Aquaculture Nutrition doi: 10.1111/j.1365-2095.2010.00801.x

2011 17; 353–360

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School of Basic Medicine and Biological Science, Soochow University, Suzhou, China; 2 School of Life Science, East China Normal University, Shanghai, China; 3 Aquaculture Protein Centre, CoE, Norwegian School of Veterinary Science, Oslo, Norway

A 6-month trial was conducted to evaluate the effects of dietary cottonseed meal (CSM) and free gossypol (FG) on allogynogenetic silver crucian carp, Carassius auratus gibelio$ · Cyprinus carpio# with 4 replicates of each treatment. Isonitrogenous and isocaloric diets were formulated with the 0 g kg)1 (control), 200 g kg)1, 400 g kg)1, and 560 g kg)1 CSM. Diets with FG were made by supplementing batches of control diet with 214 mg kg)1, 428 mg kg)1, and 642 mg kg)1. Weight gain, specific growth rate, and protein efficiency ratio increased significantly up to an inclusion level of CSM of 400 g kg)1 in the diet, with a significant decrease in food conversion ratio. Further increase in CSM to 560 g kg)1 did not cause further changes in fish performance. Free gossypol did not affect fish performance significantly at any inclusion level. Neither CSM nor FG caused significant effects in any of the other evaluated parameters such as whole body composition, haemoglobin concentration, activities of serum lysozyme, superoxide dismutase, alanine aminotransferase and aspartate aminotransferase, and histology of hepatic tissues and midgut. Our results suggested that crucian carp can tolerate at least 642 mg kg)1 FG and that it is safe to including 400 g kg)1 CSM in crucian carp feed. KEY WORDS:

allogynogenetic silver crucian carp (Carassius auratus gibelio$ · Cyprinus carpio#), blood parameters, cottonseed meal, gossypol, growth, histology Received 19 February 2010, accepted 26 April 2010 Correspondence: Yuantu Ye, School of Basic Medicine and Biological Science, Soochow University, Suzhou 215123, China. E-mail: caicf@ suda.edu.cn

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 2010 Blackwell Publishing Ltd

Cottonseed meal (CSM), a high protein by-product of the cottonseed processing industry, has been tested in numerous fish species, including Sarotherodon mossambicus (Jackson et al. 1982) and Oreochromis niloticus (Ofojekwu & Ejike 1984; El-Sayed 1990; Rinchard et al. 2000; Mbahinzireki et al. 2001; Yue & Zhou 2008), Ictalurus punctatus (Dorsa et al. 1982; Robinson et al. 1984; Robinson & Brent 1989; Robinson & Li 1994; Robinson & Tiersch 1995), Oncorhynchus tshawytscha and Oncorhynchus kisutch (Fowler 1980), Micropterus salmoides (Kurten et al. 1999), M. chrysops · M. saxatilis (Rawles & Gatlin 2000), and Oncorhynchus mykiss (Dabrowski et al. 2000, Dabrowski et al. 2001; Blom et al. 2001; Cheng & Hardy 2002; Rinchard et al. 2003; Lee et al. 2006). Although the presence of gossypol and the low lysine content in CSM limit its use in diets formulated for monogastric animals, including fish (Herman 1970; Robinson & Li 1995; Li & Robinson 2006), most of the above-mentioned studies showed that CSM can be a palatable and nutritionally acceptable substitute for fish meal in diets for many fish species, more economical than soybean meal per unit protein fish (Robinson & Li 1995). Moreover, recent research suggests that high dietary CSM supplementation up to 588 g kg)1 or complete substitution of FM protein does not impair growth in rainbow trout (Lee et al. 2006). Evaluation of effects on reproductive performance of the males showed no effects (Lee et al. 2006) too. Although several fish species have been found to accept and to utilize CSM efficiently as a nutrient source, it is necessary to evaluate other species regarding their tolerance before including CSM in the diet. In particular, should effects of the CSM antinutrient gossypol be evaluated as it is well known as an

