The Israeli Journal of Aquaculture - Bamidgeh, IIC:63.2011.

588, 8 pages

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Cottonseed Oilcake as a Protein Source in Feeds for

Juvenile Tilapia (Oreochromis niloticus): Antinutritional
Effects and Potential Detoxification by Iron
Supplementation
I. Meric1,4,a, S. Wuertz2,3*a, W. Kloas4, G. Wibbelt5, C. Schulz2,3
1

Department of Fisheries and Aquaculture, Faculty of Agriculture, University

of Ankara, Ankara, Turkey

Institute of Animal Breeding and Husbandry, Marine Aquaculture, ChristianAlbrechts-Universitt zu Kiel, Kiel, Germany
3
4

Gesellschaft fr Marine Aquakultur mbH, Bsum, Germany

Leibniz Institute of Freshwater Ecology and Inland Fisheries, Berlin,

Germany
5

Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany

(Received 12.6.10, Accepted 12.8.10)

Key words: tilapia, cottonseed, iron, fishmeal, gossypol, cottonseed, growth,

histology, sublethal, chronic
Abstract
Crude cottonseed oilcake, a protein-rich by-product of cottonseed oil
production, was used to replace fishmeal in diets for juvenile tilapia
(Oreochromis niloticus). Crude cottonseed oilcake was included in the diets at
levels of 0% (control), 15%, 30%, and 45%, as an alternative protein source.
The impact of the main antinutritional component of cottonseed oilcake,
gossypol, on growth and its chronic adverse effects on blood hematocrit and
tissue histology were assessed and compared with the documented effects of
cottonseed meal. The detoxifying effect of iron (Fe) on gossypol was
evaluated in the diets containing 30% and 45% cottonseed oilcake, which
were supplemented with ferrous sulfate heptahydrate. As the rate of oilcake
rose, the specific growth rate and protein efficiency ratio slightly, but
significantly, dropped. Ferrous supplementation compensated for this effect in
the 30% diet, but not in the 45% diet. However, iron supplementation led to
mild inflammation in the intestine (infiltration of neutrophilic granulocytes),
fatty degeneration of the liver, and a lower hepatosomatic index.
Nevertheless, due to the beneficial effect on growth, iron supplementation is
critical. No histopathological effects of the dietary cottonseed oilcake were
observed in the gills, gonads, or heart. In conclusion, the effects of
cottonseed oilcake supplementation were moderate when compared to the
effects of cottonseed meal and a replacement level of 15-30% is
recommended to obtain non-detectable effects.

* Corresponding author. Gesellschaft fr Marine Aquakultur mbH, Hafentrn 3, D-25761 Bsum,

Germany, tel.: +49-(0)4834-96539915, e-mail: wuertz@gma-buesum.de
a
The first two authors contributed equally to this work.

Meric et al.

