Aquaculture nutrition, tập 17, số 4, 2011

Aquaculture Nutrition doi: 10.1111/j.1365-2095.2010.00801.x 2011 17; 353360 1 1 School of Basic Medicine and Biological Science, Soochow University, Suzhou, China; School of Life Science, East China Normal University, Shanghai, China; Aquaculture Protein Centre, CoE, Norwegian School of Veterinary Science, Oslo, Norway A 6-month trial was conducted to evaluate the eects of dietary cottonseed meal (CSM) and free gossypol (FG) on allogynogenetic silver crucian carp, Carassius auratus gibelio$ ã Cyprinus carpio# with replicates of each treatment Isonitrogenous and isocaloric diets were formulated with the 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, specic growth rate, and protein eciency ratio increased signicantly up to an inclusion level of CSM of 400 g kg)1 in the diet, with a signicant decrease in food conversion ratio Further increase in CSM to 560 g kg)1 did not cause further changes in sh performance Free gossypol did not aect sh performance signicantly at any inclusion level Neither CSM nor FG caused signicant eects 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$ ã 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 ể 2010 Blackwell Publishing Ltd Cottonseed meal (CSM), a high protein by-product of the cottonseed processing industry, has been tested in numerous sh 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 ã 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 sh (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 sh meal in diets for many sh species, more economical than soybean meal per unit protein sh (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 eects on reproductive performance of the males showed no eects (Lee et al 2006) too Although several sh species have been found to accept and to utilize CSM eciently as a nutrient source, it is necessary to evaluate other species regarding their tolerance before including CSM in the diet In particular, should eects 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$ ã 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 benets, 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 eect 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 ll the nutritional requirements of the crucian carp The receipt of the diets is given in Table In experiment I, a diet containing 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)2ặH2O 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 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 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 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 20 14 30 10 10 240 100 20 14 30 10 10 480 100 20 14 30 10 10 720 Table Ingredient composition and estimated nutrient content of experimental diets containing different levels of cottonseed meal and free-gossypol from gossypol acetic acid 104 106 10 353 352 355 38 36 37 131 134 132 18.5 18.3 18.4 2143 4283 6423 Not detected (Nd) Mineral premix (mg or g kg)1 diet): CaCO3, 4.53 g; Ca(H2PO4)2ặH2O, 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)] 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, 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) Free gossypol was calculated as followed: level of gossypol acetic acid (GAA) ã 89.62% (freegossypol in GAA) ã 99.5% (purity) Aquaculture Nutrition 17; 353360 ể 2010 Blackwell Publishing Ltd 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 ngerlings and were then stored in a freezer until use Crucian carps (Carassius auratus gibelio$ ã 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 ltration Water source was aerated tap water, and water ow 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 25.027.5 C by a heater Water quality parameters (total ammonia < 0.1 mg L)1, nitrite < 0.1 mg L)1, and pH 7.47.7) were measured weekly to ensure the safe levels were maintained The photoperiod was set to match natural conditions After weeks of acclimatization with a commercial diet, crucian carps were randomly distributed into 28 breglass tanks, with 20 sh per tank Each diet was fed for months, three times daily, at 0830, 1230, and 1600 h, to four groups of sh The diets were fed at a ration rate of 30 g kg)1 body weight the rst week Thereafter, the amount of feed consumed by the sh in each tank was recorded daily, and rations were adjusted according to feed consumed the previous day to avoid overfeeding Excess feeding was veried by uneaten feed trapped by strainers in water outlet from the tank Aquaculture Nutrition 17; 353360 ể 2010 Blackwell Publishing Ltd 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 sh 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 sh were starved for 24 h before sampling Three sh from each tank were sampled for body composition analysis, eight sh for sampling blood, and two for intestinal tracts and hepatic tissues Sampling was conducted after sh were anaesthetized with MS-222 Whole bloods from two sh were sampled from each tank by heparinized syringes to measure the haemoglobin (Hb) concentration within 10 Whole bloods of sh from each tank were collected using non-heparinized syringes and transferred to eppendorf tubes The blood was allowed to clot for h at room temperature and for h in the cold (4 C) before centrifugation at 1500 g for at C Serum (supernatant) from each sh was sampled in equal amounts and mixed Mixed serum were separated in 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 eciency 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 sh, and surface fat and connective tissue were carefully removed Tissue samples (gut: cm, hepatic tissue: 0.2 g) were xed in phosphatebuered formalin (4%; pH 7.4) for 24 h, and then transferred to 70% ethanol for storage until processing All formalinxed tissues were routinely dehydrated in ethanol, equilibrated in xylene, and embedded in paran according to standard histological techniques Transverse sections (approximately 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 signicant dierences among treatment groups Dierences between treatment means were compared by the Tukeyếs multiple range tests Dierences were regarded as signicant when P < 0.05 The growth performance, body composition, Hb concentration, and serum enzyme activities of crucian carps that were fed diets containing dierent proportions of CSM and FG were presented in Table and 3, respectively All sh survived in all treatments Weight gain, SGR, and PER increased and peaked when dietary CSM was increased from 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 signicant changes were observed The addition of FG from a concentration of 0642 mg kg)1 diet had no signicant eects on the growth performance For the parameters whole body composition, Hb concentration, activities of LSZ, SOD, ALT, and AST, no signicant eect of either CSM or FG were observed Mortality, sh weight gain, feed conversion ratio, and protein eciency ratio were calculated as follows: Survival rate (SR, %) = 100 ã nal sh number/initial sh number Weight gain (WG, %) = 100 ã (nal weight ) initial weight)/initial weight Specic growth rate (SGR, % d)1) = 100 ã [ln (nal weight) ) ln (initial weight)] ã 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 13 159 88 34 700 10 10 159 89 36 700 8 157 93 33 707 11 95 31 182 29 95 11 26 98 31 208 30 95 19 2 90 32 190 27 97 22 93 30 211 32 100 25 Table 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 Tukeyếs multiple range tests Values in the same row with different superscripts are significantly different (P < 0.05) n = (n = 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 Aquaculture Nutrition 17; 353360 ể 2010 Blackwell Publishing Ltd Table Growth performance, body composition, haemoglobin, serum enzyme activities of crucian carp fed diets containing different levels of free-gossypol for 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 13 170 89 36 692 10 12 168 91 32 688 13 162 92 33 693 12 11 26 97 27 178 26 93 4 11 3 95 29 175 29 96 5 20 95 28 184 27 94 20 2 Data were subjected to one-way ANOVA to determine significant differences among treatment groups Differences between treatment means were compared by the Tukeyếs multiple range tests Values in the same row with different superscripts are significantly different (P < 0.05) n = (n = 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 signicant eects of dietary inclusion of CSM and FG for size of hepatic cells and their nuclei, neither in lipidic droplets Regarding intestinal tissue, all sh 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 sh 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 eect on sh 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 sh species as pointed out in the introduction Aquaculture Nutrition 17; 353360 ể 2010 Blackwell Publishing Ltd chapter For example in the study of Yue & Zhou (2008) and of Lee et al (2006), no signicant eects 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 eects 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 eects 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 eects dietary FG supplementation up to a concentration of 642 mg kg)1, are in agreement with studies of eects of gossypol on growth of rainbow trout (Dabrowski et al 2000; Blom et al 2001), channel catsh (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 catsh which showed suppressed growth when the sh 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 eciency ratio were reduced (Yildirim et al 2003) In this study, neither CSM nor FG aected the body composition or any of the observed health-related variables In channel catsh, however, CSM (Barros et al 2002) and gossypol (Yildirim et al 2003) have been observed to aect whole body proximate composition, with poor condition of sh 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 sh fed diets with gossypol containing ingredients may be because of the well-established adverse eect of gossypol on intestinal iron absorption in terrestrial monogastrics (Braham & Bressani 1975) In this study, however, no signicant dierence in haemoglobin levels was observed in crucian carp, neither in the study of eects of CSM nor in sh fed diets containing dierent levels of FG The absence of negative eects on haematological parameters in this study may reect the possible situation that crucian carp is resistant to negative eects 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 signicant dierences in non-specic 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 catsh (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 benecial eect 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 eects 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 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 signi- Figure 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 signicant dierence 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 sh 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 signicantly aected Also, our histological evaluation of gut tissue showed normal characteristics whether the sh 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 aected neither by FG nor by other