Effect of dietary level of protein and fiber on the productive performance and health status of piglets R. G. Hermes, F.

Molist, M. Ywazaki, M. Nofraras, A. Gomez de Segura, J. Gasa and J. F. Prez J Anim Sci 2009.87:3569-3577. doi: 10.2527/jas.2008-1241 originally published online Jul 31, 2009;

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Effect of dietary level of protein and fiber on the productive performance and health status of piglets1
R. G. Hermes,* F. Molist,* M. Ywazaki,* M. Nofraras, A. Gomez de Segura,* J. Gasa,* and J. F. Prez*2
*Animal Nutrition, Management and Welfare Research Group, Department of Animal and Food Science; and Centre de Recerca en Sanitat Animal (CReSA), Universitat Autnoma de Barcelona-Institut de Recerca i Tecnologia Agroalimentries, Campus de la Universitat Autnoma de Barcelona, 08193 Bellaterra, Spain

ABSTRACT: To study the interaction between the levels of protein and fiber on the productive performance and health status of piglets, ninety-six 35-d-old piglets (9.11 0.60 kg of BW) were placed in 32 pens of 3 animals each and allotted to 4 dietary treatments for 21 d. The 4 diets were based on rice, dairy products, and soybean meal in a 2 2 factorial arrangement of treatments, with 2 levels of CP (15.4 vs. 19.4%, as-fed basis) and 2 levels of dietary fiber [DF; low fiber (LF) 5.3% NDF and high fiber (HF) 7.15% NDF, as-fed basis]. The HF diet was developed by supplementing the basal diet with 40 g/kg of wheat bran and 20 g/kg of sugar beet pulp. Animal performance was obtained weekly with samples of feces collected for microbiology on the first and the last experimental day and scored from 1 (liquid) to 4 (hard). On the last day, 1 pig from each pen was sampled for blood analyses of the acutephase protein, major acute-phase protein of pigs (PigMap) and subsequently killed to register the digestive tract weight (including contents) and colon histology. Pigs fed the HF diets had greater ADG (390 vs. 457 g; P 0.001) and large intestine weight (4.4 vs. 5.4% of BW; P 0.05). This coincided with a greater (P 0.05)

short-chain fatty acid concentration (especially of acetic and butyric acids), a decrease in Escherichia coli counts (7.77 vs. 6.86 log of cfu/g of feces, P 0.05), and an increase in the ratio of lactobacilli:enterobacteria (0.76 vs. 1.37, P 0.05). However, CP level did not modify the productive performance, but 20% CP increased P 0.05) the relative weight (% of BW) of the small (6.5 vs. 7.7) and large intestine (3.8 vs. 4.3). In the large bowel, the 20% CP diet increased the numbers of goblet cells (4.6 vs. 5.4/100 m; P 0.05) and reduced the numbers of intraepithelial lymphocytes (1.8 vs. 1.3/100 m; P 0.05). In relation to health status, increasing DF was dependent of the dietary CP content. Supplementing the 16% CP diet with DF reduced the fecal score and increased the antibiotics interventions, whereas the opposite was the case in the 20% CP diet. Pigs fed the 20% CP diet showed decreased (P 0.05) PigMap concentrations than pigs fed 16% CP diets. As a whole, CP showed major effects on the gastrointestinal weight and gut barrier integrity, whereas DF increased the productive performance and promoted major changes in the microbial colonization and fermentation variables.

Key words: dietary fiber, piglet, protein, sugar beet pulp, wheat bran 2009 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2009. 87:35693577 doi:10.2527/jas.2008-1241

INTRODUCTION
Feed specifications for the newly weaned pigs are still a controversial area in the pig industry. Increased standard requirements for AA (standardized ileal digestible

1 This work was supported, in part, by the financial support of the Spanish Ministerio de Educacin y Ciencia (AGL2005-07438-C02-01/ GAN) and by the Programme Alan, European Union Programme of High Level Scholarships for Latin America, identification number E06M100404BR. 2 Corresponding author: josefrancisco.perez@uab.es Received June 17, 2008. Accepted July 23, 2009.

Lys, 12.0 to 14.0 g/kg of diet) and energy (10.4 MJ of NE/kg of diet; BSAS, 2003) are defined for piglets. However, diets containing an increased level of protein may disturb the intestinal microbiota as a consequence of protein fermentation (Bertschinger et al., 1979). Some authors have reported that reducing dietary CP with AA supplementation reduced the ammonia and short-chain fatty acids (SCFA) in the small intestine (Nyachoti et al., 2006; Htoo et al., 2007; Pierce et al., 2007), but also reduced pig performance, probably as a consequence of the lack of some essential nutrients or AA imbalance (Nyachoti et al., 2006). Alternatively, some authors have proposed to reduce protein fermentation in the small intestine by the inclusion of ferment-

