. The
storage and handling of feed grains. Federal Grain Inspection Service (USDA-GIPSA)
Sampling and testing feeds is a part of a control provides on the internet a list of approved mycotoxin
program. The accurate determination of mycotoxin tests for grains and provides excellent background
concentrations in grain and feeds depends on a number materials for the feed industry (internet address http:/
of factors. First, a statistically valid sample must be / w w w. u s d a . g ov / g i p s a / p u b s / my c o b o o k . p d f ) .
drawn from the lot (Whittaker et al., 1991). Because Laboratory methods can be found in Official Methods
mycotoxins are not evenly distributed in grains and of Analysis of AOAC International (Horwitz, 2000).
other feedstuffs, most of the error in a single analysis The potential for effective treatments has improved.
is due to sampling – as much as 90% of the error is Certain feed additives can reduce mycotoxin exposure
associated with the taking of the initial sample. Proper of animals and thus minimize their negative effects.
collection and handling of representative feed samples Some additives may be beneficial in reducing
is essential. Once collected, samples should be mycotoxin formation because they are effective in
handled properly to prevent further mold growth. reducing mold growth. Ammonia, propionic acid,
Wet samples may be frozen or dried before shipment microbial and enzymatic silage additives have all
and transit time should be minimized. The sample shown some effectiveness as mold inhibitors.
must then be finely ground and subsampled for Additives to enhance fermentation can be added at
analysis; this step is the second largest source of error ensiling. Mold growth inhibitors may be helpful as a
in an analysis. Finally, the subsample is extracted, surface treatment when capping off the silo or daily
extract purified using one of several techniques, and after silage feed-out to reduce molding of the exposed
then the toxin is measured. Toxin determination may silage feeding face. If unacceptably high levels of
be by thin-layer chromatography plates (TLC), high- mycotoxins occur, dilution or removal of the
performance liquid chromatography (HPLC), contaminated feed is preferable; however, it is usually
gas-liquid chromatography (GLC), enzyme-linked impossible to replace all of a major forage ingredient.
immunosorbent assays (ELISA), spectro- While dilution is sometimes a viable practice to
photometrically, or by other techniques. Blacklighting reduce mycotoxin exposure, reduced feeding of silage
for bright-greenish-yellow fluorescence is often used could result in such a slow feedout, that mycotoxin
as a screening technique for aflatoxin, but it is very problems within the silage increase. Ammoniation
inaccurate; newer and better methods should be used. of grains can destroy some mycotoxins, but there is
As far as we are aware, blacklighting is completely no practical method to detoxify affected forages
inappropriate for other mycotoxins. already in storage. A microbial detoxification method
Mold spore counts may not be very useful and are (Binder et al., 2000) has been identified where a
only a gross indication of the potential for toxicity, species of rumen bacterium (BBSH 797) was isolated
but mold identification can be useful to suggest which which has the ability to biotransform DON, rendering
mycotoxins may be present. Scott (1990) states that it non-toxic. Field studies have suggested that the
screening methods are needed for the Fusarium- microbial product used as a feed additive can protect
produced mycotoxins and that one approach is to test growing pigs from the effects of DON. Galvano et
first for DON, DAS, T-2 toxin and nivalenol, because al. (2001) has reviewed dietary strategies to counteract
other Fusarium mycotoxins seldom occur without one mycotoxins. Increasing nutrients such as protein,
of these four also present. Feeds could then be tested energy and antioxidant nutrients may be advisable
for other mycotoxins if necessary. (Brucato et al., 1986; Coffey et al., 1989; Smith et
Generally, laboratories provide analysis for only a al., 1971).
limited number of mycotoxins, perhaps including Sequestering agents such as clays (bentonites) added
aflatoxin, ochratoxin, deoxynivalenol, zearalenone, to contaminated diets fed to rats, poultry, swine and
fumonisin, and T-2 toxin. Minimum detection levels cattle have helped reduce the effects of mycotoxins
may be limiting because they are often directed at (Diaz et al., 1997; Galey et al., 1987; Harvey, 1988;
finding high levels that cause serious animal disease, Kubena et al., 1993; Lindemann and Blodgett, 1991;
rather than low levels which are associated with Scheideler, 1993; and Smith, 1980; 1984). In most
cases, clay has been added to the diet at about 1%. 11. Improve understanding of the ecology and
Activated carbon at 1% of the diet effectively reduced epidemiology of mycotoxin-producing fungi.
aflatoxin in milk (Galvano et al., 1996). Activated
12. Develop sound agronomic management practices
carbon fed at 0.1% of the diet did not reduce aflatoxin
to reduce mycotoxin contamination.
levels in milk (Diaz et al., 1999). A glucomannan
(Mycosorb ®) fed at 0.05% of diet dry matter or 13. Develop host-plant resistance to toxigenic fungi
bentonites at 1% of diet dry matter were similarly and to mycotoxin occurrence.
effective in reducing aflatoxin concentrations in milk
14. Develop better models to predict the potential
(Diaz et al., 1999) (Figure 1). The low inclusion
for mycotoxin formation.
rate of the glucomannan in comparison to clay-type
absorbants may be an important difference. A recent 15. Develop better sampling protocols.
review of mycotoxin binders provides more details,
but only limited comparison data are available (Huwig
References
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Pokorny, S.L. and T. Kommedahl. 1988.
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Food, nutrition
FOOD
and health
J. Diehl 257
All in good taste: creating natural savory flavorings from yeast

JOHN DIEHL
John Diehl Consulting Services, Darien, Illinois, USA
There has always been a gray area between art and grown in the Pampas and cooked in the Argentine
science that is very difficult to define. Nowhere is style. Another hydrolyzed protein system widely used
this more apparent than in the development and for many centuries is soy sauce, which is made using
preparation of food products. Master chefs, bakers, a koji-type fermentation where the protease and α−
candy makers (e.g.) have for centuries been able to amino peptidase enzymes of the Aspergillus oryzae
present cuisine that routinely defies the ability of a culture organism break down the soy protein. As food
scientist to reproduce. The nutritional aspects of the ingredients, however, the use of hydrolyzed proteins
scientist’s food may be congruent and the ingredients did not become popular until about 100 years ago.
utilized in the same ways, but the overall satisfaction
and presentation of the chef’s creation is consistently
lacking. Food, at the point of consumption, has a Yeast extracts: creation and market
complex, integrated impact on the sensory organs. development
Taste, aroma, texture, appearance, time of day, state
of health, age and mood can all affect the consumer’s In the early 1900s as the populations of major cities
experience and impressions of a given food. Of these in Europe began to grow with the Industrial
variables, the one on which food scientists focus most Revolution, the minimal (or non-existent) sewage/
of their efforts - using both science and culinary arts waste treatment systems in the cities became
– is taste. Who doesn’t enjoy an evening in a five- overloaded. Companies were no longer permitted to
star restaurant enjoying the talents of a master chef just dump waste streams from manufacturing into
and who would not also enjoy the ability to buy from the sewer. Among the big offenders of this dumping
the local supermarket prepared products closely were the yeast companies. Grown on a high sugar
simulating the cuisine of a master chef? The food nutrient source such as molasses, the resulting
industry constantly attempts to bridge this gap between Saccharomyces yeast was sold to the bread makers
gourmet restaurant cuisine and ready-to-prepare off- and brewers for uses we all know well. When a batch
the-shelf grocery products. Ingredients are being was ‘out of spec’ regarding gas or ethanol production,
made and sold to assist in this process. it was simply discarded. Now, unable to just dump
Among the key ingredients used to simulate savory rejected batches, a few companies turned to scientists
flavors are hydrolyzed protein products. Proteins, as who discovered that the protease enzyme in the yeast
they undergo hydrolysis, develop definite ‘meaty’ would lyse cell proteins when the yeast cell died,
taste characteristics that mankind has utilized for resulting in a product with a desirable meaty flavor.
centuries. Initially the proteins undergoing hydrolysis Autolyzed yeast products were thus born and the
were the meats themselves – especially beef – as they market for them continues to grow to this day.
aged between time of slaughter and time of From this beginning yeast extracts of many types
preparation. One of the fascinating observations on have been developed. For decades the terms ‘yeast
a busy main street in Buenos Aires is the large sides extract’ and ‘autolyzed yeast’ were utilized
of beef hanging in the restaurant windows slowly interchangeably, but now the differences between
aging to develop the highly desired taste of beef them are clearly defined to facilitate label statements.
258 All in good taste: creating natural savory flavorings from yeast
Autolyzed yeast has undergone the autolysis (protein at the 50-55% hydrolysis extent (measured as a ratio
hydrolysis) process but the insoluble yeast cell walls of α−amino nitrogen:total nitrogen = 0.50 to 0.55)
are still present. In contrast, a yeast extract has the desirable nondescript meaty flavor predominates.
undergone the hydrolysis and the cell walls have been
removed leaving a completely soluble, savory
flavored product. The focus of this paper is yeast TYPES OF YEAST UTILIZED AND NEW
extracts, but we will briefly also consider the value SOURCES EMERGING
of the cells walls, independent from the protein, as
adjuncts for food and nutrition. Historically, four distinct groups of yeast have been
explored for their value in yeast extract manufacture
(listed in order of importance):
Yeast extract manufacture
• Primary baker’s yeast (Saccharomyces) – grown
The process of making yeast extracts is fairly on molasses.
straightforward; and in order to be cost effective, • Spent brewer’s yeast (Saccharomyces) – grown
the key factor to control is cost of the yeast. No on wort.
wonder then that the traditional manufacturers of
yeast extracts are in either the baking yeast business • Torula yeasts (Candida) – historically grown on
(primary yeast) or operate breweries (spent yeast). paper or petroleum manufacturing wastes.
The yeast cells are killed and the intracellular protease/ • Dairy yeasts (Kluyvermyces) – grown on sweet
peptidase enzymes start the process of digestion of whey.
the intracellular macromolecules (mostly protein and
DNA). The hydrolysis of the protein proceeds until
approximately 50-55% of the α−amino linkages have Of these, the primary baker’s and spent brewer’s yeasts
been hydrolyzed. At this point, the hydrolysis is currently account for the lion’s share of yeast extract
stopped using heat to denature the enzyme. Then the manufacture. Large volumes of autolyzed torula are
cell walls are ruptured, historically by adding a large manufactured, but historically not converted to
amount of sodium chloride, so that the free amino extracts and no company has ever successfully
acids and peptides created by the hydrolysis flow out produced and sold extracts from Kluyvermyces.
into the supernatant. The cell walls are then removed The types of yeast used are beginning to change
via filtration; the remaining solution is concentrated somewhat. The availability and quality of brewer’s
and then dried. The resulting powder is very yeast has changed in recent years as large numbers
hygroscopic, so manufacturers previously added even of breweries in the traditional beer-making cities have
more sodium chloride at this point to act as a flow closed, megabreweries have been built by the
conditioner. Now, however, fluid bed driers allow a surviving companies and numerous types of beers –
more granular product with significantly reduced light, dry, traditional lager – are now made by these
hygroscopic problems. Yeast extracts that contained breweries. When the brewers were concentrated in
30-40% salt in the 1970s through the 1990s frequently cities such as Milwaukee, St. Louis, Cincinnati, etc.,
contain 15-18% salt today. The result of this process the manufacturers of the brewer’s yeast extracts could
is an off-white to brownish powder with a distinct, operate a centrally-located factory and get a regular,
nondescript, meaty flavor note. This has considerable sustained supply of spent yeast that was usually only
value when attempting to build a savory flavor a few hours old when it arrived at the facility. Now
profile. these brewer’s yeast facilities tend to have one source
Why not allow the hydrolysis to proceed beyond located nearby but are forced to transport other
50-55% of the α−amino bonds in the yeast protein? supplies over long distances in order to operate near
We know that converting the yeast protein to 100% capacity. Although these transported lots are
free amino acids would have more flavor impact per frequently chilled, the enzymatic processes that start
gram, but we also know that all enzymatic reactions following the death of the cell proceed; and the lot-
are reversible. Consequently, as the hydrolysis to-lot variability of the end products made from these
proceeds, the point where the rate of hydrolysis and yeasts is increasing. The end user market for the
the rate of synthesis of α−amino bonds are effectively brewer’s yeast extract group remains strong, but if a
equal is reached. Some of the peptides at this more reliable alternative with equal costing ever
equilibrium are very bitter (objectionably so!); but appears their use may decline significantly.
J. Diehl 259
The manufacturers of yeast extracts from primary more competitive with primary baker’s yeast extracts,
baker’s yeasts remain, with one notable exception, major users can be expected to change.
companies that make baker’s yeast for leavening as a Long-term, the spent Saccharomyces from fuel
core business. The baker’s yeast business has suffered ethanol production plants offer the potential for
in many parts of the world during the past three ‘clean-tasting’ yeast extracts (there are little, if any,
decades from over-capacity and price pressures, so off-flavor nuances from this production stream). Since
the ability to shift capacity to yeast extract this is a spent (or dead) yeast, production of the high
manufacture has been somewhat of a godsend for nucleotide enhancer yeasts will not be practical (unless
these companies. Thanks to the revelation that someone develops a technique to denature the
chloropropanols are present in acid hydrolyzed intracellular DNA), but the use of extracts for base
vegetable proteins, the use of primary baker’s yeast flavor production and Maillard reactions could
extracts grew at double-digit rates from the mid 1980s significantly cut into the current market for both
for about 15 years. Many end users however are now primary baker’s and spent brewer’s yeasts. These yeast
revisiting the risk-reward debate about the use of acid cells will be available at almost zero cost to an ethanol
hydrolyzed plant proteins (HVPs) and some will producer and joint ventures between ethanol producers
switch back for cost reasons in the next 2-3 years. and food ingredient sellers are a highly likely event
Because of the sustained rapid growth for such a long in both Brazil and the United States (and wherever
period of time, most of the key producers added else large volumes of ethanol production capacity
capacity to cope and facilitate growth. When HVPs are established). These products will almost certainly
begin to supplant yeast extracts, much of this newer have a huge cost advantage on the primary baker’s
capacity will fall idle. Initially this will allow yeasts now dominating the market and it is reasonable
companies to retire aging, inefficient older facilities, to assume that they will garner considerable market
but excess capacity will still exist. This could create share in the next 20-25 years.
price and profit pressures on the core producers at a
time when their core business – yeast for baking
Building a savory flavor
operations – is also under considerable pressure.
Where this group of products will be and the impact THE CULINARY MODEL
they will have in the market 20 years from now is
difficult to predict. Savory flavors are built in steps and have three
New sources of yeast are looming as competitors primary parts: a flavor base, a specific flavor note,
and these may bring both price and yield advantages and enhancement. Using the culinary arts as the
versus the existing yeast sources. These are Candida model, a chef would accomplish this as follows:
yeasts grown on non-waste substrates such as wood
chips and sawdust and more significantly spent Base flavor note
Saccharomyces recovered from the production of fuel
ethanol. Candida presents a very attractive profile Savory flavor bases are made using the bones, skin,
for making yeast extracts. The amino acids in the fat and sometimes the organ meats of the animal taste
Candida protein consist of a high percentage of those desired. These raw materials are braised, roasted, slow
desired for flavor development – glutamic acid, cooked, etc. to develop flavor notes, then simmered
cysteine, methionine, aspartic acid – and the RNA for an extended period to extract those flavor notes
levels are considerably higher than for Saccharomyces. and finally concentrated via reduction (evaporation).
A state-of-the-art manufacturing facility was recently The chef then uses these savory flavor bases as the
constructed in Europe and primary growth Candida first key building block when developing a flavor
is now being produced on the sawdust, not the waste, for a given cuisine. When working at home in our
coming from a paper manufacturing plant. The own kitchens, we generally utilize bouillon cubes as
resulting yeast extracts have, depending on the the source of our base savory flavor notes.
manufacturing method utilized, either an excellent
flavor profile or a very high level of 5´-active Specific flavor note
nucleotides. Currently the pricing on these products The key specific savory flavor notes we generally
is somewhat aggressive and companies have been desire are produced from Maillard reactions and the
reluctant to switch; however, should prices become complex series of Amadori rearrangements and
Stecker degradations that follow. This is the highly ribonuclease (Penicillium citrinum) under very
desired roasted/cooked flavor we associate with the exacting conditions yields the 5´-active nucleotides.
meat products we eat. When you and I cook at home Because RNA is quickly destroyed following the
we often see the brown film that forms on the bottom death of the yeast cell, these reactions must be done
of the skillet or roasting pan as a cleanup headache, on live cultures. It has been done using primary yeast
but the chef sees this as savory ‘gold’ and proceeds Saccharomyces cervisae for about 25 years and just
to ‘de glace’ the pan using wine, water, vinegar or recently this technology has been extended to primary
whatever liquid is needed for the end product and Candida strains as well (RNA content is higher than
possibly adding additional flavor notes as well for Saccharomyces). Interestingly, the ribonuclease
(mushrooms, various vegetables or herbs). enzyme does not attack the stable DNA molecules at
all (yields are <0.5%), but can attack denatured, single
Enhancement strands of DNA with yields of approximately 85%.
To date, none of the yeast extract manufacturers are
Last is the process of giving the flavor as much impact utilizing denatured DNA as a substrate for the
as possible. This is done with enhancers. The ultimate manufacture of 5´-nucleotides, but it does suggest
savory flavor enhancer has always been sodium the potential to utilize spent Saccharomyces (from
chloride, but there is a hierarchy of enhancers that brewing and alcohol production) as a source of
all contribute to the satisfaction of the end result: enhancer yeast extracts as well.
An interesting sidelight about active nucleotides is
Salt the fact they have an enhancement half-life in the
≅ mouth when consumed. When working in the lab
Free glutamate developing new savory products it is important to
≅ keep this in mind. Sequential tasting of products
Active nucleotides causes an enhancement not only of the product
(5´-IMP and 5´-GMP) containing the nucleotides, but also of the next
(5´-inosinate monophosphate and 5´-guanylate samples in the queue. You can confirm this effect by
monophosphate) making a control and an experimental sample to be
tasted. Taste the control, taste the experimental and
Salt is a wonderful enhancer, but sodium intake levels then taste the control again. Even rinsing the mouth
have become a health concern in recent years. Salt with water and eating a soda cracker will not eliminate
content can be reduced if some free glutamate is also this effect. If true randomization is needed in savory
utilized. But glutamate has also routinely come under panels, do the tests over a number of days with the
pressure from a small group of very vocal anti-MSG three tasting samples in random order. Salt and
advocates who proclaim allergies to glutamate. A few glutamate do not have this effect. Rinsing the mouth
people actually are very allergic, but most of the anti- and eating a soda cracker effectively eliminates their
MSG contingent are self-diagnosed. Many of the effect on the next savory flavor consumed.
people who claim to have this allergy suffer reactions
whenever they see MSG or hydrolyzed proteins in
label statements, but surprisingly can enjoy pasta THE YEAST EXTRACT MODEL
covered with a tomato-based sauce and parmesan
cheese with no reaction at all. Lastly, both salt and The intent of the food processor in developing a
glutamate can be reduced if active nucleotides are desirable flavor profile in a finished product is to
present. mimic as closely as possible the culinary savory flavor
Yeast extracts can contribute to two of the levels model considered earlier. Again, the flavor profile
of enhancement. First, yeast protein is very high in will be built with three parts, all of which can be
glutamic acid. As the hydrolysis proceeds the levels accomplished with yeasts although more frequently
of free glutamate become significant and the yeast other ingredients are also combined with yeast
extract can contribute to overall savory flavor extracts to produce the intended end result:
enhancement. Second, live yeast cells contain fairly
large amounts of RNA, which can be first Flavor base
enzymatically altered with deaminase (Aspergillus +
melleus) allowing deamination of adenosine to yield Maillard-reacted specific flavor(s)
inosine. Secondly, enzymatic hydrolysis with +
Enhancement
J. Diehl 261
This brings us to the three technologies behind the (HVPs). During a 30-year span from approximately
yeast extracts being manufactured and available in 1955 to 1985, HVPs were developed from wheat
the market today: hydrolysis, secondary chemical gluten, corn gliaden and numerous other plant protein
processes and Maillard reactions. sources. The hydrolysis reaction was conducted under
elevated temperature and pressure using 0.1N HCl
Degree of hydrolysis with hydrolysis proceeding to almost 100%. The
hydrolysate was then neutralized using 0.1N NaOH,
The first variable is the degree of hydrolysis, which filtered and dried. These HVPs, given the higher
is commonly measured by the α−amino- degree of hydrolysis, had more flavor impact and
nitrogen:total nitrogen (AN:TN) ratio). Since flavor were more cost effective than yeast extracts, so the
impact requires free amino acids, the objective for major soup, sauce and snack manufacturers switched
yeast extracts in flavor development is hydrolyze to away from yeast extracts. For cost reasons, the HVP
the fullest extent possible without producing bitter manufacturers could not use ‘pure’ proteins as
flavor notes. Typically this occurs at an AN:TN ratio substrates, so small amounts of the lipid fraction of
of 50-55. the plant source were also present. When researchers
When the objective is flavor enhancement however, found chloropropanols (carcinogens) present in
the goal is to minimize the degree of hydrolysis (some numerous HVP products at parts per million levels,
hydrolysis is needed for complete solubility of the many of the market leaders using HVPs made the
extract). After the desired flavor has been achieved, commitment to minimize risk by switching to the
we do not want to alter that flavor when we enhance enzymatically hydrolyzed yeast extracts. One of the
it; rather, we just wish to make the impact of that effects of this was a marked decrease in flavor impact
flavor more pronounced. The AN:TN ratios for these in the finished products. Consequently, they started
extracts are typically between 10 and 20. At this level looking for ‘label-friendly’ ways to enhance their
of hydrolysis the extract will have very little flavor products and the high nucleotide yeasts finally were
impact and will also tend to be quite light in color. ‘discovered’. A period of exponential growth
followed.
Secondary chemical processes
Maillard reactions
The other key variable is the secondary changes (other
than protein hydrolysis) either prior to or after the There is also a post-hydrolysis processing step that
hydrolysis steps. We have already considered one of creates the crucial specific flavor note products that
these, the production of active 5´-nucleotides, which are needed if we want to create the entire finished
is typically done only on enhancer (low degree of savory flavor profile from a yeast source – Maillard
hydrolysis) extracts prior to the actual hydrolysis. reactions. Flavor manufacturers have increasingly
These products now rival the volume of the traditional utilized these reactions to manufacture natural savory
yeast extracts in terms of usage in the market, but flavors since the late 1940s, but the practice is still
when they were first developed there was little not widely utilized by the yeast extract manufacturers.
enthusiasm for their use. Initially, the developers Yeast extracts are an excellent source of the amino
attempted to make a product that had a high degree acids that produce these savory flavor notes and a
of hydrolysis and contained the active nucleotides. very wide range of specific flavors can be developed
This was generally unsuccessful since yields of the using an extract as the only nitrogen source and
nucleotides declined during the protein hydrolysis combining it with different reducing sugars. Some
processing time, so they reduced the degree of of the flavors possible include:
hydrolysis, got levels of active nucleotides that would
be effective as enhancers and told their sales force to • General brown roasted flavor
go sell the product. Nothing happened! The yeast
extract sales force, business people with no technical • Roast beef
backgrounds, went to their core customers and • Roast chicken
showed the product simply as a new yeast extract.
The customers tried it, reported there was insufficient • Roast pork
flavor impact and discontinued its use. It remained • Chocolate
an R&D curiosity for over six years and probably
would still be such today if chloropropanols had not • Nutty flavors
been discovered in acid hydrolyzed plant proteins • Bread
Much of the primary baker’s yeast extract sold today trial and error. Flavor chemists generally learn this
goes to the major flavor manufacturers who use it as by working initially as a bench technician for a more
a preferred source of α−amino nitrogen for further senior colleague and then ultimately getting
reactions. Bitterness created during the hydrolysis assignments as their experience develops. The end
process is not necessarily a problem in this results are seen every day in the foods we buy, both
application, but to date none of the extract in the grocery store and at fast food type restaurants,
manufacturers have pursued making an extract with but the processes and substrates for making them are
maximized degree of hydrolysis aimed at the flavor closely guarded corporate secrets. There are a couple
reaction and fermentation markets. of flavor courses available to learn about these
Numerous factors affect the results from Maillard processes, one of which includes lab development.
reactions:
Proportion and nature of the reactants. Generally, FORMULATING THE FLAVOR SYSTEM - THE
the sugar has less influence on the sensory properties STARTING POINT
of the final flavor than the amino acid. Pentoses are
generally more reactive than hexoses. Lipids, if A flavor is typically built in steps. First a base flavor
present, can also interact in the Maillard reaction to is combined with a specific flavor(s) to approximate
modify the volatile aroma compounds present the flavor profile desired in the end product. Typically
(Farmer and Mottram, 1990). the base component will constitute 50-80% of the
blend and the specific flavor(s) 20-50% of the blend.
Solubility. Aqueous solubility can play an important The flavors made can be tested using a taste panel
role. For most substances, except gases, solubility and costs calculated to determine their acceptability
increases with increasing temperature. Reactions and then altered as needed:
occurring in phases are significantly slower than those
occurring in a single phase.
Solvent. Changing the solvent not only affects the

rate of reaction, it can change the outcome of the
reaction as well (Heinze, 2002). To date, this has
been poorly understood and solvent use is a closely Base Flavor Specific Flavor Target Flavor
guarded trade secret.
• More flavor impact – increase the ratio of specific
Water activity. Highest yields for model systems have flavor to base flavor
been reported at a water activity of 0.72 (Hartman et
al., 1984). Generally the results will show boiled • Lower cost – increase the ratio of base flavor to
meat flavor notes at higher water activities and more specific flavor(s)
roasted meat flavor notes at lower water activities.
In general, the specific flavor products made entirely
pH. Affects the rate of reaction when the reactions from yeast will cost between two and ten times the
take place in an aqueous environment (Heinze, 2002). cost of the base flavor components.
Once the desired flavor profile is achieved, then it
Temperature. Changing temperature not only affects can be added to the finished product and the overall
the rate of reaction, it also tends to change the product impact evaluated. When a savory flavor is made
distribution in the finished flavor as well (Heinze, entirely from yeast sources, it almost always requires
2002). some enhancement. Remembering that adequate
levels of salt and free glutamate must be present (the
Time. Both length of the reaction and pre-reaction yeast extracts in the flavor system will usually furnish
aging (this can be significantly modified using enough free glutamate), the enhancer yeast extract
enzymes) affect the outcome. can then be added to get the flavor’s impact in the
final product to the desired intensity. Generally the
Developing these flavors and learning to scale them starting ratio for this will be about 90% of the
up to industrial processes requires a great deal of
J. Diehl 263
compounded flavor and 10% of the high nucleotide acids, glutamic acid or higher levels of RNA will
enhancer yeast. yield strains of yeasts even more valuable for flavor
development. If we no longer have to concern
ourselves with the CO 2 production or ethanol
High production of a strain, then we can look at changing
nucleotide the chemical composition of the yeast cell to be more
yeast savory flavor friendly.
extract
Target flavor Enhanced target flavor
Bon apetite
Theoretically, you now have a finished product, with Nutritional objectives are difficult to force onto
an added flavor system made entirely from yeast that consumers. People eat what they like, not what is
the market will adopt and buy like mad because it good for them. The cereal manufacturers, for
tastes so good! example, have tried for two decades to build more
nutrition into their cereals. Consumers even say they
want this when responding to panel inquiries. But
Outside the box - what else might we be we continue to buy what our families like, not
able to do with yeast? necessarily what is good for them. Making a culinary
taste for a mass-produced food product is one of the
To date, there are only two manufacturers of yeast ways to get people to eat better, more nutritious diets.
extracts making extracts as their primary business. We have become a population with serious weight
All the others are either growing yeast to sell as an problems. Social factors are partly to blame, but
active leavening agent or to use up a waste stream excess calories are as well. The food producers and
from their core business. The use of both the ß-glucan the restaurant companies see this, too. There is a real
and mannan fractions of the yeast have been under effort right now to bring better quality with lower
study for some time in human nutrition and their serving sizes to the market. Yeast extracts and the
efficacy as probiotics is now pretty much accepted. flavor systems assist this process of making the key
Other authors at this symposium are addressing their savory portions of meals more attractive and more
nutritional benefits in non-human mammals. At culinary. Bon appetite, hopefully we can eat for both
present there is a race for use patents regarding these health and enjoyment, not one or the other.
ingredients in human foods. We will, at some point,
see the complex carbohydrate fractions and the
hydrolyzed protein fraction becoming sufficiently References
valuable that some company(s) will discontinue yeast
sales for baking and just make the derivative pieces Farmer, L.J and D.S. Mottram. 1990. J. Sci. Food
noted above. Agric. 53:505-525.
Also, genetic engineering of single cell organisms Hartman, G.J., J.D. Scheide and C.-T. Ho. 1984. J.
is fairly easy and offers the potential to make savory Food Science 49:607-613.
flavor development even better. Steps, either through Heinze, R. 2002. Flavor Reaction Training Course
strain selection or genetic code insertions that can Handbook (Flavor Knowledge Systems).
create higher levels of the sulfur-containing amino
S.J. Klahorst 265
One university’s search for intelligence in a universe of foods for wellness

SUANNE J. KLAHORST
California Institute of Food and Agricultural Research, University of California, Davis, USA
Introduction
The students in our universities this year are likely Consumer-driven industries and government agencies
to see the world population double in their lifetime follow public opinion when enacting policy. Examples
and up to 20% of the earth’s species threatened of public influence on food policy are the Dietary
(Population Council, 2004; McKee et al., 2004). Supplements Health and Education Act of 1994, the
Meanwhile, the emission of heat-trapping gases is USDA organic certification regulation, and the
predicted to cause global climate changes. To solve reluctance of large food companies to market
these problems, the experts agree that students need controversial foods. Consumers have demonstrated
to think in terms of patterns and systems, while our that they will accept policy based on tradition, cultural
universities are organized around disciplinary beliefs, food ethics, and incomplete science, while
specialization. Universities educate students as if art, they turn to health professionals, universities and the
science, ethics and the long-term future of humanity media for food and health information.
were unrelated. Planning a future for food requires To understand how education can serve food
that universities institutionalize the capacity to think systems, the University of California, Davis, located
and act across discipline boundaries as if culture, in the nation’s top agricultural state, serves as a model
science, agriculture, ethics, and the long-term were for critical examination. It is important, however, to
all related. provide the context in which UC Davis serves its
The problems of the next generation will have food and agricultural mission.
considerable impact on providing a safe and adequate
supply of appealing, health-promoting foods. An
idealistic mission for an academic center focusing The state of California
on foods for wellness might be to build consensus
for how an institution for food research will invest California’s two leading commodities in cash receipts
its limited resources for integrating food, health and are milk and grapes. California produces about 20%
agriculture in the short and long terms. Consensus of the milk receipts and 91% of the grape receipts in
would be based on sound science, but would also be the nation. California is ranked first in the US in the
subject to humanities, ethics, social sciences, and production of fluid milk, butter, ice cream and nonfat
complete knowledge. Typically, consensus begins dry milk and second in cheese (CA Dairy Research
with prior research and builds on prior success. Foundation, 2003). The American Dietetic
A university-based visioning process begins with Association claims that nine in ten women believe
campus food and agricultural leadership and extends calcium is important to their health (ADA, 2003),
outward until it exerts an influence on public opinion. yet the same number of women over age 30 consume
This influence is the sum of its faculty and their only about half the amount of calcium recommended
graduates, their activities in professional organizations per day (Institute of Medicine, 1997). In California,
and government, and their public and private as in many developed countries, consumption of
collaborators. Their influence used to be primarily nutrients does not correlate to the availability of the
regional, but is now also national and international. agricultural sources of those nutrients, although
266 One university’s search for intelligence in a universe of foods for wellness
improving yield has always been a primary goal of ingredients in snack foods made by Quaker Oats and
agricultural research. Frito-Lay. California milk goes into Kraft and Land-
Agriculture in California encompasses over 350 o-Lakes cheese and Haagen Dazs ice cream. Blue
crops. California food processors are the primary US Diamond and Hershey’s are the nation’s largest users
producers of dried and dehydrated fruits and of California almonds. Dole Vegetables uses
vegetables, and also produce a wide assortment of California lettuce and carrots. Most of these
convenient fresh and frozen fruits and vegetables. It companies have expanded their California operations
is the only state producing many specialty foods such in the last ten years.
as almonds, artichokes, raisins, prunes, olives, dates, California food manufacturing benefits from
figs and pistachios. The state supplies 45% of the proximity to consumers. California’s population in
world’s supply of processed tomato products; 100% the 2001 census was 34,501,130 and climbing (US
of the nation’s canned peaches and fruit cocktail; and Census Bureau, 2004).
100% of the supply of black ripe olives (California California’s agriculture is concentrated in the
League of Food Processors, 2004). These crops have Central Valley, which is also home to 47 endangered
been supported by 50 years of food science and animal species and 44 endangered plant species. Some
agricultural research at UC Davis. In the 1920s, when of the species are found throughout the Central Valley
fatalities from botulism in preserved olives threatened – others are found in only one county (Umbach,
the olive industry, UC developed the canned olive 1997). Agriculture is favorably viewed by California
preservation process that was later adapted to many conservation groups as a means to provide habitat,
varieties of canned fruits and vegetables. California as illustrated by the partnership between rice growers
had to become a leader in preservation technology, and local duck clubs. San Francisco’s regional
since it is the leading agricultural export state and watershed reached an agreement with local cattle
the sixth largest exporter in the world. ranchers to continue cattle production in San Mateo
County. The Nature Conservancy maintains cattle
Table 1. California’s top food commodities – 20031. ranching on its properties. California’s Marin County,
immediately north of San Francisco, established a
Value (million US$)
farmland trust (AFT, 2004) that served as a model
Milk and cream 3,812 for the American Farmland Trust, which is hosting
Grapes 2,579 their annual conference in Lexington, KY in
Lettuce 1,278 November, 2004. According to a May 2001 report
Cattle and calves 1,229
Almonds 1,190 by the Agricultural Issues Center of UC Davis, the
Strawberries 991 state lost approximately 500,000 acres of farmland
Tomatoes 926 to urban development between 1988-1998.
Oranges 559
Broccoli 488
Carrots 460
Chickens 452 The University of California
Avocados 358
Pistachios 336 The ten campuses of the University of California lead
Potatoes 307
Walnuts 305
the nation in developing new patents (439) and have
Lemons 287 for the previous ten years, according to a the US
Patent and Trademark Office. More than 1,000
1
CDFA Resource Directory, 2003. California R&D-intensive companies actively engage
in research projects with UC scientists and students.
As urban growth squeezed farming out of populated
UC trains two-thirds of California’s doctors. One of
areas, some of the large food processors were squeezed
every three California R&D firms was founded by
out of California by urban sprawl, environmental
UC scientists. It is estimated that 85% of the nation’s
regulations, energy costs, labor costs, consolidation,
biotechnology industry employs UC alumni with
or all of the above. The processors that remain are
graduate degrees. UC awards about 3000 PhDs every
tied to the state by geographic proximity to
year. UC also serves agricultural and community
agricultural production. Campbell Soup and La
development through 650 scientists at its Agricultural
Victoria Foods use locally grown tomatoes and
Experiment Station research labs and UC Cooperative
vegetables for their soups, ‘V-8’ juice and salsa.
Extension county offices throughout the state.
California-grown rice, corn and potatoes are
S.J. Klahorst 267
UC DAVIS AND THE CALIFORNIA INSTITUTE OF campus. CIFAR’s executive director, Sharon
FOOD AND AGRICULTURAL RESEARCH Shoemaker, featured nutrition research at semiannual
conferences and provided programmatic overviews
UC Davis is the largest campus in the UC System at of food research activities for visitors. Our early
5300 acres. It features a relative abundance of space conferences, like those of other campuses, tried to
for growth compared to campuses in Los Angeles, explain what these functional foods were, but we were
San Diego or San Francisco, however the City of never comfortable with the ambiguous nature of the
Davis has resisted residential growth, keeping prices term. As interest in this area has expanded, the task
high and forcing students and staff to commute from has become exponentially larger in terms of the
surrounding communities. The campus has plans for numbers of programs adding intelligence every year.
a residential community on agricultural land, Every biological department and school on campus
reflecting statewide population pressures on has its own established research collaborations in the
agricultural land use. Enrollment this academic year food system. Even though multidisciplinary grants
is 30,229 undergraduate and graduate students and and large proposals are becoming strategic to funding,
will be capped next year due to budget constraints. it is not uncommon for two UC Davis investigators
Ethnicity is 42% white, 27% Asian/Pacific, 10% to be funded by the same source, and not be aware of
Chicano/Latino, 2% African American; and 55% it until a chance meeting at a professional conference
female. Research funding in 2002-2003 was $426 in another part of the world.
million public and $72 million private. The campus The College of Agricultural and Environmental
functions much like a city with its own public Sciences (CAES) is a platform that builds a basis for
transportation system, police and fire services, waste the intelligence about food and its role in health
treatment, and energy generation. through 21 departments, 9 centers, and 40 majors
My view on the campus is from the California that teach topics such as: agricultural economics,
Institute of Food and Agricultural Research (CIFAR), agronomy, animal science, food and agricultural
founded in 1991 as a self-supporting program in the engineering, aquaculture, avian science, chemistry,
College of Agricultural and Environmental Sciences physical chemistry, enology, environmental sciences,
(CAES). CIFAR’s vision is to ensure that UC Davis environmental toxicology, food crop development,
remains connected to solving food and agricultural food product design, food processing, food
problems. Our mission is to create and enhance engineering, fermentation science, food safety and
channels for research collaboration, program regulation, food chemistry, human and community
sponsorship and technology exchange between UC development, nutrition, pomology, risk/benefit
Davis, government and private food and agricultural assessment, range science, vegetable crops,
organizations. We deliver results through viticulture, and sensory and consumer sciences.
communication, scientific and community The UC Division of Biological Sciences has six
involvement and networks. We inform faculty and sections and nine majors that collaborate with CAES
industry participants through trend forecasting, on topics relating to food production such as
conferences, targeted demonstration and research microbiology, cellular biology, biochemistry and
projects, and forums. Our program serves as a dynamic molecular biology, plant physiology, plant pathology,
catalyst for linking strategically applied disciplines: exercise biology, neurobiology, physiology, behavior
foods for health; advanced food processing and and genetics. The Schools of Medicine and Veterinary
packaging; water and energy management; and Medicine are very active in nutrition research and
biomass resource utilization. A current list of our public health research. The Graduate School of
sponsoring organizations from industry and Business Management emphasizes the food, health
government can be found on our web site and beverage businesses. UC Davis hosts the US
(CIFAR, 2004). Department of Agriculture (USDA) Western Human
Nutrition Research Center, with 50 scientists, many
of whom have adjunct appointments in nutrition and
FOOD AND HEALTH RESEARCH AT UC DAVIS medicine at the University. The UC Cooperative
The search for intelligence in a ‘universe’ of foods Extension system provides outreach for all these
for wellness tracks food from farm to fork, or more activities statewide.
accurately from farm to sewer. In 1994, CIFAR The breadth of the resources of social and science
recognized food for wellness as an emphasis on intelligence inherent in these organizations, and the
numbers of individuals that might participate in an The Functional Foods for Health Program at the
integrated initiative to improve health appears at first University of Illinois.
glance to be unmanageable. In addition to CIFAR, The UC Davis CAES initiative for foods for health
campus resources available to further research related is taking an inventory of its core competencies to
to food and health include: the Biotechnology define a short-term direction. Exploring
Program; Sustainable Agriculture Research and differentiation and areas of core competence is as
Education Program; bioinformatics; computer necessary to a university as to a business. Universities
programming; chemical and statistical analysis evaluate themselves in terms of intellectual resources,
services; the primate center, technology transfer, and physical resources, accomplishments, alumni, and
UC Connect, a campus link to venture capital and collaborations, compared with those of its
business development services. competitors. Defining core areas of expertise is
The UC Davis Genetic Resource Collections serve necessary for attracting federal and state funding, but
as depositories of many sources of food and also for gifts, corporate contracts, and endowments.
knowledge regarding their production: fruit and nut The funding success that can be achieved through
trees, tomato germplasm, yeast, seeds, insects, this exercise can be illustrated by the UC Davis
rootstocks, domestic avian species, oysters, fish, Cancer Center.
bacteria, viruses, plants and herbs (GRCP, 2004) The The UC Davis Health System invested $70 million
campus news service plays a vital role in disseminating in the cancer program over the past decade, recruiting
food/health research to the public through their link 35 new research scientists and building a new Cancer
to the national media, to UC broadcasts and web casts, Center and state-of-the-art cancer research facility.
and through their training of food researchers in Last year the center achieved a National Cancer
media communications skills. Institute designation that comes with a $1.2 million/
year grant for three years. It also expects $20 million/
Table 2. Food crop breeding programs at UC Davis1. year in extra funding from the NCI and other sources.
Alfalfa Cherry Peach Prune Wheat The NCI currently supports about $9 million/year.
Almond Chestnut Pear Rice Walnut The Cancer Center’s research partnership with
Apricot Fig Pistachio Strawberry Lawrence Livermore National Laboratories (40
Barley Grape Plum Vegetables
Bean Nectarine Potato affiliated scientists), the first of its kind in the nation,
1
was a key factor in winning the designation. Another
UC Davis Technology Transfer Center.
factor was a massive cooperative cancer research
effort created by integrating cancer investigators from
ACADEMIC CENTERS OF DEVELOPMENT OF programs in veterinary medicine, comparative
FOODS FOR WELLNESS medicine (studying similarities among humans and
other animal species), biological sciences, and
The core elements for an integrated UC Davis vision agriculture and engineering. The center describes
for the identification and development of health- itself as a “constellation of scientific expertise, focused
promoting foods are in place. Yet, UC Davis has on cancer, that doesn’t exist at other centers.” (UCD
moved more slowly, and perhaps more thoughtfully Cancer Center, 2004). A similar center for intelligence
than other campuses to define how a comprehensive in developing and selecting foods for wellness could
center might be developed, what it would offer and dovetail into existing centers to gather integrated
to whom, and who would direct and participate in its expertise to address dietary interventions for
activities. The goal of most centers is to further prevention of all types of diseases. But - will a Center
research collaboration and find creative ways to of Centers for food and health be valuable or more
strengthen the web of connections between disciplines. confusing?
In order to exploit those connections to ‘further
intelligence’, links to the interests of the public must
be apparent. A secondary goal, and perhaps a more OBTAINING PRIVATE FUNDING
urgent one in the short term, is to use that web of
connections to compete for funding. Two university UC Davis has several food and health competencies
centers that have obtained funding this way are the that have brought private funding to support food
Center for Designing Food to Improve Nutrition, and beverage research. One of the best known campus
Iowa State University, Ames; the Center for competencies is a 125-year old, internationally-
Enhancing Foods to Protect Health at Purdue, and recognized viticulture and enology program, the first
S.J. Klahorst 269
sensory science curriculum in the world, and a Wine chocolate and milk chocolate. The conclusion was
Business MBA in conjunction with the Bordeaux that a standard 1.5 oz milk chocolate bar had
Business school in France. This program, along with approximately the same quantity of phenols as a 5 oz
food science expertise, brought $25 million from glass of red wine. The article ended with the statement
Robert and Margrit Mondavi to found the Robert that “a pleasant pairing of red wine and dark chocolate
Mondavi Institute for Wine and Food Science. Funds could have synergistic advantages beyond their
will finance facilities for the viticulture and enology complementary tastes.”
and food science and technology departments and The press release was picked up by every major
new programs. newspaper in the country and kept Waterhouse in
Another competency for UC Davis is food media interviews for days. The antioxidant content
antioxidant research. Antioxidants are believed to be of chocolate has now gone mainstream. In February
important to the prevention of cardiovascular disease 2004, the Sacramento Bee contained an advertising
and cancer. UC Davis food science Professor Alloys section from California grocers Raley’s/Bel Air
Tappel was ahead of his time in understanding the entitled “Something extra, A Food and Lifestyle
significance and mechanism of oxidation in food and Publication for Enhancing Quality of Life.” Opposite
in humans. A lipid chemist, he identified Vitamin E the wine and spirits advertised specials, it stated,
as an important antioxidant and performed the first “Wine chemistry professor Andrew Waterhouse of
in vivo studies on its functions. Tappel’s pioneering UC Davis found that chocolate and red wine contain
research over three decades has become the compounds called phenolics that are thought to
mainstream of nutritional research today. Professor decrease coronary heart disease by preventing clotting
Ed Frankel, a graduate of the UC Davis dairy of the arteries.” It featured the quotable end of the
division, returned to UC Davis after a distinguished Lancet article. Since then, Waterhouse has published
career at the USDA Peoria laboratory to collaborate many additional analyses on phenolics in wine, prunes
with Tappel. Frankel, an analytical lipid chemist, co- and walnuts.
authored a Lancet publication that proposed an If wine phenolics can prevent cardiovascular
explanation for the French Paradox (Frankel et al., disease, what can they do for cancer? Susan Ebeler,
1993a). The UC Davis lipid group suspected that the viticulture and enology professor and chemist who
lower incidence of heart disease than might be studies phenolic contributions to flavor, has
expected for the high fat French diet might be due to collaborated on research to show that wine contains
the antioxidant nature of red wine phenolics. In the compounds which can delay tumor formation in an
same decade, the popularity of red wine increased animal carcinogenesis model. Ebeler and her lab
from the previous decade from 17 to 40% of the conducted studies to identify specific compounds
wine market by 2000 (CA Assoc. of Winegrape which may be responsible for this delay and to
Growers, 2002). elucidate possible mechanisms of action.
In April 1993, Frankel, Kinsella and Andrew The analytical strengths in antioxidants led to
Waterhouse, a professor in viticulture and enology, collaborations with Harold H. Schmitz, Group
published a letter in the Lancet titled Inhibition of Manager for the Analytical and Applied Sciences
human LDL oxidation by resveratrol, the polyphenol Group at M&M/MARS, who sits on the College
found in grape skins and seeds (Frankel et al., 1993b). Advisory Council and serves as affiliated faculty in
A similar level of excitement over antioxidants from the UC Davis Department of Nutrition. M&M/Mars
chocolate began with a letter published in the Lancet funded development of analytical methods to study
in September 1996 (Waterhouse et al., 1996). polyphenolic components in chocolate. Carl Keen,
Waterhouse and his graduate students analyzed cocoa chair, UC Davis nutrition department, is frequently
powder and chocolate for total flavonoid phenolics, quoted in the national publications that explain the
and compared the quantity of phenolics in hot antioxidant properties, and health benefits, of
chocolate, bakers chocolate and milk chocolate to chocolate.
those found in a serving of wine. They tested cocoa
extract for antioxidant activity by determining its
ability to inhibit oxidation of LDL cholesterol Communicating food and health research
purified from human blood just as they did for wine.
Among the three forms of chocolate, cocoa had the How do these examples illustrate the directions for
highest levels of phenols, followed by baking health-related research in the University environment?
The UC Davis nutrition department has more than “a fundamental conceptual difference exists between
18 professors, and an equal number of professional a diseased and healthy body. Simply stated, ‘diseased’
researchers and adjunct professors. They study means getting one thing wrong, while ‘healthy’ means
vitamins, trace minerals, fiber, obesity, longevity, getting everything right.” The implications of dietary
infant nutrition and lactation, fat metabolism, sports fat in obesity and artherosclerosis led to a reduction
nutrition and international at-risk populations. Their in the national fat content of foods and some
work is collectively more compelling to public health misleading dietary guidelines. Researchers are now
than chocolate research. Chocolate’s popularity, discovering that fat alone will not predict chronic
however, funded model systems and development of disease, and this has hurt the credibility of the
analytical methods that benefit antioxidant research nutrition community. German believes that the key
for many other foods from California. It also brought to understanding metabolic regulation is to assess all
the department national attention and established aspects of metabolism simultaneously (German,
chocolate as an acceptable indulgence. 2002). The future will see complex systems using
UC Davis is not unique in its ability to command multiple markers to assess disease development in
the attention of the mainstream media. Michael B. more defined and accurate methods. His former
Zemel from the Department of Nutrition, The graduate student, Steve Watkins, Chief Scientific
University of Tennessee, Knoxville was featured in Officer and cofounder of Lipomics, West
Reader’s Digest, a widely circulated US magazine, Sacramento, is collaborating with GlaxoSmithKline
explaining that calcium intake and weight gain might and Bayer to use its patented tools for lipid metabolite
be inversely related. Zemel has published eleven analysis. The application of this intelligence may lead
papers on aspects of calcium and body fat. This is to better and earlier biomarkers for diseases related
one of the first publicized findings to go against the to the human metabolism of lipids.
conventional wisdom and hints at what people have Another campus center with a vision for individual
hoped and suspected for years, that caloric intake diet recommendations is the UC Davis Center for
and activity are not the only two factors contributing Nutrigenomics, directed by Professor Ray Rodriquez.
to weight gain (Zemel, 2003). This center is studying the influence of diet on gene
expression to provide more insights on how genomics
Table 3. Foods analyzed for antioxidant content at UC Davis2. can provide optimized diets. The center is supported
by a five-year, $6.5 million grant from the National
Prune Beans Grape Tea
Onion Pomegranate Cocoa Soy Center on Minority Health and Health Disparities, a
Blackberry Cherry Wine Walnut division of the National Institutes of Health.
Peach Plum Fish Beer Nutritional genomics is the study of how foods
Green peppers Tomatoes Cloves interact with particular genes to increase the risk of
2
CIFAR Directory of Foods for Health Research diseases such as type 2 diabetes, obesity, heart disease
and some cancers. With greater understanding of
epidemiology of subpopulations and new discoveries
Developing integrated disciplines in genomics, we may be better able to link the
identification of genes with risk for disease. Some
The future for food that is ‘universally approved’ typical examples are: African-American men have a
for health will require that students learn new 60% higher risk of being diagnosed with prostate
vocabularies and cross-disciplinary boundaries cancer than Caucasian men; half of all adult Pima
without sacrificing their command of basic sciences. Indians in the US have type 2 diabetes, compared to
Metabolomics, a multidisciplinary specialty that 6.5% of adult Americans of Caucasian descent. A
identifies and quantifies metabolites in cells, is the single change in DNA in people living in Scandinavia
basis of a research concentration at UC Davis 10,000 years ago allows most Caucasian adults today
advocated by food science Professor Bruce German. to drink milk without lactose intolerance. The center’s
Also a dairy advocate, German has brought a vision press release promises “Nutritional genomics will
for individualized nutrition to the campus. He enable individuals to better manage their health and
illustrates that what is beneficial to one person is not well-being by precisely matching their diets to their
beneficial to the next, and that even the fittest unique genetic makeup.” Rodriguez is a sought-after
Olympic athletes in their sport of choice do not spokesperson who advocates this approach in his
advocate a ‘one-size-fits-all’ diet. German wrote that interviews with the media.
S.J. Klahorst 271
The role of the media and opinion leaders in At UC Davis, as at most agricultural campuses,
communicating nutrition messages is a specialty of biotechnology is central to tremendous growth in
CIFAR board member Cheryl Toner, International agricultural and biological education programs.
Food Information Council (IFIC), Arlington, VA. Many programs were based on discoveries leading
IFIC has tracked consumer reactions to nutrition to patents or advancement of a biotechnology-based
messages since 1998, using Cambridge-based Cogent agricultural economy that promised to improve the
Research. IFIC provides press kits for media and quality of life. As biotech crops captured the attention
communicators on responsible reporting, tracks media of the media, the scientific community was
coverage on emerging issues in food and nutrition, unprepared for the backlash. The issues presented by
and helps opinion leaders interpret nutritional studies activists, many based on widespread mistrust of
for the public. At the Second Annual International science, politics and regulation, left scientists
Nutrigenomics Conference in Amsterdam, the unprepared to address the underlying concerns.
Netherlands, November 2003, the Cogent Syndicated CIFAR, with a broad array of company support from
Genetic Attitudes and Trends Survey was presented. small organic food processors to large multinational
“Americans are ready and willing to buy products food and agriculture businesses, responded by
based upon their genetic information, but the science organizing forums to promote constructive dialogue.
is only in the early stages of being able to deliver,” Martina Newell-McGloughlin, Director, University
said Christy White, principal of Cogent Research. of California Biotechnology Program, and a member
“The good news is consumers aren’t looking for of CIFAR’s executive committee, became
complete diet regimens, but for individual products internationally recognized as a biotechnology
and basic recommendations.” (International Food advocate for defending the public benefits of
Information Council, 2004). biotechnology in the public arena. McGloughlin
agreed that the culture of science does little to promote
better communication between scientists and the press.
THE UNIVERSITY FORUM FOR CONTROVERSY She noted in the PEW proceedings on biotechnology
and the media, that “scientists who are adept at getting
The university campus serves as a central focal point their position across (to a broad audience) are
for airing controversy in the food industry, and has devalued in scientific circles as populists or as
been an active participant in the debate over exaggerating.” She cited the example of the late Carl
genetically modified foods; organic versus Sagan, a scientist who was enormously popular with
conventional agriculture; threats of food-borne the mass media but who was “never elected to the
disease; alternative food processing technologies like National Academy of Sciences.” (McGloughlin,
food irradiation; animal welfare and safety practices 2002). Others point out that faculty do not get grants,
in meat and poultry production; and fad diets. In tenure, or advance professionally by talking to the
most of these instances, university scientists favor media. While this may be true for food controversy,
the technological and scientific achievements in which when they bring good news to the forefront that
they are invested. Dissent against food and agricultural concerns a food to improve health, the results speak
technologies on campus is often fostered by social for themselves.
sciences, history, art, literature, international, minority Five years later, after the events of 9-11 allowed
or gender studies. However, top-notch universities the genetically engineered food backlash to cool on
encourage constructive skepticism and dissent in every a back burner, campus factions agreed to disagree,
discipline. validated by a new appreciation for values inherent
University scientists view public backlash as a in the marketplace. Participation in the debate gave
misunderstanding that can be solved through better stakeholders a new appreciation for the importance
science education. University scientists, who are of communication and transparency in the
typically accorded a great deal of respect in their implementation of new technology. The dual
fields, typically feel maligned and mistrusted when techniques of conventional breeding and recombinant
they participate in forums to air public controversy. bioengineering are likely to continue in parallel as
Many scientists resent having to defend themselves, long as the public demands alternatives. In the food
declining offers to participate. However, a new science department, award-winning toxicologist and
appreciation for the contributions of those who feel public educator Carl Winter warns consumers about
compelled to participate can be observed. the high rates of food-borne illness from organic
produce (IFT, 2002) while down the hall, ingredients typical of Chinese medicine. Shoemaker
environmental toxicologist Alyson Mitchell compares also secures funding for students to attend
the antioxidant content of conventionally and international conferences. CIFAR support has come
organically grown fruits and vegetables, reporting from China, Thailand, Denmark, Britain, Japan, and
data that sometimes favor organic methods. The Finland.
college initiative in foods for wellness has invigorated
food and agricultural research by providing a common
goal, regardless of technology. The food Conclusion
biotechnology controversy illustrates that a university
cannot afford to be single-minded in its approach to Food is an important factor in every aspect of our
problems and maintaining the traditions of academic life. Our physical and mental health, culture and
freedom ensures that it will not. rituals depend on food’s appeal, affordability, and
CIFAR continues to help prepare the campus for its ability to comfort and nurture. The nation’s
emerging controversy. A collaboration with trends headlines illustrate the complexity of the systems
forecasting partner Nuffer, Smith, Tucker, San Diego, inherent in providing the wide variety of affordable
(NST) provides opportunities for faculty to sit on an foods we now enjoy. The future promises to keep
international panel of leaders in food and agriculture this generation living longer to enjoy foods on more
to discuss issues as they emerge. The CIFAR board occasions than ever before possible. The next
benefits from this process through NST summaries generation of food scientists must feed a large, mobile
that are presented at the biannual board meetings and population in an interdependent global economy and
respond to the emerging issues through their support a changing environmental climate. Today’s students
of communications strategies, or research are entrusted with the protection of the food supply
sponsorship. from toxic contamination and new sources of disease.
And if our food traditions and cultural preferences
are equally important, students must learn to use
TEACHING FOOD, CULTURE AND LIFE emerging technologies in ways that keep foods as
appealing as the foods they remember from
An important aspect of CIFAR conferences is offering childhood. UC Davis, situated on the Pacific Rim,
the scientific community an appreciation for culinary will continue to be a leading innovator in foods for
arts. Conferences in Napa Valley at the Culinary wellness, both in education and research. The vision
Institute of America and Copia have featured speakers for foods for health and wellness that captures the
that keep scientists mindful of the taste and flavors synergistic value of all the inputs central to delivering
that rule the marketplace. The Research Chef’s benefits to the student, the public and the economy,
Association (RCA) has inspired food science students is still coming into focus as one university searches
to broaden their educational horizons to aspects of for intelligence in a small universe of foods for health.
menu development as well as science in their
education. UC Davis does not offer degrees pertaining
to the hospitality industry, however campus programs References
welcome chefs and food service professionals to learn
about food and nutrition science through short courses American Dietetic Association. 2003. Press release:
and conferences on food, health and agriculture. This American Dietetic Association survey reveals
spring we hosted our second year of continuing women are well-informed about the need for
education courses dedicated to chocolate in the food calcium but almost half still don’t think they get
science department. CIFAR also serves as a central enough. October. http://www.eatright.org Public/
resource for International collaborations. Director Media/PublicMedia_17797.cfm. Accessed March,
Sharon Shoemaker is visiting professor at three 2004.
Chinese universities and joins the Food Science and American Farmland Trust (AFT). 2004. AFT around
Technology department in co-hosting a joint
the country. http://www.farmland.org. Accessed
conference each other year with Southern Yangtze
March, 2004.
University in Wuxi, China, the leading school of food
California Association of Winegrape Growers. 2002.
science in China. Each CIFAR conference typically
features a presentation on research pertaining to Final 2001 Numbers. News Archive, http://
www.cawg.org/news/. Accessed March 2004.
S.J. Klahorst 273
California Dairy Research Foundation. 2003. International Food Information Council. 2004. http:/
California Dairy Industry Facts. http:// /ific.org/index.cfm. Accessed March 2004.
www.cdrf.org/dairy_facts/. Accessed March, 2004. McGloughlin, M. 2002. When media, science and
California Department of Food and Agriculture public policy collide. The case of food and
(CDFA) Resource Directory. 2003. California’s Top biotechnology, In: Proceedings from a workshop
20 commodities. http://www.cdfa.ca.gov/card/ sponsored by the Pew Initiative on Food and
card_new02.htm. Accessed March 2004. Biotechnology and the Joan Shorenstein Center on
California Institute of Food and Agricultural the Press, Politics and Public Policy of the Kennedy
Research. 2004. www.ucdavis.cifar.edu, accessed School of Government at Harvard University,
March 2004. November. p. 14.
California League of Food Processors. 2004. McKee, J.K., P.W. Sciulli, Fooce, C.W. and T.A.
Sacramento, CA. http://www.clfp.com. Accessed Waite. 2004. Forecasting global biodiversity threats
March, 2004. associated with human population growth,
Frankel, E.N., J. Kanner, J.B. German, E. Parks Biological Conservation, 115(1):161-164.
and J.E. Kinsella. 1993a. Inhibition of oxidation Population Council. 2004. Population growth and our
of human low-density lipoprotein by phenolic caring capacity. A Population Council Issues Paper.
sutstances in red wine. Lancet 341(8843):454-7. h t t p : / / w w w. p o p c o u n c i l . o rg / p u b l i c a t i o n s /
Frankel, E.N., A.L. Waterhouse and J.E. Kinsella issues_papers/caring_1.html. Accessed March,
1993b. Inhibition of human LDL oxidation by 2004.
resveratrol. Lancet 341(8852):1103-4. UC Davis Cancer Center, http://www.ucdmc.ucdavis.
Genetic Resources Conservation Program (GRCP). edu/cancer/. Accessed March, 2004
2004. UC Davis, http://grcp.ucdavis.edu/. Accessed Umbach, K.W. 1997. A statistical tour of California’s
March, 2004. great central valley. http://www.library.ca.gov/CRB/
German, J.B. 2002. Milk: guiding nutrition research 97/09/#Heading37.
toward wellness. California Dairy Dispatch, Winter, US Census Bureau. 2004. http://quickfacts.census.
www.cdqa.org. Accessed March 2004. gov/qfd/states/06000.html. Accessed March, 2004.
IFT. 2002. Organic foods not healthier than Waterhouse, A.L., J.R. Shirley, J.L. Donovan. 1996.
conventional. Press Release, News Archives Antioxidants in chocolate. Lancet 348(9030):834.
November 5, 2002, http://www.ift.org/cms/ Zemel, M.B. 2003. Role of dietary calcium and dairy
?pid=1000486. Accessed March 2004. products in modulating adiposity. Lipids 38(2):
Institute of Medicine. 1997. Food and Nutrition 139-46.
Board. National Academy of Sciences, Washington,
D.C.
J.C. MacRae 275
How does diet influence health – could the food chain benefit from a more
proactive approach to clinical nutrition issues?
JOHN C. MACRAE
Rowett Research Institute, Bucksburn, Aberdeen, United Kingdom
Over recent years, in the UK and Europe the food foodstuffs, with ‘animal fats’ (especially ruminant
chain and particularly its animal produce suppliers, saturated fats) being viewed with particular suspicion.
have been frequently challenged by clinicians and Indeed, it is possible to trace a timeline of awareness
public health authorities with health scares, such as about the problems and benefits of animal fats and
salmonella in eggs, E. coli in meats, BSE and FMD. their association with health problems over the last
These have, in some cases, had dramatic effects on 30 years. Starting back in the 1960/70s epidemi-
consumer choice, leading to major economic losses ologists identified strong relationships between the
in the sectors supplying the produce. Since 1999 the proportion of the dietary calories consumed as
Rowett Institute, which has a long history of nutrition saturated fat and the incidence of CHD and colon,
research on farm animals, has re-focused its research prostate and breast cancer. The CHD story related to
into the area of diet and (human) health, with the influence of saturated fat on the development of
programmes that are attempting to elucidate how high levels of cholesterol in the circulating serum
specific nutrients can help to offset the incidence of lipoproteins, with polyunsaturated fats (PUFA)
human non-communicable diseases, such as coronary seemingly modulating these rises in risk (Hegsted et
heart disease (CHD), colon cancer and obesity. It al., 1965). This led to recommendations for a
would seem to us that some important information is reduction of saturated fat in the diet. Consumers were
beginning to emerge from these biomedical not slow to take up this message, as witnessed by a
programmes that could be used by the animal sector major reduction in the full-fat liquid milk sales in
of the food chain as specific ‘health benefit’ messages the UK between 1984 (90% of total) and 1997 (<25%
in promoting animal products in terms of their ability of total).
to help prevent some of the non-communicable
diseases.
There is a vast literature linking, either through OMEGA-3 POLYUNSATURATED FATTY ACIDS
epidemiology or within more mechanistic studies,
the inappropriate consumption of specific nutrients More recently, in the early 1990s research started to
with the incidence of CHD (Krauss et al., 1996; Hu show that the different PUFA were not all equally
et al., 2001) and cancer (Wynder et al., 1997; beneficial in terms of preventing the onset of the
Department of Health, 1998). Consideration of some non-communicable disease. Inflammatory responses
of this may provide a basis for future strategies within are important components in the development of CHD
the animal sector of the feed industry. and cancer and immunologists identified that the
omega-6 (n-6) PUFA (e.g. linoleic acid, C18:2) are
less beneficial than the omega-3 (n-3) PUFA (e.g.
Fats linolenic acid, C18:3; eicosapentaenoic acid, C20:5
(EPA); docosahexanoic acid, C22:6 (DHA); Gibney
SATURATED AND POLYUNSATURATED FATS and Hunter 1993). This relates to the pro-
inflammatory ecosanoids generated during the
From the human health perspective, there has been a post-absorptive human metabolism of the n-6 fatty
history of concern about the lipid components of acids (FA). The n-3 FA have the ability to modulate
276 How does diet influence health?
this inflammation by competing with the n-6 could be an important consideration in the processing
metabolites for incorporation into immune cell of concentrates for the animal industry.
membrane phospholipids. Present recommendations
are to increase the intake of n-3 FA towards a dietary
optimum n-3:n-6 FA ratio of 0.4-0.5. However, most CONJUGATED LINOLEIC ACIDS
human foodstuffs have a ratio nearer to 0.1-0.2, hence
the health benefits of fish oil products (Leaf et al., If the n-3 PUFA were the human nutrition interest
2003), which contain high levels of n-3 FA (see of the early 1990s, over the last five years equal, if
Figure 1). not more, attention has been directed to the conjugated
The reason why fish oils are an abundant source of linoleic acids (CLA). A plethora of laboratory animal
the longer-chain n-3 FA relates to the high linolenic studies has reported apparent health benefits from
content of chloroplast lipids. The presence of the consumption of CLA. These have included
chloroplasts in marine phytoplankton is the basic reducing the severity of cholesterol-induced aortic
building block of the Marine Food Web. They are lesions in rabbits (Lee et al., 1994), reducing the
also the crucial component of fresh forages and so, incidence of carcinogen-induced mammary tumours
not surprisingly, recent research has started to identify in mice (Ip et al., 2001) and even altering the
substantial increases in the n-3:n-6 PUFA ratio in composition of body weight gain (higher protein/
the lipids of animals fed fresh forages rather than lower fat) in mice (Park et al., 1997). Recent studies
concentrates (see Dewhurst et al., 2003). Advocates at the Rowett would suggest that at least in the CHD
of the current drive for sustainability may see this as area, one of the main mechanistic attributes of the
an important longer-term positive (and specific) CLA is their ability to modulate the inflammatory
‘health’ message to help promote the sales of pasture- mechanisms at the level of adhesion molecule
reared beef and sheep and the milk from forage-fed transcript in endothelial cells. The transcription of
dairy cows. However, linseed oil also has a very high these adhesion molecules, which lead on to plaque
content of (n-3) linolenic acid (see Figure 1) and formation, is thought to be stimulated by cytokines
Lamb Soyabean oil Fish oil
Milk Linseed oil

60
g/100 g total FA
40
20
C16:0 C18:0 C18:1 C18:2n-6 C18:3n-3 C20:5n-3 C22:6n-3
C18:3n-3 is present in
the chloroplast lipids of all
Marine phytoplankton green leafy plants.
[C18:3n-3 C20:5n-3 C22:6n-3]
Marine zooplankton
[C20:5n-3 & C22:6n-3]
Marine algae & fish oil increase EPA and DHA in
poultry and meat
Figure 1. Relative concentrations of the longer-chain fatty acids in meat, milk, plant oils and fish oils and the basis of the high n-3
FA content of fish oils.
J.C. MacRae 277
generated from the oxidation of low-density Selenium

lipoproteins (LDL) in the tissue that underlies the
endothelial cells. The CLA seem to modulate these One of the underlying mechanisms associated with
cytokine signals and so reduce the inflammatory the onset of CHD and cancer is disruption of the
response (Cook et al., 1999) in a not dissimilar way normal cellular processes. In this respect, lipid
to the n-3 PUFA. oxidation is a major stressor, because the cytotoxic
The CLA are very much an attribute of ruminant hydroperoxides formed can cause membrane damage.
products, being formed as an intermediary metabolite Intracellular free radical generation, accelerated in
in the biohydrogenation of C18:2 (unsaturated) exercise, infection and even the stress of high
linoleic acid to 18:0 (saturated) stearic acid during performance, is another stressor. To modulate these
rumen fermentation. Hence, levels of CLA in milk, processes cells depend on antioxidants such as vitamin
cheese, butter, lamb and beef (4-7 mg/g total FA) E and a number of glutathione peroxidases (GSH-
are considerably higher than in non-ruminant products Px) (Figure 2). The GSH-Px are selenoproteins, and
(chicken, pork, fish, olives; <1mg/g). Unfortunately, to date 25 have been identified in the human genome.
these levels could never provide sufficient CLA intake It is not surprising therefore that early epidemiology
per day to make any meaningful contribution in terms studies indicated clear links between the availability
of offsetting inflammation in humans; at present a of selenium (Se) in the human diet (Se levels in blood)
person would need to consume over 3.5 kg of cheese and the incidence of CHD (Salonen, 1982) and cancer
per day to take in a meaningful therapeutic dose of (Clark, 1985). Some of the selenoproteins are
CLA. Currently, attention is being focused on the important also in thyroid metabolism and in the redox
regulation of biohydrogenation in rumen microbes, control of cells; and so inadequate selenium intake
either by dietary manipulations (Loch and Bauman, has been linked with impaired thyroid metabolism,
2003), or by examination of those microbes that reduced response to viral infection, infertility and,
regulate the latter steps in this process and whether in more serious situations, cardio- and skeletal-
these could be manipulated to boost the CLA content myopathies.
of muscle and milk products (John Wallace, personal One concern for clinicians in the UK and other
communication). If this could be achieved, then it parts of Europe over the last 10-15 years has been
may represent another specific health benefit with the substantial reduction in daily Se intake that has
which to make ruminant products more attractive to
the consumer.
Membrane damage, tissue necrosis
Cytotoxic hydroperoxides
Lipids more Stressors Antioxidant
readily oxidized
Cytosol PUFA in diet, Vitamin E
Exercise, infection
Free radical
generation Exercise, infection GSH-Px 1 & 2
(O2-, OH-, etc.) high performance Cu/Zn SOD
Cytotoxic
hydroperoxides
As above Mn SOD
GSH-Px 4
Organelles
Outer membrane
Figure 2. The role of selenium (glutathione peroxidases) in the antioxidant function of cells.
occurred as a result of the switch from selenium-rich dairy cows but only 15-20% in growing beef and
high-protein North American wheat to lower-Se UK sheep. Therefore, in terms of presentation of these
and European wheat for flour making in the mid products to the health-conscious, but also cost-
1980s. The volcanic and sandy soils across major conscious consumer, it may be important to consider
sectors of the UK have had a lot of their Se washed certain value-added aspects associated with the
out, leading to low levels of Se in cereals, fruit and consumption of milk and meat proteins.
vegetables; and as a result, the daily intake of Se in
the UK population (~35 µg/d) is less than half of
that in the USA (80-100 µg/d). The question of MILK PROTEINS
whether the UK should follow the example of Finland
and New Zealand and add sodium selenate to fertilisers, These comprise two distinctly different types, whey
or Se to bread flour was raised in the late 1990s (Rayman, proteins (20%) and caseins (80%). There are several
1997), but as yet nothing has been done. different types of whey protein (the protein fraction
Selenium deficiency in farm animals has been well which associates with the whey fraction during
recognised for many years. In cattle, severe deficiency cheese-making), each with its own particular
will result in myodegenerative problems such as white importance. ß-Lactoglobulin, for example, which
muscle disease, whilst marginal deficiencies have comprises about half of the total whey protein, is a
been linked to elevated levels of mastitis, scours, cystic retinol-binding protein essential for vitamin A
ovaries and retained placenta (Villar et al., 2002). absorption. It also has a high content of cysteine,
As a result Se has been included in most mineral which can help bolster glutathione production (given
supplements for farm livestock since 1978 and adequate Se availability; see above). However, it is
therefore the Se content of animal products (e.g. meat the casein fraction that makes milk also a ready source
and poultry: 100 and 160 µg/kg respectively; FSA, of calcium, phosphorus and magnesium.
2002) and particularly liver and kidney (>400 µg/kg), This property was identified many years ago (Orr,
are considerably higher than in the plant components 1928) and subsequently led to the provision of free
of the UK diet (fruit and vegetables, <10 µg/kg; milk to school children in the UK in order to alleviate
cereals, 20-25 µg/kg; and bread made from UK and the crippling disease of rickets and accelerate growth;
European wheats, 50-55 µg/kg; see FSA, 2002). It it also provided a much needed market for this
has been estimated that animal products presently agricultural product. Recent estimates have suggested
provide over 60% (18 µg per day) of this intake. that milk and dairy products supply half of the calcium
The present reluctance of governments to implement intake for most individuals in the UK plus 25-30%
supplementation policies lies in the potential toxicity of the daily requirement of the B vitamins. Milk is
problems associated with over-consumption of also a ready source of vitamin A, D, E and K, but
selenium (>900 µg per day). However, there seems these are all fat-soluble and so their provision from
no danger of reaching these dangerous levels by milk has been reduced over recent years as public
advocating the promotion of ‘health-enhancing’ perception of the health risks of saturated fat has led
animal products, (alongside the odd Brazil nut!) to to more and more consumers switching to semi-
help replete the marginal selenium intakes that have skimmed (2% fat) and skimmed milk.
developed over the last twenty years.
MEAT PROTEIN
Value-added aspects of animal proteins In the same way that milk protein can provide a
vehicle for the delivery of calcium and phosphorus,
Whilst the nutritive value of animal proteins is
meat can provide a ready source of a number of trace
recognised to be higher than that of plant proteins,
elements, including iron. Considerable clinical
(FAO, 1970), they are also more expensive. The extra
attention has been focused recently on the problems
costs of production relate predominantly to the
associated with low iron intake in pregnancy, when
inherent inefficiencies associated with conversion of
as many as one in five women are clinically iron
dietary (plant) proteins into animal products. Even
deficient and many more suffer from marginal iron
in chickens and pigs, net protein deposition represents
deficiency. If this anaemia is not corrected there is
only 30-40% of the consumed dietary protein, but in
an increased risk of poor pregnancy outcome,
ruminants the figures are much lower, 25-30% in
premature delivery and/or low birth weight. In
J.C. MacRae 279
addition, the child has a higher risk of developing differentials in meat tenderness can be correlated with
cardiovascular disease or non-insulin dependent differences in the area and frequency of the slow
diabetes later in life. Consequently, pregnant women twitch oxidative fibre in the muscle (Maltin et al.,
with anaemia are always subscribed iron supplements. 2001) with higher myoglobin content.
However, these can cause unpleasant side effects (e.g. Meat is also a major dietary source of other
gastric upset, nausea and constipation) and so micronutrients such as zinc, selenium and copper and
compliance rates are low. the vitamins folic acid and B12,; indeed B12 is only
The basic problem with inorganic iron supplements found in animal and fish products. Each of these
is one of bioavailability. Less than 10% of ingested nutrients has been linked to the prevention of non-
inorganic iron, or indeed, the non-heme iron of most communicable diseases and to the maintenance of a
plant foodstuffs, is absorbed from the gastrointestinal healthy immune system and so it is perhaps time for
tract. In comparison, heme iron, (i.e. the iron bound the animal industry to start to consider the products
up in the porphyrin ring structures of haemoglobin that it delivers to market not just in terms of consumer
and myoglobin (see Figure 3), present in meat and (organoleptic) preference, but also in terms of specific
fish, has a much higher bioavailability (20-30%, health benefits.
Roughead and Hunt, 2000). Given the clinical
problems referred to above and the experimental
information from rodent models, linking iron Conclusions
deficiency in pregnancy and the subsequent
development of hypertension problems in the growing This paper has attempted to draw on biomedical
offspring (Gambling et al., 2003), there would appear information that might be used by the animal
to be considerable potential for promoting meat as a industries in the promotion of specific health
means of alleviating anaemia, not just during messages to support niche markets. For example, the
pregnancy, but through other periods of high iron omega-3 content of meat and milk products from
requirement such as adolescence and menstruation. pasture-fed ruminants and the selenium and iron
This could be helped by the fact that the iron-carrying carrying capacity of meat products, particularly as
capacity of different qualities of meat seems to be the latter seems to be enhanced in quality breeds such
linked to eating quality as perceived by taste panel as the Scottish Aberdeen Angus. In the future,
assessment; a relationship built on the fact that particularly if the microbiologists can find ways of
Mammalian myoglobin, haem

component
X
N N
Fe2+
Fe2+
80 N N
Y
% Fe absorption
60
Tetrapyrrole ring system of

40
proporphyrin IX
20 haem iron
non-haem iron
0
0 50 100 150 200
serum ferritin µg/l
Figure 3. Comparative bioavailability of haeme and non-haeme iron.

controlling biohydrogenation in the rumen, the CLA Hu, F.B., J.E. Manson and W.C. Willet. 2001. Types
content of ruminant products could also make a of dietary fat and risk of coronary heart disease: a
powerful ‘healthy eating’ message. However, critical review. J. Am. Coll. Nutr. 20(1):5-19.
promotion of such messages may need a refocus of Ip, C., Y. Dong, H.J. Thompson, D.E. Bauman and
research effort. Over the last five years there have M.M. Ip. 2001. Control of rat mammary epithelium
been more than 1000 research papers from studies proliferation by conjugated linoleic acid. Nutr.
that focused on the tenderness, texture, juiciness and Cancer 39(2):233-238.
flavour of meat products. Perhaps, as we go through Krauss, R.M., R.J. Deckelbaum, N. Ernst, E. Fisher,
the next five years more thought should be given to B.V. Howard and R.H. Knopp. 1996. Dietary
the nutritive value of the products placed on the guidelines for healthy American adults. A statement
market. Indeed, increasingly we could be faced with
for health professionals from the Nutrition Committee,
the realisation that ‘healthy products’ are the essence
American Heart Association. Circulation 94:1795-
of an (economically) healthy industry.
1800.
Leaf, A., J.X. Kang, Y.F. Xiao and G.E. Billman.
2003. Clinical prevention of sudden cardiac death
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C.A. Moran 283
Functional components of the cell wall of Saccharomyces cerevisiae:

applications for yeast glucan and mannan
COLM A. MORAN
Introduction
In recent years, there has been increasing relation to yeast cell wall components will emphasize
biotechnological and commercial interest in yeast cell areas where they could be used as functional
wall components, including their use as biological constituents of human food products. Central to
response modifiers, anti-cancer agents, bioadsorbents, functionality of a food ingredient is its structure and
ingredients in food processing and cosmetic composition, which will be briefly discussed.
formulations, and as systems for immobilizing oral
vaccines, antibodies and enzymes of industrial
significance (Fleet, 1999). This increase in interest Cell wall composition of Saccharomyces
has occurred concomitantly with our better cerevisiae
understanding of the chemical composition, genetic
regulation and functional properties of cell wall The cell wall comprises 15-30% of the dry weight
components. of the cell with the major components being ß(1,3)
Integrity of the cell wall of Saccharomyces glucan, ß(1,6) glucan, mannoproteins and chitin
cerevisiae is required for cell viability under various (Table 1). These components are covalently linked
environmental conditions. The principle functions of to form macromolecular complexes, which are
the wall are to prevent lysis under hyper- and hypo- assembled to form the intact wall, often likened to a
osmolarity conditions, provide shape for the cell, ‘flexible building block’ (Kollar et al., 1997). In
form a permeability barrier for macromolecules, and reality, due to the cell wall components only
participate in cell-cell recognition (Orlean, 1997; occupying 10-20% of the wall volume, a better
Estrem and Skatrud, 2001). In addition, it provides analogy is that the wall is comparable to a latticework,
a matrix for a variety of enzymes involved in rather than solid structure (Figure 1) (Lipke and
hydrolytic processes, nutritional uptake, end- Ovalle, 1998). Arrangement of the cell wall is based
metabolite secretion and cellular maintenance. The on an internal skeletal layer consisting of ß(1,3)
cell wall is a dynamic structure that can adapt to glucan molecules that form a three-dimensional
physiological changes (i.e. from logarithmic to network surrounding the entire cell. This network is
stationary phase), morphological changes (conjugation, held together by local alignments between segments
sporulation or pseudohyphal growth) or environmental of ß(1,3) glucan molecules, allowing the formation
conditions (nutrient and oxygen availability, temperature of multiple hydrogen bridges. External to the skeletal
and pH) (Orlean, 1997; Kapteyn et al., 1999; Aguilar- layer, cell wall mannoproteins are linked to the non-
Uscanga and François, 2003). reducing ends of ß(1,3) glucan molecules either
The universal adoption of the yeast cell wall and directly (Pir-CWPs) or indirectly through an
its individual components, mannan oligosaccharides interconnecting ß(1,6) glucan moiety (GPI-CWPs).
and ß-glucan, as functional ingredients in animal feed Following cytokinesis, the skeletal layer becomes
has not been transferred to the human food industry. fortified by coupling of chitin chains to non-reducing
A review of the current state of knowledge with ends of ß(1,3) glucan chains. These chitin chains are
284 Functional components of the cell wall of Saccharomyces cerevisiae
found close to the plasma membrane (Kollar et al. the ß(1,3) glucan, mannoprotein and chitin (Kollar
1997). et al., 1997). Structural studies, using solid-state
nuclear magnetic resonance on intact yeast cells, have
Table 1. Major components of S. cerevisiae cell walls (adapted determined ß(1,3) glucan to possess a helical
from Lipke and Ovalle, 1998). confirmation (Krainer et al., 1994). Such helices are
Component Mean % of cell Relative composed of a single polysaccharide chain or of three
(degree of molecular mass molar ratio hydrogen-bonded chains (a triple helix) (Figure 2).
polymerization) mass (kDa) Yeast wall mannoproteins are highly glycosylated
polypeptides, often 50-95% carbohydrate by weight,
ß(1,3) glucan 240 30-50 1.0
ß(1,6) glucan 24 10 2 that form radially extending fibrillae at the outside
Mannoprotein 100-200 25-50 1.2-2.4 of the cell wall (Lipke and Ovalle, 1998; Kapteyn et
Chitin 25 1-3 0.1-0.3 al.,1999). Many mannoproteins carry N-linked
glycans with a core structure of Man10-14GlcNAc2-
ß-Glucans are generally described as polymers of Asn structures very similar to mammalian high
glucose and form the major structural polymer in mannose N-glycan chains. ‘Outer chains’ present on
the cell wall and encompass the entire cell with a N-glycans consist of 50-200 additional α-linked
microfibrillar net (Kollar et al., 1997; Lipke and mannose units, with a long α(1,6)-linked backbone
Ovalle, 1998). Three classes of ß-glucan have been decorated with short α(1,2) and α(1,3)-linked side
described: a) alkali insoluble-acetic acid insoluble chains. Until recently the identification of proteins
ß(1,3) glucan, b) alkali soluble (ß-1,3) glucan, and in the cell wall has been hampered by the complex
c) highly branched ß(1,6) glucan (Fleet, 1991; Smits nature of the cell wall structure and its relative
et al., 1999). The alkali insoluble-acetic acid insoluble resistance to simple digestion and extraction. A novel
ß(1,3) glucan is believed to be involved in maintaining method was developed to tag and identify cell surface
wall mechanical strength and shape; and the alkali proteins using a method based on treating intact cells
soluble ß(1,3) glucan has been proposed to confer with a membrane-impermeable biotinylation reagent
flexibility in the cell wall. ß(1,6) glucan plays a central that specifically reacts with free amino groups
role in cell wall organization, interconnecting with (Casanova et al., 1992). Using this method, the
Figure 1. Structural components of the cell wall of S. cerevisiae (adapted Lipke and Ovalle, 1998).
C.A. Moran 285
Yeast glucans in food and health

GLUCANS AND IMMUNE RESPONSE
Hot water extracts from various mushrooms and tree

fungi have been used for centuries as folk remedies
in Japan, China and Eastern Russia for cancer therapy
or their general health stimulating properties (Kogan,
2000). During the 1940s in Europe, a crude yeast
cell wall extract (from S. cerevisiae) called Zymosan
was investigated for its purported drug-like activities.
Research conducted by Louis Pillemer and his
colleagues demonstrated that Zymosan could non-
specifically potentiate and modulate the immune
system, regardless of the type of invader or pathogen
Figure 2. Triple helix structural conformation of ß(1,3)
(Fitzpatrick and DiCarlo, 1964). Extensive research
glucan, which forms the backbone of the yeast cell wall.
in the 1960s related the biological activity to the
presence of the polysaccharides belonging to the class
of ß-glucans having the common structure of α(1,3)
identity of approximately 20 cell wall-associated
linked backbone with the single glucosyl units
proteins was confirmed (Mrsa et al., 1997), although
attached to the backbone through (1,6) glycosidic
following a genomic approach greater than 40 have
linkages (DiLuzio, 1983). Since these early
been predicted (Smits et al., 1999). Two distinct
discoveries there have been hundreds of papers and
classes of cell wall proteins can be distinguished, GPI
patents describing the properties of fungal ß(1,3)
(glycosylphosphatidylinositol) proteins and PIR
glucans. Yeast ß(1,3) glucans belong to a class of
(proteins with internal repeats) proteins (Kapteyn et
drugs known as ‘biological response modifiers’
al., 1999). The GPI proteins are linked to other cell
(BRMs), which modify the host’s biological response
wall components through a remnant of their GPI
by stimulation of the immune system (Sandula et al.,
anchor and ß(1,6) glucan crosslinks the proteins to
1995). By stimulating the host’s defense mechanisms
ß(1,3) glucan. An example is the α-agglutinin
against disease challenge rather than attacking the
protein. The PIR proteins are less well understood,
infective or tumor-causing agent, ß(1,3) glucan
but in contrast to the GPI proteins, are not
remains non-toxic to the cells of the host organism.
posttranslationally modified by addition of a GPI
This is often not the case with synthetic or semi-
anchor, but are highly O-glycosylated (Mrsa and
synthetic therapeutics.
Tanner, 1999). The mannoproteins determine the
The main immunopharmacological activities of ß-
surface of the yeast cell and are responsible for the
glucan include: increase of the host’s resistance to
cell’s antigenic behavior.
viral, bacterial, fungal and parasitic infections, an
Chitin, quantitatively a minor component of the
anti-tumor effect and prevention of carcinogenesis,
cell wall, is glycosidically linked to non-reducing
radioprotectivity and an adjuvant effect (DiLuzio,
branches of the ß(1,3) glucan and ß(1,6) glucan
1983; Bohn and BeMiller, 1995). The protective
(Kollar et al., 1997). Addition of chitin to ß(1,3)
action of ß(1,3) glucans has been described as non-
glucan structural units is essential for the insolubility
specific immunomodulation due to involving a
of the cell wall, by transforming alkali soluble
number of different immune pathways including
material into an alkali insoluble state. The structure
macrophage activation, T-cell stimulation,
of α-chitin is similar to α-cellulose, with hydrogen-
stimulation of reticulo-endothelial system (RES),
bonded anti-parallel chains of N-acetylglucosamine
activation of natural killer (NK) cells, activation of
units (Lipke and Ovalle, 1998). Hydrogen bonds
the classical and alternative complement pathways
involving the amide groups (absent in cellulose)
and increased antibody production. Among these,
further stabilize the crystals. Due to its low
macrophages are the best characterized target for
concentration in yeast, chitin has not been
ß(1,3) glucans (Czop, 1986). Activation of
commercially exploited.
macrophages with glucan increases their size and
number, stimulates secretion of lysozyme and TNF, (Yea-Sacc1026®, Alltech Inc.) to suppress the effects
and increases the phagocytosis of antigens (Meira et of aflatoxicosis in poultry found an improvement in
al., 1996). Macrophage activation is mediated via toll- hatchability (McDaniel, 1991), improvement in
like receptor 2 (TLR2) (Underhill et al., 1999; broiler body weight gain (Stanley et al., 1993;
Pivarcsi et al., 2003). Upon stimulation with glucan, Devegowda et al., 1995) and an enhancement in the
TLR2 is recruited to the phagosome and signals the immune response (Devegowda et al., 1995). In vitro
production of TNF-α through the NF-κB pathway. studies have confirmed the dose-dependent binding
TLR2 cooperates with the CD14 receptor in response of aflatoxin to purified yeast cell wall (Devegowda
to glucan, as suggested by the high levels of NF-κB et al., 1994). Other in vitro studies have demonstrated
observed in TLR2+/CD14+ macrophages exposed to that the cell wall of S. cerevisiae is capable of binding
the ligand compared to the levels obtained in TLR2+/ a wide range of toxins; including zearalenone
CD14- macrophages. Furthermore, TLR6 and TLR2 (66.7%), fumonisin (67.0%), DON (12.6%),
were reported to coordinate macrophage activation ochratoxin (12.5%), citrinin (18.4%), T-2 toxin
by glucan (Ozinsky et al., 2000). (33.4%), DAS (12.7%) (adapted from Devegowda
et al., 1998).
Dawson and co-workers (2001) have investigated
GLUCANS AND MYCOTOXIN ADSORPTION the use of adsorption isotherms to further understand
the mycotoxin-cell wall fraction interactions,
Mycotoxins are secondary metabolites produced by including the adsorption affinity (Kd), saturation point
fungi of various genera before or after harvest, during (Ksat) and absorption capacity (Qmax) of the material.
transportation or during storage. Some of the The use of adsorption isotherms recognizes the fact
agricultural commodities affected are cereal grains, that mycotoxin adsorption in biological systems is a
soybeans, peanuts and forage crops. It has been reversible process that can be characterized as a
estimated that there are at least 300 mycotoxins known chemical equilibrium. Consequently, adsorption is a
to induce signs of toxicity in mammalian and avian concentration-dependent process influenced by
species, with more being discovered as our analytical mycotoxin concentration, the amount of adsorbent,
capabilities develop (Devegowda et al., 1998). The and the relative affinity of the adsorbent for the
most significant mycotoxins in naturally contaminated mycotoxin. Moreover, adsorption isotherms can be
foods and feeds are aflatoxins, ochratoxins, used to compare the relative binding capacity and
zearalenone, T-2 toxin, vomitoxin and the fumonisins. affinity of different adsorbents. Using this method,
Biochemically diverse with many pharmacological Dawson and co-workers (2001), compared the
effects, these toxins can have a deleterious impact on efficiency of aflatoxin B1 adsorption by a yeast cell
animal health at extremely low concentrations. wall preparation (Mycosorb®, Alltech, Inc.) to two
Various strategies have been employed to control commercial mycotoxin clay-based adsorbents (Figure
the effects of mycotoxin contamination, including 3). Mycosorb ® was demonstrated to be a more
mycotoxin adsorption to nutritionally inert sorbents effective adsorbent at low aflatoxin concentrations,
to decrease bioavailability (Piva and Galvano, 1999). had a higher affinity for aflatoxin and possessed an
The formation of a sorbent-mycotoxin complex overall greater capacity for the toxin. Researchers
reduces the availability of the toxin to be absorbed in France have recently proposed an alternative
across the gut epithelium and results in the eventual ligand-toxin model based on Hill’s equation
excretion of the toxin. Numerous inorganic sorbent (Yiannikouris et al., 2003a). They modeled data
materials have been tested including hydrated sodium from an experiment studying the interaction between
calcium aluminosilicate (HSCAS), zeolites, zearalenone and yeast cell walls and found an
bentonites, clays and activated carbons. There are a improved fit of the experimental data compared with
number of problems associated with their inclusion an isothermal saturation curve. Furthermore, they
in the diet, namely high inclusion levels, narrow range found that the new model provided clues for the
of adsorption efficiency, select number of toxins biological interpretation of toxin-cell wall
absorbed, and risk of dioxin contamination of clay interaction, an interpretation not possible using an
materials (Devegowda et al., 1998). In recent years, adsorption isotherm model. The data showed that
research into the generation of biological products to interaction between zearalenone and the cell wall is
reduce the bioavailability of mycotoxins has gained co-operative, supporting the hypothesis that the three-
momentum. Early investigations of a live yeast culture dimensional mobility of yeast cell wall is probably
C.A. Moran 287
Mycosorb®
100
Mycosorb® Clay binder No. 1
Clay binder No. 2
90
Clay binder No. 2
% of toxin bound
80
70
Clay binder No. 1

