Animal (2015), 9:7, pp 1113–1119 © The Animal Consortium 2015

doi:10.1017/S1751731115000300
animal

A dietary dairy/yeast prebiotic and flaxseed oil enhance growth,

hematological and immunological parameters in channel catfish
at a suboptimal temperature (15°C)
M. Thompsona, R. Lochmann†, H. Phillips and T. D. Sinkb
Department of Aquaculture and Fisheries, University of Arkansas at Pine Bluff, 1200 N. University Drive, Pine Bluff, AK, USA

(Received 20 August 2014; Accepted 3 February 2015; First published online 6 March 2015)

Channel catfish raised in the southern United States require two growing seasons to reach market size. Growing seasons are
separated by a cool period of about 3 months when feed intake and growth are greatly reduced. A cool-weather feeding strategy
to improve feed intake, growth or health of catfish might improve survival and reduce the time needed to achieve market size. We
conducted a feeding trial with channel catfish at a suboptimal temperature (15°C) to determine the effects of supplementing diets
with either a dairy/yeast prebiotic or flaxseed oil (high in 18:3n-3) compared with a control with soybean oil (high in 18:2n-6). The
trial was conducted in recirculating systems with 1140-l tanks containing 100 fish each (mean initial weight 61.4 g ± 0.43 s.e.m.).
A 28%-protein basal diet was supplemented with 20 g/kg cellulose and 20 g/kg soybean oil (SBO, control), 20 g/kg cellulose and
20 g/kg flaxseed oil (FLAX) or 20 g/kg of a dairy/yeast prebiotic and 20 g/kg soybean oil (PREB). Fish were fed once daily to
satiation and weighed every 3 weeks to track growth. Hematology, non-specific immune responses, proximate and fatty acid
composition of muscle were determined to assess diet effects. Catfish-fed FLAX or PREB had higher weight gain, feed consumption
and lysozyme activity than fish fed SBO. Total n-3 fatty acids in muscle were higher in fish fed SBO or FLAX than those fed PREB.
Total n-6 long-chain polyunsaturated acids were higher in muscle of fish fed PREB than those fed SBO. Fatty acids in the PREB and
SBO diets were similar, so the PREB appeared to increase elongation and desaturation of n-6 fatty acids in muscle. Flaxseed oil
and the dairy/yeast prebiotic both have potential to increase catfish performance at a low temperature.

Keywords: catfish, lipids, prebiotics, low temperature

Implications occurs primarily in the southeastern region, where water

temperature varies seasonally (MacMillan and Santucci,
Growth, feed intake and non-specific immune response of
1990). Two growing seasons separated by a winter period
channel catfish reared at a low temperature (15°C) were
(December, January and February) are required to reach
enhanced by the addition of flaxseed oil or a dairy/yeast
market size (0.6 to 0.7 kg). Catfish feed inconsistently at
prebiotic to the diet compared with a diet supplemented with
lower water temperatures (<20°C), but feeding during the
soybean oil. The use of dietary n-3 fatty acids and a prebiotic
winter may help maintain weight and health (Robinson and
to enhance growth and immune response at a suboptimal
Li, 2002). Fish are visibly active during mild winter periods
temperature could improve overall production efficiency of
when temperatures are between 13°C and 20°C, thus daily
catfish in ponds by reducing the weight loss and mortality
or every-other-day feeding is recommended (Robinson and
associated with the 3-month winter period.
Li, 2002). Economic analysis shows that this partial feeding
regime is also profitable compared with not feeding in winter
Introduction
because it reduces mortality and mitigates weight loss (Hatch
Channel catfish (Ictalurus punctatus) is the most widely cul- et al., 1998).
tured food fish in the United States. Production in ponds Few studies have focused on the potential of different
dietary lipids to enhance feed intake, feed conversion effi-
a
Present address: Aquatic Biologists Inc., N4828 US Highway 45 S, Fond du Lac, ciency and growth of channel catfish at low temperatures.
WI, USA.
b
Present address: Texas A&M University, Department of Wildlife and Fisheries
However, there are demonstrated differences in the immune
Science, Texas A&M AgriLife Extension, College Station, TX, USA. responses of catfish due both to dietary lipid source and
†
E-mail: rebecca.lochmann@yahoo.com water temperature. Channel catfish-fed diets rich in n-3 fatty