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able carbohydrates in the diet (Awati et al., 2006). Increasing the fermentable carbohydrate content, through the inclusion of wheat bran, sugar beet pulp, native starch (Bikker et al., 2006; Carneiro et al., 2007), or inulin (Wellock et al., 2008), has been shown to stimulate lactic and butyric acid production in the small and large intestine and to reduce counts of coliform bacteria. Mateos et al. (2006) and Kim et al. (2007) also described that the inclusion of moderate levels of dietary fiber, such as oat hulls, reduced the incidence of diarrhea in diets based on cooked maize or processed (cooked or extruded) rice. However, there are discrepancies in the literature about the effect of fiber on the productive performance of young piglets. Some authors reported no significant changes or even observed decreased growth performance of piglets when fermentable carbohydrates were supplemented into low CP diets (16%; Bikker et al., 2006; 16 to 18.5%, Pierce et al., 2007) or diets with increased nonstarch polysaccharide (NSP) content (greater than 10% NDF; Freire et al., 1998). However, others have reported a significant increase in the ADG and ADFI when insoluble, nonviscous carbohydrates, or lactose were supplemented to increased CP diets (21%, Pierce et al., 2007) or diets with reduced fiber content (ricebased diets, 3.43% of NDF; Mateos et al., 2006). The reasons for these discrepancies are not clear, but they appear to depend on the composition of basal diets (CP and NDF content) and the ability of the supplementary fiber to enhance the intestinal function or prevent harmful products from being released by protein fermentation. The objective of the present work was to confirm the potential interaction between the level of dietary CP and NDF on the growth performance and health status of early weaned piglets, with the aim of clarifying discrepancies among studies and understanding the physiological mechanisms involved.

(3 animals/pen). The pens were allotted to 4 treatments (Table 1) in a 2 2 factorial arrangement of treatments that included 2 levels of CP content (16 vs. 20% CP) and 2 levels of dietary fiber: low fiber (LF) vs. high fiber (HF; 5.3 and 7.15% of NDF as-fed basis, respectively). The HF content was obtained by including 40 g/kg of wheat bran, the coarse outer covering of the wheat kernel, and 20 g/kg of sugar beet pulp. Diets were isoenergetic and isonitrogenous and formulated to satisfy the nutrient requirement standards for pigs (BSAS, 2003). Each pen (3 m2) had a feeder and a water nipple to ensure ad libitum feeding and free water access. The experiment was conduced at the summer season with an average temperature in the rooms of 30C (2C).

Experimental Procedures
Animals received the experimental diets over 3 wk. Individual BW and pen feed consumption was registered weekly. Animals were checked daily to evaluate health status. Consistency of the feces samples was evaluated and assigned a ranking between 1 = liquid, 2 = soft, 3 = normal, 4 = hard feces on the first and the last day of the experiment. Piglets with clinical signs of diarrhea (watery feces) and simultaneous dehydration, fever, growth check, and apathy were daily treated (during a 3-d period) with 1 mL (intramuscular injection) per animal of marbofloxacin (Marbocyl 2%, Vtoquinol S.A., Lure Cedex, France). The number of antibiotic injections administered in each pen and mortality as a result of diarrhea were also recorded. At the beginning and on the last day of experiment, a sample of feces was taken from the rectum of 1 piglet from each pen for immediate conventional microbiological analyses. A total of 32 samples (8 per treatment) were obtained from piglets not previously treated with antibiotics. A subsample of feces was stored at 20C for further molecular microbiological analyses. On the final day of the experiment, the animal with the median BW of each pen had a blood sample taken. Briefly, 8 mL of blood was obtained by venipuncture of the cranial vena cava using a BD Vacutainer (Becton Drive, Franklin Lakes, NJ) 10-mL plasma tube with sodium heparin (17 IU/mL). The blood samples were kept at 4C until centrifugation (3,000 g, 4C, 15 min). The plasma collected was transferred in 1-mL aliquots to 1.5-mL Eppendorf (Eppendorf Ibrica, Madrid, Spain) and stored at 80C until the analytical use. Immediately after blood sampling, the animal was killed with an intravenous injection of sodium pentobarbital (200 mg/kg of BW). Animals were bled, and the abdomen was immediately opened to tie, remove, and weigh the whole gastrointestinal tract (GIT) and the liver. The stomach, small intestine, and the large intestine were separated by double ties, weighed (including digesta), and sampled. Samples of proximal colon digesta were collected and maintained at 20C for further SCFA, ammonia, and physicochemical charac-

MATERIALS AND METHODS

This experiment was performed at the Experimental Unit of the Universitat Autnoma de Barcelona and received prior approval from the Animal Protocol Review Committee of this institution. The treatment, management, housing, husbandry, and slaughtering conditions conformed to European Union Guidelines.

