Aquaculture Nutrition 2010 16; 223–230 doi: 10.1111/j.1365-2095.2009.00654.x United States Department of Agriculture, Agricultural Research Service, Sustainable Marine Aquaculture Systems, Fort Pierce, FL, USA Two experiments were conducted with Florida pompano, Trachinotus carolinus L at and 28 g L)1 salinity to determine apparent crude protein digestibility (ACPD), energy digestibility (AED) and amino acid availability (AAAA) from soybean meal (SBM), soy protein isolate (SPI) and corn gluten meal (CGM) Mean AAAA was similar to ACPD In fish adapted to g L)1 salinity, they were 81.2% and 81.9% (CGM), 93.6% and 92.2% (SBM), 93.8% and 93.1% (SPI) for AAAA and ACPD respectively In fish adapted to 28 g L)1, they were 84.5% and 83.4% (CGM), 86.5% and 87.1% (SBM), and 83.4% and 85.0% (SPI) for AAAA and ACPD respectively The AED was highest for SPI and lowest for SBM and inversely related to carbohydrate The ACPD, AED and AAAA of soy products appeared to be lower in high salinity, whereas CGM was unaffected The data suggest that SBM, SPI and CGM should be further evaluated as partial fishmeal replacements in Florida pompano diets Application of the generated coefficients can be used to develop well-balanced, low-cost diets for Florida pompano reared in low salinity or seawater KEY WORDS: amino acid availability, digestible protein, plantbased proteins, pompano Received October 2008, accepted 16 December 2008 Correspondence: Marty Riche, USDA, ARS, 5600 US Hwy 1, North, Fort Pierce, FL 39496, USA E-mail: marty.riche@ars.usda.gov Present address: Terhea N Williams, Marine Bio-Resources, University of Maine, Rogers Hall, Orono, ME 04469, USA Florida pompano, Trachinotus carolinus L is a euryhaline species representing a small marine fishery in Florida with an Ó 2009 Blackwell Publishing Ltd No claim to original US government works estimated 227 000 kg total annual catch; however, because of its highly prized taste and texture it maintains a high market demand Florida pompano tolerate a wide range of salinities, stress, readily consume pelleted rations, successfully breed in captivity, and are an excellent candidate for aquaculture (McMaster et al 2004) There is increased interest in rearing euryhaline species such as Florida pompano in freshwater or low-salinity conditions However, nutritionally balanced diets not exist for Florida pompano presenting an obstacle to development of large-scale commercial production in low salinity High quality fish meal is the best source of protein for fish, particularly for carnivorous species However, replacement of fish meal with alternative protein sources will increase sustainability and profitability (Glencross et al 2007) In addition to palatability and anti-nutritional concerns, use of ingredients as alternatives to fishmeal is limited by unknown availability of nutrients Apparent digestibility coefficients of feed ingredients exist for only a few fish species, but not Florida pompano To develop low-cost, low-polluting diets that achieve maximum efficiency, nutrient requirements and nutrient availability from dietary ingredients must be determined to implement least-cost formulation of economical and balanced diets There is also evidence that salinity affects nutrient digestibility (Lall & Bishop 1976; MacLeod 1977; Dabrowski et al 1986; Krogdahl et al 2004) Digestibility in Golden-line seabream, Sparus sarba (Forsskal) was higher in low salinity relative to isosmotic or full-strength seawater (Woo & Kelly 1995) Similarly, protein digestibility in milkfish, Chanos chanos (Forsskal) was elevated in freshwater relative to saltwater (Ferraris et al 1986) Zeitoun et al (1973) also suggested that protein requirements of rainbow trout, Oncorhynchus mykiss (Walbaum) were higher with increasing salinity We hypothesized protein digestibility and amino acid (AA) availability would be different in low-salinity adapted Florida pompano than saltwater adapted Florida pompano Therefore, the objective was to determine apparent digestibility of crude protein (CP), energy, and AA availability from soybean meal (SBM), soy protein isolate (SPI) and corn gluten meal (CGM) at both and 28 g L)1 salinity representing the known range of salinity supporting normal growth of Florida pompano Florida pompano broodstock were spawned at the USDA, Agricultural Research ServiceÕs Center for Reproduction and Larviculture, Fort Pierce, Florida, USA Postlarval juveniles were reared at 28 °C and 30 g L)1 salinity Fish were fed a commercial diet (EPAC-CW or IDL-CW; INVE Americas, Salt Lake City, UT, USA) until they were approximately g in weight Fish were subsequently transferred to a nursery system where they were held at 28 °C and g L)1 salinity and fed a commercial trout diet (Silver Cup; Nelson & Sons, Inc., Murray, UT, USA) until they were approximately 75 g in weight at which time they were acclimated to or 28 g L)1 salinity over a 1-week period Two simultaneous · Latin squares were set up to evaluate the three feed ingredients at and 28 g L)1 salinity Two 8750 L recirculating systems with sand, bead, cartridge and carbon filtration, and ultraviolet light sterilization were used Both systems were maintained at 28 °C Four 100-L tanks in each system with nominal flow rates of L min)1 served as experimental units Fish were maintained under a natural light cycle approximating 13 h light and 11 h dark A menhaden fish meal based formulation meeting the known protein and energy requirements for pompano served as the reference diet (Table 1) Solvent-extracted SBM (Rangen, Inc., Buhl, ID, USA), SPI (Archer Daniels Midland, Decatur, IL, USA) and CGM (Rangen, Inc.) were substituted at 300 g kg)1 for 300 g kg)1 of the reference diet utilizing a modified diet replacement method All diets incorporated yttrium oxide (Y2O3) at g kg)1 of the diet as an inert marker Feed ingredients were ground via hammermill (Prater Industries, Inc., Chicago, IL, USA) to pass a 250 micron screen Dry feed ingredients were mixed in a V-mixer (Patterson-Kelley, East Stroudsburg, PA, USA) Following addition of water and oil, complete diets were cold extruded and dried at 60 °C for 24 h Pelleted diets were stored at )20 °C until fed Twenty and 15 fish each, were stocked into 28 and g L)1 salinity experimental units respectively Fish were fed a Table Reference and test diets used to determine digestibility of crude protein, energy and amino acid availability from soybean meal, soy protein isolate, and corn gluten meal in Florida pompano Trachinotus carolinus Ingredient (g kg)1 dry diet) Reference diet Test diets Test ingredient Menhaden meal (low temperature)1 Soybean meal (solvent extracted)2 Corn gluten meal2 Porcine blood meal (spray dried)2 Fish solubles (dehydrated)3 Shrimp meal2 Dextrin (type-II from corn)4 Menhaden oil (stabilized)5 Sipernat 506 Mineral premix7 Vitamin premix8 Lecithin9 Ascorbyl-2-monophosphate8 a-Cellulose10 Carboxymethyl cellulose10 Yttrium oxide10 0.0 338.5 221.0 68.0 30.0 60.0 50.0 22.0 139.0 10.0 15.0 5.0 1.0 0.5 15.0 20.0 5.0 300.0 237.0 154.7 47.6 21.0 42.0 35.0 15.4 97.3 7.0 10.5 3.5 0.7 0.4 9.0 14.0 5.0 Special SelectÔ, Omega Protein, Inc., Houston, TX, USA Rangen Inc., Buhl, ID, USA International Proteins Corp., Minneapolis, MN, USA MP Biomedicals, Solon, OH, USA Alkali refined and stabilized with 500 ppm ethoxyquin, Omega Protein, Inc., Hammond LA, USA Degussa Corp., Parsippany, NJ, USA Mineral premix contained the following (g kg)1 premix): CaHPO4, 350.0; CaSO4Æ2H20, 100.0; KH2PO4, 200.0; MgSO4Æ7H20, 84.0; FeSO4Æ7H2O, 16.0; ZnSO4Æ7H2O, 3.0; MnSO4ÆH2O, 2.0; CuCl2Æ2H20, 1.0; KF, 0.23; KI, 0.1; NaMoO4Æ2H2O, 0.05; CoCl2Æ6H2O, 0.02; Na2SeO3, 0.01 Roche Vitamins Inc, Parsippany, NJ, USA USB, Cleveland, OH, USA 10 Sigma-Aldrich, St Louis, MO, USA commercial diet and allowed a 4-day acclimation to the new environment At initiation of the experiment, fish were switched to their assigned experimental diet and fed 4.7% body weight per day divided between a morning and afternoon feeding Faecal samples were collected on day and day of being fed the experimental diets Faecal samples were collected 3–4 h following the morning feeding on day of collection Prior to faecal collection, fish were anaesthetized with 75 mg L)1 tricaine methanesulphonate (MS-222; Western Chemical, Inc., Ferndale, WA, USA) Upon induction of stage IV anaesthesia, the area around the anus was dried with a towel and faecal samples collected by gentle expression of the lower gastrointestinal tract (Austreng 1978) Immature fish were used and care was taken not to contaminate samples with urine Following collection, fish were resuscitated and placed back into the experimental unit Faeces collected Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 223–230 No claim to original US government works on both day and were pooled into one sample Diets were reassigned to the experimental units and procedures were repeated until all four experimental units received each of the four diets (4 weeks) Feed and pooled faecal samples were analysed for yttrium (Y), nitrogen (N), gross energy (GE) and AA Proximate composition of reference and test diets was determined and test ingredients were analysed for each ingredientÕs contribution of nutrients to the test diet (Table 2) Coefficients were calculated as the ratio of nutrient and marker in feed and faeces (Maynard & Loosli 1969) and adjusted for nutrient concentration (Forster 1999) Test ingredients, feed and faecal samples were lyophilized to a constant weight and stored at )80 °C until analysis Nitrogen was determined following combustion (TruSpec N-elemental analyser; Leco Corp., St Joseph, MI, USA) and CP calculated as N · 6.25 GE was determined by Table Analysed composition (g kg)1) of test ingredients and experimental diets fed to Florida pompano Trachinotus carolinus adiabatic bomb calorimetry (Parr 1266; Parr Instruments Co., Moline, IL, USA) Ash was determined following incineration at 600 °C for h (AOAC 2002) Crude lipid was determined gravimetrically following chloroform : methanol extraction (Bligh & Dyer 1959) in a Soxhlet apparatus Crude fibre was determined by a commercial laboratory (Barrow-Agee Laboratories, Memphis, TN, USA) Amino acids were analysed by a commercial laboratory (Midwest Laboratories, Inc., Omaha, NE, USA) Briefly, samples were hydrolyzed with N HCl at 110 °C for 24 h A separate aliquot was analysed for cysteine (Cys) and methionine (Met) following performic acid oxidation to cysteic acid and methionine sulphone Amino acids were separated using a C-18 reverse phase HPLC column and quantified with a photodiode array detector following postcolumn derivatization with ninhydrin Test ingredient International feed no CGM1 5-28-242 Proximate components Dry matter 917.0 Crude protein 653.0 Crude lipid 22.0 Ash 26.0 Fibre 8.0 NFE4 214.0 Gross energy (kJ g)1) 20.9 Indispensable amino acids Arginine 21.1 Histidine 19.8 Isoleucine 24.2 Leucine 84.8 Lysine 10.0 Methionine 24.4 Phenylalanine 45.5 Threonine 23.9 Valine 29.2 Dispensable amino acids Alanine 66.6 Asx5 43.2 Cysteine 21.5 Glx6 170.3 Glycine 18.9 Proline 55.1 Serine 41.0 Tyrosine 35.8 SPI3 – Reference diet CGM diet SBM diet SPI diet 894.0 474.0 11.0 57.0 33.0 342.0 17.4 915.0 885.0 2.0 39.0 2.0 0.0 20.9 947.0 523.0 160.0 144.0 34.0 86.0 21.0 884.0 542.0 118.0 103.0 27.0 94.0 21.7 882.0 498.0 107.0 123.0 35.0 119.0 20.8 926.0 628.0 108.0 110.0 24.0 56.0 21.9 34.9 14.4 19.7 40.4 30.6 10.3 25.5 19.9 21.9 60.3 17.0 42.2 75.0 53.6 10.9 46.5 35.1 41.3 26.5 13.5 20.5 46.7 32.6 13.5 