60
50
10 100 1000 10000
Aflatoxin concentration (ppb)
Figure 3. Comparison of adsorption characteristics of two clay products to yeast glucan-based Mycosorb®.
important in the adsorption event. This finding is in physiochemical mechanisms involved in the
contrast to that found with inorganic binders that mycotoxin-glucan interaction.
display isothermal behavior (not co-operative), which The mycotoxin adsorption properties of yeast cell
can be ascribed to their rigid structure (Grant and wall and its derivatives have been confirmed in
Phillips, 1998; Yiannikouris et al., 2003a). In a numerous in vivo studies involving poultry (Swamy
separate study, Hill’s equation was used to compare and Devegowda, 1998; Raju and Devegowda, 2000;
zearalenone adsorption by four yeasts differing in Swamy et al., 2002a), swine (Trenholm et al,. 1994;
cell wall composition (glucans, mannans and chitin) Swamy et al., 2002b; Swamy et al., 2003) and cattle
(Yiannikouris et al., 2003b). Additionally, the four (Whitlow et al., 2000; Akay et al., 2003).
yeasts were fractionated to prepare three components
(total cell wall, alkali-soluble glucan, alkali-insoluble
glucan) to aid in the elucidation of the adsorption GLUCANS AND CHOLESTEROL
process and identify the yeast cell wall component
involved. The results clearly demonstrated that there Heart disease continues to be the leading cause of
were differences between yeast strains in their death in the US and developed world. Increasingly,
capacity to adsorb zearalenone and that the glucan medical experts advocate dietary changes to reduce
concentration in cell wall can explain 83.6% of the the risk of developing the disease. Ingestion of soluble
adsorption. The sigmoid shape of adsorption curves, ß-glucan has been shown to improve the pattern of
analyzed according to Hill’s model, indicated a co- lipids in humans and experimental animals with
operative association existing between zearalenone elevated serum cholesterol (hypercholesterolemia)
and yeast cell wall at low concentrations. (Bell et al., 1999). In clinical studies, the reversal of
Interestingly, there was a negative correlation between hypercholesterolemia has been demonstrated with
chitin content in the cell wall and the adsorptive dietary supplementation of oats or oat bran and
capacity of glucan, further suggesting that the three- purified ß-glucans from yeast. Oat ß-glucans are found
dimensional conformation is important in the in various breakfast cereals and snacks, and their
adsorption phenomenon. Future work is needed to promotion as a natural functional food with the
evaluate the adsorption of mycotoxins other than approval of the FDA has become a big business for
zearalenone by yeast glucan and further elucidate the the breakfast food industry. However, several servings
of oat-based foods containing greater than 0.75 g potential in improving the physical properties of food
(per serving) of ß-glucan are required to obtain the products; being used as a thickening, water-holding,
amount claimed to reduce the risk of heart disease emulsifying stabilizer or oil-binding agent
(Bell et al., 1999). A more concentrated form of ß- (Thammakiti et al., 2004).
glucan (>74%) can be obtained by extraction from
the yeast cell wall and has the potential to be added
to a greater variety of everyday foods or beverages Mannan oligosaccharides and health
as a nutraceutical.
In a ground-breaking study, the effect of yeast ß- RESPONSES IN ANIMALS
glucan on blood serum lipids in 15 obese
hyper-cholesterolemic (>240 mg/dL) men was Mannan oligosaccharides (MOS), in the form of Bio-
evaluated (Nicolosi et al., 1999). After a 3-week Mos® (Alltech Inc.) have been shown to be effective
period in which subjects ate their usual diet and their in improving the health and performance in a variety
baseline blood cholesterol levels were determined, of species. The use of Bio-Mos® as an alternative to
all men received 7.5 g of ß-glucan dissolved in orange antibiotic growth promoters in calf milk replacer was
juice twice daily for eight weeks. Blood analysis was first studied in the early 1990s (Newman et al., 1993;
conducted at weeks 7 and 8, and again at week 12 (4 Jacques and Newman, 1994). These investigators
weeks post-treatment). The results of the study observed a reduction in fecal coliforms, an
demonstrated that supplementation of the diet with improvement in weight gain and early dry feed intake.
15 g ß-glucan from yeast per day significantly lowered In addition, the incidence of respiratory infection was
total and LDL cholesterol and improved HDL greatly reduced in calves receiving Bio-Mos®. These
cholesterol by 16%. Further work needs to be findings sparked a great number of trials in different
conducted to optimize ß-glucan dose and investigate species to elucidate the mode of action and evaluate
long-term effects of supplementation on blood lipid the effect of MOS as a feed additive. Recently, a
chemistry. The eventual goal would combine ß-glucan series of independent meta-analyses on the data
supplementation with a special diet to reduce or gathered from global research studies over a 10 year
eliminate the necessity for cholesterol-lowering drugs period have been conducted to investigate the effect
(Nicolosi et al., 1999). of dietary inclusion of Bio-Mos® on the performance
of nursery pigs (Pettigrew, 2000); broilers (Hooge,
2004a) and turkeys (Hooge, 2004b). Meta-analysis
GLUCANS AS FUNCTIONAL INGREDIENTS is a powerful statistical tool that allows researchers
to compare results with a large database of different
Amid the competitiveness of the food manufacturing trials involving a particular product. With each meta-
industry, the manufacturer is always looking to analysis conducted, the reviewers arrived at the same
develop new ingredients to decrease the cost of raw conclusion - Bio-Mos® improves growth performance
materials. The modern health conscious consumer and should be recommended for inclusion in the
seeks natural and healthy foods. In stark contrast, it respective diet. The meta-analysis with weaning pigs,
is enough to look at the ingredients list on the label involving 55 comparisons (29 separate experiments
of most processed foods and see that today’s food and 21 research teams) demonstrated a 4.15%
reads like the Merck Chemical A –Z Index. This does improvement in weight gain, 2.34% improvement
not mean that the food is necessarily harmful, in feed conversion and 2.08% increased feed
however the consumer often perceives it that way. consumption (Pettigrew, 2000). In the broiler meta-
These chemical ingredients are often added to support analysis, the antibiotic control and Bio-Mos® diets
the physical properties of the food. Functional gave statistically equivalent performance with regard
properties such as fat binding capacity, emulsion to growth promotion and feed utilization (Hooge
capacity and control of foaming are of major 2004a). However, Bio-Mos® diets resulted in -17.2%
importance in the production of certain processed relative change in mortality averaging by treatment
foods (Romero and Gomez-Basauri, 2003). The use and -18.1% averaging by trial compared to antibiotic
of natural ingredients is often difficult to justify based controls. This indicated that Bio-Mos® improved
on high cost or high inclusion being needed, which (P<0.01) broiler livability compared to the antibiotics
may have a negative impact on the final product. evaluated (including avilamycin, bacitracin,
Yeast ß-glucan is one ingredient that has demonstrated bambermycin or virginiamycin at growth promoting
concentrations). Data in turkeys are similar to the
C.A. Moran 289
findings in broilers; a meta-analysis involving 27 in vitro studies that indicated differences exist in the
comparisons of turkeys fed Bio-Mos ® versus ability of different mannose-based sugars to block
antibiotic-free control diets demonstrated that on pathogen attachment (Firon et al., 1983). Firon et
average Bio-Mos® improved body weight (+2.09%, al. (1983) demonstrated that compounds containing
P=0.01) and FCR (-1.47%, P=0.17) and mortality both α(1,3) and α(1,6) branched mannan (as found
(P=0.016) relative to the non-medicated control in the outer cell wall of S. cerevisiae) had
(Hooge, 2004b). approximately 37.5 times the binding capacity for
E. coli as D-mannose. In another interesting study,
Fernandez and coworkers (2000) demonstrated a
MODE OF ACTION reduction in colonization of S. enterica serovar
enteritidis (PT4) in the ceca of young broiler chicks
The mannan and mannoproteins represent 25-50% receiving the cecal contents from hens fed Bio-Mos®
of the yeast cell wall and determine the cell surface (2.5% w/w) through the diet. When chick diets were
properties (Lipke and Ovalle, 1998), which are supplemented with the same Bio-Mos® level given
believed to be the basis of the three primary modes to the hens, an even greater protection was observed,
of action of MOS observed in animal and poultry as demonstrated by fewer salmonella-positive birds
studies with Bio-Mos®: 1) adsorption (agglutination) observed, 11/24 (46%) compared with those fed mash
of pathogenic bacteria containing Type 1 fimbriae; alone (17/24 (79%). The ability of Bio-Mos® to
2) modulation of the host immune response; and 3) interfere with the attachment of pathogenic bacteria
enhancement of intestinal integrity (Spring et al., in the gut raises the possibility that it could also inhibit
2000; Shane, 2001). the binding between bacteria that is required for
Perhaps the best-studied and most well understood plasmid transfer via conjugation (Newman, 2002).
mode of action involves the competitive blocking of Lou (1995) demonstrated that dietary Bio-Mos®
bacterial lectins. Adhesion of pathogens to the supplementation decreased the proportion of specific
epithelium surface of the gut (colonization) is groups of Gram-negative antibiotic resistant fecal
believed to be the first critical stage leading to bacteria in swine. Work continues in this area to
infection. Mannose-specific lectins (Type 1 fimbriae) confirm these earlier findings.
on the bacterial surface recognize glycoproteins (rich Numerous studies have investigated the effect of
in mannose) on the host cell surface. The control of Bio-Mos® on humoral and cell immunity. Whilst the
bacteria-mediated attachment has been proposed as a exact mechanisms have not been completely
possible means of reducing enteric infection. Oyofo elaborated, significant evidence has been accumulated
et al. (1989a) tested the effect of different sugars on to propose that Bio-Mos® plays a multi-purpose role
the adherence of Salmonella typhimurium to epithelial in immune modulation. Dietary inclusion of Bio-
cells from day-old chicks in vitro and found that Mos® has been shown to affect humoral immunity in
mannose and methyl-α-D-mannoside were the most turkeys by enhancing plasma IgG and bile IgA
efficient in inhibiting adherence. They reported that antibody levels (Savage et al., 1996). In another
mannose addition decreased the number of adherent study, with sows receiving Bio-Mos® 14 days pre-
bacterial cells to a defined intestinal surface by more farrowing and throughout lactation, higher
than 90% when compared to a control with no concentrations of colostrum IgG and IgM were
carbohydrate added. In three follow-up in vivo observed compared to the untreated sows (Newman
studies, Oyofo and coworkers (1989b) observed a and Newman, 2001). This increase in colostrum
significant protective effect from supplementing immunoglobulins was associated with the piglets
mannose (2.5% w/v) in the drinking water of chicks from supplemented sows being significantly heavier
for 10 days; salmonella-challenged control chickens at weaning. Non-specific cellular immunity has also
were 78, 82 and 93% colonized whereas salmonella- been positively influenced in studies investigating
challenged mannose-treated chickens were only 28, macrophage activity. The stimulation of phagocytosis
21 and 43% colonized, in three respective trials. In by Bio-Mos® has been demonstrated to be dose
other studies, addition of Bio-Mos® at significantly dependent in vitro (Sisak, 1995). This may be due to
lower dietary inclusion levels (0.4% w/w) vs. the 2.5% the presence of a mannose receptor (MR), which is
w/v mannose concentration used by Oyofo et al. involved in microbe recognition and phagocytosis in
(1989a,b) resulted in the successful reduction of the absence of specific opsonization and acts like a
salmonella and E. coli in the ceca of young broiler true lectin in the lectin phagocytosis of micro-
chicks (Spring et al., 2000). This confirmed earlier
organisms (Ofek et al., 1995). Mannose receptor is et al., 2001). Supplementation with the highest level
expressed on tissue macrophages, dendritic cells of Bio-Mos® resulted in longer (P<0.01) jejunal villi.
(mostly on Langerhans cells), endothelium, and rat The RNA content of the ileal mucosal homogenate
microglia. In addition to acting as a scavenger of was significantly greater (P<0.05) in chicks receiving
mannose-containing glycoconjugates on the surface 3.0 and 5.0 g/kg Bio-Mos® diet than in other groups.
of a wide spectrum of microorganisms such as E. The protein/RNA and RNA/DNA ratios in ileal
coli, Klebsiella pneumoniae and salmonella, MR homogenates were significantly (P<0.01) influenced
mediates their ingestion by macrophages (Mosser, by the presence of Bio-Mos® in the diet. This was
1994). MR is the main molecule involved in antigen not translated into increased mucosal growth or
recognition and the binding process in antigen- differences in digestive enzyme activities in the ileum.
presenting cells (Engering et al., 1997). Therefore, However, with Bio-Mos ® inclusion, there were
activation of immune cells by yeast-associated mannan significantly greater specific activities of maltase
may facilitate antigen processing and serve to (P<0.01), leucine aminopeptidase (P<0.05) and
stimulate the initial stages of the immune response. alkaline phosphatase (P<0.001) in the jejunum.
Recently, there has been some evidence to suggest Improvements in the intestinal mucosa with dietary
that MOS may suppress the pro-inflammatory supplementation of Bio-Mos® have been linked to a
immune response. Ferket (2002) induced an acute reduction in morbidity and mortality attributable to
immune response in turkey poults by intraperitoneal necrotic enteritis (Hofacre et al., 2003).
injection of LPS from S. typhimurium and measured
fever response. Poults fed a diet containing Bio-Mos®
showed no fever response compared with the control Future uses for yeast cell wall
(no additive) group, which experienced an increase
of +0.4oC in body temperature. Greater control of In recent years, glycobiology has become a rapidly
the immune response, particularly the fever response, developing sector of biotechnology, as researchers
can be beneficial to the host in terms of energy realize the potential for simple sugars and
savings, maintaining feed intake and reducing stress. polysaccharides to affect biological mechanisms. The
Further studies are necessary to understand the highly term ‘glycomics’ is the new buzzword of the
complex and diverse effects the yeast cell wall biological sciences, building upon the more
mannan oligosaccharides have on the immune system established areas of genomics and proteomics.
of the host. Glycomics research divulges the biological
There is increasing evidence that MOS modifies implications of mono- and polysaccharide interaction
the morphology and structure of the intestinal mucosa, with proteins and other cellular components to affect
although whether this is a direct or indirect (pathogen intercellular and cell-tissue interactions (Staples,
control) effect remains unclear. Early studies at 2003). The ubiquity and importance of carbohydrate
Oregon State University demonstrated a reduction interactions can be assessed by considering that 1%
in crypt depth of turkey poults fed diets containing of human genes encode enzymes that contribute to
0.1 % Bio-Mos® through 8 weeks of age in three glycosylation with several hundred glycosylation
sections of the intestine comprising the distal half of genes identified. The specificity and flexibility of
the duodenal loop; Meckel’s diverticulum and at the glycosylation is not lost on drug manufacturers (Dove,
junction of the jejunum and cecum (Savage et al., 2001). Biotechnology companies have long used yeast
1997). These changes in crypt depth were correlated to manufacture large quantities of proteins cheaply.
to a statistically significant increase in growth rate However, yeast glycosylate proteins contain large
through eight weeks of age, suggesting an inverse quantities of mannose, allowing the immune system
correlation between the parameters measured. Santin of the human or animal host to recognize the protein
and co-workers (2001) showed that inclusion of yeast as foreign, thereby removing it from circulation and
cell wall at 0.2% in broiler diets aided in intestinal rendering the protein therapeutically useless. On the
development with an increase in villus height during other hand, if the mannose in yeast glycoproteins
the first 7 days of life, and could be positively could be replaced using ‘mammalian like’ sugar
correlated with an improved body weight gain over groups, such as sialic acid, there would be less chance
the entire production period. Another detailed study of recognition by the host immune system and hence,
evaluated the response of the intestinal mucosa of an increased chance of successful delivery of the drug
broiler chickens to Bio-Mos® included in sorghum/ to its intended target.
lupin-based diets at 0.0, 1.0, 3.0 or 5.0g/kg diet (Iji
C.A. Moran 291
From a biotechnological viewpoint, the presence of In other research, simultaneous saccharification and
proteins at the outer surface of the yeast cell in direct fermentation of cellulose (ß-glucan) to ethanol was
contact with the environment is strategically attractive demonstrated using an engineered yeast strain
for drug manufacturers and the question of how expressing endoglucanase II (from Trichoderma
proteins are targeted to this particular location has reesei) and ß-glucosidase (Aspergillus aculeatus) on
been intensively studied (Winzeler et al., 1999; Jung the cell surface (Fujita et al., 2002). The novel
and Levin, 1999; Estrem and Skatrud, 2001). cellulose-degrading yeast strain could grow in a
Improvements in yeast expression systems, coupled synthetic medium containing ß-glucan as the sole
with the development of yeast surface display and carbon source and produced 16.5 g ethanol from 45 g
refinements in two-hybrid methodology, are ß-glucan within 50 hrs. The yield in terms of grams
expanding the role of yeasts in the process of of ethanol produced per gram of carbohydrate utilized
understanding and engineering eukaryotic proteins was 0.48 g/g, which corresponds to 93.3% of
(Cereghino and Cregg, 1999). Display on the yeast theoretical yield.
cell is particularly suited to engineering mammalian Even though this technology is still in its infancy,
cell-surface and secreted proteins that require the possibility to produce complex genetic
endoplasmic reticulum-specific posttranslational constructions resulting in functional glycoproteins
processing for efficient folding and activity displayed at specific sites on the yeast cell wall is a
(Schreuder et al., 1996; Boder and Wittrup, 1997; reality. Clearly, the application of this technology in
Guo et al., 2002). the area of feed, food and medicine will have a
Some of the most exciting opportunities are significant impact on our world in the near future.
emerging within research for fuel ethanol production. An example would include the ability to express
The production of ethanol directly from starchy or specific feed degrading enzymes on yeast cell walls
cellulosic materials has remained the ‘holy grail’ of that could be targeted to specific substrates in the
the fuel ethanol industry for many years (Dawson, large intestine. The immobilization in the yeast cell
2003). To date, the limitation has been the inability wall would render the enzymes resistant to
of S. cerevisiae to ferment complex sugars to produce degradation through the upper digestive tract or
ethanol. Construction of novel S. cerevisiae yeast through the rumen and allow for hydrolysis of a
expressing glucoamylase, α-amylase, carboxymethyl- specific substrate at a specific site. The potential to
cellulose and ß-glucosidase at the yeast cell surface maximize feed utilization, lower production costs and
has enormous potential for the industry to solve this reduce environmental pollution in the animal
problem (Ueda and Tanaka, 2000). In a series of production sector are of enormous economic benefit.
experiments, Ueda and Tanaka (2000) demonstrated In addition, the same yeast could be constructed with
the ability of S. cerevisiae to efficiently ferment starch multipurpose adhesion properties; the addition of
by introduction of genes encoding either a single genes for glycoproteins that could target non-type 1
heterologous enzyme or a multiple enzyme complex. fimbriae possessing pathogens or target specific
The gene encoding glucoamylase, from Rhizopus mycotoxins. The list of heterologous proteins
oryzae, along with its secretion signal peptide was expressed could be endless and the production of
fused with the gene encoding the C-terminal region ‘designer’ cell wall materials in the near future is
of α-agglutinin, from S. cerevisiae, and the entirely feasible.
construction was expressed in S. cerevisiae.
Glucoamylase was demonstrated to be on the cell
surface in its active form and anchored covalently to Conclusion
the cell wall. The engineered strain possessed the
ability to utilize starch as the sole carbon source, as Carbohydrates have long been known to be an
opposed to the inability of the parent strain. Further important dietary component, although traditionally
work improved the fermentation capability of this have been seen as energy yielding molecules and
engineered strain through introduction of a truncated structural components. Recently, studies have
fragment of the α-amylase gene from Bacillus demonstrated that non-digestible carbohydrates play
stearothermophilus. The engineered yeast co- an important role in animal production and human
displayed glucoamylase and α-amylase at the cell health. Moreover, there is a growing recognition that
surface and had a more rapid growth rate on starch non-digestible carbohydrates are more than an energy
as the sole carbon source than the yeast strain only source for the colonic microflora but play a vital
displaying glucoamylase.
role in cellular metabolism, protein structure and blood and tissue leukocytes in phagocytosis and
function, cell-to-cell communication and host metabolic-activation. Pathol. Immunopath. Res.
immunity. The yeast cell wall is a natural source of 5(3-5):286-296.
two of the most potent oligosaccharides, ß-glucan Dawson, K.A. 2003. Biorefineries: the versatile
and mannan oligosaccharide. The functional fermentation plants of the future. In: The Alcohol
properties of both components make them attractive Textbook, 4th Ed. (K.A. Jacques, T.P. Lyons and
for use in the human functional food sector. The data D.R. Kelsall, eds). Nottingham University Press,
generated in the multitude of animal health and UK, pp. 387-398.
production studies indicate that there is merit in Dawson, K.A., J. Evans and M. Kudupoje. 2001.
establishing clinical studies to investigate these Understanding the adsorption characteristics of
functional properties. A greater understanding of cell
yeast cell wall preparations associated with
wall composition and component functionality may
mycotoxin binding. In: Biotechnology in the Feed
lead to the development of new food, feed and
Industy, Proceedings of Alltech’s 17th International
pharmaceutical applications.
Symposium (K.A. Jacques and T.P. Lyons, eds).
Nottingham University Press, UK, pp. 169-181.
Devegowda, G., B.I.R. Aravind, M.G. Morton and
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J.M. Regenstein 297
Food animal agriculture: a few issues that will impact our future food supply
JOE M. REGENSTEIN
Cornell Kosher Food Initiative, Department of Food Science, Cornell University, Ithaca, New York,
USA
Introduction: ‘food’ versus ‘feed’

To most of us human food and animal feed, although such problems are usually masked by the large variety
supplying nutrients, are two very different kinds of of foods. Thus, getting the vitamin, mineral,
materials. This perception occasionally gets antioxidant, calorie and other nutrients required in
challenged when social scientists look at whether those the correct amounts and proportions into animal diets
folks with limited incomes are actually consuming is a challenge with feedstocks that in fact have
pet foods. The authenticity of this claim is often significant variation and which when processed can
challenged, but there does seem to be some truth to become even more variable. Thus, feed analysis is
it. Certainly canned pet foods are commercially often more critical than human food analysis and the
sterilized and meet the same regulations for low acid flexibility of the nutritional labeling system for
canned foods as regular human food. But the humans would simply not be tolerated by food animal
ingredients are obviously different and include ‘stuff’ producers. Would they permit a product that is labeled
that normally would not be considered human food. as 10 g/100 g protein to contain 8 g? What about 20
What many do not realize is that the rules governing g in a legally labeled product? The rules for human
what can be used as animal feed are at least as stringent food nutritional labeling permit those high variations
as those for human food. The assumption that many to exist!
people would probably make is that animals can eat Of course laboratory and farm animal research has
almost anything they like unless specifically banned provided a great deal of our understanding of nutrition
by the government such as the recent exclusion of – both for the specific needs of those animals – and
certain animal products from the feed supply because just as importantly for humans. Although our ability
of the incidence of BSE (mad cow disease). In fact, to extrapolate animal data to the human population
FDA can ban specific feed ingredients, but before is questionable and often controversial, the broad
anything can be fed to an animal, FDA must actively metabolic information that encompasses human
approve the ingredient. Thus, a whole infrastructure nutrition has been defined. Our nutritional needs are
exists for regulating animal feed. not exactly like those of animals, particularly not of
People working in animal agriculture often realize those animals that we normally use for research, but
that in reality the rules governing feed are more then again, we are not so far apart either, certainly in
stringent. This is not without a logical basis, as the a nutritional sense. Thus, when interpreting nutritional
feeding of animals is more complex because the science results, the normal scientific balancing act of
growers have total responsibility for the health and not overstating or understating the meaning of the
safety of their animals. And, of course, the variety in results is essential.
an individual animal’s diet is much less than in human
foods, where we probably have too many choices.
Deficiency or excess of any nutrient in an animal Food regulatory categories
feed becomes much more important and noticeable
when that feed is the sole nutrient source. For humans, When we examine the regulations for human food,
298 Food animal agriculture: a few issues that will impact our future food supply
we must recognize that the government puts these this application? Are all red meat products that use
foods and ingredients in many different categories. these ingredients covered, including those that were
Substances that have a long tradition of being not conceived of until after 1958? There apparently
consumed as foods are not subject to regulatory were no letters approving use of nitrates/nitrites in
oversight. The government does not purport to poultry meat in 1958. De facto, both of the
control the ‘food supply’. It focuses on the three main applications questioned above are covered by the prior
categories for traditional food ingredients: prior sanction regulations, although the USDA has chosen
sanctioned compounds, food additives, and food to define the amounts that can be used in many meat
components that are GRAS (generally recognized as and poultry products more narrowly. Is nitrite use in
safe). fish covered by prior sanction? Certainly fish have
Food additives go through the most stringent some very different chemistries than meat and poultry,
review. A great deal of evidence is required to so are not considered by FDA to be covered by the
establish both the safety and the need for a food prior sanction status of red meat and poultry.
ingredient. In the US this requires that a very The other category of ingestible materials is drugs,
complete petition be prepared for review by the FDA both over-the-counter (OTC) and prescription drugs.
in order to obtain approval. The process can be slow The hurdles that companies producing these materials
and very expensive. The burden of proof of safety must pass are again quite rigorous, and much stricter
and efficacy is totally with the petitioner. than those for food additives.
The GRAS approval process has changed in the As such, the US public was until recently pretty
past few years, but the onus remains on the petitioner well protected by the government from consuming
to prove the safety of the ingredient. However, in foods that were potentially dangerous. The FDA
the case of GRAS approval, the petitioner has more simply (in reality not so simple) was responsible for
access to the consensus process that occurs within reviewing data submitted that clearly proved that the
the scientific community. In recent years the FDA product was safe to place on the market.
has encouraged more companies to ‘self-certify’ an
ingredient as being GRAS. After the required
literature review a petitioning company or DIETARY SUPPLEMENTS
organization can submit a GRAS petition to the FDA,
who then have a short time in which to review the However, there is one type of material whose status
document. Unlike the Food Additive Petition process, has always been a bit confused; and the laws
if the FDA fails to respond in a timely manner, the regulating its use are not clearly covered by any of
current regulations give the GRAS petitioner de facto the above categories. This category is dietary
permission to proceed. One problem with all of the supplements. The range of available dietary
FDA procedures for foods is that a company, trade supplements is enormous, but the two issues of safety
association or other petitioner must have a strong and efficacy have never been resolved legally for
enough economic incentive to undertake the process. these products. In this wide range of products some
Thus a generic or more common material may not are clearly above reproach, but others are marginal
be worth enough to any one group to commit the from safety or efficacy standpoints. Another issue is
financial resources needed to satisfy requirements for that of potency – is the amount promised on the
FDA clearance. package actually delivered in the product?
The prior sanctioned approach to new ingredients The government has relatively recently chosen to
is basically a limited ‘grandfather’ clause that was regulate these materials; and has created an
created in 1958. It permits use in foods of materials infrastructure through the Dietary Supplement Health
for which there exists a letter from an appropriate and Education Act of 1994 (DSHEA). This act
federal agency (FDA or USDA FSIS) indicating that establishes labeling standards, including the creation
the material/process was approved by the government of a supplement nutritional labeling information panel
and can continue to be used. The only issue that that parallels the nutritional labeling panel for regular
occasionally must be clarified is how broadly is the foods. The details are similar, but must be studied
‘prior sanction’ defined? For example, nitrates/nitrites carefully in order to produce a ‘legal’ label. In
were clearly approved by the government for use in addition, DSHEA and its subsequent regulations detail
‘meats’, but was poultry also considered meat for the nature of claims that can be made, and the full
J.M. Regenstein 299
implementation of regulations for label claims is still – who could at worst find something wrong with the
in process (see below). Probably more important with product, publish it, and put FDA on the alert.
respect to consumer interest is the issue of who has Research, therefore, continues to lag at a time when
the burden of proof of safety. For dietary scientific understanding of these materials could help
supplements, unlike any other material in the food both people and animals. Despite this, study in the
supply, the law places the burden of proving the food industry will continue as companies attempt to
material unsafe on the FDA. The recent banning of move the most promising compounds into the
ephedra highlights the problems that appeared as a mainstream of foods. For many of these products,
result. Companies can sell dietary supplements as long this trend is already apparent; and FDA has responded
as the labeling, efficacy and marketing claim rules by further defining how such products can be
are obeyed – and if there are safety or health problems marketed. In some cases the FDA has precluded
with these materials, then the FDA will have to take marketing a product as a dietary supplement when
action. Given the large number of compounds that the average consumer would consider it a direct analog
might need investigating, this is a serious challenge/ to a regular food for which such ingredients are not
hardship on the FDA; and given relatively limited normally a component. Thus a ‘drink’ could be a
resources, the FDA can realistically only take action dietary supplement, but margarine with anti-
on the most dangerous of materials, and this may be cholesterol properties must be sold as a food.
a very slow process. This is essentially what happened The regulation of dietary claims will continue to
with ephedra. It is finally being withdrawn from the develop as regulatory agencies try to strike the right
market, but long after people died or became ill. balance. Currently working its way through the
Neutraceuticals and functional food ingredients are regulatory system is a plan to permit claims that do
essentially dietary supplements added to the food not meet the current high standard of significant
supply in different ways. These can be considered as scientific consensus. The FDA is trying to develop a
bioactive compounds with known (or supposed) system where claims based on less scientific consensus
biological or general physiological functions that go can be used, as long as the wording makes it clear
beyond simply providing essential nutrients. Many what consensus such claims have. The issue currently
of these compounds come from other cultures where is to determine the wording allowed for each type of
they are used in lieu of western medicine. Many have claim.
been used for years, possibly centuries. Those who Eventually the feed industry may be able to
study these compounds, either scientifically or incorporate these functional materials into animal
pragmatically, often claim remarkable properties for feed. Hopefully the additional process of getting
them. Although it would be easy as scientists to ignore materials approved for feed use once approved for
these compounds (as many scientists have and human use will be fairly straightforward, although
continue to do), others clearly feel strongly about we need to recognize that the dose fed to animals
their positive effects. As a result, many felt the need might be higher and the potential for residues in edible
to prevent the government from prohibiting their use. tissues will always make the approval process for
This lack of faith in the traditional establishment, food animals more complicated.
which includes the FDA, is clearly reflected in the Thus, the full benefit of using such materials
DSHEA law. This development is actually most remains to be developed. Thankfully companies like
unfortunate, as it suggests that these compounds could Alltech recognize the importance of this type of
not survive proper scientific testing. Many of these research for the potential benefit of the feed industry
materials have real functionality, i.e., they really are (and more recently for humans also) and have
neutraceuticals. But, on the other hand, a few are supported this type of research. Besides using the
probably outright dangerous, and a few at best have traditional peer-reviewed publication route, they have
no effect. The incentive to study them carefully is also taken a leadership role in making sure these
however lacking, although growth in consumer findings are widely reported. These meetings
interest has certainly increased the scientific sponsored by the company bring together people from
community’s involvement. With the government many different industries within the scientific
having the major responsibility for proving safety of community to review and discuss work in these
these compounds, the nutraceutical industry has a new areas.
disincentive to work with the academic community
Animal welfare
Another change occurring in animal agriculture that established that permitted producers to self-certify.
will influence the nature and future of functional In one case a program involving trained educators
foods centers on animal welfare issues. As the nature visiting the farm has been planned.
of the animal ‘experience’ changes in production The FMI/NCCR committee has been using these
agriculture, the benefits or detrimental impact of guidelines as a starting point for their work. These
various functional ingredients will change. Improved documents have been critically reviewed by a group
animal welfare reduces stress by improving housing representing a different range of interests than the
and environmental rearing conditions. Thus, those industry’s internal committies. In most cases, the FMI/
functional compounds that focus on remediation of NCCR accepted the industry standards, but in a few
excesses of current practice may not be necessary. cases found adjustments were needed. These were
On the other hand, the improved conditions may also first negotiated with the trade associations and in many
allow benefits of certain functional ingredients to be cases the two groups came to agreement and the
more fully expressed. These compounds may then industry modified its standards. This negotiating
also be used to further improve the productivity of process is ongoing.
animal agriculture in a world that is currently trying The next step is to develop an audit program, which
to feed over 6 billion people and may need to feed is outlined in Figure 1. First, the guidelines need to
over 9 billion before the century is out. be translated into audit documents. This is not an
easy task because the standards must be both
quantitative and clear. They must also be very specific
AUDIT PROGRAM so that auditors can be trained to give nearly identical
results. For this part of the program, the FMI/NCCR
What has happened on the animal welfare front? As hired an outside auditing firm, which translated the
we learned two years ago at this meeting, from Bob approved guidelines into audit documents and is now
Langert from McDonald’s, the three biggest fast food training the auditors, supervising the audits, and
chains have begun paying attention to animal welfare collecting the data. They are preparing summaries
issues. However, the supermarkets and other chain of the audit data, maintaining the data base, and
restaurants realized this was leading to establishment keeping the audit program operating. The first sets
of a number of alternative standards. Since adopting of auditors are being trained and the first audits are
a single standard would be more efficient, the Food being scheduled.
Marketing Institute (FMI) (the trade association A few important specifics need to be considered.
representing the supermarket industry) and the First, the audit firm is training ‘outside’ auditors.
National Council of Chain Restaurants (NCCR) The auditors are independent agents; and can be from
formed a scientific animal welfare committee to other auditing firms or from government agencies.
examine this important issue. The approach taken Auditors pay a training fee, take the course, and pass
was to work with the main trade associations a subsequent written test; after which they start work
representing the major commodities (beef, dairy, with an already established auditor. Auditor
pork, chicken, eggs, and turkeys), postponing others prerequisites have been established. For on-farm audits
such as fish. In each case, the major trade association the person must have an appropriate scientific college
representing that industry sector was selected as the degree (e.g., biology, animal science, etc.), while for
lead agency. Only where dairy is concerned, where the slaughterhouse audits, the person must have an
the Milk and Dairy Beef Quality Center was selected, animal science degree.
is there any question of the association being the Independent auditors bid for each ‘job’. A fee to
leading trade association for that industry. cover the infrastructure is included. The facility being
In many cases these trade associations already had audited selects an auditor and makes arrangements
or were working on animal welfare guidelines, which for the auditor’s visit. The auditors will be instructed
had resulted in guidelines established by a scientific both by the FMI/NCCR program and by the
or a scientific/industry team. The limitation with individual facility with respect to biosecurity
respect to this effort for most of the trade associations, procedures.
however, was that these guidelines are voluntary and Before undertaking a paid audit with the FMI/
non-enforceable. In a few cases, programs were NCCR program, it is strongly suggested that producers
J.M. Regenstein 301
Self assessment
(Trade association assistance)
Audit requested
Auditors notified, bids submitted

Company selects auditor
Auditor visits site

(sometimes with reviewers)
Audit Report Submitted
Appeals Board decides

Audit adjusted or not Client rejects report,
Client accepts report appeals
Changes made where needed,

Verification materials submitted
or re-audit required & conducted
Audit report posted
Audit report amended
• Client allows buyers access
for use in purchasing decisions
• Audit expires
• New audits conducted
Figure 1. General structure of an auditing program,
use the documents and guidelines of their own trade of the process, the facility will have a right of appeal
association to do a thorough self-assessment. Once and a procedure for handling appeals is being
confident of in-house performance it is then time to established.
bring in the formal auditing team.
In order to maintain consistency and skill level Who owns the data obtained by the audit?
among auditors, it is expected that a reviewer hired
by the audit firm will accompany the auditor on The data is the property of the facility audited,
approximately one out of each ten audits to evaluate regardless of who pays for the audit. The facility
the work of the auditor. To further ensure the integrity management/owner can then share the data as they
choose. Individual company data are only available Alternative and organic agriculture
to a few appropriate people in the audit firm. Only
summary data will be made available to FMI/NCCR Alternative and sustainable agriculture is becoming
or the animal welfare scientific committee. a more integral part of the US Agricultural and Land
Grant system. With this heightened interest and the
How will retailers use the data? changing demographics in the US, many commodities
are being raised and processed in new ways. For
The data will be organized so that major and minor example there is renewed interest in sheep and goat
non-compliance will be clearly indicated. rearing, which leads to new issues regarding slaughter
Retailers can then determine their own purchasing in small facilities and on-farm slaughter for the ethnic
requirements based on their standards. There will and religious markets. As part of the Cornell Sheep
NOT be a single ‘pass/fail’ standard. Each company and Goat Program and as part of a Northeast sheep
must make its own decisions. The only audit ‘failures’ initiative funded by USDA, we have been involved
will be based on gross animal abuse, in which case in efforts to improve the humane slaughter procedures
the auditor is expected to terminate the audit and for sheep and goats. This work has had three major
leave the facility immediately. thrusts. Firstly, to identify a knife appropriate for
use in religious slaughter. Secondly, we have designed
Can non-compliance issues be resolved before the a slaughter pen for small-scale slaughter of sheep
next full audit? and goats that meets modern humane animal handling
standards. Lastly, we have developed a poster that
Procedures will be put in place for correcting both
describes humane (halal) slaughter and makes
major and minor non-compliance. For most minor
suggestions on both animal handling and slaughter
non-compliance, the assumption has been made that
along with a reminder to deal with the offal in a
the facility can submit appropriate paperwork to prove
socially acceptable way, suggesting composting as a
that corrections have been made. For major non-
method most in keeping with the need for an easy
compliance the procedures will vary. Sometimes
waste management system that is consistent with
photos, paperwork and other forms of submitted
modern requirements. This poster is currently
material will be sufficient to establish that correction
available in English and Persian and will be translated
has been made, but in some cases the facility will
into Arabic, Urdu, and Spanish.
need to be re-inspected.
Although alternative agriculture does not
How often a facility should be inspected is an issue
necessarily imply organic cultural methods, certainly
still being addressed. Certainly slaughter facilities are
organic is an important subset. This grass-roots
likely to be inspected every year. Large production
movement has gone in new directions in the past 10
facilities are also likely to be inspected every year.
years, and has come under the umbrella of
On the other hand, cow-calf operations and other
government standards in the US and other countries.
small, widely dispersed operations will require less
Basically the definition of organic, and what is and
frequent inspections and the procedures for these are
is not allowed as organic or in organic products, has
still being discussed. It is possible that some sort of
been defined by the government, supposedly in
statistically based sampling regime may be necessary
conjunction with the ‘public’. However in many cases
to keep the process cost-effective.
the additional paperwork burden and costs of
participation in the government program will change
Religious slaughter
the nature of who produces organic products. The
Specific standards covering both kosher and halal facilities need to be large enough to justify the
facilities have been written and are included in the additional effort and costs. The rules are admittedly
program. Currently, these standards are based on the (the government’s own admission) stricter than all
work of Dr. Grandin and support the material in the previous rules, including those of the states that
American Meat Institute guidelines, which have been previously regulated organic labeling. In addition,
established for over 12 years. However, it is clear the political nature of the process resulted in the US
that some kosher slaughter cannot meet these standards government precluding many of the potentially
for religious reasons, and so a reconsideration of these beneficial technologies that would permit organic
needs will take place when these standards can be agriculture to flourish. By eliminating genetically
clarified and appropriate humane handling systems modified organisms and irradiation, the organic
are in place.
J.M. Regenstein 303
industry in the future will be limited in the crops

they can grow and how well they can distribute
organically-produced animal and vegetable products.
Some current data have shown that in many cases a
decrease in pesticide usage (the original emphasis of
organic farming) and the use of more benign
pesticides is possible with genetically modified crops.
However organic producers are precluded from using
GMOs. As part of the new regulations, we are seeing
organic farming become big business and we are
seeing big business take over organics – but is this
what the core organic consumer really wants? Only
time will tell how this will all settle out.
R.P. Sharma 305
Mycotoxins in the food chain: a look at their impact on immunological

responses
RAGHUBIR P. SHARMA
Department of Physiology and Pharmacology, The University of Georgia, Athens, Georgia, USA
Introduction
The immune system is an important defensive or chemicals. Some lymphocytes are natural killer
mechanism against invading parasitic organisms or cells that generally require no priming or proliferation
foreign cells. The system is highly evolved in to exert their effects. On the other hand, the
mammals and birds. In general, its complexity generalized immune system usually confers acquired
correlates with the evolutionary level of various immunity after the organism has been in contact with
animal species. In higher organisms, the system infectious organisms or other antigenic determinants.
consists of specialized cells found throughout the Lymphocytes and macrophages are cellular units of
body; these cells are localized in large quantities in both types of immune systems. The two major forms
certain organs such as the thymus, spleen, and lymph of lymphocytes, T cells and B cells, differentiate in
nodes. Cells of the immune system and cells of the the thymus and fetal liver, respectively. The T cells
hemopoietic system all originate in the bone marrow. are involved in cell-mediated immune responses, such
The bone marrow stem cells later differentiate to as delayed hypersensitivity reaction and immune
perform specialized immunologic functions. surveillance against foreign or altered cells. Several
Immunotoxicology is a relatively new discipline, subpopulations of T cells exist, e.g., cytotoxic T cells,
although the allergic responses to various chemicals helper T cells, and suppressor T cells. The B cells
have long been recognized. Chemicals, including are primarily involved in the production of a variety
mycotoxins, can either suppress or stimulate the of antibodies; however, these cells are often under
immune system. Immunosuppression likely decreases the control of T cells. The T cells interact with one
resistance to a variety of infectious diseases and may another or with other cells of the immune system via
even predispose the host to the expression and a variety of soluble factors called cytokines. In some
dispersion of cancerous cells. Stimulation of the instances direct cell-cell interactions are necessary to
immune system is not always desirable because it mount the optimal immune responses.
may lead to hypersensitivity (allergic) reactions. The Macrophages are derived from monocytes and are
mycotoxin-induced immunotoxicity has been present in various body cavities, such as pulmonary
reviewed earlier in detail (Bondy and Pestka, 2000; alveoli and peritoneum; they are also found in the
Sharma, 1985; 1991; 1993). lymph nodes and liver, as components of the innate
immune system. Other peripheral leukocytes are often
involved in various immunopathologic mechanisms.
Complexities of the immune system For acquired immunity macrophages are phagocytes
that concentrate antigens and confer specific
The immune system of mammals is highly complex, immunologic responses to various T or B cells and
and various cells of this system interact to produce also remove cell debris.
the desired effect. The immune system includes the The immune system interacts with other systems
innate immune functions that are inherent in different and is profoundly influenced by the central nervous
organs and do not require pre-exposure to antigens system, both directly via innervations of lymphatic
306 Mycotoxins and immune response
organs and indirectly via neuroendocrine various regulations limiting the exposure to
mechanisms. The cells of the immune system produce mycotoxins are in effect. On the other hand in
factors that influence the nervous system. Hormones, developing countries, where food supplies are
such as somatotrophin (growth hormone) and inadequate and storage facilities are not yet optimal,
thymosin (thymic maturation factor), stimulate the there is a greater chance for food spoilage and
immune responses, whereas steroids, including sex contamination by toxigenic molds. Even in developed
hormones, generally suppress the immune responses. countries; however, problems with mycotoxins
frequently occur in livestock health.
Mycotoxins and public health Table 1. Common mycotoxins contaminating foodsa
Mycotoxin Commodity Associated fungi

Mycotoxins are metabolites produced by certain fungi
that infest food crops and processed foods. The first Aflatoxins Peanuts, pistachios and Aspergillus flavus
recorded episode of mycotoxicosis in public health other nuts, corn, A. parasiticus
was recognized in the middle of the 19th century cottonseed, cereals
when ergotism was shown to be produced by ingestion Fumonisins Corn, other cereals Fusarium verticillioides
of rye infected with Claviceps purpurea. In the first F. proliferatum
half of the 20th century, the ‘moldy bread disease’ Ochratoxin Legumes, cereals, Aspergillus ochraceus
coffee beans Penicillium verrucosum
or alimentary toxic aleukia (ALA) was associated
with over-wintered wheat or grain in the former Patulin Apples, grapes, Penicillium expansum
Soviet Union infected with various Fusaria species. other fruits Aspergillus giganteus
The interest and intensive research in the problems other Penicillium and
Aspergillus spp.
with mycotoxins started with the incidence of Turkey-
X disease in England, where the peanut meal used in Trichothecenes Wheat, corn Fusarium tricinctum
F. poae, and other
poultry rations was found to be contaminated with Fusaria and several
aflatoxins produced by Aspergillus flavus. other species
Since the 1950s extensive research in the field of
a
mycotoxins has provided information on a number Only common mycotoxins and representative fungi and
commodities associated with them are indicated.
of fungal products that are commonly associated with
foods. The most common mycotoxins found in
various foods are aflatoxins, fumonisins and Prevalence of mycotoxins in foods
trichothecenes, with occasional contamination with
other toxins. Commonly encountered mycotoxins Mycotoxins are inadvertent contaminants in foods as
associated with various food commodities are listed the fungi that produce them are fairly widespread.
in Table 1. Although the molds that produce these In spite of a large amount of research available for
toxins are ubiquitous, the problem in foods can be various mycotoxins, information regarding their
easily managed by avoiding the fungal contamination occurrence in foods is limited. In many cases, the
and not consuming moldy or spoiled foods. Toxigenic information is limited to periods when episodes of
fungi do not always produce mycotoxins. Their adverse human or animal health effects have been
production is often aided by certain temperatures and observed. Only a few of the mycotoxins are routinely
humidity and stressful conditions to the fungi. analyzed in foods during surveys. The US Department
Avoiding storage of foods at conditions favorable to of Agriculture and Food and Drug Administration
mycotoxin production is highly valuable in have established limits for various mycotoxins in
controlling the toxic levels of these agents. foods and feeds (Food and Drug administration,
Additionally, development of fungus-resistant crops 2004). Still, food commodities sometimes exceed the
has been somewhat useful in preventing the growth allowed limits and may in fact be of concern in public
of toxigenic fungi. health. Table 2 lists the levels of selected mycotoxins
In public health, the greater problem with associated with different food commodities and their
mycotoxins is in developing countries rather than in action levels established by US Food and Drug
the developed countries. In industrialized countries Administration. Additional information and primary
the food resources are plentiful, handling and sources of mycotoxin levels in foods are available
preservation technology is well developed, and from the recent reviews or other publications
R.P. Sharma 307
(Campbell et al., 2003; Council for Agricultural Common health problems with
Science and Technology, 2003; Dutten, 1996; Gilbert mycotoxins in foods
and Vargas, 2003; Harwig et al., 1973; Osborne,
1980; Shepard, 2003; Shotwell et al., 1971; Wilson AFLATOXINS
and Nuovo, 1973).
One of the public health concerns with the presence Metabolic products of Aspergillus flavus and A.
of mycotoxins in foods is that these toxins are parasiticus occur in foods in a number of susceptible
relatively stable; once they are formed they can persist commodities. Peanuts, corn, other nuts and grains
for a long time. Only a few of them are partially are possible food commodities that may be
destroyed by cooking, pasteurization, or storage. The contaminated. Aflatoxin B1 is the most potent and
concentration of a mycotoxin may indeed increase prevalent of this group. Aflatoxin B1 is activated in
during the fermentation process or storage if the vivo by metabolizing enzymes to an epoxide, which
toxigenic fungi are present in food. The mere is very reactive and binds to various biological
presence of fungi however is not always indicative molecules, including specific bases in the DNA. After
of the occurrence of mycotoxins, since the organisms dietary exposure aflatoxin B1 is metabolized in the
produce these toxic metabolites only under certain liver, the organ with a high level of metabolizing
conditions that are determined by temperature, enzymes, and produces damage to this organ,
humidity or a lack of normal nutrients for the mold. including hepatocarcinogenesis. Indeed in some
The occurrence of mycotoxins in agricultural species, especially the rainbow trout, aflatoxin B1 is
commodities depends on factors such as geographical the most potent carcinogen known. In the short term,
region, season, and the conditions under which a aflatoxin B1 causes necrosis in the liver and possibly
certain crop is grown, harvested and stored. There damage to other organs such as kidney, heart, spleen
are no definite rules for if and when a mold will and pancreas. In animals exposed to aflatoxin B1
produce a mycotoxin. The fungal spoilage of crops decreased productivity and high mortality due to
and grains may be enhanced by drought, insect infections are often the consequence.
damage, cracked kernels during harvesting, and Several episodes of aflatoxin B 1 poisoning in
presence of excessive chaff in the harvested grain. humans have been reported. These include both
Table 2. Mycotoxin levelsa encountered in foods and their FDA action levels1.
Mycotoxin Commodity Common levels Levels with toxic

(µg/kg) episodes (µg/kg) FDA action levels
Aflatoxins Peanuts 2-6 30-125 20 ppb (µg/kg) in foods,

Peanut butter 10 14-213 0.5 ppb aflatoxin M1 in milk
Peanut candies 20
Corn >10 30-230
Fumonisins Corn products 1-12 >20,000 2 ppm in degermed dry milled corn
Corn (from various countries) 30-2,000 3 ppm in corn for popcorn
4 ppm in whole of partially degermed
corn products, corn bran and masa
Ochratoxin A Corn, barley, wheat <3 >25 (pork kidney) No action level set
210-2,900 in bread 3800 (barley in Czech
flour (lumps) Republic)
Patulin Apple juice 9-146 1,000 50 ppb (µg/kg) in apple juice or food
containing apple juice as an ingredient
Trichothecenes Wheat flour 170-400 38,000 1 ppm for deoxynivalenol in finished

(DON) Cornmeal 100-400 wheat products
Popcorn 84,000 (imports)
Bread 80
a
Levels are only from some representative reports. Wide ranges in concentrations have been reported, particularly in contaminated samples.
1
FDA, 2004.
general effects of this mycotoxin and carcinogenesis. example, dry milling of corn kernels results in bran,
Symptoms of suspected outbreaks of poisoning include flaking grits, grits, meal and flour. Fumonisins are
jaundice, rapidly developing ascites, partial found in greatest quantity in the bran fraction,
hypotension and death. In many parts of the world followed in order by flour, meal, grits and flaking
where hepatocellular carcinoma is prevalent, a high grits (lowest). This happens because fumonisins are
level of aflatoxin in the diet has been documented. concentrated in the hull and germ of corn kernel
However, many epidemiological surveys suggest the and milling fractions may contain different amounts
concurrent presence of hepatitis B virus and other of these parts. Degermed corn usually contains lower
mycotoxins such as fumonisins that may be concentration of fumonisins than whole corn.
contributory factors in addition to the aflatoxin B1
(see later).
OCHRATOXIN
FUMONISINS Ochratoxin A is a nephrotoxic fungal metabolite

produced by certain species of Aspergillus and
Fumonisins are toxins produced by Fusarium Penicillium that mainly contaminate cereals like
verticillioides and other Fusarium species commonly corn, barley, wheat and oats. This mycotoxin, along
found on corn (Riley et al., 2001). Fumonisin B1 is with another nephrotoxic mycotoxin, citrinin, was
the most toxic and prevalent of these mycotoxins; its implicated in Balkan endemic nephropathy that
presence has been demonstrated in cornmeal, breakfast affected thousands of people in the middle of the
cereals and corn tortillas. Fumonisins have produced 20th century in Eastern Europe. The disease was
fatal diseases in livestock, including equine characterized by anemia, tubular proteinuria and
leukoencephalomalacia (ELEM, a rapidly developing hematuria. This toxin has been shown to be
brain degeneration) and porcine pulmonary edema carcinogenic in rats and mice.
(PPE or hydrothorax, lethal in a few days). An Toxicity to ochratoxin A has been reported in pigs
association of the presence of the fungus producing after feeding grain with concentrations of this toxin
fumonisins and the presence of mycotoxins themselves as low as 0.2 ppm. Ochratoxin A is a potent inhibitor
has been reported with human esophageal cancer and of protein synthesis and hence is immunosuppressant.
primary liver cancer. The latter has been implicated Its teratogenic and mutagenic potential has also been
when fumonisins are co-contaminants of foodstuffs with shown in laboratory animals.
aflatoxins. Recently, carcinogenicity of fumonisin B1
has been demonstrated in laboratory animals, including
mice (hepatoma in females) and rats (renal carcinoma PATULIN
in males).
The species and gender specificity of fumonisin B1 This mycotoxin is produced by Penicillium,
is puzzling and the basis for it has not been discerned. Aspergillus and Byssochylamys molds that generally
Fumonisin B1 is a structural analog of one of the grow on apples. It can occur in significant amounts
primary sphingolipid bases, sphinganine, and thereby in apple juice or apple products. Although no major
competes for the incorporation of sphinganine into human or animal episodes have been recorded,
ceramide and ultimately in complex sphingolipids patulins are toxic in animal feeding studies. The toxin
(Riley et al., 1998, 2001). A rise in cellular levels of is stable even after pasteurization, cooking or storage.
sphinganine and sphingosine (dehydrosphinganine, Its presence has been reported in other food
another important free sphingoid base that is also a commodities, including bread, legumes, pecans,
known signaling agent) is a uniform observation in various fruits (including apricots, pears, grapes, etc.),
all tissues exposed to this mycotoxin. These free fruit juices and cheese. It was once considered a
sphingolipid bases (sphingosine and sphinganine) as potential antibiotic; however, it was abandoned due
well as their phosphate derivations are major signaling to lack of effectiveness and possible toxic
molecules that often possess paradoxical effects, consequences. It produces gastric irritation, nausea
characterized by either death or survival signals and vomiting upon ingestion. It is uniformly toxic
in cells. in all mammalian species tested. The biochemical
The occurrence of fumonisins also depends on the effects of patulin in cells include effects on
fraction of corn produced during processing. For mitochondrial respiration ultimately causing
inhibition of electron transport systems.
R.P. Sharma 309
Hazards to patulin exposure can easily be avoided by The symptoms of trichothecene toxicity include
using only tree-picked fruits. Apples or other fruits damage to skin or mucous membranes shortly after
that are damaged or rotten may not be used for contact. Weakness, dizziness and incoordination may
making juice or other food products. The Food and ensue shortly after the exposure. These symptoms
Drug Administration established an action level of are followed by bloody diarrhea, difficulty in
50 µg/kg (50 parts per billion) in apple juice or any breathing and bleeding from lungs or mucous
other foods that contain apple juice as one of the membranes after exposure to aerosol containing
ingredients. This action level is based on single trichothecenes. In livestock after a low level exposure,
strength apple juice, one which is not concentrated, loss of appetite and decreased productivity are often
or the single strength apple juice component of the reported.
food, if the food contains apple juice as an ingredient. Trichothecenes act by interfering with the ribosomes
that are important in protein synthesis. Since protein
synthesis is vital for many functions, including that
TRICHOTHECENES, INCLUDING of the immune system, the toxic manifestations of
DEOXYNIVALENOL (DON) these mycotoxins are of importance in rapidly
dividing cells and tissues. Damage to liver, spleen
One of the so-called trichothecenes, deoxynivalenol and other lymph nodes is often observed in cases of
(DON, also commonly known as vomitoxin), is DON or other trichothecene poisonings.
produced by molds of the genus Fusarium. F.
graminearum is a common contaminant of grains
including wheat, corn, barley and rye. Another major Mechanisms involved in immunotoxicity
trichothecene, T-2 toxin, was implicated in the
outbreak of alimentary toxic aleukia (ATA) that killed Little information exists on how various mycotoxins
thousands of people in post-World War II Soviet produce immunotoxicity. Some mycotoxins, such as
Union. Trichothecene mycotoxins were also involved aflatoxin Bl and fusarium T-2 toxin, inhibit protein
in the ‘red-mold disease’ intoxication in Japan. synthesis and cell proliferation (Table 3). This
Contamination of grains with trichothecenes has been inhibition may not be the primary mechanism involved
reported in the United States and Canada.
Table 3. Toxic effects of common mycotoxins and relationship to immune responses.a
Mycotoxin Species Toxic effects Effects on immune system
Aflatoxins Humans, all other Hepatotoxicity, bile duct hyperplasia, Reduced lymphoproliferation, delayed
mammals, birds, fish intestinal and renal hemorrhage, liver hypersensitivity, phagocytosis, antibody
tumors formation against T-dependent antigens,
increased infections
Fumonisins Human, pig, horse, Pulmonary edema in swine, leukoence- Decreased lymphocyte blastogenesis;
mouse, rat, phalomalacia in horses, liver and kidney decreased antibody titers after antigens or
damage, esophageal and hepatic cancer vaccines. Decreased splenic and thymic
in humans (?) cellularity and interleukin-2 production (in
female mice)
Ochratoxin A Human, swine, dog, Nephrotoxicity, enteritis, liver damage, Decreased phagocytosis, cellular depletion of
duckling, chicken, rat teratogenesis, renal carcinogenesis lymphoid organs, transient immuno-
stimulation
Patulin Birds, mammals Lung hemorrhage, capillary damage, Reduced DNA synthesis in lymphocytes,
(cat, rabbit, cattle) convulsions, brain edema, carcinogenesis decreased peripheral leukocytes, altered
(mammals and birds) distribution of lymphocyte subpopulations
Trichothecenes Human, pig, cattle, chicken, Vomiting, diarrhea, bleeding, dyspnea, Increased infectivity, decreased lymphoprolif-
horse, rat, mouse, dog itching, rash, blisters, leukopenia eration and antibody formation, decreased
macrophage cytokine production
a
Only selected symptoms and effects on immunological responses have been indicated. Many mycotoxins are also likely to stimulate
production of specific antibodies.
in their immunotoxic effects; both have selective binding of the resulting epoxide derivative of
effects on various subpopulations of lymphocytes. aflatoxin B 1 , thereby interfering with cell
Several mycotoxins are cytotoxic to lymphocytes in proliferation and protein synthesis.
vitro, perhaps because of their effects on membranes In humans, the investigations dealing with aflatoxin
(including those involving lymphocytic receptors) or B1 and impaired immune function are limited. In some
interference with macromolecular synthesis and parts of the world the exposure to this mycotoxin
function. Cytochalasins (mycotoxins isolated from can be considerably high. Exposure to aflatoxin B1
moldy rice) are highly cytotoxic and act on was reported to be high in Thailand, Swaziland, and
cytokinesis (perhaps by binding to the filamentous Mozambique (45, 43, and 222 ng/kg/day,
actin), but their immunotoxic potential has not been respectively). In Mozambique the mortality due to
ascertained. hepatoma in hepatoma B virus-antigen carriers was
Mycotoxins can indirectly influence the twice that observed in other countries. The actual
immunologic functions. Some of the compounds are risk of similar carriers is the same throughout the
neurotoxic or cause other organ pathology, and these world. It was proposed that since Mozambique had a
compounds may activate the endocrine mechanisms. high average intake of aflatoxin, the resulting loss of
The stress-induced release of corticosteroids inhibits immunoprotection may account for higher viral-
immune functions. Fusarium T -2 toxin, which acts induced hepatoma incidence (Lutwick, 1979). A
via such mechanisms, is discussed later. similar increase in primary hepatic carcinoma was
Some mycotoxins or their metabolites may be suggested in certain provinces of China when people
highly reactive in mammals and may bind to or were exposed to high levels of aflatoxin B 1 and
destroy tissues. The immune system can also respond fumonisin B1 (Ueno et al. 1997); the involvement of
to altered proteins or to other biological molecules immune system in this carcinogenesis is uncertain as
formed by binding with reactive chemicals, although both of these mycotoxins are complete carcinogens.
no experimental evidence exists of this mechanism There have been limited studies on the effect of
involving mycotoxins. Antibodies against mycotoxins aflatoxin B1 on cytokine production. The expression
conjugated with proteins have been produced and are of macrophage-derived cytokines, namely interleukin
utilized for analyses for mycotoxins using (IL)-1α, IL-6 and tumor necrosis factor α (TNFα),
immunoassays. was suppressed in mitogen-stimulated macrophages
The influence of exogenous chemicals on immune derived from aflatoxin B1 exposed mice (Dugyala
responses may be highly variable and a mycotoxin and Sharma, 1996). This report suggested that the
may increase, decrease, or fail to affect the response, effect of aflatoxin B1 was greater on macrophages
depending on the testing protocol and dose. than on other types of immune cells. Macrophages
as scavengers may engulf large amounts of aflatoxin-
bound macromolecules and thereby are selectively
Impact of mycotoxins on immune sensitive to the toxin.
responses
AFLATOXINS FUMONISINS
Immunomodulation by aflatoxin B 1 has been There have been a number of investigations involving
investigated in detail. A comprehensive description fumonisin B1 and immune responses (reviewed by
may be found elsewhere (Bondy and Pestka, 2000; Bondy and Pestka, 2000). Fumonisin B1 generally
Sharma, 1991). In most species, resistance to inhibited lymphocyte blastogenesis in cells obtained
infection is reduced by simultaneous exposure to from exposed mammals; poultry was relatively
aflatoxin B1. Effects of aflatoxin B1 are primarily on resistant. Inconsistent effects of fumonisin on immune
the cell-mediated immune functions; however, T cell- functions have been reported. Decreased antibody
dependent humoral responses are also adversely formation to injected antigens has been reported in
affected (Reddy et al., 1987; Reddy and Sharma, turkeys, pigs, calves or even rodents; however, the
1989). Generally the humoral responses that are T levels of fumonisins used in these studies have been
cell-independent are not affected by low doses of fairly large.
aflatoxins. The immunosuppressive effects of Much of the studies in laboratory animals and cell
aflatoxin B1 can be explained on the basis of DNA lines have been inconclusive with regards to
R.P. Sharma 311
fumonisin-induced immunomodulation. The results lymphocytes, diminished serum immunoglobulin

depend on the protocol of testing and sequence of concentrations, decreased delayed hypersensitivity
exposure to toxin and antigens. In a recent report it responses and increased neutrophil numbers were
was observed that fumonisin B1 altered immune reported. The observed changes in cellular phenotype
functions in female BALB/c mice, but the males were distribution of immune cells may or may not indicate
totally refractory (Johnson and Sharma, 2001). It has ultimate effects on immune functions. It is suggested
been established that fumonisin B 1 causes that a limited human exposure to foods contaminated
accumulation of free sphingoid bases, sphinganine with patulin may be of little consequence for the
and sphingosine, by interfering with their conversion immunologic functions.
to ceramide (Riley et al., 2001) and somehow
interrupts the cell cycle (Johnson et al., 2003). The
free sphingoid bases and their phosphates are TRICHOTHECENE MYCOTOXINS
important signaling agents in cells, the bases and their
phosphates usually having opposite outcomes for cell The prevalent trichothecenes, T-2 toxin, deoxynivalenol
survival. Cellular signaling is critical in mounting (DON), oxynivalenol, and diacetoxyscirpenol, have
immune responses; indeed fumonisin B1 has been used been evaluated for their immunologic effects to a
as a tool to define the role of ceramide in signaling great extent (reviewed by Sharma and Kim, 1991).
in immunocompetent cells (lymphocytes). Sphingoid This group of mycotoxins was important for
signaling in various physiological processes is immunological investigations as T-2 toxin was
becoming relevant and should be further investigated. implicated in the onset of alimentary toxic aleukia in
The possible involvement of immunological the Russian population. Leukopenia is a consistent
responses in the pathological outcome after fumonisin observation after trichothecene exposures. T-2 toxin
B1 treatment is discussed later in this report. is perhaps the only mycotoxin with known
immunologic dysfunction in humans.
Exposure to T-2 toxin and deoxynivanol results in
OCHRATOXINS severe depletion of T cells. In routine immuno-
toxicological testing in rodents, both increase and
Ochratoxin A, a nephrotoxicant widely encountered decrease of immunologic functions have been reported
as a food contaminant, has been investigated for its depending on the protocol employed. T-2 toxin was
immunologic effects. The biochemical mechanism immunosuppressive in murine in vivo and in vitro
of ochratoxin poisoning involves interference with models (Taylor et al., 1985; 1987). Although it is
macromolecular synthesis, increased lipid presumed that trichothecene mycotoxins are
peroxidation and diminished mitochondrial immunotoxic, the mechanism of their action is not
respiration. This mycotoxin is fairly cytotoxic and well understood. These mycotoxins are inhibitors of
causes atrophy of gastrointestinal lymph nodes after protein synthesis; however, the high susceptibility of
oral ingestion (reviewed by Bondy and Pestka, 2000). T cell function cannot be totally explained on this
Systemic investigations suggest that exposure to basis. The T-independent responses are generally less
ochratoxin A results in the inhibition of cellular, sensitive to T-2 toxin effects. Oral exposure to T-2
humoral or innate immune responses; however, many toxin in mice results in inflammatory lesions in the
of these effects are produced at exposure levels that forestomach (Taylor et al., 1989), leading to systemic
are also nephrotoxic. The immunotoxic effects have endotoxemia. These effects were associated with
been demonstrated in poultry, pigs, rats and mice. In increased hypothalamic catecholamine levels and
some studies immunostimulation, perhaps as a increased peripheral corticosterone concentrations,
consequence of tissue damage, was also reported. thereby implicating a possible role of hypothalamic-
pituitary-adrenal axis in the resulting immuno-
suppressive responses.
PATULIN Deoxynivalenol and T-2 toxin have been
investigated for their effects on cytokine production
Patulin has been investigated with regard to the by immunocompetent cells. However, effects were
immune system only to a limited extent (Llewellyn not consistent. T-2 toxin caused increased expression
et al., 1998). It is less toxic than other mycotoxins; and production of IL-2, IL-3 and interferon γ in
however, effects such as altered number of splenic T splenocytes from T-2 treated mice, but a decrease in
IL-1α, TNFα and IL-6 was noticed (Dugyala and Miscellaneous mycotoxins in food
Sharma, 1997). Deoxynivalenol, however, caused an
increase in TNFα production by macrophages There are a number of other mycotoxins that can
(Sugita-Konishi and Pestka, 2001; Yang and Pestka, occur in certain circumstances as food contaminants.
2002). Many of these are toxic and also influence the
The deoxynivalenol-induced nephropathy in mice immunologic functions. These include citrinin (a
can be demonstrated by passive injection of nephrotoxic mycotoxin), rubratoxins (that cause liver
deoxynivalenol-induced IgA monoclonal antibodies and kidney toxicity and diffuse hemorrhage),
(Yan et al., 1998). The findings imply that increased macrocyclic trichothecenes (a large family of
systemic IgA after deoxynivalenol may be implicated mycotoxins with varied toxicity), secalonic acid (an
in pathogenesis of this mycotoxin in kidney, antineoplastic but teratogenic toxin), cytochalasins
suggesting the involvement of an immune mechanism (mycotoxins that bind to actin and also inhibit hexose
in the ultimate pathologic outcome to this mycotoxin. transport in cells), ergotoxins (potent
vasoconstrictors), penicillic acid, etc. Few or
conflicting reports of these miscellaneous mycotoxins
Immune mechanisms in fumonisin B1- with respect to their effects on immune functions are
induced murine hepatotoxicity found in literature. Because of low probability of
their occurrence in foods at present, these are not
We have recently investigated the role of immune discussed here.
pathways in the induction of hepatotoxicity in mice
after treatment of animals with fumonisin B 1.
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Y. Xiong et al. 315
Antioxidant activity of hydrolyzed whey, soy, and yeast proteins

YOULING L. XIONG,1 LIN WANG,1 E. AIDA PEÑA-RAMOS,2 AND CHANGZHENG WANG3
1
Department of Animal Sciences, University of Kentucky, Lexington, Kentucky, USA
2
Animal Derived Food Department, CIAD, Hermosillo, Sonora, Mexico
3
Human Nutrition Program, Kentucky State University, Frankfort, Kentucky, USA
Introduction
Proteins as functional ingredients are widely used in of food products. Among these reported antioxidant
the food and animal feed industries to improve product proteins are hydrolyzed egg white albumin (Tsuge
physical attributes and to enhance nutritional value. et al, 1991), casein (Lim and Shipe, 1972; Rival et
Proteins from a variety of sources, such as soy, whey, al., 2001), capelin meat (Amarowicz and Shahidi,
yeast, and animal by-products, have also been 1997), elastin (Hattori et al., 1998), gelatin (Kim et
subjected to enzymatic hydrolysis to further enhance al., 2001), and myofibrillar protein (Saiga et al.,
their general functional performance or to impart 2003).
specific functionalities in prepared foods (Adler- The antioxidant capability of hydrolyzed proteins,
Nissen, 1986). Studies have shown improved in addition to their other physicochemical
functional properties, including emulsification, functionalities, offers potential for expanded
gelatinization, foaming, and water-binding capacity application in the food industry. In light of the
of proteins after they are partially hydrolyzed either increasing consumer demand for ‘natural’ food
by acids or by enzymes (Kinsella, 1976; Feeney and ingredients and at the same time, for high product
Whitaker, 1977). The improvement has been quality and palatability, protein hydrolysates offer
attributed to increased exposure of reactive amino an attractive alternative to other natural antioxidants
acid side chains as well as to the production of reactive that have the exclusive role of inhibiting oxidative
short peptides. processes.
While the benefit of hydrolyzed proteins (i.e.,
peptides or peptide mixtures) as texture-modifying
agents in food is well established, it was not fully Preparation and characterization of
recognized until recently that certain hydrolyzed protein hydrolysates
proteins or peptide fractions also have biological
functions, notably antioxidant activity. The
antioxidative effect of protein hydrolysates was Protein hydrolysates can be manufactured by either
initially observed by Doan and Miller (1940), who acid treatment or enzyme hydrolysis. The latter has
showed that trypsin-treated milk had improved the advantage of producing specific peptides or a
oxidative stability. Bishov et al. (1967) subsequently mixture of different peptides with a particular
presented the first report on antioxidant activity in functionality. Enzymatic hydrolysis is also more
an acid hydrolysate of soybean proteins. Later studies controllable than acid hydrolysis and does not
by Lee et al. (1980), Chen et al. (1995), and more destruct labile amino acids (Adler-Nissen, 1993). In
recently, Pena and Xiong (2001; 2002; 2003), also developing antioxidative peptides from whey (WPI)
revealed antioxidant activity of soy and whey protein and soy (SPI) protein isolates, we employed three
hydrolysates prepared by enzymatic digestion. In commercial crude proteases – Protease ‘A’
addition to soy and milk proteins, hydrolysates of (endoproteinase from Bacillus licheniformis),
other proteins have been found to exert inhibitory Protease ‘P’ (Bacillus protease complex), and Protease
effects on lipid oxidation and to extend the shelf life ‘F’ (endoprotease and exopeptidase from Aspergillus
316 Antioxidant activity of hydrolyzed whey, soy and yeast proteins
oryzae), and four pure enzymes – pepsin, papain, (IgG, serum albumin, etc.) vanished entirely.
trypsin, and chymotrypsin (Pena and Xiong, 2001, Hydrolytic products included numerous small
2002). By varying hydrolytic conditions (pH, peptides (mostly <6 kDa). Native whey protein
temperature, time, and substrate heat pretreatment), components were somewhat resistant to hydrolysis
a range of peptides varying in molecular weight, by Proteases P and F; however, with heat treatment,
hydrophobicity and solubility were produced. all whey proteins became highly susceptible substrates.
Of the three crude enzymes used, Protease A was For hydrolysis with pure enzymes, native ß-
found to be the most effective in degrading whey lactoglobulin was resistant to pepsin but became more
proteins as determined by SDS-PAGE (Figure 1). ß- susceptible after heat treatment. All the protein
Lactoglobulin, the predominant whey protein components in preheated whey samples were highly
component, was almost completely hydrolyzed after susceptible to papain, trypsin and chymotrypsin, with
30 min regardless of preheating. Within the same most degraded into fragments with molecular weights
time period, α−lactoalbumin was also mostly less than 1 kDa (Pena and Xiong, 2001).
degraded, while the higher molecular weight proteins
Native Heated
200
116
66
Protease 45
A 31
21
β-Lg
14
α-La
6
Native Heated
200
116
66
45
Protease 31
P
21 β-Lg
14 α-La
6
Native Heated
200
116
66
45
Protease 31
F
21 β-Lg
14 α-La
6
MW 0 0.5 1 6 0 0.5 1 6
Hydrolysis time (hr)
Figure 1. SDS-polyacrylamide gel electrophoresis of whey protein isolates treated with Proteases A, P and F. Molecular weight
(MW) standards shown are in kDa. ß-Lg: ß-lactoglobulin; α−La: α−lactalbumin. (Adapted from Pena and Xiong, 2001).
Y. Xiong et al. 317
Size-exclusion chromatography was performed to Table 1. Amino acid profile of yeast extracts.
separate hydrolyzed proteins into peptide fractions
Amino acid %a %b
based on their molecular weights. These fractions
were divided, based largely on their chromatographic Lysine 3.84 8.13
profiles, into three for control (nonhydrolyzed), four Alanine 3.32 7.03
for Protease A, and five for both Proteases P and F, Arginine 2.66 5.63
Aspartic acid/asparagine 5.01 10.60
with the sizes of the fractions ranging from 1.5 kDa Cysteine 0.72 1.52
to 66 kDa. Amino acid analysis showed a varying Glutamic acid/glutamine 8.89 18.81
distribution of the 20 common amino acids in the Glycine 2.37 5.02
different peptide fractions. Histidine 0.95 2.01
Leucine 3.19 6.75
As to soy proteins, both conglycinin (7S) and Isoleucine 2.24 4.74
glycinin (11S) and their subunits were susceptible to Methionine 0.82 1.74
the three crude enzymes. Hydrolysis with Protease Phenylalanine 1.78 3.77
A yielded peptides that fell into two molecular mass Proline 2.03 4.30
Threonine 2.11 4.47
groups: 60-90 kDa and less than 15 kDa (Pena and Serine 2.12 4.49
Xiong, 2002). The degree of hydrolysis increased Tyrosine 1.69 3.58
slightly with incubation time. On the other hand, Valine 2.93 6.20
hydrolysis with Proteases P and F produced a range Tryptophan 0.59 1.25
47.26 100.0
of peptides that were widely distributed (15-100 kDa).
The concentration of free amino groups in hydrolyzed a
Yeast extract dry weight basis (w/w).
b
soy protein ranged from 1.3 µmol/mg for Protease Amino acid basis (w/w).
P treatment to 5.1 µmol/mg for Protease F. Native,
unhydrolyzed soy protein contained 0.7 µmol free antioxidant activities (suppression of TBARS
amines per mg of protein. formation), depending on the enzymes involved and
Yeast protein extracts were prepared by autolysis whether or not the substrate (WPI) was preheated.
of components of a yeast strain obtained from Alltech Hydrolysis with Protease A and the four pure enzymes
Inc. During the autolytic process, endogenous (pepsin, papain, trypsin, chymotrypsin) did not
enzymes break down yeast cell proteins into peptides significantly change the antioxidant activity. However,
and amino acids, and nucleic acids into nucleotides hydrolysis of WPI by Protease P produced
(Romero and Gomez-Basuri, 2003). These pronounced antioxidant activity, which increased with
derivatives are normally used as flavor enhancers. hydrolysis time up to 6 hrs where a 32% inhibition
Electrophoretic analysis of the yeast extract with a (P<0.05) was observed (Figure 2). For Protease F
10% polyacrylamide gel detected no distinct protein treatment, a 39% reduction (P<0.05) in TBARS was
bands at >10 kDa, but the biuret measurement showed found for heated WPI that was hydrolyzed for 1 hr.
26.4% reactive peptides, suggesting that the Both the antioxidant protein hydrolysates were
hydrolytic product consisted of savory short peptides formulated (2%) into cooked pork patties before
(<2 kDa), some of which may be di-, tri- and storage, and the analysis of conjugated dienes
oligopeptides, along with free amino acids. The amino (hydroperoxides) showed a significant inhibition (up
acid composition of the yeast hydrolysates is shown to 44%) of hydroperoxides during storage (Pena and
in Table 1. Xiong, 2003). Except for the Protease P treatment,
no relationship (P>0.05) can be established between
degree of hydrolysis and antioxidant activity.
Antioxidant activity of protein Furthermore, although the heat regimen slightly
hydrolysates and peptides increased degree of hydrolysis, it did not consistently
impact antioxidant activity of the protein
WHEY PROTEINS hydrolysates.
Nonhydrolyzed WPI was slightly inhibitory of lipid For comparison, the antioxidant properties of six
oxidation, according to the TBARS assay commercial WPI hydrolysates were also analyzed.
(thiobarbituric acid-reactive substances) in a Although the procedures and conditions for preparing
liposomal oxidizing system (Pena and Xiong, 2001). the hydrolysates (e.g., proteases used) were not known,
Upon hydrolysis, the WPI samples exhibited various all the samples (8-26% degree of hydrolysis) showed
antioxidant activity. This was demonstrated by 5-24%
80 Nonheated WPI, Protease P Heated WPI, Protease F

70 Heated SPI, Chymotrypsin Heated SPI, Protease F
60
50
Inhibition (%)
40
30
20
10
0
0 1 2 3 4 5 6 7
Hydrolysis time (hrs)
Figure 2. Antioxidant effect of hydrolyzed whey (WPI) and soy (SPI) protein isolates. Antioxidant activity was expressed as
percent inhibition of TBARS formation in a liposomal assay system.
inhibition of TBARS, compared to the nonhydrolyzed these amino acids (or residues on polypeptides) to
control or the control that contained no whey proteins free radical attack seems to stem from the low energy
(Pena and Xiong, 2001). involved in the process. The reaction, in which amino
The results suggest that antioxidant activity of WPI acids are being oxidized in place of unsaturated lipids,
hydrolysates was probably inherent to the would in effect stabilize or neutralize the radicals
characteristic amino acid sequences of the peptides, and consequently, inhibit propagation of lipid
which were a function of the enzyme specificity oxidation. Østdal et al. (1999) showed that protein
(Browdy and Harris, 1997). Indeed, the antioxidant or peptide radicals tended to be remarkably stable
activity assay of the peptide fractions, prepared by and can live for a long time, e.g., >30 minutes.
size exclusion chromatography from the protein
hydrolysates that exhibited strong antioxidant Table 2. Correlation coefficient (r) between TBARS inhibition and
capability, provided compelling evidence that the amino acid concentration and peptide size of hydrolyzed whey
protein.
antioxidant power was not equally distributed in
different peptide fractions. The percent inhibition of Amino acid Correlation coefficienta
TBARS in the liposomal oxidizing system by
peptides varied from merely 8% to more than 50%. Lysine 0.894**
Alanine 0.537*
To further elucidate the nature of the antioxidant Arginine 0.583*
effect by the protein hydrolysates, TBARS inhibition Aspartic acid/asparagine 0.159
(%) was correlated with the amino acid composition Glutamic acid/glutamine 0.305
and average peptide size from each of the Glycine –0.004
Histidine 0.830**
antioxidative peptide fractions. The results showed Leucine 0.778**
that TBARS inhibition was positively correlated Isoleucine 0.825**
(P<0.05 or 0.01) with the serine, histidine, arginine, Phenylalanine 0.406
threonine, alanine, valine, lysine, isoleucine, and Proline 0.314
Threonine 0.709*
leucine content, but was overall inversely related to Serine 0.666*
the peptide size (Table 2). Tyrosine 0.325
The data were in general agreement with previous Valine 0.916
findings that certain amino acids, including most of Peptide size –0.772**
those shown in Table 2, can be antioxidative a
Significance level: *P<0.05, **P<0.001
(Marcuse, 1960; Karel et al., 1966). Vulnerability of
Y. Xiong et al. 319
Despite their antioxidant activity, none of the WPI lipid oxidation and retarding rancidity odor
hydrolysates was as effective as the propyl gallate development in ground beef during storage. It has
treatment (0.01%) that inhibited TBARS formation been shown that most soy protein isolates contained
by 85%. The results were not surprising because residual antioxidative phenolic compounds, typically
protein hydrolysates could contain both antioxidative in the 1-1.5 mg/g range, including isoflavonoids that
and pro-oxidative peptides and amino acids prepared are capable of quenching free radicals (Seo and Morr,
under certain specific hydrolytic conditions (Karel 1984). These presumable phenolics ostensibly
et al., 1966; Adler-Nissen, 1993; Chen et al., 1996). contributed to the observed antioxidant activity of
Presumably, the antioxidant effects of some peptides native SPI. For nonheated SPI, hydrolysis with
and amino acids were offset by other peptides and Protease F elicited a less antioxidative effect when
amino acids that acted as pro-oxidants. compared with heated SPI. In fact, hydrolysis of native
SPI by chymotrypsin weakened its antioxidant
potential. A similar result was obtained with papain
SOY PROTEINS hydrolysis. The type of proteases chosen and the
conditions of protein hydrolysis (substrate, time) are
All the SPI hydrolysates exhibited antioxidant ostensibly some of the most critical factors
activities, as indicated by the TBARS assay in a determining the antioxidant activity of soy protein
liposomal oxidative model system (Pena and Xiong, hydrolysates.
2002). The hydrolysates prepared from heated SPI
with chymotrypsin and Protease F were particularly YEAST PROTEIN EXTRACTS
effective in inhibiting lipid oxidation (Figure 2), e.g.,
suppressing TBARS by 59-64% by samples that were The antioxidant activity of two Alltech yeast extracts
hydrolyzed for 1 hr. When formulated into cooked was evaluated using both the FRAP assay (ferric
pork patties, both protein hydrolysates (2%) inhibited reducing/antioxidant power), as described by Benzie
hydroperoxide production by 50% (Pena and Xiong, and Strain (1996), and the TBARS measurement
2003). Comparison of hydrolyzed WPI and SPI (Sinnhuber and Yu, 1977). The FRAP analysis was
samples indicated an overall stronger antioxidant done with dilute yeast extract solutions, while the
activity for the SPI hydrolysates. TBARS analysis was conducted on cooked beef
It was of interest to note that intact SPI (no enzyme patties. The FRAP value of yeast extract 2 was higher
treatment), whether or not heated, already possessed (P<0.05) than that of yeast extract 1 at every
substantial antioxidant power (reducing TBARS by concentration level (Figure 3). Since yeast extracts 1
36%). Wu and Brewer (1994) also reported that and 2 have an identical amino acid composition (Table
unhydrolyzed soy protein was capable of inhibiting 1), the difference in FRAP values may be attributed
1500
Yeast 1 Yeast 2
1400
1300
FRAP values (µmol/g)
1200
1100
1000
900
800
700
600
0 10 20 30 40 50 60
Yeast extract (mg/mL)
Figure 3. FRAP values (obtained at 4-min assay) of yeast extracts at different concentrations.
to the peptide composition and perhaps also the (Table 2), were also high in the yeast extracts. The
nucleotides that probably differed between the two overall antioxidant power of both yeast extracts may
yeast extracts. Both yeast extracts were able to inhibit be a combined effect of the multiple active
lipid oxidation in cooked beef patties during components present in them, i.e., the individual amino
refrigerated storage, and the inhibition increased acids, the profile of the peptides, and possibly also
(P<0.05) with the level of extract addition (Figure nonprotein substances such as nucleotides.
4). At the end of storage (7 days), TBARS values in
samples with 1.5% yeast extract 1 and yeast extract
2 were 20.2% and 17.3%, respectively, less than the Possible antioxidant mechanism
control. Except for the level of 0.1%, all
concentration levels within each yeast extract group The ability of protein hydrolysates to inhibit
were able to suppress TBARS production after 3 days deleterious changes caused by lipid oxidation appears
(P<0.05). The storage study revealed no significant to be related to certain amino acid residues and the
difference between the two yeast extract samples. specific amino acid sequence of hydrolytic peptides.
The antioxidant power of the yeast extracts is Amino acids such as methionine, histidine, lysine,
presumably related to the presence of antioxidative tryptophan, and proline have previously been shown
amino acids that may be free or in a bound form. to act as antioxidative agents although in certain
For example, histidine, a widely reported particular cases they may be pro-oxidative (Marcuse,
antioxidative amino acid (Taylor and Richards, 1980; 1960; Karel et al., 1966; Jung et al., 1995; Chen et
Chen et al., 1996; Wade and Tucker, 2001), accounted al., 1996). Correlation analysis in the present study
for 0.95% (w/w) of the yeast extract weight or 2.01% indicated a positive role played also by leucine,
of total amino acid weight (Table 1). It may have a isoleucine, valine and threonine in inhibiting lipid
role in the overall antioxidant activity of the yeast oxidation. The study by Taylor and Richardson (1980)
extract samples. Furthermore, the sulfur-containing with a linoleate emulsion-hemoglobin oxidizing
amino acids cysteine and methionine, comprising system concluded that among the common amino
1.52% and 1.74%, respectively, of total amino acid acids, cysteine was the one that clearly displayed an
weight, were expected to contribute to the reducing antioxidant efficacy. The antioxidative effects of
power of the yeast protein samples. It is worth noting small peptides in oil or metal-catalyzed liposomal
that the contents of essential amino acids lysine suspensions have been demonstrated (Yamashoji and
(8.13%), leucine (6.75%), isoleucine (4.74%), and Kajimoto, 1980). In particular, carnosine and anserine,
valine (6.20%), which were responsible for much of histidine-containing antioxidative dipeptides, were
the antioxidative activity of whey protein hydrolysates capable of sequestering metal ions and scavenging
3.0 3.0
(A) (B)
2.5 2.5
TBARS (mg/kg)
TBARS (mg/kg)
2.0 2.0
Control Control
1.5 0.10% 1.5 0.10%
0.50% 0.50%
1.0 1.00% 1.0 1.00%
1.50% 1.50%
0.5 0.5
0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
Storage time (day) Storage time (day)
Figure 4. Inhibition of lipid oxidation by yeast extracts 1 (A) and 2 (B) in cooked beef patties during refrigerated storage.
Y. Xiong et al. 321
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Equine topics
EQUINE
M. Bojer 325
A novel, knowledge-based concept for performance diagnosis and training

adjustment in horses
MATTHIAS BOJER
Institut für Natursport und Ökologie, Abteilung Reitsport, Deutsche Sporthochschule Köln,
Germany
Introduction and rationale