antinutrient for several terrestrial monogastric species (reviewed by Zhang et al. 2007). Allogynogenetic silver crucian carp (Carassius auratus gibelio$ · Cyprinus carpio#) is an important fresh water species in China (Chen & Zhu 2008) and has been the second largest aquaculture species in Jiangsu, China (Lu et al. 2009); the widespread culturing of this species can be attributed to certain characteristics, such as high meat yield, high nutritive value, good tolerance to stress, and good economic benefits, which make it advantageous for commercial use. Previous studies have reported that several kinds and large quantities of plant feed material are used in crucian carp feed in China partially because protein-rich agricultural sources of protein are economical (Zhou et al. 2004). In this study, we evaluated the effect of dietary cottonseed meal and gossypol on the

growth performance, whole body composition and health aspects of crucian carp.

Two consecutive feeding trials were conducted with a total of seven isonitrogenous (CP, 350 g kg)1) and isocaloric (gross energy, 18.5 KJ g)1) diets formulated to fill the nutritional requirements of the crucian carp. The receipt of the diets is given in Table 1. In experiment I, a diet containing 0 g kg)1 cottonseed meal (CM-0) was regarded as the control. The three other diets contained 200 g kg)1, 400 g kg)1, 560 g kg)1 cottonseed meal (abbreviated CM-200, CM-400,

CSM-0 CSM-200 CSM-400 CSM-560 FG-214 FG-428 FG-642 Ingredients (g kg)1) Cottonseed meal Rapeseed meal Soybean meal Peanut cake Fish meal Blood meal Wheat bran Rice bran Corn Feed yeast Wheat glutin Flour Salt (NaCl) Ca(H2PO4)2ÆH2O Soybean oil Zeolite powder Vitamin premix1 Mineral premix2 Gossypol acetic acid (mg kg)1) Chemical composition (g kg)1 as Moisture Crude protein Fat Ash Gross energy (kJ g-1) Free gossypol (mg kg)1)

200 200 150 100 20 40 53 20 30

200 170 120 50 100 30 40 53 40 30

100 100 3 3 20 20 14 14 30 10 10 10 10 10 – – fed state) 102 104 351 357 37 36 137 124 18.4 18.5 Nd 168

400 70 50 30 100 30 34 23 70 30 10 100 3 20 10

560 00

10 10 –

10 10 –

106 363 33 114 18.3 341

104 365 34 106 18.6 473

104 30 50 11 70 20 100 3 20 12

200 200 150 100 20 40 53 20 30

200 200 150 100 20 40 53 20 30

200 200 150 100 20 40 53 20 30

100 3 20 14 30 10 10 240

100 3 20 14 30 10 10 480

100 3 20 14 30 10 10 720

Table 1 Ingredient composition and estimated nutrient content of experimental diets containing different levels of cottonseed meal and free-gossypol from gossypol acetic acid

104 106 10 4 353 352 355 38 36 37 131 134 132 18.5 18.3 18.4 2143 4283 6423

Not detected (Nd). 1 Mineral premix (mg or g kg)1 diet): CaCO3, 4.53 g; Ca(H2PO4)2ÆH2O, 11.15 g; MgSO4.7H2O, 1.3 g; KCl, 4.14 g; NaH2PO4.2H2O, 3.4 g; KAl(SO4)2, 40 mg; CoCl2.6H2O, 18 mg; MnSO4.H2O, 18 mg; KI, 3.5 mg; ZnSO4.H2O, 48 mg; Na2Se2O3, 1.5 mg; CuSO4.5H2O, 18.8 mg; FeC6H5O7.H2O, 335 mg [supplied by Beijing Sunpu Biochemical and Technology Co., Ltd. (in China)]. 2 Vitamin premix (mg kg)1 diet): vitamin A acetate (300 000 IU/g), 10 mg; vitamin D3 (400 000 IU/ g), 3.75 mg; DL-alpha-tocopheryl acetate (250 IU/g), 200 mg; menadione, 10 mg; thiamin HCl, 10 mg; riboflavin, 20 mg; nicotinicacid, 50 mg; DL Ca-pantothenate, 40 mg; pyridoxine HCl, 10 mg; vitamin B12, 0.02 mg; folic acid, 5 mg; biotin,1 mg; vitamin C phosphate ester (35%):1200 mg; inositol, 400 mg; choline chloride, 200 mg (supplied by Beijing Sunpu Biochemical and Technology Co., Ltd. (in China). 3 Free gossypol was calculated as followed: level of gossypol acetic acid (GAA) · 89.62% (freegossypol in GAA) · 99.5% (purity).

