Aquaculture nutrition, tập 18, số 1, 2012

Aquaculture Nutrition doi: 10.1111/j.1365-2095.2011.00866.x 2012 18; 111 Aquaculture Division, CIFE, Versova, Mumbai, India; National Institute of Basic Biology, NIBB, Okazaki, Japan; Riverine Fisheries Ecology Division, CIFRI, Vadodara, Gujarat, India; Aquatic Environment and Health Management Division, CIFE, Versova, Mumbai, India; Fish Nutrition and Physiology Division, CIFA, Bhubaneswar, India Six iso-nitrogenous (350 g protein kg)1) and iso-caloric (4100 kcal kg)1) diets with or without probiotics supplementation namely T1 (Basal feed (BF) without probiotics; control), T2 (BF + Bacillus subtilis and Lactococcus lactis), T3 (BF + L lactis and Saccharomyces cerevisiae), T4 (BF + B subtilis and S cerevisiae), T5 (BF + B subtilis, L lactis and S cerevisiae) and T6 (BF + heat-killed bacteria of B subtilis, L lactis and S cerevisiae) were fed to Labeo rohita ngerlings (6.0 0.06 g) for 60 days in triplicate tanks (30 sh per tank) In all probiotic-supplemented diets, the probiotic concentration was maintained at 1011 cfu kg)1 feed After 60 days of culture, the sh fed combination of three probiotics at equal proportion (T5) had higher (P < 0.05) growth, protein eciency ratio, nutrient retention and digestibility and lower (P > 0.05) feed conversion ratio over other treatment groups Total heterotrophic bacterial population in intestine was drastically reduced on 15th and 30th days of sampling than the initial value (0 day of sampling) for T3, T4 and T5 groups Except T6, the gut colonization of respective probiotics, which were supplemented through the diets, was also increased up to 30 days of culture of sh and thereafter remained constant KEY WORDS: Bacillus, digestibility, growth, Labeo rohita, Lactococcus, nutrient, probiotics, Saccharomyces Received 26 August 2010, accepted February 2011 Correspondence: Kedar Nath Mohanta, Fish Nutrition and Physiology Division, Central Institute of Freshwater Aquaculture, Bhubaneswar 751002, India E-mail: knmohanta@gmail.com ể 2011 Blackwell Publishing Ltd Sipra Mohapatra, Aquaculture Division, Central Institute of Fisheries Education, Versova, Mumbai 400061, India E-mail: mohapatra_ sipra@redimail.com With ever increasing demand for animal protein, aquaculture is emerging as one of the most viable and promising enterprises for providing nutritional and food security to the human But, most of the intensive aquaculture farms have been facing major hindrance because of disease outbreak, poor growth and low survival of sh To mitigate these problems, sh vaccine and probiotics have been used in aquaculture practices The use of probiotics in aquaculture has not only resulted the reduction of use of harmful antimicrobial compounds, particularly the antibiotics, but also improved the appetite and/or biogrowth performance of the farmed species in an eco-friendly and sustainable manner (Gatesoupe 1999; Naik et al 1999; Robertson et al 2000; Wang et al 2005) One obvious reason for the use of probiotics is that it can be supplemented even at larval and early fry stages (Muroga et al 1987) Several reports suggest that probiotics supplementation can reduce the cost of culture by improving the growth and feed utilization eciency of sh (Swain et al 1996; Bogut et al 1998; Ghosh et al 2003; Carnevali et al 2006; Wang & Xu 2006; Mazurkiewicz et al 2007; Kesarcodi-Watson et al 2008) Among the dierent probiotics that are being used in aquaculture, lactic acid bacteria (LAB) is found to be the most prominent one as it is a part of the natural intestinal microora of a healthy sh (Noh et al 1994; Brunt & Austin 2005; VaÂzquez-JuaÂrez et al 2005; Nayak et al 2007; Wang 2007, 2011; Yin et al 2007; Ramakrishnan et al 2008) It is well known that LAB often produces bacteriocins, which may inhibit the growth of Gram-negative sh pathogens In addition, the use of other microbial groups such as Lactococcus (Hagi et al 2004; Sugita et al 2009), Bacillus sp (Kumar et al 2006, 2008), Lactobacillus (Ramakrishnan et al 2008) and Saccharomyces cerevisiae (Pal et al 2007; Ramakrishnan et al 2008) as probiotics has been reported in carp culture Majority of these probiotics are nonpathogenic and non-toxic and can survive in the gut and remain potent for long period under storage and eld condition (Ramakrishnan et al 2008) Studies carried out by Mohanty et al (1996) indicated that a combination of bacteria and yeasts as probiotics resulted higher survival and better body weight gain and nutrient utilization in catla (Catla catla) Yanbo & Zirong (2006) reported that the survivability, weight gain and nutrient utilization of sh fed probiotic-supplemented diet depend on the type of probiotics used Although several authors have reported that the use of probiotics has great potential for higher sh production especially in carp culture, in most of their studies, the probiotic organisms were used as a single species, either through the diet or through culture environment (Kumar et al 2006) The literature on the use of combinations of two or more probiotic species at the same time in the diet or culture environment and their eect on growth, nutrient utilization and gut microbial population of sh are very limited Therefore, in this study, an attempt has been made to use the three probiotic microorganisms, namely B subtilis, Lactococcus lactis and S cerevisiae at dierent combinations in the diet of Labeo rohita and to evaluate the growth, nutrient utilization, digestive enzymes activities and gastrointestinal colonization of the supplemented probiotics along with the total heterotrophic bacterial (THB) population in the gut Three thousand healthy ngerlings of L rohita (average weight; 4.54 g) procured from Palghar sh farm, Maharashtra, India, were transported to the wet laboratory of Central Institute of Fisheries Education (CIFE), Mumbai As a prophylactic measure, the sh were given a salt treatment (1%) for and then acclimatized to the laboratory conditions for 15 days using ve 500 L capacity ow-through bre-reinforced plastic tanks with the provision of continuous aeration During acclimatization, the sh were fed with a sh meal-based formulated diet (350 g protein kg)1 diet and 17.2 MJ kg)1 dietary gross energy) other than the experimental diets Five hundred and forty uniform-sized healthy sh (average weight; 6.00 0.06 g) were equally distributed in six dietary treatment groups [T1 (Basal feed without probiotics), T2 (Basal feed + B subtilis and L lactis), T3 (Basal feed + L lactis and S cerevisiae), T4 (Basal feed + B subtilis and S cerevisiae), T5 (Basal feed + B subtilis, L lactis and S cerevisiae) and T6 (Basal feed + heat-killed bacteria of B subtilis, L lactis and S cerevisiae)] with three replicates each (stocking density of 30 sh per tank in 300 L of rearing water), following a completely randomized design The 500 L capacity owthrough bre-reinforced tanks (300 L water) with a ow rate of 0.5 L min)1 were used for rearing the sh Seasoned groundwater was used for rearing the sh The natural light cycle was 12 h light/12 h darkness (12 L : 12D) for the entire experimental period Pure strains of B subtilis, L lactis and S cerevisiae were procured from Microbial Type Culture Collection and Gene Bank, Chandigarh, India, and was maintained at C in the laboratory Subsequently, the microbes were inoculated into test tube containing brain heart infusion (BHI), de Man Rogosa and Sharpe (MRS) and yeast extract peptone dextrose (YEPD) broths (Himedia) for B subtilis, L lactis and S cerevisiae, respectively, and kept in incubator for 24 h at 30 C After that, a loopful of microbial culture was streaked on the respective agar media The colonies were conrmed as pure isolates of B subtilis, L lactis and S cerevisiae by performing the essential biochemical tests, and the cultures were used for mass culture for subsequent use in the experiment For mass culture, freshly grown pure inocula of B subtilis, L lactis and S cerevisiae were added to 100 mL of BHI, MRS and YEPD medium, respectively, in a 500mL conical ask and incubated at 30 C for 24 h in a shaking incubator The cultures were centrifuged at 800 g for 15 at C The supernatant was discarded, while the pellets were resuspended in phosphate buer saline (pH 7.2) The microbial pellets were washed and centrifuged similarly and then mixed in phosphate buer saline at dierent concentrations as required and added to 100 g of feed Aquaculture Nutrition 18; 111 ể 2011 Blackwell Publishing Ltd Table Composition of the ingredients used for formulating the experimental diets To determine the concentrations of the microbial probiotic inoculums to be added into the feed for the experiments, all the three probiotic microorganisms, namely B subtilis, L lactis and S cerevisiae, were streaked on BHI, MRS, YEPD plates, respectively, and incubated for 12 h at 30 C One freshly grown colony was picked up and transferred into 50 mL of respective broth and incubated under the same conditions for h A third transfer for each bacteria and yeast was carried out into 100 mL under same conditions Then, optical density (O.D) of the microbial samples was recorded at 600 nm Simultaneously, the serial dilutions were performed for each hour The dilutions were plated onto the respective agar by spread plate technique After 12 h of incubation at 28 C, the colonies were counted The data were related in graphs, obtaining the relationship cfu versus OD600 versus time Based on this, the required probiotic microorganisms were added to the feed at dierent concentrations To prepare the experimental feeds, the required ingredients such as sh meal (sun-dried miscellaneous marine trash sh, mainly the lesser sardines of family Engrolidae and ribbon sh of family Trichuridae available in the locality, neither solvent extracted nor dehulled), soybean oil cake (deoiled soybean; mechanical extraction of oil), rice polish (obtained from the local rice mill), wheat bran (the bran obtained from the wheat meal) and corn our (commercially available) were purchased from the local market (Mumbai, India) The ingredients were dried overnight at 80 C in a hot air oven and powdered by means of a California feed mill The powdered ingredients were sieved through a ne-meshed screen (0.5 mm diameter) Before formulating the experimental diets, the proximate compositions of the feed ingredients were determined (AOAC 1990) and presented in Table Six iso-nitrogenous (350 g protein kg)1 diet) and isocaloric (17.217.3 MJ kg)1 diet) experimental diets were prepared incorporating various combinations of probiotics added at equal proportions (either : or : : depending on two or three numbers of probiotics used at a time) to make the nal concentration of 1011 cfu kg)1 diet except for the control (Table 2) The required feed ingredients apart from probiotic microbes, vitamins and minerals were mixed with carboxymethyl cellulose, water and oil to make a dough and steam cooked for 20 in an autoclave at 15 psi After Aquaculture Nutrition 18; 111 ể 2011 Blackwell Publishing Ltd Chemical composition (g kg)1 on dry matter basis) Ingredients Fish meal Soybean oil cake Rice polish Wheat bran Corn flour Crude Fibre Gross energy (MJ kg)1) Dry matter Crude protein Ether extract 926 918 640.0 520.0 80.0 62.0 9.2 72.0 86.0 64.0 24.45 19.57 921 928 932 130.0 124.0 100.0 12.0 19.0 6.0 119.0 106.0 62.0 121.0 72.0 59.0 14.16 15.34 15.88 Ash cooling, the vitamins and minerals and the respective probiotic microorganisms were added Finally, the dough was pressed through a hand pelletizer to get uniform size pellets (2 mm) and kept overnight in a hot air oven (45 C) The commercially available kitchen type hand-operated pelletizer was used for preparing the experimental diets The prepared feeds were stored at C until used for better shelf life Fresh feeds were prepared in every 15 days interval to maintain the bacterial count at desired level All the treatment groups of sh were fed ad libitum to a level close to apparent satiation at 08:00, 12:00, 15:00 and 18:00 h (Mohanta et al 2009; Mohapatra et al 2010) The experiment lasted for 60 days Apparent nutrient digestibility coecients of the diets were determined by indirect method using 10 g chromic oxide (Cr2O3) kg)1 of diet (Gomes et al 1995) in the expense of wheat bran during the last 30 days of the experiment The unconsumed feed and faeces were removed h after the rst two feedings of the day at 09:00 and 13:00 h, and then, the freshly voided faeces