Animals and Housing

A total of 96 piglets [(Large White Landrace) Pietrain] were acquired from the experimental facilities of IRTA Mas de Bove (Reus, Spain). Animals were weaned at 21 d of age and fed a weaning diet for 14 d based on rice (60%), sweet milk whey (13.7%), soybean protein (10.9%), and potato protein (10%). On d 14 after weaning (35 d of age), piglets were transported to the Universitat Autnoma de Barcelona facilities, weighed (9.1 0.60 kg of BW), and placed in 32 pens

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Table 1. Ingredients (g/kg) and analyzed chemical composition of the experimental diets (as-fed basis)
16% CP Item Ingredient, g/kg of feed Rice Barley Wheat bran Sugar beet pulp Sweet whey Potato protein Soybean meal, 44% CP Animal fat Calcium carbonate Dicalcium phosphate Sodium chloride l-LysHCl dl-Met l-Thr l-Trp Choline chloride, 50% Vitamins and minerals2 Calculated content, % NE, MJ/kg Starch Crude fiber Lysine Methionine Threonine Tryptophan Isoleucine Valine Analyzed composition, % Moisture Ash GE, MJ/kg of DM Ether extract CP NDF ADF
1 2

20% CP Low fiber 361.9 200.0 147.2 83.6 146.1 30.3 6.2 12.1 2.0 2.4 1.6 0.6 0.8 1.2 4.0 10.32 36.3 2.1 1.4 0.53 0.96 0.33 0.95 1.05 10.45 5.53 18.13 4.28 19.36 5.38 2.12 High fiber 302.5 200.0 40.0 20.0 147.2 83.6 137.4 38.8 6.0 11.5 2.0 2.6 1.7 0.7 0.8 1.2 4.0 10.32 33.1 2.7 1.4 0.53 0.97 0.33 0.94 1.05 10.07 5.95 17.83 5.15 19.43 7.40 2.94

Low fiber1 442.7 200.0 147.2 40.0 101.6 28.4 6.3 12.8 2.1 6.8 2.9 2.7 1.3 1.2 4.0 10.32 42.1 1.9 1.4 0.56 0.92 0.31 0.69 0.78 10.62 5.41 17.05 4.59 15.44 5.15 2.09

High fiber1 383.7 200.0 40.0 20.0 147.2 40.0 93.1 36.8 6.1 12.1 2.0 6.8 3.0 2.7 1.3 1.2 4.0 10.32 38.8 2.6 1.4 0.57 0.92 0.32 0.69 0.78 10.66 5.55 16.97 4.75 15.43 6.89 2.70

Low fiber, 5.3% NDF, and high fiber, 7.15% NDF; as-fed basis. Supplied per kilogram of feed: 5,000 IU of vitamin A, 2,000 IU of vitamin D3, 15.0 mg of vitamin E, 1.3 mg of thiamine, 3.5 mg of riboflavin, 1.5 mg of pyridoxine, 0.025 mg of cyanocobalamin, 10.0 mg of calcium pantothenate, 15.0 mg of niacin, 0.1 mg of biotin, 0.6 mg of folic acid, 2.0 mg of vitamin K3, 80.0 mg of Fe, 6.0 mg of Cu, 0.75 mg of Co, 150 mg of Zn, 60 mg of Mn, 0.75 mg of I, 0.10 mg of Se, and 150 mg of Etoxiqun as antioxidant (Capsoquin, Itpsa, Barcelona).

terization. A 4-cm section from the proximal colon was removed, opened longitudinally, and fixed by immersion in 10% (vol/vol) buffered formalin for histological study.

Analytical Procedures
Chemical analyses of the diets were performed according to the Association of Official Analytical Chemists (AOAC, 1995) standard procedures. The viscosity of proximal colon digesta was measured in a Brookfield DV-E viscometer (Brookfield Engineering Laboratories Inc., Stoughton, MA) at a shear rate of 11 s1. For bacterial counts, fresh digesta samples were suspended (10% wt/vol) in a buffered peptone solution and subsequently homogenized. Thereafter, 10-fold

dilutions were made with a buffered peptone physiological salt solution (CM509, Oxoid Ltd., Basingstoke, Hampshire, UK; containing peptone, 10.0 g/L; sodium chloride, 5.0 g/L; di-sodium phosphate, 3.5 g/L; and potassium dihydrogen phosphate, 1.5 g/L). For the enumeration of enterococci, 100 L of each dilution was pipetted as a drop onto Chromocult EnterococciAgar (Merck KGaA, Darmstadt, Germany; dissolving 33 g of agar/L of distilled water). The plates were incubated for 24 h at 37C. The red colonies with a diameter of 0.5 to 2 mm were counted. For the enumeration of Escherichia coli and total coliforms, 1 mL of solution was pipetted onto an E. coli/coliform count plate (3M Petrifilm, Europe Laboratoires 3M Sant, Cergy-Pontoise, France) with violet red bile gel and an indicator of glucuronidase activity. The plates were

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Table 2. Effect of dietary CP and fiber (DF) on growth performance of piglets from 3 to 5 wk postweaning
16% CP Item BW, kg Initial Final ADFI, g Wk 1 Wk 2 Wk 3 Overall ADG, g/d Wk 1 Wk 2 Wk 3 Overall G:F, g/g Wk 1 Wk 2 Wk 3 Overall
1