22.9 20.4 27.5 22.6 12.5 19.3 60.9 25.3 14.3 27.3 20.2 26.5 27.6 12.7 18.4 39.4 31.4 12.2 21.9 19.5 21.0 37.6 15.9 27.3 52.5 37.6 12.7 29.4 24.6 32.9 21.9 58.8 10.4 99.6 21.9 25.5 28.1 18.1 51.5 112.0 08.4 162.0 38.8 44.5 50.5 32.2 40.4 50.1 14.5 74.6 30.8 24.5 23.2 16.2 49.8 45.5 11.9 89.8 25.7 33.4 25.7 20.8 35.9 51.2 13.2 76.9 26.6 24.1 23.4 15.7 46.4 67.7 11.2 101.0 32.5 29.9 30.6 20.6 Corn gluten meal, Rangen, Inc., Buhl, ID, USA Dehulled, solvent-extracted soybean meal; Rangen, Inc., Buhl, ID, USA Soy protein isolate, Pro-FamÒ, Archer Daniels Midland, Decatur, IL, USA Nitrogen-free extract (100 ) moisture ) crude protein ) crude lipid ) ash ) fibre) Aspartic acid + asparagine Glutamic acid + glutamine Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 223–230 No claim to original US government works SBM2 5-04-612 Differences in apparent nutrient availability were analysed using the model statement for a Latin square design: Yijk ¼ l þ Ii þ columnj þ rowk þ eijk ; Table Mean (SEM, n = 4) apparent crude protein (ACPD) and energy (AED) digestibility coefficients (%) for soybean meal, soy protein isolate and corn gluten meal fed to Florida pompano Trachinotus carolinus adapted to or 28 g L)1 salinity ACPD where I represents the main effect of test ingredient, column represents variation due to tank, and row represents variation due to week Analysis was performed using the general linear model procedure of SAS with software package version 9.1 (SAS Institute, Cary, NC, USA) Residuals were analysed to evaluate normality of distribution and homogeneity of variance Where main effect differences were detected pairwise contrasts between the three ingredients were evaluated Significance was reported at P < 0.05 unless otherwise stated Where analysis indicated row or column effects in g L)1 salinity (alanine) or 28 g L)1 (glutamic acid + glutamine) no further analysis was conducted as row and column both represent restrictions on randomization making the F-test questionable Regression analysis was performed with test ingredient protein and energy as independent variables and apparent energy digestibility (AED) as the dependent variable Total ammonia-nitrogen ranged from 0.00 to 0.21 mg L)1 and 0.01 to 0.17 mg L)1 for the low-salinity and saltwater systems, respectively Nitrite-nitrogen was 0.04–5.01 and 0.04–0.56 mg L)1 for the low-salinity and saltwater systems, respectively The pH and alkalinity ranged from 6.92 to 8.08 mg L)1 and 138 to 190 mg L)1 as CaCO3 at g L)1 salinity and from 6.62 to 7.95 mg L)1 and 86 to 139 mg L)1 as CaCO3 at 28 g L)1 salinity Values were within acceptable ranges for Florida pompano (Watanabe 1995; Weirich & Riche 2006) No mortalities occurred during the experiment Apparent crude protein digestibility (ACPD) was significantly higher in the soy products than CGM at low salinity, but not in sea water where no differences were detected (Table 3) The AED was higher from SPI than CGM and SBM at low salinity Despite a decrease in AED of the soy products at 28 g L)1, the coefficient for SPI remained higher than SBM, but not CGM Insufficient faeces necessitated reporting apparent Met and Cys availability on either two or three samples Therefore, statistical analysis was not performed on these two AA Significant differences in apparent amino acid availability (AAAA) were detected for phenylalanine (Phe) and glutamic acid + glutamine (Glx) at g L)1 salinity (Table 4) No other differences were detected at low salinity Although not AED Test ingredient g L)1 28 g L)1 g L)1 28 g L)1 Reference diet Corn gluten meal Soybean meal Soy protein isolate 72.8 81.9 92.2 93.1 74.7 83.4 87.1 85.0 71.3 77.4 70.5 93.4 72.3 77.4 62.2 78.1 (0.5) (4.2)b (2.0)a (1.9)a (1.1) (2.9)a (3.6)a (3.5)a (1.2) (4.2)b (6.5)b (2.5)a (0.7) (3.4)a (4.0)b (4.1)a Mean values within a column having different superscripts were significantly different (P < 0.05) Table Mean (SEM; n = 4) apparent amino acid availability (AAAA) coefficients (%) for soybean meal (SBM), soy protein isolate (SPI) and corn gluten meal (CGM) in Florida pompano Trachinotus carolinus adapted to g L)1 salinity Amino acids Indispensable Arginine Histidine Isoleucine Leucine Lysine Methionine1 Phenylalanine Threonine Valine Dispensable Alanine Asx2 Cysteine1 Glx3 Glycine Proline Serine Tyrosine Overall mean AAAA Reference diet CGM SBM 83.3 80.3 80.4 86.1 81.9 83.0 84.0 75.3 82.2 (1.0) (0.6) (1.2) (0.3) (1.1) (1.8) (0.5) (1.0) (2.0) 73.5 76.8 68.1 88.1 76.2 100.0 83.2 81.0 81.6 (23.3) (6.1) (10.4) (4.3) (17.9) (2.8) (6.7)b (9.4) (7.9) 102.0 103.3 91.8 92.1 100.0 110.1 97.0 92.3 85.6 81.5 73.9 84.5 80.0 71.8 73.5 79.3 83.1 80.2 (1.3) (0.3) (0.5) (0.6) (1.4) (0.7) (1.3) (1.0) (1.0) 91.5 79.3 67.8 86.5 72.8 84.2 84.9 84.8 81.2 (5.0) (7.1) (10.9) (3.7)b (9.8) (3.5) (7.3) (9.2) (2.0) 89.7 87.7 91.5 94.0 71.7 88.9 94.5 99.4 93.6 SPI (7.4) 95.3 (1.4) (9.0) 92.5 (4.5) (11.7) 96.4 (1.7) (1.1) 94.7 (1.0) (4.7) 94.1 (2.6) (6.0) 105.7 (8.1) (3.5)a 95.1 (2.7)a (6.0) 89.9 (6.4) (8.5) 98.6 (3.1) (10.9) (3.8) (3.3) (3.1)a (12.3) (2.5) (4.4) (7.1) (2.1) 96.4 90.9 82.8 93.8 88.4 93.1 93.6 92.9 93.8 (2.0) (4.1) (5.2) (3.1)a (2.7) (3.4) (3.5) (3.4) (1.2) Different superscripts across a row indicate significant differences between ingredients tested (P < 0.05) Not statistically evaluated due to insufficient material for suitable replication (n = 2) Aspartic acid + asparagine Glutamic acid + glutamine statistically different, the overall pattern suggests that AAAA appears higher from soy products than CGM at low salinity in agreement with ACPD The availability of Met approached 100% for all ingredients Overall mean AAAA was similar to ACPD for all test ingredients, they were 81.2% and 81.9% (CGM), 93.6% and 92.2% (SBM), 93.8% and 93.1% (SPI) for AAAA and ACPD respectively Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 223–230 No claim to original US government works Table Mean (SEM; n = 4) apparent amino acid availability (AAAA) coefficients (%) for soybean meal (SBM), soy protein isolate (SPI) and corn gluten meal (CGM) in Florida pompano Trachinotus carolinus adapted to 28 g L)1 salinity Amino acids Indispensable Arginine Histidine Isoleucine Leucine Lysine Methionine1 Phenylalanine Threonine Valine Dispensable Alanine Asx2 Cysteine1 Glx3 Glycine Proline Serine Tyrosine Overall mean AAAA Reference diet CGM SBM SPI 87.5 81.1 81.6 87.7 83.0 87.9 85.6 73.2 86.2 (0.6) (1.1) (1.6) (0.7) (0.4) (0.3) (0.4) (3.5) (0.6) 89.0 84.4 79.6 92.0 77.4 92.9 90.2 87.6 84.6 (3.3) 78.1 (10.2) (5.5) 88.7 (7.7) (5.4) 84.6 (17.9) (1.4) 85.6 (7.1) (7.9)b 95.6 (2.1)a (1.8) 93.9 (6.0) (0.9) 79.6 (11.8) (5.3) 105.3 (4.8) (0.8)ab 75.7 (5.3)b 79.4 83.6 91.9 85.4 83.8 90.2 85.2 87.7 88.1 (16.8) (4.6) (5.9) (5.6) (4.2)ab (4.5) (3.5) (9.3) (5.0)a 84.9 73.7 85.3 81.9 72.5 77.6 75.7 85.5 81.8 (0.7) (2.4) (1.0) (0.4) (1.5) (0.4) (3.8) (0.5) (1.3) 88.7 79.0 68.3 86.8 68.8 84.3 93.1 89.3 84.5 (1.9) (6.2) (1.7) (2.8) (9.6) (3.1) (5.5) (1.2) (1.8) 81.6 80.6 51.9 86.2 77.0 84.2 92.4 88.5 83.4 (5.3) (5.6) (8.1) (3.5) (7.3) (4.1) (6.8) (4.2) (2.2) 80.9 98.7 73.7 87.9 86.1 79.5 105.7 70.6 86.5 (10.9) (15.4) (2.4) (3.0) (16.4) (8.2) (4.8) (19.2) (2.5) Different superscripts across a row indicate significant differences between ingredients tested (P < 0.05) Not statistically evaluated due to insufficient material for suitable replication (CGM, n = 3; SBM, n = 3; SPI, n = 2) Aspartic acid + asparagine Glutamic acid + glutamine Significant differences in AAAA were detected for lysine (Lys) and valine (Val) at 28 g L)1 (Table 5) Availability of Lys was higher from SBM (95.6%) than CGM (77.4%), and neither was different from SPI (83.8%) Apparent availability of Val was higher from SPI (88.1%) than SBM (75.7%), and neither was different from CGM (84.6%) No other differences were detected at 28 g L)1 salinity As with low-salinity treatments, overall mean AAAA was similar to ACPD for all test ingredients They were 84.5% and 83.4% (CGM), 86.5% and 87.1% (SBM), and 83.4% and 85.0% (SPI) for AAAA and ACPD respectively Apparent digestibility of CP was high for all test ingredients regardless of salinity, particularly relative to the reference diet The high ACPDs suggest a potential for these plant proteins as partial replacements for fish meal in Florida pompano diets Apparent digestibilities of CP and GE from the reference diet were lower than reported for some marine species fed compounded diets (Santinha et al 1999; Peres & Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 223–230 No claim to original US government works Oliva-Teles 1999; Sa´ et al 2006) The reason is unclear; however, the values in this study are similar to previously reported values (75.8% and 73.3% for ACPD and AED respectively) for juvenile Florida pompano fed the same diet formulation (Riche, new characters, 2009) Poor digestibility is one reason attributed to low feed efficiency (FE) in Florida pompano (Tatum 1973; McMaster 1988; Lazo et al 1998; Weirich et al 2006) However, SBM digestibility and AAAA at g L)1 salinity were similar to that observed in yellowfin sea bream, Acanthopagrus latus (Houttuyn) (Wu et al 2006) and Atlantic cod, Gadus morhua L (Tibbetts et al 2006) Also, ACPD for SBM at 28 g L)1 salinity was the same as reported for gilthead seabream, Sparus aurata L (Lupatsch et al 1997) Although ACPD for SBM was similar to that reported for haddock, Melanogrammus aeglefinus L (92.2%) and Cobia, Rachycentron canadum L (92.8%), AED was approximately 18–20% lower in Florida pompano than haddock or cobia (Tibbetts et al 2004; Zhou et al 2004) Low apparent digestible energy values from SBM were also reported in European seabass, Dicentrarchus labrax L (da Silva & Oliva-Teles 1998) and red drum, Sciaenops ocellatus L (Gaylord & Gatlin 1996) De Silva & Perera (1984) suggested that lower protein digestibility occurs in diets with higher protein However, in this study no difference in protein digestibility between soy products was detected at either salinity despite 130 g kg)1 higher protein in the SPI diet Conversely, in this study AED was directly proportional to dietary protein (r2 = 1.00) and inversely proportional to dietary nitrogen free extract (NFE; r2 = 0.99) Utilization of plant starch is limited in fish, particularly carnivores Digestible energy tends to be negatively correlated to dietary carbohydrate and positively correlated to dietary protein and lipid (Sullivan & Reigh 1995) Carbohydrate digestibility in Florida pompano is about 50% (Williams et al 1985) underscoring its limited availability and impact on energy digestibility Florida pompano have short digestive tracts Intestinal transit time for a fish meal/SBM diet was reported as h in seawater at 29–31 °C (Williams et al 1985) This was later confirmed using the same dietary formulation serving as the reference diet in this study (Riche, new characters, 2009) The short transit may result in limited enzymatic contact time attenuating digestion and absorption of nutrients, possibly causing the poor FE reported for Florida pompano Faecal stripping was initiated h postprandially Consistent results with previous trials (Riche, new characters, 2009) coupled with the small SEM of coefficients in the reference diet suggests that this was appropriate for the reference diet However, the high SEM of coefficients associated with the test ingredients, particularly AAAA coefficients for SBM and CGM suggests incomplete digestion or possible interactive effects Composition, chemical, and physical characteristics of feed can affect both