Introduction
Diets are generally the largest expenditure in intensive aquaculture operations (40-70%
of the production costs), and protein is the most expensive dietary component
(Shamshak and Anderson, 2009). In view of depleting wild stocks, fishmeal, the
conventional protein source in fish feeds, needs to be substituted to allow for sustainable
aquaculture development (FAO, 2009). Thus, the replacement of fishmeal with less costly
plant feedstuff must be emphasized. However, the presence of antinutritional factors and
other active biological compounds limit the use of substitutes, and toxic effects have
been reported in fish (Francis et al., 2001; Lee et al., 2006).
Turkey is one of the biggest cotton producers in the world, producing 1,440,000 tons
of cottonseed in 2008 (FAO database). Crude cottonseed oilcake is an inexpensive and
readily available by-product of oil extraction from cottonseed. Annual production of
oilcake is projected to grow an average of 2.3% annually (Ramachandran et al., 2007).
Above all, the increased production of organic cotton over the last decade provides
pesticide-free quality oilcake, beneficial for the production of human food products (OTA,
2009). Cottonseed by-products rank third in the world among produced vegetable protein
concentrates (Chhorn, 1996) and are available at much lower costs than animal proteins.
In addition, the use of by-products is a dictate of environmental sustainability,
particularly with regard to the declining water resources in the eastern Mediterranean
region.
Traditionally, undecorticated cottonseed is crushed and screw-pressed, providing a
protein-rich crude oilcake that is characterized by high fiber and protein contents. In
recent years, additional extraction procedures (fat, protein) deliver a dry cottonseed
meal of potentially better quality (Lynn and Wedegaertner, 1986). Due to their high
protein content of up to 40% (Ramachandran et al., 2007), cottonseed products - oilcake
and meal - could be used as an inexpensive protein source in fish feeds. However,
gossypol, a secondary metabolite stored in the pigment glands of cottonseed, is wellknown for numerous antinutritional effects that limit its use as an alternative protein
source (Rinchard et al., 2003ab; Yue and Zhou, 2008).
While non-ruminant animals such as swine and poultry are particularly sensitive, fish
can tolerate relatively high levels of free gossypol (Roehm et al., 1967; Cheng and
Hardy, 2002). Studies on fish have focused on cottonseed meal supplementation,
reporting adverse effects on blood parameters (hematocrit and hemoglobin), growth, and
development (Blom et al., 2001; Rinchard et al., 2003a). The addition of iron salts to the
diet reduces gossypol toxicity (Wenegaertner, 1981; Yildirim et al., 2003). Still, reports
are controversial and the level of dietary iron necessary to prevent toxic effects has not
been conclusively determined.
Here, in contrast to other studies, we studied the feasibility of fishmeal replacement
by cottonseed oilcake (and not cottonseed meal) at a moderate level (<50%) and iron
supplementation (in diets containing 30% or 45% cottonseed oilcake) with regard to
gossypol detoxification. Focusing on chronic sublethal effects, we carried out a
comprehensive histopathology of the major organs reported for adverse gossypol impact
in order to determine a safe substitution level on the basis of the non-detectable effect
concept recently raised by animal welfare concerns.
Materials and Methods
Experimental diets. Cottonseed oilcake was obtained from a commercial feed company
(Altn Yem Co. Izmir, Turkey), ground, and used to substitute fishmeal at 0% (control),
15%, 30%, or 45% (Table 1). To assess the detoxifying effect of iron on gossypol, the
30% and 45% diets were supplemented with ferrous sulfate heptahydrate - Fe2(SO4)3 at 900 and 1350 mg Fe/kg feed, respectively, on a dry matter basis. The gossypol
content of the cottonseed oilcake was determined according to Kim and Calhaun (1995)
with modifications according to Blom et al. (2001). The free and total gossypol contents
of the oilcake were 0.08 mg/kg and 0.47 mg/kg, respectively. The six experimental diets
were isonitrogenous and isoenergetic. The dry ingredients were mixed with water and