were collected after h The faeces of the initial days were discarded, and the next 25-day samples were collected following the immediate pipetting method outlined by Spyridakis et al (1989) Pooled faecal samples of each treatment were dried at 55 C and stored at )20 C for subsequent analysis (Mohanta et al 2009) The digestibility of dry matter (DM) expressed as a percentage was calculated using the following formula: Apparent digestibility coefficient of dry matter (ADCDM ị ẳ 100 %marker in feed=% marker in faeces 100ị The apparent nutrient (protein and lipid) digestibility ẳ 100 ẵ%marker in feed % marker infaeces) %nutrients in faeces=%nutrient in feedị 100: Table Composition of experimental diets (g kg)1 on dry matter basis) Dietary treatments Ingredients T1 T2 T3 T4 T5 T6 )1 Ingredients (g kg ) Fish meal1 114 114 114 114 114 114 Soybean oil cake1 420 420 420 420 420 420 Rice polish1 130 130 130 130 130 130 Wheat bran1 110 110 110 110 110 110 Corn flour (Himedia) 100 100 100 100 100 100 Carboxymethyl Cellulose (Himedia) 10 10 10 10 10 10 Sunflower oil2 : Cod liver oil (1 : 1)3 80 80 80 80 80 80 Vitamin mineral mix4 30 30 30 30 30 30 Vitamin C (Sd-fine Chem., Mumbai, India) 1 1 1 Vitamin B complex (Sd-fine Chem., Mumbai, India) 1 1 1 BHT (Himedia) 2 2 2 Glycine (Himedia) 2 2 2 Proximate composition (g kg)1) Moisture 56.8 0.3 61.8 0.3 57.7 0.4 61.5 0.1 59.5 0.4 58.2 0.1 Crude protein 351 0.2 351 1.1 349 2.4 348 1.2 349 1.9 352 3.9 Total carbohydrate 466.1 4.4 466.4 6.8 470.5 3.4 472.8 1.3 471.6 8.5 468.8 1.3 Ether extract 96.4 1.2 95.9 1.6 92.9 1.0 91.7 6.0 92.3 8.0 91.6 4.0 Total ash 86.5 3.4 86.7 4.2 87.6 1.2 87.5 5.0 87.1 2.1 87.6 2.6 Energy (MJ kg)1) 17.3 0.02 17.3 0.03 17.3 0.02 17.2 0.01 17.3 0.04 17.3 0.01 Purchased from local dealers, Mumbai, India Marico Industries Limited, Mumbai, India Universal Medicare Private Limited, Mumbai, India Vitamin mineral mix (EMIX PLUS, Mumbai, India) (Quantity per kg) Vitamin A: 22 00 000 IU; Vitamin D3: 40 000 IU; Vitamin B2: 800 mg; Vitamin E: 300 mg; Vitamin K: 400 mg; Vitamin B6: 400 mg; Vitamin B12: 2.4 mg; Calcium Pantothenate: 1000 mg; Nicotinamide: g; Choline Chloride: 60 g; Mn: 10 800 mg; I: 400 mg; Fe: 3000 mg; Zn: 2000 mg; Cu: 800 mg; Co: 180 mg; Ca: 200 g; P: 120 g; L-lysine: g; DL-Methionine: g; Selenium: 20 ppm The proximate composition of experimental diets, faecal samples and whole body was analysed in triplicates (AOAC 1990) DM was estimated by oven drying the samples at 105 C till a constant weight and crude protein per cent were calculated by estimating nitrogen content by micro-Kjeldahl method and multiplying with a factor 6.25 Ether extract was determined by solvent extraction with petroleum ether, boiling point 4060 C, for 1012 h Total ash content was determined by incinerating the sample at 650 C for h and crude bre by acid digestion (1.25%) followed by alkali digestion (1.25%) Gross energy in diets, faecal samples and sh body was calculated by using Bomb Calorimeter (Parr, model 1341; Parr Instrument Company, Moline, IL, USA) The Cr2O3 content of the feed and faecal samples was determined as described by Furukawa & Tsukahara (1966) The total carbohydrate (%) of the feed and whole body sh was calculated as: 100 ) (Crude protein + Ether extract + Total ash) The water quality parameters were found to be in the range of temperature (25.626.4 C), pH (7.47.6), dissolved oxygen (5.86.9 mg L)1), free carbon dioxide (1.92.7 mg L)1), total hardness (156185 mg L)1), ammonia-N (0.040.07 mg L)1), nitrite-N (0.060.1 mg L)1) and nitrate-N (0.030.14 mg l)1) throughout the experiment period (APHA-AWWA-WEF 1998) While the water temperature was recorded twice daily at 0600 and 1430 h, the other parameters were analysed in every 15-day interval All the water quality parameters during the entire experiment period were found to be in the optimum range of sh rearing (Debnath et al 2007; Kumar et al 2010; Mohapatra et al 2010) The growth parameters of the L rohita ngerlings were assessed in terms of weight gain per cent, specic growth rate (SGR), feed conversion ratio (FCR), protein eciency ratio (PER), protein retention eciency, lipid productive value Aquaculture Nutrition 18; 111 ể 2011 Blackwell Publishing Ltd (LPV) and energy productive value (EPV) at the end of the experiment The experimental sh with respect to each replicate was batch weighed in every 15 days to know the body weight of sh The weight gain (%), SGR, FCR, PER, PPV, LPV and EPV were evaluated based on standard formulae as follows: Weight gain% ẳ Final weight Initial weightị= Initial weight) 100 Specific growth rate (SGR) ẳ 100loge average Final weight loge average Initial weightị= Number of culture days Feed conversion ratio (FCR) ẳ Total dry feed intake (g)= Wet weight gain (g) Protein efficiency ratio (PER) ẳ Total wet weight gain (g)= Protein fed (g) Nutrients (protein and lipid) and energy productive values (PPV, LPV and EPVị ẳ Nutrients or energy gain in body=Nutrients or energy intake Tissue homogenates of intestine were prepared with chilled sucrose solution (0.25 M), and the dierent digestive enzymes were analysed using standard procedures as described below The intestines used for digestive enzyme assay with respect to each dietary treatment were in fresh condition Protease activity was determined by the casein digestion method as Drapeau (1974) One unit of enzyme activity was dened as the amount of enzyme need to release acid-soluble fragments equivalent to 0.001A280 per minute at 37 C and pH 7.8 The lipase activity was assayed as described by Cherry & Crandall (1932) with suitable modication (Debnath et al 2007) The volume (mL) of N/20 NaOH solution required for 100 mg intestinal tissue in the experimental tube minus the volume (mL) of N/20 NaOH solution required for the same amount of intestinal tissue in the control tube represented the units of intestinal lipase activity per g tissue One unit will hydrolyse 1.0 microequivalent of fatty acid from a triglyceride in 24 h at pH 7.7 at 37 C The gastrointestinal microora analysis was carried out on 0, 15th, 30th, 45th and 60th day of feeding trial Three shes Aquaculture Nutrition 18; 111 ể 2011 Blackwell Publishing Ltd were randomly selected from each treatment (i.e one sh from each replicate group) and collected in sterile plastic bags The shes were starved for 20 h before the sampling The fresh intestine was aseptically taken out from each of these sh The intestine of all replicates of a treatment was weighed equally to make 1.0 g of sample Sample was transferred into tubes containing 9.0 mL sterile 0.85% NaCl and crushed in a homogenizer The homogenates were serially diluted up to 10)6 Nutrient agar was used for THB count Nutrient agar media, MRS media and YEPD media were used for B subtilis, L lactis and S cerevisiae, respectively In spread plate technique, 0.1 mL from highest dilution (10)6) was properly spread on the media, and the plates were incubated at 28 C for 24 h in Biological Oxygen Demand (BOD) incubator On the following day, colonies were counted and were isolated for characterization Numbers of colonies reported in the present study are an average of three replicate plates Isolated colonies (at least 10 per plate) were used for identication by morphological and biochemical tests (Bergy 1986; Guimaraes et al 2006) Each probiotic colony was counted separately and deduced from initial count to get per cent increase in colonization Comparison among all the treatments was carried out by oneway ANOVA followed by Duncanếs multiple range test Comparison was made at the 5% probability levels The data were statistically analysed by statistical package SPSS, version 16.0 In our study, we fed the shes with dierent combinations of three probiotics, and the various growth parameters, i.e weight gain, SGR, FCR, PER, PPV, LPV and EPV, were determined The growth data were substantiated by analysis of dierent digestive enzyme activities The colonization of respective probiotics in the gastrointestinal tract was analysed so as to study the eect of probiotics on growth and nutrient utilization Growth and nutrient utilization of sh are the two basic criteria that determine the production, productivity and protability of the sh culture operation The growth and nutrient utilization obtained from this study are presented in Table At the end of the experiment, it was found that the weight gain (%) was increased signicantly (P < 0.05) in all probioticsupplemented groups (except T6) than that of control with a highest value recorded in T5 (232.16) group followed by T4 (171.3), T3 (167.3) and T2 (166.3) groups Similar results were also obtained for SGR and PER The T5 group had signicantly higher (P < 0.05) SGR (0.87) and PER (1.52) values than the other groups (0.610.72 and 0.801.13, respectively) The PPV (0.15), LPV (0.28) and EPV (0.61) of T5 group of sh were also signicantly higher (P < 0.05) than the other probiotic-fed groups (0.120.13, 0.130.19, 0.340.41, respectively) Signicantly lower (P < 0.5) FCR (1.93) was also recorded in T5 group of sh as compared to other groups (2.52 3.18) However, the shes fed heat-inactivated probiotics did not show any remarkable growth and nutrient gain over the other probiotic-supplemented groups groups Similar observations were also recorded for lipase activity (Table 3) The intestinal microora of sh reects the bacterial content of ingested food and of the environment It also speculates the competitive exclusion of harmful gut microora To emphasize the probiotic colonization leading to unwanted microbial exclusion, we analysed the THB population in experimental sh gut The THB population in the intestine of L rohita ngerlings at dierent sampling days is presented in Table The THB population in the intestine was found to be signicantly higher (P < 0.05) in all sampling days (15, 30, 45 and 60th day) in T1 (control) and T6 groups compared to other treatment groups with a minimum value (2.39 cfu ã 1010 kg)1) observed in T3 on 30th day For the T3, T4 and T5 groups, on 15th and 30th day, the THB population was also drastically reduced from the initial value (0 day of sampling) and thereafter remained almost similar on 45 and 60th day of sampling But for T1 (control) and T6 groups, the THB population remained almost same for all sampling days The apparent digestibility coecient of DM (ADCDM) and protein (ADCProtein) was found to be signicantly dierent (P < 0.05) among treatment groups with highest being in T5 (52.3% for DM and 86.2% for protein) However, there was no signicant (P > 0.05) eect of probiotic feeding on apparent lipid digestibility (ADC Lipid; 73.1375.31%) in L rohita ngerlings (Table 3) To substantiate the fact that the dierence in THB is related to probiotic colonization in gut, we determined the per cent prevalence of dierent administered probiotics in the gut of L rohita ngerlings and presented in Table On feeding The protease activity of T2, T3, T4 and T5 groups was signicantly higher (P < 0.05) than the control (T1) and T6 Table Effect of different dietary probiotic supplementation on growth parameters, apparent digestibility coefcients (ADC) and digestive enzyme activities in Labeo rohita ngerlings Dietary treatment Parameter studied T1 Weight gain (%) SGR FCR PER PPV LPV EPV ADCDM ADCProtein ADCLipid Protease1 Lipase2 131.27d 0.61c 3.18a 0.80c 0.12c 0.17b 0.37b 43.62c 79.76c 73.13 0.12b 0.25b T2 7.19 0.02 0.25 0.05 0.002 0.01 0.07 1.95 0.28 0.15 0.01 0.02 166.30bc 0.71b 2.52ab 1.13b 0.13bc 0.15bc 0.40b 50.68ab 82.98b 74.92 0.20a 0.62a T3 9.39 0.03 0.08 0.04 0.01 0.00 0.05 1.27 0.46 0.39 0.03 0.17 167.35bc 0.71b 2.89a 1.01bc 0.13b 0.19b 0.41b 48.6b 83.67b 73.89 0.22a 0.68a T4 9.98 0.03 0.30 0.11 0.00 0.02 0.08 1.75 0.46 1.45 0.01 0.12 171.32b 0.72b 2.63a 1.13b 0.12bc 0.16bc 0.41b 47.22b 85.72a 74.83 0.21a 0.51a T5 6.20 0.02 0.20 0.08 0.00 0.01 0.07 1.47 0.30 1.38 0.02 0.07 232.16a 0.87a 1.93b 1.52a 0.15a 0.28a 0.61a 52.26a 86.21a 75.31 0.21a 0.66a P value T6 15.31 0.03 0.19 0.14 0.01 0.02 0.10 1.91 1.07 0.60 0.02 0.11 138.69cd 0.63c 2.96a 0.98bc 0.13bc 0.13c 0.34b 46.98b 80.67c 74.48 0.11b 0.29b 4.81 0.01 0.10 0.03 0.01 0.01 0.05 1.14 0.21 0.77 0.04 0.06 0.000 0.000 0.012 0.012 0.008 0.000 0.000 0.020 0.000 0.321 0.017 0.036 Data expressed as Mean SE (n = 3) Mean values in same row with different superscripts vary significantly (P < 0.05) Protease activity expressed as units per mg protein per Lipase activity expressed as units per mg protein per hour