Animal Feed Science and Technology 215 (2016) 124–132

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Animal Feed Science and Technology

journal homepage: www.elsevier.com/locate/anifeedsci

Effect of different dietary fiber concentrates on the

metabolism and indirect immune response in silver catfish
Taida Juliana Adorian a,∗ , Fernanda Rodrigues Goulart a , Patrícia Inês
Mombach a , Naglezi de Menezes Lovatto a , Marina Dalcin a , Mabel Molinari b ,
Rafael Lazzari c , Leila Picolli da Silva a
a
Department of Animal Science, Federal University of Santa Maria, Avenida Roraima, n◦ 1000, Cidade Universitária, Bairro Camobi,
Santa Maria, 97105-900 RS, Brazil
b
Laboratory of Histotechnology, Federal University of Santa Maria, Campus of Palmeira das Missões, Avenida Independência, n◦ 3751,
Bairro Vista Alegre, Palmeira das Missões, 98300-000 RS, Brazil
c
Department of Animal Science and Biological Sciences, Federal University of Santa Maria, Campus of Palmeira das Missões, Avenida
Independência, n◦ 3751, Bairro Vista Alegre, Palmeira das Missões, 98300-000 RS, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: This work aims at assessing the metabolic and immunologic indirect responses of the addi-
Received 30 October 2015 tion of dietary fiber as a prebiotic agent to the diets of silver catfish (Rhamdia quelen).
Received in revised form 28 February 2016 Dietary fiber was concentrated from citrus pulp, brewery yeast biomass and linseed grains.
Accepted 2 March 2016
These were prepared and included in diets, as well as a treatment control and a commer-
cial diet containing prebiotic (Actigen® ). During 50 days, 600 juveniles silver catfish were
Keywords: maintained in a water recirculation system and fed with experimental diets, three times a
Rhamdia quelen
day. At the end of the test, the animals’ blood, liver, mucus and intestine were collected for
Immunity
determination of the metabolic and immunologic parameters. The experimental design was
Insoluble fiber
Metabolism completely randomized, with five treatments and four replications; data were subjected
Prebiotic to the analysis of variance, and the means were compared by the Tukey’s test (P < 0.05).
Soluble fiber Fiber concentrates the differences in composition and physical-chemical properties, which
resulted in differences in blood parameters, intestinal goblet cell counts, hepatic metabolic
intermediates and mucoprotein production by fish. Yeast autolysate and linseed fiber pro-
vide prebiotic effects when added to silver catfish diets, bringing benefits to the metabolism
and animal’s immune system.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction

For years, dietary fiber was considered an energy depletion agent, with undesirable effects when added to fish diets.
However, recent studies have shown that adequate levels of this fraction can enhance fish growth by improving the immune
response (Cerezuela et al., 2013; Yarahmadi et al., 2014). High concentrations of fiber in diets as well as variations in their

Abbreviations: CP, citrus pulp; DFCs, dietary fiber concentrates; FBC, fat binding capacity; HC, hydration capacity; IDF, insoluble dietary fiber; LF, linseed
fiber; SCFAs, short-chain fatty acids; SDF, soluble dietary fiber; TF, total dietary fiber; YA, yeast autolysate.
∗ Corresponding author.
E-mail addresses: taidajuliana@yahoo.com.br, tj.adorian@hotmail.com (T.J. Adorian).

http://dx.doi.org/10.1016/j.anifeedsci.2016.03.001
0377-8401/© 2016 Elsevier B.V. All rights reserved.
 T.J. Adorian et al. / Animal Feed Science and Technology 215 (2016) 124–132 125

