Aquaculture nutrition, tập 17, số 5, 2011

Aquaculture Nutrition doi: 10.1111/j.1365-2095.2010.00823.x 2011 17; 469481 College of Animal Sciences, Zhejiang University, Hangzhou, China Received 18 April 2010, accepted 26 July 2010 An 8-week feeding trial was conducted to determine the eects of dietary methionine level on juvenile black sea bream Sparus macrocephalus Fish (initial body weight: 14.21 0.24 g) were reared in eighteen 350-L indoors ow-through circular breglass tanks (20 sh per tank) Isoenergetic and isonitrogenous diets contained six levels of L-methionine ranging from 7.5 to 23.5 g kg)1 of dry diet in 3.0 g kg)1 increments at a constant dietary cystine level of 3.1 g kg)1 Growth performance and feed utilization were signicantly inuenced by dietary methionine levels (P < 0.05) Maximum weight gain (WG), specic growth rate (SGR), feed eciency ratio, protein eciency ratio and protein productive value (PPV) occurred at 17.2 g methionine kg)1 diet, beyond which they showed declining tendency Protein contents in whole sh body and dorsal muscle were positively correlated with dietary methionine level, while muscle lipid content was negatively correlated with it Apparent digestibility coecients (ADCs) of dietary nutrients were signicantly aected by dietary treatments except for ADCs of crude lipid Fish fed the grade level of methionine demonstrated a signicant improvement in wholebody methionine content, total essential amino acids P P ( EAA), total non-essential amino acids ( NEAAs) and P P EAA/ NEAA ratio (P < 0.05) Regarding serum characteristics, signicant dierences were observed in total cholesterol, glucose and free methionine concentration (P > 0.05), while total protein level and triacylglycerol concentration kept relatively constant among treatments (P < 0.05) Analysis of dose response with second-order polynomial regression on the basis of either SGR or PPV, the optimum dietary methionine requirements of juvenile black sea bream were estimated to be 17.1 g kg)1 of diet (45.0 g kg)1 methionine of protein) and 17.2 g kg)1 of diet (45.3 g kg)1 methionine of protein) in the presence of 3.1 g kg)1 cystine, respectively KEY WORDS: growth, L-methionine, requirement, Sparus macrocephalus juvenile ể 2010 Blackwell Publishing Ltd Correspondence: Shao Qingjun, Aquaculutre Nutrition Laboratory, College of Animal Sciences, Zhejiang University, Hangzhou 310029, China E-mail: qjshao@zju.edu.cn Protein, especially when derived from sh meal, is the most expensive nutrient in the preparation of diets for aquatic organisms (El-Saidy & Gaber 2002) Therefore, it is important to incorporate inexpensive protein ingredients in the formulation of sh feed by taking care of essential amino acids (EAA) balances (Kaushik et al 2004; Azaza et al 2008; Mart nez-Llorens et al 2009; Sardar et al 2009) Lysine and methionine are the most limiting amino acids in feed ingredients used in diets for sh, especially, when plant protein sources are used to replace sh meal (Abimorad et al 2009; Sardar et al 2009) Methionine is known as a precursor of choline and various other metabolic processes (Ruchimat et al 1997; Kasper et al 2000) As cystine can only be synthesized from a methionine precursor, a portion of the methionine requirement can be spared by cystine in some sh species (approximately 4060%) (Moon & Gatlin 1991; Kim et al 1992; Grin et al 1994; Go & Gatlin 2004) Therefore, the requirement for total sulphur amino acids (TSAA) can be met by either methionine alone or the proper mixture of methionine and cystine (Moon & Gatlin 1991) So it is important to consider the dietary cystine content to quantify the methionine requirement of the cultured species for maximum growth and ecient feed utilization (Luo et al 2005) In a previous study, we have evaluated the optimal dietary lysine requirement for black sea bream (Sparus macrocephalus) (Zhou et al 2010a) However, so far, the quantitative dietary requirement for methionine is still unknown for this sh species Black sea bream is a valuable commercial sh species cultured in China, Japan, Korean and some other countries of Southeast Asia (Nip et al 2003; Gonzalez et al 2008) It is highly appreciated as an excellent aquaculture species for intensive culture because of its resistance to diseases, ability to tolerate a wide range of environmental conditions, high stocking densities and relative fast growth rate under intensive culture and good quality meat (Hong & Zhang 2003; Shao et al 2008) However, trash sh was used for cultured black sea bream in China, which could not meet the nutritional requirements, and was dicult to store and easy to pollute aquaculture environment Thus, the formulation of a nutritionally adequate and cost-eective feed is most important for successful and sustainable culture of this sh species Up to now, a few studies have been conducted on the nutrient requirements of black sea bream (Ma et al 2008; Shao et al 2008; Zhou et al 2010a,b; Zhang et al 2010) The purpose of this study was to quantify the dietary methionine requirement at a constant dietary cystine level for black sea bream juveniles Ingredients and proximate composition of the experimental diets are presented in Table 1, amino acid compositions (g kg)1 dry diet, L-form) of dietary ingredients in Table and the analysed EAA contents for each diet are given in Table Six diets were maintained isonitrogenous by decreasing the levels of glutamic acids as the methionine level was increased Experimental diets contained 380 g kg)1 crude protein, which was slightly lower than the optimum protein requirement suggested in our preliminary experiment (410 g kg)1, Zhang et al 2010) to ensure the maximum utilization of methionine for growth and limited catabolism for energy (Wang et al 2005; Luo et al 2005) The 280 g kg)1 dietary protein was supplied by sh meal and soybean protein concentrate, and the remaining by a mixture of crystalline amino acids (CAAs) without methionine to simulate an amino acid prole found in 380 g kg)1 whole-body protein of black sea bream The basal diet (diet 1) contained the minimum level of methionine from sh meal and soybean protein concentrate The nal levels of methionine were conrmed by amino acid analysis, and the values were 7.5, 10.9, 14.1, 17.2, 20.6 and 23.5 g kg)1, respectively, by adding incremental levels of crystalline methionine ranging from to 15 g methionine kg)1 diet (Table 3) The CAAs were coated by carboxymethylcellulose (CMC) and j-carrageenan as described by Wang et al (2005) In preparing experimental diets, all dry ingredients as well as coating CAA mixture were nely ground, weighed, mixed manually for and then transferred to a food mixer for another 15 mixing Chromic oxide (Cr2O3), which was used as external indicator for digestibility determination, was dissolved in 100 mL of distilled water (about 40%, v/w) and sprayed with an atomizer on the mash during mixing, then sh oil and corn oil were added slowly while mixing During mixing, N NaOH was added to establish a pH level of 78 (Wilson et al 1977) Distilled water was added to achieve a proper consistency, and the mixture was further homogenized and extruded through a 3-mm die by food processor (Modle L-2730026; EHSY, Co., Shanghai, China) The noodle-like diets were dried at 23 C for 72 h with air condition and electrical fan Dried noodles were broken into particles, sieved to remove particles above mm and then stored in a refrigerator at )20 C A representative sample was taken for proximate analysis (Table 1) Black sea bream were obtained from Marine Fisheries Research Institute of Zhejiang province in Zhoushan, China Prior to initiation of the feeding trial, all sh were kept in 800-L circular berglass tanks and fed with diet for weeks At the beginning of the experiment, 20 uniformsized and healthy sh (initial mean weight: 14.21 0.24 g, mean SD, n = 360) in good health and condition were stocked in each berglass tank (350-L water volume) Each experimental diet was randomly assigned to triplicate tanks in a completely randomized design Each ow-through tank was supplied with sand-ltered aerated seawater at a owing rate of L min)1 Fish were maintained under a natural photoperiod, and the temperature, ammonia-nitrogen and salinity of the seawater in tanks were 28 C, 0.02 0.04 mg L)1 and 29 g L)1, respectively pH was 8.18.3, and dissolved oxygen concentrations were above 5.0 mg L)1 at any point during the experiment by using air stones with continuous aeration Experimental sh were fed by hand twice daily (0800 h and 1600 h), which were fed slowly little by little to prevent waste of feeds When the experimental feeds were supplied, the sh would swim to the water surface to ingest the feeds As long as sh were fed to satiation, they would never come up to water surface again Hence, their apparent satiation could be judged by feeding behaviour observation, and the feed losses could also be avoided almost completely The experiment lasted for weeks and feed consumption was recorded daily Tanks were thoroughly cleaned as needed and mortality was checked daily Faeces were collected using a faecal collection column similar to the one described by Cho & Kaushik (1990) All Aquaculture Nutrition 17; 469481 ể 2010 Blackwell Publishing Ltd Table Composition and proximate analysis of experimental diets (g kg)1 diet) Diets no Diet Ingredients Fish meal 280 Soybean protein concentrate 120 Carboxymethylcellulose 40 Crystalline amino acid premix 106 Methionine Glutamic acid 15 Fish oil 40 Corn oil 80 Mineral premix1 15 Vitamin premix2 15 Others3 289 Total 1000 Proximate analysis (g kg)1 dry matter)4 Crude protein 382.6 Crude lipid 149.6 Ash 139.1 Moisture 100.1 Methionine 7.5 Cystine 3.1 Gross energy (kJ g)1 diet) 15.7 Diet Diet Diet Diet Diet 280 120 40 106 12 40 80 15 15 289 1000 280 120 40 106 40 80 15 15 289 1000 280 120 40 106 40 80 15 15 289 1000 280 120 40 106 12 40 80 15 15 289 1000 280 120 40 106 15 40 80 15 15 289 1000 385.7 145.2 137.9 99.1 10.9 3.2 15.6 383.7 147.6 140.3 99.6 14.1 3.1 15.7 381.0 145.2 135.8 99.3 17.2 3.1 15.5 385.8 150.6 133.6 100.2 20.6 3.0 15.8 380.9 146.6 134.3 100.3 23.5 3.2 15.5 Mineral premix (g kg)1 of premix): Na2SiO3, 0.4; CaCO3, 350; NaH2PO4ặH2O, 200; KH2PO4, 200; MgSO4ặ7H2O, 10; MnSO4ặH2O, 2; CuCl2ặ2H2O, 1; ZnSO4ặ7H2O, 2; FeSO4ặ7H2O, 2; NaCl, 12; KI, 0.1; CoCl2ặ6H2O, 0.1; Na2MoO4ặ2H2O, 0.5; AlCl3ặ6H2O, 1; and KF, Vitamin premix (mg kg)1 diet): retinyl acetate, 40; cholecalciferol, 0.1; DL-a-tocopheryl acetate, 80; menadione, 15; niacin, 165; riboflavin, 22; pyridoxine HCl, 40; thiamin mononitrate, 45; D-Ca pantothenate, 102, folic acid, 10; vitamin B-12, 0.9; inositol, 450; ascorbic acid, 150; Na menadione bisulphate, 5; thiamin, 5; choline chloride, 320 and p-aminobenzoic acid, 50 Others (%): a-starch, 180; sodium dihydrogen phosphate, 25; j-carrageenan, 25; a-cellulose, 54; Cr2O3, Values for the proximate analysis of the test diets are means of triplicate analyses Table Amino acid composition (g kg)1 diet) of dietary ingredients for experimental diets (excluding tryptophan) Amino acids EAAs Val Leu Ile Phe Thr His Arg Lys Met NEAAs Glu Gly Ala Tyr Asp Ser Pro Cys Supplied by 280g fish meal kg)1 diet Supplied by 120 g soybean protein concentrate kg)1 diet Supplied by crystalline amino acids Total 380 g kg)1 whole-body protein 10.6 15.0 9.2 8.7 7.1 4.4 13.0 14.7 4.4 4.8 6.9 4.2 4.7 2.8 2.0 6.0 5.2 1.3 9.5 10.6 8.7 2.6 8.5 1.0 8.6 13.0 Variable 24.9 32.5 22.1 16.0 18.4 7.4 27.6 32.9 Variable 24.9 32.5 22.1 16.0 18.4 7.4 27.6 32.9 14.5 24.5 14.7 13.0 4.9 16.7 5.6 10.4 1.6 14.5 3.4 3.5 2.2 9.1 2.8 3.6 1.5 Variable 6.4 2.5 9.6 15.5 6.7 2.8 Variable 24.5 19.0 16.7 41.3 15.1 16.8 3.1 59.5 24.5 19.0 16.7 41.3 15.1 16.8 3.0 EAAs, essential amino acids; NEAAs, non-essential amino acids Aquaculture Nutrition 17; 469481 ể 2010 Blackwell Publishing Ltd Table Analysed essential amino acid (excluding tryptophan) contents in the experimental diets EAAs (g kg)1 diet) Val Leu Ile Phe Thr His Arg Lys Met Diets Diet Diet Diet Diet Diet Diet 30.4 41.1 21.1 12.6 21.2 9.8 17.2 31.7 7.5 31.5 41.3 20.9 12.4 21.1 10.2 17.2 32.2 10.9 30.6 40.9 20.9 12.3 21.6 10.2 17.6 32.2 14.1 30.8 41.2 21.3 12.2 22.1 9.7 17.3 32.1 17.2 31.2 41.2 20.7 12.5 21.1 9.9 17.5 31.9 20.6 31.6 40.9 20.9 12.7 21.3 9.9 17.4 31.7 23.5 EAA, essential amino acid possible care was taken during feeding so that feed losses could be avoided almost completely Two hours after the nal feeding of the day, the drain pipe and faecal collection columns were thoroughly cleaned with a brush to remove the residual feed and faeces from the system Faeces were then allowed to settle overnight, and faecal samples were collected at 06:00 each morning before the next feeding Faeces collected from the settling columns were immediately ltered with lter papers to separate other materials such as dirt particles and then stored at )20 C for chemical analysis Faeces from the same tank were pooled over the sampling period to provide sucient faecal matter for analysis Ten sh at the start of the feeding trial were sampled and stored frozen ()20 C) for the analysis of proximate carcass composition At the termination of the 8-week experiment, approximately 24 h after the last feeding, all sh were counted and individually weighed Three sh from each tank were anesthetized (MS-222, Sigma, St Louis, MO, USA at 80 mg L)1) and then stored at )20 C for subsequent wholebody proximate analysis Blood samples were drawn from the caudal vein of ve sh per tank with a 27-gauge needle and 1- mL syringe Blood samples were immediately centrifuged at 836 g for 10 (4 C) to obtain serum to measure nutrient levels (Ai et al 2006), and serum was stored at )20 C until use Dorsal muscles were obtained from all the remaining sh in each tank and stored at )20 C for subsequent proximate analysis Pooled sh samples (including whole body, dorsal muscle, serum and faeces) in each tank were analysed in triplicate for proximate composition Moisture, ash, crude protein and crude lipid were determined following methods of the Association of Ocial