EPV, energy productive value; FCR, feed conversion ratio; LPV, lipid productive value; PER, protein efficiency ratio; SGR, specific growth rate Aquaculture Nutrition 18; 111 ể 2011 Blackwell Publishing Ltd Table Total heterotrophic bacteria (THB) population (cfu ã 1010 kg)1) in the intestine of Labeo rohita ngerlings at various sampling days in dierent dietary probiotic-supplemented groups Sampling days Treatment 0th day 15th day 30th day 45th day 60th day T1 T2 T3 T4 T5 T6 P value 4.54 4.44 4.36 4.63 4.61 4.49 0.06 4.37a 3.59b 3.40b 3.51b 3.37b 4.04a 0.004 4.29a 3.40c 2.39d 2.54d 2.44d 3.83b 0.000 4.25a 3.28b 2.71c 2.53d 2.42d 4.16a 0.026 4.59a 3.70c 2.83d 2.42e 2.48e 4.27b 0.000 0.19 0.06 0.11 0.09 0.08 0.04 0.16 0.12 0.14 0.09 0.09 0.06 0.17 0.17 0.11 0.08 0.06 0.07 0.06 0.01 0.09 0.08 0.07 0.04 0.12 0.10 0.08 0.07 0.10 0.06 Data expressed as Mean SE (n = 3), Unit- cfu ã 1010 kg)1 Mean values in same column with different superscripts vary significantly (P < 0.05) Table Percentage prevalence of Bacillus, Lactococcus and Saccharomyces species isolates found in the intestine of Labeo rohita ngerlings at various sampling days in dierent dietary probioticsupplemented groups Sampling days 0th day 15th day 30th day 45th day 60th day Bacillus sp Lactococcus sp Saccharomyces sp 1.9 3.0 ND 2.0 2.8 ND 2.2 3.3 ND 2.1 3.4 ND 2.2 3.5 ND Bacillus sp Lactococcus sp Saccharomyces sp 2.1 2.7 ND 16.9 18.9 ND 24.2 27.0 ND 24.2 26.9 ND 24.4 27.1 ND Bacillus sp Lactococcus sp Saccharomyces sp 2.3 3.2 ND 2.3 19.4 17.9 2.5 27.0 24.9 2.5 26.1 25.9 2.6 27.0 26 Bacillus sp Lactococcus sp Saccharomyces sp 2.2 3.1 ND 19.4 3.0 18.4 27.2 3.1 25.3 27 3.2 25.9 27.3 3.4 26.0 Bacillus sp Lactococcus sp Saccharomyces sp 2.1 2.8 ND 18.9 17.2 18.1 28.1 25.3 26.7 28.8 25.9 27 29.1 26.2 27.2 Bacillus sp Lactococcus sp Saccharomyces sp 2.2 3.3 ND 5.4 4.9 5.1 6.7 6.1 6.3 6.6 6.1 6.3 6.7 6.4 6.6 Treatment T1 T2 T3 T4 T5 T6 The percentage prevalence of probiotic microbial species in the intestine is calculated by taking three fish per treatment (one fish per replicate) in each sampling days dierent probiotics, it was observed that the specic microbial probiotics that supplemented through diets were established at much higher percentage in the gut of sh in all probiotic-fed groups (except T6 group) (Table 5) Although the increase was observed from the 15th day of feeding, the maximum gut colonization was observed on the 30th day, and thereafter, it almost remained constant Aquaculture Nutrition 18; 111 ể 2011 Blackwell Publishing Ltd In the present study, except T6, we obtained 150200% increase in weight of sh in dierent probiotic-fed groups, which was signicantly higher (P < 0.05) than the sh fed diets without probiotic supplementation (T1) This is in agreement with the earlier ndings by dierent authors (Swain et al 1996; Ghosh et al 2003; Carnevali et al 2006) Jafaryan et al (2008) also reported that the probiotic (Bacillus)-supplemented diet signicantly increased the weight, length and SGR of sh than the control diet without probiotic supplementation Although we obtained higher SGR in probiotic-supplemented groups (T2, T3, T4 and T5), not much literature is available regarding the eect of different combinations of probiotics on SGR to compare our results According to Noh et al (1994) and Bogut et al (1998), dierent probiotics act dierently to enhance the growth and nutrient utilization of various sh species We also obtained similar observations in our study Noh et al (1994) studied the eect of supplementing yeast (S cerevisiae) and bacteria (Streptococcus faecium) in the diet of Israeli carp and reported the better growth response of sh fed probiotic-supplemented diets than the diet without probiotic supplementation But they found better growth and nutrient utilization in a bacterium-supplemented diet than yeast The better food conversion (FCR), nutrient utilization (PER) and protein gain (PPV) obtained in our study in the probiotic-fed groups (except T6) as compared to control are in agreement with Bagheri et al (2008) reported for rainbow trout (Onchorhynchus mykiss) fed diet supplemented with probiotics The higher PPV, LPV and EPV values indicate better nutrient and energy retention in T5 group than the other groups (Tables and 6) This may be because of an active probiotic supplementation eect in T5 as reported by ElDakar et al (2007) Therefore, supplementation of three live probiotic microorganisms in the diet at equal proportions (1 : : 1) improves food conversion and nutrient retention, Table Whole body chemical composition (g 100 g)1 on dry matter basis) of sh in dierent probiotic-supplemented groups Dietary treatments Parameters T1 Moisture Crude protein Ether extract Total ash Total carbohydrate Energy (MJ 100g)1) 75.6 56.60b 13.54ab 19.06a 10.80c 18.91a T2 0.4 0.74 1.81 1.20 0.74 0.33 76.4 61.71a 9.04b 16.02b 13.23a 17.86b T3 0.1 0.40 1.31 0.86 0.34 0.13 T4 75.9 58.86b 11.41b 18.75a 10.98c 18.49ab 0.1 0.85 2.00 1.29 0.24 0.29 76.3 60.57a 11.03b 16.14b 12.26b 18.06b T5 0.1 0.57 0.37 0.90 0.12 0.19 76.2 56.73b 11.96b 18.91a 12.40b 18.62a T6 0.2 0.86 1.92 1.24 0.71 0.32 76.1 57.42b 15.60a 16.08b 10.90c 18.28b 0.1 1.32 1.64 0.88 0.27 0.28 Data expressed as Mean SE (n = 3) Mean values in same row with different superscripts vary significantly (P < 0.05) hence leading to higher growth of sh However, unlike Salinas et al (2008), we could not observe any growth and nutritional gain in sh by supplementing heat-inactivated probiotics in feed (T6) It is reported that the digestive organs are very sensitive to food composition and cause immediate changes in activities of the digestive enzymes (Bolasina et al 2006; Shan et al 2008), which is nally reected in sh health and growth Moreover, bacteria also secrete proteases to digest the peptide bonds in proteins and therefore break down the proteins into their constituent monomers and free amino acids, which can benet the nutritional status of the animal (MacFarlane & Cummings 1991) In the present work, except T6 (supplementation of heat-killed probiotics of B subtilis, L lactis and S cerevisiae), we observed a high protease activity in other probiotic-fed groups in relation to control (T1) Bacterial enzymatic hydrolysis has been shown to enhance the bioavailability of protein and fat (Ling & Hanninen 1992), which may result in higher growth and nutrient utilization as observed in the present study Amylase and lipase are the major enzymes related to carbohydrate and fat digestion, respectively From our results, it was observed that the lipase activity was much higher in sh fed diets supplemented with live probiotics (T2, T3, T4 and T5) than that of control (T1, diet without probiotic supplementation) and sh fed diet supplemented with heat-killed probiotics (T6), which might have resulted comparatively better growth and other growth-related parameters in all live probiotic-supplemented groups It is reported by the earlier workers that the increase in nutrient digestibility may be because of better availability of exoenzymes produced by probiotics (Vine et al 2006) or better health condition (Yanbo & Zirong 2006) when probiotic-supplemented diets are fed to the sh Bairagi et al (2002) reported that the Bacillus species isolated from the gut of Cyprinus carpio were found to have high amount of extracellular amylolytic, proteolytic and lipolytic activity Majority of probiotics are capable of secreting lipase, which triggers production and assimilation of essential fatty acids resulting higher growth and immunity in sh Feed supplementation of essential fatty acid not only boosts the immunity but also triggers the growth (Sharma et al 2009) The present data also conrm similar hypothesis The data clearly indicate that addition of probiotics signicantly increases lipase activity irrespective of species or combination of probiotics, which corroborates the ndings of Yanbo & Zirong (2006) But the exogenous enzymes produced by the probiotics represent only a small contribution to the total enzyme activity of the gut (Ding et al 2004; Ziaei-Nejad et al 2006; Zhang et al 2010) It suggests that the higher digestive enzyme activities (protease and lipase) obtained in the probiotic-supplemented sh (except T6) are mainly the outcome of stimulation by probiotic itself or exogenous enzyme produced to synthesize endogenous digestive enzyme which might have improved nutrient digestibility leading to better growth performance and feed eciency in sh Similar observations have also been reported for other shes in which the nutrient digestibility increased considerably with the use of probiotic-supplemented diet (Lara-Flores et al 2003; Yanbo & Zirong 2006) Although we did not nd any signicant variation in apparent lipid digestibility (ADCLipid), there was signicant increase in the apparent protein digestibility (ADCProtein) in probiotic fed groups The increased protease activity in probiotic-supplemented diet groups might have resulted better protein digestion and hence better growth and protein gain in sh Similar to Bagheri et al (2008), we also observed signicant reduction in THB counts when probiotic-supplemented diets were fed to the sh, which might have resulted better health and immunity of sh and hence more growth It is reported that the colonization rate of bacteria in the digestive tracts depends on the dietary bacteria level (Bagheri et al 2008) In our study, the higher degree of adhesion of specic microbes that are supplemented through diets may be the reason for enhanced growth and nutrient utilization of sh Aquaculture Nutrition 18; 111 ể 2011 Blackwell Publishing Ltd Several reports suggest that most of probiotics exert their eect through colonization in host and excretion of several growth-enhancing nutrients (Ahilan et al 2004; Bagheri et al 2008) The lower colonization in the heat-killed probiotic treatment group can be attributed to the loss of useful characteristics particularly the colonization capacity of the microbes because of the higher temperature at which it was killed This may resulted the low dietary performance of shfed heat-killed microbes as probiotics In the present work, diet supplemented with two species of bacteria (L lactis and B subtilis) and one species of yeast (S cerevisiae) in equal proportion (T5) as probiotics at the concentration of 1011 cfu kg)1 of feed showed maximum growth and dietary performance in L rohita ngerlings than the other probiotic combinations tested in the diets This indicated that combination of more probiotic organisms in the diet results better performance in sh It may be attributed to better food conversion (FCR), higher protein digestion (ADC for protein), utilization (PER) and gain (PPV), signicant reduction in THB counts in the intestine and better adhesion/establishment of probiotic microora in the gut Similar results were also obtained from Tilapia nilotica (Lara-Flores et al 2003), L rohita (Ghosh et al 2003), C carpio (Yanbo & Zirong 2006; Ramakrishnan et al 2008) and Fenneropenaeus indicus (Ziaei-Nejad et al 2006) However, we observed that the use of same combinations of heatinactivated probiotics (T6) in the diet failed to enhance growth, nutrient digestion and utilization and digestive enzyme activity The study results suggest that the live probiotic microorganisms may be incorporated while formulating the cost-eective nutritionally balanced diet of carp for its better growth performance and nutrient utilization The authors acknowledge Indian Council of Agricultural Research (ICAR), New Delhi for nancial support and Director, CIFE, Mumbai for providing all the necessary facilities to carry out this research programme Ahilan, B., Shine, G & Santhanam, R (2004) Inuence of probiotics on the growth and gut microbial load of juvenile goldsh (Carassius auratus) Asian Fish Sci., 17, 271278 AOAC (1990) Ocial Methods of Analysis of the Association of Ocial Analytical Chemists, Vol 1, 14th edn 1102 p Association of Ocial Analytical Chemists, Arlington, VA, USA APHA-AWWA-WEF (1998) Standard Methods for the Examination of Water and Wastewater, 20th edn pp, 413426 American Aquaculture Nutrition 18; 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111 ể 2011 Blackwell Publishing Ltd Table Proximate whole-body composition (g kg)1 fresh weight), hepatosomatic and visceral indices of Senegalese sole fed the experimental diets1 16 C Temperature 22 C Diets Initial 55P16L 55P8L 45P16L 45P8L SEM 55P16L 55P8L 45P16L 45P8L SEM Dry