Also, faecal collection method affects variability of availability values, with greater variability observed using faecal stripping (Yamamoto et al 1997) Digestibility coefficients are also generally lower using intestinal stripping relative to other methods (Hajen et al 1993; Yamamoto et al 1997) However, Glencross et al (2005) demonstrated feed ingredients high in carbohydrates, such as SBM and CGM, affect faecal pellet integrity and suggested that stripping is the preferred faecal collection method for plant protein digestibility trials Moreover, this method obviates diluting nutrient concentrations by external saltwater contamination of faeces Digestibility coefficients for SBM and SPI reported for Chinook salmon, Oncorhynchus tshawytscha (Walbaum) were much lower than for Florida pompano (Hajen et al 1993) Conversely, energy and N digestibility of SPI in rainbow trout (Glencross et al 2005) and Atlantic cod (Tibbetts et al 2004) were higher than for pompano, while N digestibility for SBM was the same The significant difference observed in AED between SBM and SPI was also observed in rainbow trout and Atlantic salmon, Salmo salar L (Glencross et al 2004), again supporting the negative effect of carbohydrates on digestible energy in carnivorous species Protein digestibility of the soy products was higher than CGM at low salinity, but not at 28 g L)1 Energy digestibility of CGM was similar in haddock, but ACPD in haddock was approximately 10% higher (Tibbetts et al 2004) Also, the energy digestibility coefficient of non-extruded CGM in rainbow trout was similar to that reported here, but increased substantially following extrusion (Cheng & Hardy 2003) It is likely extrusion processing would increase ADE of CGM in Florida pompano as well The AAAA from SBM in Florida pompano was similar to yellowfin seabream, Sparus latus (Houttuyn) with the exception of Lys and Phe availability being higher, and Val lower in pompano (Wu et al 2006) In cobia, AAAA from SBM was similar to Florida pompano, but that from CGM was higher ranging from 93.2% to 96.9% (Zhou et al 2004) Overall AAAA of SBM and SPI reflected CP digestibility as reported elsewhere (Yamamoto et al 1997; Allan et al 2000; Zhou et al 2004) The AAAA from CGM was 5.7–16.3% lower relative to Australian silver perch, Bidyanus bidyanus (Mitchell) for all indispensable AA except Met (Allan et al 2000) They were also substantially lower than in rainbow trout where all AAAA were >95% (Yamamoto et al 1997) Pompano fed a CGM based diet supplemented with AA to match their whole body AA profile exhibited only 60% of the weight gain of pompano fed a menhaden meal based diet with the same AA profile (Riche; unpublished data) Results from this study suggest that poor weight gain previously observed was due in part to lower AA availability from CGM Apparent availability of Met was high for all test ingredients, as it was in cobia (Zhou et al 2004) The Met availability from test ingredients evaluated in low salinity was 100–110%, suggesting enhanced availability from the other protein sources used in the test diets However, caution should be exercised in interpreting the Met values as insufficient material in some cases limited the number of samples for estimating means Significantly, lower apparent Lys availability was observed from CGM than SBM at the higher salinity (P < 0.05) and appeared to be lower than both soy products at low salinity This is similar to that reported for Australian silver perch (Allan et al 2000), red sea bream, Pagrus major (Temminck & Schlegel) (Yamamoto et al 1998), and yellowtail, Seriola quinqueradiata (Temminck & Schlegel) (Masumoto et al 1996), but the opposite of cobia (Zhou et al 2004) and Atlantic salmon (Anderson et al 1992) Lower Lys availability from CGM relative to the soy products may be an artefact of lower Lys in CGM Analysis of test ingredients indicated Lys was 53.6, 30.6 and 10.0 g kg)1 dry matter for SPI, SBM and CGM respectively At low dietary Lys, endogenous sources account for more of the recovered Lys masking true availability and depressing apparent availability The 10% increase in true Lys availability over apparent Lys availability from CGM in red sea bream (Yamamoto et al 1998) and yellowtail (Masumoto et al 1996) support this hypothesis The low CGM coefficients and high variability for Arg (SEM of 23.3%) and Lys (SEM of 17.9%) in the low salinity treatment are attributable to high recovery of these AA in one faecal sample resulting in AAAA for that replicate of 6.5% and 29.1% for Arg and Lys respectively Removal of that sample from consideration would have resulted in coefficients of 95.8% and 91.9% for Arg and Lys, respectively, which are similar to the other ingredients Although residuals of the coefficients tested as outliers (Snedecor & Cochran 1967), the coefficients were not removed from analysis because row and column effects could not be ruled out Moreover, it is possible the coefficients could represent true variability in AAAA for a marine species held at low salinity Although the experimental design precludes statistical analysis of test ingredients between the two salinities, the Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 223–230 No claim to original US government works trend was towards higher ACPD and AED from SBM and SPI for pompano reared in low salinity water relative to seawater This could result in lower FE in saltwater and suggests that dietary protein may need to be higher for production in saltwater as reported for other species (Zeitoun et al 1973; Lall & Bishop 1976) The data suggest that further research is warranted to determine if digestibility values are lower in a seawater environment In summary, the ACPD of SBM and SPI were >90% in low salinity, and significantly higher than CGM However, no differences in ACPD could be detected between the three ingredients in seawater As the ACPD coefficient for CGM was similar between the two salinities it appears protein digestibility of the soy products may be lower in seawater than freshwater, although this could not be tested The AED for the three test ingredients exhibited a parallel response to salinity as the ACPD The AED of SBM was significantly lower than SPI and was likely due to the CP/ NFE ratio as there was a positive linear relationship (r2 = 1.00) with protein and inverse relationship (r2 = 0.99) with NFE The overall AAAA from the test ingredients was similar to the ACPD coefficients and suggests that SBM, SPI and CGM should be further evaluated as partial fishmeal replacements in Florida pompano diets Application of the protein, energy and AA coefficients for SBM, SPI, and CGM generated in this study can be used to develop well-balanced, low-cost diets for Florida pompano reared in low salinity or in seawater addressing one of the obstacles to large-scale commercial production of this species The authors acknowledge David I Haley and Patrick L Tracy for their skilful technical assistance in sample collection, preparation and processing We would also like to express gratitude to Dr T Gibson Gaylord, Dr Jon Amberg and Dr Hector Acosta-Salmon for critical review and advice on preparation of this manuscript The authors acknowledge Rangen, Inc Buhl, Idaho for generously donating the plantbased proteins This work was funded in part by the Link Foundation Additional funding was provided by the USDA/Agricultural Research Service Project No 622563000-007-00D Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture All programmes and services of the US Department of Agriculture are offered on a non-discriminatory basis without Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 223–230 No claim to original US government works regard to race, colour, national origin, religion, sex, marital status, or handicap Allan, G.L., Parkinson, S., Booth, M.A., Stone, D.A.J., Rowland, S.J., Frances, J & Warner-Smith, R (2000) Replacement of fish meal in diets for Australian silver perch, Bidyanus bidyanus: I Digestibility of alternative ingredients Aquaculture, 186, 293–310 Anderson, J.S., Lall, S.P., Anderson, D.M & Chandrasoma, J (1992) Apparent and true availability of amino acids from common feed ingredients for Atlantic salmon (Salmo salar) reared in sea water Aquaculture, 108, 111–124 AOAC (Association of Official Analytical Chemists) (2002) Official Methods of Analysis of the Association of Analytical Chemists, 17th edn Association of Official Analytical Chemists, Washington, DC, USA Austreng, E (1978) Digestibility determination in fish using chromic oxide marking and analysis of contents from 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Aquaculture, 59, 93–105 Forster, I (1999) A note on the method of calculating digestibility coefficients of nutrients provided by single ingredients to feeds of aquatic animals Aquacult Nutr., 5, 143–145 Gaylord, T.G & Gatlin, D.M., III (1996) Determination of digestibility coefficients of various feedstuffs for red drum (Sciaenops ocellatus) Aquaculture, 139, 303–314 Glencross, B.D., Carter, C.G., Duijster, N., Evans, D.R., Dods, K., McCafferty, P., Hawkins, W.E., Maas, R & Sipsas, S (2004) A comparison of the digestibility of a range of lupin and soybean protein products when fed to either Atlantic salmon (Salmo salar) or rainbow trout (Oncorhynchus mykiss) Aquaculture, 237, 333–346 Glencross, B., Evans, D., Dods, K., McCafferty, P., Hawkins, W., Maas, R & Sipsas, S (2005) Evaluation of the digestible value of lupin and soybean protein concentrates and isolates when fed to rainbow trout, Oncorhynchus mykiss, using either stripping or settlement faecal collection methods Aquaculture, 245, 211–220 Glencross, B.D., Booth, M & Allan, G.L (2007) A feed is only as good as its ingredients – a review of ingredient evaluation strategies for aquaculture feeds Aquacult Nutr., 13, 17–34 Hajen, W.E., Higgs, D.A., Beames, R.M & Dosanjh, B.S (1993) Digestibility of various feedstuffs by post-juvenile Chinook salmon (Oncorhynchus tshawytscha) in sea water Measurement of digestibility Aquaculture, 112, 333–348 Krogdahl, A˚., Sundby, A & Olli, J.J (2004) Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) digest and metabolize nutrients differently Effects of water salinity and dietary starch level Aquaculture, 229, 335–360 Lall, S.P & Bishop, F.J (1976) Studies on the nutrient requirements of rainbow trout, Salmo gairdneri, grown in sea water and fresh water In: Advances in Aquaculture: FAO Technical Conference on Aquaculture, Kyoto, Japan 26 May–2 June 1976 (Pillay, T.V.R & Dill, W.A eds), pp 580–584 Fishing News Books, Farnham, England Lazo, J.P., Davis, D.A & Arnold, C.R (1998) The effects of dietary protein level on growth, feed efficiency and survival of juvenile Florida pompano (Trachinotus carolinus) Aquaculture, 169, 225– 232 Lupatsch, I., Kissil, G.W., Sklan, D & Pfeffer, E (1997) Apparent digestibility coefficients of feed ingredients and their predictability in compound diets for gilthead seabream, Sparus aurata L Aquacult Nutr., 3, 81–89 MacLeod, M.G (1977) Effects of salinity on food intake, absorption and conversion in the rainbow trout Salmo gairdneri Mar Biol., 43, 93–102 Masumoto, T., Ruchimat, T., Ito, Y., Hosokawa, H & Shimeno, S (1996) Amino acid availability values for several protein sources for yellowtail (Seriola quinqueradiata) Aquaculture, 146, 109–119 Maynard, L.A & Loosli, J.K (1969) Animal Nutrition, 5th edn McGraw-Hill Book Company, New York, NY, USA, 613 pp McMaster, M.F (1988) Pompano aquaculture: past success