Cottonseed oilcake replaces fishmeal in juvenile tilapia diets

lipids in a laboratory pelleting

machine
(Alexanderwerk
AG,
Remscheid,
Germany)
with
a
1.0
Diet
mm
diameter
die,
dried
at
room
0
15
30 30+Fe
45 45+Fe
temperature for three days, and
Ingredient
stored at 4C.
Fishmeal
417
357
297
296
236
235
Experimental setup. The 8-week
Fish oil
61
53
45
45
37
36
feeding trial was performed at the
Vit/min premix a
45
46
46
46
46
46
Wheat starch
439
311
180
179
46
46
Department of Animal Breeding and
Wheat gluten
29
29
29
29
30
30
Aquaculture in the Tropics and
Chromium oxide
9
9
9
9
9
9
Subtropics at Humboldt University,
Fe(SO4)7.H2O
0
0
0
4
0
6
Berlin. Eighteen 240-l tanks with
Cottonseed oilcake
0
195
394
392
596
592
two recirculation systems were
Proximate composition
randomly stocked with eight tilapia
Dry matter
936
927
945
929
935
934
(62.95-65.66 g), each, providing
Crude protein
311
321
326
303
329
324
triplicates for each diet. Each
Crude lipid
97
97
99
91
99
98
recirculation system comprised nine
Crude ash
132
128
128
121
128
128
tanks, a sedimentation chamber,
Crude fiber
4
52
116
99
166
170
and a trickling filter. The mean
Free gossypol
0.0 0.157 0.315 0.315 0.630 0.630
water temperature, pH, dissolved
Total gossypol
0.0 0.925 1.851 1.851 3.702 3.702
b
oxygen, and conductivity were
NFE
460
454
447
485
444
450
determined daily (WTW Multi 340 I,
Gross energy c
19.4 19.5 19.6 19.4 19.6 19.6
Weilheim, Germany). There were
P/E ratio
16.0 16.5 16.6 15.6 16.8 16.5
a
insignificant differences between the
Supplied by Vitakalk Marienfelde Mineralfutter, Marienfelde
GmbH, D-22761, Hamburg, Germany and includes (per kg
two systems (A/B: 24C/24C, pH
premix): 250,000 IU retinol, 10,000 IU cholecalciferol, 1 g 7.98/8.14, 7.17/7.25 ppm O2,
tocopherol, 0.05 g thiamine, 0.1 g riboflavin, 0.08 g
802/792 sm/cm). The fish were
pyridoxine, 1 g cyanocobolamine, 1 g ascorbic acid, 0.15 g
fed three times a day by hand to
menadione, 1 g niacin, 0.5 g calcium pantothenic acid, 5 g
biotin, 0.35 g magnesium, 0.03 g folic acid, 5 g choline
apparent satiation, which amounted
chloride, 0.15 g cobalt carbonate, 0.3 g CuSO4.5H2O, 3 g
to an average of 2.0% of the total
ZnO, 0.35 g MnO, 2.5 g Fe2(SO4)3.H2O, 0.08 g NaCl, 0.05 g
biomass, adjusted according to
NaSeO3
b
biweekly weighings.
Nitrogen free extracts including crude fiber = 100 - (crude
protein + crude lipid + crude ash)
For body composition analysis,
c
calculated according to crude protein 23.9 MJ/kg, crude lipid
three fish were sampled before the
39.8 MJ/kg, NFE 17.6 MJ/kg
trial and three fish from each
treatment at the end of the trial. The fish were ground whole and stored at -20C until
analyzed. At the end of the experiment, blood from five randomly sampled fish per tank
was drawn from the caudal vein with heparinized syringes. Plasma was immediately
separated by centrifugation (12000 g min) for quantification of the hematocrit
(determined as the mean of 3-5 readings). From each dietary treatment, 14 fishes
(seven of each sex) were dissected to determine hepatosomatic index (HSI) and
gonadosomatic index (GSI) and selected tissues (intestine, gonad, gill, heart, liver) were
sampled for histopathology. For each treatment, mortality, specific growth rate (SGR),
feed conversion ratio (FCR), protein efficiency ratio (PER), and condition factor (CF) were
calculated as the average of the triplicates.
Dietary and whole fish nutrient analysis. The experimental diets and whole body
samples were analyzed for dry matter (DM), ash, crude protein, crude fat, and crude
fiber according to Schulz et al. (2007): dry matter was determined after drying at 105C
for 24 h, ash after combustion at 550C for 4 h, crude protein (N 6.25) by the Kjeldahl
distillation method (Kjeltec System, Tecator, Sweden), crude fat after extraction with
petroleum ether by the Soxhlet method (Soxtec System HT, Tecator, Sweden), and crude
fiber in a Fibertec TM 1020 hot extractor and Fibertec System 1021 cold extractor
(Tecator TM Technology, Sweden).
Histology. After dissection, tissue samples were fixed in Bouins solution for 12 h,
transferred to an automated tissue processor (Bavimed) for dehydration, embedded in
paraffin (DDM-P064 embedding centre, MEDIM), sectioned into 5-m slices (motorized
Table 1. Formulation of the experimental diets (g/kg
dry matter).