When comparing the scientific approaches and efforts endurance, speed, power and coordination of
that are currently applied to improve performance movements are doubtless of general interest. In the
capability in all fields of human athletic competition, beginning it was hard to find the acceptance and thus
it is more than obvious that scientific approaches to enough horses to generate statistically sound data.
improve performance, training conditions or analyse Meanwhile we had the chance to start our scientific
training status of horses are dramatically ignored, research in cooperation with one of the top athletes
even by top athletes in the equestrian sport. We are of the German International Jumping Rider team, so
missing knowledge-based concepts to monitor equine the data presented have been in most cases obtained
fitness and strategies to improve performance with horses that perform in jumping events.
capabilities based on a defined and testable training
program. According to statements of top athletes, not
even the performance of our top international Exercise program to improve endurance
competitors is analysed by scientific methods during
an important sport event. This is partly due to the Our first approach was to create a training program
fact that most scientific research on horses focuses to improve endurance in horses, as endurance is the
on medical aspects. Nevertheless, it is hard to basis for all disciplines. We took advantage of
understand why in this day and age the equestrian experience from human sports. In human sports a
sport relies on traditional methods, with coach and so-called step program is the method of choice, where
rider relying alone on ‘gut feelings’ and experience. the athlete is challenged in step 1 by a time-limited
A couple of years ago, the Equestrian Research exercise (usually a run) under controlled conditions
and Study research group at the German Sports (i.e. a given speed) and has a defined resting period
University of Cologne decided to transfer the methods (walking). The athlete would then go through more
known from traditional human sport disciplines to cycles (steps 2 – x) under conditions of increasing
equestrian sports. The focus was first on the human intensity (i.e. higher speed), while the resting time is
part, the rider. But of course success in equestrian kept constant. Usually this two-step program is
sport events does not exclusively depend on the repeated four times. The performance of the athlete
capability of the rider. It is the performance of the is monitored by measuring heart rate and lactic acid
‘companion’ horse that is in most cases essential for production as described below, and the program is
success. In this respect, the focus of our research has designed in a way that matches individual abilities.
changed. We have started to develop new discipline- The design of this program, especially the challenge
specific methods of performance diagnosis and step, is usually the very sensitive part. If the athlete
training programs for the equestrian sport, with the is not sufficiently challenged, the program is useless,
horse as the centre of interest. This novel concept however overtaxing is devastating, but will be
and the analytical methods are the main focus of this revealed by physiological measurements such that the
paper. The general idea is to create concepts for all program can be adapted.
disciplines in equestrian sports, since factors such as
326 A novel, knowledge-based concept for performance diagnosis and training
Our step program for horses is as follows: Before rate increases. If the athlete is well-trained, this heart
the test begins, the resting heart rate and the resting rate drops when he returns to moderate movement
lactic acid concentration are determined. Horses are or rest. The rate of decrease in heart rate has been
allowed to warm up before the first step is started. shown to reflect a good training condition in humans.
Step 1 includes a gallop with a speed of 325 meters/ Thus our first approach was to establish a means
min for 5 min. A short rest (1-2 min) follows and of continuously recording heart rate of horses during
the first aliquot of capillary blood is taken. During exercise. As in humans, electrodes were fixed on the
Step 2, the speed is increased by 50 meters/min (i.e. body of the horse and linked to a recorder enabling
to 375 meters/minute), but the challenge time is kept us to monitor this physiological parameter during
at 5 min. Two more steps are performed, each time exercise. Figure 1 shows the simple experimental set-
increasing the speed by 50 meters/min and keeping up and a recording that was done during a regular
the challenge time constant. It should be emphasized horseback riding lesson. Heart rate reached top values
at this point, that the speed of the first step and the of 120-130 beats/min during the lesson and when
number of steps strongly depends on the endurance walking the heart rate came to steady-state levels
capability of the respective horse, and must be within 5 min. When challenging horses in the step
adjusted individually. program, heart rate values of up to 180 beats/minutes
and more were observed (Figure 2).
Maximal heart rates are certainly individual
ESTABLISHING PHYSIOLOGICAL characteristics. Even in horses we observed heart rates
MEASUREMENTS THAT DESCRIBE up to 200 beats/min and more during Grand Prix
ENDURANCE Jumping events, so they cannot be considered the
only marker of fitness.
Performance during an athletic competition certainly Lactic acid accumulation is another marker that is
depends on a discipline-specific endurance. The generally applied to assay exercise and endurance in
endurance factor is important in dressage and jumping humans (Hollmann and Hettinger 1990). The
and gains further importance in cross-country or long biochemical explanation is that during exercise,
distance racing events. Despite the differing demands needed energy is provided by two metabolic
of these events, general performance parameters can principles, which will be considered in brief.
be established to monitor endurance in each discipline. The general energy source, glucose, is metabolised
With a glance at what has been learned from human to pyruvate by a pathway called glycolysis. This 11-
athletes performing in gymnastics, heart rate and step pathway creates two molecules of ATP, the
lactic acid accumulation in the blood should be the energy currency of the body (which can be spent for
parameters of choice. It can be considered as a general energy-consuming work such as muscle contraction),
rule in sports physiology that during exercise the heart and one reduction equivalent. It is important to note
(A) (B)
200
180
160
Heart rate (bpm)
140
120
100
80
60
40
20
00:00 00:10 00:20 00:30
Time (hh:mm)
Figure 1. (A) Electrodes were fixed with tapes to the body of a horse and connected to the recorder at the rider’s wrist.
(B) Recorded time course of the heart rate of a horse during a regular riding lesson.
M. Bojer 327
200
180
160
Heart rate (bpm)
140
120
100
80
60
40
20
00:00 00:10 00:20 00:30 00:40
Time (hh:mm)
Figure 2. Time course of the heart rate of a horse challenged by our step test.
that these reactions will occur without the presence ATP-energy-producing respiratory chain is not able
of oxygen, thus they are called the anaerobic reactions to provide enough ATP for the muscle cell. Thus,
of glucose metabolism. Pyruvate is then metabolised the cell relies on its oxygen-independent pathway,
by the citric acid cycle to carbon dioxide, generating glycolysis (Figure 3). Glycolysis can only proceed if
reduction equivalents that can be used in the the reduction equivalent produced in its reaction steps
respiration chain, which uses these reduction is converted to oxidation equivalent; and only if the
equivalents to reduce oxygen to water, thus generating accumulating pyruvate, which cannot enter the citric
much more energy in the form of ATP than glycolysis. acid cycle, is removed. Thus the cell reacts by
This is the aerobic part of glucose metabolism. converting pyruvate to lactic acid with the help of
During exhaustive exercise when the rate of oxygen the reduction equivalent. The amount of lactic acid
consumption due to the work of the respiratory chain transferred to the blood can be considered as a marker
gets high, oxygen cannot be delivered by haemo- of exhaustive work.
globin in the appropriate amount, so the
100
Anaerobic
lactic Aerobic
Energy production (%)
80
60
40
20 Anaerobic
lactic
1 2 3
Time (minutes)
Figure 3. The time process of aerobic and aneraerobic reactions (Clayton, 1991).
A study by Hodgson and Rose (1994) revealed that While the assay methods described above can be
horses, independent of athletic discipline, usually generally applied, the step test can certainly be
perform under aerobic conditions. In humans it is modified. Recently we have started to develop step
known that well-trained athletes accumulate lower tests for jumping horses, which include a standardized
concentrations of lactic acid in the blood than non- course. In addition, we monitored some of our jumping
trained individuals (Hollman and Hettinger, 1990). horses during international events by measuring heart
Lactic acid is usually assayed in a small aliquot of rate and lactic acid formation to better estimate the
capillary blood in anticoagulant solution, which is kept stress during these big events.
on ice until laboratory analysis (Figure 4).
During the step test blood samples were taken to
monitor lactic acid accumulation (Figure 5). All horses Outlook
accumulated lactic acid to some extent, depending on
fitness level. From human studies it is known that the THE SPEED, POWER AND COORDINATION
blood serum lactic acid concentration influences FACTORS
performance capabilities (Heck and Rosskopf, 1994).
Humans get ‘sour’ when they reach a well known In many equestrian sports speed, power and
threshold of lactic acid accumulation and can no longer coordination are additional factors that determine
perform with coordinated movements. In humans the success. Certainly these factors are interrelated. We
threshold is 4 mM lactic acid; and we observed the have recently started to precisely monitor the speed
same threshold for horses as well. These results point and acceleration capacity of jumping horses with a
to the fact that during exhaustive exercise horses react LAVEG-speed analyser, which is based on laser
with lactic acid formation, the extent of which depends technology. The question is whether a special
on the individual performance capacity of the horse; program to improve acceleration that is known from
and this parameter must be considered during other athletic programs (i.e. short sprints followed
controlled exercise in order not to overtax the horse. by a resting period) is effective in horses as well. In
On the other hand, in the timed schedules applied, preparation are programs to improve and actually
heart rate and lactic acid accumulation will reflect monitor power with the help of weight-adjustable
the endurance capability and the progress of the sledges. But this special program must be discipline-
individual horse. specific, because horses performing in dressage,
(A) (B)
Figure 4. (A) An aliquot of capillary blood is taken from the horse and transferred to (B) Eppendorf tubes filled with
anticoagulant solution and kept on ice.
M. Bojer 329
10
9
Horse A
8 Horse B
7 Horse C
Lactate (mmol/l)
6
5 Lactic acid threshold
4
3
2
1
0
Start 1. Step 2. Step 3. Step 4. Step 5. Step
Figure 5. Time course of lactic acid formation in horses challenged with the same step test as in Figure 2
(Bojer and Schulz, 2000).
jumping or cross-country all have individual demands. video movies were recorded and evaluated (Figure
One consideration is: does the horse need maximal 6). The scientific question is whether training can
power throughout the performance, or do we need improve these kinetic parameters.
to improve ‘maximal power on the point’? In other Taken together, we have collected a broad spectrum
words, does a horse that performs in dressage for 5 of analytical methods to analyse and monitor horses
min need maximal power during all this time, or just during sport events. Training programs known to
when performing power-consuming manoeuvers like have successfully improved performance in the
passage? human sports were adapted. All these approaches
The most complex factor is coordination. To analyze definitely must be accompanied by training diaries,
the various kinetic aspects during jumping (i.e. regular veterinary check-ups, weight controls and
jumping distance to the fence, time of last contact specialized feeding programs.
with the soil or technique while jumping) high-speed
4000
3000
mm
2000
1000
0
0 1000 2000 3000 4000 5000 6000 7000
mm
Figure 6. 2-D kinetic analysis of horse and rider (Pozzo et al., 2001).
References
Bojer, M. and T. Schulz. 2000). Hämatologische Hodgson, D.R. and R.J. Rose. 1994. The athletic
Untersuchungen von Tierblut mit dem AC-T diff horse-principles and practise of equine sports
in der sportmedizinischen Forschung. In: Beckmann medicine. WB Saunders Company.
Coulter GmbH: In vitro (5), Krefeld, pp. 52-53. Hollmann, W. and T.H. Hettinger. 1990.
Clayton, H.M. 1991.Conditioning sport horses. Sport Sportmedizin – Arbeits- und Trainings-grundlagen.
Horse publication. Mason, Canada. 3 Auflage, Schattauer Verlag, Stuttgart, New York.
Heck, H. and P. Rosskopf. 1994. Grundlagen Pozzo, R., K. Lütteken and M. Bojer. 2001.
verschiedener Laktatschwellenkonzepte und ihre Kinematische Analyse der Springtechnik bei
Bedeutung für die Trainingssteuerung. In: Spitzenreitern; 6.Symposium der dvs-Sektion
Stellenwert der Laktatbestimmung in der Biomechanik Deutsche Vereinigung für
Leistungsdiagnostik (D. Clasing, H. Weicker und Sportwissenschaft Konstanz.
D. Bötting, eds). Gustav Fischer Verlag, Stuttgart,
Jena, New York, pp. 113-131.
S. Duren and K. Watts 331
Physiology and feed formulation: the proper role of carbohydrates in the

equine diet
STEPHEN DUREN1 AND KATHRYN WATTS2
1
Performance Horse Nutrition, LLC, Weiser, Idaho, USA
2
Rocky Mountain Research and Consulting, Center, Colorado, USA
Introduction
The statement, “I want a low carbohydrate feed for broken down with enzyme digestion in the small
my horse”, has become popular in recent years. The intestine and microbial fermentation in the cecum
reasons, or potential reasons, for wanting a low and colon, commonly referred to as the hindgut. Plant
carbohydrate horse feed could be many. One material (hay/pasture), cereal grain and commercial
possibility is a simple carry over from the current grain concentrate, the cornerstones of modern equine
human dietary trend of reducing carbohydrate intake. diets, consist of a wide array of carbohydrates and
The Atkins Diet is one popular means of reducing may contain up to 75% carbohydrate (Pagan, 1998).
carbohydrate intake in human meals. With millions However, not all of this carbohydrate is digested or
of dollars spent on media advertisements for absorbed in the same manner within the equine
restaurants with ‘Atkins Friendly’ menus, each of us digestive tract.
is at least reasonably familiar with the low On a simplistic basis, carbohydrates found in the
carbohydrate concept stressed in this diet. It follows equine diet can be crudely divided into two types,
then that with an estimated 64% of Americans structural and non-structural. Structural carbohydrates
overweight and at any given time 29% of men and are typically found in the cell wall of the plant and
44% of women trying to lose weight, one could are often referred to as fiber. The major carbohydrates
conceive that popular food culture may be influencing associated with the cell wall are cellulose,
the horse owner’s thought processes when buying hemicellulose and lignin. These carbohydrates are
horse feed. However, there may be more legitimate represented on a laboratory analysis report as neutral
reasons for a horse owner to seek a low carbohydrate detergent fiber (NDF). Baled hay, mature pasture
feed. Included in the list of reasons are the desires to grass, beet pulp and soybean seed coats are good
influence or modify behavior, or sensitivity to so- sources of structural (fibrous) carbohydrate (NRC,
called carbohydrate diseases including tying-up, 1989). Structural carbohydrates are resistant to
Cushing’s Disease, laminitis, insulin resistance (IR), enzyme digestion in the small intestine and must be
obesity and osteochondrosis dissecans (OCD). But fermented by bacteria in the horse’s hindgut (Frape,
before we recommend that our clients reduce or 1986). Bacterial fermentation of fiber yields volatile
eliminate carbohydrate from the equine diet, we fatty acids (VFAs). VFAs are absorbed from the
should understand more about carbohydrates and hindgut and are transported to the liver where they
their proper role in horse diets. are converted to energy substrates for the horse. The
overall digestibility of fibrous carbohydrate is quite
variable, depending on the distribution of cellulose,
Classification of carbohydrates hemicellulose and lignin in the carbohydrate fraction.
Since lignin is non-digestible by bacterial
The equine digestive tract is anatomically classified fermentation (Frape, 1986) the higher the degree of
as that of a non-ruminant herbivore (Frape, 1986). lignin present the lower the overall digestibility. Thus,
This digestive arrangement allows feedstuffs to be as plants mature and increase in lignin content,
332 Physiology and feed formulation: the proper role of carbohydrates in the equine diet
digestibility is decreased. The overall digestibility The NSC content of feeds can be determined with
of NDF in good quality forages by horses varies from modern laboratory methods (e.g. Dairy One, Forage
40-50% (Pagan, 1998). Analysis Laboratory, Ithaca, New York, USA). Feeds
Non-structural carbohydrate (NSC) is carbohydrate with large amounts of NSC include commercial grain
associated with the inner portion of the plant cell, or concentrates containing large amounts of oats, corn
plant cell contents. The plant cell includes NSC along and barley and any product containing high amounts
with protein, lipids, organic acids and soluble ash. of molasses. Forages such as pasture and baled hay
NSC is made up of sugars, disaccharides, starches also contain NSC, but the amount is quite variable,
and fructans. In warm season grasses (C4 plants), ranging from 1 to 40% of dry matter. The NSC
starch is the primary storage carbohydrate, whereas content of forages is influenced by type of plant,
in cool season grasses (C3 plants) fructan is the environmental temperature, light intensity, drought
primary storage carbohydrate. As a practical point, stress, plant fertility, and rate of drying during the
commonly fed legumes such as clover and alfalfa do curing process (Smith, 1973). Briefly, C3 (cool
not contain fructan, and store carbohydrate as starch. season) grasses are generally higher in NSC than C4
Enzymes in the horse’s small intestine break down (warm season) grasses. Cold stress, especially in C3
sugars and starch to monosaccharides (simple sugars) grasses, increases the NSC content. Photosynthetic
that are absorbed and circulate in the blood as glucose. capacity, and hence production of sugar, is directly
Fructans are resistant to mammalian enzyme correlated to light intensity and duration. NSC
digestion and must be fermented by bacteria in the concentration will be lowest in early morning if the
horse’s hindgut (Suzuki and Chatterton, 1993). Sugar night was warm enough to allow the sugars produced
and starch are highly digestible, greater than 95%, the previous day to be utilized by respiration. Further,
within the length of equine digestive tract. Bacteria shading or cloud cover of grass plants reduces NSC
located in the hindgut ferment any starch or sugar content. Drought stress is a stimulus for NSC
that is not digested by enzymes in the small intestine. accumulation in grass. Drought limits plant
Unfortunately, fermentation of sugar, starch and respiration causing the accumulation of NSC. It is
fructan by hindgut microorganisms can produce lactic widely accepted by forage researchers that nitrogen
acid, and the resulting acidosis can destroy the and phosphorus deficiency causes an increase in
environment within the hindgut leading to death of concentration of NSC in both grass and legumes.
the microorganisms and health concerns such as colic Finally, the faster plants dry after being cut for hay
and laminitis (Richards et al., 2003). production, the higher the NSC content. Plants will
The extent to which NSC is digested in the small continue to respire and burn off sugars until plant
intestine is dependent on the source of the NSC, moisture content is below approximately 40%. The
processing, level and rate of intake, time and only method to determine the NSC content of a feed
frequency of forage feeding and individual horse ingredient is laboratory analysis. Visual characteristics
digestive characteristics (Meyer et al., 1993). Briefly, and type of hay are not predicable indicators of NSC
starch originating from oat grain is more digestible content.
within the small intestine compared to starch from
corn or barley. This difference is related to the
microscopic structure of the starch granule, and thus Carbohydrate-related diseases
the surface area available for enzyme digestion.
Processing (rolling, crimping, grinding, etc.) In the last ten years, many equine disease treatment
generally increases the digestion of starch in the small protocols have investigated the role of dietary
intestine, again increasing the surface area for enzyme carbohydrates. Several disease states may be
digestion. As level of starch intake increases, the small precipitated or exacerbated when high levels of
intestine digestibility decreases due to rate of passage carbohydrate are being fed. The following is a brief
of feed material. The feeding of forage following a description of several disease conditions and the role
meal rich in sugar and starch will increase rate of that carbohydrates may play in the treatment or
passage and decrease small intestine digestibility of prevention of disease.
sugar and starch. Finally, differences in the ability Tying-up is a condition associated with the equine
of individual horses to either digest or absorb sugars musculature system. Tying-up is also known by the
from the small intestine has been reported (Richards following names: Azoturia, Monday Morning Disease,
et al., 2003). and Exertional Rhabdomyolysis. It is expressed
clinically as firm, painful muscles over the lumbar, relatively unavailable, the body fat stores, which were
sacral and gluteal regions, along with excessive never intended to become excessive, are depleted in
sweating, increased heart rate and elevated respiration order to provide energy for survival. Under many
(Valberg and McKenzie, 2002). Horses experiencing modern horse management systems, the combination
a bout of tying-up often are in extreme pain to the of feeding starch-rich rations over many years and
point they are reluctant to move or are recumbent. protracted periods of stall confinement or lack of
Tying-up is confirmed with elevated serum levels of adequate exercise tend to lead to the acquisition and
muscle enzymes including creatine kinase (CK) and maintenance of substantial body fat in the
aspartate transaminase (AST). Horses that suffer domesticated horse. The development of obesity in
frequent or chronic bouts of tying-up have been both humans and horses directly causes insulin
studied to determine possible dietary treatments. insensitivity (Johnson, 2002). Insulin insensitivity is
Dietary modification that both reduces the grain or known to lead to endothelial cell dysfunction, which
starch intake and increases the intake of fat has been is involved in the pathogenesis of vascular
shown to be effective in managing this condition complications in humans. Insulin insensitivity is
(Valberg and McKenzie, 2002). currently being studied as a cause of laminitis in
Cushing’s Disease is a result of a pituitary gland horses, and future feeding recommendations may
tumor causing an overproduction of pituitary require these horses to eat low carbohydrate (low
hormones. This disease is thought to be principally glycemic) diets.
associated with older horses (greater than 15 years Osteochondrosis is one of a number of growth
of age), but has also been described in horses as young anomalies that affect the skeleton of young horses.
as seven (Geor, 2001). The clinical signs of this Osteochondrosis can be very serious in that it often
disease include hirsutism (excessively long and curly results in debilitating lameness that in many instances
hair coat), weight loss, muscle wasting, lethargy, reduces or eliminates athletic performance (Jackson,
increased sweating, bulging of the orbit due to an 2003). The cause of osteochondrosis is multifactorial.
increase in fat deposition around the eyes, polydipsia The proposed causes of osteochondrosis include
(increased thirst) and polyuria (increased urination). genetic predisposition, rapid growth rate, mechanical
The complications of this disease are laminitis and stress and trauma, nutrition excess, mineral imbalances
diabetes mellitus, with both complications made worse and endocrine factors (McIlwraith, 1996). New
with high starch diets (Geor, 2001). research is re-focusing on hyperglycemia and/or
Laminitis is a disease condition associated with the hyperinsulinemia as a cause of osteochondrosis (Glade
hoof and its attachment to the bony structure of the et al., 1984; Ralston, 1995; Pagan 2001). Specifically,
equine foot. In a bout of laminitis, inflammation of scientists are looking to determine if certain foals that
the laminae alters tissue blood flow to the sensitive experience an exaggerated and sustained increase in blood
living tissues that attach the coffin bone to the hoof glucose and insulin in response to a carbohydrate meal
wall (Redden, 2001). This causes separation of the may be predisposed to development of osteochondrosis
non-sensitive hoof wall and the laminae such that (Pagan, 2001). Hyperinsulinemia has been reported to
the coffin bone becomes detached from the hoof wall be a potential endocrine factor that contributes to equine
and sinks toward the ground. This disease is extremely osteochondrosis (Henson et al., 1997). If this does prove
painful for the animal and often debilitating. to be a predictable cause of osteochondrosis, then feeding
Laminitis is associated with a number of risk factors, foals carbohydrate-rich diets may be contraindicated.
with overeating of grain (starch) the best known cause.
Grain overload is commonly used as a method to
induce laminitis in research animals. Minimizing carbohydrate intake
Obesity is a very common problem for modern
horses. According to Dr. Philip Johnson from the From the available information on several diseases
University of Missouri, evolution equipped the equine thought to be sensitive to carbohydrate in the diet,
metabolism for survival based on the seasonally the desire to create a low carbohydrate diet is really
variable availability of forage (grass). The temporary a need to control the amount of NSC (sugar, starch
development of additional body fat (relative obesity) and fructan) the horse consumes. Structural
at times when forage is plentiful provides a survival carbohydrate (fiber) should not be a target for
adaptation for time when conditions are harsh and elimination since fiber is essential for proper function
forage is scarce. During periods in which forage is and motility of the horse’s digestive system.
Practicing equine nutritionists commonly recommend and minerals. Since grains and grain concentrates
that horses receive a minimum of 1.5% of body with molasses contain high levels of NSC,
weight per day in dry forage to provide essential fiber. minimizing the amounts of these products included
However, some work is now being done to correlate in the diet will minimize total NSC intake.
a more predictable fiber value, neutral detergent fiber Unfortunately, many horse owners are quick to
(NDF), to voluntary dry matter intake of horses (St. eliminate all grain products from the diet and
Lawrence et al., 2001). therefore also eliminate the source of essential
If it is necessary to reduce the NSC intake in an vitamins and minerals that are associated with the
equine diet, the following steps will help accomplish grain concentrate portion of the diet. If grain is to
that goal. The first step in minimizing the amount of be eliminated from the diet, a low-intake protein,
NSC (sugar and starch) in a horse’s diet is to determine vitamin and mineral supplement should be added to
the NSC content of the feed. Unfortunately, this is provide these essential nutrients. If horses do require
not information that can be found on the feed tag. A large amounts of grain, it should be recommended
quality lab for determination of NSC is the Dairy that clients feed grain concentrates with moderate to
One Forage Lab, Ithaca, New York. The cost of low levels of NSC. The use of dietary fat, either in
analysis is approximately $15 for a combination of the feed or top-dressed onto the feed, has been shown
sugar, starch and fructan analyses and approximately to slow the rate at which the stomach empties and to
$26 for a more complete nutrient analysis including control the surge of glucose into the blood following
minerals and full carbohydrate fractions. Table 1 lists a meal (Pagan et al., 1995). Therefore, feeding high
the average NSC content of feed ingredients in the fat feeds or adding fat to the diet may help to
US as published in the Dairy One Ingredient Library. eliminate many carbohydrate problems. However,
The reader should note that NSC content of the long term feeding of fat may lead to impaired glucose
ingredients can be quite variable due to growing tolerance in ponies (Schmidt et al., 2001), and is
conditions previously outlined. With a range of up to contraindicated in obese horses.
35% of dry matter, NSC content of forages certainly The final step in reducing NSC intake is to consider
requires that we not rely on ‘book’ values for average the forage portion of the diet. Overgrazed, stressed
NSC content. pasture grass can contain an abundance of NSC and
should be avoided with horses known to have
Table 1. Non-structural carbohydrate content of feed ingredients laminitis, episodes of tying-up, insulin resistance,
(dry matter basis)1. Cushing’s Disease or obesity. Pasture grass should
Feed NSC (%) be replaced with baled hay or other fibrous
Stored forages carbohydrate sources such as hay cubes or hay pellets,
Alfalfa hay 11.4 appropriately tested for NSC content. Since visual
Mixed hay, mostly legume 12.2 characteristics and type of hay are not reliable
Mixed hay, mostly grass 13.5
Grass hay 13.3 indicators of carbohydrate content, new research is
Oat hay 23.0 focusing on methods to reduce the carbohydrate load
Straw 12.0 of hay after it has already been baled. Watts (2003)
Fiber products has reported on one method to reduce the water-
Almond hulls 45.5 soluble carbohydrate (sugar) content of hay. In that
Beet pulp 12.2 research, Watts soaked fifteen samples of various
Citrus pulp 29.0
Soybean hulls 7.2 species of hay in water. She reported the average
Cottonseed hulls 5.2 amounts of sugar reduction after 30 and 60 minutes
Cereal grains and molasses
of soaking in cold water were 18.9% and 30.7%,
Corn 73.2 respectively. The average amount of sugar removed
Barley 63.1 in 30 minutes of soaking in hot water was 29%.
Oats 50.7 This research offers the first step in methods to
Molasses 58.4
practically limit the sugar content of baled hay.
1
Dairy One Forage Laboratory, Ithaca, New York.
The second step in minimizing the amount of NSC in Summary

the diet is to only feed the amount of grain or
supplement necessary to maintain body condition and In summary, there are health concerns that dictate
ensure proper dietary fortification of protein, vitamins the need to feed low carbohydrate diets to certain
horses. Since there is an abundance of different Kentucky Equine Research, Versailles, KY.
carbohydrates in horse feed, the choice of which Meyer, H., S. Radicke, E. Kiengle. S. Wilke and D.
carbohydrates to limit is critical. Carbohydrates found Kleffken. 1993. Investigations on preileal digestion
in equine feeds can be roughly divided into two types, of oats, corn and barley starch in relation to grain
structural and non-structural. Structural carbohydrates processing. In: Proceedings of 13th Equine Nutrition
are often referred to as fibrous carbohydrates and and Physiology Conference, Gainesville, FL.
should not be eliminated from the diet. Non-structural National Research Council. 1989. Nutrient
carbohydrates (NSC) are commonly referred to as Requirements of Horses. 5th Edition NRC-NAS.
sugars, starch and fructans and can be associated with Washington, D.C.
several so called carbohydrate diseases. NSC is the Pagan, J.D., T. Rotmensen and S.G. Jackson. 1995.
carbohydrate that can be minimized in the equine
Responses of blood glucose, lactate and insulin in
diet. Methods to minimize NSC content of the diet
horses fed equal amounts of grain with or without
include analysis of feed ingredients to determine NSC
added soybean oil. In: Proceedings of the 1995 Equine
content, reduction in intake of ingredients with high
NSC content, and modification of forage intake Nutrition Conference for Feed Manufacturers.
including elimination of stressed pasture and the Kentucky Equine Research, Versailles, KY.
soaking in water of baled hay to rinse water-soluble Pagan, J.D. 1998. Carbohydrates in equine nutrition.
carbohydrate away. In: Advances in Equine Nutrition. Nottingham
University Press. UK.
Pagan, J.D. 2001. Developmental orthopedic disease:
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Nutritional Biotechnology in the Feed and Food Schmidt, O., E. Deegen, H. Fuhrmann, R. Duhlmeier,
Industries, Proceedings of Alltech’s 19th Annual and H. Sallmann. 2001. Effects of fat feeding and
Symposium (T.P. Lyons and K.A. Jacques, eds). energy level on plasma metabolites and hormones
Nottingham University Press, UK. in Shetland ponies. J. Vet. Med. 48:39-49.
Johnson, P.J. 2002. Peripheral cushingoid syndrome St. Lawrence, A., R.J. Coleman and L.M. Lawrence.
(‘equine metabolic syndrome’). In: Current Therapy 2001. Relationship between NDF and hay intake
in Equine Medicine, 5th Edition. (N.E. Robinson, in horses: A review of published studies. In:
ed). W.B. Suanders, Philadelphia, PA. Advances in Equine Nutrition II. Nottingham
McIlwraith, C.W. 1996. The equine skeleton – how University Press. UK.
does bone grow and how do abnormalities in the Smith, D. 1973. Nonstructural carbohydrates. In:
development process affect soundness? In: Chemistry and Biochemistry of Herbage. Vol. 1
Proceedings of the 1996 Short Course Equine Academic Press, London. UK.
Nutrition Conference for Feed Manufacturers. Suzuki, M. and N.J. Chatterton. 1993. Science and
Technology of Fructans. CRC Press. London. UK. Watts, K.A. 2003. Soaking hay to remove excess
Valberg, S. and A. McKenzie. 2002. Feeding fat to soluble carbohydrate. In: Proceedings of the 2nd
manage muscle disorders. In: Proceedings of the Annual Int’l. Equine Conf. on Laminitis and
2002 Equine Nutrition Conference for Feed Diseases of the Foot. Palm Beach, FL, USA.
Manufacturers. Kentucky Equine Research,
Versailles, KY.
K.H. Kline 337
Relevance of the NRC to today’s horse industry

KEVIN H. KLINE
Department of Animal Sciences, University of Illinois, Champaign-Urbana, Illinois, USA
Current status of the horse industry

The horse industry entered the new millennium during membership will continue to be cultivated by these
a period of strength and popularity not seen since organizations.
possibly the beginning of the previous century. This
upward trend followed several years of a declining
horse population beginning in the mid-1980s. The Status of the NRC Nutrient Requirements
general decline of the horse industry following a of Horses
serious economic downturn was reflected in high
horse slaughter during this time, with over 300,000 The fifth revision of the National Research Council
horses per year being processed by abattoirs. Since (NRC) Nutrient Requirements of Horses is now 15
then, horse slaughter has declined by 75-80% and years old. Released in May of 1989, it is the most
has so far shown no sign of increasing despite the current edition of recommended nutrient allowances
current economic slowdown and the general for horses. It contains several tables that present
consensus that horse populations are increasing in nutrient requirements for 19 different classes of
most parts of the United States. The improvement in horses, based upon age, rate of growth, level of
the status of the horse industry is indicated by strong physical activity, age/training interaction, stage of
feed sales by several major horse feed manufacturers pregnancy and lactation. These tables are replicated
and the continued strength of sales of equine related for ponies and horses of different expected mature
goods and services such as trucks, trailers, tack, bodyweights of 200, 400, 500, 600, 700, 800 and
pharmaceuticals and veterinary and farrier services. 900 kg.
Although the public auction sale prices of the highest The 1989 NRC publication continues to provide a
quality select horses have declined significantly since basic framework for ensuring that nutrient
2001, a reflection of world events and the depressed requirements of most categories of horses are
stock market, the entry level, companion horse, youth generally met and not exceeded to the point of
and amateur horse markets remain quite strong. Fewer toxicity. However, research findings of the past 15
low-quality horses exist today compared to two decades years and fundamental changes in the horse industry
ago, and today’s horse owner, both new and experienced, must be addressed by a new revision of Nutrient
values quality, convenience and the aesthetic properties Requirements of Horses. To their credit, the NRC has
of feeding and caring for horses like never before. The convened an expert committee to review and revise
Farm Bureau’s recent emphasis on the equine industry the publication, and the first meeting of this committee
through appointment of county, state and national equine is scheduled for April 2004.
committees suggests a fundamental change in the
perception of the equine industry by traditional
agricultural organizations. As the swine, dairy, poultry Major considerations for the new NRC
and beef industries consolidate and fewer livestock Equine Committee
producers maintain membership in professional
agricultural organizations, the horse industry Although not an exhaustive list, the following topics
338 Relevance of the NRC to today’s horse industry
will be important for the new NRC committee to populations in the cecum and large colon. Structural
consider during the development of the updated carbohydrates from forage are the most ‘natural’
Nutrient Requirements of Horses: carbohydrates for the horse to digest with assistance
from hindgut microorganisms. However, as the horse
Energy metabolism/requirements is expected to grow rapidly, lactate or perform intense
exercise for competition, the ability of structural
Carbohydrate metabolism
carbohydrates to meet the horse’s energy requirement
Protein/amino acid requirements is exceeded and starches and sugars from concentrate
mixes become necessary. Research has been
Mineral requirements
conducted to evaluate the glycemic responses of
Vitamin requirements horses to nonstructural carbohydrates from various
Water requirements types of grains as well as comparative responses to
diets that provide supplemental energy high in starch
Dietary fats and oils and sugar versus diets high in fat and fiber. Diets
Geriatric horses high in starches and sugars have been implicated in
numerous metabolic bone problems in growing horses,
Feed composition tables digestive problems related to acidification of the
Bone metabolism/growth hindgut, and to laminitis due to toxin production in
the hindgut. However, recent studies have confirmed
Dietary/genetic interactions that horses and ponies are quite capable of metabolic
Diet/exercise interactions adaptation to either high starch and sugar diets or
high fat diets (Schmidt et al., 2001; Hoffman et al.,
Nutraceutical ingredients 2003).
Nutritional/environmental interactions
Horse owner demands PROTEIN/AMINO ACID REQUIREMENTS
Limited data are available regarding the protein

ENERGY METABOLISM/REQUIREMENTS digestibility of specific feed ingredients in horses.
Amino acid absorption occurs mainly in the small
The energy requirements for most livestock species intestine of the horse, but some ammonia absorption
are given as metabolizable energy (ME), while also occurs in the hindgut. Therefore, protein
requirements for horses are currently given as requirements in the horse are expressed as crude
digestible energy (DE). Energy requirements for protein (CP). Non-protein nitrogen feed ingredients
lactating dairy cattle are given as net energy for are not efficiently utilized by the horse due to the
lactation (NEL). One of the first discussions of the location of fermentative digestion sites near the end
NRC Equine Committee regarding energy of the gastrointestinal tract. The CP requirement for
requirements might be about the merits of expressing mature, sedentary horses under maintenance
energy requirements for horses of various stages of conditions is quite low, at less than 8%. Horses at
growth, gestation, lactation or exercise as DE versus maintenance can meet CP requirements on moderate-
ME or NE. An evaluation of various equations quality forages and grains, but growing horses and
proposed in research to estimate energy requirements especially lactating mares require protein
of horses in various life stages and exercise intensities supplementation unless a high-CP forage containing
will be an important consideration of the NRC legumes is a major component of the diet.
committee. Amino acid requirements for horses are not well
defined, but some work conducted since the
publication of the last NRC Nutrient Requirements
CARBOHYDRATE METABOLISM of Horses has suggested that threonine is the second-
limiting amino acid (lysine is first-limiting) in a
The horse is fundamentally a grazing herbivore that yearling horse diet of corn, oats, soybean meal and
spends as much as 12-16 hrs per day eating forage coastal bermuda grass/hay (Graham et al., 1994).
when on pasture or rangeland. It is a hindgut There is little question that protein quality is of
fermenter with substantial cellulolytic microbial concern for growing horses and lactating mares, and
K.H. Kline 339
for the horse in general, as compared to ruminant horses. Horses under confinement conditions may
species. Adequate lysine concentrations should be lack one or both of these factors, however. Forages
present in diets high in soybean meal, but diets preserved with propionic acid or stored for extended
supplemented with cottonseed, peanut or flax meal periods of time may lose much of their vitamin A
could require additional lysine when fed to growing activity. It is not unusual for show horses or for horses
horses or lactating mares. Greater commercial being prepared for sale to be housed indoors for
availability and affordability of essential amino acids extended periods of time during summer daylight
and advances in plant breeding that alter the amino hours in order to avoid dulling of the hair coat from
acid profiles of some feeds are factors to consider the sun. These horses may lack vitamin D if good-
when making recommendations about protein/amino quality, sun-cured forage is not available. Vitamin E
acid recommendations for horse diets. Dietary protein could also potentially be deficient if poor-quality
and amino acid recommendations for working horses forages are fed for extended periods.
may some day consider the acidogenic effects of B-complex vitamins are synthesized in the horse’s
excess dietary protein (Graham-Thiers et al., 2001), hindgut and usually do not require supplementation.
although data are still limited in this area. Although no dietary biotin requirement has been
established beyond what is synthesized in the gut,
research continues into the possible use of
MINERAL REQUIREMENTS supplemental biotin for improving poor-quality
hooves. Two studies have found improved hoof
Mineral concentrations of feedstuffs may vary quality in horses with initially poor hoof quality
significantly with soil mineral concentrations, soil when fed 5-10 mg of supplemental biotin per 100 kg
fertilization, plant species, harvesting conditions and bodyweight per day for periods ranging from one to
stage of plant maturity. Special attention should be six years (Josseck et al., 1995; Geyer and Schulze,
paid to providing trace mineral supplements 1994). Another study found a 15% increase in the
formulated specifically for horses, not other species. rate of hoof growth in ponies with 0.12 mg/kg
For instance, trace-mineralized salt for sheep is bodyweight of biotin as compared to ponies on a
typically devoid of a bioavailable copper source for control diet without supplemental biotin (Reilly et
horses, since copper oxide is usually the only source al., 1998). Feeding recommendations for vitamin E
of copper. A more bioavailable source should be could be viewed both in terms of requirement levels
provided instead. Information on trace mineral needed to avoid deficiency symptoms and also as
bioavailability in the horse is not as extensive as data optimal levels for minimizing exercise-induced
collected in other species (Ammerman et al., 1995). oxidative damage to cells through free-radical
However, a substantial amount of research has been formation and for maximizing immune function.
done in recent years regarding equine mineral
nutrition that the new NRC Equine Committee will
need to evaluate. Calcium and phosphorus status of WATER REQUIREMENTS
equine bone has been found to be related to physical
activity as much as dietary status. Extreme variations A series of recent studies at Michigan State University
in the Ca:P ratio of different forages have a great (Butudom et al., 2002; 2004) found that horses
impact on the composition of supplements dehydrated by either exercise or frusemide
recommended to complement the basic forage ration. administration drank more fluid when presented as
Trace mineral nutrition has received substantial either 0.45% NaCl or 0.9% NaCl solutions as
attention from researchers in recent years, and mixed compared to regular water. Their work also supported
results have been obtained regarding the relative previous observations that horses quench their thirst
bioavailability of different trace minerals fed as faster and delay rehydration when consuming cold
inorganic versus ‘organic’ forms of trace minerals fluids after dehydration as compared to warm fluids,
that are bound to an organic molecule. and that the primary stimulus of thirst is an increase
in plasma tonicity rather than hypovolemia (Butudom
et al., 2003). Horses consumed more 20°C water than
VITAMIN REQUIREMENTS 10°C water after being dehydrated. Geor et al. (1998)
found that pre-exercise hyperhydration provided no
Fresh green forages and exposure to sunlight are all thermoregulatory advantage to horses and Sosa et
that is necessary to meet the vitamin needs of most
al. (2002) found that hyperhydration using an isotonic management strategies of geriatric horses. This is
solution at 6% of body weight resulted in arterial most likely due to the fact that so many different
hypoxemia, possibly from pulmonary edema. These age-related infirmities may be related to poor
recent studies and others will likely help to guide the nutritional performance in geriatric horses. Poor
NRC Equine Committee’s recommendations regarding teeth, malabsorption, endocrine problems such as
provision of electrolyte solutions versus water for Cushing’s disease, chronic pain, etc. may all affect
rehydration of already dehydrated horses. digestion, absorption, and metabolism of nutrients.
Such variability among geriatric horses makes
controlled study of this group of animals difficult.
DIETARY FATS AND OILS The typical feed formulation strategies being
employed by feed manufacturers include processing
The use of dietary fats and oils has been one of the of both forages and concentrates to improve foregut
most active areas of research since the publication of digestion of starches and improving hindgut digestion
the fifth revision of the National Research Council with highly fermentable fiber like beet pulp and
(NRC) Nutrient Requirements of Horses. Horses can decreasing particle size of fiber to enhance hindgut
utilize fats and oils effectively, and inclusion rates of fermentation. Addition of microbial products to
up to 15% of the concentrate ration may be well further enhance hindgut digestion and adding fats
accepted by the horse. One study found that the jejunal and oils to improve caloric density have also been
microflora of ponies was not affected by the addition employed. There is opportunity for further research
of up to 20% coconut oil in the diet (Kollarczik et in this field.
al., 1995). Research has suggested that fats and oils
in equine diets may provide benefits to horses in
addition to increased caloric density of the diet for FEED COMPOSITION TABLES
weight gain or maintenance in ‘hard keepers’. Dietary
fats and oils may also help to improve hair coat quality, As novel by-product feeds, genetically modified
spare muscle glycogen in exercising horses, reduce plants and new crops are added as potential livestock
post-exercise lactic acid accumulation, and reduce the dietary components, the NRC Equine Committee will
chance of enterotoxemia, colic and founder. Not all need to review those products that may have an
studies have found beneficial effects, however. Excess application to equine diets. Significant modification
fats and oils reaching the hindgut may inhibit fiber of the NRC feed composition tables will likely be
digestion (Jansen et al., 2000) and diets high in fat necessary.
and fiber when fed to growing foals may reduce the
absorption of calcium and other minerals necessary
for bone development (Hoffman et al., 1999). The BONE METABOLISM/GROWTH
preponderance of research data, however, appears to
support the continued expanded use of dietary fats Glade (1993) found that mid-cannon bone diameters
and oils for many classes of equines. Research of foals born to mares fed calcium-deficient diets
suggesting that added dietary fat may reduce the heat were thinner and mechanically weaker at birth than
production of performance horses working under hot in foals born to mares fed NRC recommended
and humid conditions (Kronfeld, 1996), and enhance concentrations of dietary calcium. These differences
the oxidative capacity of muscle (Orme et al., 1997) in limb bone size and strength persisted during the
continues to stimulate interest in this dietary first 40 weeks after birth. Hoffman et al. (1999)
supplement for horses. found that bone mineral content was lower in
weanlings fed a supplement high in fat and fiber
compared to weanlings fed a supplement high in
GERIATRIC HORSES starch and sugar, and they speculated that “binding
of calcium by fat and fiber may alter the availability
With more and more elderly horses represented in the of elements necessary for bone development.”
equine population every year, and more commercial Studies comparing diet manipulations and evaluating
diets being formulated for geriatric horses, this area the resulting changes in serum skeletal metabolic
of research seems to be a promising one for equine markers such as hydroxyproline (bone resorption),
nutritionists, yet little work has been done in osteocalcin (bone mineralization), C-propeptide of
determining nutrient requirements and feeding type-II procollagen (collagen synthesis) as well as
K.H. Kline 341
measurements of the physical properties of bone and decreased lipid oxidation compared with a meal
promise to further elucidate the effects of diet on of insoluble carbohydrate (alfalfa) or not feeding.
bone metabolism and growth in horses. Carbohydrate feedings did not produce a sparing of
muscle glycogen compared with fasting. Pagan and
Harris (1999) found that feeding grain before exercise
DIETARY/GENETIC INTERACTIONS reduced free fatty acid (FFA) availability and
increased blood glucose disappearance during exercise
Although feeding programs designed for horses with in Thoroughbred horses. Feeding hay either along
genetic defects should never find a large market, there with grain or ad libitum the night before exercise
has been substantial interest in managing some of resulted in reduced plasma volume and higher lactate
the more common equine metabolic disorders with production and heart rates during exercise. They
diet. The common genetic defect manifested as concluded that feeding only forage before exercise
hyperkalemic periodic paralysis (HYPP) in Quarter did not adversely affect performance. They also
Horses has been managed with both medication and suggested that grain should be withheld from horses
low potassium diets. Polack et al. (2000) found that before exercise, but that small quantities of hay should
horses with equine motor neuron disease had greater be fed to ensure proper gastrointestinal tract function.
concentrations of copper and lower concentrations
of vitamin E in the spinal cord, suggesting that an
imbalance of oxidant/antioxidant nutrients may have NUTRACEUTICALS
an effect on the occurrence of this poorly understood
disease. Recurrent exertional rhabdomyolysis (RER) The term nutraceutical was coined in the 1990s by
has been associated with a diet high in soluble Dr. Stephen DeFelice in reference to dietary substances
carbohydrate. McKenzie et al. (2003) found that affecting human health. The term has gained
horses severely affected with RER that were fed a popularity in reference to dietary products that may
high-fat, low-starch diet had dramatically lower post- impart health benefits to horses as well. Dr. DeFelice
exercise serum creatine kinase (CK) activity compared defined a nutraceutical as:
to horses fed a low-fat, high-starch diet. Firshman et
al. (2003) found that polysaccharide storage “….any substance that is a food or a part of a
myopathy (PSSM) in Quarter Horses can be managed food and provides medical or health benefits,
by following dietary recommendations of reduced including the prevention and treatment of disease.
soluble carbohydrates and supplemental fats and oils Such products may range from isolated nutrients,
combined with gradual increases in daily exercise. dietary supplements and specific diets to
genetically engineered designer foods, herbal
products, and processed foods such as cereals,
DIET/EXERCISE INTERACTIONS soups and beverages. It is important to note that
this definition applies to all categories of food
The NRC Equine Committee will have numerous and parts of food, ranging from dietary
studies to review related to the energetics of supplements such as folic acid, used for the
exercising horses. Many studies, though, deal with prevention of spina bifida, to chicken soup, taken
more than meeting additional energy demands. to lessen the discomfort of the common cold. This
Researchers have shown great interest in evaluating definition also includes a bio-engineered designer
diets that may help exercising horses to maintain vegetable food, rich in antioxidant ingredients,
proper immune function, enhance aerobic metabolism and a stimulant functional food or pharmafood.”
of fatty acids and minimize lactate production from
anaerobic glycolysis. The timing of meal feeding with
respect to exercise has received a great deal of As the number of geriatric horses increases, and as
attention in recent years. St. Lawrence et al. (2002) horses compete in more demanding competitions, the
found that 48 min of light work performed 1 hr before interest among horse owners in such nutraceutical
a meal did not affect glucose or insulin responses to products is increasing. Documentation of benefits of
the meal. Jose-Cunilleras et al. (2002) found that nutraceuticals in horse diets has been very limited.
feeding a soluble-carbohydrate-rich meal (corn) to Orth et al. (2002) found that the combination of
horses before exercise increased muscle utilization glucosamine and chondroitin sulfate inhibited the
of blood-borne glucose and carbohydrate oxidation synthesis of several mediators of cartilage degradation
in equine carpal cartilage explants in vitro. Evidence regulation of waste management in the future. Further
of benefits to equine joint health from precision in defining nutrient requirements of horses
supplementation with glucosamine and chondroitin may help to minimize unintended environmental
sulfate in vivo has been inconclusive. impacts due to excess provision of certain nutrients.
NUTRITION/ENVIRONMENTAL INTERACTIONS HORSE OWNER DEMANDS
Horses are frequently housed in facilities that are The psychology of feed buying decisions by horse
designed more for the comfort of the horse owner owners is not the primary focus of the NRC
than for the horse. Horse housing is notoriously poorly committee, but this consideration is a significant factor
ventilated to the point of being airtight in some cases, in determining feeding strategies, feed forms and
and horses are forced to breathe poor-quality air. The nutritional products that horse owners are willing to
problem is exacerbated when horses consume diets offer their horses. The adequacy of dietary energy,
containing excessive protein, resulting in high rates of protein, vitamins and minerals in a horse’s diet may
urea excretion and high aerial ammonia concentrations. receive considerably less conscious thought from the
Fear of fecal contamination of hay and/or grain with horse owner than the expression of diet sufficiency
the causative organism for Equine Protozoal Myelitis as a glossy hair coat, vigorous attitude, hearty appetite
has prompted some horse farm managers to feed all and soundness of limb and wind. The extent to which
forages and concentrates entirely as bagged cubes, today’s horse owner wants her horse to be not merely
pellets or texturized feeds. Hay cubes, complete hay/ healthy, but also fully contented, can be a great
grain cubes and completely pelleted diets are motivating force that drives feed-buying decisions.
alternative methods for providing forage for horses. This general mind-set of many modern horse owners
The primary motivation for using these forms of feed tends to drive the demand for horse feeds that are
is the extreme ease of storage and handling under both high in nutritional quality and in sensory
confinement situations or when frequent hauling of satisfaction to the horse and owner. The appearance,
animals precludes transport of bulky-type feeds. texture, aroma, ease of handling and palatability (from
Because nutrient levels in complete feeds can be the standpoint of the horse’s apparent enjoyment) are
precisely defined while still providing adequate major considerations when buying feed. The value
roughage, they are gaining favor as creep rations for of a commercial horse feed has as much to do with
rapidly growing foals and even as high-performance serving the horse owner’s emotional needs as
race horse diets as well as diets for geriatric horses. providing adequate nutrient content in a cost-effective
Hay supplies tend to be unpredictable at racetracks, manner. Successful feed companies have developed
which makes ration balancing difficult. The potential profitable new product lines based upon identifying the
for increased wood chewing, boredom, colic or motivations behind the buying decisions of horse
founder of horses on complete feeds has been cited owners. Today’s horse owner has shown a tremendous
as being of concern. However, trainers using complete willingness to pay a premium price for high-quality
hay-grain feeds have reported that horses eat the products that directly acknowledge their view of the
complete pellets slower than grain alone, and that horse as both a livestock equine partner and a companion.
boredom and health problems have not increased.
Research on a complete cubed feed tends to support
these observations (Younglove et al., 1994). References
Environmental stress, intense exercise training, large
amounts of concentrated high-energy feeds, small Ammerman, C.B., D.H. Baker, A.J. Lewis (eds).
rations of forage and a low feeding frequency per 1995. Bioavailability of Nutrients for Animals:
day are associated with gastric ulcers in horses (Feige Amino acids, Minerals and Vitamins. Academic
et al., 2002). Press.
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horse’s environment has to do with management of Kobe, B.D. Nielsen and S.W. Eberhart. 2002.
excess nutrients in fecal and urinary waste. Excess Drinking salt water enhances rehydration in horses
nitrogen, phosphorus, trace minerals such as copper dehydrated by frusemide administration and
and zinc and other nutrients fed in excess may be endurance exercise. Equine Vet. J. Suppl. 34:513.
subject to greater scrutiny and governmental
K.H. Kline 343
Butudom, P., S.M. Axiak, B.D. Nielsen, S.W. the digestibility of fibre in trotting horses. Equine
Eberhart and H.C. Schott, Jr. 2003. Effect of Vet. J. 32:27.
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Butudom, P., D.J. Barnes, M.W. Davis, B.D. Nielsen, of a meal fed before exercise alters substrate use
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administration and endurance exercise. Vet. J. horn abnormalities in Lipizzaner horses and the
167(1):72-80. effect of dietary biotin on macroscopic aspects of
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J.R. Kohnke 345
Bone biomechanics: a review of the influences of exercise and nutritional

management on bone modeling in the growing and exercising horse
JOHN R. KOHNKE
John Kohnke Consultancy Pty Ltd, Rouse Hill, New South Wales, Australia
Introduction
A strong, well-conformed and developed musculo- If the increase in loading force exceeds the rate at
skeletal system is essential to provide structural support which the cross-sectional area and mineral density
and limb soundness in exercising horses of all ages. of cortical bone can adapt, then there is a risk of
These needs are greatest in the athletic horse to bone reaction. This results in bone surface (periosteal)
withstand the loading, strain and concussive forces inflammation; and in bone subjected to the repeated
imposed on bones and joint structures. Bone and joint stress of high loading, there is the risk of bone shaft
injuries account for up to 70% of the downtime or and joint microfracture and failure (Davies, 2001).
lost training days in racing and equestrian event horses, One of the classic examples of the failure of bone
with the type of musculo-skeletal injury changing modeling to increase bone mass and resilience at an
during a horse’s athletic career (Bailey, 1998). optimum rate in response to exercise loading is dorsal
The lower forelimb below the knee of horses is metacarpal disease, commonly known as shin soreness
most likely to develop structural damage leading to or ‘bucked shins’ in young Thoroughbred racehorses.
lameness in all classes of athletic horses (Davies, (Bailey, 1998; Lawrence, 2003b; Davies, 2003). A
2003). Over the past decade, there have been similar overloading-induced reaction and metacarpal
numerous studies that have provided insight into the fracture also affects the metacarpals of racing
reactive properties of bone and limb structures as greyhounds, referred to as metacarpal periostitis,
they adapt to the increase in body weight and exercise when dogs are exercised on a ‘too fast – too early’
loading from birth and throughout a horse’s life program on a tight turn racetrack (Boemo, 1998;
(Lawrence, 2003b). Ireland 1998).
Musculo-skeletal unsoundness, particularly related The highest weight loading is imposed on the bone
to bone failure and joint injury in racing, equestrian structure of the front limbs of racing Thoroughbreds
and other athletic horses, can be linked to overloading when cornering at the gallop at speeds of up to 15
of bone structures relative to the body weight of the meters/second (Bailey, 1998). Studies have shown
horse, the age at which the young horse is first that loading forces on the front limbs of up to twice
worked, the speed of exercise and degree by which that of a horse’s bodyweight are imposed when
the bone and joint structures are able to adapt over galloping in a straight line (Lawrence, 2003b). For
time to additional body weight and loading forces example, when a horse is galloped around a corner
(Ireland, 1998; Davies, 2001). After each period of on a racetrack, an estimated combined centrifugal
exercise, even in growing horses, the bones remodel and momentum-related loading force of up to 5-10
or react to increase strength, circumference and times the animal’s body weight is placed on bone
mineral density (collectively called bone mass) while and joint structures in the lower limbs (Ireland, 1998).
internal trabecular structures adapt to withstand the Up to 80% of lameness conditions occur in the front
increased loading forces. This is a relatively slow limbs of galloping racehorses, with up to 80% of
process, which can take up to four months to complete these injuries and failures or ‘breakdowns’ focused
in a progressive remodeling process in the young on the bones, joints, ligaments and tendons below
horse exercising during the growth phase or in athletic the knee (Bailey. 1998). Failure of the musculo-
training before maturity (Lawrence, 2003b). skeletal system is most often associated with injury
346 Bone biomechanics
to the hard tissue or bone structures of the lower changes in cortical shape of bone in horses as they
limb, with tendons and joints also being subjected to progress through a training program. A technique of
‘wear and tear’ and overload in over-galloped young Radiograph Index to measure the cortical thickness
horses (Lawrence, 2003b). of the front cannon bones has been developed and is
used to assess the state of remodeling relative to the
stage of training in young Thoroughbred horses, and
Bone development in growing horses may be used to predict the extent of bone reaction
and shin soreness (Davies, 2003).
Bone is a dynamic living tissue that is responsive to
the loading forces imposed during exercise from birth,
with most response occurring in the first six months Bone adaptation to exercise
of life. A number of studies have indicated that joint
diseases or the failure of proper joint and associated One of the earliest studies in America on
bone development known as osteochondrosis, which Thoroughbred and Quarterhorse weanlings concluded
refers to a primary lesion or defect in the growth and that bone strengthens by increasing its mineral
maturation of cartilage and subchondral bone, is most density, principally by depositing calcium within
common as cartilage changes to bone as a foal grows bones (Raub et al., 1989). These researchers found
(Jeffcott, 2001; Firth, 2003a; Lawrence, 2003a). The that over an 111-day period the cannon bones of
mineralization of the cartilaginous skeletal bone weanlings exercised by trotting, initially over 400
structures commences in the unborn foal during the meters and increasing to over 4 km per day,
last three months of gestation as it doubles in size accumulated 25% more bone calcium than weanlings
prior to birth (Jeffcott, 2001). that had been stalled overnight and turned out into
At birth, the foal’s skeleton contains only 17% of pens during the day.
the mature bone mineral content, increasing to 68.5% Numerous other studies, cited by Jeffcott (2001),
by six months of age, and 76% by yearling age in Firth (2003a) and Lawrence (2003b) in a review of
athletic breeds of horses (Lawrence, 2003a; Firth, osteochondrosis and response to exercise in horses,
2003a). Studies have shown that cartilage defects have reported similar adaptive responses in foals and
occur within the cartilage of the joint surface or at weanlings up to five months of age, with a lower
the epiphyseal junction, or growth plate on the ends bone density in the cortical shaft and subchondral
of the long bones, as the skeleton develops in the bone mass in non-exercised compared to exercised
growing horse. young horses. After six months of identical exercise,
The immature bone is composed mainly of cortical the young horses in the original non-exercised group
or external wall or compact bone, and cancellous or remodeled the bone, in this case the stifle joint, to
porous, trabecular reinforcing bone within the bone’s establish an equal mineral density and cross-sectional
internal structure (Lawrence, 2003a). Mineralization area (Lawrence, 2003b).
of cortical bone with deposition of calcium, The mineralisation of the skeletal and the structural
phosphorus and magnesium, the principal bone subchondral base of joint cartilage, as well as the
minerals, continues as the body weight and exercise calcification of long bones and formation and internal
loading increases throughout the first year of life. organization of the tendon structure, is largely
Subchondral bone and structural shaft bone must completed by six months of age. It has been
continually adapt, strengthen by additional established that the amount and type of exercise has
mineralization and repair themselves when subjected a direct influence on joint cartilage, subchondral bone,
to loading, compression and concussion during bone cortical mass and maturation in young horses
exercise as the skeletal system develops in the young as they develop.
horse. After each period of exercise, the bones react It is important that young growing horses have
to remodel or adapt their internal structure and access to free paddock exercise to encourage the
strength by increasing the cortical cross-section, formation of sound cartilage and subchondral bone,
circumference and mineral density (Lawrence, 2003b; while over-exercise and excessive weight loading in
Davies, 2001). heavy weight young horses can result in damage to
The area of cortical or shaft bone increases from the developing joint cartilage and subchondral bone
around 30% at birth to 60% at six months to 80% of in joints (Firth, 2003a).
the skeletal bone structure in the mature horse (Firth, Jeffcott (2001) and Firth (2003a) concluded that
2003b). Davies (2001) describes the remodeling and the effects of high energy:nutrient ratios, confinement
J.R. Kohnke 347
to small yards and over-exercise or lack of adequate abnormal bone turnover during exercise. Studies
exercise, high weight loading and inadequate trace using radio-isotope labeling of bone calcium and other
minerals and calcium balance, all adversely influence minerals, as well as traditional radiographs of the
the cartilage development, bone mineralisation and bones, can help monitor the structure and density of
maturation of the skeleton in the formative years of the bone. Recent developments, such as scintigraphy
a horse’s life. of the bone, can map the responsive nature of bone
and identify the sites where active remodeling and
calcium deposition is occurring as new bone is formed.
INCREASED BONE TURNOVER These studies have shown that certain bones in the
horse’s skeleton undergo a high rate of repair and
One of the other major problems in bone repair is remodeling during training. If this rate of remodeling
triggered by the stress of everyday training. The bones is unable to be maintained to strengthen and rebuild
must continually adapt and remodel themselves to bone in response to exercise, the bone itself is more
maintain strength in response to training. The stress likely to fracture and fail when loaded. These include
of continuous high loading results in the bone itself the edges of the pedal bone in the hooves, the
becoming less dense during this modeling process, sesamoid bones behind the fetlocks and the knee and
triggered in response to ongoing high load exercise. hock bones in racing and hard working horses
During each hard workout, the high stress loading (Firth, 2003b).
on the bones can cause microfractures or other
microscopic changes within the cortical bone,
ultimately causing a repair response within the bone ADAPTATION TO FAST EXERCISE
(Davies, 2001).
Studies have investigated the effects of exercise on
the bone density and strength in growing and adult
BONE STRESS CONDITIONS horses at both training and racing intensities of
exercise. It has been revealed that when a horse is
Studies of shin soreness in Thoroughbred racehorses worked below its maximum speed during long
have shown that after a hard gallop over 200-300 periods, in both young and adult horses, there is no
meters (1 – 1½ furlongs), the cannon bone in a young dramatic alteration in the density or mass of bone in
horse becomes reactive and attempts to strengthen the front cannon bones (Lawrence, 2003b). However,
itself by depositing calcium within its front cortical once horses start galloping at a much faster speed in
wall for the next 10-12 days (Nunamaker et al., 1990; fast work, there is an increase of bone density relative
Davies, 2001). When too many ‘breeze-up’ or ‘all- to the speed of exercise, and a reduction in the porous
out’ gallops are given successively at 2-3 day intervals structure of the bone as more calcium is loaded into
in an accelerated ‘get fit’ or ‘too fast – too early’ the bone to strengthen it in response to exercise
training program, the bone itself cannot respond (Davies, 2001).
rapidly enough, and can become inflamed, as occurs
in shin soreness (Davies, 2001).
Once horses reach maturity at four years of age, DORSAL METACARPAL DISEASE (SHIN
the bone remodeling process is less active. Studies SORENESS OR BUCKED SHINS)
have shown that in the young horse, an injury to a
bone can affect the response within 2-5 days, but in Dorsal metacarpal disease, or ‘bucked shins’ in the
an older horse this may take up to 10-12 days, severe case, is a common example of bone overload
depending on the type and extent of the exercise and associated bone reaction. Thoroughbred trainers
overloading (Firth, 2003b). in the United States, Australia and New Zealand often
have young horses that develop dorsal metacarpal
disease. It is a widespread belief held by many trainers
ABNORMAL BONE TURNOVER in Australia that young Thoroughbreds in early race
training need to develop shin soreness to ‘toughen’
In response to high loading forces, the bone needs to their cannon bones (Bailey, 1998). This condition,
remodel to either repair itself, or to strengthen the however, is a symptom of excessive bone loading
target site where the overload has occurred. In the resulting from a program of too fast-too early pace
athletic horse, there are a number of sites that are work or breeze-ups.
more likely to fracture when working because of
Although much research has been carried out surface (moisture content and compaction) and
investigating the classic changes that occur (Nunamaker design (banking and length of straight gallop), radius
et al., 1990; Davies, 2001), many researchers fail to and crossfall of bends and end circles, seasonal
understand the relationship between speed and radius conditions and the speed and distance of fast exercise
of a circular training track. Ireland (1998) highlights in the training program (Ireland, 1998; Bailey,
the relationship between body weight, speed and radius 1998; Table 1).
of the corner on a race track that influences the rate The leading foreleg has a 20% higher risk of
of injuries and incidence of metacarpal periostitis in developing shin soreness, although both front
young greyhounds analogous to shin soreness in horses. cannons are affected in 70% of young horses (Bailey,
Ireland (1998) provides evidence of a direct 1998). Colts and fillies are affected equally. The
relationship of these factors to the degree of centrifugal direction of cornering also influences the severity
force loading that occurs when a horse or greyhound of shin soreness on the inside limb next to the running
sprints around a bend on a racetrack. When the track rail, with the leading inside foreleg cannon bone
surface has an adequate crossfall to the inside rail or developing a more severe periosteal inflammatory
is banked in proportion to the speed of movement response and remodeling reaction. The cannon bones
and radius of the track corner, the horse is able to must change their cross-sectional shape to provide
lean over to be perpendicular to the track slope and reinforcement along the stress pathways imposed by
the centrifugal force is negated, reducing its high high speed exercise (Davies, 2001). It is a slow natural
loading forces on the limbs (Ireland, 1998). process that must happen to enable the bones to
Galloping in a straight line will not induce periosteal withstand high-speed strain forces without
reaction to the same degree as occurs when the cannon developing stress related microfractures that radiate
bones are loading as a horse is galloped around a into the anterior cortex of the cannon bones.
compacted, inadequately banked bend on the racetrack.
In Australia and the southern hemisphere, dry Underlying causes
compacted track surfaces that increase limb concussion
combined with over-galloping young horses on circle Dorsal metacarpal disease has been shown to be
tracks are the major contributing causes of forelimb initiated by increased strain forces from concussion,
metacarpal periostitis and shin soreness (Bailey, 1998). increased weight forces and centrifugal turning forces
Shin soreness in young horses can vary in severity, in horses exercising at racing speeds on tracks with
but it describes the inflammation and pain that unbanked, small radius bends (Ireland, 1998). The
develops over the front surface of the foreleg cannon cannon bones change in shape and diameter as the
bones in young racehorses galloping at racing speed. horse adapts to training (Ireland, 1998; Davies,
A much better understanding of bone remodeling 2001). Very high bone strain, compression and cyclic
processes has led to revised training and management loading forces in the front third metacarpal (shin)
guidelines to reduce the risk of it developing in young bones have been recorded in early training (Davies,
racehorses. 2001).
When a young horse is worked below maximum
Incidence speed for extended periods, there is no dramatic
alteration in the density or mass in the front cannon
Surveys indicate that the risk of metacarpal remodeling bones (Davies, 2001). Generally the risk of shin
in horses (and greyhounds) is influenced by the track soreness can be reduced by adopting a revised training
Table 1. Incidence and reasons for occurrence of dorsal metacarpal disease in the UK, US and Australia1.
Incidence (%) Training influences
United Kingdom 9 – 17 Soft training surfaces, on peat moor areas, straight line gallops, long pre-race preparations
(12-14 wks) – low risk of bone strain and adequate time to adapt.
United States 65 – 70 Dirt tracks, some banking on tight end circles, 10-12 weeks race preparation – increased bone
stress and less time to adapt.
Australia 60 – 80 In Australia, most horses are trained on circle racetracks. Dry hard track surfaces, small radius
bend(s) in tracks, no banking, 8-10 weeks race preparation – high risk of bone strain and
overload, little time to adapt to racing speed with a ‘too fast-too early’ program.
1
Bailey, 1998
J.R. Kohnke 349
program allowing the cannon bones to be challenged developed a management program which can be
with controlled strain loads by galloping over short implemented by race trainers to significantly reduce
distances at an earlier stage of training (Nunamaker the risk of bucked shins, or assist the rehabilitation
et al., 1990; Boston and Nunamaker, 2000). This of horses that have developed severe periosteal
incremental loading pattern allows the bone to inflammation and cortical microfractures. This
undergo the required remodeling changes with more program is based on the research outlined in this
time to adapt to fast work at racing speeds. If a horse review and is intended as a practical guide.
is pushed too fast too early, the stress loading on the
immature bone stimulates emergency modeling with
deposition of weaker fibrous elastic bone to reinforce GENERAL INFORMATION
the bone so that it can withstand the forces of galloping
(Davies, 2001). Most Thoroughbred racehorses train at speeds much
Centrifugal force (Cf) or loading on the cannon bone slower than those at which they need to race to be
is related to the following equation (Ireland, 1998): competitive, where they can reach peak speeds of up
to 50 km/hr (15 meters/second). Therefore, a horse
Cf ≅ (BW of horse + rider) x Speed² may be modeling its cannons to withstand the loading
Radius of the track end circle imposed by slower conditioning, which is much
lower than the strains imposed at racing speed. Thus,
This equation illustrates that heavy, faster 2-year olds when the horse actually undergoes prolonged fast
galloping at racing speeds on unbanked, compacted work, such as a barrier trial or its first race, the risk
small tight circle tracks have a higher risk of bone of sudden and intense bone overload and damage is
overloading and bone reaction. increased, especially if it is galloped at speed around
When a horse is turned out and rested in the a tight circle track after being trained on a relatively
paddock after going acutely shin sore, with shortened straight track. The cannon bone has to model from a
stride, swollen, painful ‘bucked shins’ on the anterior circular, even-walled cross-section in the immature
surface of the forelimb cannon bones, the weak horse to a cross-section with a thickened inside edge,
fibrous bone is resorbed, returning the bone to its with up to twice the average wall (cortical bone)
immature cross section and lower load bearing thickness of the cannon bone shaft.
capacity over a period of 4-8 weeks (Boston and A technique of radiographic index (RI) with
Nunamaker, 2000). radiographs taken to measure the thickness of the
Ideally, the front cannon bones should be bone wall (cortical bone) on the front and inside edge
encouraged to adapt slowly from the start of training, of the front cannon bones at weekly intervals can
allowing accumulation of extra high density help monitor the adequacy of the remodeling process
mineralised cortical bone to strengthen the anterior in response to bone loading during training. If the
surface of the cannon bones to enable them to RI exceeds the established value at a set stage of
withstand concussion and increased weight bearing training, it is an indication that excessive bone
loads. Rapid reinforcement with fibrous bone involves remodeling and inflammatory reaction has occurred.
the reabsorption of calcium from the sides to In this case, the speed and distance of fast work should
strengthen the front surface, reducing the bone’s side be reduced to allow the remodeling process to catch
rigidity or ‘stiffness’ against bending under high strain up to the degree of strain loading. If the RI is below
loads. This can result in painful stress fractures the established limits at a particular stage of training,
developing on the anterior surface of the front cannon it is possible that horses that are over-galloped have
bones as they distort under extreme loading forces a high risk of developing shin soreness.
of high speed galloping and symptoms of ‘bucked Many trainers believe in long, slow work to ‘build
shins’ and risk of metacarpal fracture and failure bone’. While this program works to build and
during racing (Nunamaker et al., 1990). condition the muscles, joints and limbs, the bone that
the horse is building is more suited to long, slow
work rather than to racing. If the shins become
Practical management for prevention severely inflamed with deposition of a bone splint or
a ‘bucked shin’ swelling, the bone laid down is a
Based on my 30 years of experience as an equine weaker form of fibroelastic bone as a temporary
veterinarian and my professional role as a consultant reinforcement to prevent bone failure. This type of
to many racehorse trainers in Australia, I have bone has little residual strength; and if the horse is
rested from training, this temporary reinforcement vitamin D to provide adequate minerals for bone
‘splint’ is resorbed. The bone profile or cross-section development and modeling in response to loading as
returns to its original weaker structure that is unable the shin bones model as they thicken and adapt in
to withstand the increased loading imposed by cross sectional shape to withstand the increased stress
galloping too fast-too early when training resumes. loads.
Harness horses do not develop sore shins because
they race at slower speeds on banked tracks and have Train on the same track
two legs on the track surface at any one time to share
the additional loading. It is best to train on the same track, so that the bends
Training programs should be designed to allow the and surfaces are consistent, avoiding compacted areas
cannon bones to experience and adapt to the loading on the rails when galloping on fast work mornings.
and deformation strain imposed by higher speed The majority of racetracks have training tracks built
galloping. The leading forelimb is subjected to the inside the main grass, turf surface or sand track. These
greatest amount of strain, particularly when cornering, have smaller end circles and can impose additional
so it is recommended that the initial high speed work loading, especially on odd shaped, rather than oval
should be carried out down the straight to model the track designs, constructed to provide the length of
shins, reducing to a slower pace around the turns. straight required within the available land area.
Short gallops in early training Avoid heavy work riders
Commence a program of short, straight-line gallops Once the young horse is controllable, change to a
over 200 meters twice weekly (at least 4-5 days apart) fast work rider (50-60 kg) to reduce excessive loading
from the third week of training to progressively load as the horse is worked faster at the start of regular
the cannon bones. Short gallops in a straight line do fast work. Each extra kg of rider weight is magnified
not cause undue stress on bones or muscles after 2-3 5-10 times by centrifugal forces when galloping
weeks of initial conditioning exercise. As the horse around bends.
develops in fitness, increase the speed and distance
traveled to 300-400 metres at each fast workout. Bone Minimise concussion
modeling is a slow process as is bone fracture healing, Cushion depth and type of surface is important to
so a stepwise increase in loading over 8-10 weeks reduce concussion, vibration and ‘jarring’ on the front
will allow time and stimulate modeling in response cannon bones. Properly maintained synthetic,
to the loading forces imposed. A measurement of woodchip and grass tracks are less concussive on the
Radiographic Index to evaluate the remodeling legs than dry finely packed sand surfaces. Avoid hard
response of the bone can be carried out at weekly compacted cinder or dirt tracks with tight bends and
intervals to monitor the bone thickness in response little banking. Work out from the compacted areas
to fast work. away from the rails and choose dampened, more
cushioned areas, if possible, for galloping.
Avoid tight circle tracks
Do not gallop young horses around tight, compacted Cold therapy after galloping
curves or end circles on the track too early in their training Apply cold therapy, such as cold water hosing for
preparation. Gallop only up straights initially, then work 10-15 minutes, or preferably by ice boots or an ice
progressively faster into corners and around end circles block under a pressure bandage for 5-10 minutes,
to allow adaptation to the centrifugal sideways strain after each gallop in the later stages of training. Ice
forces, starting after 6-8 weeks of training. Avoid sudden packs can be wrapped on both front cannons during
introduction to fast work and limit speed of galloping the walk to cool-out, or during the trip home from
around unbanked, relatively tight bends initially, the track to help reduce minor inflammation and
especially on dry, compacted tracks. soreness as training progresses. A magnetic bandage
with 1500 gauss field strength wrapped on both
Adequate calcium and vitamin D
forelimb cannon bones may be helpful to increase the
Ensure high grain diets are supplemented with rate of deposition of calcium and bone minerals and
calcium, phosphorus, magnesium, vitamin A and hasten the modeling and strength of bone laid down as
J.R. Kohnke 351
it adapts to higher loading during fast work. The important in the last trimester that pregnant mares be
bandage can be wrapped in the afternoon and overnight provided with an adequate amount and balance of
when the horse is resting in its stable or yard. macrominerals, trace minerals and vitamins to ensure
the formation of cartilage and bone framework prior to
Check shins regularly for heat or soreness birth in the unborn foal. It is important that trace
minerals such as iron, copper, zinc, manganese and
Watch for abnormalities in gait and shortening of vitamins including vitamins A, D and E are
stride. If early soreness is detected, cut back on the supplemented or incorporated into feed mixes to allow
speed of fast work for 2-3 weeks to allow the shins adequate foetal liver storage prior to birth.
adequate time to model and thicken the bone along After birth, during the more rapid phase of growth,
the stress pathways. Apply cold packs after training, a low glycaemic diet should be provided, containing
or if discomfort is more severe, apply a warming adequate energy and quality protein as well as
liniment overnight or alternatively (not at the same minerals, trace minerals and vitamins to meet the
time) a clay poultice overnight until symptoms growth and mineral needs of the developing
subside. In severe cases, it is best to rest the horse for musculoskeletal structure. It is beyond the scope of
3-4 weeks until the soreness settles down, and then this paper to provide specific guidelines. However,
recommence on a revised program as outlined above. there are a number of aspects of feed formulation
When horses are rested, the cannon bone actually that should be kept in mind when blending feeds,
becomes even less rigid as modeling of new bone occurs premixes and supplement concentrates for use in
along the stress lines. growing and exercising horses. These issues are rarely
addressed by formulators or advisory consultants.
Re-program training after down time These include:
For each week that a young horse is rested because
Limit the concentration of alkaline mineral
of other problems (e.g. respiratory disease, joint
sources.
problems, severe tying-up), back step its training
program by two weeks to avoid shin soreness as the Limit calcium carbonate, magnesium oxide and
cannon bones may start to resorb calcium and weaken potassium chloride in the fines of a textured feed,
during the lighter work period. premix or supplement which also contains trace
minerals and vitamins. In textured feeds, calcium
carbonate is commonly included in the fines at a rate
Compounding feeds to promote skeletal of 1-3% by weight to provide calcium. In premixes
development and supplement concentrates, it is often used as the
carrier at 50-80% of the total batch weight. The
The requirement for a balanced and well formulated alkalinity of these carriers in the fines or bases can
diet is paramount to ensure optimum growth and reduce the bioavailability and accelerate the
skeletal development, as well as remodeling in degradation rate of vitamins surrounded by an alkaline
response to exercise, in young horses in their environment, particularly in a moist feed blend or
formative years. It is important that feeds are premix. The majority of essential vitamins, including
formulated to provide adequate digestible nutrients retinol, cholecalciferol and tocopherols, thiamin,
to meet these requirements. There is a wealth of riboflavin and cyanocobalamin are more stable when
information and nutrient recommendations from the pH is maintained between 5-6 in the fines and
equine nutritional research over the past two decades. premix carrier. Compounding a range of vitamins
Most companies that produce blended feeds, or large into a pelleted form for blending with alkaline carriers
horse breeding farms and training stables are provided would significantly reduce the destructive interactions
with up-to-date advice by equine specialists. during storage. Overages of vitamins should be
included to counteract the destructive interactions
when these alkaline carriers are used. (Kohnke, 2002).
RATION FORMULATION FOR GROWTH AND The addition of calcium hydrogen phosphate, also
DEVELOPMENT PHASES known as dicalcium phosphate (DCP), an acidic
mineral source (23-25% elemental calcium, 18%
It is important that rations are formulated to meet the
elemental phosphorus) as a dual calcium and
various phases of reproduction in mares. It is particularly
phosphorus source, blended in a 60% dicalcium Copper, zinc and manganese, along with selenium
phosphate to 40% calcium carbonate ratio, relative and vitamin E, are important factors in natural
to the mineral balance required, will maintain a more antioxidant defence. It is important to include
neutral pH environment to improve vitamin stability. adequate levels of antioxidant nutrients to limit the
Calcium carbonate (limestone carrier) has a pH of rate of lipid peroxidation and rancidity, particularly
8.41 (alkaline) when measured using 10 g in 20 ml in feeds fortified with omega-3 (n-3) and omega-6
distilled water while dicalcium phosphate has a pH (n-6) fatty acids from vegetable oils. Omega-3 fatty
of 6.68 (slightly acidic). A blend of 60% limestone acids are particularly prone to oxidation during
with 40% dicalcium phosphate (12 g + 8 g in 15 mls storage, and additional levels of antioxidants help
distilled water) has a pH of 6.83. The use of buffered protect omega-3 fatty within muscle cell membranes
propionic acid mould inhibitors, such as Mold-Zap™ once the feed or premix is consumed (Dunnet, 2003).
(Alltech Inc.) at standard addition rates, also helps Careful formulation to reduce incompatibilities,
to buffer the alkalinity of carriers to help improve chemical reactions, binding and known nutrient
vitamin stability (Kohnke, 2002; 2003, unpublished). interactions will not only improve bioavailability of
In addition, calcium is absorbed more effectively minerals and trace minerals, but minimise the need
from the small intestine in an acidified environment. for excessive overages of vitamins and extend the
shelf-life of premixes and supplements.
Mineral oxides, particularly iron oxide as well as
copper oxide and zinc oxide, should be limited in
premix blends Conclusion
Oxides generally reduce vitamin stability. Magnesium The soundness of the skeletal structure in growing
oxide appears to be less destructive to vitamins when and exercising horses is largely dependent on
used as a source of magnesium and, although it has providing an adequate diet with a balanced intake of
an alkaline pH, it can be buffered to around neutral bone and joint structural nutrients during the
by dicalcium phosphate (Kohnke, 2002; 2003). formative years of a horse’s life. Controlled exercise
will assist in the skeletal development and allow
Selenium and chromium from yeast: Sel-Plex® and remodeling in response to loading in both growing
BioChrome ® and exercising horses.
Care when formulating feeds, premixes and
These minerals, critical to antioxidant protection (Se)
supplements to ensure optimum bioavailability and
and energy utilisation (Cr) are more easily
stability of skeletal nutrients is essential to maintain
metabolised and retained than inorganic forms
long term athletic soundness in all classes of horses.
(Dunnett, 2003).
Mineral proteinates: Bioplex® trace minerals

References
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K.J. McDowell 355
Of caterpillars and horses: recent scientific progress on the cause of Mare

Reproductive Loss Syndrome
KAREN J. MCDOWELL
Department of Veterinary Science, University of Kentucky, Lexington, Kentucky, USA
Introduction
An abortigenic disease, now known as Mare Bruce Webb (Entomology, University of Kentucky),
Reproductive Loss Syndrome (MRLS), significantly Dr. Neil Williams and Dr. Mike Donahue (Livestock
impacted the horse industry in the central Ohio Valley Disease Diagnostic Center, University of Kentucky),
in late April and early May, 2001 and 2002. In 2001, and Dr. Kyle Newman (Venture Laboratories,
approximately 25% of all pregnant mares aborted Lexington, Kentucky). The studies have followed a
within several weeks (over 3,000 mares lost deductive approach, with the results of each
pregnancies), and abortion rates exceeded 60% on experiment defining the questions posed in the next
some farms. MRLS struck hard and without warning, experiment. Details of the first four experiments can
was caused by something in the environment, was be found in Webb et al. (2004). For all of these
not transmittable between animals, and was not experiments, pregnant mares were housed
associated with any known abortigenic agent or communally on grass paddocks and supplemented
disease. It is a newly recognized disease, and it cost with hay as needed, with water available ad libitum.
the state of Kentucky ~$330 million in 2001 alone Pregnancies were monitored by manual palpation and
(Thalheimer and Lawrence, 2001). Initial research real-time ultrasonography weekly during the first
in the summer and fall of 2001 was targeted toward experiment, and daily in all subsequent experiments,
specific agents, such as mycotoxins (Newman, 2003), beginning prior to the first day of treatment and
fescue toxicosis (Schultz and Bush, 2003), cyanide continuing for at least 30 days. Aborted fetuses and
(Harkins et al., 2003), mandelonitrile (Camargo et placentas, as well as blood samples from the mares,
al., 2002), weather (Priddy et al., 2003), ionic were taken to the Livestock Disease Diagnostic Center
imbalance and poison hemlock (Powell, 2002), (LDDC) for necropsy, histopathology, bacteriology,
among others. To date, none of these factors has been serology, and virology.
demonstrated to reproduce MRLS. A comprehensive
review of the history of MRLS and the initial
Eastern tent caterpillars cause mare
experiments is provided in Powell et al., 2003.
An epidemiologic survey conducted by Drs. Roberta abortions
Dwyer, David Powell and co-workers revealed a In April and May, 2002 (the first opportunity to
temporal correlation between MRLS and presence perform experiments with ETC after the 2001 MRLS
of eastern tent caterpillars (ETC; Malacosoma outbreak), two experiments were conducted to
americanum) on horse farms (Dwyer et al., 2003). determine if ETC could actually cause fetal loss in
A statistical correlation does not necessarily mean pregnant mares. For 20 days (first experiment) or
that the caterpillars caused the abortions, so several for 10 days (second experiment), for 6 hrs each day,
groups of scientists from around the country designed pregnant mares were placed on grass pasture in 16 x
experiments to determine the role ETC play in MRLS. 16 ft pens, and ETC were released into the grass in
The experiments summarized here are the results the pens. Each day all pens were moved to a new
of collaborative efforts by Dr. Karen McDowell area of pasture so that the mares would have fresh
(Veterinary Science, University of Kentucky), Dr. pasture to graze while in the pens.
356 Of caterpillars and horses: recent scientific progress on the cause of Mare Reproductive Loss Syndrome
In the first experiment, 18 of 29 mares exposed to abortion, as is typical of MRLS. This experiment
ETC and/or nest materials lost their pregnancies. In demonstrated that the abortigenic activity of ETC
the second experiment, three of seven mares exposed was stable to freezing and was destroyed by
to ETC lost their pregnancies (P<0.05), while none autoclaving. Whether the abortion in the mare fed
of six mares exposed to ETC frass (excrement) and GMC was due to treatment or would have occurred
none of the seven control mares (not exposed to ETC, regardless of treatment cannot be determined from
ETC frass or nest materials) aborted. Signs of this experiment.
abortion were consistent with MRLS, e.g. few to no
premonitory signs of impending abortion in the
mares, increased echogenicity of fetal fluids prior to The abortigenic activity is present in the
fetal death and abortion, and presence of actinobacilli caterpillar exoskeleton, and cannot be
or non-beta-hemolytic streptococci in fetal fluids and
fetal tissues. In the second experiment, blood samples
extracted with aqueous or organic
were collected from the mares daily for hematology, solutions
and every third day for clinical chemistry and serum
Two experiments, performed in May and June, 2003,
progesterone and estradiol concentrations. None of
were designed to determine which physical portion
these measurements were good predictors of which
of the insect was abortigenic, and whether the activity
mares were affected by MRLS. Serum concentrations
could be extracted with aqueous or organic solutions.
of both hormones declined associated with abortion,
ETC larvae were collected in central Kentucky in
but the decline was after changes in fetal fluid
April and May of 2003 and stored frozen at -80oC.
echogenicity were apparent. Results of the first
Pregnant mares (~45-100 days gestation) were fed
experiment were released to the public on April 26,
individually from feed buckets, with treatments
2002, before MRLS abortions were reported in the
(representing 50 g ETC larvae/mare/day for 10 days)
area, and in sufficient time for farms to take steps to
mixed in sweetfeed.
reduce exposure of pregnant mares to ETC.
In the first experiment, 35 mares were randomly
divided into seven treatment groups of five mares
each. For the first two treatments, mares were fed
Effects of eliminating pasture grass, and ETC and served as positive controls, or fed saline
freezing or autoclaving ETC and served as negative controls. For the next three
treatments, mares were fed ETC that had been
In the next experiment (August 2002), the effect of carefully dissected into three portions, the
pasture was removed by mixing ETC treatments with exoskeleton, including setae (caterpillar hairs), the
sweetfeed, and feeding the mares from feed buckets. gut, or the remainder of the internal insect tissues.
Additionally, the effects on abortigenic activity of Each of the three portions of the insect constituted a
freezing larvae or exposing them to high heat by separate treatment fed to mares. For the final two
autoclaving were tested. Finally, whether another treatments, ETC were homogenized in saline and then
hairy caterpillar, gypsy moth caterpillar (GMC, separated by size (larger than 0.45 microns or smaller
Lymantria dispar), could cause abortions in pregnant than 0.45 microns), and mares were fed either the
mares was evaluated. Pregnant mares, ~40-120 days larger sized fraction or the smaller sized fraction.
gestation, were fed ETC larvae that had been collected Fetal losses occurred in all five mares fed ETC, in
during April and May, 2002 and stored frozen (n= 5 three mares fed ETC exoskeleton (P<0.05), and in
mares), frozen ETC larvae that had been autoclaved one mare fed the larger sized fraction of the
and refrozen (n=5 mares), or frozen GMC (n=4 homogenized insects. No losses occurred in the
mares) (50 g insect/mare/day for 8 days). Blood negative control (saline) group, in mares fed other
samples were collected daily from each mare. ETC tissues (gut or internal tissues), or in mares fed
Three mares fed frozen ETC aborted (P<0.05) with the smaller sized fraction of the homogenized insects.
signs consistent with MRLS, and none of the mares Increased echogenicity of fetal fluids prior to fetal
fed autoclaved ETC aborted. One mare fed frozen death and bacteriologic findings in fetal tissues (non-
GMC aborted. Signs of abortion in the ETC-fed mares beta-hemolytic streptococci or actinobacilli) were
were consistent with MRLS, but the abortion in the consistent with MRLS as the syndrome is recognized
GMC-fed mare was not. The fetus from the GMC- in the field. This experiment demonstrated that the
fed mare did not grow at a normal rate and there was abortigenic activity was associated with the
not an increase in fetal fluid echogenicity prior to
K.J. McDowell 357
exoskeleton of the insect and that it could not be ETC larvae were collected in central Kentucky in
extracted with an aqueous solution. April and May of 2003 and stored frozen at -80oC.
The second experiment was designed to further Ten pregnant gilts, at 54-56 days of gestation at the
fractionate the ETC exoskeleton, in order to learn beginning of the project, were randomly assigned to
more about the physical and/or chemical nature of receive ETC (40 g ETC larvae/gilt/day for 10 days,
the abortigenic activity. Twenty-five pregnant mares mixed with their normal gestational ration; n=5 gilts)
were randomly divided into five treatment groups of or to serve as controls (normal gestational ration only;
five mares each. For the first three treatments, mares n=5 gilts). Gilts were then randomly paired, with
were fed dissected ETC exoskeleton, (positive one ETC-fed gilt and one control gilt in each pair.
controls), corn oil (negative controls), or ETC Two of five gilts fed ETC aborted their entire litters
exoskeleton that had been crushed with a mortar and (P=0.4444). Those two gilts and their paired controls
pestle to disrupt its physical integrity. For the were euthanized the day following fetal loss. The
remaining two treatments, lipids were extracted from remaining three ETC-fed gilts and their control pairs
the ETC exoskeleton with an organic solvent, were still pregnant at the time of euthanasia, 29 days
methylene chloride. The methylene chloride was then after the first ETC treatment. Of particular interest
evaporated through corn oil, transferring the extracted was the identification of caterpillar setae (hairs)
lipids to the corn oil. The two treatments consisted embedded in the alimentary tract of all gilts fed ETC
of the corn oil containing the extracted lipids or the but in none of the control gilts (P=0.0079). The setae
exoskeleton after it had gone through the organic were surrounded by eosinophilic microgranulomatous
extraction process. lesions and were randomly scattered throughout the
Fetal loss occurred in three mares fed ETC alimentary tract of each gilt. Caterpillar setae were
exoskeleton (positive control) (P<0.05), in one mare not found in other gilt tissues examined, and no other
fed powdered exoskeleton, and in two mares fed significant macroscopic or microscopic changes or
exoskeleton after the organic extraction. Again, disease processes were identified in the gilts or
increased echogenicity of fetal fluids prior to fetal the fetuses.
death and presence of non-beta-hemolytic streptococci This study demonstrated that domestic pigs are a
or actinobacilli in fetal tissues and fluids indicated good model for the study of MRLS. It also provided
that the losses were due to MRLS. the first indication that ETC can cause fetal death
Taken together, these two experiments demonstrate and abortion in a non-equid species, and provided
that the abortigenic activity of ETC is associated only the first indication that ETC cause physical and
with the exoskeleton of the caterpillar, it is reduced potentially harmful lesions in the digestive tract of
when the physical integrity of the exoskeleton is mammals. We have since confirmed that ETC cause
disrupted, and it is not easily extracted with either similar lesions in the alimentary tract of mares.
aqueous or organic solutions.
Where do we go from here?