Analytical Chemists (AOAC 1995) Moisture concentration was determined by drying minced samples for h in a forced-air oven maintained at 105 C Ash content was analysed by incinerating samples at 600 C for 24 h in a mue furnace Crude protein was estimated as Kjeldahl-nitrogen using factor 6.25, and crude lipid was determined by Soxhlet extraction with petroleum ether for h The concentrations of total protein, total cholesterol, triacylglycerol and glucose in the serum of juvenile black sea bream were all measured within days, using the Diagnostic Reagent Kit purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu Province, China) according to the manufacturerếs instructions Faecal samples were oven dried at 105 C to a constant weight for the determination of dry matter Then the dried samples were nely ground with a mechanical mortar and pestle and sieved using a 1-mm screen prior Chromic oxide (Cr2O3) content in diets and faeces was determined by the method of Furukawa & Tsukahara (1966) Gross energy in feeds and faeces was determined by Automatic Isoperibol Calorimeter (Modle 6300; Parr Instrument Company, Moline, IL, USA) in the laboratory in the Institute of Feed Science, Zhejiang University The amino acid compositions of all samples including ingredients, diets, whole sh body and serum were analysed following acid hydrolysis using an automatic amino acid analyser (Hitachi 835-50, Tokyo, Japan) with a column (Hitachi custom ion exchange resin no 2619) in the laboratory in Institute of Feed Science, Zhejiang University In brief, performic acid oxidation was performed prior to hydrolysis to oxidize methionine at )10 C for h to obtain methionine sulfone Then sodium metabisulte was added to decompose surplus performic acid Subsequently, amino acid was liberated from protein by hydrolysis with N HCl for 24 h Hydrolysed samples were diluted with sodium citrate buer, pH was adjusted to 2.2 and individual amino acid components were separated by ion exchange chromatography Cystine content in diets was determined from the same acid hydrolysate after treatment with dithiothreitol and sodium tetrathionate (Inglis & Liu 1970) Tryptophan could not be detected after acid hydrolysis and it was excluded from analysis at the present experiment All data were subjected to the analysis of variance and correlation analysis when appropriate using the software of SPSS for Windows (ver16.0; Chicago, IL, USA) Dierences between the means were tested by Tukeyếs multiple range test Dierences were considered signicant at P < 0.05 Broken- Aquaculture Nutrition 17; 469481 ể 2010 Blackwell Publishing Ltd (P < 0.05), although there were dierent decline extent for sh fed excessive methionine level diets The highest FER (0.89), PER (2.35) and PPV (0.43) were recorded in the sh fed diet containing 17.2 g kg)1 methionine Hepatosomatic index (HSI) was higher in sh fed 23.5 g methionine kg)1 diet than that of in sh fed diet without methionine supplement, but dierences among the other treatments were not signicant (P > 0.05) Increasing dietary methionine level decreased condition factor (CF) values although there was no signicant dierence (P > 0.05) Based on SGR, the optimum requirement of dietary methionine was estimated to be 17.1 g kg)1 of diet (45.0 g kg)1 of protein) using break-point regression method analysis (Fig 1) When PPV was plotted against dietary methionine, the optimum dietary methionine requirement was 17.2 g kg)1 of diet (45.3 g kg)1 of protein) (Fig 2) Because the experimental diets contained 3.1 g kg)1 of cystine, the corresponding requirements of this sh for TSAA (Met + Cys, TSAA) were calculated to be 21.1 g kg)1 of diet (53.2 g kg)1 of dietary protein) and 21.5 g kg)1 of diet (56.6 g kg)1 of dietary protein), respectively The eects of graded levels of dietary methionine on whole-body and dorsal muscle composition were described in Table Protein content of whole body showed an increasing trend with increasing dietary methionine levels (P < 0.05), but there was a slight decline for sh fed the diets over 17.2 g kg)1 methionine No signicant dierences were observed in body lipid, ash or moisture among the dietary line regression analysis was performed on specic growth rate (SGR) and protein productive value (PPV) to establish the dietary requirement of methionine for black sea bream The equation used in the model is Y = L + U (R ) X), where Y is the parameter (SGR or PPV) chosen to estimate the requirement, L is the ordinate and R is the abscissa of the breakpoint R is taken as the estimated requirement and U is the slope of the line for X (Robbins et al 1979) Growth performance, body-organ indices and feed utilization for juvenile black sea bream given graded levels of methionine for weeks are shown in Table Survival was the highest for sh fed the diet containing 17.2 g kg)1 methionine and showed no signicant dierence among dietary treatments, and no other nutritional deciency signs were observed in black sea bream fed methionine-decient diets Mean feed intake was signicantly lower in sh fed 7.5 g methionine kg)1 diet than those of sh fed 14.1 and 17.2 g methionine kg)1 diets, and the values were insignicantly dierent among the other groups (P > 0.05) WG and SGR increased with dietary increasing methionine level up to 17.2 g kg)1, but further additions of methionine decreased grow rate of black sea bream Fish fed diet showed the lowest feed eciency ratio (FER) (0.78), protein eciency ratio (PER) (2.10), nitrogen gain (1.05) and PPV (0.35), and those indices of sh were signicantly improved by methionine supplementation Table Growth performance, body-organ indices and feed utilization of black sea bream juvenile fed the diets with graded levels of methionine for weeks Diets (methionine level, g kg)1) IBW FBW Survival WG SGR CF HSI MFI FER PER N gain PPV Diet (7.5) Diet (10.9) Diet (14.1) Diet (17.2) Diet (20.6) Diet (23.5) 14.01 52.28 95.00 273.44 2.49 2.01 2.20 0.88 0.78 2.09 1.05 0.30 14.20 58.02 96.67 308.64 2.66 1.96 2.27 0.95 0.82 2.20 1.25 0.35 14.30 59.68 96.67 318.39 2.69 1.96 2.38 0.96 0.84 2.27 1.31 0.37 14.27 62.27 100.00 335.50 2.78 1.99 2.27 0.97 0.89 2.32 1.42 0.43 14.23 60.60 96.67 326.08 2.73 1.93 2.23 0.95 0.87 2.27 1.37 0.40 14.25 59.40 91.67 316.86 2.69 1.91 2.51 0.95 0.85 2.23 1.37 0.40 0.48 0.73c 5.00 10.56d 0.05c 0.06 0.16b 0.01b 0.02d 0.03d 0.01c 0.01c 0.18 1.47b 2.87 4.44c 0.02b 0.07 0.06ab 0.03ab 0.02c 0.02c 0.05b 0.01bc 0.26 1.48ab 5.77 4.25bc 0.03b 0.03 0.06ab 0.03a 0.01bc 0.03b 0.05b 0.02b 0.10 2.70a 0.00 6.03a 0.05a 0.13 0.13ab 0.03a 0.01a 0.03a 0.06a 0.02a 0.19 1.29ab 5.77 5.07ab 0.04ab 0.05 0.08ab 0.03ab 0.01ab 0.02b 0.03a 0.04ab 0.18 1.71ab 2.89 4.83bc 0.01b 0.25 0.10a 0.04ab 0.02b 0.01bc 0.02a 0.01ab Values are presented as mean SD (n = 3); values with different superscripts in the same row differ significantly (P < 0.05) Survival (%) = 100 ã final fish number/initial fish number; weight gain (WG) (%) = 100 ã (FBW ) IBW)/IBW; Specific growth rate (SGR) (% day)1) = 100 ã (ln FBW ) ln IBW)/day; CF (Condition