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and CM-560, respectively) replacing mainly rapeseed meal, soybean meal and peanut meal. In experiment II, three experimental diets were formulated to include 240 mg kg)1, 480 mg kg)1, 720 mg kg)1 gossypol acetic acid (GAA) (abbreviated FG-214, FG-428, and FG-642, respectively) by supplementing batches of the control CM-0 diet. Diet CM-0 was used as control diet also in Experiment II. The GAA was supplied by Beijing China Cotton-Unis Bioscience Co., Ltd (FG: 89.17%), vitamin and mineral premix by Beijing Sunpu Biochemical and Technology Co., Ltd. (in China). Other feed ingredients were supplied by Guangdong HengXing Group Co., Ltd. (in China). All feed ingredients were ground to pass through a 60-mesh sieve before use. Micronutrients (vitamins and minerals) were prediluted with ground wheat as a base before being added to the main ingredient mixture, and all the ingredients of each diet were homogenized and further blended with the addition of water. This mixture was forced through a meat grinder (TJ12-H, Henglian Food Processing Machinery Co., Ltd. China) with a hole of diameter 1.5 mm and dried under an electric fan at room temperature for 12 h. Then, the noodle-like strand was broken into small parts such that they could be fed to fingerlings and were then stored in a freezer until use.

Crucian carps (Carassius auratus gibelio$ · Cyprinus carpio#) (initial mean weight, 19.5 ± 0.97 g) were obtained from a commercial producer (Pingwang aquaculture farm, Jiangsu, China) and reared in 250-L aquaria in a closed recirculation system with mechanical filtration. Water source was aerated tap water, and water flow was maintained at approximately 2.5 L min)1. Air blowers provided aeration via air stones to maintain dissolved oxygen >6.0 mg L)1. Water temperature was maintained at 25.0–27.5 C by a heater. Water quality parameters (total ammonia < 0.1 mg L)1, nitrite < 0.1 mg L)1, and pH 7.4–7.7) were measured weekly to ensure the safe levels were maintained. The photoperiod was set to match natural conditions. After 2 weeks of acclimatization with a commercial diet, crucian carps were randomly distributed into 28 fibreglass tanks, with 20 fish per tank. Each diet was fed for 6 months, three times daily, at 0830, 1230, and 1600 h, to four groups of fish. The diets were fed at a ration rate of 30 g kg)1 body weight the first week. Thereafter, the amount of feed consumed by the fish in each tank was recorded daily, and rations were adjusted according to feed consumed the previous day to avoid overfeeding. Excess feeding was verified by uneaten feed trapped by strainers in water outlet from the tank.

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Tanks were cleaned weekly. Fish were weighed individually at the start and end of the experiment and bulk-weighed every 2nd month.