1113
Thompson, Lochmann, Phillips and Sink
acids had increased immune function at low temperatures,
but at high temperatures a diet rich in n-6 fatty acids
enhanced disease resistance (Lingenfelser et al., 1995).
Sheldon and Blazer (1991) also positively correlated dietary
n-3 concentration (particularly long-chain polyunsaturated
fatty acids, or LC-PUFA), with enhanced intracellular prone-
phros macrophage killing of Edwardsiella ictaluri in channel
catfish at optimum (28°C) and suboptimal (19°C) tempera-
tures for growth. Lysozyme activity was higher in channel
catfish maintained at 22°C and fed diets high in n-3 fatty
acids (with fish or flax oil) than those fed a diet with soybean
oil (Suja et al., 2012). Because lipids are the easiest compo-
nent of fish tissues to manipulate through the diet, their
potential to improve general fish performance as well as fish
health at low temperatures should be explored further.
Non-nutritive supplements such as prebiotics may also
have potential to enhance growth, feed efficiency and overall
health of fish at low temperatures. Prebiotics are non-
digestible dietary compounds that can be metabolized by
specific microflora in the gut (Gibson and Roberfroid, 1995).
Typical benefits include improved nutrient assimilation,
growth and immune function (Vos et al., 2007). A dairy/yeast
prebiotic had beneficial effects in golden shiners Notemigonus
crysoleucas and goldfish Carassius auratus in our laboratory
(Lochmann et al., 2009; Lochmann et al., 2011), but the same
dairy/yeast prebiotic did not enhance growth or feed conversion
of channel catfish at optimal temperatures (27°C to 28°C)
(Thompson, 2009). The gut microflora of channel catfish varies
seasonally (MacMillan and Santucci, 1990). Prebiotic effects
may vary with temperature because different bacteria have
different temperature optima for growth and survival (Panigrahi
et al., 2007). Furthermore, there is evidence that dietary lipid
and prebiotics interact in fish, as they do in mammals. In
humans, short-chain fatty acids produced by fermentation of
dietary prebiotics by the gut microflora modulate blood lipids
(Floch, 2010). Interactions between dietary lipid concentration
and prebiotic on survival of goldfish exposed to a pathogen
were observed but not explained in Lochmann et al. (2011).
These findings raise the possibility that catfish performance at
low temperatures could be manipulated with dietary lipid,
prebiotic or both to improve fish performance.
The objective of this study was to determine the effects
of supplementing diets with either soybean oil (control),
flaxseed oil (a concentrated source of n-3 fatty acids), or
soybean oil and a dairy/yeast prebiotic on the performance
and body composition of channel catfish at a suboptimal
temperature (15°C).
Material and methods
Experimental design and culture system
A 12-week feeding trial was conducted at the Aquaculture
Research Station at the University of Arkansas at Pine Bluff in
three independent recirculating systems. Each system con-
sisted of four 1140-l tanks, a sump (528 l) and a biofilter
(Ultima 2 filter, Aqua Ultraviolet, Westminster, CA, USA) to
maintain water quality. The temperature in each system was
1114
maintained at 15°C by circulating water through thermistor-
controlled heat pump and chiller units. The pH, total
ammonia nitrogen (TAN, salicylate/cyanurate method),
hardness, alkalinity and nitrite were measured every 4 weeks
to ensure suitable conditions. The pH was measured with an
electrode (Denver Instruments, Denver, CO, USA), and the
other parameters with a HachTM DR/890 colorimeter test lab
(Hach Company, Loveland, CO, USA). Un-ionized ammonia was
calculated with the results of TAN and pH measurements
(Emerson et al., 1975). Mean water quality parameters did not
differ among treatments throughout this study. Parameters
were (mean ± s.e.m.): temperature (°C), 15.1 ± 0.1; pH,
6.96 ± 0.52; total hardness (mg/l), 75.5 ± 9.1, alkalinity (mg/l),
65.6 ± 14.8, un-ionized ammonia (mg/l), 0.0043 ± 0.0013 and
nitrite (mg/l), 0.166 ± 0.028.
Feeding and monitoring
Channel catfish fingerlings were obtained onsite from the
Aquaculture Research Station. Due to the possibility of cross
contamination of different lipids, independent recirculation
systems with four tanks each were used for the three treat-
ments. Each tank within each system was stocked with
100 fish (mean weight 61.4 ± 0.43 g s.e.m.), and allowed to
acclimate to experimental water conditions and a tempera-
ture of 26°C. Over a 2-week period, the temperature in each
system was decreased 1°C daily to 15°C. This gradual
acclimation was meant to simulate water conditions commonly
found in commercial ponds in the fall, when temperatures
gradually decrease toward winter temperatures. All fish in each
tank were group-weighed every 3 weeks to monitor growth and
adjust feeding rate.
The three experimental diets were prepared by modifying
a commercially available diet that contained 280 g/kg pro-
tein (ARKAT, Dumas, Arkansas). The ingredient composition
was proprietary, but the diet was formulated to meet or
exceed all known nutrient requirements of channel catfish
(Robinson and Li, 2002). The modification consisted of
grinding the commercial diet, then adding 20 g/kg cellulose
and 20 g/kg soybean oil (SBO), 20 g/kg cellulose and 20 g/kg
flaxseed oil (FLAX), or 20 g/kg of a dairy/yeast prebiotic
(GroBiotic-A™; International Ingredient Corp., St. Louis, MO,
USA) and 20 g/kg soybean oil (PREB), before being repelleted
into sinking 6-mm pellets. The inclusion rate for the prebiotic
was chosen based on manufacturer’s recommendations.
Cellulose was included in the diets without prebiotic to
produce diets with similar total energy content. The com-
mercially available dairy/yeast prebiotic contained partially
autolyzed brewer’s yeast, dairy ingredient components and
dried fermentation products with ∼530 g/kg simple and
complex carbohydrates. The analyzed total lipid of the pre-
biotic was 36 g/kg, and 16:0, 18:1n-9, and 18:2n-6 were the
main fatty acids (comprising more than 800 g/kg of total
fatty acids). Fish were offered feed up to a maximum of 1.5%
total BW, until apparent satiation was achieved. After
45 min, the uneaten feed was collected with a siphon, and
then dried to constant weight at 50°C and weighed.