factor) (g cm)3) = 100 ã (live weight, g)/(body length, cm)3; hepatosomatic index (HSI) = 100 ã (liver weight, g)/(body weight, g); intraperitoneal ratio (IPR) = 100 ã (intraperitoneal fat weight)/(body weight); mean feed intake (MFI) = g fish)1 day)1; feed efficiency ratio (FER) = 100 ã wet weight gain in g/dry diet fed in g; protein efficiency ratio (PER) = weight gain in g/protein intake in dry basis in g; N gain (%) = 100 ã (FBW ã Nf ) IBW ã Ni); protein productive value (PPV) = protein gain in g/protein fed in dry basis in g; where IBW is initial mean body weight and FBW is final mean body weight; Nf and Ni are the N contents of the carcass at the end and at the beginning of the feeding trial Aquaculture Nutrition 17; 469481 ể 2010 Blackwell Publishing Ltd were more variable and could not be related to dietary treatments (P > 0.05) Data on nutrientsế digestibility of black sea bream fed the dierent experimental diets are presented in Table Fish fed the 17.2 g methionine kg)1 diet had higher apparent digestibility coecients (ADCs) of dry matter than that of sh fed the 7.5 g methionine kg)1 diet (P < 0.05), but no signicant dierences were found among sh fed the other diets ADCs of crude protein was lower in sh fed the diet without methionine supplement (P < 0.05), while the values in the rest groups were very stable (P > 0.05) ADCs of gross energy increased signicantly with increasing methionine level up to 17.2 g methionine kg)1 diet and then decreased slightly (P < 0.05) However, ADCs of crude lipid were unaected by dietary treatments (P > 0.05) Increasing dietary methionine level increased EAA and non-essential amino acids (NEAA) contents of whole sh P P body, and the EAA/ NEAA ratio was also increased signicantly (P < 0.05) (Table 7), and the values peaked at sh fed 23.5, 20.6 and 23.5 g methionine kg)1 diets, respectively The contents of Val, Leu and Ile in dierent groups were very stable (P > 0.05); however, the levels of rest EAAs in whole body showed increasing tendency (P < 0.05) with increasing dietary methionine level Body methionine concentration increased signicantly with dietary methionine levels from 7.5 to 20.6 g kg)1, but kept relatively constant thereafter (P > 0.05) In serum prole, total cholesterol concentration was highest in sh fed 7.5 g methionine kg)1 diet (P < 0.05), but showed no signicant dierences for sh fed other diets (Table 8) Fish supplied with 7.5 and 23.5 g methionine kg)1 diets showed higher glucose content (P < 0.05), compared to that of sh fed the diets containing 10.9 and 20.6 g kg)1 methionine Serum free methionine concentration signi- treatments (P > 0.05) Muscle protein content was positively correlated with dietary methionine level, while lipid content was negatively correlated with it Ash and moisture contents 2.85 Y = 2.782 0.0282 (17.1 X) (X 17.1) R2 = 0.9029 2.8 SGR (%/day) 2.75 2.7 2.65 Y = 2.782 0.0137 (X 17.1) (X > 17.1) 2.6 R2 = 0.9106 2.55 2.5 Xopt = 17.1 2.45 2.4 11 13 15 17 19 21 23 25 Dietary methionine level (g kg1 dry diet) Figure The relationship between weight gain (SGR, %/day, y) and dietary methionine levels (g kg)1, x) of juvenile black sea bream fed with the experimental diets for weeks SGR, specic growth rate 0.5 Y = 0.0132X + 0.1993 Y = 4.13 0.45 R = 0.9275 PPV 0.4 0.35 0.3 Xopt = 17.21 0.25 0.2 11 13 15 17 19 21 23 25 Dietary methionine level (g kg1 dry diet) Figure The relationship between protein productive value (PPV, y) and dietary methionine levels (g kg)1, x) of juvenile black sea bream fed with the experimental diets for weeks Table Effect of dietary methionine level on proximate compositions of whole body and muscle of juvenile black sea bream (% on wet matter basis) Diets (methionine level, g kg)1) Diet (0.75) Whole body Protein Lipid Ash Moisture Dorsal muscle Protein Lipid Ash Moisture Diet (10.9) Diet (14.1) Diet (17.2) Diet (20.6) Diet (23.5) 16.94 13.27 4.78 63.77 0.16b 0.34 0.29 0.16 17.52 13.36 4.80 64.08 0.18ab 1.60 0.37 1.01 17.66 13.29 4.62 64.17 0.10a 0.87 0.29 3.01 18.03 13.50 4.79 63.47 0.23a 1.27 0.21 0.15 18.05 13.63 4.86 63.40 0.31a 1.51 0.17 1.03 17.78 13.35 4.66 64.18 0.37a 1.52 0.18 1.82 19.27 5.04 1.45 73.48 0.53b 0.42a 0.08 1.11 19.76 4.57 1.41 73.52 0.43ab 0.11ab 0.03 0.73 20.36 4.48 1.39 73.52 0.70ab 0.18ab 0.03 0.48 20.49 4.03 1.43 73.89 0.30a 0.50b 0.02 0.33 20.57 4.16 1.41 73.19 0.14a 0.09b 0.03 0.54 20.46 4.19 1.41 73.19 0.16a 0.18b 0.02 0.57 Values are presented as mean SD (n = 3); values with different superscripts in the same row differ significantly (P < 0.05) Aquaculture Nutrition 17; 469481 ể 2010 Blackwell Publishing Ltd Table Effect of dietary methionine level on apparent digestibility coefcients (ADCs) of main nutrients in diets for juvenile black sea bream for weeks Diets (methionine level, g kg)1) Diet (7.5) Dry matter Protein Lipid Gross energy 85.17 95.32 96.59 88.17 Diet (10.9) b 1.24 0.50b 0.36 0.40c 87.86 97.15 97.82 89.12 ab 1.29 0.42a 0.51 0.43bc Diet (14.1) 88.18 96.65 97.98 90.29 ab 1.68 0.48a 0.35 0.30b Diet (17.2) 90.21 97.30 97.95 91.83 a 0.77 0.63a 0.34 0.21a Diet (20.6) 88.04 96.78 97.28 91.45 ab 0.54 0.22a 1.26 0.72ab Diet (23.5) 87.47 96.63 98.10 90.19 1.39ab 0.35a 0.52 0.53b ADC of dry matter (%) = 100 ã [1 ) (dietary Cr2O3)/faecal Cr2O3] ADCs of nutrients or energy (%) = 100 ã [1 ) (F/D ) DY/FY)], where F is the per cent of nutrients or energy in faeces, D is the per cent of nutrients or energy in diet, DY is the per cent of Cr2O3 in diet and FY is the per cent of Cr2O3 in faeces Values are presented as mean SD (n = 3); means with different superscript letters in the same row differ significantly (P < 0.05) Table Effect of dietary methionine levels on amino acid contents of whole body (g kg)1 on a dry matter basis) in juvenile black sea bream fed for weeks Diets (methionine level, g kg)1) Diet (7.5) EAA Val Leu Ile Lys Phe Thr His Arg Met NEAA Asp Tyr Pro Ser Glu Gly Ala P EAA P NEAA P P EAA/ NEAA Diet (10.9) Diet (14.1) Diet (17.2) Diet (20.6) Diet (23.5) 3.27 4.62 2.79 4.68 2.55 2.48 1.07 4.12 1.76 0.03 0.05 0.03 0.02c 0.02b 0.07c 0.02b 0.05b 0.03c 3.28 4.66 2.86 4.75 2.55 2.50 1.09 4.13 1.78 0.06 0.09 0.07 0.01b 0.05b 0.05c 0.02b 0.02ab 0.05c 3.27 4.93 2.85 4.81 2.67 2.59 1.10 4.17 1.83 0.05 0.04 0.07 0.02b 0.10ab 0.02b 0.03b 0.03ab 0.02c 3.30 4.80 2.84 4.92 2.71 2.66 1.09 4.18 1.97 0.03 0.17 0.09 0.01a 0.03ab 0.03ab 0.01b 0.03ab 0.06b 3.29 4.74 2.84 4.94 2.74 2.74 1.17 4.20 2.12 0.04 0.06 0.09 0.03a 0.07a 0.05a 0.03a 0.01ab 0.01a 3.35 4.75 2.89 4.99 2.77 2.72 1.19 4.23 2.14 0.03 0.05 0.07 0.04a 0.05a 0.04a 0.02a 0.03a 0.02a 4.37 1.75 3.03 1.90 7.68 5.74 4.29 27.34 28.99 0.94 0.02b 0.01 0.02 0.03 0.03c 0.03c 0.03b 0.06d 0.08d 0.09c 4.37 1.80 3.04 1.92 7.73 5.81 4.35 27.60 29.26 0.94 0.07b 0.04 0.07 0.01 0.05c 0.03c 0.06ab 0.15cd 0.27cd 0.01c 4.42 1.81 3.07 1.88 7.87 5.93 4.31 28.01 29.54 0.95 0.03ab 0.04 0.04 0.03 0.05b 0.05b 0.02ab 0.26c 0.15c 0.01bc 4.41 1.78 3.09 1.92 7.90 5.95 4.35 28.46 29.62 0.96 0.04ab 0.04 0.06 0.03 0.02ab 0.02ab 0.03ab 0.21b 0.10bc 0.02ab 4.49 1.76 3.14 1.96 7.98 6.03 