and present opportunities Aquacult Mag., 14, 28–34 McMaster, M.F., Kloty, T.C & Coburn, J.F (2004) Pompano mariculture – 2004 Aquacult Mag., 30, 25–29 Peres, H & Oliva-Teles, A (1999) Effect of dietary lipid level on growth performance and feed utilization by European sea bass juveniles (Dicentrarchus labrax) Aquaculture, 179, 325–334 Riche, M (2009) Evaluation of digestible energy and protein for growth and nitrogen retention in juvenile Florida pompano, Trachinotus carolinus J World Aquacult Soc., 40, 45–57 Sa´, R., Pousa˜o-Ferreira, P & Oliva-Teles, A (2006) Effect of dietary protein and lipid levels on growth and feed utilization of white sea bream (Diplodus sargus) juveniles Aquacult Nutr., 12, 310–321 Santinha, P.J.M., Medale, F., Corraze, G & Gomes, E.F.S (1999) Effects of the dietary protein: lipid ratio on growth and nutrient utilization in gilthead seabream (Sparus aurata L.) Aquacult Nutr., 5, 147–156 da Silva, J.G & Oliva-Teles, A (1998) Apparent digestibility coefficients of feedstuffs in seabass (Dicentrarchus labrax) juveniles Aqua Living Resour., 11, 187–191 Snedecor, G.W & Cochran, W.G (1967) Statistical Methods, 6th edn The Iowa State University Press, Ames, IA, USA, 593 pp Sullivan, J.A & Reigh, R.C (1995) Apparent digestibility of selected feedstuffs in diets for hybrid striped bass (Morone saxatilis $ · Morone chrysops #) Aquaculture, 138, 313–322 Tatum, W.M (1973) Comparative growth of pompano (Trachinotus carolinus) in suspended cages receiving diets of a floating trout chow with those receiving a mixture of 50% trout chow and 50% sinking ration In: Proceedings of the 4th Annual Meeting of the World Mariculture Society (Avault, J.W & Boudreaux, E eds), pp 125–141 Louisiana State University, Baton Rouge, LA, USA Tibbetts, S.M., Milley, J.E & Lall, S.P (2006) Apparent protein and energy digestibility of common and alternative feed ingredients by Atlantic cod, Gadus morhua (Linnaeus, 1758) Aquaculture, 261, 1314–1327 Tibbetts, S.M., Lall, S.P & Milley, J.E (2004) Apparent digestibility of common feed ingredients by juvenile haddock, Melanogrammus aeglefinus L Aquac Res., 35, 643–651 Watanabe, W.O (1995) Aquaculture of the Florida pompano and other jacks (Family Carangidae) in the Western Atlantic, Gulf of Mexico, and Caribbean basin: status and potential In: Culture of High-value Marine Fishes (Main, K.L & Rosenfeld, C eds), pp 185–205 Oceanic Institute, Honolulu, HI, USA Weirich, C.R & Riche, M (2006) Acute tolerance of juvenile Florida pompano, Trachinotus carolinus L to ammonia and nitrite at various salinities Aquac Res., 37, 855–861 Weirich, C.R., Groat, D.R., Reigh, R.C., Chesney, E.J & Malone, R.F (2006) Effect of feeding strategies on production characteristics and body compoasition of Florida pompano reared in marine recirculating systems N Am J Aquacult., 68, 330– 338 Williams, S., Lovell, R.T & Hawke, J.P (1985) Value of menhaden oil in diets of Florida pompano Prog Fish Cult., 47, 159–165 Woo, N.Y.S & Kelly, P.S (1995) Effects of salinity and nutritional status on growth and metabolism of Sparus sarba in a closed seawater system Aquaculture, 135, 229–238 Wu, X., Liu, Y., Tian, L., Mai, K & Yang, H (2006) Apparent digestibility coefficients of selected feed ingredients for yellowfin seabream, Sparus latus J World Aquacult Soc., 37, 237–245 Yamamoto, T., Ikeda, K., Unuma, T & Akiyama, T (1997) Apparent availabilities of amino acids and minerals from several protein sources for fingerling rainbow trout Fish Sci., 63, 995– 1001 Yamamoto, T., Akimoto, A., Kishi, S., Unuma, T & Akiyama, T (1998) Apparent and true availabilities of amino acids from several protein sources for fingerling rainbow trout, common carp, and red sea bream Fish Sci., 64, 448–458 Zeitoun, I.H., Halver, J.E., Ullrey, D.E & Tack, P.I (1973) Influence of salinity on protein requirements of rainbow trout (Salmo gairdneri) fingerlings J Fish Res Board Can., 30, 1867–1873 Zhou, Q., Tan, B., Mai, K & Liu, Y (2004) Apparent digestibility of selected feed ingredients for juvenile cobia Rachycentron canadum Aquaculture, 241, 441–451 Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 223–230 No claim to original US government works Aquaculture Nutrition doi: 10.1111/j.1365-2095.2009.00655.x 2010 16; 231–236 1,2 1,3 1 1 The Key Laboratory of Mariculture (Education Ministry of China), Ocean University of China, Qingdao, China; Center for Bioengineering and Biotechnology, China University of Petroleum, Qingdao, China; Guangdong Yuehai Feed Group Co Ltd., Xiashan District, Zhanjiang, China Correspondence: Kangsen Mai, The Key Laboratory of Mariculture (Education Ministry of China), Ocean University of China, Yushan Road, Qingdao 266003, China E-mail: kmai@ouc.edu.cn A 9-week feeding experiment was conducted to determine the dietary biotin requirement of Japanese seabass, Lateolabrax japonicus C Six isonitrogenous and isoenergetic purified diets (Diets 1–6) containing 0, 0.01, 0.049, 0.247, 1.238 and 6.222 mg biotin kg)1 diet were fed twice daily to triplicate groups (30 fish per group) of fish (initial average weight 2.26 ± 0.03 g) in 18 fibreglass tanks (300 L) filled with 250 L of water in a flow-through system Water flow rate through each tank was L min)1 Water temperature ranged from 25.0 to 28.0 °C, salinity from 28.0 to 29.5 g L)1, pH from 8.0 to 8.1 and dissolved oxygen content was approximately mg L)1 during the experiment After the feeding experiment, fish fed Diet developed severe biotin deficiency syndromes characterized by anorexia, poor growth, dark skin colour, atrophy and high mortality Significant lower survival (73.3%) was observed in the treatment of deficient biotin The final weight and weight gain of fish significantly increased with increasing dietary biotin up to 0.049 mg kg)1 diet (P < 0.05), and then slightly decreased Both feed efficiency ratio and protein efficiency ratio showed a very similar change pattern to that of weight gain Dietary treatments did not significantly affect carcass crude protein, crude lipid, moisture and ash content However, liver biotin concentration (0–6.1 lg g)1) significantly increased with the supplementation of dietary biotin (P < 0.05), and no tissue saturation was found within the supplementation scope of biotin Broken-line regression analysis of weight gain showed that juvenile Japanese seabass require a minimum of 0.046 mg kg)1 biotin for maximal growth KEY WORDS: biotin requirement, growth, Japanese seabass (Lateolabrax japonicus) Received April 2008, accepted January 2009 Ó 2009 The Authors Journal compilation Ó 2009 Blackwell Publishing Ltd Biotin is a water-soluble vitamin generally included in vitamin B complex and functions in several specific carboxylation and decarboxylation reactions It is part of the coenzymes of several carboxylation enzymes fixing CO2, such as propionyl CoA in the formation of propionic acid, acetylCoA carboxylase and pyruvate carboxylase Carboxylase fixation of CO2 to form methylmalonyl CoA is involved in the carboxylation and decarboxylation of tricarboxylic acids Biotin is also involved in the syntheses of fatty acids, lipids and citrulline As biotin is one of the most important and expensive vitamins added to aquafeeds, it is necessary to quantify the minimum requirement of biotin to manufacture cost-effective commercial feeds However, several factors have been proved to influence the need for dietary biotin in animals For example, high dietary lipid has been shown to obscure the effects of biotin in rats, chicks and brook trout (Jacobs et al 1970; Marson & Donaldson 1972; Poston & McCarteney 1974) The quantitative requirement of biotin for optimal growth has only been investigated in few species of fish For example, 0.1 mg kg)1 for lake trout (Poston 1976), 0.02– 0.03 mg kg)1 for common carp (Ogino et al 1970), 0.05– 0.14 mg kg)1 for rainbow trout (Castledine et al 1978; Woodward & Frigg 1989), 2.0–2.5 mg kg)1 for mirror carp (Gu¨nther & Meyer-Burgdorff 1990), 0.06 mg kg)1 for hybrid tilapia (Shiau & Chin 1999), 2.49 mg kg)1 for Asian catfish (Mohamed et al 2000) and 0.25 mg kg)1 for Indian catfish (Mohamed 2001) Japanese seabass (Lateolabrax japonicus) is an economically important food fish with fast growth and high market value, and now has been widely cultured in China However, a main constraint to Japanese seabass culture is the limited supply of trash fish that is presently the main feed source for grow-out Hence, there is an urgent need to develop a suitable practical diet for grow-out production of this fish One of the prerequisites for developing high efficient diet for Japanese seabass requires complete knowledge of its nutritional requirements A few studies have been reported on the nutrition of this seabass (Lin et al 1994; Hu et al 1995; Gao et al 1998; Hong et al 1999; Pan et al 2000; Ai et al 2004a,b; Mai et al 2006; Zhang et al 2006) To our knowledge, no information is available on its dietary biotin requirement Hence, the present investigation was undertaken to determine the optimum dietary biotin requirement on the basis of weight gain (WG) of juvenile Japanese seabass Six isonitrogenous and isoenergetic diets were formulated with graded levels of biotin (Table 1) Vitamin-free casein Table Formulation and proximate composition of the basal diet (g kg)1 dry matter) Ingredients Casein (vitamin free) Gelatin Dextrin Menhaden fish oil Soybean oil Amino acid mixture1 Lecithin Sodium alginate a-Cellulose Mineral premix2 Vitamin premix (biotin free)3 Proximate analysis (n = 3) Protein Lipid Moisture Content (g kg)1) 360 90 280 70 40 40 20 10 30 40 20 432 125 95 Amino acid mixture (g kg)1 diet): aspartic acid, 12.5 g; glycine, 0.2 g; alanine, 6.7 g; arginine, 7.3 g; cystine, 0.4 g; valine, 1.3 g; methionine, 2.9 g, isoleucine, 2.6 g; lysine, 6.1 g Mineral premix (g kg)1 permix): NaF, 0.2 g; KI, 0.08 g; CoCl2Æ6H2O (1%), 5.0 g; CuSO4Æ5H2O, 1.0 g; FeSO4ÆH2O, 8.0 g; ZnSO4ÆH2O, 3.0 g; MnSO4ÆH2O, 1.5 g; MgSO4Æ7H2O, 120.0 g; Ca (H2PO4)2ÆH2O, 750.0 g; NaCl, 10.0 g; Zoelite, 101.22 g Vitamin premix (mg kg)1 diet): B1, 25 mg; B2, 45 mg; B6, 20 mg; B12, 0.1 mg; pantothenic acid, 60 mg; niacin acid, 200 mg; folic acid, 20 mg; A, 32 mg; D, mg; E, 120 mg; K3, 10 mg; C, 2000 mg; inositol, 800 mg; choline chloride, 2500 mg; antioxidant, 150 mg; wheat middling, 14 013 mg (Sigma, Chemical, St Louis, MO, USA) and gelatin (Sigma, Chemical) were used as protein source, dextrin (Shanghai Chemical Co., Shanghai, China) as carbohydrate source, and menhaden fish oil (Food grade) and soybean oil (Food grade) as lipid sources Amino acid (Shanghai Chemical Co.) mixture was added to simulate the whole body amino acid pattern of Japanese seabass fingerling (Mai et al 2006) Biotin (Sigma) was added to the test diets at the expense of cellulose to provide concentrations of 0, 0.01, 0.05, 0.25, 1.25 and 6.25 mg kg)1 diet The biotin concentrations in the diets determined by high performance liquid chromatography (HP1100; Agilent, Palo Alto, CA, USA) (Lahely et al 1999) were 0, 0.01, 0.049, 0.247, 1.238 and 6.222 mg kg)1 diet, respectively All the ingredients were ground into fine powder through 220-lm mesh and thoroughly mixed with biotin, then with menhaden fish oil and soybean oil Finally, cold water was added to produce stiff dough, which was subsequently pelleted with an experimental diet mill [F-26 (II), South China University of Technology, China] and dried for about 12 h in a ventilated oven at 45 °C After drying, the diets were broken up and sieved into proper pellet size The sizes