Blood sample collection
At the end of 12 weeks, final group weights were taken, and
all fish were held for 2 weeks to minimize the potential
effects of handling stress on health parameters. Fish were
maintained on their experimental diets during this time. At
14 weeks, 10 fish were randomly selected from each tank for
hematological analyses. Fish were anesthetized with tricaine
methanesulfonate (MS-222, Sigma, St. Louis, MO, USA) at a
dose of 100 mg/l. Blood samples were drawn from anesthe-
tized fish by severing the caudal peduncle and collecting
blood in heparinized microhematocrit capillary tubes. Blood
from five fish from each tank was used to analyze hematocrit
(Ht) after centrifugation (3500 × g for 10 min), and hemo-
globin (Hb) following Houston (1990). Mean corpuscular
hemoglobin content (MCHC) was calculated according to the
formula: MCHC = Hb (g/dl)/ Hk. Plasma from these fresh
blood samples was used to analyze alternative complement
activity (ACH50) following Tort et al. (1996). Plasma from the
remaining five blood samples from each tank were used in
the lysozyme analysis following the procedures of Hutchinson
and Manning (1996) and Magnadottir et al. (1999).
Proximate and fatty acid analysis
Five randomly selected fish from each tank were euthanized
with an overdose of tricaine methanesulfonate (200 mg/l).
The liver of each fish was weighed for calculation of hepa-
tosomatic index (HSI = liver weight/BW). Muscle samples
were stored at −70°C and later analyzed for protein (ID
7.015) and ash content (ID 7.009) using standard methods
(Association of Official Analytical Chemists, 1995). Dry
matter was determined using AOAC procedure 7.007, except
that the drying time was reduced to 2 h because samples had
reached constant weight. Diets were analyzed using the
same procedures. Total lipids from the three experimental
diets, the basal diet, as well as muscle samples from three
fish per tank were extracted and quantified using the
chloroform/methanol procedure described by Folch et al.
(1957). Lipid extract from the chloroform/methanol proce-
dure was then used for fatty acid analysis. After methylation
of the lipid extract, fatty acid methyl esters (FAMEs) were
collected for analysis using a flame ionization gas chroma-
tograph (Varian, Model CP-3800 with a CP-8200 auto-
sampler, Walnut Creek, CA, USA) using helium as the carrier
gas. The FAMEs were separated on a fused silica capillary
column (15 m × 0.25 mm ID; Varian CP select for Fame
#CP8510). Injection volume was 1 μl, with an injector and
detector temperature of 250°C and 315°C, respectively. The
column temperature were held initially at 100°C for 10 min,
increased to 160°C at a rate of 15°C/min and held for 4 min,
then increased to 250°C at a rate of 2.5°C/min. Each sample
had a total analysis time of 60 min. The FAMEs were iden-
tified and quantified by comparing the retention time and
peak area to those of serially diluted mixtures of reference
standards (GLC-96, GLC-473b, Nu-Check Prep, Elysian, MN,
USA). Tridecanoate methyl ester (13:0) served as the internal
standard. The results of the individual fatty acids were
expressed as g/kg of total identified FAMEs.
Prebiotic, n-3 fats and catfish performance at 15°C
Statistical analysis
Percentage data were arc-sin transformed before statistical
analysis. Weight gain, survival, feed intake, FCR, hematocrit,
Hb, lysozyme, complement and proximate and fatty acid
composition data were analyzed by one-way ANOVA with
a StatView program (SAS Institute Inc, version 5.0.1) to test
for differences among experimental groups. When differences
among treatment means were significant (P ⩽ 0.05), Fisher’s
least significance difference test was used to compare means.
Results
Proximate and fatty acid composition of experimental diets
Ash and total lipid content did not differ among the three
diets (Table 1). Dry matter was higher in the SBO diet than in
the PREB diet (Table 1). CP was slightly higher (0.66%) in the
PREB diet than in the FLAX diet (Table 1), but all diets con-
tained at least 280 g/kg protein. Fatty acid composition for
the experimental diets is summarized in Table 2. Saturated
fatty acids did not differ among experimental diets. Mono-
unsaturated fatty acids (MUFAs), arachidonic acid (20:4n-6)
and total n-6 PUFAs were highest in the diet with FLAX and
lowest in the diet with SBO. Linoleic acid (18:2n-6) and total
n-6 fatty acids were highest in the diet with SBO and lowest
in the diet with FLAX. Alpha-linolenic acid (18:3n-3) and total
n-3 fatty acids were highest in the diet with FLAX and lowest
in the diet with PREB. No n-3 PUFAs were detected in any of
the diets. The ratio of n-3 : n-6 fatty acids was higher in the
FLAX diet than in both the SBO and PREB diets.
Growth, survival, and feed consumption and conversion
Mean individual weight gain and mean individual feed con-
sumption were higher in fish fed FLAX or PREB diets compared
with the SBO diet (Table 3). The FCR and survival were similar
among treatments (Table 3).
Proximate and fatty acid composition of muscle
Muscle dry matter and ash were higher in fish fed the diet
with PREB than those fed diets with SBO or FLAX (Table 4).
CP and total lipid in muscle did not differ among treatments
(Table 4). Muscle saturated fatty acids, MUFAs, 20:5n-3,
22:6n-3, total n-6 fatty acids, and n-3 LC PUFAs did not differ
among treatments (Table 4). Muscle 18:2n-6 was higher in
fish fed the diet with PREB than in fish fed diets with SBO
Table 1 Proximate composition (g/kg) of the basal diet and three
experimental diets in a 12-week feeding trial with catfish fingerlings
maintained at 15°C1
Diet Dry matter Total lipid Protein Ash
Basal 915 86 299 68
SBO 891 98 284 35
FLAX 870 96 281 43
PREB 869 94 287 46
SBO = soybean oil diet; FLAX = flaxseed oil diet; PREB = prebiotic diet.
1
All values are means of duplicate samples.
1115
Thompson, Lochmann, Phillips and Sink