4.40 28.78 30.08 0.96 0.02a 0.04 0.06 0.04 0.06a 0.05a 0.02a 0.11ab 0.19a 0.01a 4.42 1.78 3.09 1.95 7.97 6.00 4.38 29.05 29.83 0.97 0.04ab 0.04 0.02 0.04 0.01ab 0.03ab 0.05ab 0.06a 0.04ab 0.01a Values are presented as mean SD (n = 3); values with different superscripts in the same row differ significantly (P < 0.05) P P EAA, essential amino acids; NEAA, non-essential amino acids; EAA, total essential amino acids; NEAA, total essential amino acids cantly increased with increasing dietary methionine levels (P < 0.05) although there was no statistical dierence for sh fed 20.6 and 23.5 g methionine kg)1 diets (P > 0.05) Serum total protein content (ranging from 27.68 to 32.80 g L)1) showed an insignicantly increasing trend with increasing methionine level, while triacylglycerol concentration decreased from 5.47 to 3.93 mmol L)1, and no signicant differences were observed among treatments (P > 0.05) Doseresponse experiments with increasing supply of amino acid are accepted in principle as a method for determining Aquaculture Nutrition 17; 469481 ể 2010 Blackwell Publishing Ltd dietary amino acid requirements (Cowey 1995), and the model used to analyse the doseresponse relationship will inuence the estimate of requirements Because the SGR response of sh in the current study was linear, a broken-line model resulted in the lowest error term for estimating the requirement appeared to give a more precise empirical gure In previous studies, some researchers have demonstrated that cystine could spare dietary methionine portion about 60% in channel catsh (Harding et al 1977), 51% in yellow perch (Twibell et al 2000), 50% in red drum (Moon & Gatlin 1991; Go & Gatlin 2004), 42% in rainbow trout (Kim et al 1992) and 40% in hybrid striped bass (Grin et al 1994); the presence of dietary cystine reduces the amount of methionine Table Determination of serum parameters in juvenile black sea bream, fed with graded levels of methionine for weeks Diets (methionine level, g kg)1) )1 Total protein (g L ) T-CHO (mmol L)1) TG (mmol L)1) GLU (mmol L)1) Free met (mmol 100 mL)1) Diet (7.5) Diet (10.9) Diet (14.1) Diet (17.2) Diet (20.6) Diet (23.5) 27.68 9.33 5.47 9.66 8.22 30.56 7.27 5.36 6.52 8.39 31.29 6.76 4.94 6.43 8.61 32.79 6.60 4.71 6.61 8.79 30.52 6.61 3.93 6.39 8.96 32.80 6.99 4.98 7.45 9.14 1.06 0.70a 1.22 1.37a 0.06e 2.16 0.42b 0.47 1.35b 0.05d 1.94 0.21b 0.43 1.21b 0.07c 3.08 0.11b 0.49 0.58b 0.03bc 2.10 0.54b 0.47 0.74b 0.11ab 1.76 0.52b 0.96 0.68a 0.07a Values are presented as mean SD (n = 3); values with different superscripts in the same row differ significantly (P < 0.05) T-CHO, total cholesterol; TG, triacylglycerol; GLU, glucose concentration required for maximum growth Thus, Wilson & Halver (1986) suggested that sh have a TSAA (Met + Cys) requirement rather than a specic methionine requirement Methionine or total sulphur amino acids requirements have been reported varying from 22.0 to 65.0 g kg)1 of dietary protein in dierent sh species (De Silva & Anderson 1995) In the present study, the TSAA requirement of juvenile black sea bream was determined to be 53.2 g kg)1 of dietary protein (in the presence of dietary 3.1 g kg)1 cystine) based on growth performance This value was higher than that reported for some commercially important nsh species, namely, milksh (43.7 g kg)1, Borlongon & Coloso 1993), Jian carp (42.9 g kg)1, Tang et al 2009), large yellow croaker (40.2 g kg)1, Mai et al 2006), catla (35.5 g kg)1, Ravi & Devaraj 1991), yellowtail (32.8 g kg)1, Ruchimat et al 1997), grouper (32.3 g kg)1, Luo et al 2005), African catsh (32 g kg)1, Fagbenro et al 1998), Japanese ounder (31 g kg)1, Alam et al 2000) and yellow perch (30 g kg)1, Twibell et al 2000) But, the requirement of black sea bream is nearer to Indian major carp (55.0 g kg)1, Ahmed et al 2003) and rohu (55.8 g kg)1, Khan & Jafri 1993) The wide variation observed in the methionine requirement among species may be because of sh size, age, laboratory condition including feeding regime, feed allowance, water temperature, stock density and ingredients used for basal diet such as zein, casein, gelatin, gluten, soybean meal, shmeal and CAAs in various combinations (Tacon & Cowey 1985; Rodehutscord et al 1997; Forster & Dominy 2006; Nguyen & Davis 2009b) In the current work, higher levels of cystine were not tested so that it is not apparent whether this dietary level of cystine is adequate, and it could also be a possible reason for the relatively higher methionine requirement in this study However, in the study of rainbow trout, Walton et al (1982) observed similar growth performance of sh fed diets with or without cystine provided that the level of methionine was adjusted to meet the requirement level Because a portion of dietary methionine is converted to cystine the presence of dietary cystine reduces the amount of methionine required for maximum growth The replacement value of cystine for methionine and the use of methionine to provide for the total sulphur amino acid requirement of black sea bream should be further evaluated Amino acid balance in diets is necessary for optimal growth of sh (Wilson & Halver 1986), and most authors reported that sh fed dietary increasing methionine showed two dierent growth pattern: (i) increased with increasing dietary methionine and then remained constant when dietary methionine is higher than requirement (Ruchimat et al 1997; Coloso et al 1999; Alam et al 2001; Luo et al 2005; Nguyen & Davis 2009a) (ii) increased with increasing dietary methionine level and then decreased signicantly when dietary methionine is higher than requirement (Murthy & Varghese 1998; Mai et al 2006; Yan et al 2007) A few studies reported that none of the sh fed the test diets containing increased methionine showed a signicant dierence in growth (Sveier et al 2001; Espe et al 2008) In the present study, the growth response black sea bream fed increasing methionine diets is in accordance with the second pattern Reduction in the growth rate in sh fed diets with superuous level of TSAA may be because of toxic eects (Choo et al 1991), and excess amount of total sulphur amino acid in sh body would lead to extra energy expenditure towards deamination and excretion (Walton 1985; Murthy & Varghese 1998; Sveier et al 2001) FER obtained in the present study ranged from 0.78 to 0.89, which exhibited correlation with sh growth and seems satisfactory One possible reason may be that sh were slowly fed by hand little by little till apparent satiation (visual observation of sh feeding behaviour) and prevent the waste of feeds On the other hand, generally high FER suggested that black sea bream is able to utilize CAAs well in diets However, there was a marked decline in FER when sh fed methionine deciency diet or superuous methionine diets, indicating that the supplementing level of methionine should be optimized Feed intake was much lower when sh fed diet methionine-unsupplemented diets These results are similar to those reported in Aquaculture Nutrition 17; 469481 ể 2010 Blackwell