Before start of the experiment, random samples of 20 fish were anaesthetized (3-aminobenzoic acid ethyl ester, MS 222; 100 mg L)1, supplied by Xintun Aquatucal Technology. Co., Ltd. China) and stored at )18C prior to chemical analysis. At the end of the trials, all fish were starved for 24 h before sampling. Three fish from each tank were sampled for body composition analysis, eight fish for sampling blood, and two for intestinal tracts and hepatic tissues. Sampling was conducted after fish were anaesthetized with MS-222. Whole bloods from two fish were sampled from each tank by heparinized syringes to measure the haemoglobin (Hb) concentration within 10 min. Whole bloods of 6 fish from each tank were collected using non-heparinized syringes and transferred to eppendorf tubes. The blood was allowed to clot for 1 h at room temperature and for 5 h in the cold (4 C) before centrifugation at 1500 g for 5 min at 4 C. Serum (supernatant) from each fish was sampled in equal amounts and mixed. Mixed serum were separated in 8 aliquots and stored at )80 C prior to analysis. One serum sample from each tank was used for each enzyme activity determination, i.e. lysozyme (LSZ), superoxide dismutase (SOD), alanine aminotransferase (ALT), and aspartate aminotransferase (AST).

All diets and individuals of each treatment were analysed using the methods established by the AOAC (1995). Moisture content was determined by oven-drying at 105 C to constant weight. Crude protein was estimated by the Kjeldahl method after acid digestion (LNK-872, Yixing Science and instrument research institute; KDN-04III, Shanghai XianJian instruments Co., Ltd, China). Crude lipid content was determined by the ether extraction method with a Soxtec system (Yixing Science and instrument research institute, China), and ash content was determined by combustion (LNK-872, Yixing Science and instrument research institute, China) at 550 C to constant weight. Gross energy of feed was analysed by Oxygen Bomb Calorimeter (XRY-1C, Shanghai Changji Geological Instrument Co., Ltd, China). Haemoglobin concentration was measured by kits made by Shanghai Rongsheng Biotech Co., Ltd (in China). LSZ, SOD, ALT, and AST activity were determined by kits made by Nanjing Jiancheng Bioengineering Institute (in China).

Feed conversion ratio (FCR) = dry feed weight/wet weight gain. Protein efficiency ratio (PER) = weight gain (g)/protein intake (g).

The occurrence of possible histopathological damages was studied in hepatic tissues, foregut, and midgut. The gastrointestinal tract was dissected from fish, and surface fat and connective tissue were carefully removed. Tissue samples (gut: 1 cm, hepatic tissue: 0.2 g) were fixed in phosphatebuffered formalin (4%; pH 7.4) for 24 h, and then transferred to 70% ethanol for storage until processing. All formalinfixed tissues were routinely dehydrated in ethanol, equilibrated in xylene, and embedded in paraffin according to standard histological techniques. Transverse sections (approximately 5 lm thick) of the longitudinal sections of the intestines were cut and stained with haematoxylin and eosin (HE). Blinded histological examination was performed by light microscopy. Intestinal morphology was evaluated according to Baeverfjord & Krogdahl (1996).

All the data are presented as means ± SEM. Data were subjected to one-way analysis of variance (ANOVA, SPSS for Windows, version 14.0) to determine significant differences among treatment groups. Differences between treatment means were compared by the Tukeys multiple range tests. Differences were regarded as significant when P < 0.05.

The growth performance, body composition, Hb concentration, and serum enzyme activities of crucian carps that were fed diets containing different proportions of CSM and FG were presented in Table 2 and 3, respectively. All fish survived in all treatments. Weight gain, SGR, and PER increased and peaked when dietary CSM was increased from 0 to 400 g kg)1, and FCR decreased to the lowest value (1.44). When dietary CSM was increased further to 560 g kg)1, no further significant changes were observed. The addition of FG from a concentration of 0–642 mg kg)1 diet had no significant effects on the growth performance. For the parameters whole body composition, Hb concentration, activities of LSZ, SOD, ALT, and AST, no significant effect of either CSM or FG were observed.