or FLAX. Muscle 18:3n-3 and total n-3 fatty acids were higher the FLAX or PREB diets had higher mean individual weight
in fish fed diets with SBO or FLAX than in those fed the diet gain and mean individual feed consumption than fish fed the
with PREB. Muscle 20:4n-6 and total n-6 LC PUFAs were SBO diet. It is unlikely that dietary energy is the cause of
higher in fish fed the diet with PREB compared with those fed these differences in growth and consumption, because total
the diet with SBO. The ratio of n-3 to n-6 fatty acids was lipid concentrations did not differ among diets. In fish, lipids
higher in muscle of fish fed the SBO or FLAX diets than those are the major source of energy used for growth, health and
fed the PREB diet (Table 4). normal function (Sargent et al., 2002). Fish can also use
protein as a source of energy, but the slight differences in
Health indices dietary protein (<10 g/kg) would not explain the growth
Fish fed the FLAX diet had a higher HSI than fish fed diets
differences because all diets contained at least 280 g/kg,
with SBO or PREB (Table 5). Fish fed diets with FLAX or PREB
which meet catfish requirements (Robinson and Li, 2002). At
had higher MCHC and lysozyme activity compared with fish
lower temperatures, the unsaturation of lipids in fish tissues
fed the SBO diet (Table 5). Complement activity did not differ
increases as a means of maintaining normal cell membrane
among treatments (Table 5).
function (Hazel and Williams, 1990). Catfish have the ability
to elongate and desaturate shorter-chain fatty acids found in
Discussion plants into their longer-chain homologs; for instance, 18:3n-
3 → 20:3n-3 →20:5n-3. Satoh et al. (1989) showed that at
Despite the suboptimal temperature limiting growth rates
26.7ºC, catfish growth and feed efficiency were enhanced by
(35% to 44% increase from initial weights), fish fed either
dietary n-3 fatty acids. In particular, catfish fed diets with
either cod liver oil (high in 20:5n-3 and 22:6n-3) or linseed oil
Table 2 Selected fatty acid composition of the experimental diets (g/kg
(high in 18:3n-3) exhibited the highest growth rate and feed
of total fatty acids by weight) used in a 12-week feeding trial with
catfish fingerlings maintained at 15°C1 efficiency, while catfish fed diets with 18:2n-6 showed no
improvements in growth or feed efficiency. Catfish fed diets
Diets with menhaden fish oil, or a mixture of oils including fish oil
had better growth than catfish fed diets with beef tallow,
Fatty acids2 SBO FLAX PREB
corn oil or linseed oil at 28ºC (Fracalossi and Lovell, 1995).
Saturates3 213.3 220.8 216.7 However, at a lower temperature (17ºC), catfish fed diets
Monounsaturates4 285.4 301.1 292.9 with corn oil or linseed oil had similar growth to fish fed diets
18:2n-6 448.4 312.4 439.1 with menhaden oil or the mixed oil (Fracalossi and Lovell,
18:3n-3 49.0 163.7 47.3 1995). Corn oil is made up mostly of 18:2n-6, much like
20:4n-6 1.1 2.0 1.4 soybean oil, while linseed oil is similar to flaxseed oil (high in
Σn-35 49.0 163.7 47.3 18:3n-3). Yet, in the current study, catfish fed the FLAX or
Σn-66 452.3 314.4 443.1 PREB diets had enhanced growth compared with catfish fed
Σn-3 LC-PUFA 0.0 0.0 0.0 the SBO diet. Conflicting results between the study of Fra-
Σn-6 LC-PUFA7 1.1 2.0 1.4 calossi and Lovell (1995) and this study are difficult to
n-3 : n-6 ratio 0.11 0.52 0.11
explain, because there were multiple differences in the
SBO = soybean oil diet; FLAX = flaxseed oil diet; PREB = prebiotic diet; proximate and ingredient composition of the purified diets in
LC-PUFA = long-chain polyunsaturated fatty acids. their study v. the practical diets used in the current study.
1
All values are means of duplicate samples.
2
Fatty acids detected at ⩽ 1.0 g/kg of total fatty acids by weight are not included. Mixed results regarding growth and feed efficiency of fish
3
Saturates included 14:0, 16:0, 17:18:0, 19:0, 21:0, 22:0 and 24:0. fed the dairy/yeast prebiotic used in this study have been
4
Monounsaturates included 16:1, 16:1n-7, 18:1n-7, 18:1n-9 and 20:1. obtained for hybrid striped bass (Li and Gatlin, 2004), red
5
Total n-3 fatty acids included 18:3n-3.
6
Total n-6 fatty acids included 18:2n-6, 18:3n-6, 20:4n-6 and 22:2n-6. drum (Burr et al., 2009; Buentello et al., 2010), rainbow trout
7
Total n-6 LC-PUFA included 20:4n-6. (Sealey et al., 2007), golden shiner (Lochmann et al., 2011)