matter Protein Lipid Energy (kJ kg)1) Ash VI2 HSI3 189.4 140.7 21.6 40.9 32.5 243.1 158.1 57.4 57.3 26.1 72.6 20.5 228.7 164.7 38.7 51.4 24.9 67.8 18.3 249.3 161.1 61.4 59.7 25.1 66.8 20.5 223.2 164.0 34.2 48.3 27.2 67.1 19.4 0.4 0.1 0.4 0.2 0.1 0.2 0.1 247.1 165.1 59.1 60.5 23.1 60.5 15.5 236.9 174.5 43.6 58.5 22.2 55.8 16.2 257.9 163.8 70.5 64.0 23.2 60.9 18.3 233.5 169.3 41.7 55.6 23.9 53.1 13.3 0.3 0.1 0.4 0.1 0.03 0.1 0.1 Diets Variation source Temperature Diets Interaction 55P16L 55P8L 45P16L 45P8L Two-way ANOVA Dry matter Protein Lipid Energy (kJ kg)1) Ash VI HSI *** *** ** *** *** *** *** *** *** *** *** ns ns ns ns ns ns ns ns ns ns b a b bc c a b a ab ab c a b c c a ab a a a Values presented as means and pooled standard error of the mean (SEM) Visceral index: g viscera weight kg)1 body weight Hepatosomatic index: g liver weight kg)1 body weight *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant followed by diet 45P16L Protein eciency ratio (PER) and nitrogen retention (NR, % nitrogen intake) were also higher at the higher temperature Within each temperature, PER was higher and not signicantly dierent with diets 55P8L and 45P16L, while NR was higher with diet 55P8L than with the other diets Energy retention (ER, % energy intake) was also higher at 22 C and, independently of temperature, it was higher with diet 55P8L than with the other diets While with the 450 g kg)1 protein diets, ER improved with the increase in dietary lipid, the opposite was true with the 550 g kg)1 protein diets At the end of the growth trial, whole-body dry matter, protein, lipid and energy content were higher and ash content was lower in sh reared at the highest temperature (Table 3) Within each temperature, dry matter, lipid, energy and ash content were higher and protein content was lower in sh fed the high-lipid diets Visceral (VI) and hepatosomatic (HSI) indices were not signicantly aected by diet composition but were higher in sh reared at 16 than at 22 C (Table 3) Liver glycogen and lipid content were not aected by diet composition or water temperature (Table 4) Hepatic GDH, ALAT and ASAT activities were not affected by diet or water temperature (Table 4) Hepatic ME and G6PD activities were higher in sh fed the low-lipid diets G6PD activity was not aected by temperature, but ME activity was higher in sh reared at the lower water temperature Growth of Senegalese sole during this trial was very satisfactory, with DGI of 0.981.37 at 16 C and of 1.731.91 at 22 C, which compares positively with 0.861.05 or 1.22 observed at 20 C by Dias et al (2004) and Borges et al (2009), respectively According to Bureau et al (2002), DGI is relatively unaected by dierences in sh weight, although it is dependent on rearing temperature, while TGU is relatively unaected by water temperature In this study, there were however signicant dierences in TGU with water temperature, although TGU with the best-performing diets was very similar at both temperatures: 0.0590.063 at 16 C and 0.0650.068 at 22 C Protein requirement of Senegalese sole juveniles was estimated to be 530 g kg)1 based on growth performance and 600 g kg)1 based on body protein accretion (Rema et al 2008) Protein requirement may however be aected by the dietary energy density and the protein-sparing potential of conventional energy sources In the present study, best performance was achieved with diets including either 550 g kg)1 protein or 450 g kg)1 protein, depending on Aquaculture Nutrition 18; 98106 ể 2011 Blackwell Publishing Ltd Table Liver proximate composition (g kg)1 wet weight) and selected hepatic enzymes activities of Senegalese sole juveniles fed the experimental diets1 Temperature 16 C Diets 55P16L 55P8L 45P16L 45P8L 68.5 39.9 76.6 32.2 45.7 26.8 83.1 39.1 74.20 280.7 312.2 7.79 33.55 71.07 248.5 354.9 8.02 39.13 65.78 272.2 310.1 8.15 39.82 68.57 347.0 369.6 10.01 44.12 Liver Glycogen Lipid Enzyme activity (mU mg)1 protein) Glutamate dehydrogenase Alanine aminotransferase Aspartate aminotransferase Malic enzyme Glucose 6-phosphate dehydrogenase 22 C SEM 55P16L 55P8L 45P16L 45P8L SEM 0.58 0.52 53.5 34.0 75.3 38.8 63.1 78.1 55.6 22.3 0.38 0.66 1.93 20.5 15.1 0.51 2.33 84.50 380.1 374.2 4.48 32.80 72.55 341.8 377.5 7.76 54.05 72.79 305.8 317.4 5.55 30.31 67.91 253.8 387.9 8.64 58.09 2.32 26.5 14.4 0.44 3.33 Diets Variation source Temperature Diets Interaction 55P16L 55P8L 45P16L 45P8L Two-way ANOVA Glycogen Lipid Glutamate dehydrogenase Alanine aminotransferase Aspartate aminotransferase Malic enzyme Glucose 6-phosphate dehydrogenase ns ns ns ns ns ** ns ns ns ns ns ns ** ** ns ** ns ns ns ns ns a a ab bc a ab b c Values presented as means and pooled standard error of the mean (SEM) *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant dietary lipid content, suggesting a protein sparing by dietary lipids This eect of dietary lipids was however related to the dietary protein content Increasing dietary lipid level of the 450 g kg)1 protein diets improved growth and feed eciency, while in the 550 g kg)1 protein diets, the opposite occurred In Senegalese sole, the eect of dietary lipid level on high-protein diets was previously studied by Dias et al (2004) and Borges et al (2009) Present results are contradictory to the ndings of Dias et al (2004) who observed no signicant eects on growth performance or on protein deposition by increasing dietary lipids from 110 g kg)1 to 210 g kg)1 in 510 g kg)1 protein diets Our results are however in accordance with those of Borges et al (2009) who tested a wider range of lipid levels in 560 g kg)1 protein diets and concluded that dietary lipid should be limited to a maximum of 80 g kg)1 for optimal growth and nutrient utilization A protein sparing by dietary lipids was also reported in some studies with atsh (Helland & Grisdale-Helland 1998; Cho et al 2005) but not in others (Bromley 1980; Cacerez-Martinez et al 1984; Aksnes et al 1996; Lee et al 2000; Martins et al 2007) This apparent discrepancy between studies may be related to the dietary protein levels used in the dierent experiments Indeed, Andersen & Alsted (1993) referred a protein-sparing eect Aquaculture Nutrition 18; 98106 ể 2011 Blackwell Publishing Ltd by dietary lipids in turbot, which was reduced at high protein intakes There is little convincing evidence that protein requirement (expressed as percentage of diet) is aected by water temperature (NRC, 1993), although such eect was observed in a few species (Wilson 2002) Temperature aects, however, all metabolic functions and inuences feed intake and growth and, in some cases, the eciency of nutrient utilization In this study, N retention (% N intake) was higher at 22 C than at 16 C, indicating that the eciency of protein utilization improved as temperature increased There is not much information on the eect of temperature on the eciency of protein utilization in sh, and available data are controversial Our results are contradictory to those of Peres & OlivaTeles (1999a) in European sea bass (Dicentrarchus labrax) juveniles that showed higher NR at 18 C than at 25 C, but agree with those of Oliva-Teles & Rodrigues (1993) in rainbow trout (Oncorhynchus mykiss) In turbot, another atsh, protein retention was not aected by water temperature (Burel et al 1996) According to Oliva-Teles & Rodrigues (1993), the best protein retention observed at higher temperature was related to an improvement in diet digestibility This may also contribute to explain results of the present study; though, it requires to be conrmed Apparent digestibility of the diets was not determined in this study as it is a very challenging task because of low cohesion of faecal material in sole (Borges et al 2009; Dias et al 2010) Feed eciency was higher at 22 C than at 16 C, suggesting that this temperature is nearer the optimum temperature for the species as feed eciency is usually maximized at temperatures near the optimum for maximum growth of the species (Elliott 1982; Arnason et al 2009) Although it is well established that animals eat to meet their energy needs (Kaushik & Medale 1994; Bureau et al 2002) within limits, they may also adjust feed intake to meet protein requirement (Peres & Oliva-Teles 1999b) In this study, dierences in N and energy intake among groups suggest that sh adjusted feeding to protein needs rather than to energy needs, except for sh fed diet 55P16L, which showed hyperphagia Similarly, Dias et al (2004) did not observe dierences in feed intake in Senegalese sole fed isoproteic diets (520 g kg)1 DM) with dierent carbohydrate-to-lipid ratios and dierent gross energy density However, in Senegalese sole fed high-protein (560 g kg)1 DM) diets, it was observed an increase in feed intake with the increase in dietary lipid level, hence aecting intake of all other nutrients and energy (Borges et al 2009) Cases of hyperphagia in animals fed high-dense energy diets were previously reported in sea bass (Peres & Oliva-Teles1999b) and turbot but, contrary to present results, it was only observed in sh fed low-protein diets (Cho et al 2005) These results, in conjunction with those of the present study, suggest that feed intake control may be deregulated in Senegalese sole fed high-lipid diets, although such eect might be related to the dietary protein content and rearing temperature Senegalese sole is considered a low-fat sh, although dietary lipid level aects whole-body lipid deposition (Dias et al 2004; Rema et al 2008) In the present study, whole-body lipid signicantly increased with the dietary lipid level from circa 40 g kg)1 in the low-lipid diets to 62 g kg)1 in the highlipid diets, which is in accordance with previous results also in this species (Dias et al 2004; Borges et al 2009) Temperature also aected whole-body composition, with dry matter, protein, lipid and energy content being higher and ash content being lower at higher temperature An eect of temperature on whole-body composition was also observed in other atsh species (Burel et al 1996; Van Ham et al 2003; Fang et al 2010) These results should however be taken carefully, as nal sh weight was dierent at the two temperatures and this may mask temperature eect In this study, HSI values ranged from 13.3 g kg)1 to 20.5 g kg)1 and VI from 53.1 g kg)1 to 72.6 g kg)1 and were within the range of values found in other atsh species (Cacerez-Martinez et al 1984; Aksnes et al 1996; Helland & Grisdale-Helland 1998; Hamre et al 2003; Peres & OlivaTeles 2005; Martins et al 2007) They were however higher (particularly the VI) than values found in other studies with the same species (Dias et al 2004; Borges et al 2009; Silva et al 2009), and these dierences are dicult to explain at present HSI and VI were not aected by diet composition, indicating that liver and viscera were not the main sites for body fat storage Indeed, both liver glycogen and lipid contents were not aected by diet composition This is identical to observations in other atsh (Regost et al 2001; Martins et al 2007) but partially contrary to other observations in this species Indeed, Borges et al 2009 observed that both HSI and VI were higher in Senegalese sole fed high-fat diets, but while liver lipids were not aected by diet lipid content, viscera and skin lipids were higher in groups fed higher lipid levels Dias et al (2004) also observed that HSI was higher and VI not aected in sh fed high-energy diets In both cases, however, lipid deposition in both organs increased with dietary lipid or digestible carbohydrate contents The activities of the amino acid catabolic enzymes were not responsive to dierences in dietary protein and lipid contents or to temperature Aragao et al (2003) also did not observe dierences in the activity of these enzymes in Senegalese sole subject to a dietary amino acid imbalance originated by using soy protein concentrate as the major protein source Similarly, no dierences in GDH activity were observed in turbot fed diets rich in brewerếs yeast (Fournier et al 2002), plant protein (Fournier et al 2003), increased nitrogen levels (Gouillou-Coustans et al 2002) or high crystalline amino acid levels (Peres & Oliva-Teles 2005) This seems to conrm a lack of adaptation of the hepatic key enzymes involved in amino acid catabolism to changes in dietary protein level and quality Such a non-adaptative behaviour of these enzymes was advanced to be characteristic of carnivorous sh (Cowey 1995) On the contrary, dietary lipids led to an inhibition of key enzymes