ETC causes abortions in pregnant gilts in
a manner consistent with MRLS The evidence is overwhelming that ETC cause MRLS.
At present, the only known way to prevent the disease
Natural occurrence of MRLS and controlled is to avoid contact between pregnant mares and ETC.
experiments have been reported in horses, and thus On some farms that is an adequate solution; on others
far it was not known if ETC could cause pregnancy it is not. There are many unanswered questions, from
loss in any non-equid animal. In this experiment, both practical and scientific perspectives. Some of
pigs were chosen because pigs and horses share these are:
similarities in fetal development and placentation, What is the minimum level of exposure necessary
both are monogastrics, and pigs have a shorter to cause abortions (how many caterpillars does it take
gestational period, ~112-115 days compared with and for how long)? When several mares are exposed
~340 days for horses. Additionally, if MRLS could to the same levels of ETC, why do some abort while
be induced in a litter bearing species, then perhaps some do not (what makes one mare more/less
different fetuses would be affected to different susceptible than the next)? What role does the mare’s
degrees, and thereby provide valuable information immune system play? Does the abortigenic factor/
on the progression of the disease. agent persist in the environment after the several
358 Of caterpillars and horses: recent scientific progress on the cause of Mare Reproductive Loss Syndrome
weeks in the spring when ETC are present? Can other Syndrome. Proceedings of the First Workshop on
caterpillars/insects cause the disease? Can mares be Mare Reproductive Loss Syndrome (D.G. Powell,
treated before exposure to prevent the disease, by A. Troppman and T. Tobin, eds). University of
immunization, by feed supplements, or by some other Kentucky Agricultural Experiment Station, August
means? Can mares be effectively treated after 27-28, 2002, Lexington, KY, pp 66-68.
exposure? The answers to these questions, and the Powell, D.G. 2002. An update on Mare Reproductive
new knowledge gained in the process of asking, will Loss Syndrome (MRLS) in Kentucky. Proc. Ann.
certainly further our understanding and management Meet. Soc. Therio. 147-152.
of MRLS. Such studies are also likely to provide Powell, D.G., A. Troppman and T. Tobin. 2003.
new insights into the of other fetal/placental diseases, Proceedings of the First Workshop on Mare
and into the physiology of normal pregnancy in horses.
Reproductive Loss Syndrome. University of
Kentucky Agricultural Experiment Station, August
27-28, 2002, Lexington, KY.
References Priddy, T., C. Pieper and W. Long. 2003. Climatic
Camargo, F., L. Dirikolu, M. Sebastian, C. Hughes, correlations of the 2001 and 2002 episodes of mare
J. Crutchfield, J. D. Harkins, J. Boyles, A. reproductive loss syndrome. Proceedings of the
Troppmann, K. McDowell, L. Harrison, and T. First Workshop on Mare Reproductive Loss
Tobin. 2002. The toxicokinetics of cyanide and Syndrome (D.G. Powell, A. Troppman and T.
mandelonitrile in the horse and their relevance to Tobin, eds). University of Kentucky Agricultural
the Mare Reproductive Loss Syndrome. 2002. Proc Experiment Station, August 27-28, 2002,
4th International Conference of Racing Analysts Lexington, KY, pp. 37-40.
and Veterinarians Orlando, FL. Schultz, C.L. and L.P. Bush. 2003. The potential
Dwyer, R.M., L.P. Garber, J.L. Traub-Dargatz, B.J. role or ergot alkaloids in mare reproductive loss
Meade, D. Powell, M.P. Pavlick and A.J. Kane. syndrome. Proceedings of the First Workshop on
2003. Case-control study of factors associated with Mare Reproductive Loss Syndrome (D.G. Powell,
excessive proportions of early fetal losses associated A. Troppman and T. Tobin, eds). University of
with Mare Reproductive Loss Syndrome in central Kentucky Agricultural Experiment Station, August
Kentucky during 2001. J. Am. Vet. Med. Assoc. 27-28, Lexington, KY, 2002, pp. 60-63.
222:613-619. Thalheimer, R. and R.G. Lawrence. 2001. The
Harkins, J.D., L. Dirikolu, M. Sebastian, C. Hughes, economic loss to the Kentucky equine breeding
J. Crutchfield, A. Troppmann, J. Boyles, B. Webb, industry from Mare Reproductive Loss Syndrome
K. McDowell, W. Long, J. Henning, L. Harrison, (MRLS) of 2001. Department of Equine Business,
T. Fitzgerald and T. Tobin. 2003. Cherry trees, College of Business and Public Administration,
plant cyanogens, caterpillars and Mare Reproductive University of Louisville, Louisville, KY. A Report
Loss Syndrome: toxicological evaluation of a Commissioned by the Office of the Governor,
working hypothesis. Proceedings of the First Commonwealth of Kentucky.
Workshop on Mare Reproductive Loss Syndrome. Webb, B.A., W.E. Barney, D.L. Dahlman, S.N.
University of Kentucky Agricultural Experiment DeBorde, C. Weer, N.M. Williams, J.M. Donahue
Station, August 27-28, Lexington, KY, pp. 68-74. and K.J. McDowell. 2004. Eastern tent caterpillars
Newman, K. 2003. Review of mycotoxins as a (Malacosoma americanum) cause Mare
possible cause of Mare Reproductive Loss Reproductive Loss Syndrome. J. Insect Physiol.
50:185-193.
Modern
AGRONOMICS
agronomics
J.P. Bower 361
The pre-and postharvest application potential for Crop-SetTM and ISR

2000TM on citrus
JOHN P. BOWER
Horticultural Science, University of KwaZulu-Natal, Pietermaritzburg, South Africa
Introduction
Crop-SetTM and ISR 2000TM are products intended to of citrus growth within any major production area.
enhance growth, yield responses and disease resistance However, sufficient generic information relating to
in a variety of crops, including citrus. Responses such the order of growth for various plant parts is available,
as fruit size and number increases, and internal quality and can thus be used. With the exception of the fruit
improvement have been obtained by applying the maturity rate, which can vary considerably with
products to a number of cultivars, including Valencias, cultivar and environmental conditions, the cycles of
Navels, mandarins and grapefruit, at various sites in vegetative growth, root growth and flowering are
the world. However, from a commercial perspective, remarkably similar in most subtropical or
considerable confusion exists as to the most desirable Mediterranean areas of the world. There is almost
timing of applications for intended plant responses. no growth cycling in true tropical areas (Davies and
In addition, the appropriate usage of the products Albrigo, 1994), and thus these regions will not be
may not always be understood. These issues are vital considered. Therefore, a generic phenological model
for the successful usage of the products. can be used as a basis for management, suitably
An understanding of the growth pattern adapted in terms of time scale, for any production
(phenological cycle) has been useful in a number of area. Such a generic phenological cycle, based on
tree crops, and most notably in avocado (Whiley et northern hemisphere dates, is shown in Figure 1.
al., 1988) for making management decisions. If the A shoot flush is usually initiated in spring, when
growth pattern of the tree is taken into account, and the temperature rises above 12.5°C (Davies and
linked with the known constituents or perceived Albrigo, 1994) or after alleviation of water stress
activity of applied compounds, then the optimal (Mendel, 1969). This is followed by a summer flush
timing for desired effects can be determined. approximately three months later. Little substantial
The objectives of this paper are to outline the vegetative growth occurs during the rest of the year,
potential for applying the products to attain particular particularly in subtropical or Mediterranean areas.
desired effects through using a generic phenological The spring flush (March – April in the northern
cycle for citrus tree growth and fruit development hemisphere) is usually far more intense, affecting
during a cropping season, by understanding the more growing points than the summer flush (January
physiological state of the trees and matching the – February).
product applications to this. The theoretical timing Root flushes normally occur during the periods
and application of the products will be confirmed between vegetative growth flushes. Bevington and
with known effects of Crop-SetTM and ISR2000TM, Castle (1985) found the major root flushes to occur
as demonstrated by experimental evidence, where this from May through June and August through
is possible. September (the latter being the major flush) in
Florida. Stimulation of root growth is both
The phenological cycle temperature and water dependent, with a minimum
temperature of 7°C considered a threshold, although
Little has been done to outline the phenological cycle rootstock may also play a role. Perhaps of more
362 The pre- and postharvest application potential for Crop-SetTM and and ISR 2000TM on citrus
Cell Division Cell Elongation Fruit Maturation
Shoots
Roots
Buds
Growth
Flowers
Fruit Drop
Fruit Growth
Feb Apr June Aug Oct Dec

Figure 1. Schematic phenological cycle for citrus in the northern hemisphere.
importance, however, is the physiological effect of (Davenport, 1990). However, once differentiation
root activity. Wilcox and Davies (1981) reported occurs, the buds will not revert (Lord and Eckard,
considerable increases in hydraulic conductivity as 1987). The rate of bud development depends on
root temperature increased from 10 to 30°C. Nutrient climate (degree days) and inflorescence type (Lovatt
uptake in particular is affected. Thus in late winter et al., 1984), but it can be assumed that macroscopic
or early spring if root temperature is still low, differentiation occurs during December – January,
yellowing (chlorosis) may occur, which will and visible buds in February (northern hemisphere).
undoubtedly affect photosynthetic capacity of the tree Anthesis then occurs in April.
and thus fruit set, growth and retention. In addition, An important characteristic of flowering shoots in
plant growth regulators are affected, with less citrus is that the inflorescence can range from floral
movement of cytokinins from the roots to shoots and only, to predominantly leafy, with mainly new flush
thus less vegetative bud break. The consequences will leaves and few flowers. The more leafy inflorescences
be further discussed in relation to flowering. Poor result in greater fruit set or retention (Monselise,
root activity and thus water uptake and movement, 1986). This could be due to the carbohydrate
may also increase the levels of the plant growth contribution of these leaves to early fruit growth, or
regulator abscisic acid (ABA) with resultant stomatal better vascular development due to plant growth
closure (Zeevaart, 1999), and consequential decreased regulators produced by the leaves (Erner and Bravdo,
photosynthetic activity, and altered nitrogen 1983). This in turn could enhance the movement of
metabolism (King and O’Donoghue, 1995), which carbohydrates, water and plant growth regulators,
may change flowering patterns (Lovatt et al., 1998) which are of particular importance (Erner et al.,
through elevated ammonia levels. Late winter or 2000) to the fruit sink strength and thus growth. Stress
early spring, just prior to bud break, therefore presents periods, when gibberellic acid levels are low or when
an opportunity to intervene and thus modify the roles auxins decrease in the fruit as cell division slows
of available endogenous tree nutrition factors and (Cleland, 1999) would be of note. Under these
plant growth regulators such as cytokinins, thus conditions, ABA may predominate, resulting in fruit
antagonising the effects of ABA. abscission. The degree of leafiness appears related to
Citrus flowering has been extensively reviewed the stress (water or temperature) intensity of the
(Davenport, 1990; Krajewski and Rabe, 1995). induction phase (Lovatt et al., 1998). Manipulation
Flower bud induction commences during the winter of leafiness characteristics is clearly an opportunity
rest period due to cold or dry conditions. The degree for increasing fruit set.
of induction is proportional to intensity of the Once anthesis has taken place, fruit growth occurs.
inductive conditions (Southwick and Davenport, Citrus fruit has a sigmoidal growth pattern. The first
1986). During this period, reversal to a vegetative phase is one of cell division, which takes
state is possible should gibberellic acid be applied approximately six weeks, and is characterised by two
J.P. Bower 363
drop periods, one from flowering for three to four addition, the product has been fortified with
weeks, followed by a second (June drop in northern micronutrients.
hemisphere) as cell division begins decreasing. This Considering the growth pattern of citrus as outlined,
is probably related to plant growth regulator changes. the first window of opportunity would be after flower
Gibberellins may play a vital role in fruit set and induction has already occurred, but before visible
early growth (Cleland, 1999) and are particularly buds are present. This could be in approximately
important in decreasing fruit drop (Talon et al., 2000) December – January in the northern hemisphere. The
while cytokinins promote cell division and with objective at this stage would be to increase the
auxins enhance vascular development. This would leafiness of the spring growth flush, and thereby
link with the effects of inflorescence leafiness. improve the potential for fruit set (retention) and
After the cessation of cell division, fruit cell growth. The cytokinin-like activity in the product
differentiation takes place, followed by a period of could enhance the potential for bud break, as well as
cell elongation (phase 2 growth). Application of cell division, while gibberellins would enhance
auxins at the end of the cell division phase enhances leafiness and decrease flowering (Guardiola et al.,
pulp growth (Agusti, 2000) resulting in larger fruit. 1982). The nitrogen and micronutrient content of
This is due to expansion of the juice vesicles (Agusti the product could also be beneficial for fruit set, if
et al., 1995), which relies on maintenance of turgor, applied at this stage. Care needs to be taken that
which in turn requires inflow of sugars to the fruit. application is not too early, as bud reversal from
The cell expansion phase is indeed the period when flower to vegetative could occur (Davenport, 1990).
loading of sugars occurs, mainly from adjacent leaves The appropriateness of the application will also depend
(Koch and Avigne, 1984). Agusti (2000) states that on season and cultivar. The product should be used
application of auxins at the start of this phase enhances where it is expected that heavy flowering will take
sucrose deposition in the fruit, which not only place, so as to enhance the leafiness of the blossom
increases sugar content, but also ensures turgor and (El-Otmani, 2000) and as a consequence improve
larger fruit size. The cell enlargement phase varies fruit set or retention.
with cultivar and climate, but is typically three to The second window of opportunity is logically at
four months (Davies and Albrigo, 1994). Thereafter flowering. Work done by Medina (2003) on navel
fruit maturation occurs. oranges in Brazil showed that an application of Crop-
Fruit maturation is characterised by a decrease in SetTM at the end of blossoming (approximately the
juice acid, small increase in sugars and change in end of cell division) resulted in larger fruit size
rind colour. The time taken for this process varies without any decrease in fruit number. Navels are not
considerably with climate and cultivar, with sensitive to gibberellin applications at fruit set (Agusti
temperature being the predominant factor. Plant et al., 1982) although many other cultivars are.
growth regulators play a role. Application of Cytokinins applied to fruit shortly after set
gibberellins decreases the rate of chlorophyll loss (Guardiola, 2000) enhanced fruit retention. Thus, an
(Agusti, 2000), while other unpublished results application of Crop-SetTM at 100% petal fall could
appear to indicate that auxin applications at be advantageous in retaining fruitlets.
physiological drop enhance colour development. For similar reasons, the third opportunity for Crop-
Auxin applications near harvest can delay fruit SetTM application is at the end of cell division. At
senescence and prolong hanging of fruit, as is done this stage, intense competition for available
in some countries. As rind maturation progresses, so carbohydrates occurs, and application of compounds
susceptibility to postharvest pathogens increases with cytokinin-like activity may aid carbohydrate
(Angioni et al., 1998). Application of gibberellins flow to the fruits (Kreidemann, 1968). The effects
or cytokinins (anti-senescence) may slow this process. of auxins are clear, as previously discussed, and the
role of micronutrients such as zinc and boron should
not be ignored. This is also a critical period in terms
Opportunities for Crop-SetTM usage of adverse effects of environmental stress. A product
such as Crop-SetTM could mitigate the effects of
Crop-SetTM is a natural product derived from an excess ethylene production during stress periods, thus
extract primarily of the yucca plant. As such, natural improving the chances of fruit set and growth. The
plant growth regulator activity such as expected from issue of carbohydrate movement toward fruits is also
cytokinins, gibberellins and auxins are present. In important, considering that the next phase of fruit
growth (cell enlargement) coincides with the start of this were correct, then carbohydrates would have
sugar accumulation, which therefore relates to final been diverted to the leaf flush rather than the fruit,
fruit brix content. Work done by Bower (unpublished resulting in poorer size and internal quality. Thus, it
data) showed that applications of Crop-SetTM on is considered that a mid-summer application of Crop-
Valencia oranges at six weeks post-petal fall (end of SetTM should not be considered.
cell division) or at six weeks plus at 11 weeks post-
petal fall, substantially enhanced final fruit number.
Application also changed size distribution to a larger Opportunities for ISR 2000TM usage
size grouping, as indicated by the number and
percentage of total fruits in the most economical size ISR 2000TM is a product containing Crop-SetTM plus
range of 40 to 88 (15 kg carton). Results are shown a yeast cell wall component containing a mannan
in Table 1. oligosaccharide. The latter is believed to create an
Similar research was conducted in South Africa on induced resistance response in plants. Bower (2003)
grapefruit, where fruit size is a considerable problem, showed that the product, when applied to navel
enhanced by the presence of citrus tristeza. Results orange trees three weeks prior to harvest, did indeed
are shown in Table 2 for the Star Ruby cultivar, which induce a response that inhibited the growth of the
is particularly susceptible to production of small fruit. postharvest pathogen Penicillium digitatum. While
Further, similar results from South Africa and Florida the inhibition was not sufficient to be commercially
have been published by Marais and Frank (2003). acceptable on its own, efficacy was statistically
Opportunities for Crop-SetTM usage later in the fruit indistinguishable from that of Imazalil fungicide
growth cycle are questionable. A third spray in the treatment when combined with a postharvest yeast
South African Valencia trial at 16 weeks post-petal application (Table 3).
fall had no positive effects, and in fact resulted in a Considerable potential exists for the use of the
poorer fruit count distribution than the control. The product in eliminating use of traditional fungicides,
reasons are unclear, but in considering the to which consumers are becoming increasingly averse
phenological cycle (Figure 1), application would have (Lopez-Garcia et al., 2000), and solves the problem
coincided with the summer growth flush. The product of poor competitive inhibition of the pathogens by
could, as previously explained, have stimulated this most of the biological products presently available
flush (as it would have done to the leafy blossom). If for postharvest pathogen control. In addition, the
Table 1. Effect of Crop-SetTM applied at 6 weeks post-petal fall (PPF) and 6 plus 11 weeks PPF on fruit number, economic size range and total
soluble solids at harvest for Valencia sweet oranges in South Africa1.
Treatment Mean per tree Mean per tree fruit Fruit counts 40-882 Total soluble solids
fruit number number counts 40-882 (%) (%)
Control 212.6a 168a 79 9.6 a

1 spray: 6 weeks PPF 365.9b 282b 77 10.3b
2 sprays: 6 weeks plus 11 weeks PPF 353.7b 261b 74 10.1a
1
Applied at 20 ml product per 100 l water
2
Fruit counts per 15 kg carton
ab
Significance is indicated at P=0.05 by different letters within a column
Table 2. Efect of Crop-SetTM applied 4 and 8 weeks post petal fall (PPF) on fruit number and economic size range for Star Ruby grapefruit in
South Africa1.
Treatment Mean per tree Mean per tree fruit Fruit counts Number of cartons
fruit number number counts 32-562 32-56 (%) (15 kg) counts 32-56
Control 243.2a 169.5 a 69.7 3.9a

Crop-SetTM 320.5b 206.0 b 64.2 4.5b
1
Applied at 20 ml product per 100 l water
2
Fruit counts per 15 kg carton
ab
Significance is indicated at P=0.05 by different letters within a column
J.P. Bower 365
Table 3. Effect of pre- and postharvest treatments on growth of well as with cultivar and rootstock combination. By
Penicillium digitatum 21 days after application of ISR2000TM following the phenological cycle, growers can target
Infection rating1 the use of Crop-SetTM and if desired, ISR 2000TM, in
a manner which matches their needs to manipulate
Postharvest Control Imazalil ISR2000 TM ISR2000TM
treatment (2g/l) (4 g/l) the tree and fruit growth to advantage. Because tree
physiology is correctly matched with the constituents
Control 5b 1a 2.6cd 3.2c of the products, the desired responses, when required,
ISR 2000TM 2.6cd 1a 1.6ad 1.6ad are likely. Should growers follow this scheme,
1
Infection score: 1 indicates no growth; 5 indicates complete enhanced fruit size and quality can be achieved,
coverage of the fruit by fungal mycelium. without the potential for failure due to inappropriate
ab
Significance (P=0.05) is indicated by different letters. product usage, as only the physiologically correct
applications will be made.
product may, by the nature of its constituents, have a A summary of potential application windows is
positive effect on fruit integrity, by slowing the shown in Table 4.
senescence process. Thus longer shelf life, in addition
to enhanced resistance to postharvest decay, can be
expected. For these purposes, application should be
after colour break, and two to three weeks prior to
References
harvest. The product will, however, require further Agusti, M. 2000. Regulation of citrus cropping and
testing on cultivars other than Navels. Depending improvement of fruit quality using exogenous plant
upon the rate of fruit maturity, caution needs to be growth regulators. Proc. Int. Soc. Citriculture
exercised should application time coincide with
9:351-356.
flower bud induction, as the Crop-SetTM component
Agusti, M., F. Garcia-Mari and J.L. Guardiola. 1982.
may decrease blossom. This may, on the other hand,
Gibberellic acid and fruit set in sweet orange. Sci.
be advantageous if stimulation of a leafy blossom is
desired. This is indeed probably the case in Navels, Hort. 17:257-264.
due to a naturally high flower number where there Agusti, M., M. El-Otmani, M. Aznar, M. Juan and
may be benefits to decreasing flower number. Should V. Almela. 1995. Effect of 3,5,6 trichloro-2-
this not be the case in other cultivars, an alternative pyridyloxyacetic acid on fruit size at maturity. J.
product, without the component of Crop-SetTM could Hort. Sci. 70:855-962.
be used. Angioni, A., P. Cabras, G.D. Hallewin, F.M. Pirisi,
F. Reniero and M. Schirra. 1998. Synthesis and
inhibitory activity of 7-geranoxycoumarin against
Conclusions Penicillium species in citrus fruit. Phytochemistry
47:1521-1525.
The phenological cycle for citrus can be constructed Bevington, K.B. and W.S. Castle. 1985. Annual root
for any environment, and can therefore be a useful growth pattern of young citrus trees in relation to
tool for management decisions, which may vary from shoot growth, soil temperature and soil water
place to place and indeed from season to season as content. J. Amer. Soc. Hort. Sci. 110:840-845.
Table 4. Summary of potential application opportunities for Crop-SetTM on citrus taking the phenological cycle into account.
Application time Reason for application Caution or comments
After flower induction and before Enhance leafiness of the blossom and improve Do not use too early as vegetative reversion
visible flower buds (~Dec–Jan in fruit set and growth may occur. Also avoid if leafy blossom is
northern hemisphere) expected e.g. after heavy crop.
At flowering (100% petal fall) Enhance fruit size and decrease fruit drop. May be combined with second application at 6
May be particularly useful under stressful to 8 wks post-petal fall (just prior to physiological
conditions drop).
At end of cell division (6 to 8 wks Enhancement of fruit size and decrease of May be combined with a second application 4
post-petal fall) just prior to fruit drop. Enhancement of internal fruit quality wks later.
physiological drop
Bower, J.P. 2003. ISR2000: An aid to biocontrol of of flowering shoots after GA 3 treatment. Bot.
postharvest decay in citrus. In: Nutritional Gazette. 148:17-22.
Biotechnology in the Feed and Food Industries, Lopez-Garcia, B., L. Gonzalez, E. Perez-Paya and
Proc. Alltech’s 19 th Annual Symposium (KA. J.F. Marcos. 2000. Identification and
Jacques and T.P. Lyons, eds). Nottingham characterization of a hexapeptide with activity
University Press, UK, pp. 397-400. against phytopathogenic fungi that cause
Cleland, R.E. 1999. Introduction: Nature, occurrence postharvest decay in fruits. Amer. Phytopath. Soc.
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L.J. Marais and J.G. Frank 369
Citrus fruit size and quality: response to Crop-Set™ in North America

LAWRENCE J. MARAIS AND JOHN G. FRANK
Introduction
Crop-Set™ is categorized as a natural biostimulant low concentrations of saponins stimulated rapid root
by virtue of the fact that it contains a plant extract development, precocious bud formation and
belonging to a group collectively known as saponins, increased chlorophyll production and photosynthesis;
which have the ability to influence hormonal activity (iii) seed treatment with saponins affected all stages
in plants. Crop-Set™ has been used in the citrus of growth of plants e.g. seed germination, vegetative,
industries of the US, Brazil and South Africa for floral and fruiting stages. These researchers concluded
several years. The most extensive use has been in that the effect of saponins was analogous to that of
South Africa, where its application commenced almost true plant hormones, cytokinins, auxins and
seven years ago. The product has been used in the gibberellic acid (Balansard and Pellessier, 1943; 1944;
citrus industries of Brazil and the US (California, 1945). Saponins therefore affect plants at the cellular
Florida and Arizona) for almost four years. and morphogenetic levels.
To be categorized as a biostimulant a compound Biostimulants not only affect internal plant
must be able to influence the hormonal activity of a hormone levels but also promote the production of
plant. Not all biostimulants are natural. For example, antioxidants. Research has shown that environmental
the following products are also used as plant growth stresses such as drought, ultraviolet light, herbicide
regulators: benzyladenine (Accel) is a highly reactive use, high soil salt content and heat damage plants by
synthetic cytokinin; trinexapac-ethyl suppresses causing production of free radicals or reactive oxygen
gibberellin biosynthesis; gibberellic acid (GA3 – molecules. These molecules are strong oxidizing
ProGibb) and prohexadione calcium (Apogee – a GA agents and damage lipids, proteins and DNA inside
biosynthesis inhibitor); propiconazole is a fungicide the plant cells. Antioxidants are metabolites and
with growth regulatory properties; and potassium enzymes that scavenge free radicals and thereby
silicate, which also enhances plant growth. protect plant cells from damage (Zhang and Schmidt,
Natural biostimulants such as those derived from 1999).
saponins, humic acid and seaweed, are more user- Studies by plant physiologists in Brazil (Medina,
friendly and less likely to become restricted by 2003) and South Africa (J. Bower, personal
marketing agencies and consumers. The quality communication) have identified the same reactions
control procedure that Crop-Set™ is subjected to observed by Balansard and Pellessier following the
makes it unique among other natural biostimulants. application of Crop-Set™ to citrus trees. These
This fact results in the effects being consistent and include enhanced lateral bud development, stem
not fluctuating from season to season. elongation, enhanced root development, enhanced
Significant research on the effect of saponins on flower initiation, increased fruit size and weight,
the growth of plants was conducted by the French increased juice content, enhanced color and reduced
plant physiologists Balansard and Pellessier in the heat stress. The effects at the cellular level include
1940s. They made several significant discoveries viz.: increased cell division, increased elasticity of cell
(i) saponins reduced the surface tension of tissues walls, re-direction of photo-assimilates to fruit,
and modified the cellular permeability of seed; (ii) increased chlorophyll production, reduced absicissic
370 Citrus fruit size and quality: response to Crop-Set™ in North America
acid levels, increased uptake and mobilization of trials in California two entire rows (30 trees per row)
nutrients (in particular calcium) and suppression of within 11 acre blocks were treated with Crop-Set™
the effect of viruses on yield and fruit size (Jameson, and 20 trees selected at random within treated and
2000). untreated rows in these blocks. Two buffer rows were
This paper summarizes the results of field trials left between treated and untreated rows. As the
with Crop-Set™ in citrus orchards located in Arizona, mandarin trees were only 3 years old and produced
California and Florida during the 2002-2003 season. light crops, only 10 fruit per tree were measured to
assess fruit size. Statistical analysis was conducted
using Duncan’s Multiple Range Test at P = 0.05.
Materials and methods
DOSAGE RATE
Results and discussion
In all cases the dosage rate was a total of 16 oz per
acre. The amount of water used per acre varied from GRAPEFRUIT
grower to grower, but was never less than 50 gallons At the trial site in Ft. Pierce, Crop-Set™ increased
per acre when applied using a mist-blower type the yield of Marsh grapefruit in size class 27 to ≥23
spray rig. by 35.2% (Table 1). The untreated trees produced
35.5% more fruit in the smaller size class 32-40.
TIMING OF APPLICATION Crop-Set™ increased the fruit size of Ruby Red
grapefruit in the size classes 40-48 and 23-27 by 8%
Crop-Set™ applications in sweet oranges and and 5%, respectively. White grapefruit selections
mandarins were applied at petal-fall and when fruit generally produce larger fruit than pigmented
was golf ball size. The timing of applications in the selections. Certain overseas markets prefer larger
lemon trial in Arizona differed from that of the sweet grapefruit; and the increase in total percentage of
orange and mandarin studies. In one trial Crop-Set™ larger fruit in both selections increases the value of
was applied at a rate of 16 oz per acre at petal-fall the crop significantly.
and in the second trial the rate was 8 oz per acre at
petal-fall followed by 8 oz per acre at walnut size.
SWEET ORANGES
EXPERIMENTAL DESIGN AND ASSESSMENT OF Crop-Set™ increased the percentage of Valencia fruit
FRUIT SIZE in size class 56-72 at the Ft. Pierce site by 21% (Table
2). Untreated trees produced 21% more fruit in the
In the majority of the trials the experimental plots smaller size category 88-105. At the Frostproof site
were 5-10 acre treated and untreated blocks. Twenty Crop-Set™ increased the yield of Valencia fruit in
trees were selected at random in each block and the size category 48-64 by 22.5% and untreated trees
diameters of 20 fruit measured per tree, using a strap- produced 19.5% more smaller fruit in size class 88-
type caliper. The field trials conducted in Clewiston, 105. The increase in fruit size in the Valencia trial at
FL and Yuma, AZ were based on randomized block Clewiston was not as dramatic as at the previous two
designs with 6-9 blocks per treatment. Four to 10 sites but was still fairly substantial viz. 10.2 % more
trees were selected within each block for determining fruit in size class 48-72. The untreated trees produced
fruit size, yield and juice quality. In the mandarin 10% more smaller fruit in size class 88-125.
Table 1. Effect of Crop-Set™ on fruit size distribution (% of total) of grapefruit selections in Florida.
Locality and Treatment Fruit size categories
selection
>23 23 27 32 36 40 48 56 64
Ft. Pierce Control 1.0a 24.5a 34.3b 21.3b 14.5 b 4.5b 0.0 0.0 0.0
(Marsh) Crop-Set™ 24.8b 46.0b 24.5a 3.3a 1.5a 0.0a 0.0 0.0 0.0
La Belle Control 0.0 6.5a 22.0a 19.0a 32.5 a 12.0 a 3.5a 4.0a 0.5a
(Ruby Red) Crop-Set™ 0.0 14.0a 20.0a 18.0a 21.0 a 18.0 a 5.5a 3.5a 0.0a
ab
Means in a column without common superscripts differ (P=0.05).
Table 2. Effect of Crop-Set™ on fruit size distribution (% of total) of sweet orange varieties in Florida.
Locality Treatment Fruit size categories

and selection
48 56 64 72 88 105 125
Clewiston Control 0.0a 1.0 a 6.3 a 20.0a 45.2a 19.2a 8.3a

(Hamlin) Crop-Set™ 0.2 a 1.9 a 7.5 a 23.5a 43.5a 17.5a 5.8a
Clewiston Control 0.2a 4.6 a 18.3a 46.3a 26.5b 3.9b 0.2a
(Valencia) Crop-Set™ 1.3b 10.4b 25.2a 42.7a 19.4a 0.8a 0.0a
Ft. Pierce Control 0.0 0.0a 8.0 a 47.8a 40.0b 4.3b 0.0
(Valencia) Crop-Set™ 0.0 1.8b 22.5b 52.5a 22.8a 0.5a 0.0
Frostproof Control 0.0a 0.3 a 12.5a 47.3a 33.3b 6.8b 0.0
(Valencia) Crop-Set™ 1.5a 10.3 23.5b 44.3a 20.5a 0.0a 0.0
ab
The increase in fruit size in the Hamlin trial in to be one application of 16 oz. of Crop-Set™ per
Clewiston was not as dramatic as in the Valencia trials. acre at petal-fall compared with the standard
Hamlin is a mid-season variety which produces recommendation of one application of 8 oz per acre
smaller fruit than Valencia. Crop-Set ™ treated trees at petal-fall, followed up by a second application at
produced 5.8% more fruit in size class 48-72 and the the same dosage rate (Table 3). Trees that received
untreated trees 5.9% more fruit in the smaller size only one application of Crop-Set™ produced 18%
class 88-125. more fruit in total yield than the untreated trees. These
trees also produced 17% more fruit in the size class
75-95. The untreated trees produced 18% more fruit
LEMONS in the smaller size class 115-285.
Lemon trees can produce from 1-3 crops per season
depending on where they are grown. In the desert MANDARINS
areas of California only one crop is produced owing
to the harsh summer climate. In the coastal areas 3-4 The trees in these trials were only 3 years old and
crops can be produced annually. Because of the were therefore not expected to respond to treatment
‘monsoon-type’ climate in Arizona, lemons trees can to the same extent as mature trees. However, only
also produce more than one crop. Citrus growers one of the selections (No. 8) showed no enhanced
with lemon trees that produce multiple crops must fruit size, while the remaining three selections all
increase the fruit size and yield of the crop which is reacted very favorably to Crop-Set™ (Table 4). Not
most valuable to them. Timing of Crop-Set™ only did these selections show enhanced fruit size,
applications is therefore critical in this citrus variety. but all four selections showed increased Brix levels.
This is also to the benefit of the grower as he can The Brix values for the different selections treated
manipulate his trees to produce the crop desired to with Crop-Set™ increased by 1%, 1.2%, 2.9% and
meet market needs and supply the best fruit when his 4.5%, respectively for selections No. 11, No. 8, No.
competitors, e.g. Spain and Argentina, are not able 16 and No. 10. The fruit size increases for selections
to. In this particular trial, the best treatment appeared No. 10, No. 11 and No. 16 were 14.5% in size class
Table 3. Effect of Crop-Set™ on fruit size distribution (% of total) of Lisbon lemon in Yuma, Arizona.
Treatment Fruit categories Total yield

(bins/plot)
75 95 115 140 165 200 235 285
Control 16.3b 46.4ab 18.1a 10.8a 5.9a 1.8a 0.8a 0.2a 1.1b
Crop-Set™ 13.9b 41.4 b 20.9a 13.4a 7.1a 2.1a 1.1a 0.1a 1.1b
Crop-Set™* 31.4 a 50.6 a 10.5b 4.4b 4.4b 0.7b 0.2b 0.1a 1.3a
*Denotes one application of Crop-Set™ at 16 oz per acre at petal-fall.

ab
372 Citrus fruit size and quality: response to Crop-Set™ in North America
Table 4. Effect of Crop-Set™ on fruit size distribution (% of total) of different mandarin selections in California.
Selection Treatment Fruit size categories Brix

>60 60 68 81 90 105 =120
No. 8 Control 52.5a 42.0a 5.5a 0.0 0.0 0.0 0.0 7.6
Crop-Set™ 52.0a 40.0a 8.0a 0.0 0.0 0.0 0.0 7.7
No. 10 Control 64.5a 35.5b 0.0 0.0 0.0 0.0 0.0 8.8
Crop-Set™ 79.0b 21.0a 0.0 0.0 0.0 0.0 0.0 9.2
No. 11 Control 0.0 0.0 0.0 2.0a 14.5a 35.5a 48.0b 9.6
Crop-Set™ 0.0 0.0 0.0 9.0b 27.0b 37.0a 27.0a 9.7
No. 16 Control 0.0 0.5a 12.0 a 14.5a 22.0a 32.5b 18.5b 7.0
Crop-Set™ 0.0 2.5 a 23.5 b 35.5b 21.5a 12.0a 5.0a 7.2
ab
Table 5. Effect of Crop-Set™ on fruit size distribution (% of total) and quality of Minneola tangelo in Yuma, AZ.
Fruit size categories Total yield

(lb/tree)
Treatment 40 48 56 64 72 88 105 125
Control 10.1a 19.1a 20.7b 23.5a 19.4a 5.6a 1.3a 0.4a 184.3 a
Crop-Set™ 11.4a 20.8a 26.0a 22.6a 15.1a 3.4a 0.6a 0.1a 237.6 a
ab
> 60, 21% in size class 81-105, and 62% in size class Conclusions
60-81, respectively. Untreated trees produced
significantly more fruit in the smaller size classes. Crop-Set™ been has shown to consistently increase
the quality and size of fruit in the citrus varieties
discussed above. The timing of applications of Crop-
MINNEOLA TANGELOS Set™ in lemons appears to differ from that of sweet
orange varieties. It is imperative that the potential of
Minneola tangelo trees in Florida, California and Crop-Set™ to initiate flowering be utilized to its
Arizona tend to exhibit alternate bearing fullest potential in varieties that are alternate bearers
characteristics. This means that one year the crop is such as Minneola tangelo and varieties that are shy
very light and the next year a large crop may be bearers such as certain mandarin selections. This may
produced. The active ingredient in Crop-Set™ viz. mean that either an early fall application to coincide
saponin, has the ability to enhance flower initiation with the last summer flush or an application
in plants and should therefore be able to eliminate or immediately following harvest be applied.
reduce the alternate bearing characteristic of this
variety if applied at the crucial time. The orchard
currently under investigation received a third References
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trees by 29% and also increased the fruit in size class saponine du Quillaya sur le pouvoir absorbant des
40-56 by 58% (Table 5). Crop-Set™ application also graines de Pisum. Soc. Biol. Mars. 137:455.
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decreased peel thickness, though not significantly. germes de Blé isolés de la saponine du Quillaya.
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végétation. Soc. Biol. Mars. 138:622. Symposium, Lexington, KY. Nottingham
Balansard, J. and F. Pelliseur. 1945. Saponines et University Press, Nottingham, UK, pp. 407-414.
croissance chez Solanum Lycopersicum L. Soc. Zhang, X. and R. Schmidt. 1999. Biostimulating turf
Biol. Mars. 139:1098-1100. grasses. Grounds Maintenance 11:15-32.
P. Ji et al. 375
Integrated efficacy of several Improcrop compounds on bacterial wilt of

tomato plants under greenhouse conditions
PINGSHENG JI1, TIM MOMOL1 AND NECIP TOSUN2
1
North Florida Research and Education Center, IFAS, University of Florida, Gainesville, FL, USA
2
Plant Protection Department, Faculty of Agriculture, Ege University, Izmir, Turkey
Introduction
Tomatoes rank second in terms of cash receipts among The objective of this study was to evaluate the efficacy
all vegetables produced in the United States, with a of several Improcrop compounds for control of
net value of more than $1.6 billion and a total bacterial wilt of tomatoes. The compounds were used
production of 12 million tons (Swiader and Ware, for soil and seed treatments in an attempt to reduce
2002). Bacterial wilt caused by Ralstonia pathogen populations combined with application of
solanacearum is a serious disease and a major compounds that may induce resistance of tomato
constraint in production of tomatoes. This disease plants for an integrated management of this disease.
causes rapid wilt and death of the tomato plant; and
incidence can be as high as 50% or more in the
southern US and subtropical and tropical areas of Materials and methods
the world (Jones et al., 1991).
The efficacy of current strategies for control of BACTERIAL CULTURE AND INOCULUM
bacterial wilt in tomatoes is limited. Conventional PREPARATION
pesticides that are known to provide effective control
R. solanacearum tomato strain Rs5 (race 1, biovar
of the disease are not available. As a soil-borne disease
1) isolated in Quincy, Florida (Pradhanang and
with systemic infection through the xylem, bacterial
Momol, 2001) was used in this study. The bacterial
wilt cannot be controlled by foliar application of
pathogen was grown on casamino acid peptone
traditional pesticides such as copper and mancozeb,
glucose medium (CPG) (Kelman, 1954) at 28oC for
which are used for control of foliar bacterial diseases.
48 hrs. Bacterial cells were suspended in sterile
Soil treatments with general-purpose fumigants like
deionized water and concentration of inoculum was
methyl bromide did not provide satisfactory control
estimated by measuring absorbance at 590 nm. The
of the disease (Chellemi et al., 1997; Enfinger et al.,
viable bacterial population was determined following
1979). Control of bacterial wilt in infested soils is
dilution and plating on CPG agar. Soils used for
very difficult. It is generally considered that crop
tomato transplanting were infested with the pathogen
rotation with a non-host crop is of minimal value
at an initial density of 6 x 107 CFU/ml of soil.
because of the wide range of crop and weed hosts of
the pathogen (Hayward, 1991; Pradhanang and
Momol, 2001). Once the pathogen becomes
APPLICATION OF IMPROCROP COMPOUNDS
established in the field, previously productive tomato
fields may have to be abandoned due to serious Three compounds were used in the study, Stubble-
outbreak of the disease. The destructive nature of AidTM, ISR 2000TM and AgromosTM. Tomato seeds of
the disease and ineffective disease suppression of a susceptible variety (Solar Set) were treated with
current control measures have made development of Stubble-AidTM solution (50%) for 30 min before
effective disease prevention approaches desirable.
376 Integrated efficacy of several Improcrop compounds on bacterial wilt of tomato plants
seeding. The seeds were sown in Terra-lite ® height and the weight of roots and foliage were
agricultural mix (Scott Sierra Horticultural Products measured at the end of the experiment. The data were
Co., Marysville, OH) in expanded polystyrene flats analyzed using the ANOVA or GLM procedures of
with 2.5 cm by 2.5 cm cells and maintained in a the Statistical Analysis System (SAS Institute, Cary,
greenhouse. The compound ISR 2000TM was applied NC). Means were compared using Duncan’s multiple
as a foliar spray and soil drench at a concentration of range test at P = 0.05.
1 ml/L starting 15 days before tomato seedling
transplanting and once every seven days thereafter
for two more applications. Soils (the potting mix) Results and discussion
used for transplanting were either treated, or not
treated, with Stubble-AidTM solutions at the rate of
1% two hours after soil infestation with the pathogen. Application of Stubble-AidTM in conjunction with ISR
Tomato seedlings were transplanted into plastic 2000 TM and Agromos TM consistently provided
‘Cone-tainers’ (20.5 x 4.0 cm) containing the significant protection of tomato plants against
Stubble-AidTM-treated or not treated soils four days bacterial wilt. Ten days after inoculation the
after soil treatment with Stubble-AidTM. AgromosTM protection of tomato plants from R. solanacearum
was applied as a foliar spray at weekly intervals after was 100% (no treated plants wilted). Twenty-one
transplanting at a concentration of 1.2 ml/L. Nu-Film days after inoculation 24% of inoculated treated plants
17 was used as adjuvant (0.125%) in AgromosTM wilted (Figure 1). Disease incidence of treated plants
solution. Untreated tomato plants growing in the was 40% four weeks after transplanting while 100%
infested soils were used as a control. A subset of of untreated plants wilted (Table 1, Figures 2 and 3).
tomato plants treated (as described above) or not Application of these compounds also showed a
treated with these compounds, was grown in soils significant enhancement of plant growth as indicated
not infested with the pathogen to evaluate the effect by larger plant and root weight (Table 2).
of these compounds on plant growth. The plants were The results indicated that these compounds
supplied with Peter’s Peat Lite Special (15:16:17 exhibited the potential to significantly reduce bacterial
NPK) at weekly intervals at the rate of 7.5 g /L water. wilt incidence and enhance growth of the susceptible
tomato cultivar. The mode of action(s) involved in
the disease reduction by these compounds is unclear.
EXPERIMENT DESIGN AND DISEASE ANALYSIS Treatments of the soil and tomato seeds with Stubble-
Aid TM might have reduced populations of R.
A randomized complete block design was employed solanacearum in the soil and prevented colonization
with five replications and five plants for each of the root system by the pathogen. ISR 2000TM and
treatment in one replication. Disease incidence was Agromos TM may have the potential to enhance
recorded weekly after tomato transplanting and resistance of tomato plants against this pathogen and
quantified as the percentage of plants wilted. Plant reduce disease incidence by induced resistance. It is
Table 1. Effect of Stubble-AidTM and other compounds on disease incidence of bacterial wilt of tomato (cv. Solar Set) under greenhouse
conditions.
Disease incidence (% plants wilted)c,d

a,b
Treatment Day 7 Day 10 Day 14 Day 21 Day 28
Control (inoculated) 28.0 a 84.0a 96.0a 100 a 100 a

Control (no inoculation) 0b 0b 0c 0c 0c
Stubble-Aid + ISR 2000 + Agromos (inoculated) 0b 0b 16. 0b 24.0b 40.0b
Stubble-Aid + ISR 2000 + Agromos (no inoculation) 0b 0b 0c 0c 0c
a
Stubble-AidTM was used to treat seeds and soils, ISR 2000TM was applied as foliar spray and soil drench, and AgromosTM was applied as foliar
spray.
b
Inoculation or no inoculation indicates seedlings were transplanted into soils infested or not infested with R. solanacearum.
c
Values are the means of five replications (25 plants). Same letter in each column indicates no significant difference based on Duncan’s
multiple range test at P = 0.05.
d
Days after inoculation.
P. Ji et al. 377
Table 2. Effect of Stubble-AidTM and other compounds on the growth of tomato plants (cv. Solar Set) in bacterial wilt greenhouse trial.
Plant growthc,d
a,b
Treatment Plant weight Root weight Foliage weight Plant height
(g) (g) (g) (cm)
Control (inoculated) 0.53d 0.06 d 0.47c 19.34 c

Control (no inoculation) 14.57 b 2.53 b 12.04a 43.14 ab
Stubble-Aid + ISR 2000 + Agromos (inoculated) 9.21c 1.79 c 7.42b 39.96 b
Stubble-Aid + ISR 2000 + Agromos (no inoculation) 16.77 a 3.43 a 13.34a 46.16 a
a
Stubble-AidTM was used to treat seeds and soils, ISR 2000TM was applied as foliar spray and soil drench, and AgromosTM was applied as foliar
spray (see descriptions in the text for timing and dosage of applications).
b
Inoculation or no inoculation indicates seedlings were transplanted into soils infested or not infested with R. solanacearum.
c
Values are the means of five replications (25 plants). Same letter in each column indicates no significant difference based on Duncan’s
multiple range test at P = 0.05.
d
Plant growth was evaluated 36 days after inoculation.
Figure 1. All treatments: photo taken 21 days after inocula- Figure 2. Uninoculated treatments: photo taken at the end of
tion or transplanting. the experiment.
Figure 3. Inoculated treatments: photo taken at the end of the experiment.

378 Integrated efficacy of several Improcrop compounds on bacterial wilt of tomato plants
worthwhile to carry out further experiments to Enfinger, J.M., S.M. McCarter and C.A. Jaworski.
elucidate the mechanisms associated with disease 1979. Evaluation of chemicals and application
reduction by these compounds. Detailed studies will methods for control of bacterial wilt of tomato
also be necessary to investigate the timing and transplants. J. Phytopathology 69:637-640.
application dose needed to sustain the high efficacy Hayward, A.C. 1991. Biology and epidemiology of
that was observed during the early stage (10 days bacterial wilt caused by Pseudomonas
after inoculation). These studies will help optimize solanacearum. Annu. Rev. Phytopathol. 29:65-87.
the application methodology of these compounds to Jones, J.B., J.P. Jones, R.E. Stall and T.A. Zitter.
facilitate their practical use for bacterial wilt 1991. Compendium of tomato diseases. APS Press,
management in tomato production. St. Paul, MN.
Kelman, A. 1954. The relationship of pathogenicity
in Pseudomonas solanacearum to colony
References appearance on a tetrazolium medium. J.
Chellemi, D.O., S.M. Olson, D.J. Mitchell, I. Secker Phytopathology 44:693-695.
and R. McSorley. 1997. Adaptation of soil Pradhanang, P.M. and M.T. Momol. 2001. Survival
solarization to the integrated management of of Ralstonia solanacearum in soil under irrigated
soilborne pests of tomato under humid conditions. rice culture and aquatic weeds. J. Phytopathology
J. Phytopathology 87:250-258. 149:707-711.
Swiader, J.M. and G.W. Ware. 2002. Producing
vegetable crops. Interstate Publishers, Inc.,
Danville, IL.
T. Johnston et al. 379
Alternatives against Alternaria: controlling brown spot on Murcott tangors

TIM JOHNSTON1, LAWRENCE J. MARAIS2 AND L.W. TIMMER1
1
University of Florida, IFAS, Citrus Research and Education Center, Lake Alfred, Florida, USA
2
Introduction
Alternaria brown spot, caused by Alternaria resistance may be useful in the control of foliar fungal
alternata, affects Minneola tangelos, Dancy diseases of citrus. It is difficult to control these
tangerines, Murcott tangor and less frequently, diseases on rapidly expanding leaves and fruit with
Orlando tangelos, Novas, Lees and Sunburst protectant fungicides. Induced resistance could
tangerines. In rare cases it may also infect grapefruit. therefore provide systemic protection against
This disease causes serious defoliation, fruit drop and infection to substitute for, or supplement control by
fruit blemishes and is a limiting factor in the standard fungicides. Products such as Messenger
production of these cultivars in humid areas. Even (Harpin protein), ProPhyt (potassium phosphite),
in semi-arid production areas, blemishes on the peel Nutriphite (phosphorous acid), Aliette 80 WP (fosetyl-
can significantly reduce marketability of the fruit Al), KeyPlex 350DP and 445 DP (α-keto acids),
(Timmer et al., 2000). Oxycom Respond (hydrogen peroxide), ReZist,
In humid climates such as that of Florida, fungal Goemar H11 (laminarin), Serenade (Bacillus
diseases such as Alternaria brown spot are difficult subtilis), and Actigard (acibenzolar-s-methyl) have
to control. Protectant fungicides such as copper been tested against Alternaria brown spot in the
products are still the mainstay of control programs laboratory and greenhouse (Agostini et al., 2003).
for this disease (Timmer, 2002). However, copper In greenhouse studies, most of these products
accumulates in soils and can cause phytotoxicity (Alva reduced disease severity compared with the untreated
and Graham, 1991); therefore its use needs to be control, but were all less effective than the standard
limited in some plantings and in hot, dry weather. Abound fungicide. ProPhyt and Rezist have proven
The economics of citrus production preclude effective when used in early sprays for brown spot
frequent application of fungicides. Currently under control (Bhatia and Timmer, 2003).
ideal climatic conditions, growers may apply 9-10 The purpose of the current study was to assess the
sprays per season to control this disease. These costs efficacy of Agromos™ and ISR 2000™ to control
are prohibitive and can be as much as $500 per acre Alternaria brown spot under field conditions.
(N.A. Smith, personal communication). Strobilurins
have recently been registered for citrus and provide
good control of Alternaria brown spot (Timmer, Materials and methods
2004). However these fungicides must be managed
carefully to avoid future problems with the Fungicides were evaluated in an 8-year-old grove of
development of resistance. Murcott tangors near Bereah, Florida in 2003. Each
Systemic induced resistance has been investigated treatment was applied to five two-tree plots arranged
extensively for the control of many plant diseases. A in a randomized complete block design. Unsprayed
wide range of compounds such as benzothiadiazoles, guard trees were located between the plots. All sprays
salicylic acid, harpin protein, fatty acids and were applied with a handgun at 200 psi. The desired
oligosaccharides are known to be effective inducers rate per acre for mature groves was added to 125
of plant resistance to disease. Products that induce gallons of water and the trees sprayed to runoff using
approximately two gallons per tree.
380 Alternatives against Alternaria: controlling brown spot on Murcott tangors
The first application was made on March 20 when least effective and all other treatments were
the spring flush was up to 1/4 expanded to reduce significantly more effective than this product.
inoculum production on old foliage and prevent Some minor phytotoxicity occurred in the treatments
infection of new growth. A second application was where copper fungicides were applied at a rate of 4.3
made on April 11 after petal fall to reduce spore lb/acre. The phytotoxicity in combination with ISR
production on leaves and protect young fruit from 2000™ may have been as a result of too high a rate of
infection. The third application was made on May copper applied to tender leaf tissue. ISR 2000™
13 when fruit was about 1/4-1/2 inch in diameter, contains an ingredient called Crop-Set™, that enhances
approximately 4-5 weeks after petal fall. A fourth vegetative growth of citrus (Medina, 2003).
application was made on June 19 about 8-10 weeks Two applications of either Agromos™ or ISR
after petal fall to protect fruit from late Alternaria 2000™ followed by two applications of copper
infection. (Kocide 2000) were as effective as four applications
Rainfall was very high especially during May and of standard copper (Table 1). Four applications of
June. Total precipitation between the first and second copper resulted in 93% marketable fruit while the
applications was 6.2 inches, between the second and combination of copper and Agromos™ or ISR
third it was 8.5 inches and between the third and the 2000™ resulted in 91% and 92% marketable fruit,
fourth it was 20.7 inches. In the period following respectively. The untreated controls produced 62%
the last application until July 15, a total of 30.4 inches marketable fruit.
fell. Thus conditions were highly favorable for disease Agromos™ and ISR 2000™ were not tested alone,
development this season. However, Murcott is not but previous results with compounds that induce
the most highly susceptible variety and inoculum systemic resistance indicated that they do not achieve
levels were generally low at the beginning of the the same level of control as standard fungicides
season. Despite favorable conditions, damage from (Agostini et al., 2003). This may be true for
brown spot was only moderate on unsprayed controls. Agromos™ and ISR 2000™ as well, but these
In November, 50 fruit per tree were rated for products are more user-friendly than products such
severity of Alternaria brown spot on a scale of 0 = as Serenade.
no disease; 1 = mild disease, suitable for the fresh The level of control achieved by these products
market; 2-5 = fruit useful only for processing, may not be as high, but organic growers could benefit
increasing severity. The percentage of marketable from them especially if the use of copper fungicides
fruit, those rated as 0 or 1, was calculated for each is restricted. Although resistance-inducing compounds
treatment. Data were subjected to analysis of variance such as Agromos™ and ISR 2000™ may not be
and means separated by the Waller Duncan k-ratio t substitutes for current commercial fungicides, they
test, P<0.05. can be used with great effect when applied earlier to
protect rapidly expanding leaf tissue and avoid
Results and discussion inoculum buildup. Furthermore, they reduce the
number of copper sprays, which in turn reduces the
All treatments reduced disease severity significantly amount of copper reaching the soil.
compared to the unsprayed control. Serenade was
Table 1. Effect of Agromos™ and ISR 2000™ in combination with Kocide 2000 on the control of Alternaria brown spot in Murcotts.
Alternaria brown spot

Product Rate/acre Applications Severity rating Marketable Phytotoxicity
(0-5) fruit (%) (0-5)
Control – – 1.29 a 61.8c 0.00c

Serenade 4.0 lb 1, 2, 3, 4 0.92b 75.0b 0.00c
Serenade + Kocide 2000 2.0 lb + 1.0 lb 1, 2, 3, 4 0.35cd 93.2a 0.03bc
Serenade + Kocide 2000 4.0 lb + 2.0 lb 1, 2, 3, 4 0.29cd 93.8a 0.05b
Ziram 5.0 lb 1, 2, 3, 4 0.18d 96.4a 0.00c
Ziram 7.0 lb 1, 2, 3, 4 0.33cd 93.6a 0.00c
ISR 2000™ + Kocide 2000 0.63 qt 4.3 lb 1, 2, 3, 4 0.41 c 91.2a 0.22a
Agromos + Kocide 2000 0.42 qt 4.3 lb 1, 2, 3, 4 0.33 c 92.4a 0.00c
Gem 8.0 oz 1, 2, 3, 4 0.22cd 97.2a 0.00c
abc
Values in each column with a letter in common are not significantly different.
T. Johnston et al. 381
Conclusions Alva, A.K. and J.H. Graham. 1991. The role of

copper in citriculture. Adv. Agron. 1:145-170.
In conclusion, it can be stated that Agromos™ or Bhatia, A. and L.W. Timmer. 2003. Evaluation of
ISR 2000™ can be used to control Alternaria brown products for the control of Alternaria brown spot
spot when used in conjunction with a standard on Minneola tangelos, 2002. Fungicide and
fungicide such as copper. It is essential that these Nematicide Tests (online) Report 58:
products be applied early, during the emergence of MO06.DOI.10.1094/FN58. The American
the spring flush, to increase resistance and prevent Phytopathological Society, St. Paul, MN.
the buildup of inoculum. These products can be used
Medina, C.L. 2003. Comparative effects of
as tank mixes with most pesticides, which means that
gibberellic acid, Crop-Set™ and potassium nitrate
citrus growers do not need to make additional spray
on vegetative development, fruit set and quality of
applications to accommodate them. The use of
Agromos™ and ISR 2000™ will reduce the number orange fruits, Citrus sinensis (L) Osbeck. In:
of applications of conventional fungicidal sprays by Proceedings 19 th Annual Alltech Symposium,
50% and may reduce the risk of resistance Lexington, KY. (T.P Lyons and K.A. Jacques, eds.)
development when using new generation fungicides Nottingham Univ. Press. Pages 407-414.
such as strobilurins. Timmer, L.W. 2004. Florida Citrus Pest Management
Guide. Univ. Fla. Inst. Food Agric. Sci. Publ. No.
SP-43.
References Timmer, L.W., Z. Solel and M. Orozco-Santos. 2000.
Alternaria brown spot of mandarins. In:
Agostini, J.P., P.M. Bushong and L.W. Timmer. Compendium of Citrus Diseases. (L.W. Timmer,
2003. Greenhouse evaluation of products that S.M. Garnsey and J.H. Graham, eds.) APS Press,
induce host resistance for control of scab, melanose St. Paul, MN. Pages 19-20.
and Alternaria brown spot of citrus. Plant Dis.
87:69-74.
N. Tosum and H. Turkusay 383
Seed and soil treatments with a natural fungicide product against some
fungal and bacterial diseases of vegetables
NECIP TOSUN AND HUSEYIN TURKUSAY
Department of Plant Protection, Faculty of Agriculture, Ege University, Bornova–Izmir, Turkey
Introduction
Many vegetable seeds are contaminated with fungal against R. solani was 1 µl/ml, and for Pythium and
and bacterial pathogens prior to sowing. These P. syringae pv. tomato the concentration was 2 µl/ml,
pathogens usually only become active when suitable the efficacy being >97% (Tosun, 2003). The purpose
conditions occur. The stages of growth in which of this investigation was to find a practical solution
bacterial and fungal pathogens are most critical are to minimize the damage caused by the above
during germination and early seedling growth. Seed mentioned pathogens using NFP as a seed and (or)
and soil treatment is imperative to control seed-borne soil treatment.
and soil-borne diseases of vegetables. Many pathogens
overwinter in the form of special structures e.g.
sclerotia, mycelia and oospores. These pathogens gain Materials and methods
access to the seeds through cracks which occur in the
testae during germination. The isolates of R. solani, Pythium spp. P. syringae
Rhizoctonia solani and Pythium spp. occur together pv. tomato were obtained from stock cultures at the
in most soils and cause damping-off and seedling Department of Plant Protection, Ege University,
blight diseases in many plants including pepper, Turkey isolated originally from diseased tomato and
tomato and cotton. A fungicide that will control R. pepper plant materials. The fungicide standard used
solani will not necessarily control Pythium as these was Pomarsol Forte 80 WP (thiram) supplied by
fungi belong to different taxa. Pseudomonas syringae Bayer Crop Science and NFP (Stubble-Aid™) was
pv. tomato is a seed-borne bacterial pathogen of provided by Improcrop USA.
tomato causing specks on leaves and fruit. There is
currently no commercially effective seed treatment
available to control this disease. SEED TREATMENTS
Seed treatment with a mixture of thiram and
For the seed treatments with NFP, two different
alumine dust has commonly been used in practice to
methods were used: 1) seed was soaked in a 10%
control seed-borne diseases; however, poor control
NFP + water solution for 30 minutes; and 2) 500 g
of damping-off diseases in seedbeds and the field
of tomato and pepper seeds were coated with a special
has been reported over recent years, owing to the
polymer and NFP at concentrations of 60 ml polymer
dominance of R. solani, drought stress following
and 40 ml NFP, respectively, using commercial
planting and poor cultural practices. The use of
seedcoating equipment. Seed commercially pre-
natural products that exhibit elicitor properties such
treated with thiram was used as control.
as Natural Fungicide Product (NFP; Stubble-Aid™),
could be useful as an alternative to fungicides to
economically control these seed and soil-borne
SOIL TREATMENTS
diseases. Under laboratory conditions the minimum
inhibition concentrations (MIC) for this product The soil in plastic trays was watered with an NFP
384 Seed and soil treatments with a natural fungicide product
solution (2 L NFP/100 L water) prior to sowing with No commercial fungicides are sufficiently effective
untreated and treated seed inoculated with bacteria. against them under field conditions. Furrow
The trays were treated four times at weekly intervals, treatments with special fungicides are not practical
using the same solution. or cost effective to growers. Moreover, commercially
available seed fungicides are not effective enough to
control these pathogens.
PREPARATION FOR ARTIFICIAL INOCULATION P. syringae pv. tomato can cause severe bacterial
speck symptoms both in field and greenhouse
Fungal inoculum was prepared by inoculating wheat cultivated tomatoes. Copper compounds only have a
bran in bottles. Once the inoculum had grown residual activity of 7 days and are extensively used.
sufficiently, artificially inoculated soil media for However their accumulation in the soil and the crop
filling plastic trays was prepared by mixing one part are creating great concern in the public sectors and
inoculum to 49 parts of peat or soil. One hundred environmental agencies of many countries throughout
untreated and treated tomato and pepper seeds were the world. There is also growing evidence that copper
planted in the inoculated soil. Each treatment was fungicides will not be registered for use in organic
replicated five times. farming in the near future
For bacteria inoculation, the method was modified
from Krüger (1959) and Shoemaker et al. (1976).
The surface of the seeds was sterilized with 1:10 SEED TREATMENTS
sodium hypochlorite and rinsed twice with sterilized
distilled water. Bacterial suspensions were prepared In the tomato experiments the soil surface of most
at 6 x 109 CFU/ml. Four gram aliquots of tomato trays was covered with the mycelial growth of R.
and pepper seeds were infiltrated with 20 ml bacteria solani. The results revealed that coating with NFP
solution by using a vacuum pump for 30 min. was not effective against the fungal pathogens under
Untreated and inoculated seeds were sowed in NFP investigation. The mean germination of treated seed
treated soil. All tests were replicated five times. was 28% and the ratio of healthy to dead plants was
14:86 (14%). In pepper experiments the mean seed
germination was 46% but the survival of plants was
EVALUATIONS an unacceptable 33%.
More promising results were obtained with seed
The evaluations were carried out after 25 days, when soaked in NFP solution. The mean germination for
the majority of seedlings in untreated pots were tomato and pepper seeds was 39% and 54%,
recorded as dead. Re-isolations of fungi and bacteria respectively. Survival of plants for the two different
from the diseased seedlings were also carried out crops was 31% and 49%, respectively.
using selective media. In the fungal treatments the Tests with bacterial speck in tomato showed that
ratios of germinated and dead/healthy plants in treated seed treatment with NFP was very effective against
trays were compared with those of untreated trays. P. syringae pv. tomato. Significantly lower lesion
For bacteria test evaluations, the numbers of spots counts were obtained on the leaves of plants derived
on the leaves of plants derived from treated seeds from seed treated with NFP. The survival of tomato
were counted and compare to those of plants from plants was 89%.
untreated seed. In the thiram treatments the percentage germination
in pre-treated tomato and pepper seeds was 42 and
55%, respectively. The survival rate of tomato and
pepper plants was found to be 38% and 40%,
Results and discussion respectively. Thiram was therefore not effective in
controlling the diseases caused by the fungal
Isolates of R. solani and Pythium spp. virulent to pathogens under investigation.
tomato and pepper plants and P. syringae pv. tomato
isolates from infected tomato were used in the tests.
The aforementioned fungal pathogens can cause SOIL TREATMENTS
severe damping off and root rot diseases on many
vegetables and ornamental plants resulting in The most promising results were observed in the tests
significant yield and economic losses in certain years. where NFP was used as a soil drench. No mycelial
N. Tosum and H. Turkusay 385
growth was observed on the surface of the treated References

soil. Moreover, this application suppressed the
development of R. solani and Pythium spp. in the
soil. The mean germination rate for tomato was found Krüger, W. 1959. The control of tomato cancer
to be 70% and the plant survival rate 68%. The results Corynebacterium michiganence by means of
observed in the pepper trials were more impressive, antibiotic. South Africa J. Agric. Sci. 2:195-205.
here the mean seed germination was observed to be Shoemaker, P.B. and E. Echandi. 1976. Seed and
77% and the plant survival rate 81%. plant bed treatment for bacterial cancer of tomato.
In the bacterial speck trials the results were Plant Dis. Rept. 60(2):163-166.
comparable to those in the above trials. Soil drench Tosun, N. 2003. Effects of “Natural Fungicidal
treatment resulted in tomatoes having 73% less leaf Product X (NFP)” on some of the most important
specks than the plants in untreated soil. pathogenic fungi of crops in vitro and in vivo.
Presented in Agronomy Session in Alltech
International Feed Industry Symposium. Lexington,
Conclusions KY.
In summary, seed treatment with NFP was not
effective in controlling the diseases caused by the
fungal pathogens R. solani and Pythium spp., but
was effective in controlling bacterial speck caused
by P. syringae pv. tomato. It is assumed that the
bacteria were only on the surface of the seed and not
borne internally. If the bacterial pathogen was borne
within the seed, the results may have been less
impressive. The results obtained in the above study
indicate that the use of NFP (Stubble-Aid™) as a
soil treatment may be effective on its own or as an
integrated measure in controlling soilborne diseases
of tomato and pepper, in seedbeds, the greenhouse
or field.
I. Türkan et al. 387
Abiotic stresses and plant activators

ISMAIL TÜRKAN1, TIJEN DEMIRAL1, A. HEDIYE SEKMEN1 AND NECIP TOSUN2
1
Department of Biology, Faculty of Science, Ege University, Izmir, Turkey
2
Department of Plant Protection, Faculty of Agriculture, Ege University, Izmir, Turkey
Introduction: abiotic stresses

Since plants are not mobile, they have to endure abscisic acid (ABA) that causes leaves to drop. Growth
various abiotic stresses under both natural and of shallow roots is inhibited and seedling growth is
agricultural conditions. Among these, drought with reduced under water deficient conditions. Water stress
its accompanying high temperatures, is gaining more usually affects both stomatal conductance and
importance due to global warming, which is mainly photosynthetic activity as a result of which superoxide
caused by the greenhouse effect. The likely increase radicals are produced and photo-oxidation of
in the evapotranspiration potential caused by an chlorophyll (leaf bleaching symptoms) and thereby
increase in air and soil temperature brings forth other severe inhibition of photosynthesis occur. Drought
abiotic stresses such as salinity, high temperature and also reduces enzyme activity e.g.rubisco, acid
increased ultraviolet (UV-B) radiation. Abiotic invertase, nitrate reductase, nitrite reductase etc.
stresses are major constraints on worldwide crop Adaptive responses of plants against drought include
production and can account for up to 70% of the cell wall hardening, reduced plant size and growth
yield loss in crop plants. Salinity alone affects rate, ABA production and thus arrested growth,
productivity on about 80 million hectares of global stomatal closure and reproduction failure and
arable land. Abiotic stresses such as drought, salinity, accumulation of compatible solutes by the cells to
high temperatures, hypoxia conditions, nutrient provide osmotic adjustment.
deficiency, increased UV-B radiation, herbicides, etc.,
account for more crop productivity losses than any
other factor. Twelve percent of total crop yields are
SALINITY
lost due to pathogen infection, which is equivalent
to 900 million tons worldwide annually. Some of While drought and salinity, the major constraints
the abiotic stresses affecting plant growth and restricting growth and development of higher plants,
metabolism are summarized in this paper. are quite different environmental conditions, they are
frequently discussed together because they cause
similar physiological problems for plants. In both
DROUGHT stresses, water tends to be lost from plant cells. In
addition, salt stress involves both osmotic stress and
Especially in arid and semi-arid regions of the world,
ionic stress, resulting from high concentrations of
water is the major limiting factor for plant growth.
potentially toxic salt ions within plant cells. Plants
As an early response to water deficit, leaf area is
protect themselves from salt stress by excluding toxic
decreased and the growth of young leaves is inhibited.
ions from the leaves, sequestering them into the
Drought stress causes stomata to lose turgor and close
vacuoles for maintenance of turgor and synthesizing
to minimize transpiration. Cell division is normally
compounds that aid in keeping water inside the cell,
less sensitive, but cell expansion is very sensitive to
all of which also occur under osmotic stress conditions
water deficit. Drought stimulates the production of
(Hasegawa et al., 2000). These nontoxic compounds
388 Abiotic stressors and plant activators
increase the osmotic potential of the cell and allow FLOODING

normal metabolic processes to continue. Such
compounds include mannitol (a sugar alcohol), In flooded soils, the air spaces are filled with water
polyos, sugars, glycinebetaine, sorbitol and proline, and therefore lack sufficient oxygen to sustain the
which do not intrude upon normal metabolic life of roots. Oxygen deprivation causes the
processes (Hayashi and Murata, 1998) and provide production of ethylene, which causes the cells in the
osmotic adjustment by generating a more negative root cortex to undergo apoptosis. This creates air tubes
water potential, thereby helping to maintain water that allow oxygen to reach the flooded roots. Oxygen
movement into the leaf and consequently, leaf turgor. shortage in roots, like water deficit or high
concentrations of salt, can stimulate ABA production
and movement of ABA to leaves, resulting in
HIGH TEMPERATURE defoliation.
Excessive heat caused by high leaf temperature and

water deficit can denature enzymes and disrupt OXIDATIVE STRESS
metabolism. Evaporation through leaves may lower
the temperature of leaves 3-10°C below the ambient The role of reactive oxygen species (ROS) such as
temperature. In oleander (Nerium oloeander), superoxide (O2¯), hydrogen peroxide (H2O2) hydroxyl
acclimation to high temperatures is related to a greater radical (·OH) and singlet oxygen ( 1O 2)
degree of saturation of fatty acids in membrane lipids, during biotic and abiotic stress responses has been
which makes the membranes less fluid (Raison et well documented. In spite of the fact that ROS is
al., 1982). Above certain temperature (e.g. 40°C in detrimental to plants, causing lipid peroxidation,
most temperate plants), plants begin to synthesize enzyme inactivation, and oxidative damage to DNA,
large quantities of special proteins called ‘heat shock it is a key event causing the induction of systemic
proteins’. It is suspected that these heat shock proteins acquired resistance (SAR) in the hypersensitive
like chaperone proteins, help to prevent the reaction (HR).
denaturing of enzymes by creating a scaffold around Under salt or drought stress, stomatal closure limits
the enzyme. CO2 fixation and NADP+ regeneration by the Calvin
cycle in the chloroplasts thus enhancing ROS
formation. The primary source of H 2O 2 in the
LOW TEMPERATURE chloroplasts is thought to be the Mehler Reaction
(Heldt, 1997):
Damage to plant cells occurs when the water in the
cell walls and intercellular spaces freezes. Plants O2• ¯ + Fe3+(Cu2+) O2 + Fe2+(Cu+)
respond to cold stress by altering the lipid
composition of plasma membranes, e.g. more In the presence of metal ions such as iron (Fe+2, Fe+3),
unsaturated fatty acids are incorporated into the superoxide and hydrogen peroxide may react via
membrane to maintain fluidity. The lower the water Haber-Weiss reaction to produce hydroxyl radical,
potential in these areas the more water leaves the which is highly toxic to biological molecules (Bowler
cells, resulting in an increase in the concentration of et al., 1992):
solutes and lowering the freezing point of the cytosol.
Plants in cold regions increase the concentration of Fe2+, Fe3+
•
sugars in their leaves before winter. Sugars are H2O2 + O2 ¯ OH¯ + ·OH + O2
tolerated in higher concentrations than many ionic
salts. Chilling damage can be minimized if plants To control the level of ROS and to protect cells against
are first hardened (acclimated) by gradual exposure a variety of external stimuli, including biotic and
to cool, but noninjurious, temperatures. During abiotic stresses, plants have a set of defense
acclimation to cool temperatures the activity of mechanisms. These mechanisms involve multi-
desaturase enzymes increases and the proportion of component response systems such as induction of
unsaturated lipids rises (Palta et al., 1993). defense genes, induction of systemic acquired
Desaturation of fatty acids thus provides some resistance, production of pathogenesis related (PR)
protection against damage from chilling. proteins, accumulation of stress metabolites,
enhancement in the activity of enzymes scavenging
I. Türkan et al. 389
ROS (superoxide dismutase, catalase, peroxidases, naturally protect itself against a broad spectrum of
ascorbate peroxidase, dehydroascorbate reductase, pathogens. In addition, these plant pathways enhance
glutathione reductase) and a network of low molecular plant vigor and stress tolerance, and increase crop
mass antioxidants (ascorbate, glutathione, phenolic yield and quality by increasing nutrient uptake and
compounds, tocopherols). Reinforcement of cell wall photosynthesis within the plant.
and cuticle are other mechanisms of defense
(Breusegem et al., 2001; De Gara et al., 2003). There
are many studies showing that under abiotic stress NATURALLY INDUCED SAR VERSUS
conditions, which induce an overproduction of ROS ACTIVATED SAR
in cells, plant resistance is achieved by increasing
the activity of ROS-detoxifying enzymes or the In naturally induced SAR the disease continues to
biosynthesis or regeneration of antioxidant develop before SAR becomes fully effective, with
metabolites (Cakmak et al., 1993; Scandalios 1993; the first damage occurring under field conditions.
Acar et al., 2001; Bor et al., 2003). Therefore, it Natural SAR only occurs sporadically and not
seems that alteration in the expression/activity of ROS- uniformly across the field. With the application of a
detoxifying enzymes could also be a key step in the Plant Activator, the plant’s defense is activated before
activation of phytopathogen defense. the expected onset of disease. The Plant Activator
Reactive oxygen species are thought to be an induces SAR in the whole field and not only individual
important element of the HR regulation scheme, plants, offering a long-lasting protection against a
together with other secondary messengers like salicylic variety of pathogens in different crops.
acid (SA), ethylene, jasmonic acid (JA) and nitric
oxide (NO). Some of these events contribute to the
limitation of pathogen spread within the infected Conclusion
tissue, while other events, e.g. ion fluxes or changes
in protein phosphorylation state, additionally serve In the light of reported findings, we hypothesize that
as a signal initiating transcription-dependent part of plant activators can protect plants from abiotic and(or)
the local response, as well as the starting point for biotic stresses by enhancing water retaining capacity
the transduction of systemic signals to distant parts by osmotic adjustment, increasing stomatal
of the plant (Talarczyk and Hennig, 2001). The conductivity, increasing photosynthetic pigment
development of SAR is associated with the expression contents, improving photosystem-II efficiency and
of a series of genes, including those encoding thus causing an increased photosynthetic rate in plants.
pathogenesis-related (PR) proteins and phytoalexins, Also plant activators might protect many crops from
accumulation of ROS, rapid alteration in cell walls oxidative damage induced by environmental stresses
and enhanced activity of various defense related and modulate the regulation of certain defense
enzymes such as peroxidases, chitinases and ß(1,3) responses involving the antioxidative system and the
glucanases. local and systemic signal transduction pathways
operating under biotic and(or) abiotic stress situations.
With the aid of all these effects, plant activators can
provide a measure of plant protection leading to
The plant activators higher yields in plants under biotic and(or) abiotic
When pathogens attack plants, they respond by stresses. In the light of this hypothesis, we have
producing signal molecules including SA, systemin, initiated an experiment in our Stress Physiology
JA and ethylene and the coordinate-expression of a Laboratory, to investigate the effects of plant
set of genes, many of which encode pathogenesis- activators on alleviating the adverse effects of abiotic
related proteins (PR). This acquired resistance (SAR) stress. We hope that this study will contribute to the
often results in an enhanced capacity for defense- many aspects of abiotic stress-plant activator
gene activation not only at the site of initial infection, relationships.
but also in distal non-inoculated tissues, thereby
protecting the whole plant from secondary pathogen
attack. Plant activators are agents that can effectively References
‘switch on’ these plant defense mechanisms before
the plant is under attack. Once activated, a plant can Acar, O., I. Türkan and F. Özdemir. 2001. Superoxide
dismutase and peroxidase activites in drought
390 Abiotic stressors and plant activators
sensitive and resistant barley (Hordeum vulgare L.) Hayashi, H. and N. Murata. 1998. Genetically
cultivars. Acta Physiol. Plant. 23(3):351-356. engineered enhancement of salt tolerance in higher
Bor, M., F. Özdemir and I. Türkan. 2003. The effect plants. In: Stress Responses of Photosynthetic
of salt stress on lipid peroxidation and antioxidants Organisms. (K. Satoh and N. Murata, eds). Elsevier
in leaves of sugar beet Beta vulgaris L. and wild Science B.V.: The Netherlands, pp. 133-148.
beet Beta maritima L. Plant Sci. 164:77-84. Heldt, H-W. 1997. Plant Biochemistry and Molecular
Bowler, C., M.V. Montagu and D. Inzè. 1992. Biology. Oxford University Press Inc., New York,
Superoxide dismutase and stress tolerance. Annu. p. 522.
Rev. Plant Physiol. Plant Mol. Biol. 43:83-116. Palta J.P., B.D. Whitaker and L.S. Weiss. 1993.
Breusegem, F.V., E. Vranova, J.F. Dat and D. Inze. Plasma membrane lipids associated with genetic
2001. The role of active oxygen species in plant variability in freezing tolerance and cold
signal transduction. Plant Sci. 161:405-414. acclimation of Solanum species. Plant Physiol.
Cakmak, I., D. Strbac and H. Marschner. 1993. 103:793-803.
Activities of hydrogen peroxide-scavenging Raison, J.K., C.S. Pike and J.A. Berry. 1982. Growth
enzymes in germinating wheat seeds. J. Exp. Bot. temperature-induced alterations in the thermotropic
44(258):127-132. alterations in the thermotropic properties of Nerium
De Gara L., M.C. de Pinto and F. Tommasi. 2003. oleander membrane lipids. Plant Physiol. 70:215-
The antioxidant system vis-a-vis reactive oxygen 218.
species during plant-pathogen interaction. Plant Scandalios, J.G. 1993. Oxygen stress and superoxide
Physiol. Biochem. 41:863-870. dismutases. Plant Physiol. 101:7-12.
Hasegawa, P.M., R.A. Bressan, J.K. Zhu and H.J. Talarczyk, A. and J. Hennig. 2001. Early defense
Bohnert. 2000. Plant cellular and molecular responses in plants infected with pathogenic
responses to high salinity. Annu. Rev. Plant Physiol. organisms. Cell. Mol. Biol. Lett. 6:955-970.
Plant Mol. Biol. 51:463-499.
Advances in
AQUA
aquaculture
R.W. Doyle 393
Opportunities and dilemmas in molecular aquaculture genetics