components of CSM A possible cause of dierent responses between experiments even within sh 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 coecients of most nutrients in CSM obtained from dierent locations can dier signicantly between breeds and batches Therefore, the origin and Aquaculture Nutrition 17; 353360 ể 2010 Blackwell Publishing Ltd processing of dierent 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 eects 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 sta (Analytical and Testing Center of Suzhou University) for their assistance in this study Association of Ocial Analytical Chemists (AOAC) (1995) Ocial Methods of Analysis of Ocial Analytical Chemists International, 16th edn Association of Ocial Analytical Chemists, Arlington VA Baeverfjord, G & Krogdahl, A (1996) Development and regression of soybean meal induced enteritis in Atlantic salmon, Salmo salar L., distal intestine: a comparison with the intestines of fasted sh J Fish Dis., 19, 375387 Barros, M.M., Lim, C & Klesius, P.H (2002) Eect of soybean meal replacement by cottonseed meal and iron supplementation on growth, immune response and resistance of channel catsh (Ictalurus puctatus) to Edwarsiella ictaluri challenge Aquaculture, 207, 263279 Blom, J.H., Lee, K.J., Rinchard, J., Dabrowski, K & Ottobre, J (2001) Reproductive eciency and maternal-ospring transfer of gossypol in rainbow trout (Oncorhynchus mykiss) fed diets containing cottonseed meal J Anim Sci., 79, 15331539 Boatner, C.H., Castillon, L.E & Hall, C.M (1949) Gossypol and gossypurpurm in cottonseed of dierent varieties of G barbadense and G hirsutum, and variation of the pigments during storage of the seed J Am Oil Chem Soc., 26, 1925 Braham, J.E & Bressani, R (1975) Eect of dierent levels of gossypol on transaminase activity, on nonessential to essential amino acid ratio, and on iron and nitrogen retention in rats J Nutr., 105, 348355 Aquaculture Nutrition 17; 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(Linn.) fed on cottonseed-based articial diets Aquaculture, 42, 2736 Rawles, S.D & Gatlin, D.M (2000) Nutrient digestibility of common feedstus in extruded diets for sunshine bass, Morone chrysops female ã M sasatilis male J.World Aquac Soc., 31, 570579 Rinchard, J., Mbahinzireki, G., Dabrowski, K., Lee, K.-J., GarciaAbiado, M.A.R & Ottobre, J (2000) Eects of partial or total substitution of sh protein with cottonseed meal in diets on reproductive parameters of tilapia Oreochromis sp Aquaculture America 2000, Feb 25, 2000 New Orleans, p 282 Rinchard, J., Lee, K.-J., Czesny, S., Ciereszko, A., Rinchard, J., Lee, K.-J., Czesny, S., Ciereszko, A & Dabrowski, K (2003) Eect of feeding cottonseed meal containing diets to broodstock rainbow trout and their impact on the growth of their progenies Aquaculture, 227, 7787 Robinson, E.H (1991) Improvement of cottonseed meal protein with supplemental lysine in feeds for channel catsh J Appl Aquacult., 1, 114 Robinson, E.H & Brent, J.R (1989) Use of cottonseed meal in channel catsh feeds J World Aquac Soc., 20, 250255 Robinson, E.H & Li, M.H (1994) Use of plant proteins in catsh feeds: replacement of soybean meal with cottonseed meal and replacement of sh meal with soybean meal and cottonseed meal J World Aquac Soc., 25, 271276 Robinson, E.H & Li, M.H (1995) Use of cottonseed meal in aquaculture feeds In: Nutrition and Utilization Technology in Aquaculture (Lim, C & Sessa, D J eds), pp 157165 AOCS Press, Champaign, IL Robinson, E.H & Tiersch, T.R (1995) Eects of long-term feeding of cottonseed meal on growth, testis development, and sperm motility of male channel catsh Ictalurus punctatus brood sh J World Aquac Soc., 26, 426431 Robinson, E.H., Rawles, S.D., Oldenburg, P.W & Stickney, R.R (1984) Eects of feeding glandless or glanded cottonseed products and gossypol to Tilapia aurea Aquaculture, 28, 145154 Yildirim, M., Lim, C., Wan, P & Klesius, P.H (2003) Growth performance and immune response of channel catsh (Ictalurus punctatus) fed diets containing graded levels of gossypol-acetic acid Aquaculture, 219, 751768 Yue, Y.R & Zhou, Q.C (2008) Eect of replacing soybean meal with cottonseed meal on growth, feed utilization, and hematological indexes for juvenile hybrid tilapia, Oreochromis niloticus ã O aureus Aquaculture, 284, 185189 Zhang, D.L (2005) Utilization of cottonseed meal in feed for domestic animal Beifang Muye, 25 (in Chinese) Zhang, W.J., Xu, Z.R., Pan, X.L & Yan, X.H (2007) Advances in gossypol toxicity and processing eects of whole cottonseed in dairy cows feeding Livest Sci., 111, 19 Zhou, Q.C., Tan, B.P., Mai, K.S & Liu, Y.J (2004) Apparent digestibility of selected feed ingredients for juvenile cobia Rachycentron canadum Aquaculture, 241, 441451 Aquaculture Nutrition 17; 353360 ể 2010 Blackwell Publishing Ltd Aquaculture Nutrition 2011 17; 361367 1 1 doi: 10.1111/j.1365-2095.2010.00805.x Nutrition Laboratory, Fishery College, Huazhong Agricultural University, Wuhan; Key Laboratory of Freshwater Ecology and Aquaculture, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Jingzhou, China Received December 2009, accepted 18 May 2010 An 8-week feeding experiment was conducted to determine the eect of dietary betaine levels on the growth performance and hepatic intermediary metabolism of genetically improved farmed tilapia (GIFT) strain of Nile tilapia Oreochromis niloticus (mean initial body weight: 78.3 1.3 g, means SD) Six practical diets were formulated with the incorporation of betaine at the levels of (control), 5, 10, 15, 20 and 25 g kg)1 Survival showed no signicant dierences among the treatments (P > 0.05) The highest and lowest weight gain (WG) and specic growth rate (SGR) were observed for sh fed the diets containing and g kg)1 (control) betaine, respectively Feed intake showed similar trend with WG and SGR In contrast, feed conversion ratio was the lowest when dietary betaine level was g kg)1 In general, dietary betaine supplementation showed no signicant eect on hepatic composition of tilapia Condition factor and viscerosomatic index tended to increase with increasing dietary betaine levels from to g kg)1 and then decline when dietary betaine levels further increased from to 25 g kg)1 In contrast, hepatosomatic index declined with increasing dietary betaine levels (P < 0.05) Dietary betaine levels signicantly inuenced several hepatic enzymatic activities, including succinate dehydrogenase, lactate dehydrogenase, malic dehydrogenase, lipoprotein lipase and hepatic lipase, suggesting that dietary betaine addition had signicant eects on nutrient metabolism in the liver Based on the second-order polynomial regression analysis of WG, 12.5 g kg)1 of dietary betaine level seemed optimal for genetically improved farmed tilapia strain of O niloticus KEY WORDS: betaine, Genetically Improved Farmed Tilapia strain of Oreochromis niloticus, growth performance, hepatic intermediary metabolism ể 2010 Blackwell Publishing Ltd Correspondence: Dr Zhi Luo, Nutrition Laboratory, Fishery College, Huazhong Agricultural University, Wuhan 430070, China E-mail: luozhi99@yahoo.com.cn; Dr Hua Wen, Key Laboratory of Freshwater Ecology and Aquaculture, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Jingzhou 434000, China E-mail: wenhua.hb@163.com Betaine is a naturally occurring tertiary amine (trimethylglycine) present in most organisms Its application in aquafeed has increased during the last decade, often used as a feeding stimulant for several species of sh (Mackie & Mitchell 1983; Takaoka et al 1995; Knights 1996; Papatryphon & Soares 2000; Xue & Cui 2001) to enhance the growth performance In addition, betaine can play a role in the osmoregulation (De Vooys & Geenevasen 2002) and act as a methyl donor, which in turn may be used for the synthesis of methionine, carnitine, phosphatidyl choline and creatine, and plays a key role in protein and energy metabolism (Sheard & Zeisel 1989) There are also indications that betaine may play a role in lipid metabolism, and it is regarded as a lipotrope which prevents the formation of fatty liver (Wu & Davis 2005) In general, fat accumulation in sh results from the balance between dietary absorbed fat, de novo synthesis of fatty acids (lipogenesis) and fat catabolism via b-oxidation (lipolysis) Thus, for similar amount of absorbed fat, lower body fat deposition may be attributed to increased fat catabolism or diminished endogenous fatty acid (FA) synthesis or to both processes In sh, the liver is attributed preferentially to a sensitive organ reecting dietary lipid change and plays a central role in lipid metabolism (Likimani & Wilson 1982; Kiessling & Kiessling 1993) However, despite the fact that the major biochemical and metabolic pathways have been identied in sh (Cowey & Walton 1989), eects of dietary betaine levels on hepatic intermediary metabolism are still poorly understood Tilapia are widely cultured in tropical and subtropical regions of the world and form an important human protein source The genetically improved farmed tilapia (GIFT) strain of Oreochromis niloticus was developed by the International Center for Living Aquatic Resources Management (ICLARM) from the selective breeding of stocks from Africa and Asia (Tendencia et al 2006) Despite the fact that the eect of dietary betaine addition on growth performance and body composition of tilapia has been studied in recent years (Yan & Qiu 1998; Genc et al 2006), most studies are conducted on the base population for this sh species, such as Oreochromis niloticus (Yan & Qiu 1998; ElHusseiny et al 2008) and O aureus (Genc et al 2006) and small sized of sh were used in their study FernandezFigares et al (2002) suggested that genotype of experimental animals inuenced the eectiveness of dietary betaine on feed intake, growth rate and carcass quality Accordingly, it still seemed valuable to investigate the eect of dietary betaine levels on other strains (such as GIFT) of the sh species, which was expected to obtain some dierent and still valuable ndings In addition, little information is available on the eects of dietary betaine levels on hepatic intermediary metabolism in tilapia Therefore, the main purpose of this study was to determine the eect of dietary betaine levels on growth performance and liver intermediary metabolism of GIFT strain of O niloticus through examining liver activities of succinate dehydrogenase (SDH, EC 1.3.99.1), lactate dehydrogenase (LDH, EC 1.1.1.27), malic dehydrogenase (MDH, EC 1.1.1.40), lipoprotein lipase (LPL, EC 3.1.1.34) and hepatic lipase (HL, EC 3.1.1.3) (as representative enzymes involved in glycolysis and lipid catabolism) To our knowledge, this is the rst communication to demonstrate, at the biochemical level, the eects of dietary betaine levels on the enzymatic activities of liver in sh, which provides information on diet utilization and can also be used as an indicator of the nutritional status for this sh Six practical diets were formulated to contain betaine (as the free base, 99% in purity, Sigma, Aldrich Corp., St Louis, MO, USA) at the graded levels of (control), 0.5, 1, 1.5, 2.0 and 2.5%, with