of lipogenesis, a result similar to what was previously observed in another study in Senegalese sole (Dias et al 2004) but not in turbot (Regost et al 2001) Results of this study indicate that regardless of water temperature, a diet with 550 g kg)1 protein and 80 g kg)1 lipid promoted the best growth and feed eciency of Senegalese sole juveniles Increasing dietary lipid content seems to spare protein for plastic purposes in 450 g kg)1 protein diets but not in 550 g kg)1 protein diets Increasing water temperature from 16 to 22 C improved growth rate as well as feed eciency Aquaculture Nutrition 18; 98106 ể 2011 Blackwell Publishing Ltd This work was developed under the project OPTISOLE This project aims to introduce improvements in organization and management of the company and boost its presence in the international market and has a total investment exceeding 100 000 It was co-funded by the National Strategic Reference Framework, QREN, under the Regional Operational Programme North, ON2, in the amount of 343751.84 originating from the European Regional Development Fund FEDER We express our thanks to P Correia for the assistance during the growth trial Aksnes, A., Hjertnes, T & Opstvedt, J (1996) Eect of dietary protein level on growth and carcass composition in Atlantic halibut (Hippoglossus hippoglossus L) Aquaculture, 145, 225233 Andersen, N & Alsted, N.S (1993) Growth and body composition of turbot (Scophthalmus maximus (L.)) in relation to dierent lipid/protein ratios in the diet In: Fish Nutrition in Practice Les Colloques, 61, INRA, Vol 61 (Kaushik, S.J & Luquet, P eds), pp 479491 INRA, Paris, France Aragao, C., Conceicáao, L.E.C., Dias, J., Marques, A.C., Gomes, E & Dinis, M.T (2003) Soy protein concentrate as a protein source for Senegalese sole (Solea senegalensis Kaup 1858) diets: eects on growth and amino acid metabolism of postlarvae Aquacult Res., 34, 14431452 Arnason, J., Imsland, A.K., Gustavsson, A., Gunnarsson, S., Arnarson, I., Reynisson, H., Jonsson, A.F., Smaradottir, H & Thorarensen, H (2009) Optimum feed formulation for Atlantic halibut (Hippoglossus hippoglossus L.): Minimum protein content in diet for maximum growth Aquaculture, 291, 188191 Bautista, J.M., Garrido-Pertierra, A & Soler, G (1988) Glucose-6phosphate dehydrogenase from Dicentrarchus labrax liver: kinetic mechanism and kinetics of NADPH inhibition Biochim Biophys Acta, 967, 354363 Berge, G.M & Storebakken, T (1991) Eect of dietary-fat level on weight-gain, digestibility, and llet composition of Atlantic halibut Aquaculture, 99, 331338 Bergemeyer, H.U (1974) Methods of Enzymatic Analysis, Vol 4, pp 17041708 Academic Press, New York, USA Borges, P., Oliveira, B., Casal, S., Dias, J., Conceicáao, L & Valente, L.M.P (2009) Dietary lipid level aects growth performance and nutrient utilisation of Senegalese sole (Solea senegalensis) juveniles Br J Nutr., 102, 10071014 Bradford, M.M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem., 72, 248254 Brett, J.R (1979) Environmental factors and growth In: Fish Physiology, Vol VIII (Hoar, W.S et al eds), pp 599675 Academic Press, New York, USA Bromley, P.J (1980) Eect of dietary protein, lipid and energy content on the growth of turbot (Scophthalmus maximus L.) Aquaculture, 19, 359369 Bureau, D.P., Kaushik, S.J & Cho, C.Y (2002) Bioenergetics In: Fish Nutrition, 3rd edn (Halver, J.E & Hardy, R.W eds), pp 159 Academic Press, California, USA Aquaculture Nutrition 18; 98106 ể 2011 Blackwell Publishing Ltd Burel, C., Personleruyet, J., Gaumet, F., Leroux, A., Severe, A & Boeuf, G (1996) Eects of temperature on growth and metabolism in juvenile turbot J Fish Biol., 49, 678692 Cacerez-Martinez, C., Cadena-Roa, M & Metailler, R (1984) Nutritional requirements of turbot (Scophthalmus maximus): I A preliminary study of protein and lipid utilization J World Maricul Soc., 15, 191202 Cho, S.H., Lee, S.M., Lee, S.M & Lee, J.H (2005) Eect of dietary protein and lipid levels on growth and body composition of juvenile turbot (Scophthalmus maximus L) reared under optimum salinity and temperature conditions Aquacult Nutr., 11, 235240 Conceicáao, L.E.C., Ribeiro, L., Engrola, S., Aragao, C., Morais, S., Lacuisse, M., Soares, F & Dinis, M.T (2007) Nutritional physiology during development of Senegalese sole (Solea senegalensis) Aquaculture, 268, 6481 Cowey, C.B (1995) Protein and amino acid requirements a critique of methods J Appl Ichthyol., 11, 199204 Dias, J., Rueda-Jasso, R., Panserat, S., da Conceicáao, L.E.C., Gomes, E.F & Dinis, M.T (2004) Eect of dietary carbohydrateto-lipid ratios on growth, lipid deposition and metabolic hepatic enzymes in juvenile Senegalese sole (Solea senegalensis, Kaup) Aquacult Res., 35, 11221130 Dias, J., Yufera, M., Valente, L.M.P & Rema, P (2010) Feed transit and apparent protein, phosphorus and energy digestibility of practical feed ingredients by Senegalese sole (Solea senegalensis) Aquaculture, 302, 9499 Dinis, M.T., Ribeiro, L., Soares, F & Sarasquete, C (1999) A review on the cultivation potential of Solea senegalensis in Spain and in Portugal Aquaculture, 176, 2738 Dinis, M.T., Ribeiro, L., Conceicáao, L.E.C & Aragao, C (2000) Larvae digestion and new weaning experiments in Solea senegalensis In: Recent Advances in Mediterranean Aquaculture Finsh Species Diversication, Vol 47 (CIHEAM-IAMZ), pp 193204 CIHEAM, Zaragoza, Spain Elliott, J.M (1982) The eects of temperature and ration size on the growth and energetics of salmonids in captivity Comp Biochem Physiol , 72B, 8191 Fang, J., Tian, X & Dong, S (2010) The inuence of water temperature and ration on the growth, body composition and energy budget of tongue sole (Cynoglossus semilaevis) Aquaculture, 299, 106114 Folch, J., Lees, M & Sloane-Stanley, G.H.S (1957) A simple method for the isolation and purication of total lipids from animal tissue J Biol Chem., 226, 497509 Fournier, V., Gouillou-Coustans, M.F., Metailler, R., Vachot, C., Moriceau, J., Le Delliou, H., Huelvan, C., Desbruyeres, E & Kaushik, S.J (2002) Nitrogen utilisation and ureogenesis as aected by dietary nucleic acid in rainbow trout (Oncorhynchus mykiss) and turbot (Psetta maxima) Fish Physiol Biochem., 26, 177188 Fournier, V., Gouillou-Coustans, M.F., Metailler, R., Vachot, C., Moriceau, J., Le Delliou, H., Huelvan, C., Desbruyeres, E & Kaushik, S.J (2003) Excess dietary arginine aects urea excretion but does not improve N utilisation in rainbow trout Oncorhynchus mykiss and turbot Psetta maxima Aquaculture, 217, 559576 Gouillou-Coustans, M.F., Fournier, V., Metailler, R., Vachot, C., Desbruyeres, E., Huelvan, C., Moriceau, J., Le Delliou, H & Kaushik, S.J (2002) Dietary arginine degradation is a major pathway in ureagenesis in juvenile turbot (Psetta maxima) Comp Biochem Physiol A, 132, 305319 Hamre, K., Ofsti, A., Naess, T., Nortvedt, R & Holm, J.C (2003) Macronutrient composition of formulated diets for Atlantic halibut (Hippoglossus hippoglossus, L.) juveniles Aquaculture, 227, 233244 Helland, S.J & Grisdale-Helland, B (1998) Growth, feed utilization and body composition of juvenile Atlantic halibut (Hippoglossus hippoglossus) fed diets diering in the ratio between the macronutrients Aquaculture, 166, 4956 Imsland, A.K., Foss, A., Conceicáao, L.E.C., Dinis, M.T., Delbare, D., Schram, E., Kamstra, A., Rema, P & White, P (2003) A review of the culture potential of Solea solea and S senegalensis Rev Fish Biol Fish, 13, 379407 Kaushik, S.J & Medale, F (1994) Energy requirements, utilization and dietary supply to salmonids Aquaculture, 124, 8197 Lee, S.M., Cho, S.H & Kim, K.D (2000) Eects of dietary protein and energy levels on growth and body composition of juvenile ounder Paralichthys olivaceus J World Aquac Soc., 31, 306315 Martins, D.A., Valente, L.M.P & Lall, S.P (2007) Eects of dietary lipid level on growth and lipid utilization by juvenile Atlantic halibut (Hippoglossus hippoglossus, L.) Aquaculture, 263, 150158 NRC (1993) Nutrient Requirements of Fish, 114 p National Academy Press, Washington, DC, USA Ochoa, S (1955) Malic enzyme In: Methods in Enzymology, Vol (Colowick, S.P & Kaplan, N.O eds), pp 739753 Academic Press, New York, USA Oliva-Teles, A & Rodrigues, A.M (1993) The eect of high temperature and diet protein level on metabolic utilization of diets by rainbow trout In: Fish Nutrition in Practice Les Colloques INRA, Vol 61 (Kaushik, S.J & Luquet, P eds), pp 301305 INRA, Paris, France Peres, M.H & Oliva-Teles, A (1999a) Inuence of temperature on protein utilization in juvenile European seabass (Dicentrarchus labrax) Aquaculture, 170, 337348 Peres, M.H & Oliva-Teles, A (1999b) Eect of dietary lipid level on growth performance and feed utilization by European sea bass juveniles (Dicentrarchus labrax) Aquaculture, 179, 325334 Peres, H & Oliva-Teles, A (2005) The eect of dietary protein replacement by crystalline amino acid on growth and nitrogen utilization of turbot Scophthalmus maximus juveniles Aquaculture, 250, 755764 Plummer, P (1987) Glycogen determination in animal tissues In: An Introduction to Practical Biochemistry, 3rd edn, p 332 McGrow Hill Book, Maidenhead, Berkshire, UK Regost, C., Arzel, J., Cardinal, M., Robin, J., Laroche, M & Kaushik, S.J (2001) Dietary lipid level, hepatic lipogenesis and esh quality in turbot (Psetta maxima) Aquaculture, 193, 291 309 Rema, P., Conceicáao, L.E.C., Evers, F., Castro-Cunha, M., Dinis, M.T & Dias, J (2008) Optimal dietary protein levels in juvenile Senegalese sole (Solea senegalensis) Aquacult Nutr., 14, 263269 Silva, J.M.G., Espe, M., Conceicáao, L.E.C., Dias, J & Valente, L.M.P (2009) Senegalese sole juveniles (Solea senegalensis Kaup, 1858) grow equally well on diets devoid of sh meal provided the dietary amino acids are balanced Aquaculture, 296, 309317 Van Ham, E.H., Berntssen, M.H.G., Imsland, A.K., Parpoura, A.C., Bonga, S.E.W & Stefansson, S.O (2003) The inuence of temperature and ration on growth, feed conversion, body composition and nutrient retention of juvenile turbot (Scophthalmus maximus) Aquaculture, 217, 547558 Wilson, R.P (2002) Amino acids and proteins In: Fish Nutrition, 3rd edn (Halver, J.E & Hardy, R.W eds), pp 143179 Academic Press, California, USA Aquaculture Nutrition 18; 98106 ể 2011 Blackwell Publishing Ltd Aquaculture Nutrition doi: 10.1111/j.1365-2095.2011.00886.x 2012 18; 107116 College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, China An 8-week feeding test was conducted to quantify the dietary arginine requirement of juvenile largemouth bass (LMB) (25 0.4 g) Six isonitrogenous and isolipidic (459 g crude protein and 122 g crude lipid kg)1 dry diet) diets were formulated to contain graded levels of arginine (17.0 30.1 g kg)1 dry diet) Zein-coated crystalline amino acid mixtures were supplemented to simulate, except for arginine, the amino acid prole of the muscle protein of LMB Each diet was randomly assigned to quadruplicate tanks of 35 sh reared in a ow-through system Fish were fed to apparent satiation twice daily Weight gain (WG) was signicantly aected by dietary arginine level Nitrogen retention was signicantly lower in sh fed D17.0 Arginine retention signicantly decreased with dietary arginine increased Threonine, leucine and lysine concentrations in whole body were signicantly aected by dietary arginine level Serum lysozyme activity, serum protein and respiratory burst of head kidney leucocytes were signicantly aected, while complement activity (CH50) showed no dierence among treatments Based on broken-line analysis for WG against dietary digestible arginine level, the arginine requirement of LMB was 19.1 g kg)1 of dry diet (41.6 g kg)1 of crude protein) key words: arginine requirement, digestibility, sh, growth, immunity, largemouth bass Received 21 December 2010, accepted 12 May 2011 Correspondence: Naisong Chen, College of Fisheries and Life Science, Shanghai Ocean University, 999 Hucheng Huan Road, Shanghai 201306, China E-mail: nschen@shou.edu.cn Fish, like other animals, not have a true protein requirement but have a requirement for a well-balanced mixture of essential and non-essential amino acids (AAếs) ể 2011 Blackwell Publishing Ltd (Wilson 2002) An absolute requirement for 10 essential amino acids (EAAếs) (arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine) has been demonstrated in all sh species studied to date (NRC 1993) An EAA deciency may cause reduced growth performance and poor feed conversion (Wilson & Halver 1986) In certain species of sh, the deciency of an EAA, such