Publishing Ltd large yellow croaker (Mai et al 2006), Indian major carp (Ahmed et al 2003) and yellow perch (Twibell et al 2000) In more recent work, Li et al (2009) reported that methionine deciency in aquafeeds may reduce/exhaust reservoirs of antioxidants such as ascorbic acid, glutathione and vitamin E in various tissues of sh, which may result in irreversible oxidative stress, further aggravating growth retardation and feeding depression, which could also partly explain the preset results The present study showed signicantly decreased PER at high dietary methionine It may be because of unbalance amino acids composition in diets and diverting amino acids into catabolic rather than anabolic processes (Cowey & Sargent 1979) However, values of nitrogen gain and PPV decreased insignicantly at higher methionine level diets when compared with sh fed optimal methionine level diet Similar results were observed in some previous studies (Ruchimat et al 1997; Luo et al 2005; Yan et al 2007) This is to be expected as growth is largely driven by protein deposition (Sa et al 2008) Indeed, it was concluded that using maximum nitrogen gain as response criteria results in higher EAAs requirement estimates than WG in rainbow trout and Atlantic salmon (Rodehutscord et al 1997; Hauler & Carter 2001; Espe et al 2007) Results were similar to our observation; the optimal methionine for PPV was estimated to be 17.2 g kg)1 diet (corresponding to 53.2 g kg)1 of dietary protein), which was slightly higher than the optimum requirement for growth in the present study In Asian sea bass, HSI were unaected by dietary methionine level (Coloso et al 1999), and in Atlantic salmon, low methionine intake resulted in a signicantly higher liver weight relative to body weight (Espe et al 2008) However, HSI was reported to increase with increased dietary methionine up to requirement level and kept constant at higher methionine levels in yellowtail (Ruchimat et al 1997) and rocksh (Yan et al 2007), similar in the present study In addition, Walton et al (1982) pointed out that the methionine/cystine ratio aected HSI, and HSI decreased when methionine increased with the cystine level was 0.5 g kg)1 diet However, when the cystine level was 20.5 g kg)1, increasing methionine level resulted in a slight increase in HSI There were, however, no signicant dierences observed in CF of black sea bream among dietary treatments Similar result was also found in Atlantic salmon (Sveier et al 2001) and cobia (Zhou et al 2006), but in contradiction with the observation in grouper (Luo et al 2005) Methionine has three major metabolic functions: as an EAA for protein synthesis; as a sulphur source for synthesis of other sulphur-containing biochemicals; and as a methyl donor for methylation reactions (Mehler 1986) In the present study, protein contents in whole body and muscle of Aquaculture Nutrition 17; 469481 ể 2010 Blackwell Publishing Ltd black sea bream both tended to increase with dietary methionine level up to the requirement level, beyond which it remained nearly unchanged, which is in agreement with other reports (Kim et al 1992; Ruchimat et al 1997; Alam et al 2000; Luo et al 2005) De la Higuera et al (1997) suggested protein synthesis and accretion in sh required all amino acids present simultaneously at the synthesis sites, or the process is impaired or prevented The variational tendency on protein accretion was also consistent to nitrogen retention values that obtained in the present study Among amino acids, the branched-chain amino acid leucine is clearly recognized as a most eective nutrient regulator of mRNA translation and proteolysis (Kimball & Jeerson 2004; Yoshizawa 2004; Nakashima et al 2005) However, the role of methionine acting a nutrient signal to regulate protein synthesis still needs extensive studies It has demonstrated that the rst step of the initiation of mRNA translation consists of the binding of initiator Met-tRNAi to the 40S ribosomal subunit to form the 43S preinitiation complex This initiation step may be inhibited by methionine deciency (Metayer et al 2008) With regard to the potential eect of methionine on intracellular kinases, studies are sparse but indicate that this sulphur amino acid may exert a signal function by inducing 70-kDa ribosomal protein S6 kinase (p70S6K) activation in mammals (Shigemitsu et al 1999; Stubbs et al 2002) Similar results have been found in an avian myoblast cell line (QM7) of quail origin (Tesseraud et al 2003), in which the methionine or leucine regulates S6K1 phosphorylation and protein synthesis It seems there may be also a regulation of protein synthesis by methionine in sh; however, the mechanism was not clear and more investigations are needed to conrm these aspects Lipid content of dorsal muscle in the present study was higher in sh fed methionineunsupplemented diet than those of sh fed the high-methionine diets, which might be because of better utilization of protein with reduced deposition of lipid in the presence of methionine resulting lean growth of sh (Sardar et al 2009) The results were consistent with Kim et al (1992) and Schwarz et al (1998), but in contrast with the study in grouper (Luo et al 2005) With regard to the eect of methionine levels on dietary nutrientsế digestibility, studies are sparse Digestibility data are important to the sh nutritionist because the nutrients contained in poorly digested ingredients are less available to support growth and metabolism (Lin et al 2004) ADCs of crude protein in this study were higher than 95.32% and averaging 96.64%, indicating that all the nitrogen sources provided in the experimental diets were well absorbed and utilized by the juvenile black sea bream It has reported that CAAs coated with CMC and j-carrageenan used in diets can improve the utilization eectiveness by reducing its solubility in water, and encapsulation or covalent binding may also help to slow the rate of release of CAAs to transport sites in the intestinal mucosa of sh (Millamena et al 1996; Alam et al 2004), thus protecting free amino acids in the gut long enough to improve their utilization eciency (SegoviaQuintero & Reigh 2004) ADCs of crude protein was signicantly higher in black sea bream fed the test diets supplying methionine when compared to those fed the control diet, which was consistent with the ndings by Espe et al (2008) and Chi et al (in press) In the present study, ADCs of gross energy showed a similar tendency among the groups as the FER; it is according to the result observed in salmon, in which the reduced feed eciency was to a large extent explained by reduced energy and protein digestibility (Mundheim et al 2004) The ADCs of crude of lipid in all groups were relatively higher (more than 96.59%) in present study, and independent of the dietary treatments, the values compare favourably to data obtained in other sparids (Santinha et al 1996; Robaina et al 1995; Biswas et al 2007) Nevertheless, it should be noted that extraction of faeces with petroleum ether