corresponding decreases in the amount of cellulose The ingredients and proximate analysis of the experimental diets were shown in Table All six experi- Table Ingredients and proximate analysis of experimental diets Diets no Ingredients (g kg )1 diet) Soybean meal 12 12 12 12 12 12 Fish meal 16 16 16 16 16 16 Rapeseed meal 44 44 44 44 44 44 Fish oil 1.68 1.68 1.68 1.68 1.68 1.68 Soybean oil 1.6 1.6 1.6 1.6 1.6 1.6 Wheat starch 18 18 18 18 18 18 Vitamin premix 0.5 0.5 0.5 0.5 0.5 0.5 (choline free) Mineral premix 0.5 0.5 0.5 0.5 0.5 0.5 Betaine 0.5 1.5 2.5 Cellulose 3.72 3.22 2.72 2.22 1.72 1.22 Sodium alginate 2 2 2 Proximate analysis (g kg)1 on a DM basis) Moisture 57.7 67.4 63.2 64.2 63.4 70.7 Crude protein 365.6 367.1 371.2 371.4 372.9 375.3 Lipid 87.0 89.1 89.1 84.9 87.4 86.1 Ash 82.8 74.9 74.7 79.4 81.0 79.1 mental diets were produced according to the methods described in our recent study (Luo et al 2008) Briey, all dry ingredients were ground to pass 120- lm sieve, weighed and mixed to a unanimous homogeneity Then, betaine was dissolved in the preweighed distilled water and they were added to form a dough The diets were extruded at 130 C and at 30 atm using a laboratory model Muyang Extruder Machine (Jiangsu, China) for about 40 s to make the diets water stable and then ground through a 1.5-mm sieve The resulting strands were steamed for 10 and allowed to dry overnight at 80 C until the moisture was reduced to less than 10% They were then reduced to pellets of approximately mm length, packed in air-tight plastic bags and stored in a freezer at )20 C until used The experiment was conducted in an indoor static aquarium system of Panjin Guanghe Fisheries Co Ltd., Panjin, China Eight hundred GIFT strain of Nile tilapia O niloticus were obtained from a local farmer, transported to the laboratory and kept in 20 300-l circular breglass tanks for 2week acclimatization During the acclimatization period, the sh were fed with the control diet At the beginning of the experiment, 144 uniform-sized sh (mean initial weight: 78.3 1.3 g, means SD) were stocked in 18 breglass tanks, eight sh per tank Each diet was assigned to three tanks in a completely randomized design Freshwater in each tank was renewed 100% by the outlet at the bottom of the tank twice daily, before feeding They were provided with Aquaculture Nutrition 17; 361367 ể 2010 Blackwell Publishing Ltd Aquaculture Nutrition 17; e892e901 ể 2011 Blackwell Publishing Ltd No claim to original US government works 1.03 1.1 130.3 188.6 14.6 38.9 8.2 4.7 0.31 0.01 0.1 2.8a 6.9 2.3 2.5 0.3 0.1 0.11 1.10 1.2 132.4 194.1 13.2 35.7 8.5 4.3 0.67 0.18 0.2 4.2ab 7.1 0.8 1.1 0.3 0.4 0.26 1.07 1.2 137.7 208.8 10.9 39.9 8.7 5.6 0.84 0.07 0.2 0.7ab 12.4 5.3 1.2 0.4 0.2 0.62 1.15 1.2 139.5 205.9 12.3 39.6 8.8 5.1 0.80 40 0.01 0.1 5.1b 9.2 5.8 0.8 0.5 0.2 0.28 1.09 1.2 135.0 199.4 12.8 38.5 8.6 4.9 0.66 Avg 0.03* 0.07* 1.8* 3.9* 1.9* 0.7* 0.2 0.1* 0.17 57.5 160.0 1.6 100.3 150.0 76.7 30.2 9.1 93.9 0.28 8.5 4.0a 0.2 1.2a 3.8 8.8a 1.5 0.4 1.9 0.12 a 40.0 139.4 1.5 111.9 158.9 55.0 29.8 8.3 91.7 0.33 10 ab 10.8 6.0ab 0.2 0.3b 2.4 12.9ab 1.0 0.4 2.1 0.23 CN 20 CN 10 Dietary NaCl (g kg)1 diet) Dietary NaCl (g kg)1 diet) 25.0 131.2 1.8 114.6 159.5 47.4 30.6 8.9 80.5 0.46 20 2.9 19.1b 0.4 1.1b 2.1 10.4b 0.9 0.1 1.9 0.11 ab 12.5 133.8 2.0 116.7 160.2 41.0 29.5 8.8 84.2 0.51 40 9.5 18.8b 0.3 2.0b 1.4 10.7b 0.5 0.1 0.8 0.22 b 33.8 141.1 1.7 110.9 157.2 55.0 30.0 8.8 87.8 0.40 Avg 3.6 5.4* 0.1* 0.7* 1.2* 4.8* 0.4* 0.1 0.8* 0.10 1.70 1.9 113.8 169.5 16.0 35.5 9.0 6.6 0.48 0.04 0.1 2.1a 4.3 1.8 2.1 0.4 0.2 0.26 1.71 1.4 120.9 174.7 13.7 39.0 9.7 7.4 0.63 0.01 0.2 2.7b 3.5 1.2 1.3 0.3 0.1 0.18 1.63 1.6 120.8 177.0 13.2 39.0 9.5 7.4 0.55 0.02 0.5 1.5b 3.6 0.9 1.7 0.1 0.3 0.14 1.73 1.9 128.3 173.2 17.7 39.6 10.0 7.4 0.84 0.04 0.1 0.8b 5.2 2.5 0.9 0.4 0.2 10.1 1.69 1.7 121.0 173.6 15.2 38.3 9.6 7.2 0.63 0.01* 0.1* 0.9* 1.8* 0.8* 0.7* 0.2* 0.1* 0.10 27.8 91.4 1.1 102.3 154.0 89.1 31.5 8.4 92.5 0.58 11.0 16.7a 0.2 2.2a 4.1 15.3 1.2 0.2 1.0 0.32 16.7 71.1 0.8 113.3 158.5 59.4 31.6 8.2 89.2 0.71 10 6.8 14.4b 0.2 2.5ab 3.1 17.2 1.5 0.3 2.7 0.33 19.4 75.5 1.1 114.8 158.5 50.1 31.5 8.5 87.7 1.10 20 6.4 18.2ab 0.1 1.7b 5.1 19.7 1.0 0.2 3.5 0.65 13.9 69.2 0.8 118.1 163.5 53.3 31.8 8.9 83.9 0.60 40 2.8 20.2b 0.2 3.2b 2.5 12.5 1.3 0.2 1.8 0.19 19.5 76.8 1.0 112.1 158.6 63.0 31.6 8.5 88.3 0.75 Avg 3.0 7.8* 0.1* 1.1* 1.6* 7.3* 0.5* 0.1* 0.1* 0.2 Parameters reported as: mortality, %; nitrite, mg L)1; potassium, chloride, and sodium, meq L)1; cortisol, ng mL)1; haematocrit, %; Hb or haemoglobin, g dL)1; MetHb or methaemoglobin, %; ATPase, lM ADP mg protein)1 h)1 Cortisol, nitrite and MetHb increased significantly (P Ê 0.05), whereas plasma sodium, chloride, potassium, haemoglobin and haematocrit decreased significantly in channel catfish after nitrite exposure as compared to baseline, pre-exposure fish Significant differences between pre-exposure and postexposure averages are indicated by an asterisk Diet only affected plasma chloride and nitrite values Gill Na+, K+-ATPase Mortality Nitrite Potassium Chloride Sodium Cortisol Haematocrit Hb MetHb ATPase3 Avg CN 40 CN 20 Dietary NaCl (g kg)1 diet) Dietary NaCl (g kg)1 diet) 10 Postexposure2 Pre-exposure2 Table Physiological parameters (mean SE) measured in channel catsh before (Pre-exposure) and after (Postexposure) exposure to nitrite after feeding sodium chloride (NaCl) supplemented diets for 10 weeks1 Parameters reported as: mortality, %; nitrite, mg L)1; potassium, chloride, and sodium, meq L)1; cortisol, ng mL)1; haematocrit, %; Hb or haemoglobin, g dL)1; MetHb or methaemoglobin, %; ATPase, lM ADP mg protein)1 h)1 Cortisol, nitrite, potassium and MetHb increased significantly (P Ê 0.05), whereas plasma sodium, chloride and haematocrit decreased significantly in channel catfish after nitrite exposure as compared to baseline, pre-exposure fish Significant differences between pre-exposure and postexposure averages are indicated by an asterisk Diet only affected plasma chloride, nitrite, cortisol and mortality Percent mortality values with different superscripts are significantly different (P Ê 0.05) Gill Na+, K+-ATPase Mortality Nitrite Potassium Chloride Sodium Cortisol Haematocrit Hb MetHb ATPase4 Postexposure2 Pre-exposure2 Table Physiological parameters (mean SE) measured in channel catsh before (Pre-exposure) and after (Postexposure) exposure to nitrite after feeding sodium chloride (NaCl) supplemented diets for weeks1 Weeks 10 Weeks Parameter Pre-exposure Postexposure Pre-exposure Postexposure Pre-exposure nitrite-N2 Nitrite-N3 Chloride Ammonia-N Dissolved oxygen pH Temperature Hardness4 0.02 7.75 30.75 0.34 5.96 7.95 26.5 65.3 7.63 30.58 2.40 4.84 8.10 26.2 66.8 0.01 7.21 30.92 0.34 5.61 7.84 26.2 65.4 7.14 31.00 2.55 4.62 8.10 26.3 68.0 0.01 0.15 0.40 0.03 0.06 0.02 0.2 1.9 0.05 0.17 0.42 0.07 0.02 0.1 1.0 0.01 0.04 1.09 0.06 0.07 0.01 0.1 1.5 Table Water quality parameters (mean SE) before and after exposure of channel catsh to nitrite1 0.06 0.29 0.10 0.11 0.03 0.1 0.9 All parameters are expressed in mg L)1 except temperature (C) and pH (pH units) Pre-exposure, nitrite-N was measured just before addition of nitrite to aquaria Nitrite-N concentrations determined immediately after addition of KNO2 (pre-exposure) and 24 h after exposure (postexposure) The target nitrite-N concentrations were 7.70 mg L)1 at weeks and 7.18 mg L)1 at 10 weeks Hardness measured as total hardness, a measure of polyvalent cations with a charge >1, in mg L)1 Plasma nitrite levels in channel catsh were signicantly elevated after nitrite exposure in all dietary groups and tended to be lower in sh fed with diets supplemented with NaCl than in the control group Nitrite levels were almost twice as high at than at 10 weeks after nitrite exposure, most likely caused by the higher nitrite exposure concentration at weeks Plasma nitrite concentrations may reach 10 times the concentrations in the surrounding water (Lewis & Morris 1986), and we observed levels that were approximately six times (151.9 7.4 mg L)1 nitrite) the exposure concentration (25.2 mg L)1 nitrite) at weeks and about 3.5 times as high (84.9 8.7 mg L)1 nitrite) as the exposure concentration (23.6 mg L)1 nitrite) at 10 weeks Schoore et al (1995) reported a plasma nitrite concentration of approximately 110 mg L)1 after 24 h of 5.0 mg L)1 nitrite-N exposure Increases in plasma nitrite can cause a host of physiological disruptions that can lead to mortality, such as interference with ionic balance, decrease in blood oxygen transport, decreased cardiovascular function, and disturbances in endocrine function (Jensen 2003); however, toxicity of nitrite to sh is primarily attributed to methaemoglobinemia, the oxidation of Hb to form MetHb, which is incapable of binding oxygen (Urrutia & Tomasso 1987) Methaemoglobin and Hb levels were not signicantly affected by dietary supplementation with NaCl Prechallenge MetHb levels were approximately 57% of total Hb, which is near the range of 15% normally observed in channel catsh (Tucker et al 1989; Reagan & Drennan 1993) Postexposure MetHb levels increased signicantly, averaging 93% and 91% at and 10 weeks, respectively, indicating an acute level of nitrite toxicity (Lewis & Morris 1986) Blue catsh and channel catsh ã blue catsh hybrids exposed to 6.08 mg L)1 nitrite-N had MetHb levels ranging from 97.3% to 98.8% (Reagan & Drennan 1993) Channel catsh exposed to 6.30 mg L)1 nitrite-N developed approximately 15% MetHb after 24-h exposure and 37% after 48-h exposure (Urrutia & Tomasso 1987) Schoore et al (1995) reported that MetHb levels reached 80% in channel catsh after exposure to mg L)1 nitrite-N for 24 h and remained unchanged after 48 h of nitrite exposure Nitrite levels causing the development of 50% or greater MetHb are normally considered threatening to sh (Bowser et al 1983) Methaemoglobin levels of 70-80% normally cause torpor in sh, while levels approaching 100% can be lethal causing sh to die from anoxia (Lewis & Morris 1986) A high degree of variability exists in methaemoglobinemia in channel catsh exposed to nitrite, which can be aected by age, length and concentration of nitrite exposure, and water quality (Lewis & Morris 1986) In addition, catsh have a MetHb reductase enzyme that reconverts MetHb to Hb, and the activity can vary signicantly among catsh strains (Reagan & Drennan 1993), which may explain at least in part dierences in susceptibility of channel catsh to nitrite toxicity in the published literature Acute nitrite toxicity can also induce anaemia, along with methaemoglobinemia, in channel catsh, which is caused by a reduction in haemoglobin concentrations and haematocrit values (Tucker et al 1989) The authors suggest that prolonged nitrite exposure directs energy resources away from cells for use in MetHb reduction This causes a decrease in mean red blood cell (RBC) lifespan and an increased rate of cell haemolysis (Scarano et al 1984) Nitrite toxicity also Aquaculture Nutrition 17; e892e901 ể 2011 Blackwell Publishing Ltd No claim to original US government works results in a loss