Meric et al.

rotary microtome RM 2065 SuperCut, Leica), and stained with hematoxylin-eosin. To

determine iron accumulation in livers of fish fed the iron-supplemented diets, liver
sections were stained with Perls Prussian blue (Perls, 1867) and analyzed under a Leica
DMRBE microscope equipped with a digital camera.
Statistical analysis. Data are presented as meansstandard deviations (SD) of
triplicate groups or individuals evenly sampled from each replicate. Data were analyzed
for normal distribution by Kolmogorov-Smirnov and equal variance by Kruskal-Wallis
one-way analysis of variance (p<0.05) using SigmaStat 2.0 software (Jandel Scientific,
San Rafael, CA). Multiple comparison was carried out by parametric Dunnetts Test or
nonparametric Dunns Test.
Results
Growth. No acute toxicity was observed and survival was comparable in all treatments
and tanks (Table 2). Final body weight was comparable in all groups, but there were
significant differences in SGR, FCR, PER, and CF. In general, growth parameters were
low; SGR was highest in fish fed the control diet and significantly lower in fish fed diets
with high oilcake supplementation levels. Iron supplementation compensated for this
impact in the 30% diet but not in the 45% diet. Generally, FCR negatively correlated with
SGR. PER decreased as the oilcake replacement increased. Again, iron compensated for
this impact in the 30% diet but not in the 45% diet. Whole body compositions were not
significantly influenced by treatment, except for the extremely low lipid content in the
30% group.
Table 2. Growth and feed efficiency (meansSD) of Nile tilapia juveniles fed diets
containing different levels of cottonseed oilcake as a replacement of fishmeal, with or without
iron (Fe) supplementation.
0
Initial wt (g)
64.332.63
Final wt (g)
102.152.15a
Wt gain (g)
37.820.84a
1
SGR (%/d)
0.830.04a
FCR2
1.480.06a
PER3
2.330.17a
CF
1.770.10
Survival (%)
95.837.22
Whole body composition
Dry matter
2955
Crude protein
1384
Crude lipid
1072a
Crude ash
651

Diet
15
30
30+Fe
65.662.99 65.043.21
63.792.82
99.463.44a 95.675.36a
97.924.84a
ab
a
33.801.63 30.632.38
34.133.51ab
ab
b
0.740.04
0.690.03
0.760.06ab
1.610.08ab 1.730.07ab
1.550.15ab
2.100.10ab 1.880.15b
2.250.23a
1.780.02
1.840.09
1.730.03
1000.00
1000.00
1000.00
31224
1606
11217a
623

26230
13311
707b
446

30938
15617
11327a
4025

45
45+fe
62.951.13 64.214.58
93.173.96a 93.947.36a
30.224.86b 29.732.77b
0.700.10b 0.680.01b
1.730.28ab 1.790.04b
1.840.30b 1.770.05b
1.730.04
1.740.03
1000.00
1000.00
31616
16915
10217a
5819

3047
1607
1058a
5219

Means in row with different superscripts significantly differ (p<0.05).

1
Specific growth rate = 100 (ln Wtfinal - ln Winitial)/duration of experiment in days
2
Feed conversion ratio = Wttotal feed supplied/(Wtfinal - Winitial)
3
Protein efficiency ratio = (Wtfinal - Winitial)/Wdietary crude protein supplied

Gonadosomatic index (GSI), hepatosomatic index (HSI), hematocrit level, and

nutrient composition of fish. There were no significant differences (p>0.05) among
dietary treatments in terms of GSI for males or females; GSI was higher in females than
in males, though not significantly. The HSI decreased from 1.9% to 0.7% in males and
from 2.3% to 1% in females as the percent of oilcake supplementation increased (Fig.
1). Hematocrit levels varied 26.6-36.6% with no differences between sexes or
treatments.
Histological examination. No histopathological changes were evident in the gills,
gonads, or heart of fish fed oilcake supplemented diets. However, pathological changes
were detected in the liver and intestine, the most prominent was a fatty degeneration
within the liver (Fig. 2). There were highly vacuolated hepatocytes with severe fat