Mortality, fish weight gain, feed conversion ratio, and protein efficiency ratio were calculated as follows: Survival rate (SR, %) = 100 · final fish number/initial fish number. Weight gain (WG, %) = 100 · (final weight ) initial weight)/initial weight. Specific growth rate (SGR, % d)1) = 100 · [ln (final weight) ) ln (initial weight)] · days))1.

CSM-0 Survival rate (%) WG (%) SGR (% d)1) PER FCR Whole body composition Protein (mg kg)1) Lipid (mg kg)1) Ash (mg kg)1) Moisture (mg kg)1) Biochemical parameters Hb (g L)1) LSZ (U 100 mL)1) SOD (U 100 mL)1) ALT (U 100 mL)1) AST (U 100 mL)1)

CSM-200

CSM-400

CSM-560

100 296 0.74 1.64 1.73

± ± ± ±

23a 0.03a 0.05a 0.12b

100 413 0.88 1.89 1.50

± ± ± ±

50b 0.05b 0.08b 0.14a,b

100 525 0.99 1.97 1.44

± ± ± ±

33c 0.03c 0.10b 0.08a

100 495 ± 0.96 ± 1.91 ± 1.47 ±

47b,c 0.04b,c 0.06b 0.09a

166 99 35 687

± ± ± ±

8 7 3 13

159 88 34 700

± ± ± ±

10 10 3 9

159 89 36 700

± ± ± ±

8 7 4 8

157 93 33 707

± ± ± ±

7 8 3 11

95 31 182 29 95

± ± ± ± ±

11 1 26 3 5

98 31 208 30 95

± ± ± ± ±

9 3 19 2 2

90 32 190 27 97

± ± ± ± ±

6 4 22 3 4

93 30 211 32 100

± ± ± ± ±

5 2 25 3 4

Table 2 Growth performance, body composition, haemoglobin, serum enzyme activities of crucian carp fed diets containing different levels of cottonseed meal

Data were subjected to one-way ANOVA to determine significant differences among treatment groups. Differences between treatment means were compared by the Tukeys multiple range tests. Values in the same row with different superscripts are significantly different (P < 0.05). n = 4 (n = 8 in haemoglobin). ALT, Alanine aminotransferase; LSZ, lysozyme; PER, Protein efficiency ratio; FCR, Feed conversion ratio; WG, Weight gain; SGR, Specific growth rate; SOD, superoxide dismutase; ALT, alanine aminotransferase; AST, aspartate aminotransferase.

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Table 3 Growth performance, body composition, haemoglobin, serum enzyme activities of crucian carp fed diets containing different levels of free-gossypol for 6 months CSM-0 Survival rate (%) 100 WG (%) 296 ± SGR (% d)1) 0.74 ± PER 1.64 ± FCR 1.73 ± Whole body composition Protein (g kg)1) 166 ± Lipid (g kg)1) 99 ± Ash (g kg)1) 35 ± Moisture (g kg)1) 687 ± Biochemical parameters Hb (g L)1) 95 ± LSZ (U 100 mL)1) 31 ± SOD (U 100 mL)1) 182 ± ALT (U 100 mL)1) 29 ± AST (U 100 mL)1) 95 ±

FG-214

FG-428

FG-642

23 0.03 0.05 0.12

100 311 0.76 1.66 1.69

± ± ± ±

54 0.07 0.11 0.11

100 278 0.72 1.68 1.64

± ± ± ±

20 0.03 0.07 0.08

100 284 0.72 1.59 1.72

± ± ± ±

29 0.04 0.07 0.13

8 5 3 13

170 89 36 692

± ± ± ±

9 10 3 12

168 91 32 688

± ± ± ±

7 6 4 13

162 92 33 693

± ± ± ±

9 8 3 12

11 1 26 3 5

97 27 178 26 93

± ± ± ± ±

4 4 11 3 3

95 29 175 29 96

± ± ± ± ±

5 5 20 3 2

95 28 184 27 94

± ± ± ± ±

5 4 20 2 2

Data were subjected to one-way ANOVA to determine significant differences among treatment groups. Differences between treatment means were compared by the Tukeys multiple range tests. Values in the same row with different superscripts are significantly different (P < 0.05). n = 4 (n = 8 in haemoglobin). ALT, Alanine aminotransferase; LSZ, lysozyme; PER, Protein efficiency ratio; FCR, Feed conversion ratio; WG, Weight gain; SGR, Specific growth rate; SOD, superoxide dismutase; ALT, alanine aminotransferase; AST, aspartate aminotransferase.