of pellets were 1.5 · 3.0 and 2.5 · 4.0 mm All the diets were sealed in bags and stored at )15 °C until used Experimental fish were obtained from a commercial farm in Yantai, Shandong province, China Prior to the feeding trial, the fish were reared in a concrete pond (4.0 · 2.0 · 2.0 m), and fed the control diet (Diet 1) for weeks to acclimate to the experimental diet and the rearing conditions At the start of the experiment, the fish were fasted for 24 h and weighed after being anesthetized with eugenol (1 : 10 000) (Shanghai Reagent Corp., Shanghai, China) Juvenile Japanese seabass with similar size (2.26 ± 0.03 g) were randomly allotted into 18 flow-through fibreglass tanks filled with 250 L of water (three tanks per treatment) Each tank was stocked with 30 fish and provided with continual aeration The fish were fed by hand twice daily at 08:00 and 17:00 hours respectively To prevent the waste of dietary pellets, fish were slowly hand-fed little by little to apparent satiation on the basis of visual observation of fish feeding behaviour The feeding trial lasted for weeks, from weeks to 4, 1.5-mm pellets were fed; thereafter, 2.5-mm pellets were fed until the end of the experiment During the experimental period, feed consumption was recorded daily The number and weight of dead fish were recorded and a natural photoperiod was maintained Water flow rate through each tank was L min)1 Water Ó 2009 The Authors Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 231–236 Development of body weight in the respirometric unit (in %) Ar (control group in respirometric chambers) Br (barnyard grass group in respirometric chambers) 120 Cr (dried maize leaves group in respirometric chambers) BWG (%) 100 80 60 40 20 Week Development of body weight in the aquaria (in %) Aa (control group in aquaria) Ba (barnyard grass group in aquaria) 120 Ca (dried maize leaves group in aquaria) Da (fresh maize leaves group in aquaria) BWG (%) 100 80 60 experimental diet (also from the fresh maize leaves) was well utilized by the grass carp in the present study The partial digestibility of the nutrients and gross energy of the plant leaf ingredients in fish (Table 6) indicated that barnyard grass were not digestible, dried maize leaves were poorly digestible whereas fresh maize were highly digestible in grass carp There were no significant differences (P > 0.05) in the total amount of oxygen consumed by the fish of the different experimental groups during the whole experimental period as well as in the amount of oxygen consumed per unit of BWG (Table 7) Generally, the total proportion of consumed energy lost by the fish of the same treatment in both experimental systems was almost similar The partitioning of dietary energy depended on the quality of the diet fed to the grass carp The results of the energy balance are presented in Table The largest proportion of consumed energy was lost via respiration in fish fed the reference diet The proportion of total ingested feed energy allocated to growth in grass carp kept in the aquaria system was found to be low for fish fed diets B and C (6–10%), whereas this was higher for fish fed diet D Generally, the energy losses were high in the case of fish fed plant leaf-containing diets (B, C and D) and the highest part of the ingested energy was lost through faeces (groups B, C and also D), which indicated that an important part of the consumed feed could not be digested and utilized 40 20 Week Figure Weekly evolution of body weight gain of fish during the whole experimental period The HSI seemed not to be affected by the different experimental feeds The RIL of the fish fed fresh maize leaves were similar to that of the fish fed only the reference diet, but significantly lower (P < 0.05) than those of the fish of groups B and C in the aquaria unit (Table 4) The ADC of nutrients and energy obtained for the different experimental diets ranged from 61% to 94% for protein, 60% to 91% for lipid and from 44% to 96% for gross energy depending on the diet (Table 6) Generally, the diets A and D were better digested by the fish than diets B and C The protein digestibility was high for diet D (>84%), indicating that an important part of the nitrogen sources of this The protein content of maize leaves from Germany was high when compared with that of the maize leaves found in Vietnam (Dongmeza et al 2009) However, the crude fibre content was similar The protein content of barnyard grass was typical to that commonly found in Vietnam (Dongmeza et al 2009) The optimal protein content in the diet for grass carp has been suggested to be about 22–30% (Cai et al 2005) and all the experimental diets had an acceptable protein content Watkins et al (1981) examined gut contents of fingerlings ranging from 17 to 117 mm total body length (TBL) and found out that Hydrilla, Hydrilla spp., along with bank vegetation accounted for 86% of the diet of larger fish (TBL > 86 mm): the grass carp could naturally tolerate higher amounts of macrophytes in their diet and probably made good use of it The TBL of all fish selected for our present experiment was above 86 mm; thus, our experimental Ó 2009 The Authors Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 313–326 Table Growth performance, nutrient utilization, relative intestinal length (RIL) and hepatosomatic index (HSI) of grass carp fed different experimental diets Respiration boxes Aquaria Parameters Control (Ar) Br (barnyard grass meal) Initial BW (g) Final BW (g) BWG (%) SGR (%) FCR PER PPV (%) ANLU (%) ER (%) RLI HSI 17.7 29.8 68.3 0.9 1.4 1.9 37.3 115.4 38.0 1.7 2.3 18.1 23.5 29.7 0.5 6.7 0.7 14.2 41.4 9.5 1.8 1.8 ± ± ± ± ± ± ± ± ± ± ± 0.9 2.6a 14.9a 0.2a 0.3b 0.4a 6.6a 21.2a 7.2a 0.1 0.3 ± ± ± ± ± ± ± ± ± ± ± 1.3 2.9b 11.5b 0.2b 3.7a 0.3b 4.9b 10.1b 3.2b 0.3 0.5 Cr (maize leaf meal) 17.9 23.3 30.5 0.5 5.7 0.6 13.3 23.1 8.1 1.9 1.7 ± ± ± ± ± ± ± ± ± ± ± 0.8 1.0b 9.0b 0.1b 1.5a 0.2b 2.9b 12.3b 2.7b 0.1 0.6 Control (Aa) Ba (barnyard grass meal) Ca (maize leaf meal) Da (fresh maize leaves) 21.1 34.8 64.4 0.9 1.4 1.9 39.6 81.2 34.3 1.5 1.8 21.0 27.5 31.4 0.5 5.8 0.8 12.3 17.3 5.7 1.9 1.5 22.1 29.2 32.2 0.5 5.3 0.7 16.8 27.0 10.0 1.8 1.5 25.6 55.1 118.8 1.4 1.8 1.8 33.1 50.0 19.8 1.4 1.9 ± ± ± ± ± ± ± ± ± ± ± 1.3 2.7b 7.0b 0.1b 0.1b 0.2a 4.3a 15.5a 4.1a 0.3ab 0.5 ± ± ± ± ± ± ± ± ± ± ± 1.5 2.2b 14.2c 0.2c 2.0a 0.3b 16.6b 18.9c 7.2c 0.2a 0.4 ± ± ± ± ± ± ± ± ± ± ± 1.4 2.8b 10.7c 0.1c 1.6a 0.2b 2.9b 10.8c 2.7c 0.2a 0.1 ± ± ± ± ± ± ± ± ± ± ± 5.9 9.6a 19.9a 0.2a 0.2b 0.2a 3.6a 11.2b 3.2b 0.3b 0.5 Values for each experimental group in the same row followed by different superscripts are significantly different (DuncanÕs multiple-range test, P < 0.05) Values are mean (n = 5) ± SD IBW, initial body weight; FBW, final body weight BWG (body weight gain) (%) = [100 ((final body weight) – (initial body weight)) (initial body weight))1] SGR (specific growth rate) (%) = [100 (ln final body weight – ln initial body weight) (no of trial days))1] FCR (feed conversion ratio) = [(feed intake) (body weight gain))1] ANLU (apparent net lipid utilization) (%) = [100 ((final fish body lipid) – (initial fish body lipid)) (total lipid consumed))1] ER (energy retention, %) = [100 ((final fish body energy) – (initial fish body energy) (gross energy intake))1] PER (protein efficiency ratio) = (fresh body weight gain) (crude protein fed))1 PPV (protein productive value) = 100 [(body protein gain) (protein intake))1] HSI = [100 (liver weight) (body weight))1] RIL = (intestine length) (standard body length of the fish))1 Table Whole body composition of the fish at the beginning and end of the experiment in both experimental systems (g kg–1 dry matter) Respiration boxes Initial CP Lipid Ash GE (MJ kg)1) 722.10 65.6 230.4 18.5 Control (Ar) 619.5 253.7 143.1 22.8 ± ± ± ± Aquaria Br b 59.6 106.7 49.3 2.5 Cr 643.4 221.5 151.7 21.6 ± ± ± ± 37.7 30.1 16.6 1.2 ab Control (Aa) 694.1 158.2 158.3 20.9 ± ± ± ± 40.4 51.3 12.4 0.9 a 642.7 216.9 139.3 22.8 ± ± ± ± Ba c 15.6 27.3a 12.6bc 0.7a 722.7 116.7 179.7 19.9 Ca ± ± ± ± a 28.4 42.2c 16.6a 1.1b 679.1 161.9 158.5 20.9 Da ± ± ± ± b 27.3 36.8bc 17.2b 1.0b 685.6 200.8 135.0 22.6 ± ± ± ± 15.9b 27.1ab 10.6c 1.2a Values for each experimental group in the same row followed by different superscripts are significantly different (DuncanÕs multiple-range test, P < 0.05) Values determined in duplicate determination; values are mean (n = 5) ± SD CP, crude protein; GE, gross energy Treatment Whole diet mixture Aa (reference diet) Ba (diet Aa + dried barnyard grass) Ca (diet Aa + dried maize leaves) Da (diet Aa + fresh maize leaves) Plant ingredients Dried barnyard grass Dried maize leaf Fresh maize leaves Crude protein 94.1 60.9 70.5 84.7 ± ± ± ± 6.4 10.8 4.8 2.1 )71.3 ± 24.0 28.4 ± 25.4 70.6 ± 5.5 Lipid 91.3 60.7 76.8 71.8 Gross energy ± ± ± ± 1.0 12.5 7.1 4.5 )42.1 ± 24.0 40.3 ± 24.2 35.9 ± 12.0 95.9 44.5 60.6 69.1 ± ± ± ± 0.9 7.3 10.5 3.3 Table Digestibility coefficients of nutrients and energy of mixed diets and of plant leaf ingredients in the fish kept in aquaria (% dry matter nutrient or gross energy content) )21.8 ± 22.5 24.6 ± 17.1 39.5 ± 6.8 Values are mean (n = 5) ± SD Ó 2009 The Authors Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 313–326 Table Oxygen (O2) consumption during the whole experimental period by fish fed the different experimental diets and O2 uptake per unit of body weight gain (BWG) Treatment Total O2 consumed (g) O2 consumed (g)/BWG (g) Ar Br Cr 8.7 ± 2.6 9.6 ± 2.4 10.4 ± 1.9 0.7 ± 0.3 2.3 ± 1.8 2.1 ± 0.9 Values in the same column followed by different superscripts are significantly (P < 0.05) different Values are mean (n = 5) ± SD fish might also tolerate the supplementation of the reference diet with higher amounts of plant leaf material Generally, the antinutrient content of the different experimental diets was lower than the known critical values found in the literature (Hossain & Jauncey (1993); Becker & Makkar (1999); Francis et al (2001)) As could be expected, dietary quality is important and relates also to the balance of essential amino acids When compared with the essential amino acid requirement of grass carp (Wang et al 2005), the maize leaves were deficient in leucine and slightly deficient in lysine whereas barnyard grass was deficient in lysine, methionine and cysteine (Table 1) Wilson & Cowey (1985) suggested that the amino acid needs for a defined species should be very similar to, if not the same as the amino acid profile of whole body tissue of the fish being studied The strong deficiency of leucine in maize leaves suggests that for a good utilization of maize leaves, grass carp might need a supply of additional leucine In the present study, leucine from the fish meal (in diet C) could not compensate this deficiency on the one hand, while on the other hand, the digestibility of whole dietary protein (from fish meal as well as from dried maize leaves) was generally even lower The higher digestibility of whole dietary protein of diet D might have played an important role in the compensation of leucine deficiency The growth of the fish of group Da, when compared with those of group Ca, suggested that the way of presenting the maize leaf material to grass carp might have been of high importance in achieving a higher growth of the fish (Da) The reduction in water content by drying and the subsequent reduction in particle size by milling did not contribute to a better utilization of the maize leaves by the fish fed diet C It was stated that the reduced content of amino