Cottonseed oilcake replaces fishmeal in juvenile tilapia diets

deposition predominantly in the periportal region. Further, a moderate centrolobular fat

deposition was frequently observed in these samples. Iron accumulated within the
centrolobular region in groups fed iron-supplemented diets. Additionally, there was mild
infiltration of neutrophilic granulocytes within the lamina propria and tela submucosa of
the intestine in fish fed the iron-supplemented diets (Fig. 3).
a

Fig. 1. The (a) hepatosomatic index (HSI; meansSD; n = 7; asterisks indicate statistical
difference from control, p<0.05, Dunnetts) and (b) hematocrit (meansSD; n =3-5; no statistical
differences, p>0.05, Dunnetts) of tilapia juveniles (males grey, females white) fed diets with
different levels of cottonseed oilcake replacement of fishmeal.

Fig. 2. Liver histology of juvenile tilapia fed

(a) the unsupplemented control, (b) a diet
with 45% fishmeal replaced by cottonseed
oilcake, and (c) the 45% replacement diet
enriched with 1350 mg iron per kg feed. After
eight weeks of feeding, hepatocytes were
unremarkable in control fish but had moderate
(m) and severe (s) fatty degeneration (HE
staining), and mild iron accumulation (blue
color; Perls staining), in fish fed supplemented diets.

Fig. 3. Intestines of juvenile tilapia fed (a) unsupplemented control and (b) iron-supplemented
diets. There was mild infiltration of neutrophilic granulocytes (nc) in the tela submucosa of
juveniles fed the iron-supplemented diet. le = lamina epithelialis, lp = lamina propria.

Meric et al.

Discussion
With regard to the political debate on sustainable aquaculture, the use of cottonseed
oilcake as an alternative protein source is an economically promising application and an
ecological challenge. Although previous studies in a variety of fishes have shown antinutritional effects associated with feeding cottonseed meals, results vary considerably
and the potential for iron detoxification is yet unclear. In this study, crude cottonseed
oilcake derived as a first and inexpensive by-product after oil extraction was used as an
alternative protein source. This product is characterized by a relatively moderate
gossypol concentration (0.08 mg/kg free gossypol, 0.47 mg/kg total gossypol), but is as
protein-rich as cottonseed meal.
Here, 0.08% free gossypol was comparable to concentrations reported regularly for
cottonseed meal (0.07-0.18%). In contrast, total gossypol was lower in the oilcake
(0.47%) than the usual range (1.15-1.49%) reported for meal (Waldroup, 2002). Thus,
bound gossypol is substantially lower in the oilcake. Bound gossypol is considered nontoxic to animals and toxicity is attributed exclusively to free gossypol. Still, it should be
considered that bound gossypol represents mostly covalent adducts of gossypol to
proteins, from which free gossypol can be (partially) liberated by acidification or microbial
activity in the gut.
The impact on growth parameters was low and significant only in fish fed the 30%
and 45% oilcake diets. At concentrations of 315 mg/kg free gossypol (30%) and 630
mg/kg (45%), growth inhibition was comparable to early findings in tilapia where
tolerance for high levels (0.18%) of free gossypol was reported with no loss in growth,
feed efficiency, or survival when purified gossypol acetate (0.1-0.2% diet) was used
(Robinson et al., 1984). However, in the same study, growth and feed efficiency was
reduced when cottonseed meal was used as a replacement, suggesting the existence of
antinutritional factors other than gossypol. This is further supported by the substantially
reduced growth performance observed when glandless free cottonseed was used. Feed
quality and composition may have contributed to this effect. The crude, unprocessed
(press) oilcake used here did not result in reduced growth in contrast to the deoiled or
even solvent extracted meals commonly used. Growth was low in all dietary groups
including the control, similar to findings of Mbahinzireki et al. (2001) who reported
weight gains of 0.54 g/d in the control, 0.59 g/d in the 25% cottonseed meal
replacement group, and 0.25 g/d in the 100% cottonseed meal replacement group.
Depending on the parameter evaluated, the toxic range for catfish is 300-1200 mg/kg
free gossypol (Yildirim et al., 2003). In rainbow trout, 300 mg/kg free gossypol resulted
in considerable growth depression (Rinchard et al., 2003a). Compared to our results,
growth inhibition in juvenile tilapia was more pronounced in most studies (Robinson et
al., 1984; El-Saidy and Gaber, 2004). It might be that adult tilapia are less sensitive to
gossypol or exhibit an increased detoxification mechanism. Other studies suggest that
tilapia might be less susceptible than other species.
Adverse effects on blood parameters have been explained by a gossypol-mediated
impact on intestinal iron absorption and reduced iron bioavailability which may lead to
reduced hemoglobin formation and anemia (Skutches et al., 1974; Yildirim et al., 2003).
Here, hematocrit values ranged 27-36% and were comparable to normal values reported
for tilapia (Rinchard et al., 2003a; Aly et al., 2008; Giron-Perez et al., 2008; Yue and
Zhou, 2008). Some authors reported reduced hematocrit with increasing replacement
(El-Saidy and Gaber, 2004; Yue and Zhou, 2008). Our data do not provide support of
significant differences between treatments or an impact on hematopoiesis.
Iron supplementation has been suggested to compensate for gossypol toxicity by
formation of an iron-gossypol complex in the intestinal tract, preventing gossypol
absorption (Wenegaertner, 1981; Yildirim et al., 2003). This beneficial effect of iron
supplementation has been reported in a number of studies. In the iron-supplemented
30% oilcake diet, growth parameters were indeed slightly increased compared to the
unsupplemented 30% group. Still, at 45% replacement, no effect of iron was observed.
The histological findings indicate potential accumulation of iron-gossypol complexes in
the liver which may lead to chronic intoxication. The HSI was lowest in the iron-