ROGER W. DOYLE
Genetic Computation Limited, Halifax, Nova Scotia, Canada
Introduction: transgenic vs enhanced

selection
Aquaculture, like agriculture, is beset with problems (more likely than not a winner-take-all race) cannot
of cost, price, and risk. Genetic research, in yet be predicted. This paper describes recent work
aquaculture as in agriculture, promises – or dangles which is representative of the two styles, in sufficient
– solutions to many of these problems “real soon detail that the current level of sophistication of the
now”. But what kinds of solution? Investors as well science and the size of the outstanding problems can
as farmers soon notice that there are two, competing both be appreciated. The topics include possible
styles of modern genetics, which in this paper are biological limitations on transgenic growth, progress
loosely termed “transgenic” and “enhanced selection”. in transgenic growth and disease resistance, QTL
These styles are in vigorous competition for public selection, the crucial role of the major
attention and resources. History suggests that the first histocompatibility complex, and prevention of
genetic style to gain a significant lead in solving an wasteful reproduction and pointless sexual
aquacultural problem is likely to drive the other style dimorphisms.
out of the game, probably forever. Inbred/hybrid corn The winner in the commercial (as opposed to
technology effectively stopped the development of technological) race between selection and transgenics
open-pollinated, pure-line varieties of corn will depend to a large extent on the willingness of
(Kloppenburg, 1988), and the developers of four- the public to accept genetically engineered fish.
way hybrid poultry have driven pure-line poultry Transgenic fish are looked at with strong disfavour
breeding to commercial extinction in developed at the present time (Hulata, 2001), but in the long
countries. Commercialization of transgenic salmon term public opinion is an incalculable quantity. The
which grow twice as fast as existing strains could two styles of genetics also have have different long-
happen as early as this year, 2004 (Stokstad, 2002; term consequences for farmers. Marker-assisted
Hoag 2003); if this were to occur classical salmon selection and genetic engineering can benefit the
breeding programs could very well be put out of owners and developers of the technology and,
business. Transgenics with 2X or 4X current eventually, the fish-eating public. They will also give
commercial growth rates are also available for species transient economic benefits to “early adopter”
of tilapia (Rahman et al., 2001). In tilapia, however, farmers, but, in the long term, only those farmers
the rate of broodstock improvement through well- who hold exclusive franchises or own part of the
conducted selection is so rapid (e.g. Bolivar and technology are likely to benefit from it.
Newkirk, 2002) that step-wise transgenic technology
may be unable to keep up or even gain a toe-hold.
Both styles of genetics provide useful solutions to The impossibility of standing still
aquacultural problems but they have rather different
consequences for the industry as well as for the Most farmers are unwilling simply to ignore genetics,
consumer. The winning style in this technology race for two reasons. One is the obvious opportunity cost
394 Opportunities and dilemmas in molecular aquaculture genetics
– lost opportunity – of forgone genetic improvement. this initial, non-genetic, size advantage magnified by
This cost, which can be estimated from simple competition to such an extent that selection for size
economic models of the genetic process, is a becomes ineffective.
commonplace of the promotional literature of Straight-forward mass selection often does work,
aquaculture genetics. Opportunity costs calculated however. In the shrimp Penaeus stylirostris, Goyard
from projected farm revenues will, however, be et al. (2002) showed that a straight-forward mass
overtaken in the long run by the larger cost of being selection program can produce useful genetic gains
forever left behind in the technology race. This is a in growth rate. Selection intensities, which ranged
world in which there are promoters, scientists, between 4% and 18%, were not especially high. The
technology owners, early adopters, follow-on adopters program produced a 21% increase in growth rate by
and the fish-eating public. Only the swift-moving the fifth generation nevertheless. Hetzel et al. (1999)
early-adopter farmers are likely to gain any long- developed two breeding lines in another species of
term competitive advantage from new technology. shrimp, P. japonicus, one mass-selected for high
Large-scale commercial or national genetics programs weight and one for low weight. The direct response
tend to benefit the public, professional geneticists to this selection was an 8.3% gain in weight of the
and the owners of the technology. Economic benefits offspring of the high line. The low line lost about
to individual farmers who are competing with each 13%. The authors conclude that the realized
other in a commodity market are less obvious unless heritability of growth rate in mass selection is only
they become early adopters and/or own part of the moderate but the large family sizes and phenotypic
technology. The situation which confronts farmers variability (opportunity for selection) should permit
is summed up in Alice’s conversation with the Red rapid stock improvement. These remarks apply to
Queen in the Land of the Looking Glass: “ ‘Well, in all aquacultural fish and shellfish.
our country’, said Alice, still panting a little, ‘you’d Surprisingly, the least successful selection programs
generally get to somewhere else - if you ran very tend to have been those which went well beyond
fast for a long time as we’ve been doing.’ ‘A slow simple mass selection in the sophistication of their
sort of country!’ said the Queen. ‘Now, here, you statistical, quantitative genetic methodologies (i.e.
see, it takes all the running you can do, to keep in the index selection using the so-called ‘animal model’).
same place. If you want to get somewhere else, you Several of the most widely promoted projects,
must run at least twice as fast as that!’ ” continuing for decades in salmon and tilapia, have
yet to publish any peer-reviewed proof of genetic
gain. Successful index selection has in fact been
Increased growth rate through classical described in the peer-reviewed literature only twice,
broodstock selection to my knowledge (Hershberger et al., 1989; O’Flynn
et al., 1999). The realized response to selection in
Mass selection has been by far the most commonly those experiments was not, however, what the authors
used artificial selection procedure in aquaculture had predicted from their prior heritability estimates.
(Hulata, 2001). Hulata’s comprehensive review gives Selection projects that have worked best in aquaculture
instances of successful mass selection performed on have up to now been based on the simpler
many aquacultural species. Even in common species experimental designs.
like Atlantic salmon and tilapia, however, the It is not at all clear what has gone wrong with
procedure is sometimes successful (Sanchez et al., sophisticated index selection along classical lines. One
1995) and sometimes not (Teichert-Coddington and possibility is that, as we now know, the genetic
Smitherman, 1988; Hulata et al., 1986). The frequent architecture of quantitative traits differs from the
failure of well-conducted mass selection programs assumptions made when selection indices are
in aquaculture has yet to be satisfactorily explained. calculated. The genetic basis of the variation in
It is certain, however, that hatcheries that adopt a quantitative traits probably involves a few genes
‘breed the biggest’ approach to mass selection for having large effects and many genes having small
growth soon encounter practical problems arising effect (Orr, 1999), contrary to the assumptions of
from variation in spawning times and condition of the ‘infinitesimal model’ from which classical
the breeders. The biggest animals may be somewhat heritability estimates are usually derived.
older, rather than faster growing, and may then have
R.W. Doyle 395
COMPETITION EFFECTS IN SOPHISTICATED as is often the case in aquaculture, but as yet no multi-
SELECTION DESIGNS generation selection experiments based on pedigree
markers have been published. The accuracy of
A more likely explanation, specific to aquaculture, pedigree reconstruction from markers is turning out
are the statistical difficulties caused by competition not to be especially high (Thomas et al., 2002;
among individual fish within a tank, cage or pond. It Coltman and Slate, 2003; Wilson et al., 2003).
has been known for a very long time that competition The between-family component of a selection index
can have drastic effects both on the estimation of is always suspect because of non-genetic,
genetic parameters and the outcome of selection environmental effects common to members of a
programs (Doyle and Talbot, 1986; Jobling, 1983; family even when families are reared together in a
Moav and Wohlfarth, 1974; Purdom, 1974). Estimates common tank and DNA markers used to sort them
of genetic parameters such as heritabilities and genetic out (e.g. Cunningham et al., 2001; Fishback et al.,
correlations, which are used in sophisticated breeding 2002). The idea that animals in the same tank
programs, are distorted because individual error terms experience the same environment is an illusion; a
in the statistical analyses are not independent, as they family added to the tank on the last spawning day
are assumed to be in the linear statistical models used will have a smaller average size than a family added
to estimate them (Hamblin and Rosielle, 1978; Mazer on the first day and will therefore be at a competitive
and Schick, 1991). The food that one animal eats is disadvantage. It will also be relatively underfed if
not available to others; small individuals may be feed is supplied to the tank at a rate which is
intimidated by others even when food is abundant, determined by the size and weight-specific metabolic
etc. The genetic variance component due to the rate averaged across all families.
genotypes of other individuals in the population is
hidden from ordinary genetic analysis. Wolf (2003)
found a negative covariance between direct and WITHIN-FAMILY SELECTION
indirect (competitive) genetic effects, such that genes
that make an individual bigger make other individuals Successful artificial selection programs in aquaculture
smaller. This effect is surprisingly large, and tend to be those in which the effects of un-analysed
furthermore it increases as the genetic relatedness of competition are minimised operationally (e.g., by
individuals in the competing group increases. selection within families (Uraiwan and Doyle, 1986)
Thirty years ago Moav and Wohlfarth (1974) rather than statistically. In pure within-family
pointed out that in common carp non-genetic selection, the biggest animals in each (usually full-
differences in size due to an age difference of only sib) family are selected as breeders (Hill et al., 1996).
one day become magnified by competition and can Mean differences among families, such as age effects,
dominate the outcome of a selection program. At the which can be magnified by competition, are ignored.
present time, however, the lamentable truth is that Because aquacultural species usually produce large
roughly 99.9% of aquaculture genetic analyses ignore broods, selection intensities within families can be
competition despite the fact that it has major non- high. Bolivar and Newkirk (2002) described a within-
genetic effects on the variance of growth rate. There family selection experiment in tilapia, which achieved
are, of course, exceptions, including the work of a realized heritability estimate of only 0.14 over 12
Moav and his colleagues in Israel. Another recent generations. However, within-family selection
exception is the work of Brichette et al. (2001) on permitted high selection intensities of about 2%. The
competitive growth of mussels grown in trays. result was that the size of the fish at 16 weeks more
The competition problem may disappear in index than doubled after 12 generations.
selection as selection incorporates information from Families must be distinguishable in within-family
more relatives extending over more generations. selection, either by growing them in separate tanks
Since the competition effect on index and combined until they are big enough to be tagged or by using
selection has not been well studied mathematically, DNA markers for identifying families. The latter
we do not know whether to expect that happy procedure, called ‘walk-back’ (Doyle and Herbinger,
outcome or not. Information derived from DNA 1994), maximizes effective population size and can
markers may perhaps be used to generate the required achieve very high selection intensities in aquaculture
pedigree structure extending over several generations because of the large fecundities. The potential benefit
in populations that are grown and propagated together, of this procedure over ordinary within-family
selection is that families do not have to be reared for commercial production pending regulatory
separately until they are big enough to be physically approval. The review paper by Hulata (2001) discusses
tagged. the status of growth-rate transgenesis in aquaculture
Selection among families (as opposed to within in considerable detail.
families) is probably only useful when the selected An interesting example of genetic engineering
trait cannot be directly measured on the individual which has nothing to do with production is the
chosen to become a breeder. In practice, this probably development of transgenic tilapia with the potential
limits its utility to selection for resistance to or for treating human diabetes (Wright and Pohajdak,
tolerance of disease when challenge tests are used to 2001). Of course in the ordinary course of things
identify superior families. Non-challenged and tilapia have evolved to produce tilapia insulin, not
therefore non-infected siblings are used as breeders human insulin, but genetic engineering has overcome
(e.g. Argue et al., 2002; Henryon et al., 2002; Oliver this flaw in the Great Chain of Being. Wright and
et al., 2000; Sarder et al., 2001). his colleagues have developed procedures for
encapsulating and implanting fish tissue into diabetic
mice, where it accurately regulates blood glucose
Transgenics and bioengineering levels. Tilapia and human insulin differ by 17 amino
acids but Wright and Pohajdak cloned, sequenced,
Much of the current excitement in aquaculture and modified the tilapia insulin gene by site-directed
genetics – as in all other areas of genetics – lies in mutagenesis. The product was a tilapia insulin gene
transgenesis and bioengineering. This excitement is that codes for ‘humanized’ insulin while maintaining
fully justified by the rapid progress of the technology all of the tilapia regulatory sequences. They proceeded
for introducing foreign gene constructs into to develop a strain of transgenic O. niloticus that
aquacultural species, in the identification of candidate produces humanized insulin along with its normal
genes and target metabolic pathways for transgenesis, insulin. Work still needs to be done to replace the
and in the spectacular growth of genetically modified normal tilapia gene with the humanized gene by
organisms. homologous recombination, and/or to make the
The insertion of growth hormone genes has humanized gene homozygous and adjust the genetic
increased the growth of many species of fish (Devlin background.
et al., 2001; Dunham et al., 2001). Fourfold increases The objective of the project is to use the tilapia as
without obvious side effects have been reported in a source of tissue-transplant material for treatment
salmon (Devlin et al., 1994) and 2.5- to 4-fold of type II diabetes. Insulin-producing tissue is much
increases in tilapia (Rahman et al., 2001). The coding easier and cheaper to collect from tilapia than from
sequences used in transgenesis have sometimes been mammals, so insulin-producing tissue from the
exogenous, such as human or bovine growth genetically modified tilapia should have a markedly
hormone, or have sometimes been isolated from the lower production cost and, probably, enhanced safety
host species and then spliced to an exogenous, non- relative to the present mammalian xenogenetic
transcribed control region to enhance the expression transplant donors, which are usually pigs.
of the gene (Devlin et al., 1995; Martinez et al.,
2000).
Aquaculture bioengineering projects with growth POPULAR PRESSURE TO ACCEPT
hormone have been going on for more than a decade ‘FRANKENFISH’ (?)
(Fischetti, 1991) and have already resulted in patents
such as one granted to A/F Protein Limited Public anxiety over the use of genetically modified
(Anonymous, 2000). In the A/FP procedure a salmon organisms (GMOs) in food makes it impossible to
growth hormone gene is spliced to a promoter predict when transgenic aquaculture species will enter
sequence from another fish, the ocean pout, which commercial production. It is the view of most
causes the transgene to transcribe growth hormone geneticists that acceptance will be very slow, partly
in the liver. Unlike the normal salmon gene, which because the public is thought to see nothing beneficial
is expressed only some of the time, in the pituitary in the technology which would offset the perceived
gland, the transgene is continuously switched on in risks. In the case of agriculture this may very well be
the liver by its liver-specific promoter. The salmon true, at least in developed countries. The GMOs
reach a size of 8 lbs in about 1.5 years. These developed so far have been corn, soybeans etc.
transformed Atlantic salmon broodstocks are ready modified for the economic advantage of seed
R.W. Doyle 397
companies, growers and herbicide manufacturers, not extent that this turns out to be true in general, selected
the end consumers (Charles, 2001). An editorial in strains that achieve growth rates comparable to
Nature (Anonymous, 1999) sums up this attitude: transgenics may remain competitive with transgenics,
“GM soybeans? Who needs them?” The same question with much lower development costs.
can fairly be asked about genetically modified The effect of a transgene on growth may be strongly
aquacultural species, which, with few exceptions, influenced by the genetic background of the host.
have been developed with increased production in Devlin et al. (2001) compared the effect of a growth
mind. hormone transgene in slow-growing, wild rainbow
The dismissal of conventional, production-oriented trout with its effect in rainbow trout that had a long
GMOs may however be expressive of a parochial point prior history of domestication and selection for fast
of view. The need for GMO technology in some growth. They comment that “the growth response is
developing countries is actually rather obvious. The strongly influenced by the intrinsic growth rate and
answer to the rhetorical question “Who needs them?” genetic background of the host strain, and that
may be, “practically everyone in the third world”. inserting growth-hormone transgenes into highly
Trewavas (1999) makes a case for believing that a domesticated fish does not necessarily result in further
new agriculture, combining genetic modification growth enhancement.” The growth of the transgenic
technology with sustainable farming, is our best or fish speeded up 17-fold (!) but was still not faster
only hope for staving off an ecological catastrophe. than that of the highly domesticated strain. The
If we don’t use biotechnology, he says, we are going domesticated strain hardly responded to the transgene
to run out of arable land and water. The conflict at all. Cranial abnormalities were seen in the
between environmental activists and starving third- transgenic but not in the domesticated animals, which
world pragmatists made headlines at the World were growing at about the same rate, suggesting that
Summit on Sustainable Development, held in August ordinary homeostatic mechanisms were not coping
2002 in Johannesburg. Zambian President Levy with the novel pathways of growth and development
Mwanawasa (who had declared a food emergency induced by the transgene.
three months previously) announced that he had Devlin and his co-authors conclude that “The effect
stopped the distribution of 17,000,000 kg of corn of introducing a growth-hormone gene construct into
because some of it is genetically modified. “We would fish to increase growth rates appears to be dependent
rather starve than eat something toxic”, the President on the degree to which earlier enhancement has been
said, voicing the anti-GMO viewpoint of the achieved by traditional genetic selection. Such effects
developed world (Wente, 2002). Not everyone in are likely to be specific for different species, strains
Zambia agrees. Wente cites a Los Angeles Times and transgenes — in selected mice or in domesticated,
reporter who was told by a Zambian, “We don’t care rapidly growing farm animals, for example, growth-
if it is poisonous because we are dying anyway”. hormone transgenesis can have little effect on growth
Public perception, not science, is the key to the future. or it can induce pathological effects, as we have seen
Pressure to accept GMO technology which increases in transgenic salmonids.”
yield may bring GMO crops into production in the Aquaculture geneticists, like other biologists, may
poorer and more ecologically stressed parts of the be able to learn a lot from the study of mice. Bunger
world much sooner than many people expect. and Hill (1999) selected lines of mice for high and
low body weight for more than 50 generations, after
which the high and low lines had diverged
BIOLOGICAL LIMITATIONS ON TRANSGENIC approximately 3-fold in their weight at 98 days. The
GROWTH authors then eliminated growth hormone from the
metabolism of the mice by genetic ‘knock out’, which
Transgenics stands far above all other productivity- they achieved by backcrossing a defective growth
enhancing genetic technology in terms of potential hormone (GH) releasing factor receptor gene into
payoff and risk. However, there are some preliminary both lines. Control high and low lines with the normal
indications that the most direct transgenic procedure, GH gene were also maintained.
inserting a transgenic growth hormone, may be Both lines of mice carrying the knock-out gene,
relatively ineffective in lines which have already been which were thereby deficient in GH, were much
selected for high growth (Devlin et al., 2001; Parks smaller than the normal control mice at 98 days.
et al., 2000). It appears that selective improvement There is no doubt that growth hormone makes mice
and transgenesis may not combine additively. To the
grow quickly. What is surprising is that the divergence Some of the more promising transgenes for disease
of the high and low lines was almost as great in the resistance are genes encoding lectin molecules.
absence of growth hormone (2.4-fold divergence) as Lectins are small peptides (amino acid sequences)
in its presence (3.1-fold). The authors conclude that that bind to sugar molecules exposed on the surface
after appropriate scale transformation, “changes in of cell membranes. After binding, some types of lectin
the GH system contribute only a small part of the lyse the phospholipid bilayer of the membrane, killing
selection response in growth ... [and] other systems the cells. Lytic peptides are proving to be potent toxins
contributed most of the selection response”. to a broad range of bacterial, fungal and protozoan
This experiment should interest the aquaculture pathogens. Much work has gone into producing
community even though it was performed on mice. transgenic plants and mice that express enhanced
We know that transgenic fish carrying extra growth levels of lectins as built-in fungicides, bactericides
hormone genes, or modified genes that express GH and insecticides.
continuously, are fast-growing fish - sometimes very Disease-related transgenic experiments in
fast-growing. This ingenious knock-out experiment aquaculture have focused on Cecropin-B, an
on mice is a hint that the converse may not be true. antimicrobial peptide of about 35 amino acids which
Selection of fast-growing fish by classical methods is synthesized in the pupae of the silk moth in
may evoke an entirely different kind of genetic response to bacterial infection. Electroporation has
change that does not involve growth hormone. been used to incorporate cecropin-producing genes
Furthermore, it suggests that if crosses between high- into Medaka, with a resulting increase in resistance
and low-selected lines are used in searches for growth to Pseudomonas fluorescens, Aeromonas hydrophila,
QTLs, the growth hormone system will not and Vibrio anguillarum (Sarmasik et al., 2002).
necessarily provide the best candidate genes. Challenge studies showed that while about 40% of
the controls were killed by both pathogens, only up
to 10% of the F2 transgenic Medaka were killed by
TRANSGENIC DISEASE RESISTANCE P. fluorescens and about 10% to 30% by V.
anguillarum.
Resistance to disease is of particular interest in the A similar transgene construct and insertion
culture of salmonids and shrimp, a fact which is procedure greatly increased the resistance of the
reflected in the focus of ongoing projects in genetic channel catfish Ictalurus punctatus to the epizootic
engineering. Standard techniques for inserting of Flavobacterium columnare in an earthen pond
foreign genes have been difficult to apply to shrimp (Dunham et al., 2002). Fully 100% of the transgenic
because embryos of Penaeus are released from their catfish survived a natural exposure to the
mothers at a relatively advanced stage. Newly- flavobacterium, versus 27% survival of normal fish.
fertilized eggs are essentially unavailable at the When challenged in tanks with Edwardsiella ictaluri,
appropriate stage for microinjection or electro- a bacterium that causes enteric septicaemia in catfish,
poration. Sarmasik et al. (2001) may have found a survival of the transgenic fish was 41% versus 15%
way around this problem, which is expected to work for the controls.
in other crustaceans and live-bearing fish. The foreign The use of cecropin transgene in aquaculture has
gene is carried into the host by an extensively been patented (Cooper and Enright, 1999). The patent
engineered viral vector. One engineered feature of claims that “Augmentation of the host’s defences
the vector makes it unable to replicate. Other features, against infectious diseases or tumours is achieved by
derived from the hepatitis B virus and the vesicular “arming” the host’s cells with an exogenous gene
stomatitis virus (a pathogen similar to hoof and mouth encoding a natural or synthetic lytic peptide. …The
disease which infects mammals, insects and possibly transformed cells have the ability to produce and
plants), enable the vector to stick to the cell membrane secrete a broad spectrum chemotherapeutic agent that
of a wide variety of organisms. Immature gonads of has a systemic effect on certain pathogens, particularly
the crayfish were injected with a solution of the vector pathogens that might otherwise evade or overcome
about one month before the normal age of first host defences.”
reproduction. When they matured the injected
individuals were mated with normal individuals.
Sarmasik et al. (2001) provide proof of integration, MARKER-ASSISTED AND QTL SELECTION
expression and transmission of the reporter transgene
for at least three generations. The search for disease-resistance and other loci that
R.W. Doyle 399
have effects which are large enough to be useful but drive the transcription of the gene. A difference of
not large enough to be obvious by simple segregation only one base pair in the regulatory sequence accounts
analysis (quantitative trait loci, or QTLs) is following for the adaptive difference between the northern and
two approaches, marker-assisted selection (MAS) and southern populations.
the search for ‘candidate genes’. Both approaches It appears that over the long term, phenotypic
are expected to be most useful when ordinary selection for quantitative traits may give better results
quantitative genetic procedures for estimating than either MAS or QTL selection. In a simulation
breeding values have especially low accuracy – in study Villanueva et al. (2002) found that selecting
particular, when selecting for disease resistance if the for one particular gene allows the other additive
selected animals cannot be exposed to the disease. ‘background’ genes to drop out of the population by
MAS and QTL selection will be less useful in chance (inbreeding rate will usually be an indicator
selecting for growth where heritabilities are usually of this effect). Thus there is a loss of additive genetic
reasonably high and estimation of the breeding values variance with QTL and MAS selection relative to
of individuals from their phenotypes is reasonably phenotypic selection, and the ultimate selection limits
accurate. Simulation studies of selection on growth are lower. Interestingly, the reverse seemed not to
rate, a trait that is measured on all animals prior to happen in the simulation - only very rarely was the
selection, find only small gains from addition of advantageous QTL allele lost during phenotypic
marker data (Lande and Thompson, 1990). selection. However, in the short term, both QTL and
A typical example of MAS for a disease-related MAS gave a more rapid initial response than
trait in aquaculture is provided by the work of Ozaki phenotypic selection. Salmon and some other
et al. (2001) on QTLs associated with susceptibility aquacultural species have such long generation
to infectious pancreatic necrosis virus (IPNV) in intervals that rapid response could actually be worth
rainbow trout (Oncorhynchus mykiss). Backcrosses more, by an economic calculation like net present
between resistant and susceptible strains were used value, than a high selection plateau that might not be
to identify several chromosome regions containing approached for 100 years.
putative QTL genes that affect disease resistance.
Fifty-one microsatellite markers were used for the
linkage analysis. The major histocompatibility complex
Perry et al. (2001) employed a rather similar and disease resistance
approach in finding a QTL for upper thermal tolerance
in outbred strains of rainbow trout. Segregation at Interest in genetic variation in the major
the microsatellite marker for the QTL explained 7.5% histocompatibility complex of fish (MHC) is running
of the variance in thermal tolerance in the trout high these days. The diversity of the MHC loci, which
progenies. This is about what we would expect for a are the foundation of vertebrate immune systems,
relatively large QTL. appears to be driven by the diversity of pathogens in
The candidate gene approach to finding QTLs the environment (Penn et al., 2002). Fish may choose
involves looking for genetic variants in biochemical their mates to optimize the MHC genotype of their
or developmental pathways that are known, or offspring (Landry et al., 2001). The preferred
strongly suspected, to affect the trait of interest. An explanation for this ‘disassortative mating’, in which
example is the work of Schulte et al. (2000) on the fish choose mates which are genetically unlike
lactate dehydrogenase-B gene (Ldh-B) in northern themselves, is that MHC heterozygotes are
and southern populations of the fish Fundulus intrinsically more fit than homozygotes
heteroclitus. Northern (Newfoundland) fish grow (overdominant selection at MHC loci; Arkush et al.,
better at lower temperatures while fish from Florida 2002). The thought is that heterozygosity at MHC
are superior at higher temperatures. The experimental loci may enhance a host animal’s resistance to
details are too complicated to be easily summarized pathogens by increasing both the diversity of peptide
here but they include temporary transgenesis of the antigens it presents to T-cells and the diversity of the
regulatory sequences into the livers of experimental T-cells themselves.
fish; deletion studies to identify the approximate But there is also evidence (Miller et al., 2001) that
location within the regulatory sequence where the particular MHC alleles may be directionally selected,
adaptive changes in the transcript occurred; stress tests i.e. towards homozygosity, possibly on a lake-specific
of live fish to see which alleles (northern or southern) basis. Heterozygotes would not be more fit than MHC
homozygotes and disassortative mating should not population that is unusually well adapted to the
be selected in such lakes. And in an experiment in an targeted stress.
aquaculture-like environment where exposure to We would like to know what the best genetic
specific pathogen strains was controlled, particular management strategy is: selection for homozygosity
MHC alleles appeared to have a selective advantage of particular MHC alleles, or selection for MHC
but heterozygosity did not (Lohm et al., 2002). diversity per se. The answer is important both to
Along with laboratory, field and theoretical studies conservationists and to geneticists who hope to profit
of the advantages of immune-system diversity per from the development of proprietary ‘super breeds’
se, there have also been demonstrations that specific for aquaculture. Evidence from other organisms (e.g.
pathogens can exert strong selection on particular mice; Penn et al., 2002) suggests that it probably
MHC alleles in fish. Lohm et al. (2002) reported on depends on the variety and timing of challenges
the resistance to furunculosis in Atlantic salmon anticipated from pathogens. Optimal selection
originating from the Akvaforsk strain currently reared strategies for populations growing in extensive and
by AquaGen AS in Norway. Families of salmon were ‘biosecure’ aquaculture systems may be even more
mated on the basis of their MHC genotypes so as to different than we thought. MHC diversity
generate mixtures of homozygous and heterozygous considerations may become crucial, both practically
individuals within the same full-sib families, thus and politically, in the design of captive breeding and
controlling the genetic background against which aquaculture genetics programs (Arkush et al., 2002).
specific MHC alleles were expressed. Surprisingly,
in this controlled breeding experiment MHC
heterozygosity per se did not improve resistance to Diversion of resources away from growth
the furunculosis challenge test. It was particular Class and into reproduction
II MHC alleles that conferred relative fitness
differences as great as 0.5. The authors write, “This Early-maturing male salmon and trout are a problem
study clearly shows a strong survival advantage for for aquaculture because male fish are physically
individuals carrying a high-resistance allele when unappealing and their growth slow. Age-at maturation
exposed to a bacterial infection. … directional is genetically the same trait in both sexes in salmon,
selection acting on the MHC despite its high judging by the strong genetic correlation between
polymorphism stresses the importance of renewal of the males and females for both age of maturation
genetic variation at these kinds of loci, either from and weight (Kause et al., 2003). Thus it will not be
mutation, recombination or immigration from other easy to develop a strain in which males mature late
populations, when combating new or coevolving but maturation of females is unchanged. The
virulent pathogens.” heritability of both traits is sufficiently high, though,
A recent study by Cohen (2002), which delved that selection for late maturation of both sexes should
deeper into the molecular structure of the MHC work if performed on either sex.
antigen-binding sites, could be a harbinger of more There is an analogous problem in tilapia with the
powerful ways to investigate and exploit MHC difference that the female, not the male, diverts
variation in fish. A population of Fundulus resources towards reproduction at the expense of
heteroclitus was found to have adapted to an growth. An ingenious genetic work-around for sexual
environment which has been grossly polluted with dimorphism in tilapia has been found and is achieving
PCBs and other contaminants for more than half a considerable commercial success. As described by
century and which is toxic to other Fundulus. The Mair et al. (1997), the procedure involves five
population also tolerates high loads of parasites preparatory generations of progeny testing and
(helminths and others) which are rare or absent in hormonal sex reversal, both male-to-female and the
other populations. By studying this and control reverse. The final result is a set of YY ‘supermales’
populations living in more benign environments the which, when mated with normal XX females,
authors found amino acid substitutions which tend produce offspring which are nearly 100% normal
to be concentrated in different parts of the antigen- XY males. The YY supermales have a few female
binding region of the molecule. They proved that offspring, however, presumably because of the
the MHC variation is driven by selection, not drift. multifactor sex determination in this species. YY male
The first step in applying this technique in other breeders are now used commercially in many places
situations, e.g. searching for selectable QTLs in an to generate grow-out populations that consist entirely
aquaculture broodstock, would seem to be finding a
R.W. Doyle 401
of genotypically normal XY males. The lack of decisions about genetics must be made every time
females contributes to uniformity and more rapid fish or shellfish are stocked. Are these breeders the
growth and also stops unwanted reproduction in best available? Are they are even average? Would
aquaculture ponds. one of the breeds promoted on the worldwide web
The only major problem with the above procedure grow well enough to stave off bankruptcy?
is that it interferes with selection for traits such as The application of modern genetics is often listed
growth rate (in the YY male donor line, at least) as one of the highest priorities in aquaculture research
because of the five generations of preparation. The and development agendas. Nevertheless, farmer
lag could be reduced considerably if the genotypic associations and government agencies are slow to
sexes could be identified without progeny testing. invest in ‘big genetics’, as has often been noted and
There is reason to hope that commercially useful lamented by professional geneticists. This is not
sex-specific markers can be found in tilapia. Harvey because ‘big business’ is attempting to direct public-
et al. (2002) report the development of in situ sector research towards its own interests, as it is
hybridization probes to identify sex-specific sequence reputed to have done during the development of
differences in the long arm of chromosome 1 of the scientific agriculture (Kloppenburg, 1988). Individual
tilapia species Oreochromis niloticus. The binding farmers just seem to prefer activities that they can do
difference between the probe sequences from X and themselves on their own farms.
Y chromosomes is small, which is not surprising. The question a farmer asks about programs that
The authors comment that “Only limited sequence promise to provide ‘super fish’ or ‘super shrimp’ is
divergence between the X and Y chromosomes would which program will bring the largest economic benefit
be expected as YY individuals can develop into males to his own farm. As indicated earlier, modern genetics
or, if hormone treated, females that are both viable has shown itself capable of producing genetic
and fertile, although growth and survival rates are productivity gains measured not by increments but
somewhat lower in YY than XY males.... This by multiples of the existing standard. Every time a
suggests that only a very limited loss of function can farmer stocks or re-stocks his farm he realizes the
have occurred in Y-linked genes and that sequence existential horror of his predicament: the available
differences between the X and Y chromosomes are genetic information is irrelevant and incomplete in
largely confined to non-coding regions.” its most crucial practical aspects; doing nothing about
Lee et al. (2003) used bulked segregant (BS) genetics is also taking action; the appropriate emotions
analysis to search for microsatellite marker genes for the practical person are anguish and dread.
associated with phenotypic sex in tilapia. (In BS When a new agricultural technology is introduced
analysis DNA from many individuals with the same everyone involved can be considered either a
phenotype is pooled and compared to a pool of DNA beneficiary, a bystander or a victim. The immediate
from a contrasting phenotype. The contrast here was beneficiaries are easy to identify. Professional
male vs female phenotypes.) Ten markers were found, geneticists (some of them) will benefit from being
all on linkage group 8, which is therefore the (or a) the heroes of the technology and so will their research
putative Y-chromosome. The linkage of two markers sponsors. The owners of the technology when it is
with the sex-determining region was so tight that the successfully commercialized will also benefit. The
sex of offspring of two families was correctly fish-eating public will, eventually, be able to buy
predicted 95% of the time. Unfortunately the markers fish at a lower price.
were not linked to sex in the third family, which Some farmers - the early adopter farmers - will
shows that we are still some distance away from using also obtain a competitive commercial advantage
markers instead of progeny testing in the commercial which benefits them in the short term. The same
production of tilapia YY-supermales. competition, however, victimizes follow-on farmers
in the short term. Simple economic theory predicts
that farm-gate prices will go down, production will
The existential dilemma of aquaculture be up and farmers will become increasingly
farmers dependent on technology over which they have no
control, just as in modern terrestrial agriculture (Allen,
Farmers beset with problems of cost, price and risk 1984; Kloppenburg, 1988; Weller, 1999). The owners
are faced with a dilemma because ignoring genetics of the technology and the fish-eating public will be
is likely to be just as costly as paying for it. Practical sharing the benefits of the higher productivity
permitted by the new technology and farmers will question which interests Mr. James is how to invest
be in the same cost-price squeeze as they were before money. The question which interests aquaculturists
- essentially, bystanders. is how to bet the future of the fish farm, since a bet
If a new strain of fish or shellfish is available to all on genetics must be made.
producers, market competition occurs and a new If we apply James’s analysis to aquaculture genetics
equilibrium price is reached at the new and lower we conclude that companies which provide
production cost which, however, now includes proprietary products like vaccines or genetically
whatever extra fees the producers have to pay for the improved broodstock and fingerlings can potentially
new strain. It will of course benefit the technology make the highest profit but also experience the highest
developers to make their technology universally risk, in particular the risk that someone else will
available – for a price - and not restrict it to a few develop a product which is cheaper or more effective.
producers. The benefits of the increased production Purveyors of aquaculture genetic information about
efficiency accrue to the public, which may pay less genomic sequences, markers and maps are mostly
for fish, and to the sellers of the technology. but not entirely in the public sector. The opportunity
Competition for market share again requires producers to generate value from proprietary information about
to reduce their price and profits to the lowest tolerable QTLs, pathogens and broodstock genotype-
level. environment interaction in aquaculture is not being
The above reasoning leads to the conclusion that ignored, however. The commercial risk to
farmers who want to benefit from purveyors of information suppliers is that their proprietary
advanced technology should try to become early information will become ‘commoditised’ and freely
adopters and possibly insist on some sort of exclusive available. Some people are even making a moral
franchise arrangement to protect their competitive crusade out of the public right to raw genetic data.
advantage as long as possible. The farmer should also Spider Robinson neatly summed it up in the Toronto
attempt to obtain sole or part ownership of the Globe & Mail on 18 March, 2000: “It’s as though an
technology in order to continue benefiting as explorer took the first photo of a zebra - then claimed
bioeconomic equilibrium is approached. The ownership of zebras, the concept of stripedness, and
developers of the technology will be anxious to attract anything else substantially zebraic in nature”.
early adopters for promotional reasons and the Both in human genomics and in aquaculture
leverage this gives the farmer might be translated genetics there are companies that develop technology
into franchises or ownership. for use by other companies e.g. for on-farm
It should be emphasized that there is a divergence broodstock improvement. (My own consulting
of interest between the developers of the technology company falls into this category.) James notes that
and the farmers who use it. The former group wishes such companies “though in some ways offering the
to maximise the spread of the new technology, for lowest risk for investors, are always in danger of
reasons of professional prestige (including becoming generic or outdated as new ways for tackling
institutional prestige in the case of universities, a problem are developed.”
governments and international development agencies) Given the entirely rational preference that farmers
as well as commercial gain. have for technologies over which they have sole
ownership, professional geneticists could usefully
focus more effort on developing high- as well as low-
The dilemma of aquaculture genetics technology procedures that can be applied on a small
companies scale. Farmer-breeders would then have more
scientific support and information when choosing how
James (2000) recently distinguished between three to act at the farm level. They might then feel more
types of genomics companies: product providers, comfortable in their existential dilemma where any
information providers and technology providers. procedure for choosing breeders, even random
Although he wrote about companies that are working choice, has genetic consequences. Such procedures
on human health issues, his analysis applies equally would enable individuals to develop proprietary
well to genetics in aquaculture. Companies in these breeds on their own farms and should speed up the
three areas are now racing for primacy and moving application of modern genetics to aquaculture.
into each other’s commercial strategy space. The
R.W. Doyle 403
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D.F. Fegan 407
Feeds for the future: the importance of better broodstock and larval
nutrition in successful aquaculture
DAN F. FEGAN
Alltech Inc., Bangkok, Thailand
Introduction
The future of aquaculture production, as in other given the high cost of feed in a captive broodstock
animal production systems, is towards greater control maturation facility, estimated at around 50% of total
of the physical, chemical and biological variables cost, as well as a heavy reliance on fresh feed items
surrounding the production system. The trend from such as squid, mollusk meals and polychaete worms
collection of wild fry to the development of hatcheries which may vary in quality and availability, there is a
for fry production, from the use of wild spawners to demand for feeds specifically formulated for use in
the use of maturation systems and ultimately closing maturation diets. It has also been pointed out (Doyle,
the reproductive cycle to use domesticated and personal communication) that the development of
selected stocks is clear. At the same time, there is an genetically improved lines of shrimp may benefit
increasing trend towards production efficiency and from a simultaneous development of feeds specifically
cost-effectiveness, not least in the hatchery stage, tailored to individual strain requirements.
particularly where the supply – demand situation During penaeid shrimp maturation, nutrient reserves,
progresses from quantity production to quality mainly from the hepatopancreas, are mobilised to
production. These trends are becoming more evident support ovarian and testicular maturation,
in all forms of commercial aquaculture. gametogenesis and vitellogenesis (Harrison, 1990;
Broodstock and larval nutrition are key elements 1997). Tissue reserves in the hepatopancreas can be
underpinning this progress towards greater control depleted rapidly so that the diet becomes the most
and domestication. However, there is substantial work important contributor of nutrients to the developing egg.
to be done if rearing of aquaculture species is to This is particularly true when, as is the case in most
approach the level of control and understanding which captive maturation facilities, eyestalk ablation is used
are evident in the poultry, swine and ruminant sectors. to accelerate the process of gonadal maturation. The
This paper gives a brief introduction to some of the hormonal and metabolic changes that come around
key areas and issues in broodstock and larval nutrition. during such forced maturation may take place when the
Constraints of time and space mean that the paper is nutrient reserves are insufficient to support rapid ovarian
largely confined to crustacean, and particularly, development placing an even larger burden on the diet
penaeid shrimp culture. However, many of the same as a source of essential nutrients.
gaps are found when looking at the broodstock and The nutrition of penaeid shrimp broodstock has
larval nutrition of most fish species. recently been reviewed (Wouters et al., 2001a). The
authors recognize that there is a dearth of information
in the scientific literature on the subject, possibly
Broodstock nutrition due to the expense and complexity of running
sufficiently rigorous nutrition experiments on shrimp
Research into shrimp broodstock nutrition is gaining broodstock. In addition, much of the research has
importance with the increasing use of domesticated been carried out with fresh feeds, either alone or in
and genetically selected stocks for aquaculture. Also, combination with formulated diets or dietary
supplements.
408 Feeds for the future: the importance of better broodstock and larval nutrition in successful aquaculture
LIPIDS
Due to the importance of lipids in crustacean Cholesterol is the precursor of steroid hormones and
maturation, much of the work carried out to date has it is known that shrimp have a requirement for
focused on this aspect, particularly the requirement cholesterol in the diet. Cholesterol is stored in the
for highly unsaturated fatty acids (HUFA) and hepatopancreas and is mobilized during maturation.
phospholipid. During maturation, lipids are mobilized The role and mobilization of cholesterol during
from the hepatopancreas in many species and dietary shrimp maturation has been reviewed by Harrison
lipids rapidly processed for transport to the developing (1990). Some of the live feed organisms used in
ovaries. maturation diets have relatively high cholesterol
Total lipid does not appear to be important although levels (e.g. squid, clams) although to date there has
Wouters et al. (2001b) reported that excessively high been limited research into the effects of dietary
total lipid in the diet had an adverse effect on ovarian cholesterol on maturation and reproduction (Harrison,
maturation and feed consumption, possibly due to 1997; Wouters, 2001).
satiation. However, the average lipid level in During maturation, the level of triacylglycerides
commercial broodstock diets (10%) appears to be (TAG) in the ovaries increases as they are incorporated
around 3% higher than in grower feeds used in into the egg and decrease after spawning (Ravid et
commercial culture ponds. al., 1999; Wouters et al., 1999b). Triacylglycerides
Highly unsaturated fatty acids (HUFA), especially appear to be the principal energy source in eggs and
20:5n-3 and 22:6n-3, are abundant in ovarian tissues nauplii and their importance in reproduction, and egg
and are believed to be an important component of and postlarval quality has been shown (Palacios et
live and formulated maturation diets. Diets deficient al., 1998; 1999).
in n-3 HUFA have been found to have a negative
effect on ovarian development, fecundity and egg
quality (Teshima et al., 1988; Alava et al., 1993; Xu PROTEIN AND AMINO ACIDS
et al., 1992; 1994; Cahu et al., 1994; Wouters et al.,
1999a). Maturation is a time of intense protein synthesis and
Arachidonic acid (20:4n-6; AA) has been detected it is likely that the requirement for protein is higher
at high levels in the ovaries of wild shrimp and is at this time (Harrison, 1990; 1997). Wouters et al.
also abundant in some of the best fresh feed items (2001a) report that the protein content of formulated
such as polychaetes (bloodworms), clams and mussels diets in their studies was around 50% but that this
(Harrison, 1997; Wouters et al., 2001a). The n-6 was still low compared to the level in fresh feeds.
HUFA are known to be precursors of the Detailed studies into protein requirements for shrimp
prostaglandin hormones, which act in reproduction broodstock are still lacking and it has been proposed
and vitellogenesis. According to Wouters et al. that the amino acid profiles should mimic those found
(2001a), formulated maturation diets appear to be in fresh feeds (Deshimaru, 1982).
deficient in AA as well as relatively low Some studies have shown changes in protein
eicosapentanoic acid (EPA) levels. It has been content of the ovaries associated with egg
proposed that the ratio of n-3 to n-6 levels in the diet development and spawning, and with spawning
is important (Lytle et al., 1990) and that it should be success. Animulkar (1980), reported in Harrison
around 2–3 to 1 (Ravid et al., 1999; Wouters et al., (1997), found an increase in ovarian protein levels
1999b). associated with ovarian development followed by a
Phospholipids, mainly phosphatidylcholine and sharp decrease after spawning in the shrimp
phosphatidylethanolamine appear to be predominant Paratelphysa hydrodromaus and several authors have
in the shrimp ovary and there seems to be a noted a similar increase in farmed penaeid shrimp
requirement for phospholipids in the diet. Several (Read and Caulton, 1980; Castille and Lawrence,
studies (Alava et al., 1993; Cahu et al., 1994; Ravid 1989). A marked difference has also been noted in
et al., 1999; Wouters et al., 1999b) have the protein content of the hepatopancreas and ovaries
demonstrated the effects of phospholipid levels in of wild and domesticated females of Litopenaeus
the diet and it has been suggested that broodstock vannamei with good repeat spawning performance
diets should contain more than 2% phospholipid to which have been found to have significantly higher
ensure that 50% of total egg lipid is in this form protein content than females with poorer spawning
(Cahu et al., 1994). performance (Palacios et al., 2000)
D.F. Fegan 409
CARBOHYDRATES
Carbohydrates do not appear to be essential for shrimp complications (Wouters et al., 2001a). Where studies
broodstock diets although Palacios et al. (1998; 1999) have been conducted, diets were formulated with
related egg glucose levels with larval quality and mineral mixes with added calcium, phosphorus,
broodstock condition. Carbohydrates can be used as magnesium, sodium, iron, manganese and selenium
cost-effective ingredients to contribute to glycogen (Chamberlain, 1988; Marsden et al., 1997; Mendoza
accumulation in the hepatopancreas (Harrison, 1997) et al., 1997; Xu et al., 1994).
as well as providing other benefits in the broodstock Spent broodstock of L. vannamei had lower levels
diet, acting as binders and possibly playing a role in of calcium and magnesium in the muscle and lower
transport of nutrients in the hemolymph (Harrison, magnesium levels in the hepatopancreas (Mendez et
1997). al., 1997), possibly due to a combination of dietary
deficiencies and losses through moulting and transfer
VITAMINS AND MINERALS to the eggs. Copper also decreased in the
hepatopancreas, possibly through transfer to the
Detailed vitamin and mineral requirements for shrimp
ovaries, although it increased in the muscle tissue. It
broodstock diets are relatively unknown with only a
is clear that more studies need to be undertaken into
few studies on vitamins A, C and E. Alava et al.
mineral nutrition in broodstock diets.
(1993) found that ovarian maturation was slower
when fed a diet deficient in either vitamins E, A and C.
Vitamin E appears to be important in crustacean
CAROTENOIDS
broodstock nutrition. Chamberlain (1988), reported
in Harrison (1997), found a correlation between Crustaceans cannot synthesise carotenoids de novo,
vitamin E deficiency and the percentage of abnormal and a dietary source of these pigments is required.
sperm in Litopenaeus setiferus and Cahu et al. (1991) During sexual maturation, most crustaceans
found an improvement in hatching rate with accumulate carotenoids in the hepatopancreas and
increasing dietary vitamin E correlated to increasing during vitellogenesis, these are transported in the
α−tocopherol levels in the egg. Wouters et al. hemolymph as carotenoglycolipoproteins to
(1999b) found a similar correlation to that observed accumulate in the eggs as part of the lipovitellin
by Cahu et al. (1991) between spawn and hatch protein. Dall (1995) found that free astaxanthin levels
quality with α-tocopherol levels in wild spawners in the developing ovaries of Penaeus esculentus
and nauplii of L. vannamei. They found that mature increased from 2 to 34 ppm and in the digestive gland,
ovaries and nauplii contained higher levels of α− from 20 to 120 ppm.
tocopherol than immature and spent ovaries. Harrison Carotenoids, especially astaxanthin, are strong
(1997) also speculated that vitamin E in the egg yolk antioxidants and probably play a role in protecting
may also act as a natural antioxidant. the broodstock nutrient reserves and developing
Work conducted by Fisher and Kon (1958), embryos from oxidation (Dall et al., 1995; Merchie
reported in Harrison (1997), suggested the importance et al., 1998). It has also been suggested (Harrison,
of dietary vitamin A due to its accumulation in the 1997) that they act as pigment reserves in the embryos
ovaries of crustaceans during maturation. Vitamin C and larvae for the development of chromatophores
content of eggs of Fenneropenaeus indicus are also and eyespots, and as a vitamin A precursor (Dall,
influenced by the levels in the diet, and high hatching 1995).
rate has been related to high ascorbic acid levels in Wyban et al. (1997) noted a decrease in nauplius
the eggs (Cahu et al., 1995). Harrison (1997) assumes quality with successive spawns associated with a loss
that vitamin D is important in broodstock diets due of pigmentation in the ovary of L. vannamei.
to its probable role in calcium and phosphorus Addition of paprika, an inexpensive source of
metabolism in crustaceans. carotenoids, to the fresh diet at a rate of 2% (2 g
Harrison (1990) discussed the possibility that paprika per 100 g squid meat), resulted in a
mineral deficiencies or imbalances could have a significant improvement in nauplius quality (measured
negative impact on crustacean reproduction and may as survival to zoea 2 stage).
play a role in oocyte resorption, reduction in Pangantihon-Kühlmann and Hunter (1999) found
reproductive performance and egg quality. Studies that astaxanthin supplementation (50 mg/kg) of the
into mineral requirements are rare due to several diet resulted in increased egg production in Penaeus
monodon but could not demonstrate any benefit of meal, legumes (adenine is particularly high in black-
astaxanthin supplementation on either hatching rate eyed peas), yeast extracts and unicellular organisms
or metamorphosis to zoea 1 stage. such as yeasts and bacteria that are rich in RNA or
DNA (Carver and Walker, 1995; Devresse, 2000).
Devresse (2000) noted that the low digestibility of
MISCELLANEOUS FACTORS whole yeast compared to yeast extract may be related
to the protein (nitrogen) solubility as yeast extract
Hormones has much higher protein solubility than whole yeast.
It has been suggested that some of the more successful He also noted that, although fish solubles are highly
live feed organisms may provide benefits through the digestible, they leach easily in water affecting
provision of hormones or their precursors. Naessens availability.
et al. (1997) speculated that part of the reason for the Reproduction and egg development have a high
success of reproductive adult Artemia biomass requirement for RNA and DNA and it may be
supplementation of the diet of L. vannamei expected that increasing the availability of nucleotides
broodstock could be due to the presence of specific in broodstock diets may have a beneficial effect on
hormones or analogous peptides in the Artemia that egg development. Recently, research has
provoked a response in the shrimp. Bloodworms used demonstrated the effect of a nucleotide-enriched diet
in maturation have also been found to contain methyl for broodstock nutrition in aquaculture (Gonzalez-
farnesoate, an ecdysone hormone that has been shown Vecino, 2002; Gonzalez-Vecino et al., 2003).
to increase reproductive performance in the spider Nucleotide enrichment of broodstock diets for
crab Libinia emarginata (Laufer et al., 1987), L. Atlantic halibut (Hippoglossus hippoglossus) and
vannamei (Laufer et al., 1997), P. monodon (Hall et haddock (Melanogrammus aeglefinus) resulted in a
al., 1999) and the crayfish Procambarus clarkii (Laufer general trend towards better spawning performance
et al., 1998). In P. clarkii, the hemolymph titer and egg quality with the nucleotide diet. Total egg
increased from basal levels during early vitellogenesis, yield was 30% higher in the halibut fed with the
peaked during mid-cycle and then returned to basal nucleotide diet and the relative fecundity, mean egg
levels when the ovaries were in late vitellogenesis. density, hatching rate and survival of yolk-sac larvae
were also significantly improved. Haddock fed on
Nucleotides
the nucleotide-enriched diet also had significantly
higher fertilization and hatching rates. To date, no
Nucleotides, the basic building blocks of nucleic acids, work has been published on nucleotide
are recognised as important elements in mammalian supplementation of broodstock diets for shrimp but
nutrition especially during periods of rapid growth it would be interesting to conduct some trials to
or physiological stress (Uauy, 1989; 1994; Barness determine if broodstock diets enriched with
1994; Van Buren, 1994) and also appear to play a key nucleotides might offer similar benefits in shrimp
role in the immune system. Traditionally, nucleotides maturation and breeding. Similarly, the potential for
have not been considered essential nutrients although nucleotide supplementation of diets for shrimp larvae
de novo synthesis and salvage pathways are thought should also be investigated.
to be costly processes in metabolic terms. Several
studies have demonstrated that dietary sources of
nucleotides can have beneficial effects and the term Larval feeds
‘conditionally essential’ has been used to describe their
role in nutrition (Carver and Walker, 1995). Typically, marine larvae are fed with live feeds such
Exogenous sources of nucleotides are thought to as algae, zooplankton, rotifers and Artemia. In some
optimise the functions of rapidly dividing tissues, such cases, especially with rotifers and Artemia, the fatty
as those of the developing embryo and young, and acid profile is inadequate (Léger et al., 1986),
the reproductive and immune systems. especially with regard to the HUFA profile. The
Most aquaculture diet ingredients of animal and plant practice of enrichment has been developed as a means
origin contain nucleotides although there are of overcoming this nutritional deficiency.
differences in the concentration and availability. Enrichment usually involves enhancing the
Nucleotide content is particularly high in ingredients docosahexanoic acid (DHA) and EPA content of the
such as fish solubles, animal protein solubles, fish natural feed through ‘bioencapsulation’. This
D.F. Fegan 411
involves feeding the live organism with a DHA/EPA commercial hatcheries, total replacement of algae has
enriched formulation to boost the levels in the tissues proved to be more difficult. Complete replacement
and then feeding the enriched organism to the larvae. has only been achieved using ocean quality seawater
In the case of shrimp larvae, the use of enriched that is partly filtered to retain the natural bacterial
Artemia is restricted to the post-larval stages due to community (Ottogali, 1991; 1992) and reports of
the size of the first feeding instar stages of the Artemia complete replacement in commercial hatcheries
(Sorgeloos et al. 1998). appears restricted to those located in the oceanic
waters of the Pacific islands (Chim, 2003). Alabi et
al. (1997) also showed that total replacement requires
FORMULATED FEEDS the establishment of a balanced bacterial community
from either the filtered seawater or following
Although live feeds provide an excellent source of conditioning by microalgae. As a result Jones et al.
nutrition, there are several drawbacks associated with (1997; 1998) suggested the inoculation of a single
their use. Algal cultures require considerable expertise dose of live algae (SDLA) before use of artificial
to maintain them in peak nutritional condition and feeds to condition hatchery water when it is taken
facilities for their mass production can be expensive from coastal water of variable bacterial quality.
to operate. Rotifers also require considerable Wouters and Van Horenbeek (in press) summarize
expenditure in time and effort to maintain, especially the various types of commercial larval feeds available
if they, in turn, need to be provided with live feed. in the market. These include microbound diets, flakes,
Live Artemia nauplii suffer from inconsistent supply granulated feeds, microencapsulated feeds, liquid
and quality as they are obtained from cysts collected feeds (lipid-walled capsules).
in the wild environment. The bulk of cysts come from Microbound feeds are bound using a variety of
the Great Salt Lake in Utah in the US where annual different binders and produced as a small particle, or
fluctuations have been shown to cause wide as a pellet, cake or flake, which is then crumbled to
fluctuations in yield. As a result, price and quality the appropriate size. They are inexpensive to produce
can vary unpredictably (D’Abramo, 2002). but leach rapidly. Crumbles are usually used during
Such problems with live feeds have led to the postlarval stages.
development of diets specifically formulated for their Flakes are commonly used in Asia and the
replacement. However, the development of Americas. Dietary ingredients are added to water to
formulated larval diets to completely replace live obtain a dense soup. An appropriate binder is added
feeds has been an elusive goal, despite considerable and the resulting suspension is sprayed onto a steam-
effort (Langdon, 2003). The difficulties inherent in drum dryer. Temperatures can exceed 100°C. and
providing a complete nutritional package in a significant nutrient loss can occur unless passage times
sufficiently small particle to be ingested and digested are kept short. Large flakes can be crushed and passed
by the small larvae of many marine species are clear. through an appropriate mesh screen immediately
Loss of nutrients from such diets can be rapid and prior to use. They are generally used for the postlarval
result in loss of nutritional value and fouling of the stages of the shrimp.
culture medium and should be used to determine the Granulated feeds are produced using liquid binder
effectiveness of microparticulates as nutrient delivery and water sprayed onto the feed mix, resulting in
systems. On the other hand, provision of a sufficiently granules with a raspberry-like structure.
impermeable coat to prevent leaching may result in Microencapsulated feeds have an outer coat
poor digestibility and availability of the nutrients to (capsule) that retains the ingredients inside the
the developing larvae. Microbound and cross-linked particle. They can be designed to have a slow release
protein-walled capsules may be used to deliver lipids of the material or to totally prevent leaching of water-
and high-molecular weight, water-soluble nutrients soluble nutrients. Some techniques encapsulate using
such as proteins and carbohydrates whereas lipid- a cross-linked protein-wall that can be digestible yet
based particulates, including liposomes, could be capable of withstanding drying.
useful in delivering low-molecular weight, water- Liquid feeds are essentially a slurry of particles in
soluble nutrients, such as amino acids and a suspension medium. Although expensive, they are
water-soluble vitamins. claimed to cause less fouling and can be continuously
Although the use of formulated larval feeds as dosed into larviculture tanks using peristaltic pumps.
partial replacements for microalgae is common in
CONSIDERATIONS IN DEVELOPMENT OF development of the hepatopancreas, which increases

FORMULATED LARVAL FEEDS in size and complexity, and the development of the
gastric mill and gastric filter to the adult form.
The nutrition of marine larvae involves an Until the gastric mill becomes functional, the
understanding of the behavioural, mechanical and shrimp larvae are restricted to feed items that are
physiological processes of feeding in the target animal. relatively easily digestible. In the case of the
These are likely to be very different in the larval herbivorous stages, they generally filter the feed from
stages compared to the adult form. Feeding habits in the water and digestion takes place in the gut. As the
many species show a distinct change as the larvae appendages and mouthparts become stronger and
develop. In penaeid shrimp, many species change more complex, larger feed particles and live prey
from a primarily herbivorous diet in the zoea stages are held and torn apart before ingestion, rendering
to a more omnivorous diet in the postlarval stages them more easily digestible.
(Lemos and Phan, 2001). During the postlarval and There has been relatively little research into the
early juvenile stages, further changes may involve a digestive enzymes of crustacean larvae. Jones et al.
switch to a more carnivorous or detritivorous diet (1997) noted that larval enzymes differ in range and
depending on the species. There are also changes from level of activity from those that are present in adults
a planktonic existence to a benthic one and from a and that there exist species differences which may
filter feeder to an active predator to be considered, relate to the feeding strategy of the larvae. There are
all within a few days of hatching. also changes in enzyme activity patterns between
One of the key considerations is the development different larval stages and, overlaid on these, the
of the gut structure and function. Larval crustaceans possibility of diet-induced changes in enzyme levels.
have a simple gut structure, which gradually becomes In a brief review of research into enzyme activity
more complex. The physiology of the gut and gut of decapod larvae, Jones et al. (1997) reported that
enzymes also changes and, since transit times may all appeared to have strong protease activity,
be quite short (Jones et al., 1997; Jones and Kurmaly, predominantly trypsin, non-specific esterase activity
1987), designing a nutritious, easily digestible diet and amylase. In the few reports available, pepsin
is a challenge. appears to be absent in larval and most adult decapods
(Glass et al., 1989) although it has been reported in
the freshwater prawn, Macrobrachium rosenbergii
Digestive physiology of marine larvae (Lee et al., 1980). Similarly, lipase activity was only
reported in larval stages of the black tiger prawn,
The development of the digestive system of marine Penaeus monodon and the lobster, Homarus
larvae plays a fundamental role in larval nutrition. americanus although this may be open to debate.
In particular the development of gut function and Collagenase, elastase and chymotrypsin all seem to
ontogenetic changes in enzyme function are of critical be rare or absent in larval stages.
importance. Changes in enzyme activity patterns with larval
The structure of the larval gut in crustaceans has stage have been clearly demonstrated in penaeids
been studied in only a few species (Factor, 1981; (Lovett and Felder, 1990a, b; Glass et al., 1989;
Lovett and Felder, 1989; 1990a; 1990b; Abubakr and MacDonald et al., 1989; Ribeiro and Jones, 2000;
Jones, 1992). The development of gut structure Ngamphongsai, 2000; Puello-Cruz et al., 2002).
appears to follow similar patterns in most of the MacDonald et al. (1989) noted that the general pattern
species studied. The gut of early larval stages of of amylase, protease and lipase in P. monodon larvae
penaeids is relatively simple and the gastric mill and was similar with a maximum of enzymatic activity
hepatopancreas are either not present or not yet at zoea 3, possibly coinciding with the change from
functional. Feed is mixed with enzymes through predominantly filter feeding to mechanical digestion.
expansion and contraction of the midgut gland and They also noted a minimum level of all three enzymes
there is free fluxing of food between the midgut gland in the early postlarval stage, similar to that noted by
and the midgut, especially in time of food scarcity Lovett and Felder (1990a) (the so-called ‘enzyme
(Lovett and Felder, 1990b). As the larva grows, the crisis’), which was attributed to a change in gut
hepatopancreas proper develops, the lobes increasing development associated with changing feeding habits
in number and elongating during the mysis stages. as the postlarvae become more benthic. This coincides
At this point, the rudimentary gastric filter begins to with a critical period in postlarval development when
develop. The postlarval stages are marked by the rapid
D.F. Fegan 413
poor postlarval condition and high mortality may be The dietary effects of phospholipids on fish and
observed in commercial hatcheries. Puello-Cruz et crustacean larvae have been reviewed by Coutteau et
al. (2002), on the other hand, found that trypsin levels al. (1997). Although there appears to be little doubt
in Litopenaeus vannamei larvae were significantly that crustacean larvae can synthesise phospholipid
highest at the Zoea 1 stage, and declined thereafter from HUFA, the addition of dietary sources of
to the PL1 stage. They also noted that trypsin content phospholipid has been shown to be beneficial,
in zoea 2 and zoea 3 stage larvae feeding on Artemia possibly through enhancing the absorption of dietary
was significantly lower than in those feeding on algae cholesterol and triacylglycerols (Jones et al., 1997).
and suggested that L. vannamei may be Gonzalez-Felix et al. (2000), for example,
physiologically adapted to transfer to a more demonstrated that L. vannamei postlarvae fed diets
carnivorous diet during the zoeal stages than more containing phospholipid demonstrated improved
herbivorous species such as P. monodon or P. indicus growth and enhanced muscle phosphatidylcholine and
(Ngamphongsai, 2000). This accords well with phosphatidylethanolamine concentration. Coutteau et
commercial hatchery observations that L. vannamei al. (1996) however, showed that the other
is capable of feeding on Artemia from the zoea 3 phospholipids in lecithin could not compensate for
stage whereas P. monodon larvae do not become fully phosphatidylcholine deficiency in the diet of
capable of feeding on live Artemia until at least mysis postlarval L. vannamei and that, as dietary
2 or 3. Changes in the sequence of enzyme activity phosphatidylcholine increased, significantly higher
consistent with a carnivorous diet were also noted in levels of total HUFA, 20:1n-9 and 20:5n-3 were
early larval stages of Macrobrachium rosenbergii present in the shrimp.
(Kamarudin et al., 1994). In experiments carried out with larval P. japonicus,
Several authors have suggested that fish larvae may optimal metamorphosis was obtained with diets
benefit from the assistance of exogenous digestive containing 15-30 g/kg soybean phosphatidylcholine
enzymes of their live food organisms, either through and the lower level (15 g/kg) was more beneficial to
autolysis or by the action of zymogens that activate postlarvae (Camara et al., 1997). Results also
larval endogenous digestive enzymes (Kolkovski et suggested that there was no requirement for
al., 1993; 1997; Munilla-Moran et al., 1990) and phosphatidylethanolamine or phosphatidylinositol in
the same may hold for crustacean larvae. the presence of adequate dietary phosphatidylcholine.
The phospholipid requirement of crustacean larvae
from this study appears to be within the range of 1-
LIPIDS 3% given by Coutteau et al. (1997) as the range for
most marine larval species and agrees with the earlier
Much work on larval feeds has centred on the lipid figure of 3% given by Kanazawa (1990).
requirement, in particular the requirement for highly As with adult crustaceans, larvae are unable to
unsaturated fatty acids (HUFA) and phospholipids. synthesise cholesterol and have an absolute
The n-3 highly unsaturated fatty acids (HUFA) requirement for cholesterol in the diet (Teshima et
docosahexaenoic acid (22:6n-3, DHA) and al., 1983).
eicosapentaenoic acid (20:5n-3, EPA) are essential
for normal growth and development of many species
of marine fish and crustaceans (Sargent et al., 1999; PROTEIN AND AMINO ACIDS
Jones et al., 1997; Suprayudia et al., 2004). Marine
fish are also unable to synthesize arachidonic acid The optimal dietary protein level in a larval diet can
(20:4n-6; AA). be expected to vary with species, larval stage (Durruty
Quantitative requirements for essential fatty acids et al., 2002), protein source, digestibility (Le Vay et
have been rarely reported for larvae of penaeid al., 1993) and amino acid composition. Kanazawa
shrimp. It has been suggested (Kanazawa et al., 1979, (1990) recommended protein levels in larval feeds
in González-Félix and Pérez-Velazquez, 2002) that between 23% and 57%. Durruty et al. (2002)
1% n-3 HUFA in the diet could be considered as a reported differences in protein requirement according
minimal value for postlarvae although Chen and Tsai to the larval stage of L. vannamei and L. setiferus.
(1986) indicated a requirement for HUFA at 0.5-1% They estimated that protein requirement increased
of the diet for P. monodon and Xu et al. (1994) from 30% in the zoea stages up to 50% or 60% for
suggested a requirement of between 0.7% and 1% of mysis stages. Data on optimal protein:energy ratios
the diet in Fenneropenaeus chinensis.
or amino acid profiles for larval shrimp feeds have vitamin C has been available in the form of L-
not been reported (Wouters and Van Horenbeek, in ascorbyl-2-polyphosphate. Although there is little
press). work on larval nutrition using this form of the
The essential amino acids for shrimp are thought vitamin, work on the early postlarval stages of L.
to be methionine, arginine, threonine, tryptophan, vannamei has shown that high dietary levels (40 mg/
histidine, isoleucine, leucine, lysine, valine and kg)can increase resistance to salinity stress. The
phenylalanine (Akiyama, 1992). There is no reason benefit of elevated dietary vitamin C levels in
to believe that shrimp larvae have any specific amino increasing stress and disease resistance has also been
acid requirement that differs from the adult shrimp noted by other authors (Kontara et al., 1997; Merchie
(Jones et al., 1997). Although no single protein source et al., 1997; 1998).
appears to satisfy the complete requirements for
amino acids (Wouters and Van Horenbeek, in press),
Cahu (1999) stated that lower nutritional value protein OTHERS
sources can be fortified by the addition of essential
amino acids (Cahu, 1999). Nucleotides have demonstrated much potential when
fed to the young or juvenile stages of vertebrates
(Carver and Walker, 1995). The benefits of adding
CARBOHYDRATES dietary nucleotides to feeds for the rapidly developing
larval stages of crustaceans would appear to be clear.
As with adult crustaceans, there appears to be no However, there are no research data available on the
specific requirement for carbohydrates in the diet. application of exogenous nucleotides for crustacean
Carbohydrates can be used to reduce feed costs larval culture.
through protein or lipid sparing and are frequently Hatchery operators often use a mixture of additives
used as binders in larval feeds. in a typical feeding regime including additives such
as immune stimulants or probiotic bacteria. There is
still little or no scientific data to support the use of
VITAMINS AND MINERALS such additives in shrimp larval culture.
Fat-soluble and water-soluble vitamins as well as
carotenoids are essential for shrimp larvae (Wouters Conclusion
and Van Horenbeek, in press). Kanazawa (1986; 1990)
determined the vitamin requirements for P. japonicus The current state of knowledge of broodstock and
(Table 1). These levels provide a practical baseline, larval nutrition of crustaceans contains many gaps.
but Kanazawa (1990) also admits that they may be This is partly the result of the complexity and expense
affected by leaching of the vitamins from the test of conducting research into these two areas. However,
diet. In practice, formulated feeds for shrimp larvae given the increasing importance of domestication in
will contain a complete vitamin and mineral premix shrimp aquaculture particularly, there is a need for
as used in feeds for older shrimp. an increased focus on these areas. The role of nutrition
in broodstock and maturation performance and in
Table 1. Vitamin requirements of P. japonicus1. increasing larval survival and quality will be
Vitamin (mg/kg of dry diet) fundamental to obtaining optimal performance from
Thiamin HCl 4 domesticated stocks. Even in species where
Riboflavin 8 domestication is not an issue, the improvement of
Pyridoxine HCl 12 maturation performance and larval production
Nicotinic acid 40
Biotin 0.2 remains a key goal in improving the efficiency of
Choline chloride 600 production systems.
Inositol 200 It is not clear how far the goal of complete
Na-ascorbate 1,000 replacement of live feeds may be. It is likely that
Tocopherol 20
this will be attained in broodstock diets long before
1
Adapted from Kanazawa (1986; 1990) larval feeds. Indeed, given the complexities inherent
in supplying a complete nutritional package in a small
Kanazawa’s work on vitamin C requirements was particle, it may be that this goal is never reached.
conducted using sodium ascorbate. More recently,
D.F. Fegan 415
However, it may be possible to supply a range of Cahu, C. 1999. Nutrition and feeding of penaeid
diet particles (e.g. high lipid particles, high protein shrimp larvae. In: Nutrition and feeding of fish and
particles, carbohydrate particles etc.) that are aimed crustaceans. (J. Guillaume, S. Kaushik, P. Bergot
specifically at providing the right mix of nutritional and R. Métailler, eds). Springer, London, UK, pp.
elements in the culture tank that will expose the larvae 253-263.
to the appropriate nutritional mix. Alternatively, it Cahu, C., M. Fakhfakh and P. Quazuguel. 1991.
may be that, as expressed by D’Abramo (2002), the Effect of dietary α-tocopherol level on
complexity of the ontogeny of larval nutritional reproduction of Penaeus indicus. In: LARVI ’91—
physiology may mean that technical success will be Fish and Crustacean Larviculture Symposium (P.
based on a compromise between the desire to provide Lavens, P. Sorgeloos, E. Jaspers and F. Ollevier,
a complete diet package and the need to strive for
eds). Ghent, Belgium, Europ. Aquac. Soc. Spec.
simplicity in formulation and manufacture.
Pub. No. 15:242-244.
Cahu, C.L., J.C. Guillaume, G. Stephan and L. Chim.
1994. Influence of phospholipid and highly
Acknowledgements unsaturated fatty acids on spawning rate and egg
I would like to acknowledge the debt owed to tissue composition in Penaeus vannamei fed semi-
Roeland Wouters of INVE Technologies, Belgium purified diets. Aquaculture 126:159-170.
and Prof. Patrick Sorgeloos of the University of Cahu, C.L., G. Cuzan and P. Quazuguel. 1995. Effect
Ghent for their kind help and provision of access to of highly unsaturated fatty acids, alpha-tocopherol
their publications for use in writing the paper. and ascorbic acid in broodstock diet on egg
composition and development of Penaeus indicus.
Comp. Biochem. Physiol. 112:417-424.
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sp. in larval crustacean nutrition: a review. Rev. haemolymphatic vitellogenin concentration of
Fisheries Sci. 6(1&2):55-68. Pacific white shrimp Litopenaeus vannamei
D.F. Fegan 419
(Boone) fed increasing levels of total dietary lipids hatchability and fatty acid composition of Chinese
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J.W. Sweetman 421
European finfish culture: current status, recent advances and future

perspectives
JOHN W. SWEETMAN
Ecomarine Ltd, Cephalonia, Greece
Introduction
The latest review of the state of world aquaculture Finfish production at 24.4 mmt represents 50% of
from the Fisheries and Agriculture Organization the aquaculture production and over 130 major finfish
(FAO) 2003 and the latest FAO Fishstat Plus statistics species are cultured world-wide. These statistics alone
from 1950-2001 highlight the continuing growth of emphasize the scale, complexity, rate of development
aquaculture in contributing to the total fisheries catch and diversification of the global finfish aquaculture
(Figure 1). sector.
Aquaculture represented 5.3% of the total fisheries
landings in 1970 and this had increased to 34% in
2001, i.e. 48.4 million metric tonnes (mmt) of the European aquaculture
total fisheries landings of 142.1 mmt. The value of
world aquaculture production is now estimated at 61.5 Europe has a total aquaculture production of over 2
billion US$. Globally the sector has shown an mmt, and of the 210 aquaculture species cultured
average annual compounded growth rate (APR) of worldwide 60 are cultured in Europe with a value of
8.9% since 1970 compared to 1.4% for capture 4.6 billion US$. This means that while Europe
fisheries and 2.8% for terrestrial farmed meat contributes 4.4% to global production, it represents
production. 8.2% of its total value. Recently growth from
160 Total
Aquaculture
140
Fisheries
120
Million metric tonnes
100
80
60
40
20
0
80 85 90 95 2000
Year
Figure 1. World fisheries and aquaculture production (FAO, 2003).
422 European finfish culture: current status, recent advances and future perspectives
aquaculture has slowed from 7.8% p.a. in the period times the price of the fresh water species according
1980 to 1990 to 2.3% p.a. in the period 1990 to to global FAO statistics and it is primarily in this
2000. area that European aquaculture has focused and
In 2001, 1.34 mmt of Europe’s 2 mmt was developed (Tacon, 2003).
attributed to finfish production and the breakdown Table 1 shows the breakdown of the finfish species
of production among fresh, brackish and marine cultured in Europe and it can be seen that the largest
environments is shown in Figure 2. Salmon, trout, growth is in mariculture with salmon, sea bream, sea
sea bass and sea bream account for over 1 mmt or 3 bass and turbot. Currently the 15 countries of the
billion US$ in value. Salmon is by far the largest European Union produce approximately 50% of the
activity accounting for 647 thousand metric tonnes finfish total but that proportion will change with the
(tmt) and 1.86 billion US$. The marine and accession of a further 10 countries later this year.
diadromous finfish species are valued at nearly 3
900000 Marine
800000 Freshwater
Brackish
700000
600000
Metric tonnes
500000
400000
300000
200000
100000
0
1996 1997 1998 1999 2000 2001
Figure 2. European finfish production by environment (1996-2001).
Table 1. European finfish production by species (1996-2001) in metric tonnes1.

1996 1997 1998 1999 2000 2001
Freshwater species
Tilapias 320 200 200 246 180 200
Sturgeon 1,285 1,471 2,022 2,441 3,083 3,087
Eels 8,614 8,696 9,792 10,536 10,713 10,187
Catfish/perch/pike 19,321 16,389 15,437 19,903 16,583 14,868
Carps and cyprinids 175,910 172,620 180,015 191,236 197,405 210,667
Trout 279,060 293,142 304,485 301,753 301,371 331,805
TOTAL 484,510 492,518 511,951 526,115 529,335 570,814
Marine species
Sole 31 25 22 19 23 37
Halibut 2 8 13 34 93
Cod 191 304 199 157 169 763
Turbot 2,663 3,041 3,107 4,113 4,789 4,959
Amberjack and tunas 78 1 1,959 3,246 3,686 4,453
Misc. marine 740 1,205 2,153 2,733 3,031 2,275
Other breams and mullets 4,086 3,946 4,051 4,016 4,190 4,137
Sea bass 19,325 24,079 30,168 38,215 41,870 42,600
Sea bream 23,304 29,139 37626 49,601 58,163 63,370
Salmonids 416,358 473,159 510,059 611,671 617,898 647,043
TOTAL 466,766 534,901 589,352 713,784 733,853 769,730
Total European production 951,286 1,027,419 1,101,303 1,239,899 1,263,188 1,340,544
1
FAO/FEAP, 2003.
J.W. Sweetman 423
SALMON PRICE: A CONTROLLING FACTOR by what the industry feels is often unfair treatment
in the press.
Salmon prices have decreased since 2000; and in 2003 The industry, while weakened, has responded with
reached their lowest point ever at under 2 €/kg, with a positive approach. At every level the aquaculture
a recent average price of approximately 2.5 €/kg sector is promoting transparency, cooperation and
(Figure 3). This for many farms is at or below their dialogue. The Federation of European Aquaculture
production costs. Due to the importance of this Producers (FEAP) initiated the ‘Aquamedia’ project
species in the overall framework of the European and this is now providing factual, truthful and
industry, the repercussions for industry as a whole interesting information about European aquaculture
have been significant. During the same period price to the public.
decreases were also being seen in the sea bass and sea In order to focus the European effort and to
bream industry with similar effects. Overall in the promote a coherent development strategy, FEAP
EU there has been a 6.5% APR in production growth reported to a hearing on European Aquaculture held
but the overall price trend has been negative (-0.5% by the EU committee of the European Parliament in
APR) versus a positive global development (FEAP). October 2002. The following key issues were
Share prices have fallen sharply, particularly in the presented:
salmon industry; and investment confidence in
aquaculture has been shaken. Pressure has been placed • Improved economic viability, long term market
on the industry to further improve efficiency and stability and improved profit margins within the
productivity. Acquisitions and mergers have been the industry are essential in order to stimulate
order of the day and groups are consolidating in order investment and reinvestment.
to benefit financially.
Significantly, with salmon being the power house • Improved access to consolidated marketing and
of the European industry, diversification into other promotional efforts would benefit the medium
marine finfish species has been slowed due to the and smaller producers.
reluctance of financial institutions to invest in a variant • The industry should within the EU operate on an
of this troubled sector. Further, this financial pressure even playing field with regard to imported
comes at a time when consumer awareness is focusing products.
upon product quality, ease of product use, food
safety, and traceability. This is also occurring during • Food safety should be a guarantee for the
a period when the image of aquaculture has suffered consumer.
2000
4.5 2002
2002
4 2003
2004
Price (EUR/kg)
3.5
2.5
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52
Week
Figure 3. Cross section price for fresh whole gutted superior Atlantic Salmon, iced in poly boxes (Intrafish).
• Ingredient input quality to be provided by the CRITERIA FOR SPECIES SELECTION

feed manufacturers.
The selection of a species suitable for European
• The practical application of traceability and aquaculture depends primarily on its market value,
labeling to be implemented. an understanding of its biology and the ability to
• The industry should be sustainable with regard produce juveniles in significant numbers for
to environmental impact and resource issues e.g. commercial production to take place. Given these
water/feed/biodiversity. characteristics and suitable site availability with the
correct environmental conditions for culture, the
• Ensure human resources continuation through evolution to maturity of a specific species industry
training, technology and professional entry. can be summarized in the following stages: -
• Better governance and responsibility within the
industry. • Identification and selection of high value species
together with the R&D necessary to provide an
• Better acceptance of aquaculture by the public. understanding of the species biology and nutrition.
• The development of reproductive technologies
It concluded that the long-term viability of the sector to close the life cycle of the species considered
would depend upon the satisfaction of these multiple together with the acquisition, domestication and
criteria and that the development of sustainable and manipulation of broodstock to produce eggs year
responsible aquaculture will require coherent and round.
viable European actions including simplified
legislation and simplified licensing procedures. • The industrialization of technologies required for
the commercial production of juveniles and their
ongrowing.
Evolution of European finfish culture • Improvement in productivity through economies
of scale and the reduction of costs through vertical
The marine and diadromus finfish sector is of vital integration.
importance to the European industry and consists
primarily of high valued species requiring • Improvement in productivity through bio-
considerable technological and managerial technological solutions such as genetic
sophistication to culture in a sustainable manner. The advancement and nutritional engineering.
critical developments that were responsible for the • Increased market activity through commercial
commercialization of these species and their pressure with the development of quality labeling,
subsequent diversification are outlined below. Further processing and other value added activities.
areas of future development and improvement
required to ensure the continued development of the • Streamlining of the industry due to increased
industry are considered in light of the rapidly production, reduced profits resulting in grouping,
evolving requirements of modern day Europe and mergers and consolidation to remain
the consumer. commercially competitive.
While the latest FAO/FEAP statistics described • The maturation of the industry and the species
above provide a fascinating insight to the development losing its status of a high value item and
of the aquaculture industry, they obviously lack the becoming a commodity product.
benefit of real time information and the regional
focus of development promoted by the European • Species diversification in search of additional
Union, national governments and the industrial sector. profits through a recycling of the above
This has been achieved through supporting research procedure.
and development, financial support for the
establishment of the industry and by addressing TRANSFER OF TECHNOLOGY BETWEEN
marketing and consumer needs. These driving forces SPECIES
have provided a basis for development and
diversification. The transition from salmon to other marine species
is of particular interest and importance as it involved
J.W. Sweetman 425
the transfer of on-growing technologies from Norway Table 2. Comparative profitability of salmon and cod in the UK in
and Scotland to the Mediterranean for the sea bass 2003.
and sea bream industries in the 1980s to 1990s. This, Salmon Cod
however, in itself was not sufficient as the
reproductive technologies, zootechnical, nutritional Cost per kg (round) €2.04 €2.82
and environmental conditions required for the culture Cost of harvesting/gutting etc €0.423 €0.423
Yield (head on gutted) 90% 86%
of marine finfish juveniles were significantly Cost per kg (head on gutted) €2.735 €3.765
different from those of salmon. Market price (head on gutted) €2.735 €4.935
A swim up salmon fry (150 mg wet weight) is many Profit/kg 0 €1.170 (31%)
times larger in body mass than that of a sea bream Cost of filleting €0.141 €0.141
Yield after filleting (head on gutted) 68% 55%
(0.35 mg wet weight), and while the salmon fry is Cost/kg (fillet/skin on) €4.23 €9.23
capable at first feeding of ingesting and digesting
inert particulate feeds this is not the case for many
marine species. Due to the rather underdeveloped Reproductive technologies
digestive system of these larvae and their behaviour,
a sophisticated live food chain was required. It has been estimated that less than 3% of the total
Researchers and industry concentrated efforts to world aquaculture production is based on genetically
develop a species-specific nutritional strategy and to improved stocks. Norway started to work on the
identify the correct environmental conditions enabling selection of salmon in the 1960s and it was found
these very small and sensitive larvae to be successfully that there was a large genetic variability for important
reared and develop sufficiently to be weaned onto production traits between wild and culture stocks
inert diets before traditional on-growing technologies offering potential for improvement. The best strains
could then be used. were then used as a base population for a national
Since the 1980s, larval rearing technologies have genetic improvement programme. This has lead to
been continually developing for the sea bass, sea the development of salmon strains that show a genetic
bream and turbot industries and today they are well gain of around 100% for growth rate as well as other
understood. Survival rates for these species have important characteristics such as late maturing
increased from less than 1% to between 20 and 40% individuals, resulting in improved production
in 10 years. In the last few years these warm water characteristics and reduced production costs. A similar
larval rearing technologies have been adapted and breeding programme carried out using tilapia in the
introduced to Norway and Scotland, enabling the mass Philippines has shown more than a 100%
production of a marine cold water species, the cod improvement in growth rate over 10 generations.
(Gadus morhua). While production of cod is expected In both species, selection for high growth rate can
to reach only 12,000 tonnes in 2005, this species, result in a gain of over 10% per generation. Only in
which occupies the same environmental conditions the last 10 years has European Union research focused
as salmon, offers an alternative or addition to the on broodstock genetics and selection programmes for
troubled salmon industry. the sea bass and sea bream industry in the
Economically, the present viability of these two Mediterranean. Prior to this production was still based
species in the UK can be summarized as shown in on wild broodstock and selection from F1 cage
Table 2 (R. Prickett, personal communication); and production, with little monitoring to avoid inbreeding.
it should be noted that as technology and food Traditionally, preventing inbreeding has been the
conversion rates improve for cod, so should profits. largest problem in fish breeding programmes, due to
History indicates however that as volume increases large full sibling groups and the lack of individual
so market prices will reduce. identification methods. Today the identification of
It is developments such as these that offer Europe individual broodstock fish using microchip tagging
the potential to continue expanding and diversifying is commonplace. The cost of microsatelite DNA and
its existing aquaculture industry by looking forward AFLPs (amplified fragment length polymorphisms)
to new and more profitable species through the labelling has significantly decreased enabling the
adaptation and development of existing tracking of pedigrees and providing linkage maps to
biotechnologies and management strategies. identify quantitative trait loci, such as growth and
disease resistance, that have commercial importance Table 3. Commonly cultured unicellular planktonic algae.
(Agresti et al., 2000). These technologies have Bacillariophycaea Chaetoceros calcitrans
enabled individual producers to carry out selection (Diatoms) Skeletonema costatum
methods and apply them in a practical manner in the Chlorophycaeae – green algae Dunaliella tertiolecta
industrial environment. Chlorella spp.
Growth rate is generally the first characteristic of Nannochloris atomus
importance for fish farmers as it is relatively easy to Chrysophyceae Tetraselmis suecica
select and quantify. Other traits thought to be Tetraselmis chuii
genetically dependent, for example disease resistance Eustigmatophyceae Nannochloropsis oculata
and flesh quality (muscular lipid content, fat Nannochloropsis gaditana
deposition), are difficult to measure and require a Haptophyceae Isochrysis galbana
more complex approach often beyond the abilities Isochrysis spp. (Tahititan strain)
Pavlova lutheri
of the individual farmer. Various thematic R&D
programmes within the European Union are now
addressing many of these issues. The use of algae in the larval rearing tanks, the ‘green
Many species still prove difficult to spawn in water technique’, is not limited to the purely
captivity, particularly in intensive production systems, nutritional side of rotifer enrichment but has other
an example being sole (Solea senigalensis). Triggers practical applications:
for male and female maturation and ovulation are
still not well understood for this and other species • Algae can act as an antibacterial agent (Austin
such as the groupers and some tuna species. Endocrine and Day, 1990; Cooper et al., 1983). In addition,
regulation of reproduction has been effectively specific polysaccharides in the algae cell wall are
applied in some species and hormonal implants are thought to stimulate a non-specific immune
readily available (Zohar and Mylonas, 2001). More response in young larvae.
investigation of the environmental and nutritional • Algae have been reported to act as an in situ
requirements of many species is required as the biological filter removing potentially harmful
production of viable eggs is a prerequisite to the metabolites from the water by stripping off
culture of any species. nitrogenous substances. They also produce oxygen
Egg quality has a significant impact on the viability, through photosynthesis.
survival and growth of marine larvae. The
enrichment of broodstock diets with essential fatty • Algae act as a light filter and diffuser facilitating
acids and other vitamins and minerals have been an even distribution of live food and larvae within
shown to relate directly with the levels of these the tank system.
substances in marine eggs and larvae (Watanabe, • Algae act as a promoter and background for the
1993; Cedra et al., 1994; Harel et al., 1994). location of prey organisms, hence playing a
particularly important role in the critical first
feeding stage of larvae.
Marine larval rearing technologies
• Algae have been shown to stimulate the enzymatic
The very small and sensitive larvae require the synthesis and onset of feeding in young larvae.
establishment of a reliable chain of live food In recent years photo-bioreactor systems have
production consisting of unicellular marine algae, the provided an efficient alternative to traditional sack
rotifer Branchionus plicatilis and the brine shrimp culture systems for the production of unicellular
Artemia salina. This is common to both the cold water algae. These are labour saving, automated and cost
species, namely cod and halibut, and the warm water effective. Productivity using these systems can be up
species such as sea bass, sea breams, turbot and sole. to 10 times greater than that achievable with
The most commonly cultured algal species in traditional culture methods. Undoubtedly the success
Europe belong to the five taxonomic groups (Table of such systems is dependent upon light availability;
3) and the choice of species depends on the fish species and the Mediterranean climate is particularly suitable.
being cultured. The search for additional algal species continues,
and isolates of local species are being investigated in
J.W. Sweetman 427
Norway in an attempt to replace some of the selected genotypes cultured over a limited number
traditional non-indigenous species. In addition, of generations or how these cultures will be
commercial companies are marketing concentrated susceptible to changing culture conditions
algal pastes, delivered either alive with a limited shelf (Sorgeloos, 2004).
life or cryopreserved, which offer a back-up and an A better understanding of the nutritional
alternative to traditional algal production. requirements of the fish species cultured has led to
The second link of the live food chain is the rotifer. the development of a number of commercially
The duration and quantity of rotifers required varies available cultivation and emulsion type enrichment
with the species; cod, for example require up to four diets for both rotifers and artemia. Dietary research
times the number of rotifers per animal produced has indicated the importance of the (n-3) highly
when compared to the sea bream. Some species such unsaturated fatty acids, mainly eicosapentaenoic acid
as the sea bass may avoid this stage altogether first (20:5(n-3), EPA), docosahexaenoic acid (22:6(n-3),
feeding directly on Artemia nauplii, the last link in DHA) and more recently the long chain n-6 PUFA
this chain. arachidonic acid (20:4n-6, ARA) has been implicated
Rotifer production methodologies have improved as an essential fatty acid for a variety of developing
over the years from an algae and yeast-based diet marine species (Estevez et al., 1999). It is the
giving poor productivity and unpredictable results bioencapsulation of these (Figure 4) and other
to improved culture diets. The new generation of essential nutrients through the live feed chain in the
diets enables culture densities of 2000 rotifers per ratios required by the species concerned that has led
ml or more to be achieved over a 4-day batch cycle. to the alleviation of several problems such as
Further developments have resulted in additional pigmentation and some deformity issues in larval
improvements. For example, 5000 rotifers per ml rearing as well as improving survival.
can be achieved using concentrated fresh water These enrichment products today are available
chlorella algae and automatic dosing pumps; and re- together with products that for Artemia are capable
circulation systems using protein skimmers, in of altering the microbiological characteristics of the
association with novel filters enable rotifer cultures hatching and enrichment environments by reducing
to reach and be maintained at densities over 5000 Vibrio levels to 30% or less of non-treated
per ml for prolonged periods of time. These environments. This development both provides the
developments have been shown to both provide farmer with custom nutritional packages and helps
significant economic benefit and importantly improve to reduce the possibility of disease that may be
the microflora of the culture by reducing the introduced through the live food chain.
incidence of Vibrio spp. (Suantika et al., 2003).
Ongoing European Union projects have revealed
that there is considerable genetic diversity in the Larval nutrition and feed technologies
rotifer populations in European hatcheries showing
considerable difference in performance. It is not yet Larval rearing technologies are today highly intensive
clear whether it will be necessary to work with with up to 250 larvae stocked per litre. Until recently
Essential nutrients
Pigments
Prophylactics
Therapeutics
Figure 4. The bioencapsulation process.
the live food chain was entirely responsible for the The need to find additional on-growing sites for
nutrition of these larvae until weaning commenced expansion and to move away from conflicts with
at approximately 30 days post hatch. The role of the tourism and environmental concerns, particularly in
live food chain is still vitally important for many the Mediterranean region, led to the rapid migration
species, but the development of a new generation of of the industry to more off-shore locations. These
sophisticated inert co-feeding and replacement diets more remote sites had the advantage of being in
have enabled the further intensification of the larval deeper water, with better water exchange providing
rearing process, and considerably reduced reliance improved growth and hygiene conditions. The
upon the live food chain thereby simplifying structures generally employed were circular
production methodologies. polyethylene cages and these were more flexible and
Nutrition, health and performance are intimately better suited to the harsh environmental conditions
linked and the industry is placing increasing encountered. In addition, they proved to be less
importance and effort on optimizing formulations expensive and provided further economies of scale
and improving ingredient quality including the with volumes of 10,000 m3 or more.
sourcing of fresh raw materials. A cold extrusion These large cages are today fully automated with
spherizer agglomeration system has been used to computerized air blown feed delivery systems
produce diets aimed at the complete replacement of operated from a centrally moored feed barge (Figure
the live food chain and they focus on high digestibility. 5). Underwater monitoring and surveillance systems
Skretting use the above technology together with a enable operators to observe feeding behaviour, fish
patented phospolipid content of 12%. This has been health status and adjust feed delivery to ensure optimal
reported to play an important role in the reduction growth and feed conversion ratios (FCR).
of juvenile deformation and improved growth These systems have been found to be suitable for
performance. most European weather conditions, however some
It is hoped that further developments of micronised fully exposed sites are experimenting with
replacement diets will both simplify and standardize submersible cage systems to avoid extreme conditions.
future marine fish larval rearing and enable a greater These new technologies are primarily being
number of species to be commercialized. Possibly investigated in other areas of the world where tropical
given the restriction of the very small mouth sizes of storms and the lack of protected coastlines make
some marine larvae, such as the groupers, and the traditional cage systems impractical.
difficulty in maintaining extremely small strains of The future of cage on-growing technologies will
rotifers for first feeding, highly digestible diets of continue with the establishment of more offshore cage
this type may provide an alternative strategy for the systems and perhaps even larger structures will be
industry. developed to accommodate the requirements of new
species such as the tunas.
Land-based on-growing technologies in Europe can
On-growing technologies be split into open flow through raceway-type systems,
commonly used for the production of trout, and re-
European aquaculture is focused on the intensive circulation systems for eels, tilapia and turbot amongst
rearing of fish. Salmon, some trout, sea bass, sea others. The flow-through systems may exploit the
bream and other various round marine fish are grown benefits of waste warm water from power stations
in floating cage structures. Protected coastline offering where a number of species are cultured including
abundant site availability in Norway and Scotland sole, sea bass and sea breams. Semi-intensive pond
led to the development of the cage farming industry. culture of marine and brackish water species also
Following the early success and profits made in these takes place in Spain and Italy and the pond culture
areas attention turned to the Mediterranean, in of carps exists in many countries of Europe. Highly
particular the Adriatic, Ionian and Aegean seas where intensive land based systems are used for the pre-on-
similar coastlines existed. This led to the development growing of juvenile sea bass and sea bream in Spain
of the sea bass and sea bream industry. As these where the rigours of off-shore on-growing systems
industries developed and juvenile availability dictates that the size of the juveniles recruited should
increased for each species, the type of these cage be in the order of 20 g. Typically these systems
structures developed. Initially small wooden employ liquid oxygen and use where possible borehole
structures evolved to larger steel ones with the culture water, which has proved to have the benefits of
volumes increasing from 125 m3 to 3000 m3.
J.W. Sweetman 429
Figure 5. Sea cage automated response monitored feeding system (Akvasmart).
constant temperatures, low bacterial levels and high of the developing sea bass and sea bream industry in
water quality without the need for expensive pre- the Mediterranean. Initial pelletized feeds were
treatment. replaced with extruded diets and formulations adapted
Land-based systems are the only effective method to meet the demands of individual species were
of producing some flat fish species; and while in developed as production grew.
Europe land prices are at a premium, the value and Consumer confidence and food safety issues are
market potential for some species (sole 11 €/kg, important factors in aquaculture. Following the BSE
whole) is such that significant investment is already outbreaks, the European Union banned the inclusion
underway. The potential for the development of of ingredients derived from terrestrial animal by-
vertically stacked shallow raceway systems controlled products. This has stopped the incorporation of
by re-circulation technologies is therefore of interest. hemoglobin, blood meals, meat, bone and feather
meals amongst other ingredients. In addition to this,
many sales outlets and the large supermarket chains
On-growing feed technologies require European feeds to be certified GMO-free as
part of their drive to satisfy consumer demand and
Modern European aquaculture feed production perception.
developed in parallel with the needs of the salmon The newly established European Food Safety
and trout industries. Licensing restrictions based upon Authority (EFSA) runs risk analysis and risk
feed consumption and waste product release into the management and promotes an integrated approach
environment together with the commercial pressures to the responsibility of feed manufactures, farmers
of feed usage and improved growth rates have resulted and food operators on the traceability of feeds, food
in more efficient and cost effective formulations. and their ingredients.
High energy extruded feeds used in the salmon These actions, while essential to regulate the
industry today can contain fat levels of over 30% industry and address consumer and public health
and feed conversion rates have dropped to below 1:1. issues, severely restrict the formulations available for
The efficiency of these diets has been cross-checked aquaculture. This in turn places a heavy load on the
by in vivo testing using mink as a target animal to limited resource of fish meal as a protein source and
determine among other things protein digestibility. adds to the cost of feed at a time when the industry is
Excess production capacity in northern European striving to reduce production costs by all means
feed mills was quickly adapted to meet the demand possible. Fish meal and oil substitutes are emerging
now as viable, economic partial alternatives. The gut. (Bergh et al., 1994; Munro et al., 1993) and the
inclusion of digestibility enhancers and organic presence of opportunistic pathogens at this stage has
chelated trace minerals provides better bioavailability been shown to lead to disease (Grisez et al., 1996).
of important nutrients. Fish transfer from the hatchery to the pre-on-
Feed quality may also be affected by poor storage growing or on-growing facilities necessitates the
conditions or raw material quality. This can result in transfer of fish from a protected to an unprotected
the development of mycotoxins that can increase environment in which they might come into contact
disease sensibility, result in lower growth and poor with a variety of different pathogens It is possible to
FCRs (Lovell et al., 1994). Modern natural gluco- protect against certain diseases with vaccines, and a
mannan mycotoxin adsorbents are capable of limited number are available for commercial use.
absorbing a broad spectrum of mycotoxins directly Vaccination takes places prior to the transfer from
from the digestive tract preventing their absorption the hatchery facilities to the on-growing units, but
and subsequent bioavailability. due to the limited duration of protection offered to
fish further vaccination may be necessary during the
on-growing cycle.
Health and disease issues With the complexity of vaccine licensing and the
restricted use of antibiotics and some other therapeutic
Parasitic, bacterial and viral diseases cause agents the industry is turning to other methods of
considerable financial loss to the European industry. prophylaxis and control to improve the fish health
Disease problems in the marine sector originate from status. The concept of nutritional supplementation,
a diverse range of infectious agents, which have been the use and blending of selected nutrients,
reviewed by Le Breton (1996) and Rodgers and immunostimulants and immunomodulators are
Furones (1998), a list that has rapidly developed and urgently being considered by the aquaculture industry
is continually expanding. as it learns of their effectiveness in terrestrial animal
The development of molecular techniques for the culture.
identification and screening of pathogens offers the
potential to improve disease prevention and control.
The sensitivity of nucleic acid probes now enables Conclusions
the detection of subclinical carriers of some infections;
and this is an important development and tool for the European aquaculture is expected to show growth in
establishment of specific pathogen-free broodstocks. the marine sector and the success of individual
Hatchery production units try to avoid the operations will depend on the successful application
introduction of opportunistic pathogens through of a variety of multidisciplinary activities. Economic
treatment of the incoming water supplies using a viability must be linked to better marketing strategies
variety of filtration methods. Sterilization, either by and food safety. Transparency, traceability, quality
UV and (or) ozone, is common practice in both and sustainability issues are at the forefront of
freshwater and marine environments. Production European concerns and actions. Technological
scheduling now includes specific periods where either improvements are expected to continue to improve
units of, or the whole hatchery are shut down, cleaned cost efficiency and stimulate further species
and sterilized and all-in-all-out batch production diversification at a time when fisheries production is
provides regular sanitary control. Areas within the stagnant and in certain sectors in decline. Simplified
hatchery environment are kept as discrete as possible legislation and licensing procedures have been called
with the minimum of interaction by working for and continued and coherent policies for research
personnel and equipment from one area to another. and development are essential.
Isolated quarantine facilities are employed to prevent
the introduction of disease. References
In larval rearing various strategies have been
proposed for controlling the microflora of this Agresti, J.J., S. Seki, A. Cnaani, S. Poompuang, E.M.
environment. Mature water and the addition of Hallerman, N. Uniel, G. Hulata, G.A.E. Gall and
probiotics either through the live food chain or B. May. 2000. Breeding new strains of tilapia:
directly in the larval rearing tank have been used. development of an artificial centre of origin and
The larval pre-feeding and first feeding stages are linkage map based on AFLP and microsatellite loci.
critical to the establishment of the microflora of the Aquaculture 185:43-56.
J.W. Sweetman 431
Austin, B. and J.G. Day. 1990. Inhibition of prawn Le Breton A. 1996. An overview of the main
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(Hippoglosssus hippoglossus) larvae during first Sweetman and P. Lavens, eds). Presented at an
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la Higuera. 1994. Influence of nutritional Oostende, Belgium, pp. 67-86.
composition of diet on sea bass, Dicentrarchus Lovell, R.T., S. Lumlertdacha, B.W. Kemppainen,
labrax L., reproductive performance and egg and S.D. Lenz and R.A. Shelby. 1994. Researchers chart
larval quality. Aquaculture 128:345-361. mycotoxin problems in channel catfish. Feedstuffs,
Cooper, S., A. Battat, P. Marot and M. Sylvester. August 29, pp. 11.
1983. Production of antibacterial activities by two Munro, P.D., T.H. Birkhead and A. Barbour. 1993.
bacillariophyceae grown in dialysis culture. Can. Influence of the rate of bacteria colonization of
J. Microb. 29:338-341. the gut of turbot larvae on larval survival. In: Fish
Estevez, A., L.A. McEvoy, J.G. Bell and J.R. Sargent. Farming Technology (H. Reinertsen, L.A. Dahle,
1999. Growth, survival, lipid composition and L. Jorgensen and K. Tvinnereim, eds). A.A.
pigmentation in turbot (Scopthalmus maximus) Balkema, Rotterdam, p. 85-92.
larvae fed live-prey enriched in arachidonic and Rodgers, C.J. and M.D. Furones. 1998. Disease
eicosapentaenoic acids. Aquaculture 180:321-340. problems in cultured marine fish in the
European Food Safety Authority (EFSA). Mediterranean. Fish Pathol. 33:157-164.
www.efsa.eu.int. Sorgeloos P. 2004. Quality in live food. Presented at
FAO Inland Water Resources and Aquaculture the 3 rd Workshop of the PROFET Initiative,
Service. 2003. Review of the state of world ‘Hatchery technologies’. 16-17 January 2004,
aquaculture. FAO Fisheries Circular No. 886, Rev. Bordeaux, France.
2. Rome, FAO, p. 95. Suantika, G., P. Dhert, E. Sweetman, E. O’Brien
Federation of European Aquaculture Producers and P. Sorgeloos. 2003. Technical and economic
(FEAP). www.feap.org. feasibility of a rotifer recirculation system.
Grisez, L., M. Chair, P. Sorgeloos and F. Ollivier. Aquaculture 227:173-189.
1996. Mode of infection and spread of Vibrio Tacon, A.J. 2003. Aquaculture production trends
anguillarum in turbot Scopthalmus maximus larvae analysis. In: FAO Inland Water Resources and
after oral challenge through live feed. Disease Aquaculture Service. Review of the State of World
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Harel, M., A. Tandler, G.W. Kissil and S.A. 2. Rome, FAO, p. 95.
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A.G.J. Tacon 433
Fish meal and fish oil use in aquaculture: global overview and prospects for
substitution
ALBERT G.J. TACON
SEALAB Aquaculture Laboratory, Hawaiian Institute of Marine Biology, University of Hawaii at
Manoa, USA
Introduction
Although aquaculture’s contribution toward total This paper reviews the current and predicted global
world fisheries landings has increased six-fold over use and demand for fish meal and fish oil with
the past three decades, increasing from 5.3% to 34.1% compound aquafeeds for farmed finfish and
from 1970 to 2001 (Figure 1), the finfish and crustaceans, including prospects for substitution;
crustacean aquaculture sector is still highly dependent aquafeeds including commercially compounded diets,
upon marine capture fisheries for sourcing key dietary farm-made aquafeeds, and/or whole marine food/feed
nutrient inputs such as fish meal and fish oil. In fact organisms as well as fresh/frozen fish, molluscs and
when viewed in wet fish weight equivalents, although crustaceans. Particular emphasis is placed on the
only about 17.7 million metric tons (mmt) or 37% urgent need for the aquafeed-fed finfish and
of total global aquaculture production in 2001 (Figure crustacean aquaculture sector to reduce its current
2) were finfish and crustacean species whose dependence upon potentially food-grade marine
production was dependent upon the use of compound capture fishery resources for sourcing its major
aquafeeds, these species consumed the equivalent of dietary protein and lipid nutrient inputs (in the form
17 to 21 mmt of marine pelagics on a wet weight of fish meal and fish oil), and to seek alternative
basis. more sustainable feed resources; the long term
120 Total aquaculture production in 2001 was 48.4 mmt or

34.1% of total world fisheries landings of 142.1 mmt (FAO, 2003)
100
Aquaculture Capture fisheries 93.7 mmt
80
Million metric tons
60
48.4 mmt
40
20
0
1970 1973 1976 1979 1982 1985 1988 1991 1994 1997 2000
Figure 1. Contribution of aquaculture to total world fisheries landings 1970-2001.