of chloride and nitrite from RBCs and an ensuing osmotic loss of water (Jensen 1990), which most likely contributed to the lower haematocrits we observed after nitrite exposure Urrutia & Tomasso (1987) did not observe reductions in Hb content in channel catsh exposed to nitrite-N ranging from 1.31 to 4.42 mg L)1 for up to 15 days; however, after 15 days of exposure to 6.30 mg L)1 nitrite-N, signicant anaemia occurred Nitrite-induced anaemia has been reported in other sh species, such as rainbow trout (Brown & McLeay 1975) and sea bass (Scarano & Saroglia 1984) We also observed slight anaemia after nitrite exposure at 10 weeks, but Hb values at weeks did not change signicantly in channel catsh exposed to nitrite Nitrite exposure caused a signicant increase in mortality As mentioned, susceptibility of sh to nitrite toxicity is affected by a number of factors (Lewis & Morris 1986), and in channel catsh, mortality from nitrite toxicity varies considerably among strains (Tomasso & Carmichael 1991) Koniko (1975) found the median tolerance limit (giving 50% survival) of nitrite-N for channel catsh was 10.3 mg L)1 for a 24-h exposure period In nitrite toxicity LC50 tests, we found that 7.70 and 7.18 mg L)1 nitrite-N resulted in approximately 50% mortality in the control group after 24-h exposure at weeks and 10 weeks, respectively, and 9.3 mg L)1 nitrite-N produced a mortality rate of nearly 95% in the week LC50 test (data not shown) The activity level of sh can also aect susceptibility to nitrite toxicity, and severe methaemoglobinemia may not immediately prove detrimental to inactive sh, further contributing to the variability in the response (Crawford & Allen 1977) Mortality from nitrite toxicity after 24-h exposure tended to decrease with increasing concentration of dietary NaCl and became signicantly lower in catsh fed with 40 g kg)1 NaCl at weeks After 10 weeks of feeding experimental diets, mortality from nitrite toxicity was also lower in sh fed with diets supplemented with NaCl than in the control group, but the dierences were not signicant This is the rst study to examine the eects of dietary salt on the eects of nitrite toxicity in sh Treatment of nitrite toxicity in sh has primarily consisted of increasing the water chloride concentration through addition of NaCl or CaCl2 to ponds (Bowser et al 1983) The protective eect of water-borne chloride against nitrite toxicity has been attributed to competitive inhibition of nitrite uptake by the chloride cells at the gills (Lewis & Morris 1986) Bowser et al (1983) found that a : ratio of chloride/nitrite was required to prevent MetHb from reaching lethal levels in channel catsh and reported no dierence in the ecacy of NaCl or CaCl2 in preventing nitrite toxicity Sodium bicarbonate was less eective than Aquaculture Nutrition 17; e892e901 ể 2011 Blackwell Publishing Ltd No claim to original US government works both CaCl2 and NaCl Wedemeyer & Yasutake (1977), however, reported CaCl2 was superior in prevention of nitrite toxicity in salmonids Tomasso et al (1979) found that channel catsh exposed to 1.5 mg L)1 nitrite-N had MetHb levels similar to baseline sh when treated with 60 mg L)1 but not 3.54 mg L)1 NaCl; the latter NaCl treatment did not prevent methaemoglobinemia The authors suggested that 25 mg L)1 NaCl was adequate to protect channel catsh from the toxic eects of 1.5 mg L)1 nitrite-N at pH 7.0 The chloride content of rearing water is one of the most important factors aecting toxicity of sh to nitrite (Lewis & Morris 1986) and probably accounts for much of the variability in susceptibility of sh to nitrite toxicity among published reports The improvements in blood Cl and nitrite levels in catsh may at least in part be responsible for the improved survival after nitrite exposure Nitrite levels after nitrite exposure declined as NaCl increased in diet Plasma chloride levels were higher in channel catsh fed with dietary NaCl before and after exposure to nitrite, and levels tended to increase linearly with increasing NaCl in diets It is possible that the benecial eects of dietary NaCl on blood parameters were more pronounced prior to the 24-h endpoint used in this study Dietary NaCl can increase resistance of sh to the toxic eects of some water-borne cations, such as copper in rainbow trout (Kamunde et al 2003, 2005; Pyle et al 2003) Kamunde et al (2005) concluded that in rainbow trout fed with dietary NaCl, decreased susceptibility to the toxic eects of water-borne copper was because of an increase in plasma sodium The authors suggest the rise in plasma sodium concentration reduced the eux of sodium by inhibition of the branchial sodium uptake route, which is also shared by copper Nitrite and chloride also share a common uptake route, presumably the branchial apical Cl =HCO exchanger in the gill (Jensen 2003) It is possible that the elevated plasma chloride levels in NaCl-fed catsh produced a chloride concentration gradient that partially inhibited passive gill eux of chloride or active nitrite uptake by the apical exchanger, although the plasma nitrite and chloride concentrations only weakly support this hypothesis Fish maintain chloride balance by balancing active branchial uptake with passive branchial chloride eux and loss to urine During nitrite exposure, some of the active chloride uptake is shifted to nitrite (Jensen 2003); however, it is not known whether sh actually dier in their ability to distinguish between chloride and nitrite at branchial uptake (Doblander & Lackner 1996) The therapeutic eects of dietary NaCl on resistance of sh to nitrite toxicity may only be temporary under acute nitrite concentrations as in this study, and nitrite uptake may be inhibited for only a short time Furthermore, the eects of dietary NaCl on nitrite toxicity may be inuenced by feeding duration, and in this study, benets on postexposure mortality ceased somewhere between and 10 weeks, which is further supported by the fact that mortality at 10 weeks declined, but not signicantly, with increasing NaCl in diet Future research should focus on the susceptibility of NaCl-fed channel catsh to lower concentrations of water-borne nitrite, dierent durations of exposure, and feeding duration Furthermore, the dierences in plasma chloride between dietary treatments may have worked in conjunction with other mechanisms known to exclude or detoxify nitrite in channel catsh Dietary salt is known to increase the urinary ow and glomerular ltration rates in rainbow trout (Salman & Eddy 1988), and the liver has shown to be an important site of nitrite detoxication that contributes signicantly to the nitrite tolerance of sh (Doblander & Lackner 1996) Chloride cell proliferation can also increase in sh with dietary salt supplementation (Fonta nhas-Fernandes et al 2001) and nitrite exposure (Williams & Eddy 1988), in which the latter may be a compensatory response to oset chloride loss We did not examine these factors, and it is therefore unknown whether they were inuenced by dietary treatment or whether they contributed to the dierences in susceptibility of channel catsh to nitrite toxicity Cortisol values increased signicantly after exposure to nitrite at and 10 weeks There was a signicant dierence between diets at weeks, and sh fed with the control diet had a signicantly higher cortisol response after nitrite exposure than those fed with the 20 g kg)1 or 40 g kg)1 sodium chloride diet The cortisol response generally followed the mortality rate with the highest cortisol and mortality occurring in the control group with values for both decreasing as the NaCl concentration increased in the diet A similar but non-signicant trend also occurred at 10 weeks Nitrite exposure has been shown to increase the corticosteroid response in channel catsh (Tomasso et al 1981) The authors found that total corticosteroid concentration increased with increasing nitrite concentration up to mg mL)1 (the highest level tested) and time of exposure to 24 h (total length of the exposure period) They hypothesize that the increase in corticosteroids was because of physiological dysfunction brought on by methaemoglobinemiainduced hypoxia As noted, the primary cause of mortality in sh exposed to nitrite is acute methaemoglobinemia leading to eventual suocation and death if the nitrite exposure concentration is high enough Addition of sodium chloride to water is commonly used to reduce stress in channel catsh during transport (Wurts 1995) Here, we show that not only can the addition of NaCl to diets of channel catsh decrease mortality from nitrite, but it can also reduce the cortisol response as a result of nitrite stress Although cortisol and nitrite mortality followed the same general response among the dietary groups in this study, it is dicult to determine what the connection is between the two The decrease in nitrite mortality may be in part because of a NaCl-mediated reduction in the stress response and associated secondary physiological eects Although some osmoregulatory parameters (plasma ions) were improved with sodium chloride supplementation, other secondary stress eects, such as energy metabolism, were not measured Further investigation is needed to evaluate this relationship We observed mixed results in the eects of dietary NaCl level and feeding duration on the tolerance of channel catsh to nitrite toxicity A signicant decrease in mortality was seen at weeks but not at 10 weeks when dietary NaCl was supplemented in the diet at 40 g kg)1 Plasma nitrite and Cl levels and MetHb (non-signicant) trended in support of the prevailing hypotheses for the positive eects of external NaCl on nitrite toxicity Although postexposure mortality was approximately 50% or lower, the levels of nitrite used in this study caused MetHb levels that were approximately 90% for the control group, which is considerably higher than the levels (50% MetHb) deemed detrimental to sh health, and the eects of dietary salt supplementation on lower levels of nitrite toxicity should be explored in the future The authors thank Brian Peterson of the Catsh Genetics Research Unit, United States Department of Agriculture, Agricultural Research Service, Stoneville, Mississippi and Julie Bebak of the Aquatic Animal Health Research Unit, United States Department of Agriculture, Agricultural Research Service, Auburn, Alabama for critical review of this manuscript We are indebted to Mark Smith, Rashida Eljack, Todd Threadgill and Justin Brock, all of the Aquatic Animal Health Research Unit, USDA, ARS, Auburn, AL, for technical assistance in this project Curtis Day and Jeery McVicker provided wet laboratory support Use of trade name or commercial products is solely for purpose of providing specic information and does not imply endorsement by the USDA Aquaculture Nutrition 17; e892e901 ể 2011 Blackwell Publishing Ltd No claim to original US government works Bardach, J.E., Ryther, J.H & McLarney, W.D (1972) Aquaculture: The Farming and Husbandry of Freshwater and Marine Organisms Wiley Interscience, New York, NY Bowser, P.R., Falls, W.W., VanZandt, J., Collier, N & Phillips, J.D (1983) Methemoglobinemia in channel catsh: methods of prevention Prog Fish Cult., 45, 154158 Brown, B.A (1988) Routine hematology procedures In: Hematology: Principles and Procedures (Brown, B.A ed.), pp 7122 Leo and Febiger, Philadelphia, PA Brown, D.A & McLeay, D.J (1975) Eect of nitrite on methemoglobin and total hemoglobin of juvenile rainbow trout Prog Fish Cult., 37, 3643 Crawford, R.E & Allen, G.H (1977) Seawater inhibition of nitrite toxicity to Chinook salmon Trans Am Fish Soc., 106, 105 109 Doblander, C & Lackner, R (1996) Metabolism and detoxication of nitrite by trout hepatocytes Biochim Biophys Acta, 1289, 270 274 Evelyn, K.A & Malloy, H.T (1938) Microdetermination of oxyhemoglobin, methemoglobin, and sulfhemoglobin in a single sample of blood J Biol Chem., 126, 655662 Fonta nhas-Fernandes, A., Russell-Pinto, F., Gomes, E., ReisHenriques, M.A & Coimbra, J (2001) The eect of dietary sodium chloride on some osmoregulatory parameters of the teleost, Oreochromis niloticus, after transfer from freshwater to seawater Fish Physiol Biochem., 23, 307316 Huey, D.W., Beitinger, T.L & Wooten, M.C (1984) Nitrite-induced methemoglobin formation and recovery in channel catsh (Ictalurus punctatus) at three acclimation temperatures Bull Environ Contam Toxicol., 32, 674681 Hwang, P.P., Sun, C.M & Wu, S.M (1989) Changes of plasma osmolarity, chloride concentration, and gill Na-K-ATPase activity in tilapia Oreochromis mossambicus during seawater acclimation Mar Biol., 100, 295299 Jensen, F.B (1990) Nitrite and red cell function in carp: control factors for nitrite entry, membrane potassium ion permeation, oxygen anity and methaemoglobin formation J Exp Biol., 152, 149166 Jensen, F.B (2003) Nitrite disrupts multiple physiological functions in aquatic animals Comp Biochem Physiol A Comp Physiol., 135, 924 Kamunde, C.N., Pyle, G.G., McDonald, D.G & Wood, C.M (2003) Inuence of dietary sodium on waterborne copper toxicity in rainbow trout, Oncorhynchus mykiss Environ Toxicol Chem., 22, 342350 Kamunde, C.N., Niyogi, S & Wood, C.S (2005) Interaction of dietary sodium chloride and waterborne copper in rainbow trout (Oncorhynchus mykiss): copper toxicity and sodium and chloride homeostasis Can J Aquat Fish Sci., 62, 390399 Koniko, M (1975) Toxicity of nitrite to channel catsh Prog Fish Cult., 37, 9698 Larsen, H.N (1964) Comparison of various methods of hemoglobin detection of channel catsh blood Prog Fish Cult., 26, 1115 Lewis, W.M & Morris, D.P (1986) Toxicity of nitrite to sh: a review Trans Am Fish Soc., 115, 183195 Lim, C., Sealey, W.M & Klesius, P.H (1996) Iron methionine and iron sulfate as sources of dietary iron for channel catsh Ictalurus punctatus J World Aquac Soc., 27, 290296 McCormick, S.D (1993) Methods for nonlethal gill biopsy and measurement of Na+, K+-ATPase activity Can J Fish Aquat Sci., 50, 656658 Aquaculture Nutrition 17; e892e901 ể 2011 Blackwell Publishing Ltd No claim to original US government works NRC (National Research Council) (1993) Nutrient Requirements of Fish 114 pp National Academy Press, Washington, DC Prentice, R.L (1976) A generalization of the logit and probit methods for dose response curves Biometrics, 32, 761768 Pyle, G.G., Kamunde, C.N., McDonald, D.G & Wood, C.M (2003) Dietary sodium inhibits aqueous copper uptake in rainbow trout, Oncorhynchus mykiss J Exp Biol., 206, 609618 Reagan, R.E & Drennan, D.G (1993) Enzymatic reduction of methemoglobin to hemoglobin in blue catsh, Ictalurus furcatus, and channel catsh, I punctatus, $ ã Blue catsh # hybrids J Appl Aquacult., 3, 223234 Salman, N.A & Eddy, F.B (1987) Response of chloride cell numbers and gill Na+, K+-ATPase activity of freshwater rainbow trout (Salmo gairdneri Richardson) to salt feeding Aquaculture, 61, 4148 Salman, N.A & Eddy, F.B (1988) Kidney function in response to salt feeding in rainbow trout (Salmo gairdneri Richardson) Comp Biochem Physiol A Comp Physiol., 89, 535539 Scarano, G & Saroglia, M.G (1984) Recovery of sh from functional and hemolytic anemia after brief exposure to a lethal concentration of nitrite Aquaculture, 43, 421426 Scarano, G., Saroglia, M.G., Gray, R.H & Tibaldi, E (1984) Hematological responses of sea bass Dicentrarchus labrax to sublethal nitrite exposures Trans Am Fish Soc., 113, 360364 Schoore, J.E., Simco, B.A & Davis, K.B (1995) Responses of blue catsh and channel catsh to environmental nitrite J Aquat Anim Health, 7, 304311 Staurnes, M & Finstad, B (2000) The eects of dietary NaCl supplement on hypo-osmoregulatory ability and sea water performance of Arctic charr (Salvelinus alpinus L.) smolts Aquacult Res., 31, 737743 Tomasso, J.R (1986) Comparative toxicity of nitrite to freshwater shes Aquat Toxicol., 8, 129137 Tomasso, J.R & Carmichael, G.J (1991) Dierential resistance among channel catsh strains and intraspecic hybrids to environmental nitrite J Aquat Anim Health, 3, 5154 Tomasso, J.R., Simco, B.A & Davis, K.B (1979) Chloride inhibition of nitrite-induced methemoglobinemia in channel catsh (Ictalurus punctatus) J Fish Res Bd Can., 36, 11411144 Tomasso, J.R., Davis, K.B & Simco, B.A (1981) Plasma corticosteroid dynamics in channel catsh (Ictalurus punctatus) exposed to ammonia and nitrite Can J Fish Aquat Sci., 38, 11061112 Tucker, C.S & Schwedler, T.E (1983) Acclimation of channel catsh (Ictalurus punctatus) to nitrite Bull Environ Contam Toxicol., 30, 516521 Tucker, C.S., Francis-Floyd, R & Beleau, M.H (1989) Nitrite-induced anemia in channel catsh, Ictalurus punctatus Ranesque Bull Environ Contam Toxicol., 43, 295301 Urrutia, M.L & Tomasso, J.R (1987) Acclimation of channel catsh to environmental nitrite J World Aquac Soc., 18, 175179 Wedemeyer, G.A & Yasutake, W.T (1977) Clinical methods for the assessment of the eects of environmental stress on sh U.S Fish Wildl Serv., Tech Pap No 89 18 pp Williams, E.M & Eddy, F.B (1988) Anion transport, chloride cell number and nitrite-induced methaemoglobinemia in rainbow trout (Salmo gairdneri) and carp (Cyprinus carpio) Aquat Toxicol., 13, 2942 Wurts, W.A (1995) Using salt to reduce handling stress in channel catsh World Aquac., 56, 8081 Zaugg, W.S., Roley, D.D., Prentice, E.F., Gores, K.X & Wakitz, F.W (1983) Increased seawater survival and contribution to the shery of chinook salmon (Oncorhynchus tshawytscha) by supplemental dietary salt Aquaculture, 32, 183188 Aquaculture Nutrition 2011 17; e902e911 doi: 10.1111/j.1365-2095.2011.00863.x Xiamen Key Laboratory for Feed Quality Testing and Safety Evaluation, Fisheries College, Jimei University, Xiamen, China In this study, we examined the eects of the following eight experimental diets, which varied in fructo oligosaccharides (FOS), mannan oligosaccharides (MOS) and Bacillus clausii concentrations, on the Japanese ounder: control diet (no FOS, MOS and B clausii), diet F (5 g kg)1 FOS), diet M (5 g kg)1 MOS), diet FM (2.5 g kg)1 FOS + 2.5 g kg)1 MOS), diet B (107 cells g)1 B clausii), diet FB (5 g kg)1 FOS + 107 cells g)1 B clausii), diet MB (5 g kg)1 MOS + 107 cells g)1 B clausii) and diet FMB (2.5 g kg)1 FOS + 2.5 g kg)1 MOS + 107 cells g)1 B clausii) Japanese ounder, initially weighing an average of 21 g, were distributed into 24 net cages at a stocking density of 20 sh per cage Each diet was hand-fed to three groups of sh twice daily for 56 days The weight gain rate (WGR) in sh fed diets B, MB and FMB were signicantly higher than in sh fed the control diet, where the sh fed diet FMB had the highest WGR Fish fed any of the diets, except diets F and B, exhibited better feed conversion ratio than those fed the control diet Diets MB and FMB signicantly elevated intestinal protease activity compared with the control diet, but only the diet FMB promoted amylase activity Feeding diets FB and FMB increased body protein deposition; additionally, feeding diets B, MB and FMB signicantly reduced body lipid deposition Lysozyme (LSZ) activity was signicantly higher in sh fed diets B, FB, MB and FMB than in sh fed the control diet All diets, except diet M, decreased triglyceride (TG) levels compared to the control diet Low-density lipoprotein cholesterol levels in sh fed diets F, FB and FMB were signicantly lower than in sh fed the control diet Without exception, no diets aected feeding rate, condition factor, body moisture, ash contents, phagocytic activity of leuco- cytes or cholesterol or high-density lipoprotein cholesterol levels Our results suggest that diets supplemented with FOS, MOS and B clausii improved growth performance and health benets of the Japanese ounder more than other diets or the control diet KEY WORDS: Bacillus clausii, growth, innate immune response, intermediary metabolism, oligosaccharide, Paralichthys olivaceus Received 31 October 2010, accepted 16 February 2011 Correspondence: J.-D Ye, Yindou Road 43, Jimei District, Jimei University, Xiamen, China E-mail: yjdwk@sina.com; scyjs@jmu.edu.cn The rapid development of aquaculture has induced a rise in physiological stress and increased the potential risk of disease outbreaks in sh, which has led to production losses and impeded the sustainable development of the industry (Torrecillas et al 2007) Under such conditions, antibiotics are routinely the rst choice for animal therapy and prophylaxis Subtherapeutic doses of antibiotics have often been added to aquatic feeds to promote growth, which has contributed to drug resistance (Hernandez Serrano 2005) Recent restrictions on the use of antibiotics as additives in aquafeeds have prompted sh farmers to explore healthy and safe alternatives to produce safe aquatic food Currently, prebiotics and probiotics are the leading candidates for environmentfriendly feed additives in the aquaculture industry Prebiotics are non-digestible carbohydrates that are utilized by specic health-promoting bacteria, which have a positive eect on the nutrition and health of the host Probiotics are live ể 2011 Blackwell Publishing Ltd microorganisms that increase viability, improve immune and digestive systems and promote growth and general welfare of sh when consumed in adequate amounts (Ringứ et al 2010) Over the last decade, numerous studies have investigated the potential use of prebiotics and probiotics in promoting the productivity and health of cultured sh, shrimp and other aquatic organisms (Kim & Austin 2006; Wang et al 2008; Denev et al 2009; Merrield et al 2010a; Ringứ et al 2010; Dimitroglou et al 2011) In most cases, the positive eects of their use were demonstrated; however, dierences in the doses of pre- and probiotics, chemical structures and compositions of prebiotics and strains of probiotics experiments precluded the identity of consistent and benecial eects (Denev et al 2009) Nevertheless, their use in aquaculture is in its infancy, where the knowledge of the benecial mechanisms employed by prebiotics or probiotics is still limited Gibson & Roberfroid (1995) have dened the mixture of pre- and probiotics as synbiotics that exert synergistic eects in promoting benecial bacteria and the health of the gastrointestinal tract of the host, thus their potential applications have spurred attention To the best of our