physicochemical characteristics (water absorption capacity, fat binding capacity, among others) can actually improve such
responses.
Dietary fiber is a fraction that is resistant to digestion and absorption in the small intestine, with partial or total fermen-
tation in the large intestine (Association of Analytical Chemists (AOAC), 1995). Non-digestible constituents such as dietary
fiber are part of the ingredients used in the formulation of fish diets whichhave shown to possess the ability to positively
influence the intestinal microbiota, acting as a prebiotic, and reflecting metabolism, performance and health improvements
(Desai et al., 2012; Ingerslev et al., 2014). Diverse fiber fractions have shown positive results in the immune system modula-
tion, intestinal morphometry, disease resistance and fish’s intestinal pathogenic microbiota reduction (Cerezuela et al., 2013;
Yarahmadi et al., 2014). However, the effects of dietary fiber are not only associated with its solubility and dietary amount,
but also to keep identity with its origin, which determines their chemical composition and physicochemical properties (Silva
and Walter, 2012).
In agribusiness, there are several possible sources to be explored in this regard. Linseed is a rich oilseed in dietary fiber
(28–33.5 g/kg), widely used for oil extraction, which creates an underutilized residue in Brazil (Morris, 2001; Hutchins et al.,
2001). Likewise, citrus pulp generated from orange juice and brewing yeast are residues with high fiber content (54.8 and
31.4 respectively) which characterize them as potential sources for use in fish diets (Macagnan et al., 2014; Yamada et al.,
2003). Thus, diverse effects on metabolism and immune system can be observed with the use of different sources of fiber,
even at similar dietary levels on the diets.
High stocking density and metabolite excretion characterize intensive fish farming systems, resulting in water quality
deterioration and, thus, animal stress, causing an increase of cortisol concentration and a decrease of disease resistance in
fish (Harris and Bird, 2000). The most common way to treat these problems is using antibiotics, to which environmental
contamination, pathogen resistance and residual accumulation in fish meat are reported (Denev et al., 2009; Rico et al.,
2012). These facts have led to an increasing interest in the use of environmentally suitable substances, such as prebiotics,
which provide greater resistance to disease, immunity, intestinal modulation and performance in fish (Soleimani et al., 2012;
Hoseinifar et al., 2013, 2014). Given the above, the purpose of this study is to examine the effect of administration of diverse
dietary fiber concentrates from diverse dietary fiber sources, with different solubility and physicochemical characteristics,
on the metabolism and immunologic indirect responses of juveniles silver catfish (Rhamdia quelen).

2. Material and methods

The study was conducted at the Laboratory of Fisheries of the Department of Animal Science of the Federal University of
Santa Maria (UFSM), Rio Grande do Sul, Brazil (Latitude: 29◦ 41 03 S; Longitude: 53◦ 48 25 W), after being approved by
the Ethics Committee on Animal Trials of this University, under the process number 23081.009051/2014-53.

2.1. Test materials and preparation of dietary fiber concentrates

Three sources of dietary fiber from different origins (linseed, orange and brewer’s yeast) were selected for extraction and
concentration of this nutrient, because they have different proportions of soluble and insoluble fiber. In order to optimize
their addition in the experimental diets, total dietary fiber from these sources was concentrated to obtain ingredients with
at least 500 g/kg total dietary fiber, producing Dietary Fiber Concentrates (DFCs), which were added to the experimental
diets.

2.1.1. Linseed fiber

Linseed fiber is obtained in two distinct stages. In the first stage, soluble fiber of linseed (mucilage) was obtained by
soaking the whole grain in water at a concentration of 10% w/v, maintaining the reaction between 60 ◦ C and 80 ◦ C under
constant stirring for 150 min. Subsequently, the soluble fiber was separated from the grains by sieving, followed by addition
of ethanol, for the precipitation of this fraction following the method described by Goulart et al. (2013). In the second stage,
the demucilaged grain was defatted with hexane at a ratio of 1:2 (w/v), performing for 30 min washes. After defatted, the
protein content of the residue was reduced by dispersion in distilled water at room temperature at the ratio of 1:30 (w/v),
sifted and dried in an air circulating oven at 55 ◦ C for 24 h. The fibers obtained in both stages were mixed in order to get all
the fiber contained in linseed and ground in a micro-grinder (Marconi, model MA-630/1) to obtain an average particle size
of 590 ␮m, thus obtaining the linseed DFC.

2.1.2. Citrus pulp

Citrus pulp was obtained after the orange juice extraction of the Valencia variety. The remaining residue composed by
flavedo, albedo, seeds and membranes was washed with water at room temperature, titrated in cutter (Metvisa, model CUT
2.5) for 20 s and dried in air circulating oven at 55 ◦ C for 24 h, followed by grinding (Marconi, model MA-630/1) to obtain
an average particle size of 590 ␮m, resulting in the citrus pulp to be used in the test. This process aimed at simulating the
industrial obtainment of the citrus pulp.
126 T.J. Adorian et al. / Animal Feed Science and Technology 215 (2016) 124–132

Table 1
Fiber content (g/kg) and some physicochemical properties (/g sample) of the tested dietary fiber concentrates.