acids in sun-dried sweet potato leaves could be due to the action of endogenous plant enzymes during the initial phase of the drying process In sun-dried sweet potato leaves, the contents of some essential amino acids such as histidine, lysine, cystine, tyrosine and non-essential amino acids such as glycine were lower than in the fresh material (An et al 2004) Such effects cannot be completely excluded in the present study as the maize material was oven-dried Reasons other than the essential amino acid deficiency might have caused the poor utilization of the plant leaf ingredients of the diets Cai et al (2005) also suggested that the lower fibre content of the diets could explain the better growth performance and ADC of some groups of grass carp in their study It is generally believed that fibre decreases energy availability by hastening transport through the gut and hence increases nitrogen and fat in the feaces (Hajra et al 1987); this might have been the case in our study for fish fed diet B or C Li (1996) suggested that the crude fibre content of diets for juvenile grass carp should be less than 100 g kg)1 In the present study, the fibre content of diets B, C and D were all higher than the critical value indicated before However, it is reasonable to suggest that the cell wall Table Energy allocation by fish fed the different experimental diets in the experimental systems Respiration unit Aquaria unit Parameter Ar (control) Br (barnyard grass) Cr (dried maize leaves) Efeed (kJ) R (kJ) RE (kJ) Efaeces (kJ) Balance (kJ) R (% of Efeed) RE (% of Efeed) Efaeces (% of Efeed) Balance (% of Efeed) 331.5 151.8 126.9 13.3 265.6 45.9 38.3 4.1 81.5 542.6 149.1 57.1 212.8 407.9 27.7 10.3 39.4 75.5 578.5 169.3 43.2 190.0 391.4 29.4 7.4 32.8 67.6 ± ± ± ± ± ± ± ± ± 8.7 28.9 32.9 0.4 36.8 9.2 9.7 0.0 9.2 ± ± ± ± ± ± ± ± ± 45.2 37.9 19.7 15.5 49.3 7.5 3.0 0.0 7.1 ± ± ± ± ± ± ± ± ± 18.6 23.3 20.9 5.4 17.2 5.0 3.5 0.0 4.1 Aa (control) Ba (barnyard grass) Ca (dried maize leaves) Da (fresh maize leaves) 362.5 ± 22.4 604.7 ± 24.5 687.8 ± 36.6 1014.0 ± 140.3 124.7 ± 17.9 14.4 ± 1.5 34.3 ± 43.5 322.5 ± 43.5 69.2 ± 21.7 278.3 ± 69.9 200.8 ± 38.7 311.4 ± 34.4 34.3 ± 4.1 4.1 ± 0.6 5.7 ± 7.2 53.6 ± 8.5 10.0 ± 2.7 39.4 ± 10.5 19.8 ± 3.2 30.9 ± 3.3 Values are mean (n = 5) ± SD Efeed, total energy fed; RE, energy retained as growth in the body; R, total energy expenditure calculated from O2 consumption; Efaeces, energy content of faeces Ó 2009 The Authors Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 313–326 of the fresh maize leaves are easily broken by the fish, which could thus make better use of the cell contents In the present study, all the diets contained the same proportion of reference feed; this indicates that the fish of groups B, C and D were supposed to grow at least as good as or better than those fed the reference diet only (group A) in the aquaria and in the respiration units In previous studies, grass carp fed diet supplemented with aquatic macrophytes grew faster than those fed only the commercial ration (Shireman et al 1978; Shelton et al 1981) The importance of factors other than protein was suggested as possible reasons for this faster growth Mgbenka & Lovell (1986) reported that addition of 19% alfalfa meal and 3% soybean oil to the catfish diet formula greatly improved the growth of grass carp Protein in their low-alfalfa diet (27%) was lower than for the commercial diet used in that study (32–35%; practical-type catfish diet) In our study, the fish fed diet A (reference diet only) generally grew better than those fed diets B and C and the values of the parameters such as %BWG, ER, ANLU, PER, PPV as well as the SGR for the group A were significantly (P < 0.05) higher when compared with those of groups B and C Stroganov (1963) reported different values of FCR and stated that this coefficient can be as poor as and in that case a higher proportion of animal food was contained in the feed In the present study, the FCR of diets containing dried plant leaves were 5.8 and 5.3 for the fish of groups Ba and Ca, respectively The FCR of the fish fed diet D (1.8, which was very low for a diet containing a high proportion of plant leaves) was significantly lower than that of the fish fed diet C, and almost similar to that of the fish fed the control diet (1.4) Consequently, the fish of group D had a significantly higher BWG (119.9%), which was almost double than that of the fish fed only the reference diet (64.4%), although they also had a higher feeding rate than fish fed only the reference diet This result supports the fact that the supplemented fresh maize leaves were more efficiently utilized by grass carp than the dried ground maize leaves or barnyard grass The digestion coefficients of the nutrients of our reference diet were found to be similar to those reported by Law in 1986 (90% for protein, 100% for lipid and 98% for energy) who fed commercial feed containing 38% of protein to grass carp The supplementation of dried barnyard grass and dried maize leaves to the reference feed (A) led to a poorer growth of those fish; in this case, the hypothesis that there is an interaction between the plant leaf material and the reference diet has to be considered (Sugiura et al 1998) The supplementation of the control feed with fresh maize leaves effectively led to an additional BWG in fish of group D when compared with those of group Aa, showing that fresh maize leaves were better utilized than dried maize leaves and barnyard grass The results of this study are in contradiction with the conclusion of Law (1986), who suggested that grass carp could digest fresh grass and dry grass equally In the present study, dry plant materials seem to be less digestible and could even inhibit digestion and utilization of other nutrients contained in the reference diet The metabolic costs seemed to be higher for the fish fed diets containing dried leaf material (B and C) In the present study, the energy expenditure through oxygen consumption seemed to be higher for the fish fed diets supplemented with plant material The oxygen uptake per unit of BWG was relatively higher for fish fed the diet containing dried barnyard grass or dried maize leaves when compared with the control group, indicating higher metabolic cost needed for the digestion of these diets (as compared with growth and excretion processes) The difference in the proportion of absorbed energy allocated to the feeding metabolism might also have been important in causing the growth difference among the different experimental fish groups (group A compared with groups B and C) Quantities of energy metabolically combusted can exceed the quantity absorbed from the plant component of the experimental diet To compensate these deficits, nutrients of animal component of the experimental diet are used; this could be expressed by the poor growth of the fish (treatments B and C) Huisman (1976b) and Huisman & Valentijn (1981) suggested that growth rate and oxygen consumption rate are closely related This was, however, not confirmed in this study, specially for the fish of groups Br and Cr Cui & Liu (1990) constructed average energy budgets for six teleost species fed ad libitum (at approximately the maximum daily intake) and found that heat loss (R) was always the largest component, 50–69% of consumed energy, whereas the energy used for growth was much smaller: 21–35% In the present study, this was the case for the fish fed diet A for which the energy retained for growth (RE) was between 34% and 38% Ó 2009 The Authors Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 313–326 In the aquaria unit, the fish fed the control diet allocated 34.3% of the total feed energy to growth The energy allocated to growth by the fish fed diet D (20%) was higher than that allocated for the same purpose by the fish of groups Ba (6%) and Ca (10 %); this showed that diet D containing fresh maize leaves was better used than diet C, which contained dried and ground maize leaves Consequently, the highest proportion of energy loss from the total energy consumed was found in the groups Ba and Ca (94% and 90%, respectively) in the aquaria units and 91% and 92% for groups Br and Cr, respectively in the respiration unit system, which also reflected and explained their poor growth In an experiment conducted by Fischer & Lyakhnovich (1973), fish gained weight when fed on animal-derived food and lost weight when fed plants The authors suggested that either respiration increased considerably in fish fed plants, or assimilation decreased by the same fish In the present study, both of these two suggestions could be confirmed for the fish fed diets containing dried barnyard grass and dried maize leaves On the one hand, their oxygen consumption per unit of BWG was higher than that of the fish fed only the reference diet On the other hand, the assimilation of feeds B and C was lower than that of the control feed (reference feed) When the plant component of the feed is digested better, the needs of the fish for metabolic processes are met and the energy gained from supplemented plant material can contribute to a supplemental growth of the fish as it is the case for fish of group D in the present study In the present study, 51% of the assimilated energy was channelled to heat production and 49% to growth in the fish of group Ar; these values were between those obtained for common carp: 45.6% and 54.4% of assimilated energy for growth and heat production, respectively (calculated from the data published by Focken et al 1997) and those obtained for tilapia: 54.5% and 45.5% of assimilated energy assigned to growth and to heat production, respectively (calculated from the data obtained by Santiago et al 2001) These results suggested that grass carp has a low growth efficiency and high metabolic expenditure These results contradict the conclusion of two previous studies on grass carp (Stanley 1974; Wiley & Wilke 1986), which suggested that grass carp compensated for low absorption efficiency with a low metabolic rate to achieve a better growth rate This was more accentuated in fish fed diets B and C in which the proportions of assimilated energy channelled to growth were lower (26% and 23%, respectively) and the proportion of assimilated energy channelled to heat production was higher (74% and 77%, respectively) This shows that the energy partition in the experimental fish was affected by the nature and quality of feed consumed, which affected their metabolism In the present work, the energy balances for different experimental diets ranged between 68% and 82%, which were in the same range as those obtained by Carter & Brafield (1991) who obtained energy balance values varying from 61.3% to 103.6% for different experimental diets with grass carp; this suggests that the methods used here are sound; however, the energy balance values obtained in the present study were lower than the optimal 100% value Those differences in balances from the consumed energy might probably have been because of some underlying experimental errors According to Brafield (1985) and Carter & Brafield (1991), the accuracy and errors that can be made in such studies during the estimation of each channel in the energy budget should always be evaluated The measurement of energy consumed Efeed was probably very accurate, as we could record the exact amount of feed fed to each fish As faeces were collected by siphoning in the present study, it would be expected that errors might occur in the estimation