does not extract the fatty acids bound as calcium soaps, and this extraction will therefore to some extent underestimate the amount of lipids in the faeces and overestimate lipid digestibility (Santinha et al 1996) In general, few investigations with eects of methionine supplemented on nutrientsế digestibility were reported Di- and tripeptides are absorbed into the enterocytes without any hydrolysis by microvillous peptidases (Jose et al 1997), and c-glutamyltransferase (c-GT) is involved in peptide transport (Grith & Meister 1980), and in the study with Jian carp, Tang et al (2009) found that there was signicant improvement in intestine and hepatopancreas weight, as well as c-GT activity by dietary methionine level Also in the study by Tang et al (2009), methionine was reported to improve intestinal creatine kinase activity, which plays a role in energy transfer in tissue Anyhow, it is necessary to conduct further studies to make clear this aspect To support the requirement estimated by growth response, whole sh body EAA contents of experimental black sea bream were analysed (Table 7) Dietary amino acid proles are known to inuence the postfeeding levels of free amino acids in sh tissues, such as plasma, liver, muscle and whole body (Twibell et al 2000; Zhou et al 2006; Espe et al 2007) Both the total EAA and NEAA content of whole sh body showed marked increase with increasing dietary methionine level, which was similar to other results which have demonstrated that dietary methionine level aects tissue free amino acids in sh (Grin et al 1994; Luo et al 2005) It was probably because dietary restriction of one EAA led to an increase in oxidation of other essential and non-essential amino acids present at normal levels in the diets (Ronnestad et al 2000; Ozorio et al 2002) Mai et al (2006) suggested methionine deciency in diet could inhibit methionine participating in protein synthesis and reduce its level in amino acid pool in tissues; however, in the present study, methionine concentration in sh body increased signicantly with dietary methionine levels, similar result was also observed in group (Luo et al 2005) According to Espe et al (2007), the P P ratio of EAA to NEAA was held close to to maximize amino acid utilization; however, in the present trial, the P P EAA/ NEAA ratio was observed to increase signicantly with dietary methionine level It has been demonstrated that excessive intake of one amino acid or disproportionate amounts of one amino acid aect the utilization of the other amino acids (Choo et al 1991; Coloso et al 1999), but the P P maximum value of EAA/ NEAA ratio in whole sh body occured in sh fed the highest methionine level diet The reason for this discrepancy is uncertain, and black sea bream might be able to assimilate excess dietary amino acids into tissues within a short period Currently, little information is available on the eect of dietary methionine on serum characteristics The free amino acid content in the blood initially reects the content of amino acids in the dietary protein following absorption (Yamada et al 1981; Simmons et al 1999), and methionine may accumulate in blood when the total dietary methionine supply exceeds the requirement (Mambrini et al 1999) In corroboration with the present ndings, Harding et al (1977), Schwarz et al (1998) and Zhou et al (2006) all observed that free methionine concentration in blood increased with increasing dietary methionine Plasma or serum methionine levels have been used to conrm dietary TSAA requirements derived from WG and FER data in some sh species (Harding et al 1977; Grin et al 1994); however, Cowey (1995) considers the free amino acids in plasma to be relatively unsuitable for the determination of amino acid requirements, because no close relationship exists between intake and concentration in animal tissues In the current study, serum methionine concentrations were positively related to the dietary supply; consequently, it could not be used evaluating methionine requirement for black sea bream There was an increasing tendency in serum protein concentration with dietary methionine level, which was similar with Luo et al (2005) The observation of higher serum cholesterol content for sh fed low-methionine diets compared with high-methionine diets was also Aquaculture Nutrition 17; 469481 ể 2010 Blackwell Publishing Ltd Aquaculture Nutrition 2011 17; 570577 doi: 10.1111/j.1365-2095.2011.00855.x 1 USDA ARS National Cold Water Marine Aquaculture Center, Franklin, ME, USA; USDA, Agricultural Research Service, Hagerman Fish Culture Experiment Station, Hagerman, ID, USA; U.S Fish and Wildlife Service, Bozeman Fish Technology Center, Bozeman, MT, USA Alternative protein sources for aquafeeds need to be indentied in order to increase the eciency of production Many studies have examined terrestrial plant meals/protein concentrates as alternatives Recently the focus has turned to aquatic protists and plants as well as by-products from other industries, such as breweries Atlantic salmon, Salmo salar, and Arctic charr, Salvelinus alpinus, were fed diets containing canola meal, soybean meal, corn gluten meal, soy protein concentrate, barley protein concentrate, and solar dried algae included at 30% of the test diet Barley protein concentrate had the highest apparent protein digestibility values for both species (96.3% for Atlantic salmon and 85.1% for Arctic charr), followed by corn gluten meal Algae had the highest organic matter digestibility value for arctic charr (80.1%) while corn gluten meal had the highest organic matter digestibility value for Atlantic salmon (88.4%) Algae had a high energy apparent digestibility coecient (82.4 salmon, 82.7 charr) along with corn gluten meal (78.5 salmon, 82.7 charr) for both species In general, Atlantic salmon had higher apparent digestibility coecients compared to Arctic charr for most of the tested ingredients Both corn gluten and barley protein concentrate appear good candidates as alternative protein sources with both species KEY WORDS: algae, amino acid, Atlantic salmon, Arctic charr, digestibility, nutrients Received 23 July 2010, accepted 30 January 2011 Correspondence: Gary S Burr, USDA ARS National Cold Water Marine Aquaculture Center, 25 Salmon Farm Rd., Franklin, ME 04634, USA E-mail: gary.burr@ars.usda.gov The inclusion of plant protein sources in aquafeeds is expanding due to the limited amount of shmeal available for production of animal feeds (e.g Glencross et al 2005; Gatlin et al 2007) One of the greatest challenges is to increase the amount of plant protein in the diet of carnivorous shes Carnivorous shes have been shown to have decreased growth and protein utilization when plant proteins such as soybean meal have been utilized to replace shmeal protein in the feeds (Refstie et al 2006; Aslaksen et al 2007) Soybean meal and other plant feedstus have been widely used in diets for omnivorous nsh (Gatlin et al 2007); however, sh meal is still a major source of protein in prepared feeds for carnivorous