as methionine or tryptophan, leads to pathologies (NRC 1993) Therefore, it is very important in preparing well-balanced diets to investigate EAA requirements of a cultured sh species Arginine is involved in many metabolic pathways like protein synthesis, urea production, metabolism of glutamic acid and proline, and synthesis of creatine and polymines (Kaushik et al 1988) Dietary arginine deciency signs reported in sh include reduced growth and protein deposition (Tibaldi et al 1994; Alam et al 2002) Dietary arginine requirements have been quantied for a number of sh species, and the data before the year of 2000 were summarized by Wilson (2002), ranging from 32 to 65 g kg)1 of dietary protein Since then, dietary arginine requirements of some other sh species have been reported, such as Indian major carp (Ahmed & Khan 2004) and hybrid Clarias (Singh & Khan 2006) Arginine has been reported to have an eect on immunological functions in mammals (Li et al 2007) However, few data on this aspect are available in sh The supplementation of dietary arginine could modulate some innate immune mechanisms, and a moderate supply of arginine was ideal in enhancing phagocytosis in channel catsh (Buentello et al 2007) Buentello & Gatlin (2001) found that the survival rate of channel catsh challenged with Edwardsiella ictaluri critically depended upon the dietary arginine level Largemouth bass (LMB) shows promise as an aquaculture species of great commercial value in China, where a main constraint to the culture is the limited supply of trash sh that is presently the main feed source for grow-out So it is urgent to develop a highly eective practical diet for grow-out production of LMB To our knowledge, there are a few studies on the nutrition and immunity for this sh (Tidwell et al 1996; Portz et al 2001; Portz & Cyrino 2003; Subhadra et al 2006; Dairiki et al 2007) No published information is available on the qualitative and quantitative dietary arginine requirements and the eect of dietary arginine on immunity of this sh Hence, this study was aimed to determine the dietary arginine requirement and assess the eect of dietary arginine on immunity of juvenile LMB Six isonitrogenous and isolipidic (459 g crude protein kg)1 and 122 g crude lipid kg)1 of dry diet) diets were formulated with graded levels of arginine ranging from 17.0 to 30.1 g kg)1 of dry diet (D17.0, D19.4, D22.3, D25.2, D27.6 and D30.1) at about 2.7 g kg)1 increment by adding coated crystalline L-arginine Dietary arginine was quantitatively increased at the expense of non-essential AA mixture Coated crystalline LAA mixture was added to obtain the dietary AA prole similar to that of muscle AA of LMB (average body weight, 25 0.6 g), except for arginine under investigation Mineral and vitamin premixes were supplemented in the diets; 4.5 g kg)1 chromic oxide (Cr2O3) was incorporated in the diets as an inner marker to estimate the digestibility of dietary arginine (Table 1) The AAếs composition of the experimental diets and the muscle were shown in Table To reduce crystalline AA leaching, single crystalline AA or mixture used in this study was coated with alcohol-soluble zein, which at a 5% incorporation level was previously dissolved in 90% alcohol to get a zein solution of 10% (w/v) Ground crystalline AA was added to the solution with blending, and then the mixture was dried at 60 C The dried mixture was ground into ne powder before mixing with the other ingredients Solid ingredients were ground into ne powder (420 lm mesh) with a grinder All the ingredients were mixed with sh oil and soybean oil, and 45% water was added to produce sti dough The dough was extruded with an electric mincer into semimoist pellets with suitable sizes and then stored at )20 C until used Largemouth bass juveniles were obtained from a local commercial husbandry and were transported to an indoor ow-through aquarium system at Nonghao Feed Co Ltd, Shanghai, China Before the beginning of the experiment, sh Table Composition and proximate analysis of the experimental diets (g kg)1 dry diet) Diets D17.0 D19.4 D22.3 D25.2 D27.6 D30.1 985.8 0.0 14.2 985.8 2.9 11.3 985.8 5.7 8.5 985.8 8.5 5.7 985.8 11.3 2.9 985.8 14.2 0.0 453.2 121.8 84.6 896.4 21.62 17.0 454.6 121.7 84.6 896.2 21.50 19.4 459.0 122.6 84.3 893.7 21.54 22.3 459.8 12.2.4 84.3 895.1 21.63 25.2 463.1 122.3 83.7 888.1 21.71 27.6 464.1 123.9 84.1 891.8 21.70 30.1 )1 Ingredients (g kg ) Basal ingredients1 Coated arginine2 Coated NEAA mixture3 Proximate analysis (g kg)1 dry diet) Crude protein Crude lipid Ash Dry matter (DM) Gross energy (kJ g)1 DM) Arginine Basal ingredients (g kg)1): fish meal, 50; shrimp meal, 30; corn gluten, 90; casein, 216; soybean meal, 72; squid viscera meal, 45; yeast, 18; wheat gluten, 45; a-starch, 110; calcium biphosphate, 9; fish hydrolysate, 18; brewers yeast extract, 18; soybean phospholipid, 18; marine fish oil, 36; soybean oil, 54; a-cellulose, 24.95; carboxymethyl cellulose, 18; vitamin C (35% ascorbic acid), 0.9; vitamin premix *, 9; mineral premix **, 9; zeolite powder (used as inert filler), 36; chromic oxide, 4.5; coated amino acid mixture, 54.45 (L-threonine (98%), 3.03; L-valine, 0.63; L-methionine (98%), 2.85; L-isoleucine, 0.36; L-histidine, 1.71; L-lysine (78%), 17.31; L-aspartic acid, 10.26; L-glycine, 8.37; L-alanine, 7.20; zein, 2.73) Coated arginine (%): L-arginine, 95; zein, Coated non-essential amino acid (NEAA) mixture (%): L-aspartic acid, 37.74; L-glycine, 30.78; L-alanine, 26.58; zein, * Vitamin premix (mg or IU kg)1 diet): vitamin A, 18 000 IU; vitamin D, 9000 IU; vitamin K, 16.56; thiamin, 20.03; riboflavine, 54; pyridoxine, 33.21; cyanocobalamine, 0.27; tocopherol acetate, 180; ascorbic acid (35%), 900; niacinamide, 89.1; calcium-D-pantothenate, 82.8; folic acid, 7.2; biotin, 0.72; inositol, 360; choline chloride, 1350; L-carnitine, 90 ** Mineral premix (mg kg)1 diet): Cu (CuSO4), 1.79; Zn (ZnSO4), 30.18; Mn(MnSO4), 5.58; Fe (FeSO4),18.98; I (Ca(IO3)2), 1.46; Se (Na2SeO3), 0.16; Co (CoCl2), 0.22; Mg (MgSO4ặH2O), 47.43 Aquaculture Nutrition 18; 107116 ể 2011 Blackwell Publishing Ltd Table Amino acid composition of the experimental diets (% dry diet) and sh muscle (relative to 460 g kg)1 protein)1 460 g kg)1 muscle D17.0 D19.4 D22.3 D25.2 D27.6 D30.1 protein Diets Amino acids Essential amino acids Threonine 1.69 1.74 Valine 1.95 1.81 Methionine 1.12 1.14 Isoleucine 1.69 1.65 Leucine 3.50 3.45 Phenylalanine 1.84 1.82 Histidine 1.07 1.05 Lysine 3.51 3.56 Arginine 1.70 1.94 Non-essential amino acids Aspartic acid 4.21 4.27 Serine 1.91 2.02 Glutamic acid 7.67 7.79 Glycine 2.40 2.34 Alanine 2.56 2.55 Tyrosine 1.75 1.76 Proline 3.18 3.04 1.75 1.86 1.14 1.61 3.50 1.85 1.08 3.52 2.23 1.79 1.94 1.18 1.67 3.63 1.91 1.07 3.54 2.52 1.77 1.97 1.19 1.72 3.63 1.91 1.06 3.52 2.76 1.82 1.94 1.16 1.67 3.64 1.91 1.07 3.55 3.01 1.76 2.00 1.20 1.71 3.29 1.70 1.09 3.69 2.26 4.22 2.03 7.87 2.35 2.49 1.78 3.08 4.14 2.06 8.05 2.33 2.50 1.83 3.22 4.07 2.02 8.05 2.21 2.42 1.83 3.13 4.03 2.12 8.17 2.08 2.37 1.85 3.16 4.02 1.63 6.23 2.13 2.42 1.28 1.24 Values are means from duplicate samples of experimental diets and fish muscle were acclimated to experimental conditions for weeks by feeding D17.0 Then, following fasting for 24 h, each group of 35 sh with a similar size (average initial body weight, 25 0.4 g) was bulk-weighed and assigned into an 800-L cylindrical glass-bre tank Each experimental diet was randomly assigned to four tanks The tanks were provided with a continuous ow of sand-ltered freshwater (2 L min)1) with continuous aeration to maintain the dissolved oxygen level close to saturation The sh were fed by hand to apparent satiation twice daily at 08:00 and 1600 h, respectively The feeding trial lasted for weeks with suitable diet pellet sizes according to sh sizes The amount of feed consumed in each tank was recorded daily The number and weight of dead sh, if any, were recorded During the experiment period, water temperature was maintained at 28 C; pH, 7.27.6; ammonia nitrogen, lower than 0.3 mg L)1 The natural light cycle was adapted At the beginning of the experiment, 35 sh fasted for 24 h were sampled for an analysis of initial body composition After 1-week feeding, faecal samples were collected with a collection column attached to the tank, as described by Lee (2002) When feed and faeces residues in the tank and collection column were cleared, faeces were allowed to settle in Aquaculture Nutrition 18; 107116 ể 2011 Blackwell Publishing Ltd the collecting device for h, immediately ltered and kept frozen at )20 C till being freeze-dried Dried faecal samples were sieved to remove scales before analysis At the end of the experiment, following fasting for 24 h, sh in each tank were bulk-weighed Five sh from each tank were used as a pooled sample for proximate analysis of whole body Another ve sh were used for the calculation of hepatosomatic index and viscerosomatic index, and the dorsal muscle was pooled and then frozen at )80 C until analysis In addition, approximately 1.5 mL of blood was drawn from the caudal vein of each of ve sh per tank with a 2.5 mL syringe and allowed to clot at C for h Following centrifugation (836 g, 10 min, C), the serum was removed and frozen at )80 C until analysis The remaining sh were kept being fed in respective diets, and three sh per tank were randomly sampled for head kidneys as needed AA concentration of diets, faeces and carcasses was analysed in an automatic amino acid analyser (L-8800; Hitachi, Tokyo, Japan) after samples were hydrolysed with M hydrochloric acid under a nitrogen atmosphere at 110 C for 22 h Tryptophan was not measured in this method Proximate analysis of moisture, crude protein and ash was performed according to the standard method (AOAC 1995): moisture was determined by oven-drying at 105 C until constant weight; crude protein (N ã 6.25) by the Kjeldahl method after acid digestion using an Auto Kjeldahl System (Kjeltec 2200; Foss, Hillerứd, Denmark); ash by a mue furnace at 550 C until constant weight Crude lipid was determined by a chloroformmethanol extraction method (Lee et al 1996) Gross energy was determined by an adiabatic bomb calorimeter (Parr 6200, Moline, IL, USA) Chromic oxide concentration in diets and faeces was evaluated following the method described by Divakaran et al (2002) with some modication Briey, specimens were oxidized in thick-walled Pyrex boiling tubes with mL of concentrated nitric acid, added mL of perchloric acid and then heated to 210220 C until chromic oxide was converted to dichromate After the solution was cooled to room temperature, it was transferred and made up to 50 mL in a volumetric ask by rinsing repeatedly with distilled water To mL of the volume-adjusted sample placed in a test tube, 1.5 mL of N sulphuric acid and 3.5 mL of distilled water were added, and the mixture was well mixed in a vortex shaker Then, 0.5 mL of 0.25% diphenylcarbazide solution was added to each tube, immediately mixed as above The absorbance of the nal solution was read by a spectropho- tometer at 540 nm Absorbance values for known serial dilutions were used to generate a regression equation to calculate unknown concentrations of chromic oxide in the samples Serum lysozyme activity was determined by turbidimetric assay adapted to 96-well microplates (Sitja`-Bobadilla et al 2003) Briey, Micrococcus lysodeikticus (lyophilized, M3770; Sigma, St Louis, MO, USA) suspension (0.3 mg mL)1) in 0.05 M sodium phosphate buer (pH 6.2) was used as a substrate for serum lysozyme Triplicates of test serum (diluted 1:2, 10 lL) were added to 200 lL of the suspension, and the reduction in absorbance at 450 nm was measured after 0.5 and 4.5 A unit of lysozyme activity was dened as the amount of enzyme that caused a decrease in absorbance of 0.001 min)1 Serum protein was assayed according to Bradford (1976) Classical complement pathway (CCP) activity in serum was assayed according to Inglis et al (2008) with some modication Sheep red blood cells (SRBCs, Shanghai Life Science Reagent Co Ltd) were sensitized with rabbit antiSRBC hemolysin (S8014; Sigma) and then used as target cells in the CCP hemolysis assay A series of volumes of the diluted serum (1 : 8) ranging from to 50 lL was dispensed into 96-well plates The total volume in each well was made up to 150 lL with barbitone buer (pH 7.4) in the presence of Mg2+ and Ca2+, and then 50 lL of 2% sensitized SRBC suspension was added to each well Incubated for 60 