Cottonseed oilcake replaces fishmeal in juvenile tilapia diets

supplemented 45% cottonseed group, indicating adverse effects on the liver, but further
research is needed. Liver abnormalities, including liver necrosis, have been reported in
rainbow trout fed a diet containing 0.0531% free gossypol (Herman, 1970). The
symptoms observed here might precede such toxic effects. Mild to severe fat
degeneration of the liver was observed with increasing oilcake inclusion. No other
histopathological changes were observed in gills, heart, or gonads.
Gossypol can have a severe impact on reproductive parameters. Cottonseed meal
significantly reduced GSI of female fish associated with depressed growth and a
consequent delay in gonad maturation while males were not affected (Dabrowski et al.,
2000; El-Saidy and Gaber, 2004). Estrogenic compounds, e.g., quercetin and other
isoflavones, have been reported in cottonseed (Tacon, 1997), and these might
compensate for some of the effects of gossypol. Histopathology of gonad tissue and GSI
revealed no differences between groups or abnormalities (e.g., ovotestis).
In conclusion, moderate replacement of up to 45% cottonseed oilcake resulted in
slightly reduced growth while 15-30% of the fishmeal can be replaced by cottonseed
oilcake without impact. Above 30% cottonseed oilcake, the impact on growth must be
considered, although no evidence of adverse chronic effects was detected in the blood
parameters or histopathology of the major organs. Taking into account the variability of
results reported when cottonseed meal was used as a replacement, one may speculate
that the quality of cottonseed as a protein source depends on the use of pesticides,
although this issue has not yet been considered.
Acknowledgements
This study was supported by the Leibniz-Institute for Freshwater Ecology and Inland
Fisheries (Berlin, Germany). The authors thank Altn Yem Co. (Izmir, Turkey) for
providing the cottonseed oilcake, Monika Heller, Saman Rahat El-Tor, and Peter Lschau
for technical assistance in the laboratory as well as during sampling.
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