Actigen Linseed fiber Citrus pulp Yeast autolysate

Dietary fiber concentrates

IDF 98.80 438.60 358.60 335.80
SDF 420.70 194.70 207.70 343.60
TF 519.50 633.30 566.30 679.40
Crude protein – 165.60 41.00 143.00
Ether extract – 75.10 76.50 52.80
Dry matter – 879.6 887.50 894.50
Physicochemical properties
HC (g wader) 2.16 ± 0.03d 11.37 ± 0.03a 5.22 ± 0.03b 2.64 ± 0.005c
FBC (g fat) 0.79 ± 0.01c 1.60 ± 0.005a 1.20 ± 0.02b 0.99 ± 0.04bc

TF: Total dietary fiber (g/kg); IDF: Insoluble dietary fiber (g/kg); SDF: Soluble dietary fiber (g/kg); HC: hydration capacity; FBC: fat binding capacity. Values
are expressed as mean ± standard deviation. Different letters on the rows indicate significant difference by the Tukey’s test (P < 0.05).

2.1.3. Brewer’s yeast autolysate

The brewer’s biomass (Saccharomyces cerevisiae) donated by a beer manufacturer was collected and centrifuged twice
at 1200g for 15 min and then washed three times with 1:1 (w/v) distilled water to remove impurities and residual alcohol,
followed by another centrifugation. The clean yeast biomass was subjected to autolysis in water bath with stirring for 8 h at
49 ◦ C, according to the method described by Matiazi, 2006. Subsequently, it was dried in an air circulating oven at 55 ◦ C for
48 h and grinded in a micro-grinder (Marconi, model MA-630/1) to obtain an average particle size of 590 ␮m, resulting in
the Yeast Autolysate.

2.1.4. Chemical and physicochemical analysis

Chemical analyses of DFCs for determination of total dietary fiber (TF), insoluble fiber (IDF) and soluble fiber (SDF) was
carried out according to the gravimetric enzymatic method number 991.43 of Association of Analytical Chemists (AOAC)
(1985) (Table 1). Physicochemical analyses to determine the hydration capacity and fat binding were carried out according
to the methodology proposed by Wang and Kinsella (1976) (Table 1). The chemical composition was determined based on
analyses of dry matter, crude protein (Association of Analytical Chemists (AOAC), 1995) and fat (Bligh and Dyer, 1959).

2.2. Experimental diets

Five experimental diets (Table 2) were formulated to achieve the nutritional requirements of juvenile silver catfish,
according to Meyer and Fracalossi (2004). The experiment comprised the following treatments: a control diet based on fish
meal and maize starch (without addition of prebiotic agents); a diet containing 2.50 g/kg of commercial prebiotic Actigen®
based on mannan-oligosaccharides (Alltech Inc., Nicholasville, Kentucky, USA); and diets containing concentrated fiber of
Linseed (LF), Citrus pulp (CP) and Yeast autolysate (YA). The diets were produced in the Aquaculture Laboratory of UFSM.
The dry ingredients were weighed and manually homogenized, then water was added and pelleting with matrix of 4 mm in
diameter. They were dried in an oven with forced air circulation for 24 h at a temperature of 55 ◦ C. After drying, the diets were
milled and selected according to the fish ingestion capacity. Diets were stored under a temperature of −20 ◦ C throughout
the experimental period. The diets composition and physicochemical properties were determined based on analyses of dry
matter, ash, crude protein (Association of Analytical Chemists (AOAC), 1995), fat (Bligh and Dyer, 1959), hydration capacity
and fat binding capacity (Wang and Kinsella, 1976).

2.3. Animals and feed

Six-hundred juveniles of silver catfish with an average initial weight of 3.54 ± 0.53 g were distributed randomly in 20
polypropylene tanks with 230 liters capacity (30 animals per experimental unit). Each tank had individual water inlet and
outlet, arranged in a water recirculation system comprised of a decanter, two mechanical and biological filtering and a water
reservoir with a capacity for 2000 liters, equipped with a heating system. During the experimental period, the fish were fed
with the experimental diet until apparent satiation three times a day (8:00, 13:00 and 17:00 o’clock) for 50 days.