434 Fish meal and fish oil use in aquaculture: global overview and prospects for substitution
Total production by weight

48.41 mmt
Aquatic plants
21.8%
Total fish and crustaceans

Finfish 24.4 mmt
Crustaceans 2.0 mmt
Finfish
50.5%
Molluscs Fed species

18 mmt or 37% of total
23.3%
aquaculture production in 2001
Other Crustaceans
0.3% 4.1%
Figure 2. Total world aquaculture production in 2001 (FAO, 2003).
evaluation and use of single cell proteins (SCP) being similar dependency also exists for fish oil (as the
particularly encouraged and holding much promise. main source of dietary lipids and essential fatty acids
in compound aquafeeds) for the above species, with
crustaceans currently being less dependent than
Current fish meal and fish oil usage carnivorous finfish due to the lower levels of dietary
lipids generally used in crustacean feeds. In addition
As mentioned previously, the finfish and crustacean to the above, fish meal and fish oil are also commonly
aquaculture sector is currently heavily dependent upon used as a secondary source of dietary protein (usually
capture fisheries for sourcing key nutrients and feed included at low dietary inclusion levels) and lipid
ingredients for compound aquafeeds. These include for many omnivorous cultured finfish species,
high quality animal proteins and feeding attractants including freshwater carps, tilapia and catfish (Tacon,
(fish meal, and to a lesser extent fish solubles, shrimp 2003b).
meal, squid meal, fish/squid liver meals, fish/ Table 1 shows the estimated global use and demand
crustacean hydrolysates, and krill meal) and essential for fish meal and fish oil within compound aquafeeds
dietary lipids (fish oil, and to a lesser extent fish and according to the author, with estimates from the
squid liver oils); Hardy and Tacon, 2002; New and International Fish Meal and Fish Oil Manufacturers
Wijkström, 2002; Tacon, 2003a). This dependency Association (IFFO; Pike and Barlow, 2003) given in
upon fish meal and fish oil is particularly strong for italics. From these data it can be seen that the total
those higher value species feeding high on the aquatic production of industrially compounded aquafeed
food chain, including all carnivorous (i.e. fish/ production in 2001 was about 16.7 mmt (Figure 3),
invertebrate animal-eating) finfish species and most with aquafeed production currently representing only
omnivorous/scavenging crustacean species. about 3% of total global industrial animal feed
Finfish and crustacean species that are currently production (estimated at 612 mmt in 2003; Figure 4).
reliant upon fish meal as the main source of dietary The major species groups dependent upon
protein in compound aquafeeds include: finfish - all compound aquafeeds in 2001 include the non-filter
farmed marine finfish (excluding mullets and feeding carps (8.0 mmt of aquafeeds used in 2001),
rabbitfish); diadromous species - salmonids (salmon, marine shrimp (2.1 mmt), salmon (1.56 mmt), marine
trout, char), eels, barramundi, sturgeon; freshwater finfish (excludes mullets; 1.21 mmt), tilapia (1.16
species - mandarin fish, pike, pike-perch, snakehead, mmt), trout (0.74 mmt), catfish (0.60 mmt),
certain freshwater Clarias catfishes; and crustaceans freshwater crustaceans (0.52 mmt), milkfish (0.42
- all marine shrimp, crabs, and freshwater prawns. A mmt) and eels (0.37 mmt; Figure 3).
A.G.J. Tacon 435
Table 1. Estimated global use and demand for fish meal and fish oil within compound aquafeeds (2001 species group aquaculture production
estimates taken from FAO, 2003)
2001 2002 2005 2010
Species group Thousand metric tons (tmt, dry as-fed basis)
MARINE SHRIMP
Total production, tmt 1 1,271 - 1,387 1,771
IFFO estimate, tmt 2 - 1,353 - 1,999
3
Predicted growth, APR, %/year 5 - 5 -
IFFO estimate, %/year - 5 5 -
Percent on feeds, % 4 83 - 87 92
IFFO estimate, % - 80 - 90
Species economic FCR 5 2.0 - 1.8 1.6
IFFO estimate - 1.8 - 1.6
Total aquafeeds used, tmt 6 2,110 - 2,172 2,607
IFFO estimate, tmt - 1,948 - 2,878
Average fish meal content, % 24 - 14 4
Average fish oil content, % 2 - 1 1
Total fish meal used, tmt 506 - 304 104
IFFO estimate, tmt - 487 - 576
Total fish oil used, tmt 42 - 22 26
FRESHWATER CRUSTACEANS
Total production, tmt 7 514 - 809 1,188
Growth, APR, %/year 12 - 8 -
Percent on feeds, % 42 - 45 50
Species economic FCR 2.4 - 2.2 2.0
Total aquafeeds used, tmt 518 - 801 1,188
Average fish oil content, % 2 - 1.5 1.5
Total fish oil used, tmt 10.4 - 12.0 17.8
MARINE FISH
Total production, tmt 8 974 - 1,386 1,926
IFFO estimate, tmt - 703 - 1,172
IFFO estimate, %/year - 5-22 - 5-22
IFFO estimate, % - 60-90 - 80-100
IFFO estimate - 2-2.1 - 1.7-2
Total aquafeeds used, tmt 1,208 - 1,746 2,465
IFFO estimate, tmt - 907 - 1,894
IFFO estimate, % - 45-55 - 40-45
IFFO estimate, % - 8-12 - 10
SALMON
Total production, tmt 1,200 - 1,633 2,186
IFFO estimate, %/year - 5 - 5
Table 1. Continued
2001 2002 2005 2010
I FFO estimate, % - 28 - 20
TROUT
Total production, tmt 567 - 689 839
-
IFFO estimate, tmt 500 - 633
Total aquafeeds used, tmt 737 - 827 923
EEL
Total fish oil used, tmt 15.0 - 11.2 8
MILKFISH
A.G.J. Tacon 437
Table 1. Continued
2001 2002 2005 2010
Average fish oil content, % 1 - 0.5 0
FEEDING CARP
Total production, tmt9 10,549 - 13,947 18,664
IFFO estimate, % - 0 - 0.5
TILAPIA
Total production, tmt 1,385 - 1,694 2,376
Species economic FCR 2 - 1.8 1.6
IFFO estimate, % - 1 - 0.5
CATFISH
Table 1. Continued
2001 2002 2005 2010
10
CARNIVOROUS FRESHWATER FISH
Total production, tmt - 551 - 946
Growth, APR, %/year - 7 - 7
Percent on feeds, % - 20 - 60
Species economic FCR - 2.4 - 2.0
Total aquafeeds used , tmt - 264 - 1,135
Average fish meal content, % - 15 - 10
Average fish oil content, % - 6 - 7
Total fish meal used, tmt - 40 - 114
Total fish oil used, tmt - 16 - 79
TOTAL GLOBAL ESTIMATES 2001 2002 2005 2010

Total fed production, tmt 11 17,690 - 22,836 30,472
Total aquafeed production, tmt 16,700 - 21,111 28,596
Total estimated fish meal used, tmt 2,614 - 2,294 1,550
Total estimated fish oil used, tmt 594 - 478 447
Total fish meal + fish oil used, tmt 3,208 - 2,772 1,997
IFFO calculated this paper, tmt - 2,949 - 3,807
Equivalent pelagics used, tmt 12 16,040 - 13,860 9,985
1
Total reported farmed species group production for 2001 (FAO, 2003), and estimates for 2005 and 2010 (Tacon, 2003b)
2
International Fish meal and Fish Oil Organization (IFFO) estimated farmed marine shrimp + crab production for 2002 and estimates for
2005 and 2010 (Pike and Barlow, 2003)
3
Estimated Annual Percent Rate of Growth of farmed species group production 2001/2002 to 2010 (APR, %)
4
Estimated percent of total species group production on aquafeeds
5
Estimated average species group economic food conversion ratio (total food fed / total species group biomass increase)
6
Estimated total species group aquafeed used (total species group production x FCR)
7
Freshwater crustaceans includes freshwater prawn, river crab and crayfish
8
Marine finfish species group excludes mullets
9
Feeding carp species excludes filter feeders such as silver carp, big head carp and catla
10
Includes Chinese bream, mandarin fish, yellow croakere, long-nose catfish (carnivorous/omnivorous) but excludes eel (Pike and Barlow,
2003)
11
Excludes filter feeding fish species (5,878 tmt in 2001), freshwater fish species (species unknown: 2,259 tmt in 2001), marine crabs and
other marine crustaceans (200 tmt), Mandarin fish (116 tmt in 2001), and other miscellaneous freshwater fish species
(including climbing perch, snakeheads, colossoma, gourami ca. 165 tmt in 2001).
12
Using a mean fish meal+fish oil to pelagics conversion ratio of 1:5 (FAO conversion factor for fish meal).
Concerning fish meal and fish oil usage, it is estimated On a species group level salmonids consumed the
that the compound aquafeed sector consumed about largest proportion of fish meal and fish oil in 2001
2.62 mmt of fish meal and 0.59 mmt of fish oil in (29.4% and 64.5% of totals used in aquafeeds,
2001 (Table 1), or equivalent to 43.1% and 53.6% respectively), followed by marine fish (22.6% and
of the total global production of fish meal (6.08 mmt) 20.3%), marine shrimp (19.3% and 7.0%), feeding
and fish oil (1.10 mmt), respectively (FAO, 2003). carp (15.3% in the case of fish meal) and eels (6.9%
A.G.J. Tacon 439
Freshw ater Others

crustaceans 3%
3.1% Aquaculture
Marine shrimp 3%
Tilapia 12.6%
6.9%
Catfish
3.6%
Salmon Cattle
9.3% Poultry
24%
38%
Trout
4.4%
Milkfish
2.5%
Eel
2.2%
Marine fish
Carp 7.2%
48.0%
Pigs
Total estimated compound aquafeed
32%
production in 2001- 16.70 mmt
Total estimated industrial animal
feed production in 2003 - 612 mmt
Figure 3. Estimated global compound aquafeed production Figure 4. Estimated global industrial animal feed production
in 2001 for major farmed species (values expressed as % in 2001 for major farmed species (values expressed as %
total aquafeed production, dry as-fed basis). dry as-fed basis).
and 2.5%; Figures 5 and 6). The total use of fish produced through farming in 2001 included marine
meal and fish oil in compound aquafeeds is almost eels (current pelagic input per unit of production 3.4-
certainly higher than the figure given above, as an 4.2), marine fish (2.9-3.7), salmonids (2.6-3.3),
additional 2.6 mmt of finfish and crustacean marine shrimp (1.7-2.1), freshwater crustaceans (1.0-
production (equivalent to 10% total finfish and 1.3), whereas, net fish producers included milkfish
crustacean production) was not included in these (0.33-0.42), catfish (0.28-0.35), tilapia (0.24-0.29),
calculations (includes unknown freshwater fish and feeding carp (0.15-0.19).
species (2.26 mmt in 2001), marine crabs and other Moreover, coupled with the use of trash fish as a
marine crustaceans (0.2 mmt), Mandarin fish (0.12 direct food source for farmed fish and crustaceans in
mmt), and other miscellaneous freshwater fish some Asian countries (China reportedly using 4 mmt
species). According to IFFO (Pike and Barlow, 2003) of trash fish as feed for marine finfish and crustaceans
fish meal and fish oil usage within compound in 2000; D’Abramo et al., 2002), it is estimated that
aquafeeds in 2002 was estimated to be 2.22 mmt and the aquaculture sector consumed the equivalent of
0.73 mmt, respectively (Table 1). 17-20 mmt of fish as feed in 2001 (either in the form
The total estimated use of fish meal and fish oil in of fish meal, fish oil or trash fish, expressed in live
aquafeeds (3.2 mmt in 2001, dry basis) was equivalent weight equivalents) for the total production of 17.69
to the use of 12.8 to 16.1 mmt of pelagics (using a mmt of aquafeed-based farmed fish and crustaceans
dry meal/oil to wet fish weight equivalents conversion in 2001. However, in contrast to the 8 to 11% annual
factor of 4 to 5) for the production of 17.69 mmt of growth rate of the aquaculture sector over the past
the major farmed-fed finfish and crustacean species decade, the proportion of the global fish catch
in 2001. Cultured species groups currently destined for non-food uses (including for reduction
consuming more fish through feeding than is being into fish meal and fish oil, or for direct animal
Catfish Freshw ater

crustaceans Tilapia Freshwater
0.9%
4.5% 1.9% crustaceans
Tilapia Marine shrimp Milkfish
1.7%
2.7% 19.4% 0.7% Catfish
Eels 1.0% Marine shrimp
Carp 7.1%
2.5%
15.3%
Marine fish
Marine fish 20.4%
Milkfish 19.4%
1.4%
Eels
6.9%
Salmon
47.3% Trout
Trout 17.3%
Salmon
22.7% 6.8%
Total estimated fish meal used in aquafeeds in 2001 was Total estimated fish oil used in aquafeeds in 2001 was
2,614 TMT (43.1% of total world fish meal production) 594 TMT (53.6% of total world fish oil production)
Figure 5. Estimated global use of fishmeal in compound Figure 6. Estimated global use of fish oil in compound
aquafeeds in 2001 by major cultivated species (expressed as aquafeeds in 2001 by major cultivated species (expressed as
% total fish meal used within aquafeeds, dry as-fed basis). % total fish meal used within aquafeeds, dry as-fed basis).
feeding) has remained relatively constant, in recent Future fish meal and fish oil usage
years fluctuating from a low of 25.3 mmt in 1998
(strong El Niño year) to a high of 34.8 mmt in 2000 It follows from the above discussion that for those
(Figure 7); total capture fisheries in 2001 reported aquaculture species and exporting/importing countries
as 92.4 mmt, including 61.1 mmt destined for direct currently dependent upon the use of these relatively
human consumption and 31.3 mmt or 33.9% destined finite fishery commodities as feed inputs,
for non-food uses (FAO, 2003). consumption of these commodities will have to
120
Aquaculture Capture - feed
Capture - food Total capture
100
92.4 mmt
80
Million metric tons
60 61.1 mmt
40 37.8 mmt
31.3 mmt
20
0
1970 1973 1976 1979 1982 1985 1988 1991 1994 1997 2000
Figure 7. World finfish and shellfish production from capture fisheries and aquaculture, and disposition of the catch 1970-2001
(expressed in million metric tons, live weight equivalents; FAO, 2003).
A.G.J. Tacon 441
increase if current dietary inclusion levels are to be Chile, Brazil) D’Abramo et al., 2002; FAO, 2003;
maintained. For example, according to IFFO (Barlow Tacon, 2003a; Figure 4),
and Pike, 2003) the aquaculture sector’s consumption
• A global trend toward increasing fish meal and
of fish meal and fish oil is expected to increase from
fish oil prices with increasing market demand for
34% (2,217 tmt) and 56% (732 tmt) of the total global
these valuable commodities (Hinrichsen, 2003),
production of fish meal and fish oil in 2002, to 48%
(2,854 tmt) and 79% (953 tmt) in 2010, respectively • Increasing consumer awareness for increased
(Table 1); this increase being equivalent to a 29-30% food/feed safety and traceability in the food
increase in global fish meal and fish oil usage by the production process (Best, 2002; Soponpong,
aquaculture sector from 2002 to 2010. 2002), the environmental, ecological and social
The above predictions by IFFO differ from those sustainability of our food production (Costa-
of the present author and others (Hardy and Tacon, Pierce, 2003; Hole, 2004; Naylor et al., 1998;
2002; Tacon and Forster, 2000), who estimated that Raven, 2002; Tacon, 1997), and consumer
fish meal and fish oil use by the aquaculture sector demand for leaner and healthier food products.
will actually decrease rather than increase in the long
term (Table 1). Thus, from the data presented in Table Moreover, as a result of increased aquaculture
1 it is expected that usage will decrease by 40% in production and decreasing fish/shrimp market prices
the case of fish meal (from 2,614 tmt in 2001 to (Harvey, 2003; Hinrichsen, 2003), nutritionists and
1,550 tmt in 2010) and 25% in the case of fish oil feed manufacturers alike have been forced to reduce
(from 594 tmt in 2001 to 447 tmt in 2010, Table 1). feed costs (through the development of fish meal and
The main reasons why fish meal and fish oil use fish oil replacers) and/or by improving on-farm feed
by the aquaculture sector is expected to decrease in performance so as to maintain profitability.
the long term is due to a combination of increasing
economic/market pressures placed upon the fish meal
and fish oil manufacturing industry and animal feed
compounder on the one hand; and the consequent
Sustaining aquaculture production:
search, development and use of lower cost and more substitution prospects
sustainable alternatives by the aquafeed
Clearly, in view of the fact that wild fish stocks have
manufacturing sector on the other hand so as to
remained relatively static over the last decade (Figure
maintain profitability and sustain the growth of the
7), the aquaculture sector has no choice but to reduce
feed-dependent aquaculture sector. Examples of
its dependence upon capture fisheries for sourcing
increasing economic/market pressures placed upon
its dietary protein and lipid nutrient inputs if it is to
the fish meal/aquafeed manufacturing sector include:
sustain its annual growth rate of 8.8% per year since
1970 (Figure 1). Although this will be a relatively
• The increasing market demand for the production
simple task for omnivorous/herbivorous finfish and
and use of less environmentally contaminated fish
crustacean species (in view of their more flexible
meals and oils (through the selection of less
feeding habits/food preferences and available feed
contaminated fish stocks and/or through
resources), this will be more difficult for carnivorous
increasing legislative controls limiting fish meal/
species.
fish oil use in aquafeeds; Hites et al., 2004; Jacobs
To date the majority of recent effort has focused
et al., 2002; Pike, 2002; Smith et al., 2002),
on the development and use of the following major
• The increasing global demand for the use of fish meal replacers:
potentially food-grade pelagics (including
mackerel, sardines, herring, pilchards, anchovies) Terrestrial vegetable proteins (TVP), includes
for direct human consumption rather than for protein-rich oilseed and grain by-product meals
reduction into fish meal and fish oil (Wray, 2001), including soybean, rapeseed, corn gluten, wheat
gluten, and to a lesser extent pea and lupin meals
• Increased global competition for available stocks
(Abdelghany, 2003; Abery et al., 2002; Barros et al.,
of fish meal and fish oil by the rapidly emerging
2002; Bautista-Teruel et al., 2003a, 2003b;
aquafeeds and compound animal feed
Borlongan et al., 2003; Cheng and Hardy, 2002a;
manufacturing sector in developing countries
Cheng et al., 2003; Cremer et al., 2003; Du and Niu,
(including China, Thailand, Indonesia, India,
2003; Farhangi and Carter, 2001; Francis et al., 2001; Single cell proteins (SCP), includes unicellular and
Glencross et al., 2003a, 2003b, 2003d; Grisdale- filamentous algae, yeasts and bacteria (Lara-Flores
Helland et al., 2002; Hari and Kurup, 2003; Jahan et et al., 2003; Li and Gatlin, 2003; Nates and Tacon,
al., 2003; Lee et al., 2002; Maina et al., 2003; Mente 2003; Nates et al., 2002; Olvera-Novoa et al., 2003).
et al., 2003; Opstvedt et al., 2003a, 2003b;
Penaflorida, 2002; Pereira and Oliva-Teles, 2002; Of the above-mentioned feed sources, considerable
Peres et al., 2003; Refstie and Tiekstra, 2003; Richter further work still needs to be undertaken concerning
et al., 2003; Rinchard et al., 2002; Dabrowski et al., the use and suitability of SCP as dietary fish meal
2003; Siddhuraju and Becker, 2003; Singh et al., replacers for both finfish and crustaceans. These
2003; Takagi et al., 2001; Thiessen et al., 2003a, products hold particular promise by virtue of their
2003b). According to the FAO agricultural statistical ability to be produced from renewable resources and/
database the total production of plant oil cakes and or agricultural/petrochemical waste streams/
meals in 2002 was over 177 mmt (Figure 8), as substrates, their rapid growth rate, high dietary
compared with a total global production of just over protein content and nutritive value (generally devoid
7 mmt for fish meal and fish oil. of anti-nutrients and overt nutrient imbalances), and
the ability to tailor their nutritional composition to
Terrestrial animal proteins (TAP), includes animal approximate needs of the cultured target species
by-product meals: poultry by-product meal, meat (through direct nutrient modifications during the
meal, meat and bone meal, and to a lesser extent fermentation or growth process).
feather meal and blood meal (Abdel-Warith et al., Finally, total replacement of fish oil with
2001; Bharadwaj et al., 2002; Cheng and Hardy, commercially available plant and animal oils has been
2002b; Cheng et al., 2002; Forster et al., 2003; more problematic, especially within carnivorous
Mendoza et al., 2001; Menasveta et al., 2003; marine and diadromous finfish species. Studies where
Millamena and Golez, 2001; Millamena, 2002; Tan plant and animal oils (including soybean, rapeseed,
et al., 2003; Williams et al., 2003a, 2003b; linseed oils and terrestrial animal fats) have achieved
Woodgate, 2004a, 2004b; Zhu and Yu, 2003). some success as fish oil replacers (depending upon
Coconut
1.5%
Palmkernel
Maize Linseed
2.2%
Ricebran 1.3% 1.1% Others
2.7% 0.7%
Groundnut
3.9%
Sunflowerseed
5.4%
Cottonseed
7.2%
Soybean
63.1%
Rapeseed
10.9%
Total production of plant oil cake and meal in 2002: 177.7 mmt
Figure 8. Estimated global production of plant oilseed cakes and meals in 2002 (source: FAOSTAT Agriculture Database).
A.G.J. Tacon 443
the species farmed) have included: Ballestrazzi, 2003; Abdel-Warith, A.A., P.M. Russell and S.J. Davies.
Bell et al., 2002, 2003a, 2003b; Bransden et al., 2003; 2001. Inclusion of a commercial poultry by-product
Bureau, 2004; Grisdale-Helland et al., 2002b; meal as a protein replacement of fish meal in
Glencross et al. 2003c; Martino, 2003; Montero et practical diets for African catfish Clarias gariepinus
al., 2003; Ng et al., 2003; Raso and Anderson, 2003; (Burchell 1822). Aquacult. Res. 32(1):296-305.
Regost et al., 2003a, 2003b; Tocher et al., 2002; Abery, N.W., R.M. Gunasekera, S.S. DeSilva. 2002.
Turchini et al., 2003. However, as in the case of Growth and nutrient utilization of Murray cod
dietary protein sources, considerable potential also Maccullochella peelii peelii (Mitchell) fingerlings
exists concerning the further development and use fed diets with varying levels of soybean meal and
of SCP for the production of dietary lipid blood meal. Aquacult. Res. 33(4):279-289.
supplements and ingredients rich in long chain
Ballestrazzi, R., S. Rainis, F. Tulli and A. Bracelli.
polyunsaturated fatty acids and other essential lipid
2003. The effect of dietary coconut oil on
soluble nutrients, including carotenoid antioxidants,
reproductive traits and egg fatty acid composition
phospholipids, specific steroids etc. (Nates and Tacon,
2003b). in rainbow trout (Oncorhynchus mykiss). Aquacult.
Clearly, the long term sustainability of the Int. 11(3):289-299.
aquaculture and compound aquafeed sectors will only Barros, M.M., C. Lim and P.H. Klesius. 2002. Effect
be ensured if ingredients can be sourced whose of soybean meal replacement by cottonseed meal
production can keep pace with the growth of the sector and iron supplementation on growth, immune
(including the competing terrestrial livestock meat response and resistance of channel catfish (Ictalurus
production sector; Figure 9), and whose nutritional puctatus) to Edwardsiella ictaluri challenge.
composition and characteristics are flexible and can Aquaculture 207(3):263-279.
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Creating alternative protein sources for aquafeeds using applied enzyme

technologies
S. L. WOODGATE
PDM Group Ltd, Greenleigh, Kelmarsh Rd, Clipston, United Kingdom
Introduction Application principles for upgrading by-

products
One of the great challenges of the next decade will
be to provide sufficient high quality proteins for the WHAT ARE THE PROBLEMS WITH BY-
rapidly growing aquaculture industry. A wide range PRODUCTS?
of species to be farmed will necessitate sourcing feed
proteins that are able to meet the specific requirements Poor nutritional value. Many potential sources of
of the target species. Fish meals have traditionally protein have a low nutritional value. This may be
provided a large proportion of the protein required manifest as a poor or low digestibility of the protein
by aquaculture species, and even if there is no or amino acids, or because the amino acid profile is
immediate shortage of supply, there are requirements not suitable for the target species.
for alternatives for a variety of reasons. One of the Antinutritional factors. Certain (mainly vegetable)
key aspects is the high cost of fish meals, mainly proteins contain anti-nutritional factors; the best
caused by an imbalance of supply and demand. known of which is probably trypsin inhibitor, a
Although this may vary from year to year because of digestive enzyme inhibitor present in raw processed
supply variation, the trend toward higher prices has soya beans. Many antinutritional factors can be
been steadily increasing because of growing demand. neutralised by a process step, and in the case of
There are bountiful supplies of many protein trypsin inhibitor, it is inactivated by a heat processing
sources that may, in principle, be suitable for stage.
inclusion in aquafeeds. However, just because an Stigma/prejudice. Some feed ingredients have a
ingredient has a high crude protein level does not long history of having of little or no value as an
mean that it is a viable alternative, and a much greater animal feed ingredient. In this situation, additional
depth of knowledge must be attained before a product promotional work will be required if efforts to
can be accepted. Of course over the last decade, many increase value in terms of enhanced nutritional value
researchers have been considering the need for fish have succeeded, but stigma still exists.
meal alternatives, and how they can fit into cost Environment. Some products have an adverse
effective and environmentally balanced feeds (Bureau environmental impact if they cannot otherwise be used,
and Viana, 2003). either in an unmodified or added value state. This
This paper discusses a more fundamental stepwise may manifest itself (in the worse case) as disposal in
approach to the problem of supplying alternative a landfill, which results in both a loss of nutrients
protein sources using, where appropriate, applied and a negative environmental impact by leaching
enzyme technology. This stepwise approach will be pollutants into groundwater or by increasing biogas
described, followed by a case study following the production.
same principles, using enzyme processed feather Legislation. In some cases legislation may ban the
protein for aquaculture feed as an example. use of certain products for use in animal feeds. In
Europe, a temporary ban on certain processed animal
proteins (OJEC, 2000; 2000/766/ec), has resulted in
450 Creating alternative protein sources for aquafeeds using applied enzyme technologies
a temporary loss of protein resource for animal feeds. for both targeting the parameters to be upgraded and
It is hoped that the temporary ban will be lifted in for defining value to the target species.
stages during 2004/5, resulting in some key animal
proteins being available for farmed animals such as
aquaculture species. THE KEY PROCESS PARAMETERS
Supply/demand imbalance. If the supply/demand
considerations do not initiate interest in upgrading, The parameters discussed below are all factors that
then development of an upgrading strategy is more require a stepwise approach to the optimisation of
difficult. While the current situation of uncertain and the process. Here the benefit of using laboratory and
expensive supply failing to meet growing demand is pilot scale development equipment cannot be
not critical, these circumstances must be addressed overestimated. The iterative approach required is
when developing an upgrading protocol. possibly achieved most successfully in applied
No specific target species. If there are no specific research establishments such as the Alltech Bioscience
target species for the by-product, then making an Centres.
economic model for the upgrading process is First the substrate must be defined. For example,
particularly difficult. Identification of key species as the protein content and configuration of a raw
the target market for the upgrading protocol can be material should be known before any attempt to
crucial to the success of an upgrading project. upgrade is made. Any potential interaction with
another nutrient such as complex carbohydrate that
might affect protein or amino acid availability should
FOCUS ON TARGET SUBSTRATE also be considered.
In general, particle size should be as small as
Protein and nitrogen compounds. These are the key practical to ensure that the largest surface area possible
parameters. Much of the nutritional value analysis is available for enzymic hydrolysis. If an additional
will depend on protein, particularly if protein is a pre-milling or size reduction step is required, then
major nutrient in the overall nutrient profile of the this requirement needs to be confirmed and included
product. Major emphasis is now placed upon the in in the process development steps.
vivo digestible amino acid profile of ingredients, so As a rule, sufficient active water content is needed
this is a vital parameter to consider. to ensure optimal hydrolysis. The optimum range
Complex carbohydrates. Although protein is the may be narrow or wide, and this optimisation also
major focus for aquaculture feeds, the presence of requires previous knowledge or new development
complex carbohydrate may limit how much positive research, possibly using a pilot plant. When
effect can be achieved by targeting protein alone. considering moisture, processes that operate for all
Therefore, if complex carbohydrates are present, an or some of the time in a high moisture environment
enzyme step to minimise any potentially negative may have a significant advantage over those that are
actions on protein digestibility might be introduced. dehydrated before any enzymic action might be
Fats, oils and waxes. Fats and oils may inhibit possible.
enzyme activity in some circumstances. If the natural It is well known that most enzymes are somewhat
level of fat is high or there is major contamination, sensitive to pH, and this range must be considered
then steps may be taken to reduce the potentially when matching the enzyme and substrate, particularly
negative effect that the fat may have on the proposed if any pH adjustment is likely to be required.
protein upgrading step. As with pH, temperature and its control are critical.
Minerals. Minerals present in high quantities may Too low a temperature may mean processing time is
affect any proposed protein upgrading step by too long to be commercially viable. If temperature
interaction with the substrate or enzyme. Thus the is too high, there are potential problems with enzyme
effect of certain minerals on both of these parameters inactivation when critical limits are reached. Of
should be understood and compensated for if possible. course many commercial enzymes have a broad range
Potential interactions. It is critical to know in detail of temperature optima, so the challenge is to match
the nutrient profile of the target product. To be able the optimum enzyme temperature with the process
to move the upgrading project forward, an temperature without having to make excessive
understanding of the sample analysis and its alterations to the latter.
provenance is essential. These details will be required Mixing is a vital feature that may be overlooked,
particularly when additional equipment is needed to
S.L. Woodgate 451
facilitate optimal enzyme hydrolysis. If mixing is processing. Energy requirements are one such consid-
inadequate, then the desired improvements may not eration. Many processes using enzyme technology
be realised, and the project may fail. Therefore the will require less energy input in a variety of forms
importance and scope of any mixing needs to be e.g. mechanical or electrical power, and steam
considered as early in the project as possible, as generated to provide heat for processing and
additional capital may be needed if a mixing stage is dehydration. Likewise, environmental impact of
needed. conventional and enzyme processes differs. Emissions
The enzyme of choice will be determined by the to the environment in the form of solids, gases, or
substrate, but many of the above mentioned factors liquids must all be controlled and in the future
may need to be taken into account before settling on minimised. The process and product that can deliver
one or more enzymes to be used. environmental benefits over a conventional method
In some circumstances enzymes alone may not be will be more valuable in the long term.
completely effective and great benefit could be A product nutrient comparison of conventional and
obtained by introducing a co-factor. For products with enzyme-processed products may not reveal major or
a complex chemical composition, the addition of a dramatic differences using the proximate analysis.
co-factor designed to achieve a specific purpose may In reality, much of the value added by enzyme
allow the enzyme to be greatly more effective. processing must be considered using other evaluation
Stabilization is critical. It is assumed that the techniques such as in vivo response or animal
materials of interest are of a high or unstable moisture performance. In vivo response is the penultimate
content after the enzyme activity is complete. The evaluation method, but may involve both costly and
choice of stabilization method depends on a number time-consuming techniques. Notwithstanding these
of factors, including the possible environmental potential negatives, a number of in vivo tests are
challenges of one route vs another, the cost of nearly always required as a prelude to a smaller
equipment, operating (fuel and drying) cost and number of animal performance trials. An in vivo
finished product transport cost. evaluation may not however be possible in the prime
The application of heat to dry the product will both target species, and a surrogate may be necessary as a
inactivate any residual enzyme activity and reduce first step, particularly if more than one target species
water activity to a level where the product is stable. has been identified. Palatability, mortality, feed
Wet preservation is generally achieved by the addition conversion and growth must be evaluated. This type
of acid or alkali with the purpose of inactivating the of evaluation will allow an economic value to be
enzyme and inhibiting any possible spoilage ascribed to the product in question, and therefore a
organisms. The moisture content is unchanged from cost benefit analysis of the upgrading technology step
the starting level, and therefore transport of finished can be completed.
product includes cost of transporting water as well
as the product.
NEXT: FITTING INTO FEED FORMULAS
COMPARING CONVENTIONAL AND ENZYME The animal performance evaluation will yield data
PROCESSES that will allow nutritionists and feed formulators to
fully include the upgraded product into commercial
In the context of processes that are suited for the formulas. The choice at that point is between an
application of enzyme technology, there are existing inclusion strategy that targets cost savings or one that
processing conditions usually described as focuses on performance enhancement, or a mixture
‘conventional’. Here, benchmarks for a variety of of both. Many nutritionists will set up a formula to
process and product parameters are already known, compare the conventional and enzyme products such
and these can be used to evaluate the differences and that there will be a direct comparison. Alternatively,
improvements that enzyme technology can bring. In both products could be used as substitutes for a higher
most cases a monetary value change can be ascribed value ingredient, such as low temperature (LT) fish
to the differences seen, although this is not always meal, and the costs of diets compared. This approach
possible with concepts such as product prejudice. has great value if the formulator is able to make
There are a number of key benchmarks that can be equally nutritious diets that give equal performance.
considered when comparing conventional and enzyme Here the full economic value change can be
determined under realistic commercial conditions.
Sustainability is particularly important if the surfactants. In the case of keratin protein, there are
conventionally-manufactured product is prohibited (in numerous sulphur bridges. A reducing compound
extreme cases ) or restricted to a lower level that does may be effective in assisting the enzyme to break
not allow use of all of the product. Notwithstanding down these structural bonds.
economics, if enzyme upgrading allows full utilisation
of a product, this can be seen to improve sustainability
of the whole livestock sector. PROCESS PARAMETERS
The proof of any economic advantage can only come
after performance trials have been completed and the In the case of feather proteins there are a range of
improvements indicated by previous evaluations have conventional or steam hydrolysis systems in use
been proven. Under these circumstances, the enzyme around the world. In most cases the most effective
technology can be endorsed as providing the expected way forward has been to adapt conventional systems
benefits. In the case of advantages both at the process by addition of an extra step or by simply altering
and the inclusion (in feed) level, then the economic the processing profile.
advantage may be shared, according to the local
circumstances.
LABORATORY AND PILOT SCALE
PRELIMINARIES
Case study: Enzyme hydrolysed feather During the development of the enzyme system key
protein for aquaculture feeds process parameters must be controlled. The following
parameters were evaluated in laboratories such as
PROBLEM TO BE SOLVED the Alltech Bioscience Centers followed by pilot plant
Hydrolysed feather protein has in the past been poorly work, initially at PDM Group, Ltd. in the UK.
regarded by nutritionists around the world. Its status Feathers are naturally present in a wide variety of
as a variable product with low digestibility and an shapes and sizes, ranging from small down-type
absence of essential amino acids has probably justified feathers from broilers to large primary turkey wing
this opinion in the past. However on the positive side, feathers up to 18 inches long. The development work
the high level of crude protein, and high levels of has concluded that no pre-size reduction is necessary,
some key amino acids might make it a valuable as long as all other parameters are met satisfactorily.
ingredient for certain target species. Process temperature and time are vital parameters.
Feather protein is comprised of keratin, a complex Temperature must be controlled below a maximum
polymer that is difficult to solubulise. As a result, of 55oC. A target of 50oC is recommended, for a
typical processing using steam to hydrolyse the feather time of 30 minutes. If a temperature of 50oC is not
often results in an overprocessing which destroys part possible, then 40oC for 60 minutes or 30oC for 120
of the chemical constituents. minutes are viable alternatives.
In consideration of the nature of feather protein it A dose of 0.5 kg of Allzyme™ FD (Alltech Inc.)
was decided to offer maximum upgrading potential. plus 2.0 kg of Allzyme™ CoPack per tonne of raw
A stepwise approach was adopted, commencing with feather is recommended. This addition rate was
laboratory studies and continuing through pilot plant established following a series of dose response
tests and into full-scale production. The trial goal of experiments.
targeting aquaculture feeds as the most appropriate The method of mixing should be able to distribute
use of the upgraded protein was kept in mind from the enzyme and Allzyme™ CoPack throughout the
the early stages of the project. feathers. A ribbon blender type design is particularly
suitable, either as integral to the batch processor (as
used in this study) or as a separate unit in a continuous
TARGET SUBSTRATE system.
An adequate level of moisture is required to
Protein is the major component of feathers comprising provide the appropriate water activity. In the case of
~85% of the dry matter, but oil/waxes are also feathers the natural moisture content is about 70%
naturally present at ~5%. These fatty components may after removal from the bird and transporting around
interfere with protein hydrolysis, so their presence the slaughterhouse using a water fluming system.
must be dealt with by use of appropriate enzymes or Some operators press excess water out of the feathers,
S.L. Woodgate 453
resulting in a moisture level of ~50%. However this

is still an adequate level of moisture for enzyme action Raw feathers
to take place.
Sterilization is important for hygiene standards to
be attained, and a pressure / temperature combination
of 2 bar/122°C for 20 minutes is sufficient to kill
pathogenic microorganisms, such as Salmonella spp.,
and also vegetative states of bacteria such as Clostridia Enzyme process
spp. 50°C for 30 min
The moisture level following sterilisation is similar

to the starting level, so dehydration is required to
stabilise the product to ~5% moisture. There are
several choices, including indirect steam drying or Sterilization process
direct air drying. There is anecdotal evidence that 2 bar, 120°C for 20 min
the latter is preferable in terms of enhanced nutritional
value, but the former may have advantages in terms
of lower energy demands and the fact that
environmental controls of lower volumes of air are
somewhat easier.
Dehydration
For the enzyme process to be introduced into a full
scale batch production, only changes to the processing
profile will, in all probability, be necessary. As a
result, no additional investment in plant and
machinery is required. For continuous systems an
additional enzyme process step is required which takes Cooling and milling
the form of an enzyme pre-hydrolyser unit. Both
systems are outlined in Figure 1.
EVALUATING RESPONSES
Dispatch
In the case of enzyme processing of feathers there
are two main desired effects. Firstly there are process
and environment benefits, and secondly there are Figure 1. Flow diagram showing production of enzyme-
nutritional improvements. hydrolysed feather protein.
Process and environmental responses include
significantly lower energy inputs required, as can be addition, much preliminary work was necessary in a
seen by the contrast in time and temperature profiles species that was not expected ultimately to be the
in Figure 2. The lower input of energy in the enzyme major target species. For example, in the case of
process also results in lower environmental pollution feather proteins, much in vivo evaluation was
because there is less breakdown of the nitrogen and completed in chickens, although the substrate is not
sulphur-containing protein. As a consequence, lower expected to feature in poultry diets in the future. The
levels of polluting chemicals such as ammonia and typical analytical profiles of hydrolysed feather
hydrogen sulphide need treating by the environmental proteins compared to fish meal, including in vivo data,
control equipment, resulting in lower environmental are shown in Table 1.
control costs. In the case of hydrolysed feather protein, legislative
Nutritional evaluation requires assumptions to be limitations must be considered. In particular, the
made which may or may not always be valid. A major European Union (EU) regulation on the use of animal
issue arises for feather protein when it is assumed by-products (OJEC, 2002) is a limitation to utilization
that in vitro and in vivo measurements of nutritional both in Europe and in countries that export to Europe.
value are positively related. This is not the case for Woodgate (2004b) describes the practical implications
feather protein digestibility and therefore only in vivo of this regulation.
measurements are valid (Woodgate, 2004a). In
140
Standard Process
(30 min. @ 3 Bar 135°C)
Enzyme (Allzyme™ FD)
120
Standard
100
Enzyme Process
°C
(20 min. @ 2 Bar 122°C)

80
60
Enzyme Process
(30 min. @ 50°C)
40
20
00:00 01:00 02:00 03:00 04:00 05:00
Time (hours)
Figure 2. Comparison of standard and enzyme-hydrolysed feather protein processes.
Table 1. Typical analysis of enzyme- and steam-hydrolysed feather Table 2. Digestibility data for enzyme- and steam-hydrolysed
proteins and fish meal1. feather protein (HFP) sources compared to low temperature fish
meal in rainbow trout.
Steam- Enzyme- Fish
hydrolysed hydrolysed2 meal Crude Digestible Digestible Indigestible
protein (%) protein protein protein
Crude protein, % 85 85 65 (in vivo) (%) (%)
True ME, MJ/kg 13.5 15.5 12.5 (% of protein)
Crude fat, % 5.0 6.0 8.0
Calcium, % 0.65 0.65 5.5 Fish meal 66 91.6 60.5 5.5
Phosphorus, % 0.35 0.35 2.8 Enzyme HFP 85 88.9 75.6 9.4
Steam HFP 85 73.0 62.1 22.9
Digestible amino acids
Lysine, % 1.2* 1.5* 4.5
Met + Cys, % 2.8* 4.5* 2.5
Table 3. Amino acid digestibility (%) for enzyme- and steam-
Threonine, % 2.5* 3.2* 2.2
hydrolysed feather protein (HFP) sources compared to low
Arginine, % 3.0* 3.8* 3.6
temperature fish meal in rainbow trout.
1
In vivo determination in poultry of feather protein sources (not
Amino acid Steam HFP Enzyme HFP Fish meal
fish).
2
Allzyme™ FD.
Histidine 91.63 84.62 91.43
*Derived from in vivo data.
Arginine 76.55 87.71 95.32
Threonine 52.20 73.62 92.12
Response to feather protein meal in aquaculture Valine 55.61 71.51 93.23
Methionine 98.94 88.15 92.88
Hydrolysed feather protein (HFP) in aquaculture feed Lysine 96.37 91.96 95.68
meets both the requirements of the EU regulation Isoleucine 70.12 79.07 92.76
and potentially offers a supply of highly digestible Leucine 63.75 79.98 94.18
Phenylalanine 60.05 84.26 92.60
amino acids to a farmed species which has a
particularly high demand for them. Preliminary
digestibility studies were completed in rainbow trout The digestibility data obtained in this trial were then
(Serwata et al., 2004) and a comparison of enzyme- used to formulate a series of diets for rainbow trout
hydrolysed HFP with steam-hydrolysed HFP, together that were isonutritive in terms of crude protein,
with a reference protein (low temperature fish meal, digestible amino acids and energy. The growth trial
LTFM). Table 2 indicates the in vivo digestibility of was completed over 10 weeks, with the increase in
protein and Table 3 shows the in vivo digestibility live weight over the period shown in Figure 3. There
values for essential amino acids for all three protein were no significant differences in growth between
sources.
S.L. Woodgate 455
200 Control Enzyme-hydrolysed Steam-hydrolysed
150
Liveweight (g)
100
50
0
0 2 4 6 8 10 12
Weeks
Figure 3. Effect of feather meal processing method on liveweight gain of rainbow trout.
the treatments, thus confirming the in vivo nutritional Table 5. Summary of feeding Trial 1: Tilapia (O. niloticus).
value determinations, and most importantly indicating
Diet (inclusion) Enzyme HFP Steam HFP
that successful formulation based upon digestible
amino acids is both practical and feasible. This allows Fish meal (low temp), g/kg 146 146
more precision in formulation and lowers the waste Enzyme HFP, g/kg 243 -
protein in the diet that could be a potential source of Steam HFP, g/kg - 243
Protein in diet, % 27 27
pollution. Table 4 shows a comparison of the supply
of indigestible protein (from digestibility data) which, Performance
Start weight, g 5.46 5.56
when supplying equal amounts of digestible protein, Final weight, g 22.13 16.36
reveals a significant difference between the steam- Weight gain, % 305 194
hydrolysed and enzyme-hydrolysed feather protein FCR 1.84 2.17
meal. These differences are important, as minimising PER 1.49 1.11
the potential for nitrogen pollution is now critical
for the aquaculture industry. Table 6. Summary of feeding Trial 2: Red tilapia (O. niloticus x O.
mossambicus).
Table 4. Feed trial formulations including substitution of fish meal
Diet (inclusion) Enzyme HFP Steam HFP
protein with feather protein and the indigestible protein content of
the diet (kg/mT). Fish meal (low temp), g/kg 128 128
Enzyme HFP, g/kg 224 -
Ingredient Fish meal HFP Digestible Indigestible Steam HFP, g/kg - 224
protein protein Protein in diet, % 33 33
Performance
Fish meal 600 — 363 33
Start weight, g 2.03 1.91
+ Enzyme HFP 200 320 363 41
Final weight, g 6.03 5.18
+ Steam HFP 200 390 363 100
Weight gain, % 190 170
FCR 2.17 2.20
PER 1.30 1.33
Feeding trials on two tilapia species have been
completed at the University of Plymouth in the UK
(Davies and Fasakin, 2004), and preliminary data compared with the conventional steam-hydrolysed
are shown in Tables 5 and 6. In both of these species, feather protein. Increased weight gain and improved
a positive response to the inclusion of enzyme- FCR was seen with the diet including the enzyme-
hydrolysed feather protein was observed when prepared product; and this improvement is currently
being converted into economic value.
Further research is ongoing in a range of other References

freshwater and marine species, including sea bass,
sea bream and turbot. Plans are to extend studies to Bureau, D.P and M.T.Viana. 2003. Formulating cost
shrimp and eel in the future. effective and environmentally friendly feeds based
on improved knowledge of nutrient requirements
and utilisation. Intl. Aquafeed 6(3):20-21.
Conclusions Davies, S.J. and E.A. Fasakin. 2004. The use of
animal by-products in Tilapia diets. In press.
The advent of practical enzyme technology for the Official Journal of the European Communities
upgrading of key raw materials for farm animal (OJEC). 2000. Council Decision (2000/766/EC)
feeding has given great opportunity for new or concerning certain protection measures with regard
advanced products to be introduced into aquaculture to the feeding of animal protein. L306/32.
feeds. The benefits of the enzyme technology as
Official Journal of the European Communities
applied to the processing of feathers has given rise to
(OJEC). 2002. Regulation (EC) No. 1774/2002 of
a sustainable technology that gives economic benefits
the European Parliament and of the Council, laying
to both process and product alike. The introduction
of formulation techniques that use digestible amino down health rules concerning animal by-products
acid data has been beneficial for the enzyme processed not intended for human consumption. L273/1.
product, which contains significantly higher levels Serwata R.D., S.J. Davies and K Jauncey. 2004.
of most digestible amino acids as measured in vivo. Partial replacement of fish meal based on digestible
This nutritional enhancement can be directly protein and energy coefficients for rendered animal
translated to economic benefit, which can be used in protein sources for rainbow trout (O. mykiss).
either the poultry or aquaculture sectors. World Aquaculture Symposium, March 1-5,
Using the techniques described above, many more Honolulu, Hawaii.
animal or vegetable raw materials should be evaluated Woodgate, S.L. 2004a. The relationship between in
for potential upgrading with enzyme technology. vivo and in vitro nutritional evaluation methods for
These materials could include alfalfa, shrimp shells/ hydrolysed feather proteins. In press.
heads, fish heads, distillers dried grains, and sugar Woodgate, S.L. 2004b. Update: New European Union
beet pulp. A programme to evaluate some of these regulations on animal by-products. Intl. Aquafeed
feed materials is currently underway at Alltech 7(1):29-32.
Bioscience Centres.
R.P. Subasinghe 457
From farm to fork: the challenges that fish farming faces as a responsible
supplier of global food
ROHANA P. SUBASINGHE
Fisheries Department, Food and Agriculture Organization of the United Nations, Rome, Italy
“World production, total consumption, food demand use of by-catch for human consumption, e.g., use of
and per capita food consumption will increase over at least part of the catch going for reduction to fish
the next three decades; however, the rate of these meal and fish oils. Better management of fishery
increases will slow over time. World capture resources and enhanced efforts to protect fishery
production is projected to stagnate, while world resources from accelerating environmental
aquaculture production is projected to increase, albeit degradation may well contribute to sustained, if not
at a slower rate than in the past. enhanced, fish supplies in the medium- to long-term.
Aquaculture appears to have stronger potential to
With a view to predicting future fisheries and fish meet the increasing demands for aquatic products in
production, Food and Agriculture Organization of most regions of the world. Potential contributions
the United Nations (FAO) commissioned several from aquaculture to local food security and
studies. Based on economic models of demand, trade livelihoods can be highly significant, especially in
and the supply of fish in main markets, these studies many remote and resource-poor rural areas. The
were helpful in providing an analysis of plausible challenge is to develop approaches to increase the
trends in production, consumption and trade. The contribution of aquaculture, which are realistic and
above statements of trends in production and achievable, within the context of current social,
consumption of aquatic food for the period up to economic, environmental and political circumstances.
2030 emerged from the said analyses. It was also Such approaches should not focus only on increasing
suggested that in developed countries consumption production; they should also focus on producing a
patterns will reflect demand for, and imports of, high- product that is affordable, acceptable and accessible
cost/high-value species, whilst in the developing to all sectors of society.
countries, trade flows will reflect the exportation of
high-cost/high-value species and the importation of
low-cost/low-value species. Let’s look at the Global aquaculture production and
aquaculture sector, which is the fastest growing food
trends
producing sector in the world, its contribution, future
potential, opportunities and challenges as a Aquaculture is the fastest growing food-producing
responsible supplier of global food. sector in the world. A great proportion (over 90%)
of this production comes from the developing world.
In contrast to terrestrial farming systems, where the
Introduction bulk of global production is based on a limited
number of animal and plant species, the aquaculture
The world population is on the increase, as is the sector comprises of over 200 different species. This
demand for aquatic food products. Production from large number of species cultivated reflects the diversity
capture fisheries at a global level is levelling off and of the sector, particularly the wide variety of candidate
most of the main fishing areas have reached their species cultivated and different production systems used.
maximum potential. Global fish supply could be Currently all major aquaculture producing countries are
increased through reduction of discards and better in Asia. Six Asian countries (China, India, Indonesia,
458 From farm to fork: the challenges that fish farming faces
Japan, Bangladesh and Thailand) contributed 83.3% Congo, Cambodia, Sri Lanka, Philippines). The
to the global production in the year 2002. International Conference on Sustainable Contribution
The main species groups reared in fresh water are of Fisheries to Food Security, held in Kyoto in 1995,
finfish while high value crustaceans and finfish recognized that aquatic products contribute
predominate in brackish water, as molluscs and aquatic meaningfully to the maintenance of good nutrition.
plants do in marine waters. Of these three Fish are important sources for many nutrients,
environments, freshwater aquaculture could be including protein of very high quality, retinol
considered as the most important in terms of (Vitamin A), vitamin D, vitamin E, iodine, selenium.
contributing to achieving food security. Marine Evidence is increasing that the consumption of fish
aquaculture, particularly of sea weeds and molluscs, enhances brain development and learning in children,
also contributes to food security and poverty protects vision and eye health, and offers protection
alleviation, as most of its products are produced from cardiovascular disease and some cancers. The
within small to medium scale operations. Albeit fats and fatty acids in fish, particularly the long chain
brackish water shrimp culture is generally aimed at n-3 polyunsaturated fatty acids (n-3 PUFA), are
producing a high value export commodity, coastal highly beneficial and difficult to obtain from other
shrimp culture also plays an important role in rural food sources.
livelihoods and food security. Increasing the worldwide availability of good
quality animal products is necessary if the per capita
supplies are to keep pace with the increase in demand.
Fish, human health and nutrition Aquaculture has an important role to play in this effort
to fulfil the demand for animal products. Despite a
Fish is an important part of the diet for a large lack of quantitative information concerning the role
proportion of the people living in the developing of rural aquaculture in achieving food security, there
world. Many types and forms of fish and aquatic are undoubtedly benefits related to fish consumption;
products are available at affordable prices in a) fish have a highly desirable nutrient profile, b)
developing countries. Economic affordability is a key fish is an excellent source of high quality animal
factor as to why aquaculture is making an essential protein and with variable amounts of dietary energy,
contribution to human health in the developing world. c) fatty fish, in particular, are an extremely rich source
More ‘food fish’ is consumed globally on a per capita of omega-3 PUFAs, fat soluble vitamins (A, D and
basis than any other type of meat or animal protein. E) and water soluble vitamins (B complex), and d)
In terms of animal protein supply, food fish fish is an important source of minerals (calcium,
represented 16.5% of total supply in 1997 (total global phosphorus, iron, iodine and selenium).
animal protein supply was reported as 27.1 g daily
per capita in 1997), followed by pig meat (14.7%),
beef and veal (13.6%), and poultry meat (12.5%). It Fish, food security and rural
is interesting to note that farmed aquatic meat development
production in China currently ranks second to pig
meat; per capita availability of food fish in China Hunger and malnutrition remain amongst the most
increasing from 6.3 kg in 1984 to 25.5 kg in 1998. devastating problems facing the world’s poor. A
The main reason for the high demand for staple considerable portion of the global population is
food fish within most developing countries is their currently suffering from one or more forms of
greater affordability to the poorer segments of the nutrient deficiency. This remains a continuing travesty
community. At present food fish represents the of the recognized fundamental human right to
primary source of animal protein (contributing more adequate food and nutrition, and freedom from
than 25% of the total animal protein supply) for about hunger and malnutrition, particularly in a world that
1 billion people within 58 countries worldwide, has both the resources and knowledge to end this
including many developing countries and LIFDCs catastrophe.
(value excludes China). Latest estimates from FAO’s Report on the State
In the diets of many countries, fish contributes more of Food Insecurity 2002 indicate that 799 million
than or close to 50% of total animal proteins (e.g. people in the developing countries (840 million
Gambia, Ghana, Equatorial Guinea, Indonesia, Sierra worldwide) regularly go to bed hungry. This is only
Leone, Togo, Guinea, Bangladesh, the Republic of about 20 million people less than the benchmark figure
R.P. Subasinghe 459
set eight years ago by the World Food Summit and is the number of people active in aquaculture over the
far short of the pace needed to reduce the number of last decade have shown that aquaculture production
hungry people by half by 2015. Of the 11 million systems ranging from extensive to highly intensive
children under the age of five who die each year, can be economically feasible.
more than half (6 million) die of malnutrition and
hunger-related causes. The challenge is to rapidly
accelerate the pace by which hunger and malnutrition Aquaculture and foreign exchange
are eliminated. This goal can be accomplished by
improving access to food by the poorest and International trade in seafood is a multi-billion dollar
nutritionally most vulnerable population groups and sector, with global volumes expanding from around
individuals. This needs to be done in a manner that $7 billion in 1976, to $55.3 billion in 2000.
is socially acceptable and environmentally Developing countries dominate seafood exports,
sustainable. This requires a dual approach whereby contributing over 50% to the internationally traded
increased production and productivity gains are seafood, with developed countries accounting for 80%
combined with improvements in the use and or more of the imports, and Asia supplying 50% of
management of natural resources while also ensuring all seafood exports. Asia produced 80% of the world’s
a more equitable access to food and other resources farmed shrimp in 2000. Trade in live food fish,
by the poor. especially reef species, an increasing volume of which
The objective of rural development is to facilitate is from farms, is concentrated in Asia and is virtually
a sustainable rural economy and to secure a one-way flow from Oceania, Southeast Asia and
improvements in the welfare of rural populations. South Asia to mainly China. The foreign exchange
The opportunities for the integration of fish farming earned through international trading of aquaculture
into rural development are characterized by diverse products is a considerable contribution to many
aquatic resources and a wide range of stakeholders national economies.
with diverse livelihoods. Objectives may further range
from food production, income generation, and wild Major issues and challenges
stock enhancement to recreation (ornamental fish or
sport). The scale may be intensive commercial or Aquaculture is an income generating activity.
subsistence management within developed and less- However, rapid sector growth has, in some instances,
developed economies. outstripped planning and regulatory activities. As a
The aquaculture sector provides worldwide result, many areas have seen a regulatory rebound,
employment to millions of people. Total employment with disproportionate requirements as resource use
in the aquaculture sector is highest in China where conflicts have occurred, resource scarcities have
almost 4 million people are employed full-time in become more constraining and demand for product
aquaculture production, and the annual growth rate quality and safety has increased significantly.
for aquaculture employment rate is reported as 6%. Increasingly, some markets will consider additional
In many countries the average market prices of fish product attributes, like environmental and social
are lower than those of other animal products such impacts of production. In some regions, aquaculture
as chicken, pork and red meat. Especially in Asia the faces a considerable problem with public perception.
low prices of aquaculture commodities such as carps In some cases, aquaculture development has failed
and tilapias make fish highly accessible to even the to keep up with, or meet, many environmental and
poorest segments of the population. socio-economic issues and expectations. Future
Studies have shown that practicing aquaculture is aquaculture development needs to produce a product
economically feasible under many different which is not only acceptable to public and consumers
circumstances. Many types of low-cost, low-risk, in terms of price, quality, and safety, but also in terms
simple, aquaculture technologies have emerged in of environmental cost.
recent years. Comparative studies between rice, rice- Although it has been said that aquaculture has a
fish and fish farming systems in sub-Saharan Africa significant potential for improving food security and
demonstrated that farmers investing in aquaculture alleviating poverty, the role of aquaculture in food
increased their household incomes considerably with security has been a major concern of the sector for
only minor investments. In Europe, USA, China and many years. From the point of view of production,
other Asian countries the increases in production and it has been in the increase for many years, although
at a reducing rate. Overall, the driving force behind efficiency in feed formulation, c) improving food
the relative increase in production and decline in value conversion ratio thus increasing net energy
appears to be declining prices for luxury and conversion, d) containment and recycling of wastes
commodity products as markets are becoming in cages and flow-through systems, e) increased land
saturated and competition is increasing. and water use efficiency, f) improvement to health
management and reduction of chemical and
veterinary drug use, and g) domestication and genetic
Maintaining environmental sustainability improvement towards reducing negative impacts on
aquatic biodiversity.
Certain forms of aquaculture have a bad reputation. While production is increased, the global
The argument mainly comes from the use of feed and population is also on the increase and the land and
seed resources, disease control and chemical and water use for aquaculture production is also
veterinary drug use, accumulation of environmental increasing. Aquaculture production is increasing, not
contaminants, escapees and point source contamination necessarily due to intensification but mainly due to
of wild resources, negative or low net energy conversion expansion. The global increase in production, at an
during farming of top carnivores, mangrove clearance average rate of 9%, is mainly due to the increase in
and land degradation, etc. Some of the arguments are production rate in China; and if the trend is
true and worthy of considering but the quantum to considered without China the rate is much less. Use
which the issues are highlighted is certainly biased. of resources such as fish and fish products (fish meal)
Traditional or extensive aquaculture, which is a low- for producing fish is being questioned. The net energy
tech, low-input aquaculture practice invented by the conversion in certain forms of aquaculture appears
Chinese some 3000 years ago, is still in practice, to have negative ecological impacts thus requiring
producing large volumes of fish feeding low in the urgent rectification. Reduction in fish meal in fish
food chain. Over 80% of the fish produced by feed requires more attention and research.
aquaculture are herbivorous or omnivorous, mostly
produced in low-intensity systems for local
consumption. They, undoubtedly support livelihoods Keeping up with safety and quality
of people, provide food, alleviate rural poverty and
improve health among less fortunate communities. As mentioned earlier, international trade in seafood
However modern-day aquaculture, mainly the is a multi-billion dollar sector. Developing countries
production of high value carnivorous fish or shellfish dominate seafood exports, contributing over 50%
destined to import markets is a different kettle of fish to the internationally traded seafood, with developed
altogether. This sector uses a considerable quantity of countries accounting for 80% or more of the imports.
natural resources and also produces a considerable Asian developing countries top seafood production
quantity of effluent and waste. The sector’s statistics. As the current production through capture
sustainability and environmental acceptability has been fisheries is somewhat static and in order to respond
increased significantly over the past decade through to the increasing demand for seafood, aquaculture is
research involving development of technically now contributing significantly to global seafood trade
specialised conditions. and the share is increasing. While the quantity of
However, the environmental, social and economic trade is increasing, the consumer demand for safer
landscape within which aquaculture has performed seafood is also increasing and as a result the
well up to now is changing. In particular, competition international trading environment is changing, with
will increase as barriers to trade decline through the food safety issues in particular receiving considerable
process of economic globalization. In addition, the attention.
negative environmental and social impacts of Traditionally, food safety in seafood has been
aquaculture that occur in some situations will increase concerned with post-harvest handling and processing.
public scrutiny and criticism that could well alter the Now, with importing countries and consumers
policies that have so far fostered growth. The trend concerned about residues, attention has shifted
has been to improve the environmental acceptability toward the way seafood is produced. This requires
or sustainability of the sector through several aquaculturists to work together with food safety
interventions and developments such as; a) reduced experts to develop systems for farming aquatic
reliance on fishmeal in fish feed, b) increased animals that assures food safety, (particularly the
R.P. Subasinghe 461
species which are destined to international trade), oriented solutions to the problems faced by the small-
based on internationally accepted, science-based scale sector, allowing the sector to benefit from the
quality control mechanisms, such as risk assessment development opportunities offered through trade,
and HACCP and Good Hygienic Practice (GHP). while reducing exposure to the associated risks.
Food safety concerns are leading to new demands Asian aquaculture systems have many traditional
for traceability of aquaculture products. This will and diverse advantages in safe, healthy and sustainable
not be easy with the large number of small-scale farmers seafood production, such as some ecologically sound
engaged in production, and the fragmented supply chains integrated farming systems. Collaborative research
operating in many countries. Substantial institutional and development should be used to encourage both
reorganization, legal and policy development, awareness the traditions and innovations in aquaculture farming
raising and capacity building efforts will be essential that can give the region comparative advantage in
among the diverse stakeholders in public and business this new trading environment.
sectors to make this work.
International food safety standards are being set
with minimum inputs from the region, in particularly Combating disease and managing health
from the producing sector, for various reasons. Asia
needs to enhance and organize better its inputs to Disease has become a primary constraint to sustainable
international food safety standard setting bodies such aquaculture production and product trade. A multitude
as Codex Alimentarius, given the importance of such of factors has contributed to the health problems
standards for future trade in aquaculture products currently faced by aquaculture. As note above, over
from the region. With most countries in Asia giving the years aquaculture has expanded, intensified, and
increased attention to food safety, there is a growing diversified, based heavily on movements of live
proliferation of product certification systems, “good aquatic animals and animal products (broodstock,
aquaculture practice” guidelines, Codes of Conduct, seed, and feed). This trend has been triggered by
and other mechanisms/schemes intended to provide changing circumstances and perspectives, especially
a basis for safe and sustainable seafood production. world trade liberalisation.
Without some harmonization among regional New outlooks and directions have accelerated the
countries, this proliferation of certification schemes accidental spread and incursion of diseases into new
has potential to confuse consumers, importing populations and geographic regions, for example,
countries, lead to increased costs, and potentially through movements of hatchery produced stocks, new
constrain trade. species for culture, enhancement and development
Asian domestic and intra-regional trade in of the ornamental fish trade. The impacts of trans-
aquaculture products, services and inputs such as feed boundary aquatic animal diseases on international
and chemicals, is growing, in line with increasing trade, as well as socioeconomic and biodiversity
free-trade agreements between countries. This opens implications are considerable. Different measures are
new opportunities for trade and development, perhaps being used to deal with diseases of fish and shellfish,
helping to avoid some of the complex procedures of such as; international codes, regionally oriented
other importing regions, but also poses challenges. guidelines, national programs and legislation,
This further emphasizes the need for harmonization technology for diagnostics, therapy and information
of food safety assurance procedures among trading communication.
partners in Asia. Such cooperation may also avoid The aquatic animal health management programs
problems of residues being transferred from one carried out in different parts of the globe are different
country to another. with respect to efficacy of disease prophylaxis/control
Applying new food safety standards and traceability and pathogen detection/disease diagnostics, inherent
poses special organizational difficulties for the large problems with national legislation and international/
community of small-scale farmers in the region, and regional codes, and the effectiveness of programs on
as a consequence, some of the poorest farmers might education, training and extension services. Health
be at risk due to difficulties in participating in such management problems which pose risks to rural
schemes. There is a need therefore to better small-scale aquaculture require special consideration.
understand the implications of new food safety There is a need for effective communication at all
standards and international trading standards for levels of the production systems. There are roles to
small-scale farmers, and develop suitable market- play by the state, private sector (e.g. aquaculturists,
industry associations, cooperatives, etc.) professional international trade obligations and requirements,
societies, diagnosticians and researchers, education, environmental concerns, etc. More emphasis on
training, and other related extension services. investment, research, information, and public
The current trend to meet the demand for more aquatic education are needed. Challenges for increasing
food, through expansion, intensification, and aquaculture’s contribution to food security, poverty
diversification, will continue to provoke the emergence alleviation and rural livelihoods will have to be met.
and recurrence of disease challenges. How industry, Aquaculture development, especially if it is to be
government and other stakeholders rise to meet these sustainable for food security goals, may need to be
challenges will dictate how aquaculture survives and stimulated, at least in the beginning, so there should
achieves true sustainability. The options are not always be a key point on increasing access to credit for
easy. The varying levels of political, economic and farmers, producers and local marketing. It is
social development among countries, the trans- important to understand the investment opportunities
boundary nature and commonality of many major in the sector.
disease problems, and the need to harmonise In an era of globalisation, it is imperative to
approaches, all complicate effective cooperation and emphasise national and international trends of trade.
consultation. The current situation offers a big Trade of aquaculture produce, input supplies, capital
challenge and an opportunity to all concerned but, if and information are all important to acknowledge.
maintained at the present level, major epidemics will Aquaculture is dependent on key natural resources
continue to threaten, break out and impact the such as water, land, seed, and nutrients. There is
ultimate goal of aquaculture sustainability. strong pressure for production and marketing systems
that are more efficient and more effective in terms
of resource utilisation. In this respect, we should
Conclusions invest in research on developing production and
marketing systems with better resource utilisation and
Globalization, stringent food safety standards, more efficient performance.
realization of environmental and social responsibility During production, there should be emphasis on
of aquaculture production has begun to change the targeting the consumers. We must emphasise the
way aquaculture development and management has difference between mass production and production
been pursued, in particular the practices which for the masses. For example, trends of formerly
brought global controversy. The ‘aquaculture expensive produce such as salmon and shrimp are
industry’ will benefit from this and noticeable increasingly becoming affordable by larger segments
changes will take place in countries where high value of the population. We should compete with and
commodities are farmed, especially for foreign complement other food producing sectors and
markets. providers. Aquaculture produce should be acceptable
States will respond to improving legislation and to all sectors of the society. Tremendous gains will
regulatory frameworks for better practices, farmers be possible through improved biotechnology, genetic
themselves will get together to police and regulate modification, improved nutrition, probiotics, disease
their practices, in the face of less state regulation. diagnosis and treatment. However, the problem of
Markets and profits will continue to drive the consumer resistance to perceived risks stemming from
international trade and as in agriculture sector, larger ‘unnatural’ products, ethical problems and fear of
and more vertically integrated systems will increase unknown technologies will affect potential gain.
their share of the international market. Maybe small As mentioned, environmental and human health
numbers of large producers will eventually produce issues will slow development or reduce market access.
huge quantities of a few species destined for foreign Strategic solutions are required. We should emphasise
markets. Smaller farms, to make profits, will have biosafety issues, development and promotion of
to look for specialised products. However, as global biotechnology that conserve the environment; we
economy increases, the viability of these small-scale, should promote policies that support ethical issues
profit-oriented farms will increase and will benefit of welfare and autonomy and emphasise labelling
from parallel development of the corporate sector, and transparency for production process and
thus ensuring market synergy and co-existence. beneficiaries. There is a need to increase the impact
Aquaculture will continue to grow but must address of research to understand technical and other
the costs of production, quality and safety of products,
R.P. Subasinghe 463
constraints and to enhance the applicability and use Conference on Aquaculture in the Third Millennium
of research results in the development of strategies (R.P. Subasinghe, P. Bueno, M.J. Phillips, C.
to overcome these challenges. Hough, and S.E. McGladdery, eds). Bangkok,
Thailand. 20-25 February 2000. NACA, Bangkok
and FAO, Rome, Italy, p. 471.
References New, M. 1999. Global aquaculture: current trends
and challenges for the 21 st century. World
FAOSTAT Agriculture Data. Food Balance Sheets, 1 Aquaculture 30(1):8-79.
June 2000. Pedini, M. 2000. Bridging the gap: can aquaculture
FAO. 1995. Code of conduct for responsible fisheries. meet the additional demand for fishery products?
Food and Agriculture Organization of the United FAO Fisheries and Aquaculture Newsletter 24:4-9.
Nations, Rome, Italy. Subasinghe, R.P., M.G. Bondad-Reantaso and S.E.
FAO. 1995. Aquaculture development trends: McGladdery. 2001. Aquaculture development,
perspectives for food security. Background health and wealth. In: Aquaculture in the Third
document prepared by FAO for the International Millennium (R.P. Subasinghe, P. Bueno, M.J.
Conference on Sustainable Contribution of Fisheries Phillips, C. Hough and S.E. McGladdery, eds).
to Food Security, Kyoto, Japan, 1995.FAO. 2002. Technical Proceedings of the Conference on
The State of World Aquaculture 2002. Rome, Italy, Aquaculture in the Third Millennium, Bangkok,
p. 150. Thailand, 20-25 February 2000, pp. 167-191.
FAO. 2003. Review of the State of World NACA, Bangkok and FAO, Rome, Italy.
Aquaculture. FAO Fisheries Circular 886(Rev. 2). Tacon, A.G.J. 2001. Increasing the contribution of
Rome, Italy, p. 95. aquaculture for food security and poverty
FAO. 2003. The role of aquaculture in improving alleviation. In: Aquaculture in the Third Millennium
food security and nutrition. Committee on World (R.P. Subasinghe, P. Bueno, M.J. Phillips, C. Hough
Food Security. 29 th Session. 12-16 May 2003. and S.E. McGladdery, eds). Technical Proceedings
Rome, Italy. of the Conference on Aquaculture in the Third
Fish to 2020: supply and demand in changing global Millennium, Bangkok, Thailand, 20-25 February
markets (C.L. Delgado, N. Wade, M.W. Rosegrant, 2000, pp. 63-72. NACA, Bangkok and FAO, Rome,
S. Meijer and M. Akhmed, eds). International Food Italy.
Policy Research Institute, Washington D.C. and The promise of a blue revolution. The Economist,
WorldFish Centre, Penang. August 7, 2003.
Hishamunda, N. and R. Subasinghe. 2003. Ye, Y. 1999. Historical consumption and future
Aquaculture development in China: the role of demand for fish and fishery products: exploratory
public sector policies. FAO Fisheries Technical calculations for the years 2015/2030. FAO Fisheries
Paper. No. 427. Rome, Italy, p. 64. Circular, No. 946. Rome, Italy, FAO, p. 31.
NACA/FAO. 2001. Aquaculture in the third
millennium. In: Technical Proceedings of the
Companion
PETS
animals
G. Aldrich 467
USA poultry meal: quality issues and concerns in pet foods