of digestibility; nutrient digestibility can be overestimated, owing to the leaching of water-soluble nutrients (Windell et al 1978; Smith et al 1980; Lied et al 1982) Such an effect can be large and may explain the high digestibility of nutrients and the low Efaeces obtained in the present study It was already pointed out that faeces collection (from water) was not accurate and this was especially true for grass carp fed plant leaf material (duckweed), as those fish might produce faeces of a loose texture (Cui et al 1992; Glencross et al 2005) This loose texture of faeces might have accentuated the leaching of water-soluble nutrients in the present study where the faeces produced by the fish fed the diet containing dried maize leaf (diet C) were very instable and quickly disintegrated in water whereas the feaces excreted by fish fed diet B showed higher integrity (were more compact); this might have resulted in increased errors in the estimation of the digestibility coefficient of diet C The increased leaching might have resulted in an overestimation of energy digestibility determined from faeces of fish fed diet C and consequently in lower energy content in the faeces of these fish; thus, the energy balance obtained for these fish might have been lowered (68% only) This might also alter the evaluation of the digestibility of the nutrients of dried maize leaves Glencross et al (2005) suggested that faeces collection by faecal stripping might be recommended in such a case; however, in the present work we used relatively small (juvenile) fish and the same fish were also used for growth and performance estimation and for nutrient utilization determination as well Sugiura et al (1998) also pointed out that Ó 2009 The Authors Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 313–326 the collection of faeces by stripping might encounter different problems such as the risk of collecting incompletely digested materials and the possible contamination of faeces with brownish intestinal fluid and urine that could lead to erroneously low digestibility values Errors could also be associated with the estimation of heat loss (R) where composite Qox values (14.85 kJ g)1 O2 in the present study) are used in the assumption that substrates are respired in the same proportions as they occur in the feed (Brafield & Llewellyn 1982) No significant difference (P > 0.05) was found in the HSI of the fish fed the different experimental diets This is understandable as none of the diets contained levels of toxic/ antinutritive substances higher than the known critical values However, the HSI of fish of group D were higher (but not significantly) than those of groups B and C This might have been because of the higher fat absorption and consequently increased fat retention in their whole body (see Table 5), particularly in the livers of the fish of group D The RIL of the fish fed diets B and C were found to be significantly (P < 0.05) higher than that of the fish of group D The higher RIL could be related to the quality of fibre contained in diets B and C Kihara & Sakata (1997) suggested that highly fibrous or poorly digestible diets cause elongation of the intestine The results of the present study indicated that the fresh maize leaves could support the growth of grass carp Thus, fresh maize leaves, a cheap nutrient source, have good potential for use as a supplement in fishmeal-based diets for grass carp These results confirm previous findings that grass carp could make good use of some macrophytes Both dried maize leaves and dried barnyard grass have a negative impact on the reference diet utilization, which resulted in a slower growth of grass carp Thus, it would be recommended to the farmer to avoid feeding barnyard grass to grass carp; a better use of this grass could be made by other animals (ruminants) The findings clearly show that grass carp utilizes fresh and freeze-dried maize leaf materials differently Further studies are needed to determine the reasons for this Feed based on fresh leaf material as well as selected plant ingredients with high nutrient and low crude fibre content are better suited for supplementing grass carp diets E B Dongmeza is thankful to the ÔKatholischer Akademischer Ausla¨nder DienstÕ, Bonn, for providing him a scholarship to conduct his PhD in Germany The authors also thank the staff of the aquaculture and analytical laboratory facilities at Hohenheim University for the support in the execution of this study and Dr H.P.S Makkar for his valuable assistance during the redaction of this paper Aksnes, A., Hjertnes, T & Opstvedt, J (1996) Comparison of two assay methods for determination of nutrient and energy digestibility in fish Aquaculture, 140, 343–359 An, L.V., Hong, T.T.T & Lindberg, J.E (2004) Ileal and total tract digestibility in growing pigs fed cassava root meal diets with inclusion of fresh, dry and ensiled sweet potato (Ipomoea batatas L (Lam.)) leaves Anim Feed Sci Technol., 114, 127–139 AOAC (1980) Official Methods of Analysis, 13th edn Association of Official Agricultural Chemists, Washington, DC, 125–142 AOAC (1990) Official Methods of Analysis, 15th edn Association of Official Analytical Chemists, Arlington, VA Bassler, R & Buchholz, H (1993) Amino acid analysis Section 4.11.1 In: Methodenbuch (Naumann, C., Bassler, R., Seibold, R., & Birth, C eds), Die Chemische Untersuchung von Futtermitteln VDUFA-Verlag, Darmstadt, Germany Becker, K & Makkar, H.P.S (1999) Effects of dietary tannic acid and quebracho tannin on growth performance and metabolic rates of common carp (Cyprinus carpio L.) 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313–326 Smedes, F (1999) Determination of total lipid using non-chlorinated solvents Analysts, 124, 1711–1718 Smith, R.R., Peterson, M.C & Allred, A.C (1980) Effect of leaching on apparent digestion coefficients of feedstuff for salmonids Prog Fish-Cult., 42, 195–199 Stanley, J.G (1974) Energy balance of white amur fed Egeria Water Hyacinth Control J., 12, 62–66 Steinbronn, S., Tuan, N.N., Focken, U & Becker, K (2005) Limitations in fish production in Yen Chau/Son La Province/Northern Vietnam Presented at Deutscher Tropentag, Stuttgart, Germany, October Steinbronn, S., Dongmeza, E.B., Tuan, N.N., Hong, N.T.L., Focken, U & Becker, K (2006) Livelihood from weeds and leaves – nutrient flows in the crop/aquaculture system of Black Thai farmers in Yen Chau/Son La Province/northern Vietnam Presented at symposium, ‘‘Towards Sustainable Livelihoods and Ecosystems in Mountainous Regions’’, Chiang Mai, Thailand, March Stroganov, N.S (1963) The food selectivity of the amur fishes Problems of the Fisheries Akad Nauk Turkmensk SSR., Ashkhabad from Ref Zh Biol 1964, No 3132 (transl from Russian), pp 181–191 Sugiura, S.H., Dong, F.M., Rathbone, C.K & Hardy, R.W (1998) Apparent protein digestibility and mineral availabilities in various feed ingredients for salmonid feeds Aquaculture, 159, 177–202 Tan, Y.T (1970) Composition and nutritive value of some grasses, plants and aquatic weeds tested as diets J Fish Biol., 2, 253–257 Vaintraub, I.A & Lepteva, N.A (1988) Colorimetric determination of phytate in unpurified extracts of seeds and the products of their processing Anal Biochem., 175, 227–230 Van Soest, P.J., Robertson, J.B & Lewis, B.A (1991) Methods for dietary fiber, neutral detergent fiber and non starch polysaccharides in relation to animal nutrition J Dairy Sci., 74, 3583–3597 Venkatesh, B & Shetty, H.P.C (1978) Studies on the growth rate of the grass carp Ctenopharyngodon idella (Valenciennes) fed on two aquatic weeds and a terrestrial grass Aquaculture, 13, 45–53 Wang, S., Liu, Y.-J., Tian, L.-X., Xie, M.-Q., Yang, H.-J., Wang, Y & Liang, G.-Y (2005) Quantitative dietary lysine requirement of juvenile grass carp (Ctenopharyngodon idella) Aquaculture, 249, 419–429 Watkins, C.E., Shireman, J.V., Rottmann, R.W & Colle, D.E (1981) Food habits of fingerling grass carp Prog Fish-Cult., 43, 95–97 Wen, Z (1990) Studies on aquatic vegetation and ration carrying capacity of grass carp, Ctenopharyngodon idella Val., in Lake Niushanhu M.Sc Thesis, Institute of Hydrobiology, Academia Sinica Wiley, M.J & Wilke, L.D (1986) Energy balances of diploid, triploid and hybrid grass carp Trans Am Fish Soc., 115, 853–861 Wilson, R.P & Cowey, C.B (1985) Amino acid composition of whole body tissue of rainbow trout and Atlantic salmon Aquaculture, 48, 373–376 Windell, J.T., Foltz, J.W & Sarokon, J.A (1978) Methods of feed collection and nutrient leaching in digestibilities studies Prog Fish-Cult., 40, 51–55 Ó 2009 The Authors Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 313–326 Aquaculture Nutrition doi: 10.1111/j.1365-2095.2009.00668.x 2010 16; 327–333 1 1 1 Nutrition Laboratory, Institute of Aquatic Economic Animals, School of Life Science, Sun Yat-Sen University, Guangzhou, China; Laboratory of Aquaculture Nutrition, College of Fisheries, Ocean University of China, Qingdao, China KEY WORDS: carbohydrate : lipid ratio, enzymes, feed utilization, grass carp, growth Six isonitrogenous (390 g kg)1) and isoenergetic (16.2 kJ g)1) diets with varying carbohydrate : lipid (CHO : L) ratios (202.5–1.74), were fed to triplicate groups of 25 fish in indoor recirculation system Over 8-week-growth trial, best weight gain (WG), specific growth rate, feed conversion ratio, protein efficiency ratio and protein production value (P < 0.05) were observed in fish-fed diets with CHO : L ratio of 7.5 Fish fed either the lowest (1.7) or highest (202.5) CHO : L ratio tended to produce lower (P < 0.05) growth and feed conversion efficiencies The values of viscerosomatic index, hepatosomatic index and intraperitoneal fat ratio increased as dietary CHO : L ratios decreased There were no significant differences in whole body and liver crude protein among dietary treatments Whole body and liver lipid increased as CHO : L ratios decreased Plasma cholesterol and triacylglyceride levels increased linearly as dietary CHO : L ratios decreased Activities of glucokinase and pyruvate kinase were stimulated by elevated levels of dietary carbohydrate; however, activities of lipase (LPS) and alkaline phosphatase were stimulated by elevated levels of dietary lipid Based on a second-order polynomial regression analysis of WG against dietary carbohydrate and lipid levels, 275 g kg)1 of carbohydrate and 59 g kg)1 of lipid, corresponding to a CHO : L ratio of 4.7, in a diet holding 390 g kg)1 of crude protein and 16.3 kJ g)1 of gross energy, proved to be optimal for grass carp These results indicated that utilization of dietary lipid and carbohydrate was moderate in grass carp, but the fish were a little more capable of utilizing lipid compared with carbohydrate Ó 2009 The Authors Journal compilation Ó 2009 Blackwell Publishing Ltd Received 16 December 2008, accepted 11 March 2009 Correspondence: Professor Li-Xia Tian, Institute of Aquatic Economic Animals, School of Life Science, Sun Yat-Sen University, No 135 XinÕgang Xi Road, Guangzhou 510275, China E-mail: edls@mail.sysu.edu.cn Protein, essential for tissue growth and maintenance, is an expensive component of formulated diets When insufficient energy is available in a diet from non-protein sources, protein may be catabolized to meet the energy requirements at the cost of nutrient supply somatic growth (Capuzzo & Lancaster 1979; Sedgwick 1979) The most efficient diets contain sufficient non-protein energy sources (lipid and carbohydrate) that are metabolized and spare protein to meet general