2.4. Water quality

Prior to the first and last meals (7:30 and 15:00 o’clock), fecal residues were removed from the tanks by siphoning twice a
day. During the experimental period, the water quality parameters were monitored using colorimetric kits and maintained
as follows: morning temperature of 20.94 ± 2.08 ◦ C; afternoon temperature of 22.01 ± 1.93 ◦ C; pH: 7.25 ± 0.27; alkalinity:
55 ± 13.95 mg CaCO3 /L; hardness: 64.83 ± 11.28 mg CaCO3 /L; total ammonia: 0.1 ± 0.06 mg L−1 ; nitrite: 0.12 ± 0.08 mg L−1
and oxygen: 8.43 ± 0.88 mg L−1 .
 T.J. Adorian et al. / Animal Feed Science and Technology 215 (2016) 124–132 127

Table 2
Formulation and centesimal composition of experimental diets (g/kg).

Treatmentsa

Control Actigen LF CP YA

Ingredients
Fish mealg 610.10 610.10 570.00 600.00 570.80
Maize starch 118.00 118.00 114.00 121.00 120.00
Actigen® b 0 2.50 0 0 0
Linseed fiber 0 0 146.00 0 0
Citrus pulp 0 0 0 177.00 0
Yeast autolysate 0 0 0 0 138.00
Cellulose 106.00 106.00 0 0 0
Soybean oil 60.00 60.00 42.00 46.00 55.00
Salt 5.00 5.00 5.00 5.00 5.00
Melbondc 20.00 20.00 20.00 20.00 20.00
Vitamin and mineral mixtured 30.00 30.00 30.00 30.00 30.00
BHTe 0.10 0.10 0.10 0.10 0.10
Inert (sand) 49.90 47.40 72.90 0.90 53.90
Total 1000 1000 1000 1000 1000
Analyzed nutrient
Crude protein 360.40 360.40 361.00 360.70 360.30
Ether extract 114.70 114.70 113.90 113.30 114.10
Total dietary fiber 100.20 100.20 100.40 100.70 100.90
Soluble fiber – – 31.20 37.30 50.90
Insoluble fiber 100.20 100.20 69.70 63.10 49.80
Dry matter 895.80 889.80 890.50 899.50 888.90
Ash 143.60 141.10 158.40 122.70 140.60
Digestible energy (MJ kg−1 )g f 13.43 13.43 13.40 13.43 13.42
Physicochemical properties
Hydration capacity 1.51 1.48 1.76 1.61 1.21
Fat binding capacity 0.89 0.87 0.89 0.87 0.86
a
Treatments: Control; Actigen: addition of commercial prebiotics Actigen® ; LF: linseed fiber; CP: citrus pulp; YA: yeast autolysate.
b
Commercial prebiotics based on mannan-oligosaccharide (Alltech, Inc, Nicholasville, Kentucky, USA).
c
Binder.
d
Composition (Migfish® /Mig-Plus, Casca, Rio Grande do Sul, Brazil (mg/kg diet)): Folic acid: 299.88; Ascorbic acid: 15,000.12; Pantothenic: 3,000.10;
Biotin: 12.06; Niacin (B3): 9,000.32; Hill (B4): 103,500.00; Vit. A: 1000000.00; Vit. B1: 1500.38; Vit. B2: 1500.0; Vit. B6: 1500.38; Vit. D3: 240000.00; Vit. E:
10000.00; Vit. K3: 400.00; Inositol: 9999.92; Iron: 6416.80; Manganese: 8000.40; Copper: 1000.00; Zinc: 13999.50; Iodine: 45.36; Cobalt: 60.06; Selenium:
60.30; Magnesium: 5.10; Chlorine: 2.30%; Sulfur: 0.01%.
e
Butyl hydroxy toluene (BHT).
f
Digestible energy: calculated digestible energy: [(Crude protein × 5.65 × 0.85) + (Fat × 9.4 × 0.9) + (Carbohydrates × 4.15 × 0.7)] (adapted from Meyer
and Fracalossi (2004)).
g
Fish meal of waste flour tilapia/Copisces-Paraná/RS Brazil.