20% CP Low fiber 9.18 16.7 570 765 890 742 338 374 444 385 0.49 0.49 0.53 0.54 High fiber 9.20 18.45 596 833 1,121 849 371 439 594 466 0.62 0.54 0.55 0.57 SEM 0.60 1.04 79 215 252 158 56 86 81 44 0.10 0.11 0.12 0.07 CP 0.415 0.659 0.994 0.739 0.948 0.908 0.928 0.876 0.700 0.773 0.615 0.797 0.611 0.858

P-value DF 0.785 0.001 0.949 0.827 0.361 0.709 0.272 0.131 0.001 0.001 0.207 0.078 0.056 0.001 CP DF 0.836 0.465 0.317 0.281 0.108 0.136 0.616 0.673 0.494 0.378 0.737 0.658 0.191 0.237

Low fiber1 8.97 16.89 598 875 1,032 835 350 382 453 395 0.60 0.46 0.45 0.50

High fiber1 9.07 18.01 568 774 967 769 362 417 563 447 0.66 0.55 0.59 0.60

Low fiber, 5.3% NDF, and high fiber, 7.15% NDF; as-fed basis.

incubated for 48 h at 37C, and all blue E. coli colonies and total coliform colonies were counted following the manufacturers instructions. The DNA from feces was extracted and purified using the commercial QIAamp DNA Stool Mini Kit (Qiagen, West Sussex, UK), and enterobacteria and lactobacilli were quantified by realtime PCR using SyBR Green dye (Castillo et al., 2005). The lactobacilli:enterobacteria ratio was calculated by subtracting log 16S rDNA gene lactobacilli copies/g of feces minus log 16S rDNA gene enterobacteria copies/g of feces (as-is basis). Short-chain fatty acids and lactic acid were determined by gas chromatography, after submitting the samples to an acid-base treatment followed by an ether extraction and derivatization, as described by Jensen and Jorgensen (1994). Ammonia was determined with the aid of a gas-sensitive electrode (Crison ISE-9665, Crison Instruments, S.A., Barcelona, Spain) combined with a digital voltmeter (Crison GLP 22). Three grams of digesta were diluted (1:2) with 0.16 M NaOH, and after homogenization, samples were centrifuged for 10 min (1,500 g at room temperature). The ammonia released was measured in the supernatant as different voltage in millivolts (adapted from Diebold et al., 2004). Tissue samples for morphological measures were dehydrated and embedded in paraffin wax, sectioned at 4 m, and stained with hematoxylin and eosin. Morphological measurements were performed with a light microscope (BHS, Olympus, Spain; Nofraras et al., 2006). Goblet cells and the number of intraepithelial lymphocytes were measured in 10 well-oriented crypts using a linear ocular micrometer (Olympus, Ref 20935040, Microplanet, Barcelona, Spain). These variables were expressed per 100 m. Differences among the nuclei of enterocytes, mitotic figures, goblet cells, and lymphocytes were clearly distinguishable at 400 magnifica-

tion. All the morphological measurements were made by the same person, who was blind to the treatments. Pig major acute-phase protein (Pig-MAP) concentration was determined by a sandwich type ELISA (PigMAP kit ELISA, PigCHAMP Pro Europa S.A., Segovia, Spain), according to the manufacturers instructions (Pieiro et al., 2009).

Statistical Analyses
Data on productive performance, organ measurements, microbial counts, SCFA, digesta viscosity, and intestinal morphology were subjected to factorial analysis, with initial BW as a covariate (whenever significant), using the GLM procedure (SAS Inst. Inc., Cary, NC). For all data analyzed, pen was considered the experimental unit. The level used for the determination of significance for all the analysis was 0.05. Data are presented as least squares means and SEM.

RESULTS Animal Performance and Health Status

In Table 2, the BW, ADFI, ADG, and G:F are presented. No significant differences were observed in the ADFI among treatments. Changes in the level of protein did not result in effects on animal performance. However, supplementation of fiber increased (P 0.01) the overall ADG and final BW. Furthermore, supplementation of fiber increased overall feed efficiency (P 0.001). In Table 3, the diarrhea incidence and antibiotic interventions during the 3-wk experimental period, and the fecal score and serum concentration of Pig-MAP on d 35 after weaning are presented. A significant in-

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Table 3. Effects of dietary CP and dietary fiber (DF) on the diarrhea incidence and antibiotic interventions in piglets from wk 3 to 5 after weaning, and the fecal score and serum pig-major acute phase protein (Pig-MAP) concentration on d 35 after weaning
16% CP Item Diarrhea incidence Fecal score3 Antibiotic interventions4 Pig-MAP,5 mg/mL
a,b 1 2

20% CP Low fiber 1.8 2.96ab 2.75ab 0.93 High fiber 1.1 3.29a 1.88b 0.84 SEM 1.4 0.74 2.81 0.31 CP 0.222 0.490 0.054 0.018