knowledge, extensive investigation into the eects of dietary synbiotics on the growth performance and health benets of sh (Rodrigues-Estrada et al 2009) and shrimp (Li et al 2009) has not been performed Fructo oligosaccharides (FOS) and mannan oligosaccharides (MOS) are two frequently used prebiotics with unique chemical structures that may improve the gut health and ecosystem of the host in dierent ways (Denev et al 2009; Ringứ et al 2010) To date, the eects of the combination of FOS and MOS on the growth and health benets in sh have not been evaluated The Japanese ounder is an important marine cultured species with great economic interest (Seikai 2002), but the information on the ecacy of pre- and probiotics in the culture of this sh has been limited (Taoka et al 2006) Therefore, we investigated singular and synthetic eects of FOS, MOS and a newly isolated probiotic Bacillus clausii on growth, feed utilization, body composition, digestive enzyme activity, non-specic immunity and lipid metabolism parameters of Japanese ounder We had previously isolated the probiotic strain B clausii from grouper (Epinephelus coioides) intestine (Sun et al 2009) We identied the strain based on 16S rRNA gene sequencing (Sun et al 2009) and on the morphological, Aquaculture Nutrition 17; e902e911 ể 2011 Blackwell Publishing Ltd physiological and biochemical characteristics described in the Bergeyếs Manual of Systematic Bacteriology (Holt et al 1994) The strain showed antagonistic eects on suspected pathogenic Vibrio species (i.e V parahaemolyticus, V metschnikovi and V alginolyticus) in vitro (Sun et al 2009) Juvenile grouper exhibited improved growth and immune responses when the probiotic was consumed (Sun et al 2010) The bacteria were grown in nutrient broth (10-g peptone, 3-g beef extract and 5-g sodium chloride to 1-l deionized water) at 28 C for 48 h Colonies from the culture plates were subcultured in fresh broth in a shaking incubator for 18 h Cultures were harvested by centrifugation at 5031 g for 10 at C, washed in sterile saline twice and resuspended in sterile saline The number of bacteria in the suspension was determined by plate counting on tryptone soy agar at 28 C for 48 h (3.8 ã 1010 cells mL)1) Bacterial purity and identication were monitored throughout experiments according to this protocol (Nikoskelainen et al 2003) Fructo oligosaccharides and MOS were obtained from Quantum Hi-tech Biological Engineering Co Ltd., Jiangmen, China and Alltech Inc., Nicholasville, KY, USA, respectively Steam-dried sh meal (rst feed grade) was produced by Shine Biotech Co Ltd., Shandong, China Soybean meal, peanut meal, wheat our, shrimp head meal and other feed ingredients were obtained from Fuxing Feed Co., Ltd., Xiamen, China Commercial ingredients were used to formulate a basal diet composed of 485 g kg)1 crude protein and 85 g kg)1 crude lipids The diet was supplemented with FOS, MOS and B clausii alone or in combination on an as-fed basis MOS, FOS or a combination of the two was supplemented at the expense of wheat our The concentrations of the administered supplements in the eight experimental diets (Table 1) were as follows: control diet (no FOS, MOS and B clausii), diet F (5 g kg)1 FOS), diet M (5 g kg)1 MOS), diet FM (2.5 g kg)1 FOS + 2.5 g kg)1 MOS), diet B (107 cells g)1 B clausii), diet FB (5 g kg)1 FOS + 107 cells g)1 B clausii), diet MB (5 g kg)1 MOS + 107 cells g)1 B clausii) and diet FMB (2.5 g kg)1 FOS + 2.5 g kg)1 MOS + 107 cells g)1 B clausii) The ingredients of each diet were blended in a 3D mixer from Punaier Drying Equipment Co Ltd., Changzhou, China and sterilized water was added to the mixture and mixed uniformly by hand The probiotic diet was prepared by gently spraying the required amount of bacterial suspension on diets F, M or FM and controls and mixing it part-by-part in a mixer to obtain a nal probiotic concentration of 1.0 ã 107 cells g)1 The nal mixture was extruded through a 3-mm die to form strands and pelleted to a suitable size using a multifunctional spiral extrusion machinery from CD4XITS, South China University of Table Ingredients and proximate composition of the experimental diets Diets1 Ingredients )1 Fish meal (g kg ) Soybean meal (g kg)1)3 Wheat flour (g kg)1)3 Peanut meal (g kg)1)3 Shrimp head meal (g kg)1)3 Soybean phospholipid (g kg)1)3 Fish oil (g kg)1)3 Ca(H2PO4)2 (g kg)1)3 Vitamin premix (g kg)1)4 Mineral premix (g kg)1)5 FOS (g kg)1) MOS (g kg)1) Bacillus clausii (cells g)1 feed) Nutrient level (g kg)1) Dry matter Crude protein Crude lipid Ash Control Diet F Diet M Diet FM Diet B Diet FB Diet MB Diet FMB 550 130 198 50 30 20 10 10 1 550 130 193 50 30 20 10 10 1 550 130 193 50 30 20 10 10 1 550 130 193 50 30 20 10 10 1 2.5 2.5 550 130 198 50 30 20 10 10 1 107 550 130 193 50 30 20 10 10 1 107 550 130 193 50 30 20 10 10 1 107 550 130 193 50 30 20 10 10 1 2.5 2.5 107 926.5 485.5 84.5 95.4 925.9 486.1 86.2 96.5 927.4 486.3 85.6 95.7 926.6 485.2 87.2 96.4 926.1 486.7 86.8 94.2 927.1 487.4 86.5 93.5 927.8 487.7 85.1 94.7 925.6 484.8 87.6 96.1 Values are expressed as fed basis Control diet (no FOS, MOS and B clausii), diet F (5 g kg)1 FOS), diet M (5 g kg)1 MOS), diet FM (2.5 g kg)1 FOS + 2.5 g kg)1 MOS), diet B (107 cells g)1 B clausii), diet FB (5 g kg)1 FOS + 107 cells g)1 B clausii), diet MB (5 g kg)1 MOS + 107 cells g)1 B clausii) and diet FMB (2.5 g kg)1 FOS + 2.5 g kg)1 MOS + 107 cells g)1 B clausii) Steam-dried FM (first feed grade) was produced by Shine Biotech Co Ltd., Shandong, China Soybean meal, peanut meal, soybean phospholipid, fish oil and calcium dihydrogen phosphate were obtained from Fuxing Feed Co Ltd., Xiamen, China Vitamin premix (g kg)1 mixture): retinyl acetate, 12; cholecalciferol, 3.6; dl-a-tocopheryl acetate, 120; Menadione sodium bisulphite, 10; thiamin hydrochloride, 15; riboflavin, 10; pyridoxine hydrochloride, 16; D-calcium pantothenate, 18; folic acid, 5; biotin, 0.3; cyanocobalamin, 0.02; inositol, 200; L-ascorbate-2-polyphosphate, 200; choline chloride, 390.08 Mineral premix (g kg)1 mixture): ferrous sulphate, 253.83; zinc sulphate, 358.82; potassium iodine, 0.53; copper sulphate, 8; manganese sulphate, 47.1; sodium selenite, 0.45; cobalt chloride, 3.3 g; magnesium sulphate, 327.67 FOS, fructo oligosaccharides; MOS, mannan oligosaccharides Technology, China The pellets were dried at 40 C in an oven and stored at C in sealed plastic bags Juvenile Japanese ounder, initially weighing an average of 21 g, were obtained from a commercial sh farm in Zhaoan County, Fujian, China The sh were acclimated in an indoor concrete pond (6.0 m ã 3.0 m ã 1.5 m) with a ow-through seawater system for weeks at a private farm in Zhangpu County, Fujian, China Following acclimation, the sh were fasted for 24 h, and similar sizes were selected and stocked at a density of 20 sh per cage in 24 net cages (60 ã 50 ã 60 cm) in an indoor concrete pond (10 ã ã 1.5 m) The pond was approximately 1.5 m deep Air was continuously supplied from pipes with holes on the bottom of the pond, which were placed at 1-m intervals Three cages were allocated for each experimental diet During the 8-week feeding period, the sh were hand-fed until satiated at 08:00 and 15:00 daily Fish were considered satiated when two to three pellets were not consumed within of being oered at every meal The water temperature, salinity, pH and dissolved oxygen during the feeding trial ranged from 22.6 to 25.4 C, 3236 g L)1, 7.88.1 and 5.67.7 mg L)1, respectively At the end of the feeding trial, sh from each cage were batch-weighed after 24 h of starvation Four sh were randomly sampled from each cage and sacriced with an overdose of MS 222 (tricaine methanesulfonate) solution, pooled in plastic bags and stored at )20 C for whole body composition analysis Another four sh from each cage were randomly sampled, anesthetized with MS 222 (100 mg L)1) and weighed individually Blood was collected via venipuncture and divided into two fractions One aliquot was allowed to clot at C for h, and the serum was obtained after centrifugation at 6261 g for 10 at C Collected serum was pooled by cage and stored at )80 C for subsequent analysis of blood biochemical parameters The rest was injected into a centrifuge tube containing heparin, and Aquaculture Nutrition 17; e902e911 ể 2011 Blackwell Publishing Ltd this heparinized blood was used to determine leucocyte phagocytosis After haemospasia, the sh were killed by a sharp blow to the head, and their length was recorded for condition factor determination The intestines were also removed, washed with chilled distilled water, weighed and pooled by cage The contents were stored at )80 C for later ascertainment of digestive enzyme activities Whole sh samples were autoclaved at 121 C for 20 min, homogenized and dried at 65 C for 24 h All the samples, including feed ingredients and experimental diets, were analysed for proximate composition determination (i.e moisture, crude protein, crude lipid and ash) according to standard methods (AOAC 1995) Crude protein was determined by measuring the nitrogen levels (N ã 6.25) using Kjeltecễ 8400 Auto Sample Systems (Foss Tecator AB, Hoăganaăs, Sweden) Crude lipid was analysed with an ether extract using a Soxtec Avanti 2050 (Foss Tecator AB) Dry matter was determined by drying in an oven at 105 C, and ash was determined by combustion in a mue furnace at 550 C Triglyceride (TG), cholesterol (CHO), high-density lipoprotein cholesterol (HDL-C) and Low-density lipoprotein cholesterol (LDL-C) were assessed according to the methods described by Bucolo & David (1973), Allain et al (1974), Warnick et al (1982) and Friedewald et al (1972), respectively All parameters were determined using an automatic biochemical analyzer (Hitachi 7020, Tokyo, Japan) The pooled intestines for each sample were homogenized in chilled saline at a ratio of : (w/v) for 10 The homogenate was centrifuged at 6261 g for 15 at C before the supernatant was collected Aliquots of the supernatant, designated as the crude extract, were used to estimate the activities of amylase and protease Amylase activity was measured according to the procedure in the manual book (Medical chemical laboratory of Shanghai 1979) using an iodine solution to reveal non-hydrolysed starch Briey, the enzymatic reaction mixture consisted of 1.0 mL of phosphate buer (pH 7.0), containing 1/2500 (w/v) soluble starch as the substrate, and 0.2 mL of the crude extract Test and control samples were incubated for 7.5 at 37 C A 1.0-mL Aquaculture Nutrition 17; e902e911 ể 2011 Blackwell Publishing Ltd aliquot of a 10-mM iodine solution was immediately added to the reaction mixture, followed by the addition of 6-mL distilled water The absorbance was recorded at 660 nm using a UV-2800S spectrophotometer One unit of enzyme activity was dened as 10 mg of starch hydrolysed per 30 per g of wet tissue Protease activity was elucidated based on the method described in the manual book (Biological department of Sun Yat-sen University 1979) using the Folin phenol reagent The enzymatic reaction mixture, consisting of g kg)1 alkaline casein (pH 7.5) substrate