2.5. Data collection and variables analyzed

At the end of the experimental period, the fish were fasted for 18 h. Subsequently, blood samples were collected randomly
(twelve fish/treatment) by tail vein puncture using heparinized syringes. The samples were placed in microcentrifuge tubes
for centrifuging (1000g, 10 min at room temperature). The plasma was stored and refrigerated (8 ◦ C) to determine the
concentrations of total circulating proteins (g/dL), albumin (g/dL), globulin (g/dL) (globulin (g/dL) = total protein−albumin),
glucose (mg/gL) and cholesterol (mg/gL). Eight fish/treatment were used for total blood sampling and determination of
hemoglobin. All tests were carried out using commercial kits made by Doles® .
After blood collection, the animals were euthanized by benzocaine overdose (10%, ≥250 mg/L) according to the American
Veterinary Medical Association (Association American Veterinary Medical, 2013) and collected the liver tissue. Hepatic
metabolites were determined in the liver samples (50 mg), which were heated to 100 ◦ C with KOH to estimate the protein
content according to the technique described by Bradford (1976). In an aliquot of this extract, ethanol was added to hydrolyze
and precipitate glycogen, and after centrifugation at 1000g for 10 min, the glucose content was determined (Park and Johnson,
1949). The liver samples (50 mg) were homogenized in 10% trichloroacetic acid (TCA) and centrifuged (1000g, 10 min), and
the supernatant was used for glucose quantification (Park and Johnson, 1949).
In addition to the liver, the anterior intestine was collected and prepared for light microscopy. Histological samples were
fixed in 10% formalin and preserved in 70% ethanol and subjected to the histological routine, following the method described
by Gressler et al. (2015). The material was sent to go through the histological routine for dehydration in increasing ethanol
series (70%–99% alcohol) and embedded in methacrylate glycol resin (Technovit 7100). From this material, slits of 2 ␮m were
obtained from rotary microtome (LEICA RM2245) to subsequent coloration with hematoxylin-eosin. For morphological
examination, the slides were observed and documented in light microscopy (ZEISS PrimoStar with AxioCam ERc5s) and
analyzed through the software ZEN LITE (Carl Zeiss). Goblet cells were counted in 500 ␮m of villus and the results expressed
in ␮m. The slides were thoroughly examined in order to determine the presence of histopathological alterations.
128 T.J. Adorian et al. / Animal Feed Science and Technology 215 (2016) 124–132

Table 3
Effect of dietary fiber concentrates on blood parameters in silver catfish (Rhamdia quelen).

Control Actigen Linseed fiber Citrus pulp Yeast autolysate SE P-value

Glucose (mg/dL) 73.07 78.11 77.99 76.30 74.90 2.19 0.220

Cholesterol (mg/dL) 75.32b 85.29ab 95.52a 87.66ab 93.59a 2.63 0.010
Total proteins (g/dL) 2.72b 3.09ab 3.24a 3.22a 3.28a 0.07 0.009
Albumin (g/dL) 0.24 0.29 0.28 0.30 0.26 0.01 0.234
Globulin (g/dL) 2.48b 2.80ab 2.96a 2.91ab 3.02a 0.07 0.014
Hemoglobin (g/dL) 4.76 5.08 5.37 5.32 4.42 0.14 0.185

Values are expressed as mean. Different letters on the rows indicate significant difference by the Tukey’s test (P < 0.05).

Table 4
Effect of dietary fiber concentrates on intestinal goblet cell counts (cells/g) in silver catfish.

Treatments

Control Actigen Linseed fiber Citrus pulp Yeast autolysate SE P-value

b ab a ab ab
Goblet cell counts (cells/g) 19.75 22.25 26.50 21.50 20.50 1.17 0.044

Values are expressed as mean. Different letters on the rows represent significant difference according to the Tukey’s test (P < 0.05).

The mucus of twelve fish/treatment was scraped from the skin for determination of the levels of mucoprotein (glycopro-
tein), using a Bioclin® commercial kit. The principle of this methodology is the protein precipitation in a solution of perchloric
acid, resulting in a glycoprotein fraction denominated seromucoid and/or mucoproteins. These are, then, precipitated in the
filtrate with phosphotungstic acid and subsequently dissolved and dosed by means of the tyrosine content.

2.6. Statistical analysis

A completely randomized design was used, with five treatments and four replications. The data were subjected to analysis
of variance and the means were compared by the Tukey’s test. Differences were considered significant at P < 0.05 level.

3. Results

3.1. Composition and properties of DFCs

The concentrates indicated total dietary fiber (TF) contents higher than 50% (Table 1), with distinct ratios of fiber sol-
uble/insoluble fractions (0.44 for LF, 0.58 for CP and 1.02 for YA) (Table 1). Linseed fiber has the highest insoluble fiber
content between the test ingredients, since the highest soluble fiber content is observed in the Yeast autolysate, followed
by Actigen. All fiber concentrates exhibit full dietary fiber content than the commercial prebiotic. The DFCs and commercial
prebiotics showed significant differences regarding the physicochemical characteristics of hydration capacity (P = 0.001) and
fat binding capacity (P = 0.024), with higher values for LF. The crude protein of concentrates were similar between Linseed
fiber (165.60 g/ kg) and Yeast autolysate (143.00 g/ kg), while Citrus pulp has a lower amount (41.00 g/ kg). The fat and dry
matter was very similar between the fiber concentrates.