P-value DF 0.622 0.890 0.114 0.776 CP DF 0.092 0.041 0.018 0.293

Low fiber1 1.5 3.16ab 2.25ab 1.09

High fiber1 2.6 2.87b 6.38a 1.25

Different superscripts in the same row denote a significant difference (P 0.05). Low fiber, 5.3% NDF, and high fiber, 7.15% NDF; as-fed basis. 2 Mean values of animals having shown diarrhea per pen at the overall experimental period. 3 Fecal score: 1 = liquid; 2 = soft; 3 = normal; 4 = hard; at the last experimental day. 4 Mean values of intramuscular injections of Marbofloxacin (1 mL, Marbocyl 2%, Vtoquinol S.A., Lure Cedex, France) per pen at the overall experimental period. 5 At the last experimental day.

teraction (P 0.05) was observed between protein and fiber levels for fecal score and antibiotic interventions. Supplementing the 16% CP diet with fiber reduced the fecal score and increased the antibiotic interventions, whereas the opposite was observed in pigs fed the 20% CP diet. Furthermore, pigs fed on the 16% CP diet showed an increase (P 0.05) in the serum concentration of Pig-MAP compared with 20% CP diet.

Physicochemical Characteristics of Digesta and Fermentation

In Table 5 the viscosity, ammonia concentration, and SCFA (concentration and profile) of the proximal colon digesta are presented. A significant interaction (P 0.05) between protein and fiber levels was observed on the viscosity values of proximal colon digesta. Supplementation of fiber increased the viscosity of the colonic digesta in pigs fed on the 16% CP diet and decreased it with the 20% CP diet. Supplementation of fiber in the 16% CP diet also tended to increase the ammonia concentration of the proximal colon digesta, but decreased it when it was supplemented in the 20% CP diet (interaction, P = 0.073). High levels of protein (P = 0.066) or fiber (P 0.05) increased the concentration of the total amount of SCFA in the colonic digesta. Protein level had no effect on the SCFA profile, but fiber supplementation decreased (P 0.05) the proportion of propionic acid. An interaction was observed in the isoacids (sum of isobutyric and isovaleric acids) proportion, where the fiber supplementation decreased (P 0.05) their concentrations in the 20% CP diets, but increased their concentrations in the 16% CP diets.

Gut Development and Morphology of the Intestinal Wall

Table 4 shows the results of the relative full intestinal tract weight and proximal colon morphology measurements at 35 d postweaning. Changes in the protein and fiber level promoted shifts in the relative weight of the full intestines. A high protein level increased (P 0.05) the relative weight of the small and large intestine, whereas a greater fiber level increased (P 0.05) the relative weight of the large intestine. No differences between treatments were observed in relative liver weight (mean of treatments: 3.2% of BW, data not presented). A greater level of protein increased (P 0.05) the number of goblet cells and reduced (P 0.05) the intraepithelial lymphocytes.

Table 4. Effects of dietary CP and dietary fiber (DF) on gastrointestinal percentage of BW and the proximal colon morphology of piglets on d 35 after weaning
16% CP Item Intestinal weight (with content) Small intestine, % of BW Large intestine, % of BW Morphology Goblet cells/100 m IEL,2 cells/100 m
1 2

20% CP Low fiber 7.6 4.3 5.1 1.2 High fiber 7.8 5.4 5.6 1.5 SEM 1.10 0.90 0.35 0.17 CP 0.004 0.048 0.043 0.020

P-value DF 0.346 0.018 0.130 0.423 CP DF 0.158 0.486 0.772 0.252

Low fiber1 6.2 3.8 4.3 1.8

High fiber1 6.8 4.5 4.9 1.7

Low fiber, 5.3% NDF, and high fiber, 7.15% NDF; as-fed basis. IEL = intraepithelial lymphocytes.
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Table 5. Effect of dietary CP and dietary fiber (DF) on the physicochemical characterization of colon digesta and the amount and profile of short-chain fatty acids (SCFA) in the proximal colon digesta of piglets on d 35 after weaning
16% CP Item Viscosity, log of cP, SR11 Ammonia, mM/kg of fresh matter SCFA, mmol/L Profile, % SCFA Acetic Propionic Butyric Isoacids2 Lactic
a,b 1

20% CP Low fiber 3.1 9.2 146

ab

P-value SEM 0.44 2.52 20.2 4.47 4.29 3.49 0.31 0.513 CP 0.515 0.365 0.066 0.936 0.947 0.806 0.28 0.452 DF 0.559 0.479 0.048 0.153 0.011 0.223 0.653 0.333 CP DF 0.033 0.073 0.376 0.235 0.531 0.095 0.010 0.877

Low fiber1 2.8 8.4 126

ab

High fiber1 3.1 10.8 147

a

High fiber 2.6 8.2 155

b

51.2 30.8 12.3 0.7b 0.1

55.5 27.6 11.7 1.1a 0.3

53.0 31.7 10.5 0.9ab 0.2

63.4 26.5 14.2 0.6b 0.4

Different superscripts in the same row denote a significant difference (P 0.05). Low fiber, 5.3% NDF, and high fiber, 7.15% NDF; as-fed basis. 2 Sum of isobutyric and isovaleric acids.