Table 3 Effect of diets supplemented with SBO, FLAX or SBO and a dairy/yeast PREB on weight gain, survival, feed consumption and FCR of channel
catfish fingerlings in a 12-week feeding trial at 15°C1
Pooled
SBO FLAX PREB s.e.m. P-value

Weight gain (g) 21.55y 27.07z 26.51z 0.67 0.039

Mean individual feed consumption (g) 63.88y 71.50z 75.39z 115.92 0.005
FCR2 3.37 2.95 3.28 0.06 0.080
Survival (%) 99.25 98.50 99.50 0.16 0.201
SBO = soybean oil; FLAX = flaxseed oil; PREB = prebiotic; FCR = feed conversion ratio.
1
All values are means of four replicate tanks, each containing 100 fish initially. Means within rows with different letters are different (P ⩽ 0.05) as determined by one-
way ANOVA.
2
FCR is the mean value for the calculations from each replicate. FCR = mean individual consumption/mean individual weight gain.

1116
 Prebiotic, n-3 fats and catfish performance at 15°C

Table 4 Effect of diets supplemented with SBO, FLAX or SBO and a dairy/yeast PREB on muscle proximate composition (g/kg) and selected fatty acid
composition (g/kg of total fatty acids by weight) of channel catfish fingerlings in a 12-week feeding trial at 15°C1
SBO FLAX PREB Pooled s.e.m. P-value

Dry matter 242.02y 245.47y 253.08z 0.25 0.031

Total lipid 53.28 58.43 63.63 0.26 0.065
Protein 174.99 177.28 176.71 1.00 0.207
Ash 48.11y 42.62y 50.55z 0.17 0.024
Fatty acids2
Saturates3 268.63 272.16 274.38 0.17 0.136
Monounsaturates4 436.72 431.06 431.53 0.27 0.504
18:2n-6 188.79y 189.56y 202.79z 0.41 0.052
18:3n-3 38.00z 32.33z 17.59y 0.41 0.009
20:4n-6 15.90y 20.36yz 20.40z 0.09 0.010
20:5n-3 2.24 2.40 1.57 0.02 0.080
22:6n-3 7.77 10.54 8.32 0.06 0.061
Σn-35 68.81z 68.64z 51.03y 0.44 0.027
Σn-66 224.03 226.35 241.41 0.52 0.101
Σn-3 LC-PUFA7 13.78 17.28 13.18 0.11 0.102
Σn-6 LC-PUFA8 17.73y 22.67yz 22.95z 0.10 0.002
n-3 : n-6 ratio 0.32z 0.32z 0.21y 0.03 0.027
SBO = soybean oil; FLAX = flaxseed oil; PREB = prebiotic; LC-PUFA = long-chain polyunsaturated fatty acids.
1
All values are means of duplicate samples of five individual fish from each of four replicate tanks per treatment. Means within rows with different letters are different
(P ⩽ 0.05) as determined by one-way ANOVA.
2
Fatty acids detected at ⩽ 1.0 g/kg of total fatty acids by weight are not included.
3
Saturates included 14:0, 16:0, 17:0, 18:0, 19:0, 20:0 and 21:0.
4
Monounsaturates included 16:1, 16:1n-7, 18:1(n-7 + n-9), 20:1, 22:1 and 24:1.
5
Total n-3 fatty acids included 18:3n-3, 20:3n-3, 20:5n-3, 22:5n-3 and 22:6n-3.
6
Total n-6 fatty acids included 18:2n-6, 18:3n-6, 20:4n-6, 22:2n-6 and 22:4n-6.
7
Total n-3 LC-PUFA included 20:5n-3, 22:5n-3 and 22:6n-3.
8
Total n-6 LC-PUFA included 20:4n-6 and 22:4n-6.