3.2. Blood parameters

Supplementation with DFCs affected significantly (P < 0.05) the plasma parameters for cholesterol, total protein and glob-
ulin (Table 3). However, plasma glucose, albumin and hemoglobin did not change. The use of Linseed fiber, Yeast autolysate
and Citrus pulp in the diet resulted in higher values (P < 0.05) of total circulating protein, when compared to the results of
the control diet (Table 3). Cholesterol and serum globulins were higher (P < 0.05) in the animals fed with diets containing
Linseed fiber or Yeast autolysate, compared to the control group.

3.3. Count of goblet cells

Distinct counts of intestinal goblet cells were found in the intestine of fish fed with the experimental diets (P < 0.05)
(Table 4). The animals fed with Linseed fiber presented a higher goblet cell number than the control group (Table 4). The
other DFCs and the commercial prebiotics did not lead to significant differences when compared to the control group.

3.4. Hepatic parameters

The control and Actigen® diets yielded higher liver glycogen scores (Table 5). The animals supplemented with linseed
fiber showed the highest levels of hepatic protein (P < 0.05). Supplementation with the DFCs or with commercial prebiotic
did not alter the hepatic glucose concentration (Table 5).
 T.J. Adorian et al. / Animal Feed Science and Technology 215 (2016) 124–132 129

Table 5
Effect of dietary fiber concentrates on hepatic metabolic intermediates (/g tissue) in silver catfish (Rhamdia quelen).

Treatments

Control Actigen Linseed fiber Citrus pulp Yeast autolysate SE P-value

Glucose (␮mol glucose/g tissue) 168.34 164.38 128.43 156.07 163.14 6.97 0.382
Glycogen (␮mol glucose/g tissue) 8.55a 7.27a 5.28bc 4.93bc 4.07c 0.40 0.001
Protein (mg protein/g tissue) 6.39b 7.27ab 8.78a 6.95b 7.40ab 0.21 0.004

Values are expressed as mean. Different letters on the rows indicate significant difference by the Tukey’s test (P < 0.05).

3.5. Mucoprotein production

Production of mucoprotein in the silver catfish skin was strongly influenced (P < 0.05) by the experimental diets. Diets
with Linseed fiber or Yeast autolysate led to a higher production of mucoprotein (P = 0.013) compared to the control diet
(Fig. 1).

4. Discussion

The current study shows that the fiber concentration processes yielded products (DFCs) with similar contents of total
dietary fiber, but different solubility and physicochemical characteristics (Table 1), which can be exploited in a targeted way
to promote animal health. Usually, a higher hydration capacity by the fiber is related to its solubility, which is influenced by
the fiber composition and chemical structure.
The small difference in the amount of crude protein between fiber concentrates is not sufficient to cause differences in
diet, especially because these are isoproteic. Likewise, the fat and dry matter has little difference among concentrates. For
diets isonutritives, small differences in the composition of the ingredients are not sufficient to affect or mask the effects of
the test, since there is a balance of basic nutrients such as protein and fat. Thus, these differences are consequences of the
differences inherent to fiber and its physicochemical properties.
The concentrates of Linseed fiber and Citrus pulp had similar contents of soluble fiber, but a difference of approximately
two times in their respective hydration capacity (Table 1). The soluble fraction of Linseed fiber (mucilage) is made up of
polysaccharides formed by six different monosaccharides (Qian et al., 2012), structured in a highly branched chain, which
are capable of holding a great volume of water per molecular unit. On the other hand, the soluble fraction of Citrus pulp
consists of distinct pectin molecules, made up of at least 65% of galacturonic acids and sixteen different monosaccharides
(Voragen et al., 2009), with a less branched structure than that of mucilage, which explains its lower water holding capacity.
Already the Yeast autolysate and Actigen, which have the highest levels of soluble fiber, reflected in lower hydration capacity
thereof. This result may be related to the predominant presence of mannans and glucans in the fiber content thereof, since
they are derived from brewery yeast biomass.
The increased levels of soluble fiber in the diets containing DFCs did not have impact on the animals glucose level (Table 3).
In contrast, other studies indicate that a higher intake of soluble fiber leads to increase viscosity and luminal transit time,
thus reducing the rate and amount of glucose absorbed by the body (Fabek et al., 2014; Abirami et al., 2014). An increased
viscosity is associated with the food hydration capacity, which in the present study was higher in the diets containing
concentrates of Linseed fiber and Citrus pulp (Table 2). Although the variations of the water absorption capacity found in the
tested diets might have affected the digest viscosity, the effects on digestion and nutrients absorption were not expressive
enough to cause glucose variations.
The current results show that the silver catfish supplemented with 138.00 g/ kg of Yeast autolysate or 146.00 g/ kg of
Linseed fiber showed higher levels of plasma cholesterol compared to the control group (Table 3). Such finding can be