The histological examinations of liver tissues did not show any significant effects of dietary inclusion of CSM and FG for size of hepatic cells and their nuclei, neither in lipidic droplets. Regarding intestinal tissue, all fish showed normal villous and brush borders of foregut and midgut. No changes in the width of lamina propria or submucosa were noticed at any level of CSM or FG. Intraepithelial lymphocytes were mostly observed near the basal lamina and nuclei located base of enterocytes (Fig. 1, a: hepatic tissue, CM-0; b: hepatic tissue, FG-642; c: hepatic tissue, CM-560; d: Midgut, CM-0; e: Midgut, FG-642; f: Midgut, CM-560).

Rather than reducing performance, as observed in some fish species, dietary inclusion of CSM up to a concentration of 400 g kg)1 improved growth performance and feed utilization of crucian carp. The CSM showed better nutritional quality than the mixture of rape seed, soybean, and pea nut cake meal it replaced. The improving effect on fish performance seemed to level out above 400 g kg)1. Our results indicate that the crucian carp can tolerate a wide range of CSM in their diet, in agreement with results obtained with some other fish species as pointed out in the introduction

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chapter. For example in the study of Yue & Zhou (2008) and of Lee et al. (2006), no significant effects of CSM were observed in juvenile hybrid tilapia and rainbow trout with dietary levels of 337.6 and 588 g kg)1, respectively. However, CSM have shown negative effects in rainbow trout and other species in other experiments. Inclusion level up to 200 g kg)1 was observed to impair weight gain and feed conversion ratio in rainbow trout (Cheng & Hardy 2002). Inferior negative effects of CSM on performance have also been observed in tilapia fed diets with 240 and 470 g kg)1 cottonseed product (Robinson et al. 1984). Our results, showing no negative effects dietary FG supplementation up to a concentration of 642 mg kg)1, are in agreement with studies of effects of gossypol on growth of rainbow trout (Dabrowski et al. 2000; Blom et al. 2001), channel catfish (Robinson 1991) as well as tilapia (Jackson et al. 1982; Robinson et al. 1984). They are, however, in contrast to observations made with juvenile channel catfish which showed suppressed growth when the fish were fed diets added FG at a level of 300 mg kg)1. At levels of 600 mg kg)1 and higher also feed intake and protein efficiency ratio were reduced (Yildirim et al. 2003). In this study, neither CSM nor FG affected the body composition or any of the observed health-related variables. In channel catfish, however, CSM (Barros et al. 2002) and gossypol (Yildirim et al. 2003) have been observed to affect whole body proximate composition, with poor condition of fish fed diets with high levels of gossypol. Moreover, Herman (1970), Dabrowski et al. (2000) and Blom et al. (2001) working with rainbow trout, Mbahinzireki et al. (2001) working with tilapia and Yildirim et al. (2003) reported reduced haematocrit and haemoglobin values as indicators of gossypol toxicity. The anaemia developing in some fish fed diets with gossypol containing ingredients may be because of the well-established adverse effect of gossypol on intestinal iron absorption in terrestrial monogastrics (Braham & Bressani 1975). In this study, however, no significant difference in haemoglobin levels was observed in crucian carp, neither in the study of effects of CSM nor in fish fed diets containing different levels of FG. The absence of negative effects on haematological parameters in this study may reflect the possible situation that crucian carp is resistant to negative effects of gossypol. Another possible explanation is that our diet may contain high levels of certain nutrients that may interact with free gossypol to reduce its toxicity (Jones 1987). Further research is needed to reach a conclusion whether the crucian carp is less sensitive to gossypol than other species. No significant differences in non-specific immunity parameters of LSZ and SOD were observed in this study.