nsh species Due to various antinutritional factors in soybean meal and other plant feedstus, the diets of most carnivorous nsh species contain a relatively low percentage of these feedstus In order to increase the amount of plant feedstus in the diet of carnivorous species, protein utilization must be increased and the eects of the various anti-nutritional factors must be mitigated Recently, multiple studies have reported the digestibility of various plant feed ingredients for Atlantic salmon (Salmo salar) (Storebakken et al 2000; Glencross et al 2004; Refstie et al 2005; Aas et al 2006; Refstie et al 2006; Aslaksen et al 2007; Denstadli et al 2007; Kraugerud et al 2007) The digestibility of plant proteins was not signicantly lower for the plant feed ingredients compared to shmeal; except for bacterial protein meal, extracted soybean meal, oat, rapeseed (canola), and sunower (Table 1) There have been variable results reported for soybean meal with some studies showing decreased protein digestibility compared to shmeal while other studies did not detect any change in protein digestibility (Refstie et al 2005, 2006; Aslaksen et al 2007; Kraugerud et al 2007) When the plant feed stu was further Published 2011 This article is a U.S Government work and is in the public domain in the USA Table Reported apparent digestibilities of plant protein ingredients compared to sh meal Feedstuff level (%) Protein Lipid Organic Starch matter Reference Bacterial Protein Meal Bio processed Soybean meal Corn Gluten Faba, Dehulled Bean Faba, Whole Bean Fremented ESBM Lupin protein conc Lupin protein conc Lupin protein conc Lupin Lupin kernal meal Lupin Kernal meal Oat Pea Rapeseed Soybean meal Soybean meal Soybean meal Soybean meal Soybean meal Soy protein conc Soy protein conc Soy protein isolate Sunflower Wheat Wheat Gluten Wheat Gluten Various 20 ) + ) ) na + na na Aas et al 2006 Refstie et al 2005 20 20 20 20 30 30 30 20 30 30 20 20 20 50 20 30 20 30 60 30 30 20 20 6.25, 12.5 25, 50 = = = + = = = = = ) + ) = ) = ) = = = = ) = = + = = = ) + na na + + na = = + ) (ns*) = = ) na na na na + = na na ) = ) + na na na + na na = = = ) + na + na na na na = = na na = = = na + = + + ) ) ) = = ) ) ) na = na = + = = na na Aslaksen et al 2007 Aslaksen et al 2007 Aslaksen et al 2007 Refstie et al 2005 Refstie et al 2006 Glencross et al 2004 Glencross et al 2004 Aslaksen et al 2007 Refstie et al 2006 Glencross et al 2004 Aslaksen et al 2007 Aslaksen et al 2007 Aslaksen et al 2007 Kraugerud et al 2007 Aslaksen et al 2007 Refstie et al 2006 Refstie et al 2005 Glencross et al 2004 Denstadli et al 2007 Glencross et al 2004 Glencross et al 2004 Aslaksen et al 2007 Aslaksen et al 2007 Storebakken et al 2000 Storebakken et al 2000 + increased digestibility ) decreased digestibility = did not alter digestibility processed to make protein concentrates, digestibility was unaected (Glencross et al 2004; Denstadli et al 2007) The variability observed among studies in protein digestibility of soybean meal could be due to the use of dierent genetic varieties of soy that have dierent amounts of bber and carbohydrates Variability in the levels of anti-nutritional factors (i.e saponins, lectins, and oligosaccahrides) may also be an important factor Additionally, soybean meal induced enteritis has been demonstrated in Atlantic salmon Salmon with enteritis have been shown to have elevated trypsin expression in the distal intestine that could contribute to this condition (Lilleeng et al 2007) Temperature is an environmental factor that can increase the appearance of enteritis; sh kept at higher temperatures had more severe enteritis than sh kept at lower temperatures (Uran et al 2007) A wealth of information is available on the digestibility of nutrients from a wide range of ingredients that are both commonly utilized in Atlantic salmon feeds as well as ones that may have future potential Novel ingredients and product lines developed from historical ingredients such as soybeans, corn, canola, etc are continuing to become avail- able as aquafeed ingredients Information on the nutritional value of these ingredients including gross nutrient content as well as digestibility and availability will be necessary to assess their nutritional and economic value in aquafeeds Therefore, the purpose of this study was to examine macronutrient digestibility of several plant and novel feed ingredients with Atlantic salmon and Arctic charr Sixteen Atlantic salmon (average weight of 745 117 g), or 20 Arctic charr (average weight of 315 82 g) were stocked into a recirculating system consisting of twenty-four 0.265 m3 berglass tanks, a drum lter, bio-lter, 1200-L sump, and pumps The rst study was conducted with salmon and the second trial was conducted with charr The salmon were removed from the system and the charr were stocked in the system weeks prior to start of the trial For both trials, make up water (brackish well water $3 g L)1) was added to the system at the rate of 19-L min)1 and water quality was monitored weekly to ensure that a healthy environment is Aquaculture Nutrition 17; 570577 Published 2011 This article is a U.S Government work and is in the public domain in the USA Table Composition (g kg)1 dry weight) of the basal diet )1 Ingredient name g kg Anchovy meal Fish oil Barley Corn Gluten meal Vitamin premix1 Choline CL Stay-C Trace mineral premix2 Chromium oxide Analysis Crude protein Crude fat Dry matter 505.2 271.3 141.2 65.3 8.0 6.0 2.0 1.0 10.0 406.0 324.0 983.0 Contributed per kilogram of diet: vitamin A (as retinol palmitate), 10 000 IU; vitamin D3, 720 IU; vitamin E (as DL-a-tocopherylacetate), 530 IU; niacin, 330 mg; calcium pantothenate, 160 mg; riboflavin, 80 mg; thiamin mononitrate, 50 mg; pyridoxine hydrochloride, 45 mg; menadione sodium bisulfate, 25 mg; folacin, 13 mg; biotin, mg; vitamin B12, 30 lg Contributed in mg kg)1 of diet: zinc, 37; manganese, 10; iodine, 5; copper, maintained during the trial Dissolved oxygen and temperature were monitored daily to ensure that the oxygen concentration remains at or near saturation Temperature was $12 C for the duration of the study Fish were fed the basal diet (Table 2) for week prior to being fed the experimental diets Each of the experimental diets was randomly assigned to three tanks The basal diet contained 400 g kg)1 protein and 320 g kg)1 lipid with an estimated digestible energy of 19.6 kJ g)1 (Table 2) The methods of Cho et al (1982) and Bureau et al (1999) were used to estimate apparent digestibility coecients Chromic oxide served as the inert maker A complete reference diet meeting or exceeding all know nutritional requirements for Atlantic salmon (NRC 1993) (Table 2) was blended with the test ingredients (Table 3) in a 70:30 ratio (dry weight basis) to form test diets All of the diets were produced using commercial manufacturing methods All ingredients were ground to a particle size of [...]... challenges Aquaculture, 1 55, 401– 417 Aquaculture Nutrition 17; 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