Fig. 1. Effect of dietary fiber concentrates on mucoprotein production (mg/dL) in silver catfish (Rhamdia quelen). Values are expressed as mean ± standard
error. Different letters in the figure represent significant difference by the Tukey’s test (P < 0.05).
130 T.J. Adorian et al. / Animal Feed Science and Technology 215 (2016) 124–132

associated with the fibers fermentation and increased production of short-chain fatty acids (SCFAs) in the colon, particularly
acetate, which causes enhanced synthesis of cholesterol (Kihara and Sakata, 1997; Hugenholtz et al., 2013). Studies point
to cholesterol as the precursor of hormones associated with protection against stress and vitamin D synthesis (Deng et al.,
2013; Cuesta et al., 2007). For this reason, plasma increase has been used as an indicator of health and improved immunity
of fish (Maita et al., 2006, 1998; Laporte and Trushenski, 2012). Cortisol levels in the plasma of fish fed diet containing
177.00 g/ kg of Citrus pulp did not differ from those who received the control diet (Table 3). This effect may be related to the
composition of soluble fiber source which is composed of high pectin content, a substance that has its consumption related
to reduction of plasma cholesterol levels (Judd and Truswell, 1982).
The immunomodulatory effect of diets supplemented with DCFs and the increased levels of plasma globulin could indicate
an immunomodulatory effect of diets by the increased levels of total protein and plasma globulin (Table 3), parameters that
are associated with the innate immune response and indicate an increased immunological resistance in animals (Andrews
et al., 2011). Total proteins and fractions are important in the assessment of the animals’ health condition because they
act on the regulation of the inflammatory response and response to infections (Ballow, 1997, 2011). Globulins are key for
maintaining the immune system in good condition, once they are the source of nearly all immunologically active proteins
in the blood (Choudhury et al., 2005; Jha et al., 2007).
Studies found in the literature did not investigate the effect of supplementary dietary fibers on the levels of protein and
plasma globulin, but rather carbohydrates in general, e.g. dextrin and corn starch, which provide a positive immunological
response in animals (Zhou et al., 2014; Wang et al., 2014). In addition, Dügenci et al. (2003) report an increase of total
proteins in rainbow trout (Oncorhynchus mykiss) fed with diets supplemented with immunostimulant basis for yeast RNA.
According to Choudhury et al. (2005); the levels of plasma globulin in fishes fed with immunostimulants are always higher
than the control. This statement corroborates the results found in this study and suggest that, when added to the diet in the
quantities tested, Yeast autolysate and Linseed fiber are beneficial to the silver catfish immune system.
The positive result of DCFs intake can also be observed in the intestinal goblet cells count (Table 4). The results from the
current study show that the silver catfish fed with diets with 146.00 g/ kg of linseed fiber had more goblet cells compared
to the control diet (Table 4), indicating a higher mucus production in the intestine. This result corroborates the higher skin
mucus production in the fishes that were fed with Linseed fiber diet (Fig. 1).
The number of goblet cells (Table 4) in the intestine is an important immunological indicator, once these cells are respon-
sible for the secretion of the mucus that covers the intestinal epithelium, forming the first defense line of the colon. The
mucus produced by the goblet cells provides chemical protection against the aggression caused by antigens, toxins and
digestive enzymes existing in the intestinal lumen (Gaudier et al., 2009), besides having bactericidal properties, reducing
the bacterial population in direct contact with the epithelial surface (Corfield et al., 2000; Hoebler et al., 2006). Thus, it is
suggested that the intestinal mucosa of fish that received Linseed fiber in the diet is the most protected of possible infections.
As the fish’s gastrointestinal tract is the gateway to numerous key pathogens, a greater amount of goblet cells can provide
resistance to intestinal pathogens benefiting animals. When studying the prebiotic Pediococcus acidilactici in diets for rain-
bow trout exposed to Vibrio anguillarum, Harper et al. (2011) observed an increased count of goblet cells in the animals that
received the prebiotic, which diminished the epithelium damages caused by V. anguillarum.
The positive effect of dietary supplementation with DFCs has also been found in the production of mucoproteins (Fig. 1),
which are responsible for the formation of a protective layer of mucus in the fish skin. It is well known that mucus is
primarily composed of water and glycoproteins, being part of the innate defense system and acts in preventing the pathogens
adherence in the skin due to its thickness and composition, but mainly because it is continuously produced and discarded
by the animal (Shephard, 1994; Guardiola et al., 2014; Tort et al., 2003; Nigam et al., 2012). The direct contact of fishes with
the environment makes them to be continually exposed to external hazards, such as pathogens and pollutants (Benhameda
et al., 2014; Coscia et al., 2014). Therefore, feed constituents that provide an increase in mucus production by the fishes can
be beneficial to their defense mechanisms against pathogens, being characterized as an important promoter of the immune
system.
Although studies assessing the effect of dietary fiber on fish mucus production have not been found, Sheikhzadeh et al.
(2012) investigated the effect of fermented Saccharomyces cerevisiae on growth and immunological components of the skin
mucus of rainbow trout, concluding that yeast supplementation has effectively contributed to improved growth perfor-
mance and non-specific immunological parameters of the mucus. In a work with the same species, Yarahmadi et al. (2014)
reported that dietary fiber could be considered an useful supplement in the positive regulation of the immunity-related
genes, stimulation of the immune response and increase of disease resistance, pointing out that the form of action of the
fibers on the immune system has not been established yet. Both works support the idea of the dietary fiber potential as a
prebiotic agent in diets and its beneficial effects on the fish immune system.
The results of the current study show that the fishes fed in the control diet and Actigen, Citrus pulp and Yeast autolysate
diets presented lower liver protein stock (Table 5) and control diet and Actigen diet showed a reduction in the levels of
total proteins in the plasma (Table 3), which indicates a lower immune status of the animals. The liver is responsible for
the synthesis of the components of the innate and adaptive immune response. Plasma proteins, of recognized importance
to the animal immunity (Andrews et al., 2011; Ballow, 1997, 2011), are synthesized in the liver, and higher hepatic protein
concentrations indicate an increase of the total circulating proteins in the plasma and improved immune system of the
animals. Reduced levels of hepatic protein can indicate that the fishes are using protein to provide their energy needs to
maintain blood glucose.
 T.J. Adorian et al. / Animal Feed Science and Technology 215 (2016) 124–132 131