GREG ALDRICH
Pet Food & Ingredient Technology, Inc., Topeka Kansas, USA
Introduction
Poultry protein meal represents a substantial portion eggs, and intestines, exclusive of feathers, except in
of the high quality protein and fat in modern such amounts as might occur unavoidably in good
companion animal diets. Poultry protein meal is processing practices. The label shall include
commonly included at 5 to 40% and can contribute guarantees for minimum crude protein, minimum
in excess of 85% of the dietary protein and 30% of crude fat, maximum crude fiber, minimum
the dietary fat. Thus, changes to the quality or phosphorous (P), and minimum and maximum
composition of the protein or fat in poultry protein calcium (Ca). The calcium (Ca) level shall not exceed
meal can have profound effects on the nutritional the actual level of phosphorous (P) by more than 2.2
value of the diet. The process of producing poultry times. If the product bears a name descriptive of its
protein meal is a daunting task undertaken to stabilize kind, the name must correspond thereto.
the raw material from microbial deterioration.
Unfortunately, during this process protein and fat 9.71 Poultry Meal is the dry rendered product from
quality are affected. a combination of clean flesh and skin with or without
accompanying bone, derived from the parts of whole
carcasses of poultry or a combination thereof,
Defining poultry meal exclusive of feathers, heads, feet, and entrails. It shall
be suitable for use in animal food. If it bears a name
The availability of fresh poultry and rendered poultry descriptive of its kind, it must correspond thereto.
products coincided with the commercialization and
industrialization of poultry production in the 1940s In short, poultry by-product meal (PBPM) differs
and 1950s; and feed values for poultry by-product from poultry meal (PM) only by the inclusion of
meal were first established in the 1950s (Fuller, heads, feet, and entrails. However, no quality
1996). The volume of rendered poultry proteins in considerations are described in the definitions. A
2003 was estimated at 3,073.5 million lbs; and on proposal in 1998 to drop the ‘by-product’ designation
average the companion animal industry consumes from poultry products and replace it with the term
about 23% (Pearl, 2003). ‘protein’ was rejected by AAFCO. The newest
The collective term ‘poultry protein meal’ covers proposal requests that the ‘by-’ be dropped from by-
both poultry by-product meal and poultry meal from product. Proponents of this request are asking for a
chicken, turkey, or other poultry origin. The official level playing field with meat meal, fish meal, and
definitions according to the Association of American lamb meal; while opponents claim that this would be
Feed Control Officials (AAFCO, 2004) are: misleading to the consumer. At issue is the name on
a label and how it is represented. However, there is
9.10 Poultry By-Product Meal consists of the little evidence in the literature to support differing
ground, rendered, clean parts of the carcass of names. In a comparison of chicken meal (CM) and
slaughtered poultry, such as necks, feet, undeveloped chicken by-product meal (CBPM) using a chick
468 USA poultry meal: quality issues and concerns in pet foods
protein efficiency ratio (PER) model, no difference edible stream. The rendering plant accepts and
was observed in protein quality (Aldrich and processes all raw materials received from the animal
Daristotle, 1998). Bednar et al. (2000) fed dogs diets processing plant and must ‘render’ them stable so as
containing either PBPM or PM and reported that ileal to avoid public health problems. The process
digestibility of protein and amino acids was not transforms raw unused poultry parts into a form that
different between treatments; however, total tract can be easily stored and transported. Rendering, in
protein digestibility was 6.74% lower for dogs its simplest description, is a sterilization, dehydration,
receiving PBPM than for dogs receiving PM. From and resizing process (Miller, 1996). In the US,
the perspective of chemical composition, there is a standard rendering is a ‘high temperature’ process.
fair amount of overlap between PM and PBPM This involves extensive heating (approximately
(Table 1). 280°F), which drives water and fat from the bone
Several different grades of rendered poultry and tissue. The fat is removed by pressing and the
products are available. Feed grade is seldom used in remaining ‘cake’ is ground in a hammer mill to a
pet food because it contains a higher level of ash and uniform particle size. The fat goes to storage vessels
lower protein content. Standard pet food grade where it can be further processed or sold. Likewise,
contains less than 14% ash; and low ash poultry meal the ground meal is conveyed to storage silos for
and (or) poultry by-product meal contain less than cooling and eventual sale, or further processed in an
11% ash. The latter is available in limited quantities effort to ‘improve’ its chemical composition.
at a premium price and typically reserved for low
ash cat formulas (Miller, 1996).
Protein quality
Rendering Fresh meats would be a preferred material with which

to construct petfood diets, but this is not always
‘Rendering’ is required to stabilize the mass of poultry practical for several reasons: 1) expense associated
co-products that have been removed from the human with freezing and chilling, 2) expense involved with
Table 1. Nutrient composition (dry matter basis) of poultry meal (PM) and (or) poultry by-product meal (PBPM) and low ash (LA) PBPM or
PM.
PBPM PBPM PBPM-LA PM-LA PM
Murray Johnson Johnson Clapper Yamka
(1997) (1998) (1998) (2001) (2003)
Protein 67.6 64.6 68.8 74.5 69.3
Ash 13.9 17.0 7.5 9.6 9.0
Fat 11.6 12.5 19.8 15.0 11.6
Essential amino acids
Arg 4.4 4.6 4.8 4.9 4.4
His 1.6 1.2 1.5 1.8 1.7
Ile 2.6 2.3 2.9 2.8 2.3
Leu 4.6 4.4 5.4 5.2 4.8
Lys 3.8 3.6 4.2 4.7 3.1
Met 1.1 1.2 1.4 0.8 1.1
Phe 2.5 2.6 3.0 2.8 2.7
Thr 2.5 2.6 3.1 2.8 3.0
Trp NR NR NR NR 0.5
Val 3.1 2.9 3.6 3.3 3.2
Nonessential amino acids
Ala 4.1 4.5 4.5 4.4 4.3
Asp 5.2 5.8 6.3 6.3 5.6
Cys 1.8 0.9 1.0 1.0 1.3
Glu 8.5 8.7 9.8 9.7 8.6
Gly 5.7 7.3 5.6 5.5 6.5
Pro 5.3 5.0 4.4 4.4 NR
Ser 2.7 3.1 3.4 3.2 4.4
Tyr 1.8 1.7 2.3 2.1 1.8
NR: values not reported
G. Aldrich 469
transportation of high amounts of moisture, 3) most in PBPM (16.3% vs. 7.2%) did not affect ileal
extrusion processes will not handle more than 25% digestibility of protein or amino acids or total tract
fresh meat in a formula, 4) fresh meat reduces protein digestibility in dogs, amino acid digestibility
production efficiency, and 5) fresh meat diets can be in cecectomized roosters (Johnson et al., 1998), or
more difficult to stabilize. Therefore, the use of dry protein quality (PER) in chicks (Johnson and Parsons,
meals with concentrated protein is often necessary. 1997). Feeding dogs increasing amounts of low ash
Achieving this dry meal requires rendering; PM (10.4% to 32.5% of the diet) did not affect
however, the rendering process can have a substantial protein or amino acid digestibility at the ileum
impact on nutritional quality. Murray et al. (1997) (Yamka et al., 2003a). Thus, increasing consumption
reported that protein and total amino acid digestibility of ash from poultry sources does not negatively affect
at the ileum in dogs fed a diet containing rendered nutrient quality. If not ash, then it is likely that the
poultry by-product (meal) was reduced by greater lower protein quality of bone/cartilage and feet is
than 10% when compared to a diet containing fresh associated with high levels of connective tissue and a
poultry by-product. However, no differences in total reduction in the ratio of essential to non-essential
tract protein digestibility were detected. Energy and amino acids. Bone/cartilage can be a component of
amino acid digestibility of animal by-product meals either PM or PBPM, and feet a component of PBPM;
can be negatively affected by different rendering regardless, adding these components to the raw
processes, high rendering temperatures, extended material mix would likely reduce the quality of either
residence times (Wang, 1997), and high rendering meal.
vessel pressures (Shirley and Parsons, 2000). To assure the protein quality of poultry protein
Poultry protein meals can have a better (Bednar et meal, the focus of the pet food manufacturer should
al., 2000; Yamka, 2003b), equal (Bednar et al., be on a solid relationship with the supplier/renderer
2000), or poorer (Clapper et al., 2001) protein and an understanding of their business processes.
digestibility than soybean meal. Unlike vegetable Measurement of amino acids and amino acid
proteins, poultry meal is not fraught with some of digestibility and utilization can be a good source of
the anti-nutritional components; however, information for general trends in quality; however,
composition (Locatelli and Hoehler, 2003; Dozier et this is too slow, laborious, and costly for day-to-day
al., 2003) and performance can be quite variable. As decisions. Numerous tests have been described to
an example, van Kempen et al. (2004) reported that provide rapid estimates. They range from nitrogen
the variability in digestible lysine and methionine in solubility tests in various buffers, to in vitro tests with
PBPM was 3 times that of soybean meal. Locatelli enzymes and acids, to the use of Near Infrared
and Hoehler (2003) reported that protein and amino Reflectance (NIR) and Fourier Transform Infrared
acid concentrations of 409 PBPM samples from 1999 Reflectance (FTIR) technology (van Kempen et al.,
to 2002 varied widely within years and the mean 2004). Each provides information supporting
concentrations changed by several percentage points purchase and use of consistent poultry protein meal;
from one year to the next. however, the rapid methods must be validated
From where does this variability arise? Besides periodically with animal test data.
variation in processing conditions, the components
making up the raw material mix can also change from
day to day, week to week, and season to season. To Fat quality
illustrate this point, one must look at the protein
quality of the different raw material components. In Fat in dog and cat diets is used to support the energy
a study comparing protein quality of various rendered needs of the animal, meet its essential fatty acid
chicken parts, Aldrich and Daristotle (1998) reported requirements, aid absorption of fat soluble vitamins,
that the PER value of feet and bone/cartilage were impart flavor, aroma, and texture to the product, and
0.87 and 1.22, respectively; whereas heads, viscera, enhance product appearance. Poultry meal contains
and gizzards/livers/hearts were 2.50, 3.04, and 3.08, about 15% fat, the portion that is left after the
respectively. The protein quality of these latter parts extraction process. In one of the few reports in the
was comparable to backs/breastplate and whole birds literature for PBPM, the predominant fatty acids were
without feathers (2.88 and 3.43, respectively). Based oleic (18:1n-9), palmitic (16:0), and linoleic (18:2n-
on these data, one might conclude that the level of 6) at 41, 21.7 and 20%, respectively (Table 2;
ash (bone residue) would have the greatest impact Kirkland and Fuller, 1971). This agrees fairly well
on nutritional quality of the meal. However, ash level with the poultry fat data of Pesti et al. (2002) and
the USDA National Nutrient Database (USDA-ARS, The high level of linoleic acid present in poultry
2003). Whether there is a real difference between protein meal fat complements the nutrient
the fatty acid profiles of poultry protein meal and requirements for dogs and cats (1% and 0.5% of diet
poultry fat has not been reported; but one might DM, respectively). In addition, poultry fat is well
speculate that the structural lipids remaining in poultry accepted by both dogs and cats; and its flavor is
protein meal would be slightly different than those preferred over a number of other fat sources.
present in bulk poultry fat. Differences between However, the fat present in poultry protein meal is
chicken and turkey fat appear to be small. Chicken susceptible to oxidative rancidity. Rancidity is the
fat is comprised of 65.6% mono- and poly- irreversible oxidation of fat initiated by catalysts such
unsaturated fatty acids with 37.3% as oleic and 19.5% as light, transition metals (iron and copper), heat,
as linoleic compared to turkey fat with 66% as mono- and free radicals (molecules possessing an unpaired
and poly-unsaturated fatty acids and 35.9% as oleic electron; Figure 1). Once the reaction is initiated, it
and 21.2% as linoleic (USDA-ARS, 2003). It has becomes autocatalytic and proceeds unabated until
been suggested that the proportion of linoleic acid the reactants are completely exhausted. The point in
has been increasing as more unsaturated vegetable time at which the rate of the reaction becomes
fats are incorporated into poultry diets. This has autocatalytic corresponds to the induction point (IP)
implications for the nutritive value and the stability and from this point forward, the rate of the reaction
of the fat; however, no data were found in the proceeds rapidly (Figure 2). This rapid deterioration
literature to support this claim. is classically termed propagation and concludes, as
described, once the reactants are exhausted.
Table 2. Fatty acid profile of poultry by-product meal (PBPM), While heat, pressure, mechanical grinding, and
poultry grease, chicken fat, and turkey fat. mixing are necessary components of the rendering
Kirkland Pesti USDA USDA process, unfortunately they contribute to the course
(1971) (2002) (2003) (2003) of fat oxidation. During rendering, the inherent
Fatty acids PBPM Poultry Chicken Turkey animal cellular defense mechanisms are disrupted or
grease fat fat destroyed. Lipids are intimately mixed with metabolic
enzymes, transition metals, and water. Transition
Myristic (14:0) 1.3 0.61 0.9 0.9
Palmitic (16:0) 21.7 22.83 21.6 20.6 metals, such as iron, are key catalysts to initiate the
Palmitoleic (16:1) 6.4 8.98 5.7 6 oxidation process. Interestingly, chicken and turkey
Stearic (18:0) 7.6 4.67 6 6.2 lose a greater proportion of heme iron during cooking
Oleic (18:1) 41 43.4 37.3 35.9 than do the red meats (Lombardi-Boccia et al., 2002).
Linoleic (18:2) 20 16.88 19.5 21.2
Linolenic (18:3) 1 1.14 1 1.4 These factors all combine to accelerate oxidation of
Arachidonic (20:4) 1 0.36 0.1 0.3 the fat in the poultry protein meal.
R=R
T0, uV,
• O2 Fe, *R
Catalyst
*R - R
Antioxidant R' R"

Figure 1. Schematic of the catalyst induced (T° = temperature, uV = ultraviolet light, Fe = transition metals like iron and copper, and *R,
.O2 = free radicals) oxidation of an unsaturated fat (R=R) to intermediate (*R-R) and secondary (R′ and R′′) oxidation products.
G. Aldrich 471
12 Peroxides Secondary oxidation products
10
8
Oxidation
4 IP
0
1 2 3 4 5 6 7 8 9 10 11 12
Time
Figure 2. Formation of peroxides and secondary oxidation products in a model fat system undergoing oxidation.
Monitoring fat quality depends upon gathering and 2003). In many cases, rancidity in the diet can be
utilizing data from several different methods. The overcome with high levels of supplemental vitamin
elapsed time from raw material to finished product E. However, this is an expensive solution and one
can have an impact on the production of free fatty that neglects the root problem.
acids (FFA). Free fatty acids are produced as a result The nutritive value of poultry fat is compromised
of lipase enzyme activity on the triglycerides. The with elevated levels of oxidation; wherein, the
cleavage of fatty acid(s) from the glycerol backbone essential fatty acids and energy value have been shown
produces non-esterified or ‘free’ fatty acids. These to decline over time. The proportion of linoleic acid
FFAs are more susceptible to oxidation. The heat in unstabilized PBPM fat declined from 20% to
process of rendering denatures the lipase enzymes, 11.8% over a 12-week storage period and
thus monitoring FFA levels provides an indication corresponded to an elevation in peroxide values (PV;
of pre-rendering raw material handling. The initial Figure 4; Kirkland and Fuller, 1971). In this study,
peroxide value (iPV) is commonly measured. linoleic acid levels did not change for stabilized
However, its relevance is valid only as an initial post- PBPM. Likewise, linoleic acid declined in puppy diets
rendering indication of quality. This is because and subsequently in serum as the level of dietary
peroxides break down to secondary oxidation aldehydes increased (Figure 3; Turek et al., 2003).
products. The secondary oxidation products include In poultry grease, an elevated level of oxidation was
aldehydes, ketones, epoxides, etc. It is these secondary associated with lower dietary metabolizable energy
oxidation products that result in rancidity and off- (Pesti et al., 2002). There have been suggestions that
aromas. Standard methods for their quantification the fatty acid concentrations and oxidative conditions
include p-anisidine values, GC-headspace analysis, of poultry protein meal vary with season. This may
and reaction with thiobarbituric acid (TBARS). It is be possible as seasonal temperatures, poultry feeding
these secondary oxidation products and other reactive practices, and holiday poultry consumption changes;
oxidation species that can be detrimental. As an however, no reports were found in the literature that
example, consumption of oxidized poultry fat has supports this claim.
been reported to decrease growth, lower hematocrit Oxidation of fat can be retarded, but not eliminated.
counts, decrease half-life of intestinal cells, and affect The goal of most poultry protein meal suppliers is to
IgA and lymphocyte proliferation in chicks and pigs delay the onset of oxidation through good
(Dibner et al., 1996). Feeding diets with moderate management practices, quick product turnover, and
levels of aldehydes was also shown to retard puppy the judicious application of antioxidants early in the
growth and suppress immune function (Turek et al., rendering process. The idea is to slow or delay the
30 Diet Serum
25
18:2n-6 (mol/100 ml)
20
15
10
0
Control 100 ppm 500 ppm
Total aldehydes (ppm)
Figure 3. Proportions of linoleic acid (18:2n-6) in diet and serum from puppies fed oxidized diets (100 and 500 ppm total
aldehydes; adapted from Turek et al., 2003).
30 PB 18:2 PBS 18:2 100
PB PV PBS PV
25
80
20
Peroxide (meq/kg)
60
18:2n-6 (%)
15
40
10
20
5
0 0
0 3 6 9 12
Storage (weeks)
Figure 4. Effects of storage time on peroxide value (PV) and linoleic acid (18:2n-6) concentration in unstabilized (PB) and
stabilized (PBS) poultry by-product meal (adapted from Kirkland and Fuller, 1971).
IP for as long as possible. Poultry protein meal have fallen out of favor with purchasers of pet foods.
suppliers and some pet food manufacturers use the The newer challenge to stabilizing poultry protein
antioxidant ethoxyquin for this purpose. Ethoxyquin meal is the request for natural preservatives. Over
has been shown to be effective at reducing oxidation the past decade there has been a substantial increase
(Kirkland and Fuller, 1971), and is approved for use in the number of products that carry a ‘natural’ claim.
on animal feeds (21CFR573.380). Other synthetic This eliminates use of the synthetic antioxidants
antioxidants such as BHA, BHT, TBHQ, and propyl mentioned previously. Poultry protein meal can be
gallate are used separately or in various combinations effectively stabilized with natural antioxidant
with general success. However, synthetic antioxidants
G. Aldrich 473
systems; however, it is much more costly and requires References

more attention to the details. Most natural antioxidant
systems are built on a backbone of mixed tocopherols AAFCO. 2004. American Association of Feed Control
carried in a vegetable oil with added chelators, spices, Officials. Official Publication.
and emulsifiers. Because the natural antioxidants are Aldrich, C.G. and L. Daristotle. 1998. Petfood and
oil miscible and mix poorly with water, there are the economic impact. Proceedings: California
other physical properties (e.g. viscosity) that make Animal Nutrition Conference, pp. 140-148.
them more difficult to work with. The goal is to Bednar, G.E., S.M. Murray, A.R. Patil, E.A.
match the amount of natural antioxidant needed with Flickinger, N.R. Merchen and G.C. Fahey, Jr. 2000.
the desired shelf-life of the poultry protein meal. Selected animal and plant protein sources affect
Uniform application as early in the rendering process nutrient digestibility and fecal characteristics of
as possible and thorough mixing so that the ileally cannulated dogs. Arch. Anim. Nutr. 53:127-
antioxidant is intimately associated with the lipid in 140.
the meal is preferable. Constant monitoring of Clapper, G.M., C.M. Grieshop, N.R. Merchen, J.C.
application systems and efficacy is required.
Russett, J.L. Brent, Jr. and G.C. Fahey, Jr. 2001.
Rapid and (or) predictive analytical tests are
Ileal and total tract nutrient digestibilities and fecal
available to assist in decision making about the
characteristics of dogs as affected by soybean protein
relative freshness of poultry protein meal samples
and the efficacy of the antioxidant system. Most of inclusions in dry, extruded diets. J. Anim. Sci.
the methods rely upon an external factor to accelerate 79:1523-1532.
the rate of oxidation, i.e. heat, oxygen, or pro- Dibner, J.J., C.A. Atwell, M.L. Kitchell, W.D.
oxidants, etc. The most appropriate tests for poultry Shermer and F.J. Ivey. 1996. Feeding of oxidized
protein meal are the Schaal oven, oxygen uptake fats to broilers and swine: effects on enterocyte
(Oxygen Bomb), and pro-oxidant stress tests. Each turnover, hepatocyte proliferation and the gut
of these methods requires the determination of the associated lymphoid tissue. Anim. Feed Sci.
relationship to real-time storage. Once this Technol. 62:1-13.
relationship is known, they can provide very rapid Dozier III, W.A., N.M. Dale and C.R. Dove. 2003.
and reliable information about the stability of poultry Nutrient composition of feed-grade and pet-food-
protein meal. grade poultry by-product meal. J. Appl. Pou. Res.
12:526-530.
Fuller, H.L. 1996. Utilizing Rendered Products:
Summary Poultry. In: The Original Recyclers (D.A. Franco
and W. Swanson, eds). The Animal Protein
Very few studies have been reported in the literature Producers Industry, The Fats & Proteins Research
relating changes in protein and fat quality of poultry Foundation, and The National Renderers
protein meal to performance and health of dogs and Association. USA.
cats. Because the rendering process by which the meal Johnson, M.L. and C.M. Parsons. 1997. Effects of
is produced requires heat and mechanical mixing,
raw material source, ash content, and assay length
the protein and fat quality can be dramatically
on protein efficiency ratio and net protein ratio
affected. The protein quality of poultry protein meal
values for animal protein meals. Pou. Sci. 76:1722-
can be affected by heat damage and dilution with
non-essential amino acids; and the fat quality can be 1727.
negatively affected by oxidative rancidity. Each result Johnson, M.L., C.M. Parsons, G.C. Fahey, Jr., N.R.
in a lower than expected nutritional value associated Merchen and C.G. Aldrich. 1998. Effects of species
with the poultry protein meal, and can have harmful raw material source, ash content, and processing
effects on the dog and cat. It is recommended that temperature on amino acid digestibility of animal
constant monitoring to assure the quality of the protein by-product meals by cecectomized roosters and
and fat be practiced in coordination with the renderer/ ileally cannulated dogs. J. Anim. Sci. 76:1112-
supplier. Further, judicious use of antioxidants and 1122.
close monitoring of results will assure a safe and Kempen, T. van, S. Peak and Y. Qiao. 2004. In vitro
nutritionally adequate poultry protein meal for the digestibility could meet quality control needs.
companion animal industry. Feedstuffs. 76:11-20.
Kirkland, W.M. and H.L. Fuller. 1971. The effect Shirley, R.B. and C.M. Parsons. 2000. Effect of
of ethoxyquin on the chemical and nutritional pressure processing on amino acid digestibility of
changes of poultry by-product meal and poultry meat and bone meal for poultry. Pou. Sci. 79:1775-
offal fat during storage. Pou. Sci. 50:137-143. 1781.
Lombardi-Boccia, G., B. Martinez-Dominguez and Turek, J.J., B.A. Watkins, I.A. Schoenlein, K.G.D.
A. Aguzzi. 2002. Total heme and non-heme iron Allen, M.G. Hayek and C.G. Aldrich. 2003.
in raw and cooked meats. J. Food Sci. 67:1738- Oxidized lipid depresses canine growth, immune
1741. function and bone formation. J. Nutr. Bioch. 14:24-
Locatelli, M.L. and D. Hoehler. 2003. Poultry 31.
byproduct meal: consider protein quality and U.S. Department of Agriculture, Agricultural
variability. Feed Management 54:6-10. Research Service. 2003. USDA National Nutrient
Murray, S.M., A.R. Patil, G.C. Fahey, Jr., N.R. Database for Standard Reference, Release 16.
Merchen and D.M. Hughes. 1997. Raw and Nutrient Data Laboratory Home Page. http://
rendered animal by-products as ingredients in dog www.nal.usda.gov/fnic/foodcomp.
diets. J. Anim. Sci. 75:2497-2505. Wang, X. 1997. Effect of Processing Methods and
Miller, T. 1996. Utilizing Rendered Products: Raw Material Sources on Protein Quality of Animal
Petfood. In: The Original Recyclers. (D.A. Franco Protein Meals. PhD Thesis, University of Illinois,
and W. Swanson, eds). The Animal Protein Urbana-Champaign.
Producers Industry, The Fats & Proteins Research Yamka, R.M., U. Jamikorn, A.D. True and D.L.
Foundation, and The National Renderers Harmon. 2003a. Evaluation of low-ash poultry
Association. USA. meal as a source in canine foods. J. Anim. Sci.
Pearl, G. 2003. President, Fats & Protein Research 81:2270-2284.
Foundation, personal communication. Yamka, R.M. 2003b. Evaluation of low-
Pesti, G.M., R.I. Bakalli, M. Qiao and K.G. Sterling. oligosaccharide and low-oligosaccharide low-
2002. A comparison of eight grades of fat as broiler phytate whole soybeans and soybean meal in canine
feed ingredients. Pou. Sci. 81:382-390. foods. PhD Dissertation, University of Kentucky,
Lexington.
K.S. Swanson and G.C. Fahey 475
The role of yeasts in companion animal nutrition

KELLY S. SWANSON AND GEORGE C. FAHEY, JR.
Types of yeast
Several strains of the yeast Saccharomyces cerevisiae commonly grind and cook corn using enzymes to
are used in the baking, brewing, distilling, and wine convert starch to sugar. A rapid continuous
production industries (Sumner and Avery, 2002). fermentation process at temperatures between 35 and
Although these strains share common features such 38°C then is employed to maximize substrate
as efficient sugar utilization, high ethanol tolerance utilization and ethanol production yielding 9-12%
and production, high yield and fermentation rate, and alcohol. Although not fully researched, many of the
genetic stability, they also possess properties specific differences in brewer's dried yeast and yeast from
to each group (Trivedi et al., 1986; Benítez et al., corn wet milling ethanol production [e.g., fat content,
1996). hops products (caryophyllene, humulene), and sugar
Eight official and one ‘tentative’ yeast products profiles] are likely factors that affect palatability.
are currently defined by the Association of American
Feed Control Officials (2003) and are differentiated Table 1. Chemical composition of brewer's yeast and corn wet
milling yeast
by source of yeast and characteristics such as moisture
and crude protein concentrations, and fermentative Item Brewer's yeast Corn wet
activity. Brewer's dried yeast is the dried, non- milling yeast
fermentative, non-extracted yeast of the botanical
Dry matter, % 95.3 91.2
classification Saccharomyces resulting as a by-product Organic matter, % of DM 94.2 92.8
from the brewing of beer and ale. It must contain Crude protein, % of DM 43.1 46.8
not less than 35% crude protein and be labeled Total dietary fiber, % of DM 22.5 19.1
according to its crude protein content (AAFCO, Ash, % of DM 5.9 7.2
Fat, % of DM 3.0 9.4
2003). As defined, brewer's dried yeast must originate Total monosaccharides, mg/g (DMB) 460.8 277.8
from a brewery and the brewing of beverages, beer Glucose, mg/g (DMB) 369.7 168.1
or ale for human consumption, and should not be Mannose, mg/g (DMB) 81.4 60.6
confused with corn wet milling yeast that is used in Sugar alcohols, mg/g (DMB) 9.7 49.1
Uronic acids, mg/g (DMB) 7.0 7.2
industrial ethanol production. Caryophyllene, µg/g 0.31 ND1
Brewer's dried yeast and corn wet milling yeast Humulene, µg/g 0.65 ND
are different in terms of chemical composition and
1
organoleptic properties, which is likely due to Not detected
differences in the fermentation processes and in the
COMPOSITION OF BREWER'S YEAST
substrates used (Table 1). In the brewing industry,
wort derived mostly from malted barley is fermented Commercially available brewer's yeast is typically
slowly at temperatures of 10 to 20°C using a batch dried from a yeast slurry to a dry powder of less than
fermentation process that yields a beer with an 10% moisture to facilitate handling, storage, and
alcohol content of approximately 6%. In contrast, transport. Brewer's yeast is relatively high in crude
during wet milling ethanol fermentation, distillers protein and carbohydrate concentrations, while the
476 The role of yeasts in companion animal nutrition
concentrations of fat and ash are relatively low. This skeletal framework and often are linked to cell wall
is not surprising because yeast synthesizes protein proteins.
and vitamins while absorbing minerals from the beer Mannose polysaccharides are linked to proteins to
wort during the fermentation process. The relatively form a mannoprotein layer localized at the external
low fat content of brewer's yeast compared to yeast surface of the yeast cell wall. Two classes of
from commercial wet milling ethanol fermentation covalently linked cell wall proteins have been
likely is due to substrate differences (the low fat identified. The first class consists of glycosyl
concentration of barley compared to corn) and phosphatidylinositol proteins that form a complex
differences in the fermentation processes. with ß(1,3)- and ß(1,6)-chains (Kollár et al., 1997).
Fiber concentration of yeast depends greatly on the The second class of cell wall proteins, the protein
method used (Table 2). Although the method of with internal repeats, are linked directly to ß(1,3)
measuring crude fiber (AOAC, 1980) is used for glucans. Mannoproteins are strictly regulated in
regulatory purposes, results are misleading as several response to changes in external conditions (e.g., heat
fibrous compounds are solubilized with this shock, hypo-osmotic shock, carbon source) and
procedure, resulting in a large underestimation of internal changes during the cell division cycle (Horie
fiber content. The neutral detergent fiber (NDF) and Isono, 2001).
method of Robertson and Van Soest (1977) results in While glucans and mannoproteins are main
solubilization of viscous fiber components and components of the cell wall and found in
recovery of cell wall constituents. Because brewer's approximately equal amounts, chitin constitutes only
yeast contains a considerable amount of protein that ~1-3% of the cell wall. Although present in small
becomes viscous when partially hydrolyzed during quantities, it is a major component of the primary
the NDF procedure, filtration problems and inflated septum and is involved in the separation of mother
recoveries result in overestimated fiber concentrations and daughter cells, making it essential for cell division
(Merchen et al., 1990). For proteinaceous feeds such (Shaw et al., 1991). The remaining components of
as brewer's yeast, the method of Prosky et al. (1992) yeast, excluding the cell wall, are collectively referred
used to measure total dietary fiber (TDF) is most to as yeast cell extract and contain numerous
accurate. nucleotides, enzymes, vitamins, and minerals.
Table 2. Fiber composition of brewer's yeast1

SELENIUM YEAST
Item % (DM basis)
Crude fiber 0.5

The essential trace element, selenium (Se), has been
Total dietary fiber (TDF) 25.1 heavily studied recently as it is thought to play a role
Neutral detergent fiber (NDF) 48.2 in cancer prevention. Selenium is an integral part of
Acid detergent fiber (ADF) 6.8 the enzyme glutathione peroxidase, which functions
Acid detergent lignin (ADL) 2.9
to prevent oxidative damage. In the past, sodium
1
Merchen et al., 1990. selenite (inorganic form) was commonly used as the
Se supplement for livestock feeds. However, recent
YEAST CELL WALL COMPOSITION studies have identified organic sources (e.g.,
selenomethionine) present in plants and selenized
The cell wall of Saccharomyces cerevisiae constitutes yeast as highly digestible alternatives (Yoshida et al.,
approximately 15-30% of the dry weight of the cell 2002; Gunter et al., 2003). Although growth
and consists primarily of mannosylated proteins, ß- conditions and yeast strain may influence the
glucans, and chitin (N-acetylglucosamine), which are proportion of selenocompounds present in selenized
covalently linked. The glucan portion consists of yeast, the bulk of the Se is in the form of
ß(1,3)- and ß(1,6)-chains. Beta (1,3)-glucans, which selenomethionine (Ip et al., 2000; Zheng et al., 2000;
form the internal skeletal framework of the cell, are Yoshida et al., 2002). Other Se forms present in
the major structural components and are largely selenized yeast include selenite and selenoamino acids
responsible for its mechanical strength. This form of (selenocystine, selenolanthionine, selenocystathio-
glucan is highly branched and possesses multiple non- nine, Se-methylselenocysteine, Se-adenosyl
reducing ends that function as attachment sites for selenohomocysteine, and γ-glutamyl-Se-methyl-
other components of the cell wall (Kollár et al., 1997). selenocysteine (Ip et al., 2000). The presence of these
Beta (1,6)-glucans are found primarily outside the highly digestible forms of Se may be partly
responsible for the beneficial effects often observed corn wet milling yeast (included at 1% of diet). In
with selenoyeast supplementation. these experiments, consumption ratios of brewer's
yeast were 1.9:1 – 2.1:1 compared to corn wet milling
yeast. In each experiment, a panel of 20 dogs or cats
Use of yeasts in companion animals was used to test food preference with a standard 4-
day palatability test. Each day on test, both diets were
Brewer's yeast is used in companion animal foods offered simultaneously for a period of 1 hr. To
because it is a high quality protein source rich in B- account for right-left bias, the placement of diets was
vitamins, amino acids, and minerals. Inclusion of alternated each day. After the 1 hr feeding period,
brewer's yeast (included at 1% of diet) in companion both diets were removed simultaneously and weighed
animal diets has been shown to increase (P<0.05) to calculate intake.
palatability in both dogs (Figure 1; Kennelwood Inc., Although few have studied the effects of selenized
unpublished data; Ontario Nutri Lab Inc., yeast on companion animal health, one recent
unpublished data) and cats (Figure 2; Kennelwood experiment reported its beneficial effects on prostate
Inc., unpublished data) compared to diets containing health in aged dogs (Waters et al., 2003). In that
100
Corn wet milling yeast Brewer's yeast
75 P<0.05 P<0.05
% of consumption
50
25
0
Experiment No. 1 Experiment No. 2
Figure 1. Four-day canine palatability tests. Consumption of dog food including brewer's yeast was greater (P<0.05) than that of
a food including corn wet milling yeast (n = 20 dogs in each experiment).
100
Corn wet milling yeast Brewer's yeast
75 P<0.05
% of consumption
50
25
0
Experiment No. 1
Figure 2. Four-day feline palatability test. Consumption of cat food including brewer's yeast was greater (P<0.05) than that of a
food including wet milling yeast (n = 20 cats).
study, 49 elderly (8.5 to 10.5 yr) sexually intact males and concentration of mycotoxins in pet foods
were randomly assigned to one of five treatments commercially available in the US, and to determine
and fed for 7 months: 1) control diet containing 0.3 whether these concentrations are cause for concern.
ppm Se; 2) control diet + 3 µg/kg/d selenomethionine; If that is the case, inclusion of glucomannans in pet
3) control diet + 6 µg/kg/d selenomethionine; 4) foods, especially those containing high concentrations
control diet + 3 µg/kg/d high-Se yeast; or 5) control of grain, may be prudent. Glucomannans may also
diet + 6 µg/kg/d high-Se yeast. Although no play a role in colon cancer prevention because of
carcinomas were observed during histopathologic their antimutagenic and antioxidative activity
examination, Se supplementation decreased (Chorvatovicová et al., 1999; Krizková et al., 2001).
(P<0.001) DNA damage and increased (P=0.04)
number of apoptotic cells in prostate epithelia.
Because DNA damage and apoptosis may be Se- MANNANS
responsive events that are important regulatory points
in prostate carcinogenesis, selenized yeast Mannans, also referred to as mannan oligosaccharides
supplementation may prove to be protective against (MOS), are composed of short chains (attached
its development. mainly by α(1,2) and α(1,3) bonds) and long chains
(attached mainly by α(1,6) linkages with branches
linked by α(1,2) and α(1,3) bonds) (Spring and
Use of yeast components in companion Dawson, 2000). Mannans have been studied for their
ability to agglutinate and interfere with intestinal
animal foods binding and colonization of harmful microbial
‘Functional foods’, ‘nutraceuticals’, and species. Numerous E. coli and Salmonella strains
‘phytochemicals’ are terms commonly used to refer possess mannose-specific fimbriae, agglutinate
to foods or compounds in foods that possess properties mannans in vitro, and colonize in lower concentrations
that may benefit the human in ways other than in animals supplemented with mannans. Fimbrial
providing nutritive value. Although use of these adhesins specific for mannan residues are referred to
ingredients began in the human food industry, there as Type-1 adhesins. Mannans aid in the resistance of
is interest in including them in pet foods as well. pathogenic colonization by acting as receptor
Many functional ingredients are thought to decrease analogues for Type-1 fimbriae and decrease the
the incidence of certain disease states or extend the number of available binding sites (Oyofo et al.,
lifespan of pets by possessing antioxidant activity, 1989).
antimicrobial action, or immuno-enhancing Mannans are capable of modulating the immune
properties. Several components present in yeast may system and influencing microbial populations in the
be classified as being functional, including gut. Mannans (Bio-Mos ®) have been reported to
glucomannans, mannans, mannoproteins, ß-glucans, increase (P=0.14) serum IgA concentrations in dogs
and nucleotides. (2.33 vs 1.93 g/L, Swanson et al., 2002a). In adult
dogs, mannan oligosaccharides as Bio-Mos ®
beneficially altered microbial ecology by increasing
GLUCOMANNANS (P=0.13) lactobacilli populations (9.16 vs 8.48 log10
CFU/g fecal dry matter) and decreasing (P=0.05) total
Glucomannans, extracted from the inner cell wall of aerobe populations (7.68 vs 8.67 log10 CFU/g fecal
yeast, may prove to be beneficial in animal foods dry matter, Swanson et al., 2002a).
because of their ability to bind mycotoxins.
Mycotoxins are naturally occurring toxic chemicals
produced by molds under certain environmental MANNOPROTEINS
conditions. Sharma and Márquez (2001) tested 12
pet foods commercially available in Mexico for Recent experiments have suggested that mannoproteins
frequency and concentration of aflatoxins. In that could be promising vaccine candidates for individuals
experiment, seven aflatoxins and aflatoxicol were with compromised T-cell function (e.g., AIDS,
detected in most samples, with aflatoxin B1 being lymphoma). Mansour et al. (2002) determined that
present in the highest frequency and concentration. mannoproteins are ligands for the macrophage
In all contaminated samples, maize was the main mannose receptor, which serves as a link between
ingredient. Research is needed to measure incidence innate and adaptive immunity (Sallusto et al., 1995).
Mannoproteins also have been shown to elicit delayed- effects of yeast or yeast fractions on dog and cat
type hypersensitivity reactions and induce production health, with mannans being the only fraction studied
of cytokines important in decreasing fungal pathogens to any extent. Vickers et al. (2001) used canine fecal
(Chaka et al., 1997; Pietrella et al., 2001). inoculum to determine the fermentability
characteristics of mannan oligosaccharides. In that
experiment, moderate concentrations of total short-
ß-GLUCANS chain fatty acids were produced after 6 (0.49 mmol/
g of organic matter), 12 (1.45 mmol), and 24 hrs
Although much of the ß-glucan research has focused (2.40 mmol/g) of in vitro fermentation. The microbial
on oat bran, experiments using ß-glucans derived species responsible for mannan oligosaccharide
from yeast have resulted in similar findings. Of all breakdown were not determined in this experiment.
the beneficial properties that have been reported, the Hussein and Healy (2001) also performed an in
lipid-lowering effect of ß-glucans probably has been vitro experiment using canine and feline fecal
the most popular. An experiment using free-living, inoculum to determine fermentability of mannan
obese, hypercholesterolemic men demonstrated that oligosaccharide in Bio-Mos ® (Alltech Inc.).
yeast-derived ß-glucans were well tolerated and Differences were not observed in fermentability
decreased (P<0.05) blood total cholesterol between dog and cat fecal inoculum. By examining
concentrations similar to the effect of oat products dry matter and organic matter disappearance, it
(Nicolosi et al., 1999). Yeast-derived ß-glucans also appeared that mannan oligosaccharide was highly
appear to possess antimicrobial and antitumor fermented. Dry matter disappearance after 6, 12, 18,
properties by enhancing immune function. The and 24 hrs of in vitro fermentation was 54.3, 57.9,
binding of ß-glucan to its receptor present on 60.7, and 61.3%, respectively. Organic matter
macrophages results in phagocytosis, respiratory disappearance was similar to that of dry matter (56.8,
bursts, and secretion of TNF-α (Chen and Hasumi, 60.7, 63.7, and 64.1% after 6, 12, 18, and 24 hrs of
1993; Lee et al., 2001). Finally, ß-glucans are readily fermentation). Dry matter and organic matter
fermented in the large bowel and serve as a fuel source disappearance do not always reflect microbial
for microbial populations. fermentation due to the disappearance of soluble
carbohydrates present in the substrates that are not
retained during filtering. Although soluble
NUCLEOTIDES carbohydrates are available for fermentation,
In contrast to the components listed above, nucleotides gravimetric methods cannot determine the proportion
are present in yeast extract rather than cell wall. used by the microbes as an energy source. Therefore,
Although endogenously produced by the body, dietary the measurement of dry matter and organic matter
nucleotides may be essential in certain life stages or disappearance is not as accurate as the measurement
in certain health conditions (e.g., neonates, immune- of the fermentation end-products (i.e., short-chain
compromised) (Sánchez-Pozo and Gil, 2002). In fatty acids and gas), which is a direct measurement
addition to stimulating the development of the small of fermentation. Concentrations of total short-chain
intestine (Bueno et al., 1994) and liver (Sánchez- fatty acids, acetate, and propionate increased linearly
Pozo et al., 1998), exogenous nucleotides have been over time. Moderate concentrations of total short-
shown to enhance immune function by increasing chain fatty acids (10.1, 26.8, 36.7, and 49.7 mM)
production of immunoglobulins, improving response were produced after 6, 12, 18, and 24 hrs. In
to vaccines, and increasing tolerance to dietary comparison to total short-chain fatty acids, lactate
antigens (Maldonado et al., 2001). Because of their concentrations were fairly high (7.7, 8.7, 7.6, and
importance in neonatal nutrition, the inclusion of 5.9 mM), suggesting fermentation by a lactate-
nucleotides in human infant formulas is under producing species (e.g., lactobacilli, bifidobacteria).
investigation (Cordle et al., 2002; Ostrom et al., In agreement with the work of Vickers et al. (2001),
2002). these data suggest that mannan oligosaccharide is
moderately fermentable by canine and feline
microflora. The lactate produced during fermentation
IN VITRO RESEARCH ON YEAST COMPOUNDS
suggests that lactate-producing species are able to
utilize mannan oligosaccharide, possibly by acting
Limited research has been performed testing the as a prebiotic for these species.
In vitro data from our laboratory indicated that pH, ammonia and short-chain fatty acid
mannan oligosaccharide was highly fermentable by concentrations, blood glucose, and fecal consistency.
canine fecal inoculum (Swanson, unpublished data). Besides minor changes in short-chain fatty acid
After 0, 4, 12, and 24 hr of in vitro fermentation, concentrations, the only relevant finding was a
organic matter disappearance was 38.1, 40.5, 41.5, decrease (P = 0.07) in Clostridium perfringens
and 60.4%, respectively. The relatively high organic populations in dogs fed Bio-Mos® (4.48 log10 CFU/
matter disappearance at 0 hrs was not due to microbial g) vs dogs fed xylooligosaccharides (5.16 log10 CFU/
fermentation, but rather to the soluble carbohydrates g) or oligofructose (4.74 log 10 CFU/g). Because
present in mannan oligosaccharide not being retained clostridia species do not possess mannose-specific
during filtering. After 4, 12, and 24 hrs of fimbriae, another mechanism is likely occurring. The
fermentation, corrected total short-chain fatty acid lack of any significant findings may be due to the
concentrations were 1.18, 2.71, and 4.60 mmol/g low dose of prebiotics consumed (only ~1.3 g/d) or
organic matter, respectively. These short-chain fatty to the use of soybean meal in the control diet, which
acid concentrations were approximately twice as high supplied an estimated 10 g/kg of naturally occurring
as those reported by Vickers et al. (2001) at the 12 oligosaccharides, mainly galactooligosaccharides.
and 24 hr time points. In agreement with short-chain Any beneficial effects resulting from Bio-Mos ®
fatty acid data, gas was produced in relatively high consumption may have been masked by the presence
amounts (corrected gas values were 17.4, 58.3, and of these naturally occurring oligosaccharides.
100.9 mL/g organic matter after 4, 12, and 24 hrs of Zentek et al. (2002) used 4 dogs in a 4 x 4 Latin
fermentation). square design to determine the effects of mannan
oligosaccharide (Bio-Mos ®), transgalactosylated
oligosaccharides, lactose, and lactulose on fecal
CANINE RESEARCH ON YEAST COMPONENTS characteristics, total tract digestibility, and
concentrations of microbial end-products in feces and
O’Carra (1997) performed two experiments urine. Carbohydrate supplements were administered
examining mannan oligosaccharide in Bio-Mos® at a rate of 1 g/kg BW/d. Mannan oligosaccharide
(Alltech Inc.) and its effects on immune function in supplementation decreased (P<0.05) fecal pH (6.6
dogs. In the first experiment, adult beagles were fed vs 6.9), fecal ammonia excretion (78.4 vs 116 µmol/
diets containing 0, 1, 2, or 4 g Bio-Mos®/kg diet. g feces), and apparent dry matter (81.9 vs 85.0%),
Changes in plasma protein and IgG measurements crude protein (79.8 vs 82.5%), and nitrogen-free
were not observed after 15 or 31 days of supple- extract (83.1 vs 94.8%) digestibilities. By decreasing
mentation. In the second experiment, Border collie fecal pH and ammonia, mannan oligosaccharide
pups were fed diets containing 0 or 2 g Bio-Mos®/ supplementation appeared to improve indices of
kg. After a 7-day adaptation period, a vaccination colonic health. However, the decreases observed in
protocol was initiated. All dogs were vaccinated apparent nutrient digestibilities resulting from mannan
against parvovirus, leptospirosis, adenovirus, and oligosaccharide supplementation would increase fecal
distemper. Vaccine boosters were applied on day 21 quantity and the cost of feeding the animal. The dose
for leptospirosis and on day 35 for parvovirus. Blood of carbohydrate supplements fed in this experiment
characteristics were measured over a 9-wk period. (1 g/kg BW/d) was very high. Smaller doses of
No changes were observed in weight gain, lysozyme mannan oligosaccharide may not have such negative
activity, plasma protein concentration, or plasma IgG effects on nutrient digestibility.
concentration. Neutrophil activity was numerically Using ileal cannulated adult dogs, Swanson et al.
increased in pups fed the diet containing Bio-Mos® (2002a) examined the effects of supplemental
after vaccination [approximately 18 vs 14 Nitroblue mannan oligosaccharide (Bio-Mos ® ) and (or)
tetrazolium (NBT)+ cells/slide]. However, due to low fructooligosaccharides (FOS) on colonic microbial
animal numbers (n = 3/group), statistical significance populations, local and systemic immune function,
was not reached. fecal protein catabolite concentrations, and ileal and
Using adult ileal cannulated dogs, Strickling et al. total tract nutrient digestibilities. A 4 x 4 Latin square
(2000) compared a control diet to those containing 5 design with 14-day periods was used. Twice daily,
g oligosaccharide/kg diet, one of which was Bio- dogs were offered 200 g of dry, extruded, kibble
Mos®. Researchers measured ileal and total tract diet and given the following treatments orally via
nutrient digestibilities, microbial populations, ileal gelatin capsules: 1) control (no supplemental MOS
or FOS), 2) 1 g FOS, 3) 1 g MOS, or 4) 1 g FOS + Grieshop et al. (2004) tested the effects of chicory
1 g MOS. Mannan oligosaccharide supplementation and (or) mannan oligosaccharide on nutritional and
beneficially influenced microbial populations, immunological characteristics of geriatric dogs. After
decreasing (P=0.05) total aerobe (7.68 vs 8.67 log10 a 4-wk baseline period, 34 senior dogs (beagles: 9-
CFU/g fecal dry matter) and tending to increase 11 yr old; pointers: 8-11 yr old) were randomly
(P=0.13) Lactobacillus populations (9.16 vs 8.48 log10 allotted to one of four treatments: 1) control (no
CFU/g fecal dry matter). Mannan oligosaccharide chicory or Bio-Mos®); 2) 1% chicory; 3) 1% Bio-
also increased serum IgA concentrations (2.33 vs 1.93 Mos®; or 4) 1% chicory + 1% Bio-Mos ®. Dogs
g/L, P=0.14) and lymphocyte numbers (20.4 vs remained on treatment for 4 wks. Increased (P=0.07)
15.6% of total white blood cells, P<0.05). A tendency food intake by dogs fed chicory + MOS and MOS
for decreased ileal dry matter (55.0 vs 67.7 %, P=0.15) alone resulted in increased (P<0.05) wet fecal output.
and organic matter (63.6 vs 74.1%, P=0.15) Dry matter, organic matter, and crude protein
digestibility also was observed from Bio-Mos ® digestibilities were unchanged due to treatment.
supplementation. The combination of FOS and MOS Supplementation of Bio-Mos® increased (P<0.05)
supplementation enhanced immune characteristics, fecal bifidobacteria populations and decreased
increasing ileal IgA concentrations on a dry matter (P<0.05) fecal E. coli populations compared to
basis (4.90 vs 3.40 mg/g ileal dry matter, P=0.06) control. Supplementation of chicory + Bio-Mos®
and crude protein basis (12.22 vs 8.22 mg/g ileal tended to increase (P<0.10) neutrophil concentrations,
crude protein, P=0.05). Supplementation of FOS + while Bio-Mos® (P = 0.06) and chicory + Bio-Mos®
MOS also decreased (P<0.05) total fecal indole and (P<0.05) decreased lymphocyte concentrations.
phenol concentrations (1.54 vs 3.03 µmol/g fecal dry Finally, prebiotic supplementation altered proportions
matter), compounds partially responsible for fecal of lymphocytes expressing CD4 and CD8 cell surface
odor and detrimental to intestinal health. This markers. Chicory + Bio-Mos ® supplementation
experiment was performed using healthy adult dogs, decreased (P=0.07) CD8-specific lymphocytes.
which would not be at the highest risk for intestinal Results of this experiment support findings from
irregularities. It is likely that the health benefits of previous experiments that in addition to altering gut
feeding mannan oligosaccharide alone, or in microbial ecology, Bio-Mos® supplementation may
combination with FOS, would be more beneficial to affect immune status.
populations of elderly dogs, young weanling puppies,
or stressed animals.
In a follow-up study, Swanson et al. (2002b) ACTIVE COMPONENTS IN YEAST
supplemented ileal cannulated dogs with either 1 g COMPONENTS
sucrose (placebo) or 2 g FOS plus 1 g Bio-Mos®.
Fecal, ileal, and blood samples were collected at the Our laboratory has analyzed several commercially
end of each 14-day period to measure microbial available sources of mannan oligosaccharides and
populations and immune characteristics. found considerable differences in crude protein, fat,
Supplementation of FOS plus MOS increased total dietary fiber, and monosaccharide concentrations
(P<0.05) fecal bifidobacteria (10.04 vs 9.42 log10 (Table 3). Most of the monosaccharides are present
CFU/g fecal dry matter) and lactobacilli as part of polysaccharides rather than as free sugars.
concentrations in feces (9.75 vs 8.24 log10 CFU/g fecal Source B was unique in that it contained a
dry matter) and ileal effluent (8.66 vs 7.55 log10 CFU/ considerable amount of galactose in addition to
g ileal dry matter). Dogs fed FOS plus MOS also glucose and mannose. The presence of galactose in
tended to have lower (P=0.08) blood neutrophils that source may suggest that guar gum or locust bean
(62.99 vs 66.13 % of total white blood cells; 6.40 vs gum, which contain galactomannans, are also present
7.22 x 10 3 cells/µL) and greater (P=0.06) blood in this source of MOS. Although marketed as a source
lymphocytes (19.95 vs 17.29 % of total white blood of MOS, these products are very complex and also
cells) vs placebo. Serum, fecal, and ileal contain glucans, mannoproteins, phosphate, and
immunoglobulin concentrations were unchanged several other compounds that apparently are not
(P>0.05) by treatment. Supplementation of FOS plus excluded in the crude extraction process. Because the
MOS beneficially influenced indices of gut health composition of MOS is complex, the components that
by improving ileal and fecal microbial ecology and result in beneficial effects are not known. Although
altered immune function by causing a shift in blood the mannan portion of MOS is generally thought to
immune cells. be responsible for the pathogenic resistance effect
by acting as a receptor analog for Type-1 fimbrial References

adhesions present on species such as E. coli and
Salmonella, it is possible that a different fraction Association of American Feed Control Officials:
present in MOS is responsible for its effects on Official Publication. 2003. Association of American
immune function. For example, mannoproteins and Feed Control Officials, West Lafayette, IN.
ß-glucans taken from yeast cell walls have been AOAC. 1980. Official Methods of Analysis. (13th Ed).
reported to enhance immunity. Therefore, more Association of Official Analytical Chemists,
research is needed in order to determine whether Washington, DC.
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OM, %1 91.6 82.2 93.0 93.5 98.1 J. Scharringa, J. Verhoef, H. Snippe and A.I.
CP, %1 33.7 39.0 34.4 42.2 42.9
TDF, %1 37.0 21.4 40.8 NA4 NA
Hoepelman. 1997. Induction of TNF-α in human
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Mannose, mg/g 119.3 58.6 99.7 90.7 144.0 human mannose binding protein. J. Immunol.
Galactose, mg/g ND2 35.9 ND ND ND
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NA = not analyzed.
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T. Phillips 485
The expanding pet food industry: where are the opportunities?

TIM PHILLIPS
PETFOOD INDUSTRY Magazine, Watt Publishing Co., Mt. Morris, Illinois, USA
Introduction
The pet food market is full of dynamic and diverse It seems likely that in the near future, ingredients
opportunities, according to a report by Lisansky like omega-3 fatty acids and other ‘healthy’ human
(2003). The global market has boomed with new food ingredients will become more common in pet
products, new presentations and new focus on pet food. Probiotics and prebiotics, separately and
population sectors. There is solid and steady growth combined as synbiotics, now popular for humans and
in pet foods, particularly in dry cat and dog food in animal feed, are likely to become components of
areas. Pet ownership has also increased, and pet food. Many botanicals, known for their effects
improving pet nutrition through consumption of on humans, are also going to become ingredients of
commercial pet foods and treats is on the rise. new ‘healthy’ pet food.
Geographic expansion is also booming with the Specialist pet foods for specific health conditions
areas of southern Europe becoming more affluent already exist and should become more popular as
and converting to manufactured pet food, opening pets live longer and humans spend more to protect
up manufacturing and distribution opportunities for them. Low protein, low ash and easily digestible pet
local manufacture. foods are useful for owners wanting to keep pets
Pet food has become more targeted on specific healthy.
life stages. For example, there are foods for kittens, Major pet food producers are becoming more
adults, queens and seniors. Packaging has become specific in their requirements and are vigorously
more convenient and more expensive with single- communicating their needs to ingredient companies.
meal cans and pouches replacing large cans. They want fewer commodity ingredients and more
Pet food now comes in several forms. There are specialty ingredients for both existing and new
homogeneous mixtures, chunks, some with jelly and products. They are willing to pay more for the ‘right’
some with gravy. Dry pet food is no longer the ingredients since they can pass on the cost to
standard issue kibble left for pets between meals. It consumers who will buy the ‘right’ products. Such
is now available over a much wider range of flavors specialty ingredients should provide improved
and prices. Up-market dry pet food, first sold in technical functionality, supply health benefits or
veterinary surgeries and then in specialist stores, has perhaps both. The growth in dry foods in Europe
now moved into mainstream supermarkets, suggesting and North America, although threatening to erode
much more is being sold. the market for wet food to some extent, may also
Pet food has the advantage over human food in open up opportunities for new ingredients including
that people often love their pets more, and are more ‘healthy’ ingredients. For example, hydrocolloid
willing to spend extra money to keep them happy companies who may lose some market in wet pet
and healthy. Many owners do not buy on price, but foods could take the opportunity to regain market in
on pet acceptability, giving ingredient manufacturers dry pet food, perhaps by attempting to develop health
superb opportunities to enter the market with better ingredients based on soluble fiber or to use
products. hydrocolloids as carriers for healthy ingredients.
486 The expanding pet food industry: where are the opportunities
In the food industry, the development of the can carry out these investigations themselves and
‘functional food’ market continues, with food usually do an excellent job, but the work is time
producers making health claims for a variety of consuming and often must be done by a deadline.
products. With the increasing consumer awareness Consultants can provide the extra resources required
of health benefits delivered in food and beverage to ensure a thorough and timely review. They also
products, it is likely that the trend for ‘healthier’ can investigate new markets without alerting
foods will also have an impact on the consumer’s competitors or potential customers. This anonymity
choice of pet foods. Pet owners are likely to pay is a big advantage. Lastly, consultants have an
particular attention to the dental, digestive, circulatory objective viewpoint and can tell companies what they
and immune health of their pets in order to improve need to know rather than what a senior executive
longevity and quality of life. wants to hear.
A systematic and scientific approach should be used
to explore the possibilities of new markets. It is wise
Pet food ingredient types
to determine the nature of the opportunity before
Pet food ingredients fall into three principal taking action. One should find the most cost-effective
categories: commodities, semi-commodities, and approach and select ways of approaching it that
specialties. The commodity ingredients, many also reduce the business risk while simultaneously
used in food manufacture, include starches and fats, increasing strategic choices.
bulk proteins, fibers and other bulking agents. First, research should be used to determine the
Marketing strategies for these products rely on current market situation. Not only is the market
providing relatively low prices while maintaining volume and value important, the past and future
product standards, specifications and safety. The trends should be identified in the context of the
opportunities for expanding markets include moving economic and regulatory environments. Experts
geographically into areas of increasing pet food use should be consulted from as many sources as possible
as well as taking market share from competitors with – from customers, suppliers and competitors to get
a better offering. as close to the ‘truth’ of the market as possible.
Semi-commodity items include flavorings like The most important aspects of market analysis are
protein hydrolysates, texturizers such as gluten and identifying the key factors for success and the barriers
gums, and nutritional items like vitamins, minerals to entry. This applies whether one is looking for a
and premixes. Marketing strategies are oriented better strategy for market penetration, to expand
towards performance and value for money combined geographically or to launch new products. Also useful
with reliability and customer services. Many is a detailed ‘SWOT’ analysis of one’s company –
opportunities exist for market expansion of semi- strengths, weaknesses, opportunities and threats. Once
commodities by creating points of differentiation with the data are analyzed and the opinions considered,
competitors; this requires both product development then usually it is possible to generate conclusions and
and customer support. The higher margins available recommendations for the best options.
can justify the investment and effort.
Specialty ingredients, including specialty fats and
oils, high-quality palatants and flavors, custom STRATEGY FOR GROWTH BY GEOGRAPHIC
premixes, fermentation and cheese products, have the EXPANSION
best margins and the lowest volumes. Specialties are
Geographic expansion is one of the first things
what most companies strive to sell, as they have high
companies think about when considering expansion.
barriers to entry. However, developing and proving
Most companies have a geographic area with which
the value of specialties can be a lengthy and expensive
they are familiar. They understand the subtle dynamic
task; the strategy for market expansion should be
of doing business there – how many times new
carefully planned and costed at the start.
customers need to be seen before they buy, what kind
of service customers want and are prepared to pay
Exploring new markets for, what are the key success factors in making a
sale. Companies often think that going into a new
GETTING STARTED geographic region will be an extension of their
previous way of doing business. This is almost never
Experience suggests that there is hard homework to
true.
do that requires a lot of data and analysis. Companies
T. Phillips 487
In the US, companies, customers and suppliers want of ‘store brands’ can be contracted out and handled
to talk about the price, the specification, the delivery entirely by a contract supplier.
criteria, the guarantees and occasionally about Because the US is so big and heterogeneous, most
customer service, but are wary of having to pay for retailers in the US are regional, while many food
it. They will often trade loyalty for price, as in and pet food companies are national or multinational.
strategic supplier alliances, where qualified suppliers The two groups of companies are different in size
get minimum assured orders in exchange for reduced and in how they perceive and manage the ultimate
prices. In the US, especially in the ingredients customers. Retailers are at the ‘point-of-sale’ with
business, price remains overwhelmingly the most customers but their economic success may not depend
important criterion on which supplier offers are very much on what those customers think.
judged. By contrast, in the EU where the countries are
In the EU, the situation can be quite different. much smaller and within each country much more
Products are sold on price, but other factors can culturally homogeneous, retailers have become
contribute significantly to making sales. One example national and larger than in the US. They compete
from well outside the pet food market was a company for market share within their own countries; they
making road-paving equipment in the US and in the organize the logistics of national supply and
EU. Machines in both regions were sold on price, distribution. They make money, less from renting
but in the US the machines had an unfinished and space, and more from selling products. Successful
economical appearance, whereas in the EU all the EU retailers earn a much higher return on investment
metal had to be smoothed and carefully painted before than retailers in the US.
a customer would consider buying it. The company’s Consequently, EU retailers make greater efforts to
US staff could not understand why machines made cater to consumers and will dictate their requirements
to the US specification would not suffice for the EU to food manufacturers, rather than allow the
market, nor could they imagine themselves selling manufacturers to determine what products are sold
machines ‘tarted-up’ like those made in Europe. The in their stores. There is less shelf space available,
cultures were different and irreconcilable with one and that space must be used with maximal efficiency
model. To be successful, the company had to adapt for the company to make a profit.
its production and sales methods to suit two different
groups of customers.
The EU will tolerate higher ingredient prices and INVEST IN EXPANSION
higher prices for finished products than can be
obtained in the US, if the performance of the product There are many different ways for a business to
will attract more sales. This represents an opportunity expand—all businesses are different and most have
for US companies to earn higher profits in the EU, different objectives. The optimal strategy depends
but they have to tailor the marketing and sales not only on the type of product, but also on the
approach to fit these customers. dynamics of the target market. Successful geographic
Conversely, the US remains the land of opportunity market development relies on good local knowledge
for many EU companies. However, the requirements in terms of consumer preferences, regulation and
of US customers can be very different from those in marketing practices as well as an appraisal of local
the EU. competition and the barriers to entry.
Why should this be true? The following observations Many companies fail to take full advantage of an
are perhaps over-stated generalizations, but they are existing or a new market because of inadequate
made to illustrate that differences exist and should groundwork and preparation. Companies must invest
be considered when planning market expansion. more time, money and resources in order to carry
In the food and pet food businesses, one important out a thorough investigation of opportunities to expand
difference between the US and the EU is the strength their businesses.
of the retailer. In the US, it is commonly accepted
that the retailer is a ‘realtor’ renting shelf space to
manufacturers to display their products. The retailer INDUSTRY CHANGE
makes a small margin and may have relatively little Globalization means accelerated growth for the pet
interest in whether the product sells or not. Displays food industry. Factors that are leading to
and stock management can be left to the globalization include:
manufacturers. Even the production and management
• Falling trade barriers. As barriers fall, pet food Table 1. Estimates of the sales of various pet food companies in
US$ billions, retail sales prices1,2.
companies can use more efficient plants to serve
groups of countries vs building a plant in each Company 2000 2002
country.
1. Mars Inc. 6.8 7.9
• Homogenized preferences. Pet owners are 2. Nestlé SA 7.8 7.8
becoming similar in their pet food wants and 3. Iams Co., The 2.1 2.6
needs. Increasingly, they are exposed to the same 4. Hill’s Pet Nutrition 1.9 2.0
5. Del Monte Foods Co. 0.0 1.2
media messages (news, entertainment, sports, 6. Doane NA 0.88
advertising). 7. Nutro Products Inc. 0.52 0.60
8. Agrolimen SA 0.0 0.35
• Big technology investments. Pet food product 9. Uni-Charm Corp. 0.22 0.24
development is becoming more sophisticated. 10. Meow Mix Co., The 0.0 0.23
Manufacturing process research is also becoming 11. Heinz Co., HJ 1.2 0.04
more important. 12. Royal Canin SA 0.56 0.0
13. Private label 3.01 3.2
• Easier movement. It is getting easier, faster and 1
Euromonitor estimates that global pet food sales (retail, except
cheaper to move information, money and people Doane) were US$32 billion in 2002.
around the world. Thus, the cost of adopting a 2
Euromonitor and company sources.
global strategy is lower.
Does it have to be this way? Not necessarily. A trend

What does globalization mean to the pet food
towards greater size is not inevitable, and certainly
industry? Pet food companies are likely to become
does not spell oblivion for smaller competitors.
larger, more international and more sophisticated in
When companies expand, they require more
terms of formulation, processing and packaging.
resources and capabilities to meet the needs of new
Globalization means consolidation, expansion and
markets, new product lines, and greater scope of
accelerated market development.
operations. For example in the international arena,
one of the biggest barriers to a firm’s growth can be
a lack of managers with the appropriate training and
Growth strategies experience. In fact, research by Nitsch (2003)
It would be easy to look at the pet food industry suggests that the availability of managerial resources
today and say, “If I’m not Nestlé or Mars, I might as is the strongest determinant of a company’s decision
well give up.” Large, dominant firms seem to have on whether to establish a joint venture or a wholly
all the marketing clout, financial leverage, production owned subsidiary in a new location. A lack of
economies of scale, channel power, R&D capability managers is linked to a preference for joint venturing,
and ability to respond to new opportunities, leaving to a significantly greater degree than a lack of financial
smaller competitors with few options but to wait for or technical resources. Nevertheless, a lack of
the inevitable hostile takeover or market oblivion. technology or money can also be a motivation for
But such a defeatist attitude is premature, to say the seeking a partnership instead of going it alone.
least. Anti-trust action aside, the fact is that the top Nitsche’s research supports a view of strategic
five firms (counting Nestlé and Ralston Purina as a management that is based on resource availability. A
single company) accounted for a little more than half firm, confronted with market opportunities, should
the market share in the US. Market dominance has decide to pursue those that it can execute effectively,
clearly not occurred yet (Table 1). not those which generically seem to be the most
A look at the future, however, may be more attractive. It is more important to do things right
ominous. Of thirteen major new product introductions than to do the right things. If this is true, then a firm
in 2000 and 2001, ten (or 77%) came from one of presented with strategic alternatives has two choices
these top five firms. This statistic, while far from when it comes to assembling the resources necessary
conclusive, nevertheless suggests that innovation is to implement them: either develop or supply the
occurring at a more rapid rate in deeper-pocketed resources internally, or obtain them from outside
companies, and that increasing market dominance by the firm.
larger firms may be just around the corner.
T. Phillips 489
THE POTENTIAL OF ALLIANCES between the principals. The management costs, both
in terms of actual expenditures and in terms of
All forms of business activity, with the exception of management time and focus, can be enormous.
wholly owned subsidiaries, can be broadly called Nevertheless, alliances of all kinds are increasing in
strategic alliances. The partners may be manufacturer popularity across all industries. This is because the
and distributor, or licensor and licensee, in which potential benefits can more than offset the costs. The
case there is a contractual alliance. Alliances can also opportunity to form an alliance can often make the
entail an exchange or pooling of equity capital, used difference between taking advantage of a market
for the creation of a new venture or the purchase of opportunity and not being able to enter the market
an existing business in the foreign country. Equity at all.
joint ventures are thus a special case of strategic Skill in forming and managing alliances in a fast-
alliances. In the pet food context, all such relationships moving industry like pet food can also determine the
are a means for the participants to gain access to difference between companies that are able to survive
resources that they do not possess themselves, as a and prosper, and those which fade away into
means of obtaining a competitive advantage. irrelevance.
If the managers in a firm lack the needed market
knowledge, for example, a joint venture with a local
partner, licensing to a local manufacturer, or exporting Noteworthy trends
to a local distributor may be a wiser choice than
attempting to go it alone. Similarly, a need to upgrade The global market for pet food and pet care products
the firm’s skills technologically might lead to a rose 2.6% to reach US$46 billion in 2002 (retail
decision to create a joint venture with a partner that sales), recording a stronger increase compared to the
has the desired capabilities, and from whom the firm two previous years (Crossley, 2003). Sales growth
can learn. The reverse is also possible, that is, a local was depressed in 2000 and 2001, partly as a result of
company can be a licensee to a foreign firm, or weak currencies against the US dollar. In volume
become a local distributor for someone else’s product. terms, the market also recorded growth reaching 16.7
Some companies are both licensors of some of their million tons.
own products, and licensees for those of others.
Alliances can thus represent an opportunity for a
relatively small player in an industry to ‘punch above MAJOR MARKET SALES
its weight’ in the competitive arena. Technology
lacking? License someone else’s product to earn North America is the largest region, with 41% of
incremental profits or fill a market niche. Not big global market value in 2002, 39% coming from the
enough to get maximum scale economies? Have your US. The US remains by far the largest national market
product produced by a larger competitor with excess in terms of total value sales, and has by no means
capacity. Can’t afford the R&D to do what you need? reached saturation. While over 90% of cats are
Pool resources with others to amass the research bench currently being fed prepared food, the penetration
strength necessary. levels of prepared dog food continue to be lower,
Obviously, there is no free lunch. Alliances, standing at less than 80% in 2002. Consequently, there
especially equity joint ventures, represent a remains strong possibility for growth, in particular
collaboration out of which all partners must be able for dog food. A continuing rise in the cat population
to gain something. This is one of the key things to ensures a growing consumer base in this sector.
remember in the search for an appropriate alliance Growth is moreover a result of consumers moving
partner. It can be useful to think not in terms of who to premium food and spending money on non-
is the ideal partner, but how you can be a better essential products such as treats or pet care products.
partner yourself. Total market value rose by nearly 5% in the US during
Alliances can also be challenging to manage well. 2002 (Figures 1 and 2).
The sources of potential conflict are endless, ranging Western Europe accounts for a further 30.5%.
from questions about the scope of activity covered Similarly to North America, a rising pet population
by the alliance, to disputes over distribution of the and growing expenditure per pet resulted in strong
gains, staffing issues, managerial authority, strategic value growth in the region.
goal misalignment, and even personality differences
Treats and
mixers
100
Dry food
90
80 Wet food
70
60
%
50
40
30
20
10
0
Dog food Cat food
Figure 1. Pet product breakdown (Combelles, 2003).
Dog and cat food

6%
Pet care products
Other pet food
22%
72%
Figure 2. Breakdown of dog and cat foods (Combelles, 2003).
Japan, the next largest market, represents 13%, of rising disposable incomes, and supported by
although this market is currently stagnating as a result consumer education on pet nutrition, are the main
of the declining pet population and weak economic reasons for particularly strong growth in this now
environment. Pets purchased during the ‘pet boom’ major market.
years of the 1990s gradually died off, and the weak Although China’s value sales seem marginal in
economic environment resulted in fewer consumers comparison with major markets of Western Europe
being willing to replace their pets. or Latin America, the country’s sales increased
Mexico, which ranks ninth in terms of total value rapidly and its growth potential is indeed remarkable.
sales, recorded one of the strongest growth rates. Value In 2002, value rose by nearly 13%, as rising
growth of over 25% in 2002, and nearly 140% from disposable incomes and a gradually improving retail
1998 to 2002. Rapidly rising penetration levels of infrastructure enable more and more pet owners to
industrially prepared dog and cat food, partly a result purchase prepared food as well as pet care products.
T. Phillips 491
GROWTH STIMULANTS expected to continue to rise in the foreseeable future,

as urbanization and hectic lifestyles often result in
Manufacturers lead growth in mature markets people living on their own, turning to pets as
In mature markets of North America, Western Europe companions.
and Japan, manufacturers lead market growth, often
Rising pet ownership levels in emerging markets
through new product launches. As penetration levels
of prepared dog and cat food are high and even close In emerging markets, growth was also a result of
to saturation in some markets, product innovation is rising pet ownership levels. Value growth was,
of paramount importance to expansion, encouraging however, not dependent on consumers moving to
consumers to move to higher value products utilizing premium varieties, but a result of rising penetration
advanced formulations for food, health care and levels of industrially prepared dog and cat food.
hygiene products. Product segmentation strategies Rising disposable incomes, as well as efforts by
also stimulate growth, via premium brands for various manufacturers and trade associations to educate
life stages, lifestyles and breeds of pets. consumers on pet nutrition, play a major role in the
increasing acceptance of prepared food in emerging
‘Humanization’ of pets markets.
The ongoing trend towards ‘humanization’ of pets
Growth surge in premium and superpremium dry
has also proven instrumental in sustaining growth in
food
even the most saturated of markets, with consumers
inclined to spend excessively on products echoing Growth in most mature markets was underpinned by
human tastes. This was most evident in the growth the rise of higher-priced dry premium and
of gourmet flavors of pet food, particularly for cats, superpremium pet foods, particularly in the core dog
mirroring human taste in cuisine, but was also evident and cat food sectors. A trend towards such products
in the growth of treats, gifts and other extravagant was noticeable in North America, Western Europe as
luxury items purchased for animals by their owners. well as Japan. It was partly a result of manufacturers’
Humanization is also increasingly prevalent in product innovation being concentrated in the
emerging markets, as evident by the growing premium end of the market—products which in turn
popularity of nonessential pet care products in were very well received by consumers who are
countries such as China and South Korea. increasingly concerned about pet health and well-
being.
Improvements in distribution add value to global
sales Economy and mid-priced brands maintain a large
customer base
In the major markets of North America, Western
Europe and Japan, growth of the pet superstore made Although premium dog and cat food enjoyed more
pet accessories and – most importantly – premium dynamic growth, economy and mid-priced brands
and superpremium dry cat and dog food easily continue to have a large customer base. For many
accessible to a mainstream audience. This trend was cost-conscious consumers, mid-priced products in
compounded with the introduction of the particular are regarded as offering sufficiently high
superpremium brand Iams into supermarkets in the quality to ensure adequate nutrition for their pets.
US and parts of Western Europe. Meanwhile, the Moreover, many mid-priced products now also
growth of supermarkets in major emerging markets incorporate the life stage/lifestyle concept previously
such as China and Brazil served to increase usage of only available within the premium segment. In most
prepared pet food. major markets, in volume terms, sales of mid-priced
dog and cat food continue to be higher than those of
Increase in the pet population ensures customer premium food.
base growth
A worldwide increase in the pet population – Wet vs dry

particularly in cats and dogs – ensures that the In most developed markets, dry dog and cat food
customer base will continue to grow, resulting in enjoyed more dynamic growth compared to wet
strong growth in volume terms. Pet ownership is
varieties, due to health and convenience reasons. The and 2001. Mars and Nestlé are primarily present
trend towards premium, life stage- and lifestyle- within the mid-price segment, but both companies
specific products also led to the increasing popularity focused strongly on establishing themselves also
of dry food. As dry products can be bought in bulk, within the premium segment, partly through product
are relatively easy to store and do not emanate reformulations, and partly through the takeovers.
unpleasant odors, dry food enjoyed stronger growth
compared to wet food for both dogs and cats. Dry
dog and cat food also dominate in emerging markets, Global outlook
albeit for different reasons. As the unit prices for
wet food are very high, most pet owners who are The ‘recession proof’ pet products market is set to
willing and able to purchase prepared food opt for continue to grow strongly in developed and mature
dry products. Wet food has premium status in markets. Over the 2002-2007 period, value sales are
emerging markets and is thus only purchased by a forecast to grow to US$51 billion, as a result of
minority, i.e., the most affluent sections of the increasing pet ownership levels, rising consumption
population. Nevertheless, wet food enjoyed strong levels of industrially prepared food in developing
growth in some emerging markets, most notably markets and consumers moving to premium products
Mexico. in developed markets (Table 2, Figure 3).
Mars/Nestlé ‘duopoly’ Table 2. Pet products: global future outlook.
The global market for pet food and pet care products • Growing market (+16% or US$8 billion)
remains concentrated in the hands of two main • New products in North America and Western Europe
manufacturers—Mars and Nestlé. Recent takeovers,
• Functional pet food
i.e., that of Ralston Purina by Nestlé during 2001
and that of Royal Canin by Mars one year later, further •· China to drive growth in Asia Pacific
added to their already prevalent ‘duopoly’.
• Educational programs
Nevertheless, growth surge in premium and
superpremium cat and dog food saw other • Still a large potential to explore for further growth
manufacturers gain share in 2001, most notably
Procter & Gamble, which benefited from an
expansion of Iams into supermarkets during 2000
60
50
40
Billion US$
30
20
10
0
1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008
Figure 3. Sales of pet products in the world market: forecast to 2008 (Combelles, 2003).
T. Phillips 493
References
Combelles, E., 2003. Market report: Global trends.
Proc. Petfood Forum Europe 2003. pg 80.
Crossley, A., 2003. The future of the petfood industry.
Proc. Petfood Forum 2003. pg 213.
Kvamme, J., 2004. Global players. Petfood Industry,
Watt Publications, Mt. Morris, IL. 46(1):6.
Lisansky, S., 2003. Exploring new markets. Proc.
Petfood Forum Europe 2003. pg 123.
Nitsch, D., 2003. Mergers, acquisitions and alliances.
Proc. Petfood Forum 2003. pg 206.
K.S. Swanson 495
Using nutritional genomics to study canine obesity and diabetes

KELLY S. SWANSON
Incidence of obesity and diabetes

According to the 2000 National Health and Nutrition whereas the second (type 2) is due to a combination
Examination Survey (NHANES), approximately of resistance to insulin action and an inadequate
64% of American adults (>20 years of age) are either compensatory insulin secretory response (ADA,
overweight (>25 body mass index (BMI)) or obese 2002). Type 2 diabetes, also referred to as non-insulin
(>30 BMI) (CDC, 2003). Although a genetic dependent diabetes, is by far the most common form,
component exists, behavioral patterns and food accounting for approximately 90% of cases (Warram
availability are the major factors in the recent increase et al., 1994). Diabetes increases mortality and
in obese populations. Americans are eating more than morbidity rates, as it is associated with several
previous generations due to the availability of a wide complications including heart disease and stroke,
variety of good-tasting, inexpensive, energy-dense blindness, amputations, renal disease, and damage to
foods. Lower amounts of exercise at work, school, the nervous system.
and home are also to blame. Obesity has been equated The parallels that exist between dogs and humans
with aging 20 years, being more strongly linked to as regards recent lifestyle changes, increased lifespan,
chronic diseases than living in poverty, smoking, or and increased incidence of obesity and associated
drinking (Sturm, 2002). Obesity is a major risk factor diseases are remarkable. Obesity is likely the most
for several life-threatening diseases such as heart common disease found in companion animals today.
attack, stroke, non-insulin dependent diabetes, and Up to 40% of dogs presented to veterinarians in the
certain cancers (e.g., colon, breast), leading to US are now overweight, which is significantly higher
approximately 300,000 obesity-related deaths each than just a few decades ago (Sunvold and Bouchard,
year in the US (Marx, 2003). 1998). Because the anomalies associated with canine
Closely associated with obesity is diabetes, which obesity and diabetes are intertwined, it is not
continues to increase in the United States and other surprising that the incidence of diabetes also has
developed nations. Approximately 6% of Americans increased by 3-fold in this same period (Guptill et
have diabetes and an equal number of people are al., 1999). Immune-mediated diabetes (type 1) is the
thought to be in a ‘pre-diabetic’ state (Marx, 2002). most common form found in dogs (Hoenig and Dawe,
Diabetes mellitus is a group of metabolic diseases 1992). Although the type 2 (non-insulin dependent)
characterized by chronic hyperglycemia and form accounts for only 1 in 5 canine diabetic cases
disturbances in carbohydrate, fat, and protein (Feldman and Nelson, 1996), the etiology and gross
metabolism associated with absolute or relative clinical signs are similar to those of humans (Hoenig,
deficiencies in insulin production by pancreatic ß- 1995). This similarity will prove to be beneficial for
cells and/or insulin action at target tissues (Bennett, both species, as dogs are important biomedical models
1994). Several pathologies are responsible for the for several human diseases. Conversely, clinical and
development of diabetes, which can take several nutritional information collected from human
forms. The majority of human diabetes cases fall into experiments may be applied to canines to improve
two broad etiopathogenetic categories. The first (type nutritional and health status.
1) is due to an absolute deficiency of insulin secretion,
496 Using nutritional genomics to study canine obesity and diabetes
The dog has been crucial in understanding glucose This relationship is corroborated by the fact that over
metabolism, pancreatic function, and diabetes, as it 80% of human diabetics are also obese. Knowler et
was the first animal to become diabetic experimentally al. (1990) reported that individuals having a BMI
and was the animal used to determine pancreatic >35 were 100-300% more likely to develop diabetes
function (Mering and Minkowski, 1889). The cause compared with those having a BMI <25. The presence
of obesity in dogs is similar to that in humans; of obesity is not always a positive predictor of
inadequate daily exercise and excessive intake of high diabetes, however, as only 10% of obese people are
quality foods (Mason, 1970). Many of the negative diabetic (Beck-Nielsen and Hother-Nielsen, 1996).
health outcomes of obesity observed in humans also Diabetes resulting from over-nutrition is also common
are present in the dog. Weight gain is associated with in dogs, as most dogs diagnosed with diabetes are
increases in blood pressure, heart rate, plasma volume, overweight. In addition to total caloric intake, the
cardiac output, and fasting insulin concentration type of food consumed also may affect the
(Rocchini et al., 1987). Many of the complications development of diabetes. Several animal experiments
associated with diabetes in humans, including have demonstrated that glucose metabolism and
hypertension (Struble et al., 1998), hyper- homeostasis are influenced by high fat consumption.
cholesterolemia (Barrie et al., 1993), atherosclerosis Several researchers have measured the glycemic and
(Sottiaux, 1999), and retinopathy (Wyman et al., insulinemic responses of pet foods having different
1988), also are present in canines. In fact, the dog is carbohydrate (e.g., starch, soluble and insoluble
a popular model for ocular manifestations because dietary fiber), fat, and protein contents or prepared
diabetes causes cataracts and is the leading cause of using different processing methods (e.g., canned, soft
blindness in dogs (Wyman et al., 1988). Finally, moist, dry) (Holste et al., 1989; Nguyen et al., 1998).
clinical signs of diabetes are similar to those of These experiments usually were performed to identify
humans, with polydipsia and polyuria being the most diets useful for managing dogs suffering from obesity
common signs in newly diagnosed diabetic dogs and diabetes. However, little has been done to identify
(Plotnick and Greco, 1995). ingredients that may contribute to the development
of canine diabetes.
Finally, genetics plays an important role in the
Factors influencing incidence of obesity development of diabetes, as its incidence in racial
and diabetes groups differs substantially. The incidence of type 2
diabetes in African Americans, Hispanics, and whites
The pathogenesis of obesity and type 2 diabetes is a living in the US is approximately 13, 10.2, and 6.5%,
complex process that occurs over an extended period respectively (Marx, 2002). In comparison,
of time in both humans and dogs. Age, genetic approximately 50% of the Pima Indians of Arizona
profile, and dietary/lifestyle habits are highly have the disease. Numerous genetic loci affecting
associated with these metabolic disease states. In diabetes susceptibility, unrelated to racial
humans, diabetes affects only 0.2% of people under categorization, also likely exist. Genotype (genetic
the age of 20, 8.6% of those between 20 and 65, and makeup) also plays an important role in canine
20.1% of people over 65 years old (www.cdc.gov/ metabolic disease states such as obesity and diabetes.
diabetes/pubs/estimates.htm). Dogs may develop Breeds such as Labrador retrievers, Cairn terriers,
diabetes at almost any age, but it is most common cocker spaniels, long-haired dachshunds, Shetland
from 7 to 9 years of age (Nelson, 1995). Mattheeuws sheepdogs, basset hounds, Cavalier King Charles
et al. (1984) reported that as dogs increased in age spaniels, and beagles, have a greater prevalence of
and became heavier in weight, regulation of plasma obesity than other breeds (Mason, 1970; Edney and
glucose and insulin worsened, which is similar in the Smith, 1986). In another experiment, Samoyeds,
human (Glass et al., 1981). Other reports also have miniature schnauzers, miniature poodles, pugs, and
suggested poor regulation of glucose metabolism in toy poodles were found to be at high risk for
geriatric dogs (Sheffy et al., 1985; Mosier, 1989). developing diabetes, while German shepherds, golden
Nutrition also plays a major role in the development retrievers, and American pit bull terriers were at low
of diabetes. Over-consumption of food by normal- risk (Hess et al., 2000). Specific genetic mutations
weight subjects, resulting in weight gain and resulting in diabetes at a young age also have been
adiposity, has been shown to induce hyperinsulinemia identified in dogs. Inherited insulin-dependent
in animal models and humans (Simms et al., 1973). diabetes has been identified in keeshond and Samoyed
K.S. Swanson 497
breeds (Kramer et al., 1980; Kimmel et al., 2002). regulatory sites, and regulatory elements such as
A mutation in the glucose-6-phosphatase gene, a key insulin and glucagon have been described. However,
regulatory enzyme in glucose homeostasis, also has completely understanding the regulation and
been identified in dogs (Feng et al., 1997; Kishnani interaction of metabolic pathways is a complex
et al., 1997). As more genes and genetic problem that will not be easily solved. Energy
polymorphisms (genetic variants) are identified in balance and whole body metabolism is a complex
the canine genome, other populations prone to obesity process involving numerous genes and proteins that
and diabetes may be identified. interact to determine metabolic status. The fact that
most mammalian genomes are estimated to contain
only ~30,000 to 40,000 genes suggests that in
Metabolic diseases are highly complex addition to the overall number of genes present in a
and difficult to study genome, other factors such as temporal and spatial
gene expression patterns, alternative splicing, post-
Despite substantial investments of time and money transitional modification, and protein-protein
by many laboratories, the identification of genes interactions, greatly influence phenotype (physical
contributing to type 2 diabetes has been difficult characteristics). Recent advances in molecular
(Elbein, 1997; Neel, 1999). Type 2 diabetes biology, including high-throughput tools used for gene
frequently goes undiagnosed for several years because and protein functional analyses, will be instrumental
hyperglycemia develops slowly and is not severe in describing and characterizing normal and diseased
enough in early stages for the patient to notice the metabolic states.
classical signs associated with the disease (e.g.,
polyuria, polydipsia, polyphagia, weight loss) (ADA,
2002). Type 2 diabetes often is not diagnosed until Importance of canine genome sequencing
overt diabetes, an irreversible part of the disease cycle,
is present. This may be especially true for companion Because genomic data are often needed to implement
animals because owners may not notice clinical signs the use of the emerging molecular biological
of disease until it has progressed to an irreversible techniques, canine genome sequencing is crucial.
stage. Therefore, screening tools are needed to Because of their similarities to human physiology
identify the progression of diabetes that is undetected and genome structure, the dog is a powerful
by serological indices (e.g., glucose and insulin biomedical model. Canine models of human diseases
concentrations). Although fasting plasma glucose and are important in identifying disease-causing genes,
other serological indices may be helpful in that they analyzing gene and protein function, and developing
are able to detect glucose intolerance and early stages treatment strategies. As approximately 50% of canine
of diabetes, they are not able to predict its genetic diseases have a human counterpart,
development. Screening tools able to predict the researchers have demonstrated the dog’s utility as a
disease are needed so nutritional and exercise biomedical model of numerous human diseases
strategies may be implemented to prevent or prolong including obesity and diabetes (O’Brien et al., 2002).
its development. Gene expression profiling may be a The importance of the dog as a biomedical model is
method to detect early stages of diseases such as indicated by its rank in the National Human Genome
diabetes before gross clinical signs are noticeable. Research Initiative (NHGRI) genome sequencing
Because previous efforts to identify genetic loci scheme (http://genome.gov). The dog is the first
responsible for the complex pathogenesis of obesity nonrodent mammalian animal model to be sequenced
and type 2 diabetes have had little success, a better and is expected to be completed (6.5 X coverage) by
understanding of metabolic pathways in healthy June 2004 (www.genome.wi.mit.edu/media/2003/
individuals may be needed before confronting such pr_03_tasha.html). In addition to the canine
complex metabolic diseases. Nutritionists are sequencing funded and coordinated by NIH, a
beginning to use the powerful molecular biological privately-funded project recently sequenced ~70-80%
techniques available, but the nutritional genomics of the canine genome (Kirkness et al., 2003). In
field is still in its infancy. Metabolic pathways such addition to the continual supply of sequence data
as the glycolytic pathway, which was completely added to the public databases, researchers have access
elucidated in 1940, have been studied to a great extent to canine genetic maps. Breen et al. (2001) published
(Stryer, 1995). Substrate and enzyme structures, the first fully integrated, comprehensive map of the
canine genome. This 1800-marker map (containing regards genome sequencing has been astounding, the
320 genes and 1078 microsatellites) covered >90% vast amount of information it provides comes without
of the canine genome. Shortly thereafter, Guyon et interpretation. Genome structure is important, but
al. (2003) published another map of the canine the function, regulation, and interaction of genes and
genome that contained 3270 markers. These gene products (proteins) have the major influence
established genetic maps are crucial in ordering genes on phenotype. Therefore, assessing gene function by
in the canine genome as the sequence information analyzing RNA and protein expression, localizing
continues to be added to public databases. proteins, and determining the significance of protein-
Comparative mapping with human and mouse protein interactions are of major importance
genome sequences, which have already been (International Human Genome Sequencing Consort-
completed, will increase the speed at which this ium, 2001). As the field of functional genomics and
process occurs in dogs. proteomics (study of protein profiles) matures, our
Although sequencing the dog genome was initiated understanding of gene and protein function, cellular
because of its importance as a biomedical model for function, and physiology will be greatly enhanced.
humans, dogs and their owners also will greatly This newfound knowledge will allow scientists to
benefit from this information. The most immediate fully utilize genome sequence data and lead to an
impact of genome sequencing may be the detection improved understanding of the biological systems of
and elimination of canine genetic diseases by the body and subsequently develop effective
identifying genes responsible for them. At present, prevention and (or) treatment therapies for disease.
approximately 450 canine genetic diseases have been This area of research also will be very important for
identified (http://www.angis.org.au/Databases/BIRX/ the field of nutritional sciences. If applied correctly,
omia). Test results may be used to eliminate carriers nutritional genomics will enhance our understanding
from the breeding population in order to decrease or of metabolic pathways and aid in maximizing dog
eliminate incidence of disease. Due to high sequence nutritional and health status.
homology among dogs, humans, and mice, Dietary constituents can up-regulate or down-
comparative mapping will be helpful in correctly regulate gene expression directly, as is the case for
placing the genes in the canine genome. Once certain vitamins and minerals, or by indirect means
sequence information is known, genetic (e.g., dietary fiber) through hormonal signaling,
polymorphisms may be identified. Further testing will mechanical stimuli, or metabolites produced from
be required to identify which of these polymorphisms gut microflora (Cousins, 1999). Changes in gene
are important in health and disease. Some common expression have been used to study a broad range of
genomic terms are presented in Table 1. topics, including caloric restriction (Lee et al., 1999),
vitamin (Nur et al., 2002) and mineral deficiencies
Table 1. Common genomic terms. (Blanchard et al., 2001), glucose metabolism (Uyeda
Functional genomics: the study of the function of every gene et al., 2002), and diseases affecting nutritional status
encoded in a genome. (Gannon and Nuttall, 1997). However, the effects of
Genome: the totality of all DNA in an organism. nutrition on gene expression in companion animals
Genomics: the study of genomes, including genome mapping and
sequencing. have not yet been tested. Several areas of companion
Genotype: the genetic makeup of an organism. animal nutrition are not well studied, but experimental
Nutritional genomics: the study of nutritional effects on gene work may be enhanced by using nutritional genomic
expression. techniques, including the determination of minimal,
Phenotype: the physical characteristics of an organism.
Polymorphism: a variation in DNA sequence. optimal, and toxic concentrations of all nutrients,
Proteomics: the study of the full complement of proteins found in efficacy and toxicity testing of novel ingredients,
an organism. effects of nutrition on development, prevention, and
Transcription: the creation of a messenger RNA strand from a treatment of complex diseases, and genetic
DNA strand.
Translation: the creation of a protein from a messenger RNA polymorphisms important for nutritional metabolism
strand. and requirements and susceptibility to diseases.
Genomic technology may be used to identify
genetic and dietary factors responsible for the
Utility of functional and nutritional development of a complex disease and aid in the
genomics development of prevention and treatment strategies
and therapeutics. Continued advances in
Although the progress made in the past decade as
K.S. Swanson 499
biotechnology have provided scientists with powerful been available only on a proprietary basis at an
research tools that will generate more informative extremely high cost. However, increased knowledge
and less invasive research. Many of these new tools in both key areas (canine genome, microarray
have been in the area of functional genomics, the technology) in the past decade is enabling the
study of assessing gene function. One of these production of canine microarrays using gene-specific
techniques, microarray technology, has become very probes. Because options are currently limited, canine
popular because it enables researchers to measure the microarrays are still very expensive ($400 to $500/
expression of hundreds to thousands of genes slide). However, as competition in the marketplace
simultaneously (Pease et al., 1994; Schena et al., increases, these costs will likely decrease. By
1995). This snapshot of gene expression provides a generating gene expression profiles of canine blood
global perspective of the cellular or tissue metabolic and liver samples already known to have metabolic
state, rather than measuring only a few genes at a differences (but in healthy animals), relationships
time, which limited classical techniques. By using between these datasets may be established. Along with
this technology, gene expression profiles may be understanding gene function and the effects of gene-
generated and correlated with metabolic indices from gene interactions, these relationships will lay the
blood or tissue samples to identify relationships foundation for future experiments studying complex
important to understanding whole body metabolism. diseases such as obesity and type 2 diabetes.
To be less invasive and provide more accurate
analysis, several microgenomic techniques also have
been developed in the past decade. Laser capture Illinois canine nutritional genomics
microdissection (LCM) enables researchers to isolate experiment
and study pure cellular populations from a
heterogeneous tissue. In combination with RNA Our laboratory recently conducted a 12-month
isolation and amplification procedures, LCM has experiment evaluating the effects of diet on gene
become a very important tool for researchers and expression in healthy elderly (11 years old at baseline)
clinicians with small sample sizes (Simone et al., and weanling (8 weeks old at baseline) dogs. Due to
1998). Because LCM allows researchers to perform the lack of knowledge in this area, this initial
cell-specific genomic analysis on small samples such experiment was designed to evaluate major differences
as biopsies, this technique has become very popular in age and diet on canine gene expression profiles.
in cancer and gastrointestinal research. Because In this experiment, an animal product-based diet was
individual cells may be selected for analysis using compared with a plant product-based diet. Blood and
LCM, results are not skewed or diluted by unwanted liver biopsy samples were collected over the course
cell populations in the sample. Our laboratory is of the experiment and tissue samples were harvested
currently using LCM and microarray technology to at its end for isolation of RNA to be used for
compare gene expression profiles of intestinal villus microarray analysis. In addition to RNA isolation,
and crypt cell populations. blood samples were used for determining serum
Rather than attempting to tackle complex metabolic metabolite concentrations and complete blood count.
diseases such as obesity and diabetes immediately, Fecal and colonic digesta samples were used to
comparison of gene expression profiles from simple measure nutrient digestibility and fermentative end-
differences in dietary status or composition (e.g., product concentrations. Tissue samples of various
fasted vs fed, animal vs plant-based ingredients) may regions of the gut also were collected for the
be the place to begin. Once relationships of normal measurement of gut morphology and for RNA
metabolic status are established, gene expression isolation using LCM, which will generate intestinal
profiles or ‘signatures’ of disease may be identified villus cell- and crypt cell-specific gene expression
in future experiments. These signatures will reveal profiles. Our primary goal was achieved, as several
abnormalities in metabolism that may be important metabolic differences due to age and diet were
for understanding disease development, developing identified. Nutrient digestibility, hematology, and
biomarkers of disease, and choosing effective serum chemistry data were recently submitted for
therapies. Due to the lack of knowledge regarding publication (Swanson et al., 2004).
the canine genome and the expense involved with We are currently in the process of generating gene
microarray technology in the past, canine microarrays expression profiles that may be correlated with the
containing a limited number of known genes have metabolic data already collected. Significant
correlations may identify mechanisms responsible for Lippincott-Raven, Philadelphia, PA.