Table 5 Effect of diets supplemented with SBO, FLAX, or SBO and a dairy/yeast PREB on HSI, MCHC, lysozyme, and
alternative complement activity (ACH50) of channel catfish fingerlings in a 12-week feeding trial at 15°C 1
Pooled
SBO FLAX PREB s.e.m. P-value

HSI2 1.97y 2.44z 2.10y 0.20 0.004

MCHC3 15.17y 16.88z 17.20z 0.40 <0.000
Lysozyme (units/ml serum) 8.81y 34.72z 26.60z 6.57 0.004
ACH50 (units of activity)4 13.21 14.86 14.87 0.76 0.100
SBO = soybean oil; FLAX = flaxseed oil; PREB = prebiotic; HSI = hepatosomatic index; MCHC = mean corpuscular hemoglobin
concentration.
1
All values are means of four replicate tanks. Five individual fish per tank (20 per diet) were used in the analysis of MCHC, ACH50, and an
additional five individual fish per tank were used in the analysis of lysozyme activity. Means within rows with different letters are
different (P ⩽ 0.05) as determined by one-way ANOVA.
2
HSI is calculated by the following formula: HSI = fish liver weight (g)/BW (g) × 100 from five individual fish from each of four replicate
tanks per treatment.
3
MCHC is calculated by the formula: MCHC = Hb concentration/hematocrit fraction.
4
ACH50 is reported as the reciprocal of the serum dilution causing 50% lysis of rabbit red blood cells.

and goldfish (Savolainen and Gatlin, 2009). In channel cat-
fish reared at an optimal temperature for growth (27°C),
there were no differences in general performance of fry or
fingerlings fed the dairy/yeast prebiotic (Thompson, 2009).
Therefore, growth improvement in catfish fed the prebiotic
in this trial at a suboptimal temperature was encouraging.
Prebiotics are thought to alter the microbial community in the
intestine, which in turn can enhance dietary nutrient utilization
by the fish. Red drum (Sciaenops ocellatus) fed a dairy/yeast
prebiotic had increased apparent protein and organic matter
digestibility over fish fed the basal diet (Burr et al., 2008). In cool
months of the year, characterized by inconsistent feeding, poor
growth and increased FCR, the use of a dietary prebiotic may
enhance the ability of catfish to utilize the smaller amount of
feed that they consume. However, nutrient digestibility was not
measured in this trial and the mechanism of growth enhance-
ment by prebiotics in catfish at a low temperature needs to be
addressed with additional research.