at 30 C with a constant shake, the plates were centrifuged at 400 g for at C The supernatant (160 lL) in each well was carefully transferred to new 96-well plates, and the absorbance was read at 540 nm in an ELISA reader (FLUOstar OPTIMA; BMG, Oenburg, Germany) The values of maximum (100%) and minimum (spontaneous) haemolysis were obtained by adding 200 lL of distilled water or the buer to 50 lL of 2% sensitized SRBC suspension, respectively The degree of haemolysis (Y) for each diluted specimen was estimated by measuring the absorbance Three replicate wells per specimen were run The lysis curve was obtained by plotting Y/(1 ) Y) against the volume of serum added (mL) on a loglog scaled graph The volume of serum producing 50% complement haemolysis (CH50) was determined, and the number of CH50 unit mL)1 was obtained for each specimen Isolation of head kidney (HK) leucocytes and respiratory burst assay were performed as previously described by Secombes (1990) with some modications Briey, after bleeding, head kidneys were excised, cut into small fragments and suspended aseptically in RPMI-1640 (Gibco, Carlsbad, CA, USA) medium supplemented with 10 IU mL)1 heparin (Shanghai Chemical Reagent Co Ltd, Shanghai, China), 100 IU mL)1 penicillin (Sigma, USA), 100 lg mL)1 streptomycin (Sigma, USA) and 2% foetal calf serum (Gibco) Cell suspensions were prepared by forcing the head kidney through a 100-lm stainless steel mesh The resultant cell suspensions were enriched by centrifugation (297 g, 20 and C) on 14.1% Nycodenz (Axis Shield, Oslo, Norway) density gradient medium prepared according to the manufacturerếs instruction The band of cells at the gradient interface was collected and washed twice with the RPMI1640 medium The cell concentration was adjusted to ã 107 mL)1 in the medium Cell viability was determined by a trypan blue exclusion method Production of intracellular superoxide anion was evaluated using nitroblue tetrazolium (NBT; Shanghai Chemical Reagent Co Ltd) reduction Hundred microlitre of the cell suspension was added to each well of 96-well plates with 100 lL of 0.1% NBT and lg mL)1 phorbol-1, 2-myristate-1, 3-acetate (PMA; Sigma) dissolved in 0.9% NaCl solution After incubated for 30 at 28 C in a humid chamber, three replicate wells per sample were run Absolute methanol was added to terminate the reaction Following washing three times with 70% methanol, the wells were air-dried One hundred and twenty microlitre M KOH and 140 lL dimethyl sulfoxide (DMSO; Sigma) were added, and then the colour was subsequently measured at 630 nm with a microplate reader using KOH/DMSO as a blank Data are presented as mean SEM and subjected to oneway analysis of variance (ANOVA) using SPSS 13.0 (Chicago, IL, USA) Duncaếs multiple range test was used to evaluate the dierences among treatments Statistical signicance was examined at P < 0.05 unless otherwise noted The brokenline analysis model (Robbins et al 1979) y = U ) L(R ) x) was employed using the software by Vedenov & Pesti (2007) to estimate the optimum arginine requirement Growth performance and feed utilization eciency of sh fed dierent diets for weeks are presented in Table Survival rates of sh fed diets with graded arginine levels were above Aquaculture Nutrition 18; 107116 ể 2011 Blackwell Publishing Ltd Table Growth performance and feed utilization efciency of sh fed the test diets1 Diets (digestible arginine, g kg)1 dry diet) Initial body weight (g) Final body weight (g) SGR (% day)1)2 WG (g kg ABW)1 d)1)3 FI (g DM fish)1)4 PER5 NR (% N intake)6 FCR7 Survival (%) D17.0 (13.9) D19.4 (16.6) D22.3 (19.1) D25.2 (22.1) D27.6 (24.5) D30.1 (26.9) 25.1 60.5 1.57 14.74 29.02 2.44 42.02 0.92 98.57 25.0 63.9 1.68 15.64 29.55 2.61 47.34 0.85 99.29 24.8 68.8 1.83 16.79 31.65 2.71 47.90 0.81 100.00 24.8 67.9 1.80 16.60 30.30 2.81 49.74 0.77 99.29 24.7 67.27 1.79 16.52 31.24 2.61 48.08 0.83 99.29 25.2 68.3 1.78 16.46 33.26 2.50 47.21 0.87 100.00 0.3 1.0c 0.04c 0.29c 0.83b 0.15 2.56b 0.11 1.43 0.6 0.7b 0.04b 0.29b 1.36b 0.14 1.27a 0.04 0.71 0.2 1.4a 0.03a 0.21a 0.42ab 0.05 0.95a 0.03 0.00 0.3 0.9a 0.02a 0.13a 0.90ab 0.07 1.22a 0.04 0.71 0.4 0.9a 0.02a 0.18a 0.41ab 0.05 0.93a 0.03 0.71 0.4 0.8a 0.02a 0.20a 1.45a 0.09 1.61a 0.06 0.00 Values are means SEM of four replicate tanks Values within the same raw sharing a common superscript are not significantly different determined (P > 0.05) Specific growth rate (SGR, % per day) = 100 ã (ln (mean final weight) ) ln (mean initial weight))/experimental days Weight gain (WG, g kg ABW)1 d)1) = (final body weight ) initial body weight) (g)/ABW/experimental days ABW (average body weight) = (initial body weight + final body weight) (kg)/2 Feed intake (FI, g dry diet fish)1) = consumed (g)/[1/2(nf + ni)], where nf and ni represent the numbers of fish per aquarium at the end and beginning of the experiment, respectively Protein efficiency ratio (PER) = body weight gain (g, on fresh basis)/protein intake (g) Nitrogen retention (NR, % N intake) =100 ã [(final body weight ã final body nitrogen content) ) (initial body weight ã initial body nitrogen content)]/nitrogen intake Feed conversion ratio (FCR) = (feed consumed (g, dry diet))/body weight gain (g) 98%, and no pathological signs were observed among treatments No dierence in voluntary feed intake occurred between the diets except D30.1, which caused a higher value than D17.0 and D19.4 (P < 0.05) Nitrogen retention (NR) was signicantly lower in sh fed D17.0 than in sh fed the other diets (P < 0.05) Weight gain (WG, g kg ABW)1 d)1) and specic growth rate (SGR) increased signicantly with increased dietary arginine from D17.0 to D22.3 (P < 0.05) and then levelled o Feed conversion ratio (FCR) and protein eciency ratio (PER), however, were not signicantly aected by dietary arginine level (P > 0.05) The apparent digestibility of arginine in the diets is showed in Table The digestibility signicantly increased with dietary arginine level increasing (P < 0.05) The broken-line analysis based on WG (y) against dietary digestible arginine level (x) showed a function y = 16.59 ) 0.358(19.1 ) x) (R2 = 0.9787, Fig 1) The optimum dietary arginine requirement for juvenile LMB was estimated to be 19.1 g kg)1 dry diet (corresponding to 41.6 g kg)1 crude protein), which was corrected by the apparent digestibility for dietary arginine There were no signicant dierences in condition factor (K), hepatosomatic index (HSI) and viscerosomatic index (VSI) among the treatments (P > 0.05, data not shown) Moisture, ash and lipid concentrations in whole body were not aected (P > 0.05) No signicant dierences occurred in protein, moisture, ash and lipid concentrations of muscle among the treatments (P > 0.05) But the protein content in whole body increased with increased dietary arginine level Fish fed Table Apparent digestibility of arginine in the experimental diets (in %)1 Diets D17.0 ADC (%) D19.4 e 81.76 0.66 85.27 0.31 D22.3 d D25.2 cd 86.51 0.52 87.57 0.75 D27.6 bc 88.58 0.09 D30.1 ab 89.14 0.25a Values are means SEM of four replicate tanks Values within the same row sharing a common superscript letter are not significantly different (P > 0.05) Apparent digestibility coefficient (ADC, %) = 100 ã [1 ) (feed inert marker content ã faecal nutrient content)/(feed nutrient content ã faecal marker content)] Aquaculture Nutrition 18; 107116 ể 2011 Blackwell Publishing Ltd WG (g kg ABW1 day1) 20 was found in sh fed D17.0 and D19.4 compared with that in sh fed the other diets, except that arginine retention decreased with increasing dietary arginine levels (P < 0.05) y = 16.59 0.358 (19.1 x), x 19.1 y = 16.59, x > 19.1 R = 0.9787 18 16 14 12 11 16 21 26 Dietary digestible arginine level (g kg1 31 dry diet) Figure Relationship between weight gain (WG) and dietary digestible arginine level as tted by broken-line model Serum lysozyme activity and respiratory burst activity of head kidney leucocytes in test sh were correlated with the dietary arginine level (P < 0.05, Table 8) CCP activity (CH50) showed no signicant dierence among the treatments (P > 0.05), although the higher CH50 was observed in sh fed D25.2 Serum protein concentrations were signicantly higher in sh fed D19.4 and D22.3 than those fed with the other diets (P < 0.05) D17.0 showed signicantly lower whole-body protein than those fed D27.6 and D30.1 (P < 0.05, Table 5) The eects of dierent dietary arginine levels on EAA composition of whole body are shown in Table Lysine, leucine and threonine concentrations were signicantly affected by dietary arginine level (P < 0.05) The highest lysine, leucine and threonine concentrations were observed in D22.3, while the lowest concentration was observed in D17.0 and D19.4 The retention of individual EAAếs in the whole body was signicantly aected by the dietary treatment (P < 0.05, Table 7) Signicantly lower retention for individual EAAếs In the present study, LMB fed arginine-limiting diets showed reduced SGR and NR Adequate supplementation of crystalline L-arginine to the diets improved growth performance and dietary protein utilization in the sh (Table 3) These indicate that exogenous arginine is essential for optimal growth and protein deposition of LMB, although endogenous arginine synthesis and no or low maintenance requirements were reported in other sh species (Buentello & Gatlin 2000; Fournier et al 2002) Supplemental arginine did not only aect the total amount of protein retained by LMB but also the EAA composition in gained body protein (Table 7) Threonine, leucine and lysine concentrations, expressed as g per 16 g N, were signicantly higher in the whole-body protein of sh fed the optimal arginine diet (19.1 g kg)1 dry diet) Similarly, Rodehutscord et al (2000) found that an increase in Table Proximate composition (g kg)1) of whole body and muscle of sh fed the test diets1 Diets (digestible arginine, g kg)1 dry diet) D17.0 (13.9) Whole body Crude protein Ash Moisture Crude lipid Muscle Crude protein Ash Moisture Crude lipid D19.4 (16.6) D22.3 (19.1) D25.2 (22.1) D27.6 (24.5) D30.1 (26.9) 173.3 29.6 730.2 62.1 1.8c 0.5 2.0 1.4 178.8 29.0 738.3 60.4 1.1abc 0.6 2.1 2.9 176.1 29.6 729.2 60.3 1.4bc 0.3 0.5 1.3 176.1 28.8 732.7 59.6 3.1bc 1.0 3.7 3.0 180.7 30.8 721.6 60.0 2.0ab 1.4 3.7 1.4 183.7 28.1 730.4 61.6 3.0a 1.2 5.5 4.5 197.1 12.1 779.7 13.6 3.9 0.1 2.6 1.0 201.9 12.2 781.5 12.1 1.6 0.2 0.8 0.9 201.1 12.6 778.0 13.1 2.4 0.1 2.9 1.2 205.7 12.1 779.2 12.1 5.0 0.1 1.5 0.8 202.5 12.3 782.5 11.3 0.4 0.1 0.9 0.4 201.9 12.2 780.3 13.0 1.1 0.1 0.8 0.4 Values are means SEM of four replicate tanks Values within the same raw sharing a common superscript are not significantly different (P > 0.05) Aquaculture Nutrition 18; 107116 ể 2011 Blackwell Publishing Ltd Table Essential amino acid (EAA) concentrations of whole body (g per 16 g N) in sh fed the test diets1 Diets (digestible arginineinine, g kg)1 dry diet) EAA Threonine Valine Methionine Isoleucine Leucine Phenylalanine Histidine Lysine Arginine D17.0 (13.9) 4.03 3.87 2.34 3.44 6.38 3.68 2.22 7.18 5.86 D19.4 (16.6) bc 0.05 0.09 0.04 0.10 0.10b 0.03 0.04 0.11b 0.14 3.98 3.86 2.35 3.45 6.43 3.60 2.18 7.17 5.62 D22.3 (19.1) 0.10c 0.14 0.04 0.13 0.17b 0.11 0.05 0.19b 0.16 4.33 4.06 2.59 3.66 6.98 3.87 2.33 7.80 6.07 D25.2 (22.1) a 0.05 0.05 0.04 0.08 0.09a 0.03 0.04 0.08a 0.24 4.17 4.08 2.31 3.63 6.64 3.76 2.35 7.47 6.11 D27.6 (24.5) abc 0.03 0.05 0.17 0.07 0.09ab 0.02 0.01 0.08ab 0.13 4.21 4.13 2.48 3.72 6.79 3.81 2.31 7.62 5.96 ab 0.07 0.09 0.05 0.09 0.13ab 0.06 0.05 0.14a 0.08 D30.1 (26.9) 4.10 4.06 2.46 3.67 6.70 3.72 2.27 7.50 5.86 0.09bc 0.08 0.06 0.08 0.16ab 0.08 0.04 0.17ab 0.17 Values are means SEM of four replicate tanks Values within the raw sharing a common superscript are not significantly different (P > 0.05) Table Retention of dietary EAAếs in the whole body of sh fed the test diets1 Diets (digestible arginine, g kg)1 dry diet) EAA D17.0 (13.9) Threonine Valine Methionine Isoleucine Leucine Phenylalanine Histidine Lysine Arginine 44.68 35.80 38.35 36.18 33.58 37.35 40.38 39.23 63.48 2.61c 2.40c 2.19 2.91b 2.32c 2.34c 1.89c 2.55c 1.36a D19.4 (16.6) 47.07 42.87 42.37 44.63 38.30 40.50 42.93 43.53 57.73 D22.3 (19.1) 2.58c 3.15ab 1.91 3.32a 2.23bc 2.51c 2.51bc 2.40bc 3.54a 56.55 49.95 51.73 52.18 45.70 47.68 50.38 51.33 60.65 D25.2 (22.1) 0.98a 0.72a 1.14 1.59a 0.38a 0.83a 1.63a 0.46a 3.75a 54.93 47.90 44.48 48.88 42.55 45.83 51.38 49.10 57.00 D27.6 (24.5) 1.82ab 1.97ab 5.69 2.63a 1.94ab 1.52ab 1.27a 2.00ab 1.11a 53.78 46.53 