changes observed in biological systems (e.g., Bennett, P.H. 1994. Definition, diagnosis, and
metabolic pathways, immune function) due to age classification of diabetes mellitus and impaired
and diet. These results also will be used to design glucose tolerance. In: Joslin’s Diabetes Mellitus
future experiments in our laboratory evaluating the (Kahn, C.R., and G.C. Weir, eds.). Lea & Febiger,
effects of nutrition on obesity and diabetes. We plan Malvern, PA. Pages 193-200.
to identify biomarkers of disease and test dietary Blanchard, R.K., J.B. Moore, C.L. Green, and R.J.
regimens that may prevent disease or improve Cousins. 2001. Modulation of intestinal gene
nutritional and health status. expression by dietary zinc status: Effectiveness of
cDNA arrays for expression profiling of a single
nutrient deficiency. Proc. Nat’l Acad. Sci.
Conclusions 98:13507-13513.
Now that pets are living longer lives due to improved Breen, M., S. Jouquand, C. Renier, C.S. Mellersh,
nutrition and veterinary care, the incidence of C. Hitte, N.G. Holmes, A. Chéron, N. Suter, F.
complex metabolic diseases such as obesity and Vignaux, A.E. Bristow, C. Priat, E. McCann, C.
diabetes has increased. These complex diseases are André, S. Boundy, P. Gitsham, R. Thomas, W.L.
difficult to study experimentally because of the time Bridge, H.F. Spriggs, E.J. Ryder, A. Curson, J.
they take to develop and the various environmental Sampson, E.A. Ostrander, M.M. Binns and F.
and genetic factors involved. However, emerging Galibert. 2001. Chromosome-specific single-locus
genomic technologies are enabling scientists to FISH probes allow anchorage of an 1800-marker
perform more definitive and less invasive research integrated radiation-hybrid/linkage map of the
that may enhance our understanding of normal and domestic dog genome to all chromosomes. Genome
diseased metabolic states. Nutritional genomics may Res. 11:1784-1795.
enable researchers to identify biomarkers of these CDC, 2003. Healthy weight, overweight, and obesity
diseases so they may be detected in early stages and among US adults. National Health and Nutrition
treated appropriately. The current canine genome Examination Survey. http://www.cdc.gov/nchs/
sequencing initiative will be crucial to improving data/nhanes/databriefs/adultweight.pdf.
canine health, as it will allow researchers to locate Cousins, R.J. 1999. Nutritional regulation of gene
and determine the function of genes and identify expression. Am. J. Med. 106:20S-23S.
genetic polymorphisms that influence metabolism, Edney, A.T.B., and P.M. Smith. 1986. Study of
immune status, and other biological systems. These obesity in dogs visiting veterinary practices in the
technologies will not only identify populations prone United Kingdom. Vet. Record 118:391-396.
to disease, but will play an important role in developing
Elbein, S.C. 1997. The genetics of human non-
strategies for prevention and (or) treatment.
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A. Thompson 503
A peek into the new NRC for dogs and cats

ANGELE THOMPSON
Thompson PetTech, New Providence, New Jersey, USA
Introduction: new directions for the NRC

book for companion animals
Three years ago, I spoke to this audience with great the references are given. There are entirely new
anticipation of the release of the National Research sections. The discussions include not only essential
Council’s Nutrient Requirements for Dogs and Cats. nutrients, but also non-essential nutrients. There are
I identified areas of nutrition research where gaps synergies in the combination of the two species
still existed in our knowledge and tried to predict allowing for the combination of basic information.
some of the new information that might appear in However that same combination is often confusing
the new NRC publication. While the release of the in its organization, leaving the reader struggling to
final document has been delayed until mid-2004, a be sure which species is under discussion.
pre-publication document was made available in Each edition of NRC publications has a new and
November of 2003 (NRC, 2003). My excitement different expert committee chosen to write the
diminished after looking at the pre-publication. There publication. Each committee gets a slightly different
is a tremendous amount of good work in the pre- charge for its job. The 2003 committee was told to
publication; but there were parts that gave me, as an use only peer-reviewed documents. The committee
end user of the document, serious concern. Let me writing the 1986 NRC for cats was able to use
stress to anyone attempting to use the pre-publication ‘published research and practical experience’ (NRC,
document, that it is NOT the final version. The NRC 1986). As quoted from the 2003 pre-publication
staff has been resolute that the final document will overview, “This edition differs from the others
look exactly like the pre-publication document with because it contains the latest data on requirements,
the exception of editorial changes. I cannot predict based on the utilization of nutrients in ingredients
what will be deemed an editorial change and what commonly produced and commercially available in
will not. My comments are based on the pre- dog and cat food rather than only on purified diets.”
publication version with caveats. I have quoted the The nutrient requirement tables in the 1985 dog book
pre-publication document (as NRC, 2003) and these and the 1986 cat book were based on purified diets
cited sections might be changed in the final version. and at least in the cat publication, “A margin of safety
It has been nearly 19 years since the 1985 NRC was intentionally not incorporated…” (NRC, 1985;
Nutrient Requirements for Dogs was released. That 1986). Thus, the nutrient requirement tables in the
edition contained 79 pages. The 1986 NRC Nutrient two earlier editions could not be put into practical
Requirements for Cats contained 78 pages. The 2003 use without adjustments and could not be used directly
pre-publication version of the new combined dog and for regulatory purposes. This is one reason that the
cat edition contained 447 pages and had no index AAFCO Dog and Cat Food Nutrient Profiles were
(NRC, 2003). The difference between the editions developed (AAFCO, 2004). In the text of the new
shows the immense amount of work done by the edition, comments noted that for some values
contributors to the document. The text was greatly adjustments have been included to account for
expanded. The review of literature was extensive and bioavailability, but not for all values. So, the 2003
504 A peek into the new NRC for dogs and cats
publication appears to be related to practical diets paragraphs on obesity in dogs and cats in the more-
but not in a consistent manner. It is important to than-400 pages, but the vast majority of the document
remember that the nutrient values are is NOT about obesity. As you will see below, it is
recommendations for healthy animals and normal about much more.
function, not for animals in disease states. The 2003
committee was made aware that the publication would New topics included
be promoted for a wide variety of audiences:
professionals in the industry, academia, students, Dogs and cats are complex organisms with many
government officials and pet owners. Thus, “…the factors influencing their nutrient needs. This new
committee chose to err on the side of caution and edition tries to tell the entire story. The animal must
include adequate detail from the literature cited to eat the food, digest the food, absorb the components
provide a clear roadmap for how the recommendations of the food and the body has to metabolize those
were derived” (NRC, 2003). The publication is a components. In addition, nutrient needs are controlled
serious scientific document addressing the complexity by physical activity and various environmental
of nutritional science for dogs and cats. While there factors. Four entirely new topics have been included:
are knowledgeable individuals in the public audience, comparative digestive physiology, feeding behavior,
the publication is not casual, accessible reading. special considerations for laboratory animals, and
There is a vast amount in this new edition to digest. physical activity and climate. The new topics
The initial review of the pre-publication version by emphasize the broader view of this publication
industry professionals found errors and raised serious compared to past ones. No longer are the minimum
questions about parts of the document. As with requirements for the essential nutrients the entire
previous editions, the tables in this book must not be purpose of the document. All of these factors are
used in isolation from the text. The values in the covered in the new edition, which should be
tables are based on certain assumptions about energy considered a strength of the publication.
content and do not translate directly to most products
in the marketplace. This is especially important for
government regulators, both in the US and in other Changes to the tables of requirements
countries, to know and understand. The Pet Food
Institute has already received numerous inquiries from The traditional table of requirements, which in the
other countries wishing to use the publication for past listed minimum requirements for growth and
regulatory purposes. Regulators in the US are maintenance, has become multiple tables expanded
planning a review of the practical, usable AAFCO to include reproduction. Each table now has a
Dog and Cat Food Nutrient Profiles, once the final complicated four-category system of values,
NRC document is available (AAFCO, 2004). That depending on the information available: minimal
does not necessarily mean that the AAFCO Nutrient requirement, adequate intake, recommended
Profiles will change. It means that the latest allowance and safe upper limit. The requirements
information will be considered and may be have new assumptions for specific body weights, and
incorporated. Other countries, in the past, have not in lactation for specific numbers of kittens/puppies.
used the AAFCO Profiles, but continued to rely on Footnotes are given for adjustment of the values when
the NRC minimum requirement values to judge the body weights or numbers of offspring change. There
nutritional adequacy of pet products. The values in are new units appearing in the tables as well. Where
the tables in this new edition of the NRC document the previous edition utilized percentages or parts per
should not be used directly for regulatory purposes. million on a dry matter basis plus amounts per 1000
This is a scientific document, not a regulatory kcal ME; the new edition has four types of units.
document. I cannot state that strongly enough. The requirements are represented as: amount per kg
Finally, some of you will have seen the newspaper dry matter, amount per 1000 kcal ME, amount per
headlines the morning after the press conference on kg body weight and amount per kg metabolic body
the pub-publication release. “Pudgy Pets are Packing weight (BW 0.75). These extra units were included to
on Pounds,” cried the Wall Street Journal (Foley, assist veterinarians in customizing dietary support for
2003). “Chew on This: Pets are Pudgy, Too” individual patients. In short, the tables look
exclaimed the Los Angeles Times (Mestel, 2003). significantly different from those of past editions.
Yes, the publication does include a handful of
A. Thompson 505
Essential nutrients sufficient for all breeds.” Why then the footnote?
Perhaps the second part of the footnote refers to only
In my previous paper, I identified the following gaps one column of the table, which then would be more
that I thought might be covered in the new edition: consistent with the text.
taurine for cats (would such be listed for dogs?); While there is a long discussion of fatty acids in
linoleic acid for dogs; levels of calcium and the text (about which more will be discussed later),
phosphorus for large breed dogs; copper requirements minimal requirements are not set for linoleic acid in
for reproducing cats; and finally, vitamin K for cats. either species, nor are minimal requirements given
I suspected that some of the other gaps in nutrient for arachidonic acid for cats. Adequate intakes are
requirements would remain gaps. These included given for linoleic and arachidonic acids for both
manganese for dogs, iodine and selenium for cats, species. As I said three years ago, this gap in research
the effect of interactions of minerals on availability, on known essential fatty acids seemed so basic, and
and vitamin requirements for reproduction. yet the needed research just was not done to allow
Taurine was included as an essential nutrient for the establishment of minimal requirements.
cats with values given for minimal requirements. Despite some relevant research, a minimal
Taurine has been extensively researched in the years requirement for copper is not set for cats during
since the last NRC edition. This research is well reproduction citing “…the absence of specific
recognized in the publication. Taurine needs in dogs experimental data…” (NRC, 2003). The available
are discussed in the text, but a minimal requirement data were reviewed, deemed not specific enough and
is not included in the table. The committee did not only an adequate intake is listed in the table. Many
find peer-reviewed, dose-response experiments that of the trace minerals are in the same situation,
supported the establishment of a minimal adequate levels are set but not minimal requirements.
requirement. They did comment on the practice of This is the case for both dogs and cats. The greatest
inclusion of taurine in lower protein diets for dogs, number of minimum requirements for trace minerals
which allows the maintenance of taurine pools. is set for growth in both species. Clearly, trace mineral
Calcium and phosphorus levels in puppies of research is still an open area.
different breeds are discussed, which is good news. Several vitamin requirements remain open for both
But the bad news is that the user of the tables must species. I had speculated that a vitamin K requirement
carefully read the text in order to set values in diets. might be set for cats, but while there are some data,
For example, a minimum calcium requirement for the committee’s reports were not enough to set a
puppies is set in the table, with a footnote which minimal requirement value. Only one minimal
states “For calcium and phosphorus, the need may vitamin requirement is set for dogs, which is for
decrease by up to 20% per unit energy between 60 riboflavin in adult maintenance.
and 100% of mature body weight. Values are for giant Although research had been done for various
breeds and may be different in others (see text)” essential nutrients, the experiments did not fulfill the
(NRC, 2003). This is an example of the complications committee’s criteria needed to set the minimal
inherent in setting nutrient requirements. The table requirements.
of requirement values for growth of puppies is set
using a model 3-month old puppy weighing 5.5 kg,
consuming 1000 kcal ME per day, but the footnote Non-essential nutrients
says the calcium and phosphorus values are set for
giant breed puppies. The last 3-month old St. Bernard One of the two biggest categories of non-essential
puppy I saw weighed closer to 18 kg than 5.5 kg. A nutrients identified for consideration three years ago
reading of the text shows “…a minimum requirement was omega-3 fatty acids (referred to in the text as n-
of 2.0 g Ca per 1000 kcal ME (8.0 g Ca per kg, 4000 3 fatty acids). The 2003 pre-publication did recognize
kcal ME per kg) may be set as the minimum the importance of this area. The text discusses not
requirement of dietary Ca that will support normal only the details of the n-6 fatty acids: linoleic and
growth in puppies of both large and small breeds.” arachidonic acids, but also includes the n-3 fatty acids:
The table value for minimal requirement is indeed alpha-linolenic (ALA), eicosapentaeoic (EPA), and
2.0 g Ca per 1000 kcal ME. The text additionally docosahexaeonic (DHA). The text states “Both n-6
says that the recommended allowance of 3.0 g Ca and n-3 fatty acids are essential for dogs and cats.”
per 1000 kcal ME (the value in the table) “should be However, it also states “A specific requirement for
long-chain n-3 PUFAs (EPA and DHA) in adult dogs carbohydrates section, the 2003 committee made a
has not been identified to date.” Under some great effort in the energy chapter. The four pages in
circumstances certain fatty acids were noted as the 1985 NRC for dogs and the three pages in the
conditionally essential. All of the above fatty acids 1986 NRC for cats have become 26 pages in the 2003
are listed in the tables for both species and adequate edition. (NRC, 1985; 1986; 2003) The Holy Grail
intake values, (not minimal requirements) are set for has been one energy requirement calculation equation
all. Does inclusion in the tables recognize all these for each species, which is clearly not appropriate based
fatty acids as essential or not? on the research. This is reflected in the tables, where
There is a listing for the combination of EPA and energy requirement calculation equations for each
DHA in each of the tables. The tables for adult dogs, species at each life stage are provided. These
reproducing dogs and adult cats include a footnote equations are much more sophisticated than those
regarding a general recommendation about the given in earlier editions. This approach is entirely
appropriate mixture of EPA and DHA for diets, at consistent with the goal of providing end users the
30% of each. What then should the other 40% of n- ability to individualize energy delivery to animals.
3 fatty acids be? How was this ratio established? The However, for regulatory purposes in labeling products
following statement is in the text section on adult with feeding directions, this level of complexity
maintenance of dogs “A general recommendation is presents problems. The AAFCO Model Pet Food
that EPA and DHA should contribute approximately Regulations require that feeding directions be given
one-third each of the total dietary n-3 LCPUFAs.” on all pet foods labeled as complete and balanced for
No reference is given as to the source of this general any and/or all life stages (AAFCO, 2004). These
recommendation and yet the charge to the committee directions must be consistent with the intended use
was to only consider peer-reviewed publications. of the product. This means that if a product is labeled
The second big category identified for consideration for all life stages, then feeding directions must be
three years ago was carbohydrates and fiber. The 2003 given for all life stages. In the 2003 NRC, there is
committee did a great deal of good work in this area. one equation for weaned puppies and one equation
Carbohydrates as a nutrient category were covered for adult dogs but six multiplication factors
in less than four pages total in both previous editions. determined by the dog’s activity level and breed. For
That has now been expanded to 35 pages with a late gestation, there is a two-part equation, while for
complete discussion of all groups in the carbohydrate lactation, there is another two-part equation that
family. The category was divided into four groups: depends on the number of puppies and week of
absorbable, digestible, fermentable and non- lactation. Those equations are all scientifically sound,
fermentable. The text is quite detailed in its discussion but it will be difficult for a non-expert regulator to
of the utility, nutritive value, effects on growth and judge the appropriateness of the feeding directions
reproduction and physiological effects of each group on a label. In addition, how extensive do label feeding
of carbohydrates. It is interesting to note that while directions need to be? This will be for AAFCO Pet
many important nutritional aspects of carbohydrates Food Committee and industry to work together to
are discussed, there are no carbohydrates included in balance accurate science with usability for the
the tables; thus leaving the impression that while they consumer. This is another reason why the nutrient
play a definite role in dog and cat nutrition, they are requirement tables in the 2003 publication should
still not considered essential nutrients. From the US not be put directly in to use as regulation.
regulatory perspective, the only recognition of
carbohydrates in regulation is as crude fiber, a term
not used in the text of the carbohydrates chapter. So Safe Upper Limit (SUL)
carbohydrates are important in nutrition, but not
classically essential, and not a simple nutrient class This is the first appearance of SULs in the NRC for
for regulatory attention. dogs and cats. In fact, SULs have not appeared in
previous NRC publications such as those for swine,
dairy cattle or beef cattle (NRC, 1998; 2000; 2001).
Energy Those documents used the carefully defined words
“maximum tolerable concentration” and did not
The topic of energy received vast research attention include values in the requirement tables. There were
between editions of the publication. As in the too many conditions related to every value that had
A. Thompson 507
to be considered when applying it to a diet. The 2003 would be 1000 times the dietary requirement (NRC,
NRC dog and cat publication defines the SUL as “the 1987). The text states “There are no reports of toxicity
maximal concentration or amount of a nutrient that resulting from excessive oral intakes of thiamin, but
has not been associated with adverse effects…” The intravenous thiamin in high doses can cause
inclusion of SULs in the tables is of great concern to neuromuscular and ganglionic blockade” (NRC,
industry professionals. The concept of upper limits 2003). If the SUL is for parenteral nutrition and not
is not foreign to the industry. The AAFCO Dog and practical commercially available pet foods (the stated
Cat Food Nutrient Profiles include a few maximum intent of the publication), then why is the value
levels for nutrients where valid concerns about toxic included or why is it not at least footnoted to
levels exist (AAFCO, 2004). Industry review of the acknowledge that fact?
pre-publication raised serious doubts about the usage Also of great concern is the inclusion of an SUL
of SULs as they appear in the 2003 NRC. for total fat in each of the tables. This indicates that
The logical interpretation of ‘safe upper limit’ by dietary levels above the stated value would be harmful
government officials and the public is that any product to the animal. But the SUL for total fat was based
containing nutrients at levels higher than those given only on the amount of fat that would result in a
in the publication will harm animals. But that is not reasonable amount of space for protein at the given
the way the values were developed. This is a major caloric content, not a direct safety concern for fat. In
disconnect. Perhaps at least the name should be fact, the footnote for the SUL for total fat states “The
changed to better reflect the concept as used by the upper limit of fat may be greater than values indicated
NRC. The origin of the values throughout the because it is limited by protein concentration
publication appears inconsistent, which indicates that requirement in a diet designed to be 4 kcal ME per g
the meaning of the SUL is inconsistent. For example, DM. Any higher fat content would exceed this
three of the applications of the SUL concept are: assumed caloric density.” (NRC, 2003). However for
someone just looking at the fat level in the diet and
• absolute maximum where toxicity exists; looking at the table, a higher level of fat would be
castigated as harmful. Perhaps this issue should be
• upper level tested in research;
dealt with by inclusion of the footnote without a
• an upper level tested in research with a safety potentially misleading value. In the tables for dogs,
factor added. there is a footnote for ALA in the SUL column. This
footnote indicates a recommended ratio of LA to ALA
In some cases, the SUL represents the highest level and the ratio varies by life stage. In its discussion of
tested in the research, but often no higher level was SUL for LA and ALA, the text states “…so the ratio
tested or shown to result in adverse effects. Thus, of LA to ALA is within the recommended range (see
the highest level tested was set as the SUL, without Table 5-1) “. There are no recommended ranges given
knowledge as to whether or not a higher level would in Table 5-1. Table 5-1 is entitled “List of Abbreviations
result in adverse effects. This seems misleading. of Selected Fatty Acids…” The reference regarding the
Some of the SUL values in the tables show a ‘greater ratios may be in the text; it is certainly difficult to find.
than’ symbol in front of the value. Examples are zinc One very troubling issue in the carbohydrates
for adult cats, amino acids for kittens, lysine for chapter was the last table, which set SULs for various
puppies, sodium and iodine in adult dogs, etc. This ingredients for maintenance of adult animals. This is
appears to mean that the SUL is some unknown value a clear departure from the rest of the publication.
above the value given. I do not see that including SULs in the rest of the document are set for nutrients,
such a value in the table is reasonable. No limit is not ingredients. The inclusion of SULs for ingredients
defined. Perhaps a footnote without a value noting can easily result in the included ingredients being
that there is concern about potentially toxic levels identified as ‘unsafe’. This is a disservice to very
for that nutrient would be appropriate. healthy and useful ingredients. The definition used
Another concern with SUL is that some values here for SUL is “a level of dietary inclusion at which
were set based on a general rule of thumb, which is no adverse effects can be expected.” (NRC, 2003)
not equivalent to the “maximal concentration…not However, of the 23 SULs set for dogs in the 2003
associated with adverse effects.” For example, the NRC publication, 13 of the levels had the following
SUL for thiamin in both species was set based on a footnote, “Higher levels were not tested.” Therefore
suggestion by NRC that the presumed safe upper limit there really are no data to say whether these 13 values
are or are not the SULs. This table also raises the in the overview, “ Gaps in our knowledge of specific
potential for products currently in the marketplace, requirements are noted in the text and by absence of
with histories of safe use, to be deemed ‘unsafe’, data in the requirement tables.” One of the goals for
with no evidence that they are unsafe. this NRC publication is for “…identifying new topics
The concerns about SUL are not limited to the for research.” I think those gaps are evident. Nutrition
above, but they are some of the most visible issues. research for dogs and cats remains an active arena.
This concept and its use are of great concern. It is a We all have many opportunities and a lot of work
serious, troubling possibility that government still to do.
regulators or lay public could use SULs in
inappropriate ways. SUL values, taken directly from
the tables, cannot be utilized in a simple, across the References
board application to all products for the purpose of
judging safety of the products. Association of American Feed Control Officials.
2004. Official Publication. AAFCO, Oxford, IN.
Foley, R.J. 2003. Pudgy Pets Are Packing on Pounds.
Conclusion Wall Street Journal Sept. 9, 2003.
Mestel, R. 2003. Chew on This: Pets Are Pudgy, Too.
I would like to thank the expert committee that put Los Angeles Times. Sept. 9, 2003.
in many hours on a volunteer basis toward the National Research Council. 1985. Nutrient
completion of the publication. They were given a Requirements of Dogs. Revised. National Academy
huge task. Much of the pre-publication document is Press, Washington, DC.
worthwhile. However, there are areas of concern to National Research Council. 1986. Nutrient
at least one end user group, i.e., industry professionals.
Requirements of Cats. Revised ed. National
These areas of concern have been raised to the NRC
Academy Press, Washington, DC.
staff and members of the industry have offered to
National Research Council. 1987. Vitamin Tolerances
work with the expert committee and NRC staff to
help make this publication more sound and usable of Animals. National Academy Press, Washington,
by the various target audiences. The NRC staff DC.
refused these offers. National Research Council. 1998. Nutrient
I would also like to thank a group of very Requirements of Swine. 10th revised ed. National
experienced nutrition professionals who dove in and Academy Press, Washington, DC.
reviewed the pre-publication document when it was National Research Council. 2000. Nutrient
released. These professionals came from all sectors Requirements of Beef Cattle. 7th revised ed. Update.
of the industry. Their comments were valuable to National Academy Press, Washington, DC.
me and I hope will be valuable in the future use of National Research Council. 2001. Nutrient
the final NRC publication. Requirements of Dairy Cattle. 7 th revised ed.
We now await the final publication of the NRC National Academy Press, Washington, DC.
Nutrient Requirements for Dogs and Cats. I think National Research Council. 2003. Nutrient
the peek at the pre-publication document gives us a Requirements of Dogs and Cats. Prepublication
very vital piece of information. There are still gaps copy. National Academy Press, Washington, DC.
in our knowledge of dog and cat nutrition. As noted
L. Tucker 509
The importance of antioxidant protection: demonstrating and branding

benefits in pet food
LUCY TUCKER
Alltech Inc., Stamford, Lincolnshire, United Kingdom
Animal nutrition in the last decade has seen great as oxygen is utilised in respiration, metabolism and
advances in the understanding and application of the energy production. However, oxygen is highly
correct balance of nutrients required in the diet and reactive and potentially toxic (Knight, 1998); and
the quality of the raw materials used. In the fundamental mechanisms such as respiration result
companion animal sector, the focus on specific dietary in the production of oxidizing compounds called free
requirements now means that pet owners have a huge radicals or ‘reactive oxygen species’ (ROS), the
choice of commercial diets of varying specificity and majority being in the chemical form O2-. These are
good nutritional quality available. Often the quality highly unstable and reactive and facilitate various
of pet food is given the same status as human food, chain reactions that damage cell membranes and
as it is bought using the same purchasing decisions tissue function. Free radicals have been identified as
as the family’s weekly groceries. Scares regarding causative agents for pulmonary hypertension
the use of chemicals and drugs in animal feed have syndrome, inflammation, cancerous tumour
heightened consumer worries regarding the safety and development and reproductive disorders. Essentially,
health benefits of their pet’s diet. This, coupled with free radicals reduce cell function and interfere with
the rise in interest in feeding for animal well-being, cell growth by damaging DNA, proteins, lipids (and
longevity and preventative medicine, makes the role hence membranes) and carbohydrates, which is why
of dietary antioxidants increasingly important as a antioxidant deficiency is linked to a variety of
maintainer of health status as well as a potential disorders. Under normal, unstressed physiological
marketing opportunity. conditions, between 3 and 5% of cellular oxygen is
Recently consumers have become more concerned transformed into free radicals (Singal et al., 1998),
with the quality and ingredients that are used in both an amount which increases dramatically under stress
human food and animal feed. The idea that natural conditions. If the immune system is stimulated, free
or organically derived ingredients can help us and radicals are produced in large amounts during the
our pets remain healthy is particularly attractive, as proliferation of immune cells and can be used to
these components are seen as being, by inference, damage and inactivate invading pathogens (Schwartz,
safe to use, whilst having multi-functional but non- 1996; Kettle and Winterburn, 1997; Surai, 2002),
pharmaceutical benefits. Antioxidants fit very well but will also cause further problems for the animal
as natural products strongly associated with health unless they are removed efficiently.
benefits, and are regarded by many as vital ingredients
in animal diets. There is a growing body of research
evidence into how antioxidants work and which Preventing oxidation damage with
dietary sources are the most potent, which lends antioxidants
technical weight to the justification for using such
ingredients in commercial production. Organisms have evolved specific antioxidant
protective mechanisms that helped them to survive
Importance of counteracting oxidation when oxygen concentration in the atmosphere was
rising (Halliwell and Gutteridge, 1999). As a result
Oxidation is a necessary phenomenon of cell functions there are thousands of naturally occurring compounds
510 The importance of antioxidant protection: demonstrating and branding benefits in pet food
possessing antioxidant properties to disable free have bound, reactivating the antioxidant. Two of the
radicals. These compounds work in various ways, as key enzymes involved in antioxidation are superoxide
shown in Table 1. dismutase (SOD) and glutathione peroxidase (GSH-
Px). Certain minerals are integral components of these
Table 1. Description of antioxidant compounds and precursors enzymes and are required in sufficient amounts from
(adapted from Surai, 2002) the diet to ensure synthesis in cell organelles to
Antioxidant Antioxidant function maximise the efficiency of antioxidation. For
example, GSH-Px synthesis is highly dependent on
Vitamin E Chain-breaking antioxidant, prevention of ROS selenium availability. There are two major forms of
proliferation, binding ROS SOD in the cell: manganese SOD, which is located
Vitamin C Restriction of ROS propagation, Vitamin E in mitochondria, and copper or zinc SOD which is
recycling, ROS scavenging
found in the cytosol. This enzyme transforms the O2-
Carotenoids Restriction of ROS propagation, immune free radical to form hydrogen peroxide H2O2 rather
function, mopping up excess ROS
than the more reactive radicals such as hydroxyl free
Glutathione Prevention of ROS formation by enzyme GSH- radical OH*, which would occur otherwise.
Px
Selenium Prevention of ROS formation by enzymes: GSH-
Px, thioredoxin reductase and other
O2-+O2- + 2H+ SOD
H2O2 +O2
(Zn, Mn)
selenoenzymes, recycling vitamin E, DNA repair
Zinc Prevention of free radical formation (superoxide
Hydrogen peroxide is more stable, but still toxic and
dismutase enzyme) must be removed from the cell by a further reaction,
Copper Prevention of free radical formation (superoxide which is facilitated by either GSH-Px or catalase
dismutase enzyme). Must be bound to protein to (CAT). Because GSH-Px is found in many cellular
reduce oxidative potential locations while CAT is located mainly in
Manganese Prevention of free radical formation (superoxide peroxisomes, the efficacy of H2O2 removal from the
dismutase enzyme) cell is greater with GSH-Px (Surai, 2002).
Iron Prevention of free radical formation (catalase
enzyme). Must be bound to protein to reduce H2O2 GSH-Px
H2O
oxidative potential (Se)
As this first level of antioxidant defence in the cell is

The main modes of antioxidant action can be split not sufficient to completely prevent free radical
into three. The first is those compounds that prevent formation and lipid peroxidation, therefore a second
free radical formation in the first place. They do this level of antioxidant defence includes fat-soluble
by binding to co-factors required for formation of (vitamins A, E, carotenoids, ubiquinols) and water-
free radicals, such as iron. However, as is obvious soluble (e.g. ascorbic acid, glutathione, uric acid)
from the immune example above, most reactions that antioxidants. These antioxidants are potent chain-
generate free radicals are very important, so this is breaking compounds, which prevent free radical chain
not always desirable. The second mode of action is formation and propagation. This mechanism is not
the ability to neutralise excess free radicals once they straightforward. For example vitamin E reacts with
have been formed, as seen in compounds such as a lipid peroxyl radical (LOO*), the antioxidant is
vitamin E, and prevent the initiation of cascade oxidised and lipid hydroperoxide (LOOH) is
reactions. The properties of such antioxidants can be produced in the following reaction:
measured via a standard test known as a Trolox
Equivalent Antioxidant Capacity (TEAC) analysis, Vitamin E + LOO* Vitamin E-radical
where their chelating strength is measured against +LOOH
vitamin E in a standard free radical solution. These
compounds, however, can only bind a finite amount Lipid hydroperoxide products can react with metals
of free radicals, after which they become inactive. A such as iron to form cytotoxic products such as
constant dietary supply of antioxidant nutrients is aldehydes, alkoxyl radicals (LO*) and peroxyl
therefore essential. This leads to the third mode of radicals (LOO*):
action, which is via the important antioxidant enzyme
system in cells. These compounds work in concert LOOH + Fe2+ LO* + Fe3+ + OH-
with the chelating antioxidants by ‘recycling’ them LOOH + Fe3+ LOO* + Fe2+ + H+
– i.e. removing and neutralising the free radicals they
L. Tucker 511
Again, GSH-Px is required to deal with lipid been found to be less well retained compared to
hydroperoxides. Therefore selenium as an integral organic sources of these metals. This is because
part of GSH-Px belongs to the first and second levels animals have evolved mechanisms to extract organic
of antioxidant defence. Even the second level of forms from the diet, which are usually bound to amino
antioxidant defence is not able to prevent lipid acids (Surai, 2000). An example of this is the organic
peroxidation and some biological molecules are selenium synthesised and stored in certain yeasts,
damaged. In this case the third level of antioxidant which is primarily in the form of selenomethionine
defence deals with the repair of damaged molecules, (as found in plant materials). This compound is
consisting of specific enzymes such as proteases or readily absorbed and stored in the body, making it
lipases (Surai, 2002). an effective source of selenium for synthesis of
Whilst each level of antioxidant protection is antioxidant enzymes during times of stress. Certain
important in its own right, it is only by ensuring botanical ingredients have shown strong antioxidant
they all work in harmony that complete protection properties, and are the focus of research in animal
can be afforded to the animal. It also ensures the and human health as potential food and feed
efficient use of antioxidant resources that are often supplements. Hops, rosemary, thyme, turmeric and
poorly stored in the body or in limited supply. An grapeseed extracts show interesting levels of
example is the efficient recycling of vitamin E in the antioxidant activity (Bagchi et al., 1998; Tucker,
presence of sufficient vitamin C and selenium. 2002).
Animal requirements for antioxidants vary with age,
breed, health, work intensity, physiological status and
Implications of oxidative stress for
environmental stress. Exposure to disease, air
pollution (also cigarette smoke) generates oxidative companion animals
stress. This makes it essential to supply adequate
Concerns regarding sources and effects of oxidative
levels of antioxidant and precursor components for
stress in companion animals center on cumulative
SOD, GSH-Px and catalase in forms that are readily
effects associated with aging and age-related diseases.
taken up, and preferably stored in the body. When
Cancer is a major killer of pet dogs and cats, primarily
the oxidative stress level changes, tissue reserves leave
due to their long lives and exposure to oxidative
the animal prepared to neutralise free radicals
stress, which can lead to the failure of correct DNA
effectively before any serious damage occurs.
replication and tumour growth. Increases in oxidative
stress can come from varying sources, and some
Antioxidant sources examples are given in Table 2. The majority of work
concerning the relationship between oxidation,
Most antioxidants are delivered via the diet, either selenium and cancer has been conducted on humans,
naturally occurring in raw materials or via however the mechanisms are not species-specific. An
supplementation. Fat-soluble antioxidants such as excellent review of the relationship between cancer
vitamin E and carotenoids operate in lipid-rich and selenium intake showed that incidence of cancer
environments such as membranes, whereas water- in vulnerable subjects (human and small animal
soluble ascorbic acid (vitamin C) and glutathione, studies) can be reduced by 30-50% (Whanger, 2004).
which are synthesised in the body, are found in the
cell cytosol. All require adequate supplies of basic
components for synthesis from dietary sources. Table 2. Major sources of oxidative stress in companion animals.
Deficiency (and, in some cases, excess) of these Internal oxidising sources External oxidising sources
elements causes oxidative stress and damage to cellular
Respiration – mitochondria Cigarette smoke
function. Selenium deficiency is a particular problem, Immunity - phagocytes Radiation
as the main dietary sources are plant-based food Reactions with transition metals UV light
ingredients that must grow in selenium-rich, alkaline (e.g. Fe) Pollution
soils to accumulate the element. In many parts of the Pro-oxidant inorganic elements Certain drugs
Arachidonate pathways Chemical reagents
world soils are very low in selenium, and intakes in Peroxisomes Industrial solvents
both the human and animal populations have declined Under/over exercising Mycotoxins
alarmingly in recent decades, making the need for Inflammation Oxidised dietary fat
supplementation critical. Vaccination Disease challenges
Inorganic sources of elements such as zinc can often Adapted from Furst, 1996.
meet the immediate needs of the animal, but have
Oxidative effects are associated with inflammatory e.g. immune stimulation through vaccination. Body
activity. An example of this is the antioxidant extract stores of such compounds are especially important
silymarin, which has been widely demonstrated to since many stresses are associated with decrease in
reduce arthritic and oedemal inflammation in both food consumption. In contrast to inorganic selenium,
humans and rodents (Figure 1). Trials were conducted the selenoamino acid from organic sources fed to
with mice whereby inflammation was introduced by the dam is transferred to milk and colostrum,
latex injection into the ear. The width of the resulting ensuring nursing neonates obtain a continuing supply.
inflamed area was then compared between groups of Trials on pigs have shown that feeding a suitable
mice receiving various levels of the natural antioxidant source of organic selenium (Sel-Plex®) to lactating
(Gupta et al., 1999). Results indicated that inflammation animals can increase the levels of Se in milk, which
caused by the latex injection was reduced by nearly 30% is subsequently passed to the young. Furthermore
in mice fed 50 mg/kg antioxidant or more. this supplementation of selenium in the milk can
Reproductive organs are also sensitive to antioxidant then be used to generate stores in tissue such as liver.
status. Spermatozoa are rich in polyunsaturated fatty (Table 3, Figure 3).
acids and require adequate membrane protection. The enzyme iodothyronine deiodinase, which is
Therefore, if antioxidant protection is compromised, involved in thyroid hormone activation and
reproductive success will be compromised as well. influences basal metabolic rate and thermoregulation,
This effect has been well documented in agricultural is seleno-dependent. Young animals are less able to
species such as pigs and poultry. Trials conducted in maintain body temperature, especially during disease
poultry (Surai, 2002) have shown important challenge. Improving the capacity for tissue retention
reductions in lipid peroxidation in sperm from birds of selenium could potentially improve neonate
fed combinations of vitamin E and organic selenium livability.
(Sel-Plex®). Figure 2 shows that peroxidation levels
in sperm decreased when males were fed increasing Table 3. Selenium content of tissues of neonatal piglets from sows
levels of vitamin E and organic selenium. receiving different selenium supplements.
Very young animals are particularly vulnerable to Control Inorganic Se Sel-Plex® Combined
stresses that can be regulated by antioxidant enzyme Se, ppm 0 0.15 0.3 0.15 0.3 0.3
precursors. Selenium reserves in muscle are released Liver, mg 0.197 0.238 0.279 0.348 0.424 0.344
Muscle, mg 0.037 0.046 0.046 0.066 0.107 0.074
under stress conditions for the synthesis of Total, mg 0.075 0.079 0.100 0.123 0.200 0.116
selenoprotein enzymes such as GSH-Px to prevent
Mahan, 2002.
damaging effects of free radical overproduction from,
1.25
Relative inflammation (inflamed area, mm)
1.20
1.15 2
y = 6E-05x - 0.0086x + 1.2009
2
R = 0.9275
1.10
1.05
1.00
0.95
0.90
0.85
0.80
0 20 40 60 80 100 120
Added antioxidant level (mg/kg)
Figure 1. Reduced inflammation in rodents supplemented with natural antioxidant sources (Gupta et al., 2000).
L. Tucker 513
30
Peroxidation (MDA nmol/g) 25
20
15
10
0
Control 20 + 0 200 + 0 20 + 0.3 200 + 0.3
®
Vitamin E (IU) + Sel-Plex Se (ppm)
Figure 2. Effect of Sel-Plex® and vitamin E supplementation on fresh sperm peroxidation (adapted from Surai, 2002).
0.12
Litter 1 Litter 2 Litter 3 Litter 4
0.10
0.08
Milk se (ppm)
0.06
0.04
0.02
0.00
Control Inorg Inorg Sel-Plex® Sel-Plex®
0.15 0.3 0.15 0.3
Figure 3. Effect of selenium level (ppm) and form on sow milk selenium content (Mahan, 2002).
Stability of pet food

A major use of antioxidants, especially those of a and animal by-products in formulations, as well as
chemical nature, is as a food preservative to increase the inclusion of polyunsaturated fats in premium pet
shelflife and ensure palatability. Recently there has foods, require specialised antioxidant programs to
been increasing interest in using more natural forms ensure food quality. The high levels of processing
of antioxidants in pet foods – both as a marketing experienced, especially in extruded or canned foods,
opportunity and to allay consumer worries regarding increase exposure to heat and air, promoting auto-
the use of chemicals. The established use of meat oxidation reactions and facilitating rancidity. Any
antioxidant, whether chemical, tocopherol-based or the combination of natural antioxidants in Nature

derived from natural plant sources, must be stable at Ban™ resulted in the lowest peroxide values over an
high temperatures to be practical under such extreme extended storage period at high temperature. Such
conditions. results demonstrate that the use of natural alternatives
Losses of natural antioxidants from extrusion are can ensure both the quality of pet food and the
frequently reported as being 50% or higher, making demands for more natural ingredients by purchasers.
heat tolerance a key attribute for natural alternatives
replacing synthetics. Research at the University of
Santiago in Chile (Valenzuela, 2003) demonstrated Branding antioxidant benefits
that a commercial antioxidant system containing
natural mixed tocopherols and rosemary extract Branding and marketing pet food typically follows
(Nature Ban™) was thermally stable for 1 hr at 200°C the same consumer decisions as for human food.
and for 2 hrs at 150°C when tested on refined sardine Recent trend analysis in retailing has shown that the
oil. growth market is for high quality and added value
Commercial experience indicates that certain food (Hughes, 2003), so opportunities also exist for
natural antioxidants show less than 30% losses after higher quality pet food formulated with functional
extrusion. Figure 4 shows the comparative stability ingredients such as antioxidants. As people realise
of chemical and natural antioxidants, including the the importance of antioxidants in their own diets,
commercial product Nature Ban™. Natural products they increasingly look for similar benefits for their
show similar levels of protection as the chemical pets. An increase in public awareness regarding the
products when applied at similar doses. However a importance of antioxidant-containing foods as well
combination of natural antioxidants, as supplied via as mineral supplementation and ‘eating for health’ is
Nature Ban™, gave the longest stability period. already responsible for changes in many buying
Comparisons between chemical and natural decisions.
antioxidants for preventing rancidity in animal fats A key factor in the marketing of antioxidant-
have also been conducted. Prevention of peroxide supplemented pet foods is effective communication
generation was monitored in chicken fat samples with the person making the purchase. The plethora
treated with chemical or natural antioxidants of pet foods available is often bewildering, and, while
(Valenzuela, 2002). The results clearly showed that it is well established that people are willing to pay
Control
BHT (400 ppm)
BHA (400 ppm)
TBHQ (400 ppm)
Tocopherol mix (30% α-T, 60% γ-T) (400 ppm)
Rosemary extract (400 ppm)
Nature Ban™ (400 ppm)
Nature Ban™ (800 ppm)
0 2 4 6 8 10 12 14
Hours
Figure 4. Comparison of chemical and natural antioxidants on the induction period required for oxidation of highly refined fish
oil under Rancimat test conditions.
L. Tucker 515
60 BHT (0.3%)
BHA (0.3%)
Com. Brand (0.3%)
50 Nature Ban™ (0.3%)
40
Peroxides (meq/kg)
30
20
10
0
0 200 400 600 800 1000 1200 1400 1600
Hours of storage at 75°C
Figure 5. Effect of chemical and natural antioxidants on peroxide generation in chicken fat (Valenzuela, 2002).
for perceived higher quality, it is essential to put the Halliwell, B. and J.M.C. Gutteridge. 1999. In: Free
message across regarding the added value antioxidants Radicals in Biology and Medicine, 3rd Ed. Oxford
bring to the diet. This should be done in conjunction University Press, Oxford.
with making a bold statement via packaging that Hughes, D. 2003. Competing and communicating in
antioxidants are included. Some manufacturers make an enlarged Europe and in a global marketplace.
claims for antioxidants based solely on the standard Egg and Poultry Industry Conference, Blackpool,
practise of adding vitamin E to the diet. Hence it is UK, November 2003.
of key importance to use and promote the explicit Kettle, A.J. and C.C. Winterburn. 1997.
benefits of well-established antioxidant products that Mierloperoxidase: a key regulator of neutrophil
add health and welfare benefits, backed up with oxidant production. Redox Report 3:3-15.
proven science.
Knight, J.A. 1998. Free radicals: their history and
current status in ageing and disease. Ann. Clicial
Lab. Sci. 28:331-346.
References Mahan, D. 2002. personal communication.
Bagchi, D., A. Garg and R.L. Krohn. 1998. Protective Schwartz, K.B. 1996. Oxidative stress during viral
effects of grapeseed proanthocyanidins and selected infection: a review. Free Rad. Bio. Med. 21:641-
antioxidants against TPA-induced hepatic and brain 649.
lipid peroxidation and DNA fragmentation, and Singal, P.K., N. Khaper, V. Palace and D. Kumar.
peritoneal macrophage activation in mice. Gen. 1998. The role of oxidative stress in the genesis of
Pharm. 30(5):771-776. heart disease. Card. Res. 40:426-432.
Furst, P. 1996. The role of antioxidants in nutritional Surai, P.F. 2000. Effect of the selenium and vitamin
support. Proc. Nutr. Soc. 55:945-961. E content of the maternal diet on the antioxidant
Gupta, O.P., S.K. Banerjee, S. Bani, B.D. Gupta, system of the yolk and the developing chick. Brit.
S.S. Handa, S. Malhotra, N. Sharma and S. Sing. Poult. Sci. 41:235-243.
2000. Anti-inflammatory and anti-arthritic Surai, P.F. 2002. In: Natural Antioxidants in Avian
activities of silymarin acting through inhibition of Nutrition and Reproduction. Nottingham University
5-lipoxygenase. Phytomedicine 7(1):21-24. Press, UK.
Tucker, L.A. 2002. Botanical ingredients for petfood of a new food-grade antioxidant: PreventoxÔ In:
- the technical edge. Feed Compounder, Petfood Nutritional Biotechnology in the Feed and Food
Supplement, Iss. 3, Autumn. Industries, Proceedings of the 19 th Alltech
Valenzuela, A. 2002. Natural antioxidants: from food International Symposium. (T.P. Lyons and K.A.
safety to health benefits. In: Nutritional Jacques, eds). Nottingham University Press, UK.
Biotechnology in the Feed and Food Industries, pp. 379-384.
Proceedings of the 18 th Alltech International Whanger, P.D. 2004. Selenium and its relationship
Symposium. (T.P. Lyons and K.A. Jacques, eds). to cancer: an update. Brit. J. Nutr. 91(1):11-28.
Nottingham University Press, UK. pp. 323-332.
Valenzuela, A., S. Nieta, J. Sanhueza and J. Gomez-
Basauri. 2003. Chemical and physical properties
J. Zentek 517
A changing landscape: the pet food market in Europe

JÜRGEN ZENTEK
Institute of Nutrition, Department of Veterinary Public Health and Food Science, Veterinary
University of Vienna, Austria
Introduction The European pet population in 2004

The European pet food market has continuously The number of households with pets is estimated by
expanded during recent decades. This can be explained the European Pet Food Industry Federation
by a long lasting growth in pet ownership, by the (FEDIAF) to have reached 55 million. The number
decreasing use of table scraps and the general trend of cats is higher than the number of dogs; and
toward convenience foods. In the past years the estimates are that 47 million cats and 41 million dogs
situation in Western Europe has changed and the are kept in the Western European countries (FEDIAF,
growth rate of the market has slowed down in many 2004). A population of 35 million pet birds, 9 million
countries so that a mature market situation has aquaria and 36 million other pets, mainly rodents
developed. The market split into different segments. and small mammals, but also reptiles, must be
Pet owners’ demands for premium products and considered as another important segment of the
sophisticated accessories for their animals have led market. Over the last years, certain trends have been
to an increase of sales in those sectors. On the other observed. The number of dogs tends to be stable or
hand, it is obvious that other pet owners have a in some countries slightly decreasing while the
requirement for pet foods that fulfil the nutritional number of cats is increasing. Based on a total number
needs of the animal at reasonable prices. This trend of households in Western Europe of 155.6 million,
has been strengthened by the unfavourable economic it can be estimated that 21% keep dogs and 20%
situation of the last years. Another important aspect keep cats with an average number of 1.1 and 1.4
for the pet food market is the fact that the European dogs and cats, respectively (FEDIAF, 2004). The
Community has expanded and will expand in the number of cats has increased and the trend to keep
future. The applicant countries offer promising more than one cat per household is observed in many
opportunities due to the lower level of calorie countries (ZZF, 2001). This trend reflects the
coverage by prepared pet food and the expected popularity of cats as a ‘lifestyle adapted’ pet, and
growth of the markets. But even in the old member may also be explained by the recommendations to
states of the European Union there exist considerable facilitate socialisation by keeping more than one cat,
differences among the countries. Although the especially in households where cats must stay alone
standard of living in those countries is more or less over longer times during the day. The trend for the
comparable, there are obvious differences in the habits number of dogs to decrease can be explained by the
of keeping pets and the traditions of pet feeding that fact that lifestyles of pet owners have changed. In
affect the market. Europe is not uniform. The addition, the selection of dogs as pets has also been
European countries differ in population density of subject to other influences. External factors have had
dogs and cats, the calorie coverage by commercial great impact on the public opinion towards the role
pet foods and the balance between commercial foods of dogs as pets. In recent years it has become obvious
and the traditional ways to feed pets with table scraps. that keeping dogs as pets is a very sensitive topic for
the public. Discussions have come up on the role of
518 A changing landscape: the pet food market in Europe
dogs in urban environments regarding problems for Regarding the age structure of dogs in Western
public hygiene. Additional controversies developed Europe, it is estimated that 9% of the dogs are younger
after some unfortunate accidents with dogs that than two years and 16% older than 10 years. The
induced a negative picture of ‘dangerous’ dogs and number of older animals in the United Kingdom
dog-keeping in general in the media. For instance, (23%), the Netherlands (21%), France (18%), and
in Germany a discussion along these lines developed Germany (17%) is above the European average.
after a series of bite attacks by dogs was picked up Lower numbers are seen in Portugal (7%), Norway
by the media including one severe accident that killed (11%), Italy and Spain (12%) (Euromonitor, 2001).
a young child. This unfortunate event focused public In contrast to the dog population the number of
opinion transiently but strongly against ‘aggressive’ cats has increased steadily in recent years. This
and ‘dangerous’ dogs and consequently induced a development has huge economic consequences for
general discussion on dog keeping. This had a negative the related industries. Data on the total cat population
impact on the number of dogs, as documented by the in Europe differ, but in most countries of Western
statistical data (Table 1) of the German Kennel Club Europe the number of cats is considered to be either
(VDH, 2003). These figures show the trend of the stable or increasing. France, United Kingdom, Italy
dog population in Germany, which is one of the five and Germany are the countries with the biggest cat
countries (together with France, the United Kingdom, populations (Euromonitor, 2001). Regarding the
Italy and Spain) in Western Europe with strong number of cats per household, differences are obvious.
economic impact on the feed industry. The estimated average number of cats in Western
Europe is 1.4 per household. Households with more
Table 1. Number of puppies registered by the German Kennel than one cat are seen more frequently in Austria (2.2),
Club. Switzerland (1.8), Belgium (1.8), Norway (1.8),
Year Number of puppies Spain (1.7), the United Kingdom (1.6), and Italy
(1.6). Regarding the age of domestic cats, 26% of
2002 90257 the total cat population is younger than two years
2001 89822 and 18% is older than 10 years. Countries with a
2000 88659
1999 97860 higher population of older cats are the United
1998 106815 Kingdom (29%), the Netherlands (23%), Germany
1997 114670 (22%), Sweden (21%), and Switzerland (19%).
1996 117893 The situation in the application countries and in
1995 119460
1994 114690 Eastern Europe is difficult to predict because no
1993 109785 statistical data have been documented in the public
1992 106490 literature. Assuming that the density of dogs and cats
is comparable to Western Europe, it can be expected
VDH, 2003; 2004
that these countries offer a dynamic and promising
area for future economic activities. The markets for
The stable or negative trend in the dog population is pet food and care products are relatively undeveloped
also observed with regard to dog breed and dog size and the use of prepared pet food is much lower
preferences. In Europe the number of small sized compared to the Western European countries.
dogs (<10 kg BW) was estimated to be 34%, the
medium and large sized dogs make up 37 and 29%
of the total population (Euromonitor, 2001). The Demographic and social factors
southern countries (France, Italy, Spain, Portugal)
Several facts must be taken into consideration when
have a certain preference for smaller dogs, while other
speculating about the future of the European pet food
- especially the northern - countries seem to prefer
market. The current population dynamics in Europe
larger breeds. This preference is seen in Germany,
show negative trends in many countries. Birth rates
Belgium, Netherlands, Denmark, Norway, Sweden,
have slowed or are decreasing, and the median age
Austria, and Switzerland. However, the number of
of the population is increasing. In many countries a
large dog breeds is decreasing and a shift in the dog
trend toward urbanisation with higher numbers of
population can be expected towards smaller breeds
households and lower numbers of persons per
in many of these countries.
J. Zentek 519
household is observed. In addition, lifestyles have Pet food industry and market
changed. Individualization is important for many expectations
young people, and the length of time a person is out
of the house over the day or for longer periods has The market for pet food and related products is mature
increased. Traditionally, mobility has been lower in in most Western European countries. Volumes of dog
many countries of the European Union compared to food are falling in several countries, inducing a
other parts of the world, but due to the changes in general fall of pet food volumes, but not necessarily
economic conditions and lifestyles of young people, on a sales basis. FEDIAF estimates the total number
it has increased in recent years. Statistical data are of pet food companies in Europe at 450 and the
available to support these assumptions. Taking figures current volume of all pet food is estimated at 5 million
for Germany as an example (ZZF, 2001), most pets tons with an estimated sales value of 8.5 billion €
are kept by owners aged 35-49 years (39% of all (FEDIAF, 2004). The growth rate of the pet food
pets) with a clear relation to the number of family industry was 3% over the last three years (2001-
members. Pets are often kept in families with at least 2003), which is a reasonably good result compared
one child (46% of all pets), lower frequencies are to stagnation or negative growth rates in other
found in single households (22%) and with couples industries. The number of employees in the pet food
without children (32%). Based on a recent statistical industry was 21,000 persons working directly in the
evaluation in Germany (ZZF, 2003) with 30,500 industry. Another 30,000 persons are estimated to
interviews, this trend must be interpreted in light of work indirectly for this industry. The significance of
some new aspects. According to this investigation, the pet food industry for the total agricultural sector
couples without children and singles represent on a is becoming increasingly important. The yearly
household basis 60% of cat owners, 59% of dog purchase of agricultural by-products in the European
owners, 58% of the pet bird owners and 48% of the Union was 2.75 million tons (FEDIAF, 2004).
aquarium owners. Singles seem to prefer cats as pets, Manufacturers will have to continue to be innovative,
dogs are only second choice. This fact is important, because innovation will be an important feature of
because in Germany and in other countries it can be successful companies. However, in recent years
expected that the number of households, either singles moderate increases in quantity have been accompanied
or couples without children, will increase and will by falling returns in the market for ready-mixed pet
make up about 72% of the total number of households food (Siessegger, 1997). The distribution and retail
in 2010. It can be expected that the number of cats channels have changed significantly in the past years.
will be increasing in the next years because cats are Traditionally, pet food was either sold in grocery
more suitable for this household structure than are stores or in many European countries by small,
dogs. The population of dogs, pet birds, and also of traditionally structured ‘over-the-counter shops’.
small mammals, which is currently still expanding, Many of these offered a good standard of products
will probably decrease. and had a specific strength in advising customers.
These important demographic trends are On the other hand, small shops have lost significant
comparable in many other member states. On the market share all over Europe due to the competitive
other hand, changing lifestyles of pet owners have price situation. The relative significance of grocery
other effects on the development of pet markets. stores and specialised pet food markets has increased.
Importantly, these demographic changes also affect The small solitary shops have and will probably
the position of the animal in society. Humanisation maintain a certain niche with higher priced products
of pets is obvious, and can be explained by the and with products that need more explanation for
demographic changes bringing pets into a specific the consumers. Another specific target group for this
role as social partners. This development has been sector is non-experienced owners that need more
noted and further supported by the marketing continuous advice. While the volume of pet food has
strategies of pet food and supplier companies. Seeing reached a certain plateau in Europe, the situation in
pets as beings with equal or at least similar the individual member states differs. It has become
requirements as humans means that many owners are obvious that customer demands are increasingly
willing to spend more time and money to indulge orientated toward premium products, treats, and in
their animals. the segment of complete diets toward dry food in
dogs (Figure 1). Similar trends can be observed for
cats.
520 A changing landscape: the pet food market in Europe
Consumer expectations
Pet owners have different and specific expectations plants to assure optimum quality of the ingredients.
that pet food should fulfil. Consumers have a demand Future developments are difficult to predict, but it
for high quality standards in each price segment. can be assumed that the trends of the last five years
Safety, nutritional adequacy and health promoting will continue: a considerable and growing part of
effects of pet food products are an important issue. the consumers will demand premium quality and will
‘Healthiness’ is a major aspect for many consumers, be prepared to pay adequate prices. Quality, safety
especially those who are prepared to pay higher prices and nutritional adequacy will be an ongoing issue
for premium products. Another group of consumers for this group. Functionality of pet food is debated
do not consider health aspects a priority, but they and will result in new products, but in this aspect the
expect that the pet enjoys eating a specific product European perspective seems to differ from other parts
or that they themselves are enjoying feeding the of the world. In Europe, the use of dietary
specific product to their pet. A third group of pet supplements is less common in humans and therefore
owners is mainly interested in getting access to consumers are less willing to use functional
reasonable products at economic prices. All will supplements for pets. On the other hand, many
expect not only high nutritional value and adequacy, benefits of functional pet foods will be appreciated:
but also high palatability and digestibility of the food. palatable treats with specific functionality, products
In many of the western European countries issues with proven efficacy in the promotion of well-being,
have been raised around the ethical aspects of pet health and longevity, and products of natural origin.
food. Since the BSE crisis certain raw materials are
no longer used by the pet food industry and have
been discarded from the pet food chain earlier than References
from the human food chain. The ingredients of animal
origin must be approved by the veterinary service Euromonitor. 2001. http://www.euromonitor. com/
and must be fit for human consumption, otherwise Petfood. Accessed 02/22/2004.
they cannot be used for pet food production. In the FEDIAF. 2004. www.fediaf.org/pages/figure.html.
higher price segment it is expected by consumers that Accessed 02/22/2004.
manufacturers conduct additional controls in their PFMA. 2004. www.pfma.com. Accessed 02/22/2004.
Moist
Semi-moist
600 Dry
Mixers
Treats
500
400
× 1000 tons
300
200
100
0
1987 1989 1991 1993 1995 1997 1999 2001
Year
Figure 1. Market trends of moist, semi-moist, dry pet food, mixers and treats for dogs in the United Kingdom (PFMA, 2004).
J. Zentek 521
Siessegger, A. 1997. The German market for ready- ZZF. 2001. www.zzf.de/presse/markt/markt2001.html.
mixed pet food. Der deutsche Markt fur Heimtier- Accessed 02/22/2004.
Fertignahrung. Kraftfutter 3:108-109. ZZF. 2003. www.zzf.de/zza/031226.html. Accessed
VDH. 2003. Geschäftsbericht zum Jahr 2003. 02/22/2004.
Verband für das deutsche Hundewesen, Dortmund.
VDH. 2004. http://www.vdh.de/cgi-bin/vdh_gesamt/
welpenzahlen. Accessed 02/22/2004).
Index of topics 523
Index of topics
AAFCO 467, 503 Antinutritional factors 75

Abiotic stress 387-389 Antioxidants
Abscisic acid 362, 387 Enzymes
Acidifiers 61-62, 69, 94 catalase 510
Acidosis 221 GSH-Px (see selenoproteins proteins)
Acquired resistance (SAR) 389 SOD 510
Adhesins 30, 60, 289 Human foods 269
Adsorbents (see Mycosorb®) chocolate and, 269-270
AGPs (see antibiotics) red wine phenolics 269
Agrimetrics 13 Mechanisms 83, 143-144, 277, 470-472, 509-511
AgriStats® 13 Natural v. synthetic 315, 472
AgromosTM Nature BanTM 514-515
Alternaria 379-381 peptide hydrolysates
Bacterial wilt 376-378 mechanism 320-321
AIDS 478 soy proteins 319
Allergens 260, 305 whey proteins 316-319
Aleukia (ALA) 306 yeast protein extracts 319-320
Alternaria 379-381 Oxidative stress 388-389, 511
Allzyme FDTM 452-456 Pet foods 471-473, 509-515
Allzyme Vegpro™ 1-2 Vitamin C 83, 510
AmaizeTM 198-201 Vitamin E 83, 143-144, 278, 510
Amylase 71, 197 (see AmaizeTM) Aquaculture (see also shrimp)
Amylose 28, 71 Artemia 410-411
Animal welfare audits 300-302 Bioencapsulation 410-411, 427
Anemia, 278-279 Carotenoids 409
Anthrax 27 Cold extrusion spherizer 428
Antibiotics Consumer issues 429
Alternatives/replacers 11-19, 57, 59-64, 69, 93-100, Disease control strategies 430, 461-462
159, 288 Disease resistance
Avilamycin 57, 288 MHC variations 399-400
Avoparcin, 57 transgenics 398
Bambermycin 288 European aqua
Benefits 57-59 salmon price impact 423-424
BMD 30, 57, 288 species cultured 422, 424
EU ban 57-59, 69, 93 Fish meal/oil
Modes of action 58 current usage 434-440
Monensin 57 future demand 440-441
Resistance 58-59, 63, 159, 289 replacements for 441-443, 449-456
Salinomycin 57, 103 Flake feeds 411
Spiromycin 57 Food safety issues 460-461
Tylosin 57 Food value 458
Virginiamycin 63-64, 288
524 Index of topics
Genetics Mode of action 4, 29, 62-64, 289

adoption of 393-394, 401-402 Mucin production 64
consumer/market issues 396-397 Pigs 16-19, 61, 288
mass selection vs transgenics 393-399, 425-426 Resistance 63
sexual dimorphism 400-401 Salmonella 29, 289
Industry statistics Turkeys 63-64, 288
global 421, 433-434, 457-458 vs antibiotics 63-64
Europe 422 BioplexTM
Larval technologies Absorption of 5
formulated feeds 427-428 Copper 152-153
live feeds 426-427 Environmental impact 152-153
Lipids 408, 413, 427 Horses 352
Mycotoxins 430 Pigs 152
Nucleotides 410, 414 Structure 5
On-growing technologies 428-430 Zinc 5
Rotifers 410-411, 427 Biorefinery 6-7
Rural development 459 Bioterrorism 27
Safety issues 460-461 BSE 1, 3, 11, 297, 520
Shrimp Biostimulants (see also Crop-SetTM)
broodstock nutrition 407-410 Natural vs synthetic 369
larval feeds 410-414 Blood type antigens 29
larval gut physiology 412-413 Botanicals 94, 485
nucleotides 414 Brewers dried yeast 475-77
vitamin requirements 414 Brush border 197
Sustainability 460 Butyric acid 198
Aspergillosis 232 By-products
Astaxanthin 409-410 Animal origin ban 6, 69, 429
Autolysis 257-259 Distillery sources 6-7
Avian flu 1 Poultry sources 467-473
Upgrading 6-7, 449-456
Bacterial wilt 375

AgromosTM 376-378 Campylobacter
ISR 2000TM 376-378 Bio-Mos® and 30
Stubble-AidTM 376-378 California
Beef cattle Agricultural production 265-266
Selenium 211 CIFAR 267
Sel-Plex® response 211-219 University of 266-269
Betahydroxybutyrate 198 Calves
Bifidobacteria 30, 58, 62, 165 AmaizeTM response 198-202
BioChrome 7, 352 Bio-Mos® in, 288
Bio-Mos® 6, 7 Digestive enzymes 195-198
Broilers 63-64, 288 Rumen development 197-198
Calves 288 Cancer 268, 495
Campylobacter 30 Antioxidants and 269, 277
Cats 479 Prostate cancer 270, 477-478
Dogs 478-481 Candida albicans 232
E. coli 29, 30 Carboxypeptidase 196
Gut morphology 64, 290 Carotenoids 510
Immune response 61, 63-64, 289-290, 478, 480 Cellobiose 28
Meta-analysis 4, 288 Chitin 283-285, 476
Microbial ecology 478, 479-480 Chocolate 270
Index of topics 525
Cholesterol Production stats (US) 205-206, 265

Humans 287-288 Rumen protein degradability 188
Shrimp requirement 408 Selenium 9
Chondroitin sulfate 341-342 Sel-Plex® response 211
Chymosin 196 Systems 206-207
Citrus (see Crop-SetTM, ISR 2000TM) Damping off 383
Citrus tristeza 364 Diabetes
Claviceps 233, 306 Dogs 495-499
Clostridia 30, 58, 61, 62, 69, 75, 165 Humans 495-496
Cold stress 388 Dietary supplement status 298
Colostrum 132 Dioxins 286
Companion animals (see pets, dogs) DHA (see also Omega-3 PUFA) 141, 145-146
Competitive exclusion 60 Distillers grains 6
Conjugated linoleic acids 275-277, 280 Dogs
Consumer issues 3-4, 11-12 Bio-Mos® 478-481
Lifestyles and food 142, 205, 495-496 Genomics
COOL 12 canine genome sequencing 497-498
Copper functional foods for antioxidants
BioplexTM Cu 152-153 nutritional genomics 498-500
Pig fecal content 154 Drought stress 387
Supranutritional levels 4 DSHEA 265, 298
Cornell-Penn-Miner model 188-192
Coronary Heart Disease 269, 275, 277-279
Carotenoids 409-410 E. coli 60, 62, 165
Crop-Set™ Bio-Mos® 30
Grapefruit 364, 370 Selenium/Sel-Plex® 40
Lemons 371 Edwardsiella 30
Mandarins 371-372 Endophyte 233, 239, 242-243
Minneola tangelos 372 Enterobacter 30
Navel oranges 363, 369 Enzymes (See also VegproTM, AllzymeTM, FD AmaizeTM)
Phenology and application 363, 365 Antioxidant (see antioxidants, selenoproteins)
Saponins 369 By-product upgrade 6-7, 449-456
Valencia oranges 364, 370-371 Digestive
Cytokinins 362, 363 brush border 196
calves 195-198
crustaceans 412-413
Dairy Exogenous
Amino acid sources 192 AmaizeTM 198-201
BST 205 aquaculture 413
Dairy nutrition software 189-191 glycosylation 27
Educational needs 207-209 heat processing 72
Environmental concerns 206 poultry & NSPs 61, 69, 73-74
Factors limiting milk yield 209 phytase 21-23
Hemorrhagic bowel syndrome 231 VegproTM 1-2
Herd longevity 2-3 Hydrolysis of whey, soy, yeast 315-316
Herd size 206-207 Rubisco 387
Milk Environmental impact
proteins 278 BioplexTM Cu 152-153
Se content 9 Dairy 206
vitamins in 278 Manure 21
Mycotoxins 231-245 Pigs 149-155
Nutritional models for 187-192 Essential oils 62
526 Index of topics
Ethanol production 6-7, 291, 475 Customer service 128

European Food Safety Authority 429 Management 128-129
European Union Commission 94 Mergers/acquisitions 123
Equine Research application 124-127
Anaerobic/aerobic work 326-327 US top ten 121-122
Bone Feed mills
adaptation to exercise 346-347 Milling technology 123
development 346 Multi vs single species 123
diet effects 340-341, 351-352 Premix 123
Coordination 329 Toll milling 123
Carbohydrates Feed processing 73, 123
forms in equine diets 331-332, 334 Fescue toxicosis 233, 239, 242-243
low CHO diets 333-334 Fish (see aquaculture)
metabolism 338, 341 Fish meal
Cushing’s Disease 331, 333 Current usage 434-440
Disorders Future demand 440-441
bucked shins (dorsal metacarpal disease) 345, Replacements for 441-443, 449-456
347-351 Fish oil
turnover 347 Omega-3 content 276
weight loading 345 Usage 434-440
Environment and nutrition 342 Flavors 257-263
Exercise physiology Flooding 388
diet and 341 Food regulations 297-298
endurance training 325-328 Free radicals (ROS) (see antioxidant function)
heart rate 325-326 Fructooligosaccharides
lactic acid 326-328 Humans 30
power 329 Mode of action 30, 62
speed training 328-329 Fucose 29, 30
Fats and oils 340 Fungicide 383
Geriatric diets 340 vs Stubble-AidTM 383-385
Industry status 337 Fusarium (see molds, mycotoxins)
Insulin resistance 331 Fusobacterium 30
Laminitis 331, 333
Minerals
BioChrome® 352 Galactomannan 30
BioplexesTM 352 Galactose 30, 197
pH 351-352 Genetically modified organisms 396-397
requirements 339 Genomics 270
Sel-Plex® 352 Canine genome sequencing 497-498
MRLS 351-358 Nutritional genomics 498-500
Myositis (tying up) 331, 332-333 Gibberellins 362
Neutraceuticals 341-342 Global warming 387
NRC review 338-342 Glucan 6-7, 283-285, 476, 479
Obesity 331, 333 Cholesterol 287
OCD 331, 333 Functional properties in food 288
Protein/AA requirements 338-339 Immune response 285
Vitamin requirements 339 Mycotoxin adsorption 286-287
Water 339-340 Glucomannan (see mannan, Bio-Mos®)
FDA 3, 36, 37, 93, 94, 131, 211, 297-299 Glucosamine 341-342
Feather meal 449-456 Glucose 27, 30, 197
Feed companies Glutamic acid 260, 263
Brands 123 Glutathione peroxidase (see Selenoproteins)
Index of topics 527
Glycolysis 326-327 Least cost formulation 13

Glycomics 27-31, 289 Lifestyles and food 142, 205, 495-496
Glycosaminoglycan (GAG) 28 Linoleic acid 143 (see Omega-3 PUFA)
Glycosylation Linolenic acid 143 (see Omega-3 PUFA)
Disorders of 27-28 Lipase 196
Enzymes 27 Longevity 2-3
Yeast cell wall 284, 290
GRAS status 298
Gut microbial ecology 13-14 Maillard reactions 261-262
Mannan oligosaccharides (See yeast cell wall composition,
Bio-Mos®)
HACCP 11, 461 Mannoproteins 478-479
Halal 302 Mannose adhesions 30
Heat processing 72 Mannose binding protein 29
Heat shock proteins Manure
Plants 388 P content 21-24
Poultry 40-43 Zn, Cu content 21
Hemorrhagic bowel syndrome 231 Mare Reproductive Loss Syndrome (MRLS) 355-358
Herbs 62 Meat
Horse (See Equine) Iron content 278
HSCAS 286 Se content 217-218
Hunger 458-459 Sel-Plex® and 216
Protein 278-279
USDA grade 213
Immunity Microbial ecology 13
Bio-Mos® and 18-19, 63-64 Molecular techniques 16-17
Function 29, 305-306 Milk
GALT 18, 60-61 Milk proteins 278
Inflammation 60, 64 Production stats 265
Maternal 14 Sows 134, 512-513
Mycotoxin impact on 309-312 Milk replacer, 197
Induced systemic resistance 364, 379 Modeling
vs acquired resistance 389 Cornell-Penn-Miner model 188-192
Iron Nutritional models for cattle 187-188
Deficiency anemia 278-279 Pronutrient antibiotics 95-100
Supplement bioavailability 279 Response to yeast supplements 221-228
ISR 2000™ MMA 131-132
Alternaria 379-381 Molds
Bacterial wilt 378-378 Aspergillus spp. 231-233, 306
Phenology and application 364-365 Distribution 233
vs imazalil for oranges 364-365 Fusarium 103, 231
Growth conditions 233-234
In silages 242-243
Klebsiella 30 Penicillium 233
Koji 1-2, 257 Mold-ZapTM 352
Kosher 302 Monosodium glutamate 260
Mucin barrier 5, 60, 64
Labeling foods 297, 299 Mycosorb® 6, 7, 8, 103,
Lactase 197 Adsorption kinetics 286-287
Lactobacillus brevis 14-16 Broilers 103-107
Lactobacilli 58, 60, 62, 165 Dairy cattle 239-240, 241
Lactose 30 Layers 103-107
528 Index of topics
Turkeys 103-107 Milk content 161

Mycoses 231-232 Pets 479
Mycotoxins Pigs and 164-165
Adsorbents 6, 8, 286 Roles of 163-164
Aflatoxins 6, 8, 232, 235, 239-240, 307-308, 310 Shrimp 410, 414
Citrinin 238, 312 Sources 160
Contamination of Synthesis 162-3
human foods 238, 306-309 NuPro™ 6, 7
milk 239-241 Pigs 19-20
silages 242-243 Nutraceuticals 299, 341-342
DAS 104
Dicoumerol 239
Distribution, 232 Obesity
DON 8, 103-107, 236-237 Horses 331, 333
poultry 103-107 Humans 495-496
Ergot alkaloids 232, 239, 242-243, 306 Pets 495-499, 504
FDA Oligosaccharides (see Bio-Mos®, FOS)
feed action levels 235, 237, 239 Omega-3 PUFA
food action levels 306-307 Pork 141, 145-146, 275-276, 408, 413, 427, 458, 505-506
Fumonisin 231, 232, 237-238, 306, 308, 310-312 Omega-6 PUFA, 275, 408, 413, 427, 505-506
Fusarenone 104 Optigen®1200 179-185
Fusaric acid 104 Organic foods
Fusarium toxins 234 Production 302-303
distribution 234 USDA certification 265
Gliotoxin 231-232 Organic minerals (See also Bioplex™)
HT-2, 104 Oxidative stress
Immunotoxicity mechanisms 309-312 Pets 511
Ochratoxin A 232, 238, 306, 308, 311 Plants 388-389
Patulin 238, 306, 308, 311
Penicillic acid 312
PR toxin 238 Palatability 477
Safe levels 241-242 Papillae 198-201
Silages 242-243 Pelleting 73
Symptoms in cows 234-235 Pets and pet food
Synergism 103-104 Bio-Mos®
T-2 8, 237 cats 479
Trichothecenes 232, 306, 308 (see also DON, DAS, T-2) dogs 478-481
Turkey X Disease 287 Brewers yeast 475-477
Vomitoxin (see DON) Fat quality 469-470
Zearalenone 8, 103, 231, 236 oxidation products 471
Myoglobin 279 Flavors 486
FOS 480-481
Genomics
Nature BanTM 514-515 canine genome sequencing 497-498
Necrotic enteritis 69, 75, 290 nutritional genomics 498-500
NFP (see Stubble-AidTM) Globalization 487-488
NPN 179 Mannan oligosaccharides (see Bio-Mos®) 478
Nucleotides, 6, 75, 159-165 Mannoproteins 478
Biochemistry 151-152 Markets
Flavors 260-261 company sales 488
Human infants 159 European market 517-520
Metabolism 161-162 expansion strategies 486-492
Index of topics 529
major markets 489-490 Weaners 115-117, 126-127

trends 485-486, 491-492 Plant activators 389
Nature BanTM 514-516 Plasma protein 19
Nucleotides 479 vs NuProTM, 19-20
NRC update 503-508 Population issues 300, 458-459
Obesity and diabetes 495-499, 504 Pork
Omega-3 PUFA 485, 505-506 Consumption 141
Oxidative stress 511-512 Omega-3 PUFA 141-
Palatability 469 Meat quality 114, 117
Pet population 517-518 Poultry
Poultry protein products 467-473 Age and nutrient digestibility 70-71
Processing effects 470-473 Broiler breeders & Se 81-89
Selenium Egg shell quality 84, 85
cancer and 477-478 Embryonic mortality 87-89
Stability 471-472, 513-515 Enzyme supplementation 72, 74 (See also VegproTM)
SULs 506-508 Feed processing 73
Taurine 505 Fiber 73-74
Vitamins 486 Hatching egg quality 82-83
Yeast glucans and mycotoxins 478 Heat processing of feeds 72
Phenological cycle 361-363 Heat shock proteins 40-43
Phytase Immunity development 74-75
Pigs 151-152 Insoluble fiber 71
Pigs Layers 74
Abortions due to ETC 357 Male fertility 84-89
Bio-PlexTM Cu 152-153 Mycosorb® response 103-107
Bio-Mos® 16-19 Non-starch polysaccharides 73-74
Density of pig populations 149 Post-hatch nutrition 69-70
Environmental issues 114, 117-118, 151-155 Rice utilization 71
farm nutrient plan 150 Starch digestion 71, 72
manure composition 150, 152-155 Whole grain feeding 73
N & pigs, 151, 154-155 Poultry by-product meals 467-473
P & pigs, 151-152 Prebiotic 94, 485
S & pigs 155 Premier Pig ProgramTM 113-119
trace minerals 152-153 Probiotics 94, 485
Fiber 155 Pronutrient 94
Grow-finish 117-118 Proteinates (see BioplexesTM)
Gut microbial ecology 13-19 Pseudomonas syringae 383-385
Inventory (US) 141
Lysine needs 124-125
Meat quality 114, 117 Ractopamine 13
Minerals Radical oxygen metabolites 46
digestion vs retention 150 Radio Frequency Identification Device 11
Nucleotides 164-165 Ration balancing software 189-191
NuProTM 19-20 Rendering 468
Phase feeding 152-153 Rheumatoid arthritis 28
Phytase 151 Rhizoctonia solani 383
Production targets 113-114 Rubisco 387
Segregated early weaning (SEW) 13-14 Rumen/rumen function
Sow Acidosis, 211
productivity 115-116 Bacteria growth 188
selenium and 131-138 Biohydrogenation and CLAs 277, 280
Trypsin inhibitor 75 Development 197-198
530 Index of topics
NPN 178-185 GSH-Px 38, 40, 131-132, 277, 510-511

Optigen® 1200 Thioredoxin reductase 41, 46-48
Papillae length 198-201 Selenoamino acids 83
Protein degradability 188 Sel-Plex® (see also selenium, selenoproteins) 2-3
Response to yeast supplements 221 Beef cows and calves 3, 211
Beef finishing cattle 214-219
Dairy 2, 9
Saccharomyces cerevisiae (see yeast) FDA 3, 37, 133
Salinity 387-388 Horses 352
Salmonella 30, 60 Meat Se content
Bio-Mos® 29-30, 63 Beef 217-218
typhimurium 29, 59 Milk, 9
Sanitation 59 sows 134, 512-513
Seedling blight 383 Pigs
Segregated early weaning 13 meat Se content 144
Selenium 2-3 (see Sel-Plex®, Se-proteins) neonates 132, 512
Cancer 9, 20, 477-478 sows 133-138, 512-513
Cattle weanlings 136-137
colostrum 132-133 Poultry
Chemistry 35 broiler breeders 3, 45, 83-89
Coronary heart disease 277-279 broilers 3, 37-48
Distribution 35 embryonic mortality 87-89
Horses 352 male fertility 83, 84-89, 511-512
Human diets perivitelline sperm hole assay 84-89
amounts in foods 277-278 shell quality 84
intake 277-278, 511 layers 3
Immunity role 44, 512 meat quality 45
Inorganic vs organic sources 20 (See Sel-Plex®) selenium in 37, 211
Interactions with vitamin E toxicity vs selenite 39
In yeast 3-4, 476 Semen production 86
Male fertility 83, 511-512 Shigella 30
Meat quality 45 Shrimp and prawns
Metabolism of 36, 38-40 Broodstock nutrition
Pigs carbohydrates 409
fetus, 132 carotenoids 409
hair 137 hormones 410
neonates 132-133 lipids 408
sows 131-138 nucleotides 410
Poultry protein/AA 408
bioavailability 36 vitamins/minerals 409
deficiencies 36 Larvae
egg content 83 digestive physiology 412-413
requirements 35-36 feeds
tissue levels 36 DHA/HUFA 410-411
Pro-oxidative selenite 37 lipids 413
Role of 38, 510 live vs formulated 411-412
Thyroid hormone and 43-46 protein/AA 413-414
Tissue levels 38-39 vitamin requirements 414
Toxicity 39 Silage
Vitamin E and 131-132, 144 Inoculants and 171-176
Selenoproteins 3, 40-48, 83 Mycotoxins 242-243
Deiodinase 40, 41, 43-46, 277, 512 Sorghum silage 171-176
Index of topics 531
Sil-AllTM VegproTM 1-2

Economics 176 Veterinarians 207
Sorghum silage 172-176 VFAs 64
Soybean Vitamin E (see also antioxidants)
production 2 Pigs 131
Optigen®2000 vs 179-180 Shrimp 409
VegproTM 1-2 VitaporkTM 141-145
Spices 62
Streptococcus 30
Stubble-AidTM Wastage 154
bacterial wilt 376-378 Watershed 21-24
damping off 383-385 Wellness foods
Swine (see Pigs) Pets 509
SWOT analysis University programs for 267-272
West Nile virus 30
Whey proteins 316-319
Tay-Sachs Disease 28
Thyroid hormone 43-46
Tomatoes Xylanase 72
Bacterial wilt 375-378
Damping off 383-385
Traceability 11 Yea Sacc ® 7, 286
Trace minerals (see also Bioplexes) Yeast
Absorption 5 Cell wall
Environmental concerns 5, 152 composition 283-285, 476
Pigs functions 283
BioplexTM Zn and Cu in finishers 5 glucan 6-7, 283-285
digestion and retention 150-152 mannans 478 (see Bio-Mos®)
supranutritional levels 4-5 Extracts
Turkey X Disease 287 Flavor development 257-263
Trehalose 28 Hydrolysate antioxidant activity 317, 319-320
Trypsin inhibitor 75 Types of 258, 201, 475
Urea 179 (See also Optigen®2000, NPN) Zinc

Bioplex Zn 5, 21, 150
Proceedings of Alltech’s 20th Annual Symposium
Re-imagining the Feed Industry
Natural Technologies for Food Production
C an we re-invent feeding animals? Feed companies? Ingredients and nutrient supplies? Can
we change how food animal products and crops are perceived by, and marketed to,
consumers?
The theme of the 20th Annual Alltech Symposium focuses on doing exactly that: re-defining
how we feed animals and re-imagining our agribusinesses to create the compelling force
behind an exciting future.
The basic and applied research is done. Natural feeding strategies can reduce environmental
impact, replace antibiotics, make producers more competitive and change how consumers
view and value food animal products. There is a shift in the dynamic of the industry as well.
Change is no longer something ‘happening to’ the feed industry. Change is something the
industry makes happen.
Nutritional
Biotechnology in the
Feed and Food Industries
Lyons &
Jacques Edited by T.P. Lyons
2004 and K.A. Jacques

Re-Imagining The Feed Industry

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Proceedings of Alltech’s 20th Annual Symposium

Nutritional Biotechnology in the Feed and Food Industries

Nutritional Biotechnology in the Feed and Food Industries

Nutritional Biotechnology in the Feed

Edited by TP Lyons and KA Jacques

Nottingham University Press

First published 2004

All rights reserved. No part of this publication

Typeset by Nottingham University Press, Nottingham

Re-imagining the feed industry: focus on price, perception and policy 1

Future of the feed/food industry: re-inventing animal feed 11

Glycomics: putting carbohydrates to work for animal and human health 27

Selenium sources and selenoproteins in practical poultry production 35

Alternatives to antibiotics in poultry production: responses, practical experience and 57

Optimizing the replacement of pronutrient antibiotics in poultry nutrition 93

Successful feed companies in the future 121

The role of selenium and Sel-Plex® in sow reproduction 131

Dairy and Beef Cattle

Optigen® 1200: controlled release of non-protein nitrogen in the rumen 179

Dairy nutrition models: their forms and applications 187

Gastrointestinal development in dairy calves 195

Meeting the educational needs of dairy clientele in 2020 205

Rumen acidosis: modeling ruminant response to yeast culture 221

Food, Nutrition and Health

Antioxidant activity of hydrolyzed whey, soy, and yeast proteins 315

Relevance of the NRC to today’s horse industry 337

Alternatives against Alternaria: controlling brown spot on Murcott tangors 379

Abiotic stresses and plant activators 387

Opportunities and dilemmas in molecular aquaculture genetics 393

The role of yeasts in companion animal nutrition 475

Using nutritional genomics to study canine obesity and diabetes 495

A changing landscape: the pet food market in Europe 517

Index of topics 523

Re-imagining the feed industry: focus on price, perception and policy

Replacement heifer cost, USD 1400

PERCEPTION 1: ALL ANIMAL FEED CONTAINS

Villus height 600 µm

Separation, Solids Solubles

Solids Organic acids

Pharmaceuticals Specialty Distillers DDGS Condensed

Figure 5. From distillery to biorefinery.

Yea-Sacc1026® Fuel ethanol

Yeast component products

Figure 6. A yeast biorefinery.

Future of the feed/food industry: re-inventing animal feed

methodologies have created the first opportunity for

Extract DNA PCR with a 3' 5'

Recognition of Fragment separation

Control Bio-Mos ® SEM P=

Figure 6. Effect of manure fertilization on zinc concentration in runoff.

Treatment Cu (µg/L) lbs %

1. Unfertilized 2.5 Total P delivered in feed 8,222

Table 14. Farm nitrogen balance.

Glycomics: putting carbohydrates to work for animal and human health

Figure 1. α- and ß-bonded (1-4) glucose units.

diverse nature of carbohydrate chemistry to differentiation from bone marrow in supplemented

Conclusions microbial lectins and agglutinins. In: Microbial

Tanaka, M., Y. Machida, S. Niu, T. Ikeda, N.R. Jana,

Selenium sources and selenoproteins in practical poultry production