Table 6 presents the results of standard microbiological testing for plate counts of enterococci and coliforms, and molecular microbiology using real-time PCR for counts of 16S rDNA gene copies of enterobacteria and lactobacilli on the last day of the experiment. On the first day of sampling, no significant differences were observed between treatments (data not shown). On d 35 after weaning, protein level did not promote changes in the counts of bacteria in feces. However, the increase in the level of fiber reduced (P 0.05) the coliform counts measured by conventional microbiology and enterobacteria measured by molecular microbiology. An increase (P < 0.05) was observed in the lactobacilli:enterobacteria ratio after fiber supplementation.

DISCUSSION Influence of the Level of CP on the Productive Performance

Previous reports have described the possibility of reducing CP level in piglet feeds in association with an adequate AA supplementation without affecting BW

gain and protein deposition (Le Bellego and Noblet, 2002). These authors described a reduction in the N excretion and an increase in the feed intake when CP was reduced from 22.4 to 20.4%. However, the practical application of this strategy to reduce levels of dietary protein may be limited by the availability of essential AA in relation to the requirements of the piglets (Figueroa et al., 2002). In a recent study, Nyachoti et al. (2006) observed a significant reduction on the ADG and ADFI of piglets when received diets containing 19% or less CP. The authors suggested that Ile, Val, or some other essential AA might have limited piglet performance. In the present study, pigs fed on the 16% CP diet showed similar performance than those pigs fed on the 20% CP diet, but showed less intestinal weight and fewer mucus-containing goblet cells. These results are similar to those presented by Le Bellego and Noblet (2002), who described that protein restriction (22.4 vs. 16.9% CP) in association with an adequate AA supplementation did not affect the level of performance of the piglets, but decreased the contribution of the internal organs (including the GIT) to the empty BW. As stated previously, diets used in the present study were formulated

Table 6. Effects of dietary CP and dietary fiber (DF) on the counts of bacteria measured by conventional microbiological analyses (enterococci and coliforms) and by quantitative real-time PCR (qPCR, enterobacteria and lactobacilli) in the feces of piglets on d 35 after weaning
16% CP Item Plate count, log of cfu/g Enterococci Coliforms qPCR, log 16S rDNA gene copies/g Enterobacteria Lactobacilli Ratio lacto:entero2
1 2

20% CP Low fiber 5.3 8.3 11.1 11.7 0.61 High fiber 5.3 7.0 10.3 11.8 1.46 SEM 0.92 1.20 1.13 0.36 1.131 CP 0.183 0.254 0.623 0.391 0.828

P-value DF 0.222 0.005 0.061 0.463 0.036 CP DF 0.256 0.122 0.406 0.917 0.388

Low fiber1 5.3 7.5 10.7 11.6 0.91

High fiber1 6.0 7.1 10.4 11.7 1.28

Low fiber, 5.3% NDF, and high fiber, 7.15% NDF; as-fed basis. Log 16S rDNA gene lactobacilli copies/g of feces minus log 16S rDNA gene enterobacteria copies/g of feces (as-is basis).
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to satisfy requirement standards for pigs between 10 to 30 kg of BW (BSAS, 2003) on a NE, Lys, Met, Thr, and Trp basis, but no corrections were performed for other essential AA, such as Ile or Val. The nutritional state appears to be a key factor in regulating GIT development, the mucous layer, and gut barrier integrity. Some AA, such as Leu, Pro, and Gln, which were at reduced quantity in the 16% CP diet, are recognized as essential substrates for the intestinal anabolic and catabolic processes (Wu, 1998; Buddington et al., 2001) and for the development of an adequate intestinal enzymatic activity (Zhang et al., 1998; Adeola and King, 2006).

Influence of the Level of Fiber on the Productive Performance

Results from the study indicate that the supplementation of the piglet diet with wheat bran (40 g/kg) and sugar beet pulp (20 g/kg) improved the productive performance and the feed efficiency of piglets from wk 3 to 5 after weaning. Differences in the BW at 5 wk after weaning ranged from 16.8 to 18.2 kg with the LF and HF diets, respectively. However, it should be pointed out that the increased BW gain with the HF diets was also due in part to the increased weight of the internal organs, possibly by a greater weight of the gut contents. Differences for the full small and large intestine weight averaged from 1.99 to 2.43 kg in piglets fed the LF and HF 20% CP diets respectively, which account for approximately 30 to 40% of the BW differences. Variable results are reported in the literature regarding the effect of dietary fiber in the daily BW gain and feed efficiency of piglets. It is generally accepted that fiber in the growing pig diets may reduce voluntary intake and fat and energy digestibility. Thus, in some studies, supplementation of the diets with NSP has been associated with a decrease in the daily BW gain of growing animals, even if diet formulations were calculated to provide a similar calculated NE supply. Wellock et al. (2008) reported a decrease in the BW gain of piglets (wk 1 and 2 after weaning) fed diets that had NSP content increased from 9.4 to 17.7% of NSP with increased amounts (15%) of inulin and highly purified cellulose. Similar results were observed by Freire et al. (1998) where piglets were fed diets supplemented with 15% of wheat bran (from 10 to 15% of NDF). In contrast, other studies support the idea of no adverse effects on BW gain or feed efficiency when piglets are fed moderate amounts of nonviscous fibrous sources. Longland et al. (1994) and Gill et al. (2000) reported no significant differences in BW gain and apparent digestibility of N and GE when 4- to 8-wk-old piglets were fed a cereal-based diet containing 0 or 15 to 18.5% of sugar-beet pulp (NSP from 13.8 to 21.8%, NDF from 12.1 to 16.5%). Negative effects were observed the first week after weaning, when HF diets tended to restrict voluntary intake and reduced daily BW gain. Mateos et al. (2006) showed an interaction between the type of