46.83 48.00 41.95 44.48 48.18 48.80 47.33 D30.1 (26.9) 0.72ab 1.37ab 1.17 1.78a 0.98ab 0.74ab 1.25ab 1.00ab 0.50b 49.25 45.35 46.95 47.6 40.48 42.40 45.53 46.55 41.85 3.03bc 2.71ab 3.16 2.81a 2.63ab 2.64bc 2.60abc 2.91ab 3.21b Values are means SEM of four replicate tanks Values within the same raw sharing a common superscript are not significantly different (P > 0.05) Essential amino acid (EAA) retention (%) =100 ã [(final body weight ã final EAA content) ) (initial body weight ã initial EAA content)]/ (EAA intake) Table Classical complement pathway (CH50), lysozyme and respiratory burst activities and serum protein of sh fed the test diets1 Diets (digestible arginine, g kg)1 dry diet) )1 CH50 activity (unit mL ) Lysozyme activity (unit lL)1) Respiratory burst activity (OD630) Serum protein (mg mL)1) D17.0 (13.9) D19.4 (16.6) D22.3 (19.1) D25.2 (22.1) D27.6 (24.5) D30.1 (26.9) 220 2.85 0.15 3.34 232 4.44 0.31 5.05 253 4.42 0.32 4.99 362 4.07 0.36 4.01 264 6.18 0.32 3.46 237 5.12 0.46 3.59 39.2 0.34c 0.05c 0.16c 33.6 0.49abc 0.05b 0.23a 59.6 0.30abc 0.06ab 0.21a 51.7 0.25bc 0.04ab 0.24b 32.6 0.56a 0.03ab 0.30bc 26.1 0.10ab 0.04a 0.39bc CH50, 50% complement haemolysis Values are means SEM of four replicate tanks Values within the same raw sharing a common superscript are not significantly different (P > 0.05) crystalline lysine in a wheat gluten-based diet fed to rainbow trout resulted in an increase in the concentrations of histidine, isoleucine, lysine and phenylalanine in gained protein Helland & Grisdale-Helland (2006) found that in halibut, histidine, lysine and methionine concentrations as a percentage of the sum of AAếs were changed by dietary wheat gluten incorporation These ndings challenge the Aquaculture Nutrition 18; 107116 ể 2011 Blackwell Publishing Ltd common assumption that the AA composition of whole sh is constant Diet appears a signicant factor inuencing tissue AA composition in sh Another concern is reliability or accuracy when using the ideal protein concept for estimating EAA requirements in sh (Wilson 2002) Comparing arginine requirements for dierent teleosts, expressed as per kg dietary protein, reveals some similarities but even wide variation The present study suggested that dietary digestible arginine requirement of juvenile LMB was 41.6 g kg)1 of the dietary protein (19.1g kg)1 of the dry diet) when estimated by broken-line analysis on WG (g kg ABW)1 d)1) The value is similar to the requirements of 4349 g kg)1 for yellow perch (Twibell & Brown 1997), 42.9 g kg)1 for channel catsh (Robinson et al 1981) and 40.842 g kg)1 for Japanese ounder (Alam et al 2002), but is lower than the values with 49 g kg)1 for coho salmon (Luzzana et al 1998), 44.550.0 g kg)1 for Hybrid clarias (Singh & Khan 2006), 5459 g kg)1 for rainbow trout (Ketola 1983), 5051 g kg)1 for Atlantic salmon (Berge et al 1997), 52.5 g kg)1 for milksh (Borlongan & Coloso 1993), 68 g kg)1 for silver perch (Ngamsnae et al 1999) and 55 g kg)1 for grouper (Luo et al 2007) The apparent analogy or dierence among the sh species cannot explain the reliability of the estimates, given that a number of factors such as test diet composition, data analysis procedure, response criterion, sh size or age and culture conditions have frequently been claimed as a major issue aecting reliability of EAA requirement estimates in sh (Cowey 1995) In doseresponse studies with sh EAA requirements, a large amount of crystalline AA is generally incorporated in test diets to obtain an optimal AA prole and a range of graded-level AA under test The incorporation, however, often leads to depressed growth performance and biased AA requirement estimates because of crystalline AA leaching, dierent absorption peaks of crystalline AAếs and poor diet palatability (Zarate & Lovell 1997; Peres & Oliva-Teles 2005; Ambardekar et al 2009) Coating and encapsulation techniques can reduce the solubility and non-synchronous absorption of free AAếs (Chen et al 1992; Segovia-Quintero & Reigh 2004) In the current study, we coated crystalline AA used with alcohol-soluble zein and successfully avoided these problems Resultantly, the sh had higher SGR and NR, and lower FCR, as compared with those in the study by Portz et al (2001) There are dierent viewpoints on the AA prole of basal diets used to assess AAếs requirements in sh Alam et al (2005) suggested that the AA prole of whole body could be used as an appropriate reference of dietary AAếs for red sea bream In Japanese ounder and European sea bass, AA prole of sh meal could be more suitable as a reference AA prole in the diet (Alam et al 2002; Peres & Oliva-Teles 2007) However, Gomez-Requeni et al (2003) found that resembling the muscle indispensable AA prole was important in diet for gilthead sea bream for the best growth performance Compared between the AA proles of roes, muscle tissue and carcass of LMB, the AA proles of the muscle tissue and carcass seemed to be a much better reference index for estimation of the AA requirements of this species (Portz & Cyrino 2003) In this study, the AA prole of the muscle was chosen as a reference AA prole in the diet Resultantly, the parameters on NR and FCR (Table 3) were satisfactory, indicating that the muscle AA prole used in test diets is suitable for the assessment of AA requirements in LMB Thus, there seems a species-specic AA prole for reference diets in sh The mathematical model used to t doseresponse relationship also inuences the estimate of EAA requirement in sh There are four models to be used: an exponential model, a four-parameter logistic function, a saturation kinetics model and a broken-line model Which one would be more suitable for tting doseresponse data has been debated (Rodehutscord & Pack 1999; Bureau & Encarnacáao 2006) In this study, the broken-line model was used to determine the requirement estimate, because there was the highest correlation coecient (R2, 0.9787) of the four models above The broken-line model has, by far, been the most popular model used in sh nutrition study although non-linear models seem in accordance with the biological principle often referred to as ễlaw of diminishing returnế In the present study, the actual arginine concentration of the experimental diets was corrected by the apparent digestibility coecients, which resulted from the digestibility study to obtain an accurate estimate of the true requirement The use of semipuried diets in estimating AA requirements of sh gives raise to some concerns about AA digestibility, which, if low, may articially elevate the estimated requirement (National Research Council (NRC) 1993) Luzzana et al (1998) observed that the arginine requirement of coho salmon, corrected by the apparent digestibility coecients, was more conservative than the values reported by Klein & Halver (1970), which did not take AA digestibility into account Therefore, the use of digestibility coecients would allow a more reliable and conservative estimate of the true AA requirement of sh studied In higher animals, numerous studies have shown a modulating role of dietary arginine in immune function Sharma et al (2004) reported that arginine supplementation in stressed rats and mice resulted in enhancement of humoral and cellular immune responses Tayade et al (2006) demonstrated that arginine supplementation in chickens immunized with intermediate plus strain of infectious bursal disease vaccine improved the humoral immune response Similarly, dietary arginine supplementation enhanced the immune status of pregnant sows, neonatal pigs and weaned pigs, thereby reducing morbidity and mortality in response Aquaculture Nutrition 18; 107116 ể 2011 Blackwell Publishing Ltd to infectious pathogens (Tan et al 2009) In sh, however, little information has been available about the eect of dietary arginine intake on sh immunity In the present study, high survival rates with above 98% (Table 4) and no pathological symptoms (in the text) were found in LMB fed dierent dietary arginine levels, and CH50 activity was not aected by the dietary treatment Judged from these, it seems that LMB would have a much lower dietary arginine requirement for immunity than for growth However, serum protein content, serum lysozyme activity and respiratory burst activities of head kidney leucocytes were improved with increased dietary arginine to a dierent extent (Table 7) Further insight is needed into whether longer-term arginine deciency would react in the similar manner in LMB In conclusion, arginine is essential for growth of LMB The dietary digestible arginine requirement for this sh was 19.1 g kg)1 of the dry diet (41.6 g kg)1 of the dietary protein) when estimated by broken-line analysis on WG Some aspects of immunity were sensitive to dietary arginine level The data generated in the present study will be useful in developing well-balanced practical diets for this species This work was nanced by a grant (No 2006-6-4) from the Shanghai Science & Technology Tackling Key Problems in Agriculture and a grant (No 10320503100) from the Shanghai Science & Technology Committee The authors thank the sta of Shanghai Nonghao Feed Co Ltd for their logistic support during this study We also thank Jianzhong Ma, Jie Zhou, Qinglang Liang and Wenwen Xiao for their assistance with sampling Ahmed, I & Khan, M (2004) Dietary arginine requirement of ngerling Indian major carp, Cirrhinus mrigala (Hamilton) Aquac Nutr., 10, 217225 Alam, M.S., Teshima, S., Koshio, S & Ishikawa, M (2002) Arginine requirement of juvenile Japanese ounder Paralichthys olivaceus estimated by growth and biochemical parameters Aquaculture, 205, 127140 Alam, M.S., Teshima, S., Yaniharto, D., Sumule, O., Ishikawa, M & Koshio, S (2005) Assessment of reference dietary amino acid pattern for juvenile red sea bream, Pagrus major Aquacult Int., 13, 369379 Ambardekar, A.A., Reigh, R.C & Williams, M.B (2009) Absorption of amino acids from intact dietary proteins and puried amino acid supplements follows dierent time-courses in channel catsh (Ictalurus punctatus) Aquaculture, 291, 179187 Aquaculture Nutrition 18; 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Aquaculture, 295, 614 Ytteborg, E., Bổverfjord, G., Torgersen, J., Hjelde, K & Takle, H (2010) Molecular pathology of vertebral deformities in hyperthermic Atlantic salmon (Salmo salar) BMC Physiol., 10, 12 Aquaculture Nutrition 18; 1220 ể 2011 Blackwell Publishing Ltd Aquaculture Nutrition doi: 10.1111/j.1365-2095.2011.00872.x 2012 18; 2134 1 1 4 1 1,2 1 3 1 Aquaculture. .. and growth in the Indian white shrimp Fenneropenaeus indicus Aquaculture, 252, 516524 Aquaculture Nutrition 2012 18; 1220 doi: 10.1111/j.1365-2095.2011.00871.x 1,2 5 1 3 6,7 1 3 4 1 1 2 NIFES, National Institute of Nutrition and Seafood Research, Bergen, Norway; MarinPet AS, Stavanger, Norway; 3 Institute of Marine Research, Matre Aquaculture Research Station, Matredal, Norway; 4 Institute... Anim Feed Sci Technol., 118, 109119 Zhou, Z., Liu, Y., He, S., Shi, P., Gao, X., Yao, B & Ringứ, E (2009) Eects of dietary potassium diformate (KDF) on growth performance, feed conversion and intestinal bacterial community of hybrid tilapia (Oreochromis niloticus $ ã O aureus #) Aquaculture, 2 91, 8994 Aquaculture Nutrition 18; 2134 ể 2011 Blackwell Publishing Ltd Aquaculture Nutrition doi:... ễP120ế is a polygon block (kneading element) Screw conguration 2 (SCF2) was similar to SCF1, except that the two ễ60L20ế elements were continuous to each other In Exp 1, the untreated FFSBM was extruded using SCF1, whereas the pretreated FFSBM were extruded using SCF2 In Exp 2, SBM-based diets were extruded using SCF1, and the extruder temperature was increased from 110 to 130 and 150 C by increasing the... 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Aquaculture, 2 61, 715728 Fjelldal, P.G., Hansen, T.J & Berg, A.E (2007) A radiological study on the development of vertebral deformities in cultured Atlantic salmon (Salmo salar L.) Aquaculture, 273, 721728 Fjelldal, P.G., Hansen, T., Breck, O., Sandvik, R., Waagbứ, R., Berg, A & ỉrnsrud, R... 463472 Vagsholm, I & Djupvik, H (1998) Risk factors for spinal deformities in Atlantic salmon (Salmo salar L.) J Fish Dis., 21, 4753 Vielma, J & Lall, S.P (1998) Phosphorus utilization by Atlantic salmon (Salmo salar) reared in freshwater is not inuenced by higher dietary calcium intake Aquaculture, 160, 117128 Waagbứ, R (2008) Reducing production-related diseases in farmed sh In: Improving Farmed Fish Quality