(a)

(b)

(c)

(d)

(e)

(f)

This is in contrast to results obtained with channel catfish (Yildirim et al. 2003) which showed increased serum lysozyme activity at a dietary level of 900 mg kg)1 gossypol or higher. It was speculated that gossypol or other compounds present in CSM may have a beneficial effect by improving the immune response (Barros et al. 2002). On the other hand, reduced SOD activity has been observed in crucian carp fed CSM hydrolysate at 50 g kg)1 (Gui et al. 2010). The effects of CSM and FG on immunity responses were not explained. Herman (1970) observed necrotic changes in the liver cells, thickening of the glomerular basement membrane, and accumulation of ceroid pigment granules in rainbow trout liver, and a dietary level of 1 g kg)1 gossypol resulted in quick development of severe focal fatty degeneration in the liver and extensive kidney damage in the same study. In this study, however, histological examination showed no signifi-

Figure 1 Histological sections of the heptic tissue and midgut for crucian carps that were fed the control diet (a and d); diet containing 642 mg kg)1 free-gossypol (b and e) and diet containing 560 mg kg)1 cottonseed meal (c and f). No significant difference was observed in the size of hepatic cells and their nuclei and lipid droplets (a, b and c). The villous tips and the brush border of midgut appear normal in the midgut of all individuals. Intraepithelial lymphocytes were usually located near the basal lamina, and increased numbers of lymphocytes were not observed in fish that were fed diets containing 642 mg kg)1 freegossypol (e) and diets with 560 mg kg)1 cottonseed meal (f). Bar = 20 lm.

cant change in hepatic cell structure in any of the treatments. Neither ALT nor AST activities, indicators of liver function, were significantly affected. Also, our histological evaluation of gut tissue showed normal characteristics whether the fish had been fed CSM or FG. All our results indicating health aspects were in agreement with the growth performance results, suggesting that crucian carp is not negatively affected neither by FG nor by other components of CSM. A possible cause of different responses between experiments even within fish species upon feeding with CSM may be related to breed and production season of the CSM. The amount of gossypol content in CSM is dependent on environmental factors (Cheery et al. 1978) and species of cotton (Boatner et al. 1949). As Cheng & Hardy (2002) have pointed out, e.g. apparent digestibility coefficients of most nutrients in CSM obtained from different locations can differ significantly between breeds and batches. Therefore, the origin and

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processing of different CSM sources might contribute to the above controversy between results. Besides this, FG tolerance seems species-dependent. Commercially available CSM in China has FG levels ranging from about 410 mg kg)1 to about 1450 mg kg)1 (Zhang 2005). It means even if a diet contains 400 g kg)1 CSM, the possible highest FG content was 580 mg kg)1 diet or so. According to the results in this experiment, it should not be necessary to pay attention to gossypol level in CSM when used in crucian carp feed. Conclusion: crucian carp tolerated free gossypol up to 642 mg kg)1. High dietary inclusion of CSM, up to 560 g kg)1, did not cause any detrimental effects on growth, body composition, or on the investigated health parameters of crucian carp. Cotton seed meal seemed to be a good protein source which can be used at levels up to 400 g kg)1 CSM in diets for crucian carp.

This work was supported by grants from the Social Development Fund of Jiangsu Province (No. BS2006021; No.BS2007140) and the Agricultural Basic Research Fund of Suzhou City (No. YJG0912). We are grateful to the staff (Analytical and Testing Center of Suzhou University) for their assistance in this study.

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Aquaculture Nutrition 17; 353–360  2010 Blackwell Publishing Ltd