cereal and a moderate amount of oat hulls inclusion (2 to 4%) on feed intake and BW gain of piglets from wk 1 to 5 after weaning. Additional fiber increased performance in piglets given rice, but reduced it in piglets given maize. In both cases, supplementation of oat hulls increased the apparent total tract digestibility coefficients of OM, CP, and energy. Mateos et al. (2006) suggested that young pigs may have a minimal requirement for fiber of 6% NDF for 6 to 12 kg piglets that was not reached in the rice diets (3.43% of NDF). The BSAS (2003) nutrient requirement standard for pigs suggests a NDF content of 7 to 13% for weaned pigs of 10 to 30 kg of BW. In our study, the increase on the NDF content from 5.3 to 7.15% was associated with increased growth performance of the animals and confirms the observation that pigs may have a minimal requirement for fiber. Diets with reduced dietary fiber content and mean particle size, below requirements for a healthy development of the GIT of young pigs, may constrain the voluntary intake and the efficient utilization of nutrients.

Influence of the Level of CP and Fiber on the Intestinal Health

Our experiment showed an interaction between the protein and fiber level in the incidence of diarrhea and antibiotic interventions. The incorporation of fiber increased diarrhea in pigs fed on the 16% CP diet, but reduced it in pigs fed on the 20% CP diet. Interestingly, this interaction was not observed on the productive performance or the microbial counts in the colonic digesta, which may likely be explained by the greater antibiotic interventions applied to piglets fed on the HF 16% CP diet or by changes in other bacteria species not accounted for in our microbial analysis. Similar results regarding the interaction between CP concentration and fermentable carbohydrates on the productive performance or the intestinal microbiota have been reported by other authors. Bikker et al. (2006) reported that a HF diet (19.5 vs. 13.2% NSP) reduced growth performance of pigs from 4 to 8 wk of age, when fed on a low CP diet (15.2%) but not on a high CP diet (21.6%). Likewise, Pierce et al. (2007) reported that increasing concentrations of lactose in the postweaning diet from 3 to 6 wk of age allowed significant increases in the ADFI and ADG of piglets fed on a 21% CP diet, but reduced it when supplemented to a 16% CP diet. The authors also reported significant increases with the lactose supplementation in the lactobacilli and Bifidobacterium concentration in feces, and significant decreases in the E. coli counts, especially in the high protein diet. Protein fermentation in the digestive tract is considered as a potential risk provoking an intestinal dysbiosis and the proliferation of pathogenic bacteria (Prohaszka and Baron, 1980; Ball and Aherne, 1987). Some of the products released by protein fermentation contribute to SCFA production, but also release potential irritants of