1117
Thompson, Lochmann, Phillips and Sink
Fish fed the diet with PREB had an increase in both fillet
dry matter and ash content. Prebiotics can enhance calcium
and magnesium uptake through several possible modes of
action, such as decreasing gastrointestinal pH, bacterial
community shifts and production of short-chain fatty acids
such as acetate and propionate (Griffin and Abrams, 2008).
The fatty acid composition of muscle differed among the
three treatments in many ways. Fish have some ability to
modify fatty acids consumed in the diet (Sargent et al.,
2002), which is one mechanism used to facilitate physiolo-
gical adaptation to different environmental temperatures
(Bell et al., 1986). The FLAX- and SBO-fed fish had increased
proportions of total n-3 fatty acids in the muscle tissue
compared with fish fed the PREB diet. However, the pro-
portions of the individual n-3 fatty acids differed slightly. Fish
fed the FLAX or SBO diet had higher concentrations of 18:3n-
3 over those fed the PREB diet, but all three treatments
resulted in similar muscle concentrations of 20:5n-3, 22:6n-3
and total n-3 LC-PUFA. This suggests that different rates of
elongation and desaturation of the dietary n-3 fatty acids
occurred among treatments. Suja et al. (2012) found that
catfish reared at 22°C fed diets supplemented with soybean
oil, flaxseed oil or menhaden fish oil had similar fatty acid
profiles (including longer-chain fatty acid homologs) to those
of their respective diets. The concentration of total n-6 fatty
acids in muscle tissue in this study did not differ among
treatments. However, the concentration of 18:2n-6 in muscle
of fish fed the PREB diet was higher than that of fish fed the
FLAX or SBO diets. Concentrations of 20:4n-6 and total n-6
LC-PUFA were also higher in muscle of PREB-fed fish compared
with those fed the SBO diet without prebiotic. The differences in
fatty acid composition of the SBO and PREB diets were very
minor, and do not explain the higher concentrations of n-6 fatty
acids (18:2n-6 and 20:4n-6) found in muscle of fish fed the
PREB diet compared with the SBO diet. These results indicate
the dairy/yeast prebiotic altered the metabolism of dietary fatty
acids in channel catfish at a low temperature. Prebiotics sti-
mulate the gut microflora to produce short-chain fatty acids
such as acetic and propionic acid that can affect serum cho-
lesterol, triglycerides and phospholipids (Floch, 2010). However,
further research is needed to determine the effects of prebiotics
on the metabolism of individual fatty acids, particularly at
suboptimal temperatures.
The HSI was higher in fish fed the FLAX diet than in fish fed
the SBO or PREB diet. Larger livers in fish fed the FLAX diet
may be linked with higher concentrations of total hepatic
enzymes, as production can increase in fish at a low tem-
perature to compensate for a lower metabolic rate (Kent
et al., 1988). The livers were smaller in fish fed either diet
with soybean oil, indicating that the same adaptation did not
occur in fish fed the SBO or PREB diets with more n-6 fatty
acids. The FLAX- and PREB-fed fish had significant increases
in both MCHC and lysozyme activity over the SBO treatment.
Complement activity followed the same trend, but did not
differ among treatments. The FLAX diet contained the high-
est proportion of n-3 fatty acids as 18:3n-3 relative to the
other diets, which might have been a contributing factor to
1118