In the animals fed diets containing DFCs, maintenance of the plasma glucose has likely occurred at the expense of the
liver glycogen stores (Table 5), which indicates a higher energy demand of these animals to produce the immunological
indicators assessed. This condition can be the consequence of the animal’s preference for using the energy stored in the form
of glycogen to keep the immunological status in the fasting condition. According to Klasing (1998); the immune system is
the priority met by nutrients readily available, such as glycogen, where diets that maximize performance generally provides
adequate substrate for the immune system to function satisfactorily.
According to Gibson and Roberfroid (1995), prebiotics are indigestible nutritional ingredients that beneficially affect the
host by improving the health of its host. Thus, it can be deduced from the results of the current study that Linseed and
Yeast autolysate fiber concentrates could act as prebiotics in diets to silver catfish since they provide benefits to the immune
system, causing equivalent or higher effects than those obtained by the administration of the commercial prebiotic that was
tested (Table 3–4 and Fig. 1). These results demonstrate the possibility of manipulating diets into the search for benefit the
health of animals, ensuring food safety, once they minimize the need for antibiotics. Further researches are necessary in
order to associate the dietary fiber with the prebiotic effects, such as the intestinal microbiota modulation and resistance to
diseases.

Acknowledgements

The authors express their gratitude to the National Council of Scientific and Technological Development (CNPq) for the
grant of the research productivity scholarship (Leila Picolli da Silva), to CAPES for the Master’s scholarship (Taida Juliana
Adorian), and to Alltech® , Agropecuária Giruá, Giovelli & Cia Ltda and Santamate Indústria e Comércio Ltda for the ingredients
provided as courtesy.

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