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Hermes et al.
Apparent nutrient digestibility and incidence and severity of diarrhea. Can. J. Anim. Sci. 67:11051115. Bertschinger, H. U., E. Eggenberger, H. Jucker, and H. P. Pfirter. 1979. Evaluation of low nutrient, high fiber diets for the prevention of porcine Escherichia coli enterotoxaemia. Vet. Microbiol. 3:281290. Bikker, P., A. Dirkzwager, J. Fledderus, P. Trevisi, I. le HuerouLuron, and J. P. Lalles. 2006. The effect of dietary protein and fermentable carbohydrates levels on growth performance and intestinal characteristics in newly weaned piglets. J. Anim. Sci. 84:33373345. BSAS. 2003. Nutrient Requirement Standards for Pigs. Br. Soc. Anim. Sci., Penicuik, Midlothian, UK. Buddington, R. K., J. Elnif, A. A. Puchal-Gardiner, and P. T. Sangild. 2001. Intestinal apical amino acid absorption during development of the pig. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280:R241R247. Carneiro, M., M. Lordelo, L. F. Cunha, and J. Freire. 2007. Microbial activity in the gut of piglets: II. Effect of fiber source and enzyme supplementation. Livest. Sci. 108:262265. Castillo, M., S. M. Martn-Ore, M. Roca, E. G. Manzanilla, I. Badiola, J. F. Prez, and J. Gasa. 2005. The response of gastrointestinal microbiota to the use of avilamycin, butyrate and plant extracts in early-weaned pigs. J. Anim. Sci. 84:27252734. Diebold, G., R. Mosenthin, H. Piepho, and W. C. Sauer. 2004. Effect of supplementation of xylanase and phospholipase to a wheatbased diet for weanling pigs on nutrient digestibility and concentrations of microbial metabolites in ileal digesta and feces. J. Anim. Sci. 82:26472656. Figueroa, J. L., A. J. Lewis, P. S. Miller, R. L. Fischer, R. S. Gomez, and R. M. Diedrichsen. 2002. Nitrogen metabolism and growth performance of gilts fed standard corn-soybean meal diets or low-crude protein, amino acid-supplemented diets. J. Anim. Sci. 80:29112919. Freire, J. P. B., J. Peiniau, L. F. Cunha, J. A. A. Almeida, and A. Aumaitre. 1998. Comparative effects of dietary fat and fiber in Alentejano and Large White piglets: Digestibility, digestive enzymes and metabolic data. Livest. Prod. Sci. 53:3747. Gill, B. P., J. Mellange, and J. A. Rooke. 2000. Growth performance and apparent nutrient digestibility in weaned piglets offered wheat-, barley- or sugar-beet pulp-based diets supplemented with food enzymes. Anim. Sci. 70:107118. Htoo, J. K., B. A. Araiza, W. C. Sauer, M. Rademacher, Y. Zhang, and M. Cervantes. 2007. Effect of dietary protein content on ileal amino acid digestibility, growth performance, and formation of microbial metabolites in ileal and cecal digesta of earlyweaned pigs. J. Anim. Sci. 85:33033312. Jensen, B. B., and H. Jorgensen. 1994. Effect of dietary fiber on microbial activity and microbial gasproduction in various regions of the gastrointestinal tract of pigs. Appl. Environ. Microbiol. 60:18971904. Kim, J. C., B. P. Mullan, D. J. Hampson, and J. R. Pluske. 2007. Addition of oat hulls to an extruded rice-based diet for weaner pigs ameliorates the incidence of diarrhea and reduces indices of protein fermentation in the gastrointestinal tract. Br. J. Nutr. 99:12171225. Le Bellego, L., and J. Noblet. 2002. Performance and utilization of dietary energy and amino acids in piglets fed low protein diets. Livest. Prod. Sci. 76:4558. Longland, A. C., J. Carruthers, and A. G. Low. 1994. The ability of piglets 4 to 8 weeks old to digest and perform on diets containing two contrasting sources of non-starch polysaccharide. Anim. Prod. 58:465480. Mateos, G. G., M. Martin, M. A. Latorre, and R. Lazaro. 2006. Inclusion of oat hull in diets for young pigs based on cooked maize or cooked rice. Anim. Sci. 82:5763. Murata, H., N. Shimada, and M. Yoshioka. 2004. Current research on acute phase proteins in veterinary diagnosis: An overview. Vet. J. 168:2840. Nofraras, M., E. G. Manzanilla, J. Pujols, X. Gibert, N. Majo, J. Segales, and J. Gasa. 2006. Effects of spray-dried porcine plasma

the intestinal mucosa (e.g., ammonia, skatol, or indol). In our study, we detected the greatest concentrations of isoacids and ammonia in the colonic digesta of the LF 20% CP diets and HF 16% CP. Our results confirm that a greater dietary CP content (20% CP) was associated with greater protein fermentation in the colon; whereas a simultaneous increase in the amount of fiber into high CP diets increased the fermentation of carbohydrates (i.e., the SCFA concentration) and allowed a reduction in the protein fermentation and the enterobacteria counts in feces. In contrast, it is remarkable that the incorporation of fiber into the 16% CP diet increased the isoacid and ammonia concentrations, the viscosity of the colonic digesta, and the incidence of diarrhea. It could be speculated that a low protein diet coupled with a greater fiber diet might have altered GIT function and increased the amount of substrates that escape digestion in the small intestine, predisposing pigs to diarrhea (Pluske et al., 2002). Unfortunately, we did not measure foregut digestibility or small intestine digesta variables. However, as described above, we observed a reduced relative weight of the small and large intestine, and fewer mucus-containing goblet cells in piglets fed the low CP diet vs. the high CP diet, with likely implications on the gut barrier function. These results were coupled with a greater count of intraepithelial lymphocytes in the colon epithelium and a greater serum concentration of the PigMAP in the low CP diets. Acute-phase protein serum concentration is known to increase after inflammation caused by tissue damage or pathogen challenge (Pieiro et al., 2009), the magnitude of the response being in general related to the degree of lesions or extent of the disease (Murata et al., 2004). Present results suggest that a moderate inclusion of dietary fiber in the diet may increase productive performance and gut maturation of the piglets. However, effects on the intestinal health may depend on the level of protein and essential AA in the diet, which appear to have a key role in the development and function of the intestinal tract. Therefore, protein levels of 20% CP supplemented with wheat bran (4%) and sugar beet pulp (2%) showed positive effects on animal performance and less incidence of diarrhea and antibiotic interventions.

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Dietary protein and fiber for piglets

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