the increased lysozyme observed in FLAX-fed fish. In general,
there is a correlation between the concentration of n-3 fatty
acids in the diet, temperature and the immunocompetence of
fish (Bowden, 2008). In Suja et al. (2012), catfish reared at
22°C and fed diets with flaxseed oil or menhaden fish oil had
increased lysozyme activity compared with fish fed the con-
trol diet with soybean oil. Catfish reared at 18°C fed a diet
high in total n-3 fatty acids from menhaden fish oil had
enhanced immune parameters compared with catfish fed
diets with catfish oil and a low level of n-3 fatty acids (Lin-
genfelser et al., 1995). However, fish fed the PREB diet in the
current study had lower concentrations of total n-3 fatty
acids than fish fed diets with FLAX or SBO, yet fish fed the
PREB diet also had increased lysozyme activity compared
with those fed the SBO diet. Red drum fed a dairy/yeast
prebiotic also had increased lysozyme activity (Buentello
et al., 2010), but catfish fed the same prebiotic at optimal
temperature for growth did not have increased lysozyme
activity compared with the control (Thompson, 2009).
Ambient temperature clearly affects many physiological
conditions in fish, including gut microflora, health status and
immunocompetence (MacMillan and Santucci, 1990; Bow-
den, 2008). Seasonal trends in intestinal microflora are found
in many species, including channel catfish (MacMillan and
Santucci, 1990; Hagi et al., 2004), hybrid tilapia Oreochromis
niloticus × Oreochromis aureus (Al-Harbi and Uddin, 2004),
silver carp Hypophthalmichthys molitrix, common carp
Cyprinus carpio and crucian carp Carassius cuvieri (Hagi
et al., 2004). At low temperatures when catfish feed intake is
reduced significantly the gastrointestinal tract may even
become devoid of all microbes (MacMillan and Santucci, 1990).
However, as long as fish are feeding they should retain a
microflora, which can be affected by dietary prebiotics or lipids,
both of which can modulate the immune response (Gibson and
Roberfroid, 1995). Further studies are needed to characterize
the microbial community in catfish gastrointestinal tracts at
different temperatures, as well as the specific effects of pre-
biotics and dietary fatty acids on that community.
In summary, the growth, feed intake, lysozyme and MCHC
of catfish reared at a low temperature were enhanced by the
addition of flaxseed oil or a dairy/yeast prebiotic to the diet
compared with a diet supplemented only with SBO. Lipids
have a well-documented role in the maintenance of immu-
nocompetence, especially at lower temperatures, and the
inclusion of n-3 rich lipids should be considered for winter diets
for catfish. In contrast, prebiotics have had mixed effects on the
immune response and overall performance in fish, and their
modes of action are poorly understood. In addition, the gut may
be sparsely colonized by microbes at low temperatures. There-
fore, the ability of prebiotics to enhance nutrient utilization and
immune response of catfish during this vulnerable part of the
production cycle should be explored further.
Acknowledgments
The authors thank the Arkansas Catfish Promotion Board for
partial support of this study, International Ingredient

Corporation for donating the GroBiotic®-A, and Bioriginal Food
and Science Corporation for contributing flaxseed oil. Andrew
Goodwin, David Heikes, and Alf Haukenes reviewed the
manuscript.
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