and mL of the enzyme extract, was incubated for 15 at 37 C The addition of 1/10 (w/v) trichloroacetic acid stopped the reaction, and the resultant mixture was centrifuged at 22 940 g for Using a UV-2800S spectrophotometer, the absorbance of the supernatant was recorded at 680 nm against a tyrosine standard One unit of enzyme activity was dened as 1-lg tyrosine released per per g of wet tissue Lysozyme (LSZ) activity was assayed using the turbidimetric method described by Parry et al (1965) with some modications Briey, the lyophilized Micrococcus lysodeikticus suspension (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) was prepared at a concentration of 0.2 mg mL)1 in 40 mM phosphate buer (pH 6.2) Two millilitres of the suspension was mixed with 200 lL of serum, and the optical density was read at 450 nm After 60 of incubation at 37 C, the optical density of the suspension was recorded again One unit of LSZ activity, expressed as unit per millilitre, was dened as the amount of enzyme that reduced absorbance by 0.001 per millilitre of serum per minute Phagocytosis activity for leucocytes was assayed according to the in vitro method described by Li et al (2009) with some modications Staphylococcus aureus replaced yeast as a source of foreign particles The strains obtained from our laboratory were cultured in nutrient broth at 28 C for 48 h, and the colonies were collected after centrifugation at 6261 g for 10 The bacteria were killed in 1/200 (v/v) formalin, rinsed in sterile distilled water three times and resuspended in sterile distilled water to a concentration of approximately 108 cells mL)1 One hundred microlitres of formalin-killed S aureus suspension was mixed with 200 lL of blood The mixture was incubated at 28 C for 60 min, and blood was smeared onto glass slides, air-dried and xed with methanol for 10 to allow for cell attachment The slides were washed with water, air-dried and stained with Giemsa stain for h For each sample, 100200 cells that were in the eld Diets1 IBW2 Control Diet F Diet M Diet FM Diet B Diet FB Diet MB Diet FMB 21.4 21.5 21.5 21.4 21.2 21.3 21.2 21.2 FBW2 0.1 0.2 0.2 0.3 0.2 0.2 0.1 0.2 73.5 74.4 75.5 75.2 73.9 75.0 75.3 75.5 FI2 0.9b 2.1ab 0.6a 0.5a 0.8ab 0.4ab 0.7a 0.9a 60.0 60.1 60.7 60.5 61.0 60.0 61.0 60.7 WGR2 0.4 0.9 0.6 0.3 0.1 0.3 0.5 0.3 242.7 245.8 251.2 252.0 248.5 251.8 255.2 255.5 FCR2 6.4b 11.2ab 2.8ab 5.3ab 5.8ab 4.5ab 2.5a 1.8a 1.17 1.15 1.13 1.13 1.16 1.12 1.13 1.12 Table Growth performance of Japanese ounder fed the experimental diets CF3 0.01a 0.04ab 0.01bc 0.01bc 0.02ab 0.01c 0.02bc 0.02c 0.85 0.82 0.84 0.85 0.85 0.81 0.82 0.82 0.06 0.06 0.04 0.05 0.02 0.03 0.02 0.01 Control diet (no FOS, MOS and Bacillus clausii), diet F (5 g kg)1 FOS), diet M (5 g kg)1 MOS), diet FM (2.5 g kg)1 FOS + 2.5 g kg)1 MOS), diet B (107 cells g)1 B clausii), diet FB (5 g kg)1 FOS + 107 cells g)1 B clausii), diet MB (5 g kg)1 MOS + 107 cells g)1 B clausii) and diet FMB (2.5 g kg)1 FOS + 2.5 g kg)1 MOS + 107 cells g)1 B clausii) Data from each dietary treatment are mean SD of triplicate cages Data from each dietary treatment are mean SD of 12 fish Values with different superscripts within a column indicate significant differences (P < 0.05) FOS, fructo oligosaccharides; MOS, mannan oligosaccharides; IBW, initial body weight (g per fish); FBW, final body weight (g per fish); FI, feed intake (g); WGR, weight gain rate (%); FCR, feed conversion ratio; CF, condition factor (%) of view were counted under oil immersion at 100 ã magnication for three slides Phagocytic percentage (PP) = 100 ã (number of phagocytizing leucocytes/total number of leucocytes counted) Phagocytic index (PI) = number of bacterial cell engulfed/ number of ingesting phagocytes Weight gain rate (WGR) = 100 ã (Wf ) Wi)/Wi Feed conversion ratio (FCR) = FI/(Wf ) Wi) Condition factor (CF) = 100 ã Wb/l3 In the formulas above, Wi (g) is the initial body weight, Wf (g) is the nal body weight (FBW), FI (g) is the total amount of the diet intake per sh in each tank, Wb (g) is the FBW of the sampled sh, and l (cm) is the nal body length of the sampled sh Statistical analysis was performed with SPSS 11.5 statistical software (SPSS, Chicago, IL, USA) Eects of dietary treatments on these parameters were tested using one-way ANOVA, and Duncanếs multiple range tests, at the 5% level of signicance, were used to compare the mean values Prior to statistical analysis, percentage data were subjected to arcsine or log transformation respectively All sh maintained active ingestion, exhibited proper growth during the feeding period and survived each dietary treat- ment The control group exhibited the lowest FBW and WGR of all dietary treatments (Table 2) Diets M, FM, MB and FBM increased FBW, whereas diets MB and FMB increased WGR (P < 0.05) However, there were no signicant dierences (P > 0.05) in FBW and WGR among any FOS, MOS and B clausii treatments In contrast, the sh that were fed diets M, FM, FB, MB or FMB had signicantly lower FCR than those fed the control diet (P < 0.05) Neither FI nor CF dier among any of the dietary treatments (P > 0.05) Table Body composition of Japanese ounder fed the experimental diets Diets1 Moisture Crude protein Control Diet F Diet M Diet FM Diet B Diet FB Diet MB Diet FMB 784.5 781.0 782.9 781.4 784.6 779.3 786.2 782.4 142.0 146.4 144.7 147.1 145.3 149.1 145.0 147.7 6.8 5.5 4.5 5.2 1.3 2.0 5.5 5.0 4.3b 6.1ab 4.2ab 6.0ab 2.2ab 1.3a 4.2ab 7.0a Crude lipid Ash 3.6a 1.7ab 2.4a 4.5ab 3.2b 1.5ab 2.2b 2.9b 37.8 37.7 37.1 37.8 36.9 37.3 37.0 36.3 30.2 28.0 30.1 28.7 26.0 26.9 26.5 26.0 2.3 1.1 3.6 1.9 2.5 2.6 2.9 1.8 Values are expressed as wet body basis FOS, fructo oligosaccharides; MOS, mannan oligosaccharides Control diet (no FOS, MOS and Bacillus clausii), diet F (5 g kg)1 FOS), diet M (5 g kg)1 MOS), diet FM (2.5 g kg)1 FOS + 2.5 g kg)1 MOS), diet B (107 cells g)1 B clausii), diet FB (5 g kg)1 FOS + 107 cells g)1 B clausii), diet MB (5 g kg)1 MOS + 107 cells g)1 B clausii) and diet FMB (2.5 g kg)1 FOS + 2.5 g kg)1 MOS + 107 cells g)1 B clausii) Data from each dietary treatment are mean SD of triplicate cages Values are expressed on a wet weight basis (g per kg) Values with different superscripts within a column indicate significant difference (P < 0.05) Aquaculture Nutrition 17; e902e911 ể 2011 Blackwell Publishing Ltd There was an increase in body protein content in sh fed a FOS-, MOS- and/or B clausii-containing diet compared to the control (Table 3) Fish fed diets FB and FMB also exhibited signicantly higher body protein content than sh fed the control diet (P < 0.05) Body lipid content demonstrated an opposite trend to body protein content, where sh fed diets B, MB and FMB presented with signicantly lower levels than sh fed the control diet (P < 0.05) Neither body moisture nor ash content were aected by any dietary treatments (P > 0.05) Dietary treatments aected LSZ activity dierently (Table 4) LSZ activity was signicantly higher in sh fed diets B, FB, MB and FMB compared with those fed the control diet (P < 0.05) There was no signicant dierence in LSZ activity between diets F, M and FM and between diets FB, MB and FMB (P > 0.05) The PP and PI of leucocytes in sh fed diets supplemented with FOS, MOS and/or B clausii, were similar to those of sh fed the control diet (P > 0.05) Serum biochemical parameters are presented in Table The TG level was lower or tended to be lower in sh fed the FOS, MOS and/or B clausii-containing diets versus the control diet Diets supplemented with FOS, MOS and/or B clausii, except diet M, signicantly decreased TG levels compared with the control diet (P < 0.05) By comparison, the TG level in sh fed diet FM was lower than in sh fed diet M (P < 0.05) but similar to the levels in sh fed diet F (P > 0.05) There were no dierences in TG levels among diets FB, MB and FMB (P > 0.05) The LDL-C response was similar to TG levels However, feeding diets F, FB or FMB yielded signicant decreases in LDL-C levels compared with the control diet (P < 0.05) Signicant dierences (P > 0.05) were not observed in CHO or HDL-C levels among all dietary treatments The eects of dietary pre- and prebiotics administration on intestinal protease and amylase activities of sh are shown in Fig Protease activity was signicantly higher (P < 0.05) in sh fed diets MB (37.65 7.66 U g)1) and FMB (38.37 9.24 U g)1) than in sh fed the control diet (27.18 4.93 U g)1) but similar to the activity in sh fed other pre- and/or probiotic-containing diets (P > 0.05) Fish fed the diet FBM (0.303 0.016 U g)1) exhibited the highest amylase activity, which was signicantly higher (P < 0.05) than in sh fed diets FM (0.252 0.033 U g)1), B (0.249 0.009 U g)1) and control (0.257 0.023 U g)1) The results in this study have illustrated that a single administration of FOS, MOS and B clausii did not improve the WGR of the Japanese ounder compared with the control diet Although some studies have reported a similar response with other sh species (Hidalgo et al 2006; Salze et al 2008; Buentello et al 2010; Dimitroglou et al 2010; Peterson et al 2010; Ringứ et al 2010), a positive WGR response to these substances has been observed in other sh studies (Torrecillas et al 2007; Aly et al 2008; Merrield et al 2010b) Our current study and several recent reports (Staykov et al 2007; Torrecillas et al 2007; Grisdale-Helland et al 2008) showed no feed eciency response to FOS or MOS In contrast, both FOS and MOS improved feed eciency (Buentello et al 2010), which was dierent from our Table Values of immune and blood parameters of Japanese ounder fed the experimental diets Immune parameter Diets Control Diet F Diet M Diet FM Diet B Diet FB Diet MB Diet FMB LSZ 36.35 38.71 38.29 40.10 43.08 44.69 44.47 46.58 Blood parameter PP d 3.59 4.02cd 1.14cd 4.05bcd 3.93ab 6.63ab 4.82ab 2.44a 49.40 50.73 49.67 54.13 54.93 54.27 53.87 55.40 PI 2.80 2.64 7.85 2.14 2.16 2.40 2.44 3.34 3.98 4.01 4.07 3.96 3.98 4.12 4.19 4.27 TG 0.07 0.06 0.28 0.25 0.12 0.39 0.12 0.14 2.49 2.34 2.48 2.28 2.38 2.31 2.33 2.28 CHO a 0.22 0.07bc 0.09ab 0.13c 0.14b 0.13c 0.14bc 0.11c 8.73 8.07 8.33 7.95 8.57 7.59 7.68 7.74 LDL-C 0.99 0.71 2.24 0.58 0.80 0.67 0.99 0.42 2.81 2.51 2.60 2.61 2.69 2.46 2.69 2.54 HDL-C a 0.26 0.14b 0.42ab 0.14ab 0.18ab 0.16b 0.09ab 0.12b 1.54 1.69 1.61 1.59 1.58 1.71 1.72 1.68 0.18 0.11 0.24 0.20 0.25 0.15 0.23 0.23 FOS, fructo oligosaccharides; MOS, mannan oligosaccharides Control diet (no FOS, MOS and Bacillus clausii), diet F (5 g kg)1 FOS), diet M (5 g kg)1 MOS), diet FM (2.5 g kg)1 FOS + 2.5 g kg)1 MOS), diet B (107 cells g)1 B clausii), diet FB (5 g kg)1 FOS + 107 cells g)1 B clausii), diet MB (5 g kg)1 MOS + 107 cells g)1 B clausii) and diet FMB (2.5 g kg)1 FOS + 2.5 g kg)1 MOS + 107 cells g)1 B clausii) Data from each dietary treatment are mean SD of triplicate cages Values with different superscripts within a column indicate significant difference (P < 0.05) LSZ, lysozyme (U mL)1); PP, phagocytic percentage (%); PI, phagocytic index; TG, triglyceride (mM); CHO, cholesterol (mM); LDL-C, low-density lipoprotein cholesterol (mM); HDL-C, high-density lipoprotein cholesterol (mM) Aquaculture Nutrition 17; e902e911 ể 2011 Blackwell Publishing Ltd 50 Protease (U g1) 40 35 a ab 45 c bc Control F bc bc bc M FM bc 30 25 20 15 10 B FB 0.35 Amylase (U g1) 0.3 b ab ab b MB ab ab FB MB FMB a b 0.25 0.2 0.15 0.1 0.05 Control F M FM B FMB Figure Activities of protease and amylase in intestine of Japanese ounder fed the experimental diets FOS, fructo oligosaccharides; MOS, mannan oligosaccharides Control diet (no FOS, MOS and Bacillus clausii), diet F (5 g kg)1 FOS), diet M (5 g kg)1 MOS), diet FM (2.5 g kg)1 FOS + 2.5 g kg)1 MOS), diet B (107 cells g)1 B clausii), diet FB (5 g kg)1 FOS + 107 cells g)1 B clausii), diet MB (5 g kg)1 MOS + 107 cells g)1 B clausii) and diet FMB (2.5 g kg)1 FOS + 2.5 g kg)1 MOS + 107 cells g)1 B clausii) Data from each dietary treatment are mean SD of triplicate cages Bars bearing dierent superscripts indicate signicant dierence (P < 0.05) observations and those of Grisdale-Helland et al (2008) that demonstrated that feeding MOS led to improved feed eciency, whereas feeding FOS did not when compared with feeding the control diet There is a paucity of research regarding dierences in growth performance between prebiotics as well as between prebiotics and synbiotics In a comparison between FOS and MOS eects, Buentello et al (2010) revealed that sh fed the MOS diet exhibited a better feed eciency FOS and MOS are oligosaccharides that display dierent chemical structures They may help promote gut health of the host in dierent ways (Denev et al 2009), which result in dierences in growth performance However, we did not observe a signicant improvement in feed eciency in sh fed diets supplemented with FOS and MOS No feed eciency response to B clausii alone was observed in this study, which is supported by some previous studies (Hidalgo et al 2006; Aly et al 2008; Sun et al 2010; Wang 2011) but not others (Bagheri et al 2008; Ghosh et al 2008; Merrield et al 2010b) In this study, sh fed diets supple- mented with MOS + B clausii and FOS + MOS + B clausii displayed a higher WGR Furthermore, FOS + B clausii-fed sh exhibited a better feed eciency than those fed FOS alone Feeding FOS, MOS or B clausii alone, or in various combinations, improved WGR and feed eciency, which was more pronounced in sh fed the synbiotics than those fed pre- and probiotics alone Similar synergistic eects were observed in studies with MOS + Enterococcus faecalis fed rainbow trout (Oncorhynchus mykiss) (RodriguesEstrada et al 2009) and symbiotic-fed (isomaltooligosaccharides + Bacillus OJ) Pacic white shrimp (Litopenaeus vannamei) (Li et al 2009) Taken together, our results, in combination with previously published work, demonstrated that FOS, MOS and B clausii dietary supplements, alone or in combination, inconsistently aected the growth performance of farmed sh The positive eects of pre- or probiotics on WGR and/or feed eciency may be associated with improved nutrient digestibility, which could be a result of a promotion of the digestive enzymes that would allow the host to degrade more nutrients De Schrijver & Ollevier (2000) observed increased protein degradation in the proximal intestine of turbot (Scophthalmus maximus) when fed a Vibrio proteolyticus diet Enhanced digestive enzyme activity in sh fed a pre- or probiotics diet was reported in multiple studies (Wang & Xu 2006; Ghosh et al 2008; Suzer et al 2008; Wang 2011), which was inconsistent with our current results However, our study illustrated that protease and amylase activities tended to be enhanced by diets supplemented with FOS + B clausii, MOS + B clausii and FOS + MOS + B clausii, with the diet containing FOS + MOS + B clausii eliciting the strongest activities The eects of pre- and/or probiotics on protease were larger than on amylase activity Additionally, the enhanced protease activities in sh fed diets supplemented with MOS + B clausii and FOS + MOS + B clausii also promoted WGR; however, digestive enzyme activity did not exhaustively mimic feed eciency Diets MB and FMB signicantly promoted protease activity, whereas diets M, FM, FB, MB and FMB improved feed eciency To our knowledge, the microvilli structures of the gut were improved when sh were fed FOS, MOS and probiotic-treated diets (Yilmaz et al 2007) Therefore, an improvement in gut morphology, resulting in better absorption of nutrients in gut digesta, may account for the improved feed utilization in the sh fed FOS, MOS and B clausii-containing diets in this study Dietary MOS administration has been demonstrated to increase body protein deposition in rainbow trout (Yilmaz et al 2007) and hybrid tilapia (Oreochromis niloticus ã O aureus; Aquaculture Nutrition 17; e902e911 ể 2011 Blackwell Publishing Ltd Gencá et al 2007) but to lower body protein deposition in Atlantic salmon (Salmo salar; Grisdale-Helland et al 2008) In contrast, we did not observe an eect on body protein deposition in MOS-fed Japanese ounders Additionally, feeding FOS did not aect body protein deposition in the current study and in Atlantic salmon of another study (Grisdale-Helland et al 2008) Although Bacillus subtilis dietary supplementation has been shown to increase body protein deposition in Nile tilapia (O niloticus; EL-Haroun et al 2006), rainbow trout (Bagheri et al 2008) and in all the ornamental shes (Poecilia reticulata, Poecilia sphenops, Xiphophorus helleri and Xiphophorus maculatus; Ghosh et al 2008), we did not observe this eect with sh fed B clausii Signicantly higher body protein deposition was observed in sh fed the diets supplemented with FOS + B clausii and FOS + MOS + B clausii compared to diets with FOS or FOS + MOS only FOS, MOS and FOS + MOS-supplemented diets aected body lipid deposition similarly to the control diet, which was also reported in Atlantic salmon (Grisdale-Helland et al 2008) However, feeding B clausii led to decreased body lipid deposition, which was consistent with what has been reported in Nile tilapia (EL-Haroun et al 2006), rainbow trout (Bagheri et al 2008) and all the ornamental shes (Ghosh et al 2008) The addition of prebiotics to a B clausii-supplemented diet did not further decrease body lipid deposition FOS, MOS and B clausii dietary supplementations did not aect body dry matter and ash deposition, which is supported by the results of Grisdale-Helland et al (2008) but contradicted by Ghosh et al (2008), who reported an increase in sh body ash deposition Probiotics and prebiotics stimulate the host immune system (Dimitroglou et al 2011) The variance in their immunomodulatory eects in animals is attributed to the origin or form of the pro- or prebiotics In this study, FOS, MOS and FOS + MOS dietary supplementations did not alter LSZ activity compared with control Similar results have been reported with European sea bass (Dicentrarchus labrax; Torrecillas et al 2007) and Atlantic salmon (Grisdale-Helland et al 2008) However, other studies have reported an increase in LSZ activity when diets were supplemented with only FOS or MOS (Staykov et al 2007; Buentello et al 2010) In contrast, LSZ activity was lower in sh fed a MOS diet than in sh fed the control diet (Grisdale-Helland et al 2008) In this study, feeding B clausii promoted LSZ activity, which is similar to what has been reported in rainbow trout (Newaj-Fyzul et al 2007; Merrield et al 2010b), Nile tilapia (Aly et al 2008) and grouper (Sun et al 2010) Moreover, sh fed a symbiotic diet (FOS + B clausii, MOS + B clausii or FOS + MOS + B clausii) exhibited Aquaculture Nutrition 17; e902e911 ể 2011 Blackwell Publishing Ltd signicantly higher LSZ activity than sh fed diets supplemented with the individual prebiotics and tended to enhance activity compared to those fed the B clausii diet, which suggests that the dietary administration of FOS or/and MOS combined with B clausii synergistically modulates LSZ activity We did not observe an eect on phagocytic activity in the Japanese ounder when diets were supplemented with FOS, MOS or B clausii alone or in combination In contrast, dietary MOS incorporation at g kg)1 improved phagocytic activity in European sea bass (Torrecillas et al 2007) and rainbow trout (Rodrigues-Estrada et al 2009) Dietary administration of Bacillus has also been reported to enhance phagocytic activity in gilthead sea bream (Sparus aurata; Salinas et al 2005), rainbow trout (Newaj-Fyzul et al 2007) and grouper (Sun et al 2010) Single dietary administration of FOS, MOS or probiotics has been shown to induce a hypocholesterolaemic eect in terrestrial animals and humans, wherein the supplement reduced TG, CHO and/or LDL-C levels and increased HDL-C levels (Yamashita et al 1984; Fiordaliso et al 1995; Yalcáinkaya et al 2008; Wang et al 2009) There has been a lack of adequate research investigating the eects of dietary pre- and probiotics administration on lipid metabolism in sh In this study, both TG and LDL-C levels were predominantly lower in sh fed the pre- and/or probiotic-containing diets compared to those fed the control diet However, we did not observe dierences in CHO and HDL-C levels between supplemented diets and the control, which was partially consistent with what has been demonstrated above in terrestrial animals and humans These phenotypes may be accredited to lipid metabolism alterations in the liver by short-chain fatty acids that improve the gastrointestinal tract environment (Fiordaliso et al 1995; Denev et al 2009) As mentioned previously, pre- and/or probiotics dietary supplements tended to decrease body lipid deposition in the Japanese ounder, which may have resulted from a decrease in TG and LDL-C levels when sh were fed a pre- and/or probiotic-containing diet Contrary to our results, Panigrahi et al (2009) reported elevated TG and CHO levels in Lactobacillus rhamnosus -fed rainbow trout The mechanisms underlying the apparent dierences in lipid metabolism in sh are unclear and 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Inland Fisheries and Aquaculture, Wojska Polskiego 71c, 60-625 Poznan´, Poland E-mail: karp@up.poznan.pl Ó 2010 Blackwell Publishing Ltd Carp are the most widely cultivated fish species in the world In Europe, common carp (Cyprinus carpio L.) is an omnivorous warm water fish that plays an important role in pond aquaculture After the enlargement of the European Union in 20 04, common carp became... ascertain if palatability was a problem at the higher SPI inclusion rates Two groups of fish were used in a randomized complete block cross-over design One group received Aquaculture Nutrition 17; 368–379 Ó 2011 Blackwell Publishing Ltd No claim to original US government works Table 3 Formulation and analysed composition of diets containing SPI substituted for 200, 400, 600, 800 or 1000... the end of the final 2 weeks Feed intake, weight gain and survival were recorded Feed intake was standardized to intake g)1 fish and analysed as mean daily intake period)1 Aquaculture Nutrition 17; 368–379 Ó 2011 Blackwell Publishing Ltd No claim to original US government works Diets and fish samples were dried to a constant weight at 105 °C and finely ground prior to analysis Nitrogen (N)