energy requirements, leaving an organism to direct the maximum level of available dietary protein into growth (Sedgwick 1979; Bautista 1986) Lipids are well utilized by most fish, but at high dietary levels, lipids may reduce fish growth (Page & Andrews 1973; Garling & Wilson 1977; El-Sayed & Garling 1988; Ellis & Reigh 1991) Carbohydrates, on the other hand, appear to be poorly utilized, especially by carnivorous fish such as salmonids, which digest and utilize starch less efficiently than omnivorous or herbivorous fish (Kitamikado & Tachino 1960; Lin et al 1978; Bergot 1979; Furuichi & Yone 1981; Hofer & Sturmbauer 1985) However, carbohydrates are more readily available and much less expensive than lipids, but excess dietary carbohydrate may lead to fat deposition by stimulating lipogenic enzymes Therefore, it is a prerequisite that the optimum ratio between carbohydrate and lipid in fish feeds is carefully defined Production of grass carp (Ctenopharyngodon idella) constitutes the largest aquaculture industry of finfish in China As a typical herbivorous finfish without stomach, the natural food of grass carp is water plants However, only some nutrient requirements have been investigated (Dabrowski 1977; Dabrowski & Kozak 1979; Lin et al 1989; Carter & Brafield 1991, 1992a,b; Lin 1991; Cui et al 1992) There is no exact information about optimal carbohydrate-to-lipid (CHO : L) ratios of grass carp until now Therefore, the aim of the present experiment was to examine the effect of various dietary CHO : L ratios on growth performance, body composition, nutrient utilization and hepatic enzyme activities of grass carp Table Ingredient composition and proximate analysis of the experimental diets Ingredients (g kg)1) Diets Casein 380 Gelatin 40 Cellulose 40 Corn starch 400 Fish oil Corn oil Vitamin mix1 20 Mineral mix2 80 others3 40 Proximate analysis (g kg)1 dry Dry matter 935 Crude protein 389 Crude lipid Ash 98 Carbohydrate 405 Carbohydrate/lipid4 202.5 Cross energy (kJ g)1) 16.2 P/E (g MJ)1) 24.0 380 40 65 355 15 20 80 40 matter) 938 387 21 95 358 17.1 16.1 24.0 380 40 90 310 10 30 20 80 40 380 40 115 265 15 45 20 80 40 940 943 389 385 42 61 97 99 314 267 7.5 4.4 16.2 16.1 23.9 23.9 380 40 140 220 20 60 20 80 40 380 40 165 175 25 75 20 80 40 948 391 81 100 223 2.8 16.3 24.0 951 383 102 96 177 1.7 16.1 23.8 Vitamin mix (mg kg)1 of diet): thiamine, 50; riboflavin, 50; vitamin A, 25 000 IU; vitamin E, 400; vitamin D3, 24 000 IU; menadione, 40; pyridoxine HCl, 40; cyanocobalamin, 0.1; biotin, 6; calcium pantothenate, 100; folic acid, 15; niacin, 200; inositol, 2000; and cellulose was used as a carrier Mineral mix (g kg)1 diet): calcium biphosphate, 9.8; calcium lactate, 37.9; sodium chloride, 2.6; potassium sulphate, 13.1; potassium chloride, 5.3; ferrous sulphate, 0.9; ferric citrate, 3.1; magnesium sulphate, 3.5; zinc sulphate, 0.04; manganese sulphate, 0.03; cupric sulphate, 0.02; cobalt chloride, 0.03; potassium iodide, 0.002; and cellulose 42 Others (g kg)1 diet): taurine, 5; choline chloride (50%), 6; betaine, 4; carboxymethyl cellulose, 20; vitamin C, Carbohydrate-to-lipid ratio on weight basis (g g)1) An 8-week growth experiment was carried out to determine the effect of various dietary CHO : L ratios on growth performance, body composition, nutrient utilization and digestive enzymes activities of herbivorous grass carp (C idella) Prior to use, all feed ingredients were analysed for their proximate composition and the data obtained were used as a basis for the formulation (Table 1) Six isonitrogenous and isoenergetic diets, with approximately 390 g kg)1 crude protein and 16.2 kJ g)1, were prepared with varying CHO : L ratios by adjusting lipid and carbohydrate levels Casein and gelatin were protein sources used Corn starch was used as a carbohydrate source, and fish oil and corn oil were used as sources of lipid Diet ingredients were ground through a 60-mm mesh Distilled water and oil were added to the premixed dry ingredients and thoroughly mixed until homogenous in a Hobart-type mixer The 2- and 3-mm diameter pellets were wetextruded, dried overnight at 60 °C in a forced draught oven, sealed in plastic bags and frozen stored ()18 °C) until used Juvenile grass carp were obtained from a local hatchery Prior to the present study, the fish were acclimated to a commercial diet for weeks After the acclimatization, fish were sorted by weight and absence of physical abnormalities into uniform groups The fish were randomly distributed to the experimental 200-L fibreglass tanks at an equal stocking rate of 25 fish per tank connected to a recirculation system The initial body weight averaged 2.27 g Five of the remaining fish were sacri- ficed to provide an estimate of initial whole-body chemical composition Photoperiod was held to a constant 12 : 12 h light–dark cycle The fish were fed for 56 days with a daily ration of 5% of body weight divided into meals day)1 The culture tanks were cleaned weekly Water quality parameters were monitored daily between 10:00 and 17:00 h During the experimental period, temperature ranged from 28 to 30 °C; dissolved oxygen was 7.6–7.8 mg L)1; total ammonianitrogen was 0.097 ± 0.05 mg L)1 and pH was 7.8 ± 0.09 At the termination of the 8-week feeding trail, fish in each tank were weighed and sampled for tissue analysis 24 h after the last feeding Nine fish from each tank were randomly collected for proximate analysis, three for analysis of whole-body composition and six for blood collection and to obtain weights of individual whole body, viscera, liver and mesenteric fat The liver were dissected and frozen immediately in liquid nitrogen Ó 2009 The Authors Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 327–333 and stored at )70 °C until used and the plasma was separated by centrifugation and also stored at )70 °C until analysed Gross energy content was calculated using 23.6 kJ g)1 for protein, 39.56 kJ g)1 for lipid and 17.2 kJ g)1 for carbohydrate (NRC 1993) Crude protein, crude lipid and moisture in diets, liver and whole body were determined by standard methods (AOAC, 1995) Moisture was determined by ovendrying at 105 °C for 24 h; crude protein (n · 6.25) was analysed by the Kjeldahl method after acid digestion using an Auto Kjeldahl System (1030-Auto-analyzer, Tecator, Ho¨gana¨s, Sweden); crude lipid was determined by the etherextraction method using a Soxtec System HT (Soxtec System HT6, Tecator, Sweden) The concentrations of plasma triacyglyceride and total cholesterol were assayed by enzymatic procedure using automatic biochemical analyser and attached kit (Hitachi 7170; DAICHI, Tokyo, Japan) Carbohydrate content of diets was analysed by the 3,5-dinitro salicylic acid method (Yu et al 1998) Reductive monosaccharide is prepared from hydrolysis of hydrochloric acid The absorption spectrum of 3,5-dinitrosalicylic acid reducing product with reducing sugar has been studied The result shows a maximum absorption at 540 nm Lipase activity was determined by the method of Pan & Wang (1997), while the activity of alkaline phosphatase (ALP) was measured following the method of Bessey et al (1946) Enzyme-specific activities were expressed as lmoles of substrate hydrolyzed per minute, per mg of protein (i.e U mg protein)1), 30 °C for lipase and 37 °C for ALP The protein concentration of the supernatant solutions was determined by the biuret method, using bovine serum albumin as the standard The GK (high Km hexokinase or hexokinase IV) activity was measured according to Panserat et al (2000) at 37 °C To measure PK activities, the supernatant was centrifuged at 10 000 g for 20 and the resultant cytosolic fraction was used for enzyme activity measurements according to Foster & Moon (1985) GK and PK activities were expressed as mU mg protein)1 The data were subjected to one- and two-way ANOVA to test the effects of dietary protein and carbohydrate Differences for all analyses were considered significant at P < 0.05 The data are presented as mean ± SD of the replicate groups All analyses were conducted using SPSS Version 10.0 (SPSS, Illinois, USA) All diets were accepted by the fish and survival was high in all groups Growth performance of fish fed the diets containing varying CHO : L ratios (1.7–202.5) differed significantly (P < 0.05) producing a quadratic growth pattern (Table 2) Best (P < 0.05) weight gain (WG) and specific growth ratio (SGR) were observed in grass carp fed 310 g kg)1 dietary carbohydrate and 40 g kg)1 lipid, corresponding to a CHO : L ratio of 7.5 (Table 2) Fish fed either the lowest (1.7) or the highest (202.5) CHO : L ratio tended to produce lower (P < 0.05) growth and feed utilization (Table 2) Best Table Growth, feed utilization and biometric parameters of grass carp-fed experimental diets at the end of the growth trial CHO : L WG1 202.5 17.1 7.5 4.4 2.8 1.7 344 449 544 501 481 438 ± ± ± ± ± ± SGR2 20a 9.6b 6.6d 16c 3.6c 16b 2.66 3.04 3.32 3.20 3.14 3.00 FCR3 ± ± ± ± ± ± 0.08a 0.03b 0.02d 0.07c 0.01c 0.03b 1.75 1.40 1.28 1.34 1.45 1.59 PER4 ± ± ± ± ± ± 0.13c 0.05a 0.08a 0.09a 0.04ab 0.14bc 1.47 1.84 2.02 1.95 1.77 1.65 PPV5 ± ± ± ± ± ± 0.11a 0.07bcd 0.12d 0.13cd 0.05bc 0.15ab 0.17 0.20 0.22 0.21 0.18 0.17 VSI6 ± ± ± ± ± ± 0.01a 0.01bc 0.02c 0.01c 0.01ab 0.02a 10.3 10.9 11.7 12.9 12.8 12.4 HSI7 ± ± ± ± ± ± 1.17a 0.86a 1.33b 1.01c 1.27c 1.07c 2.37 2.96 3.20 3.88 3.75 4.19 IPF8 ± ± ± ± ± ± 0.51a 0.47b 0.40b 0.60c 0.71c 1.16c 2.63 2.29 3.32 4.08 4.19 4.51 ± ± ± ± ± ± 1.04a 0.76a 0.63b 1.10c 0.87c 1.05c Values represents means ± SD of three replicates and values with the same column with different letters were significantly different (P < 0.05) Weight gain = 100 · (final body weight ) initial body weight)/initial body weight Specific growth ratio = 100 · ln(final weight/initial weight)/days of the experiment Feed conversion ratio = g dry feed consumed/g wet weight gain Protein efficiency ratio = fish wet weight gain/protein intake Protein production value = (final body protein ) initial body protein)/total protein fed Viscerosomatic index = 100 · (viscera weight/whole-body weight) Hepatosomatic index = 100 · (hepatosomatic weight/whole-body weight) Intraperitoneal fat ratio = 100 · (IPF weight/whole-body weight) Ó 2009 The Authors Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 327–333 CHO : L ratios decreased Triacylglyceride values of fishfed diets with CHO : L ratios higher than 4.4 were significantly (P < 0.05) lower than those of the other groups The activities of hepatic enzymes are presented in Table Activities of GK and PK were stimulated by elevated levels of dietary carbohydrate Higher (P < 0.05) activities of lipase (LPS) and ALP were observed in fish-fed diets with CHO : L ratios lower than 7.5 The relationship between WG and dietary carbohydrate and lipid levels were expressed by the polynomial second-order regression model; the regression lines y = )0.0102x2+5.6047x )248.3R2=0.9001 and y=)0.0532x2 + 6.2439x + 340.7R2 = 0.902 were obtained respectively Growth maxima were observed when the dietary carbohydrate and lipid levels were 274.7 and 58.7 g kg)1, corresponding to a CHO : L ratio of 4.68 protein efficiency ratio (PER) was observed in fish-fed diet with CHO : L ratio of 7.5, which was not significantly different from those fed CHO : L ratios of 17.1 and 4.4, but apparently better than those fed CHO : L ratios of 202.5, 2.75 and 1.74 (Table 2) FCR and PPV followed similar trend as PER The values of viscerosomatic index (VSI), hepatosomatic index (HSI) and intraperitoneal fat ratio (IPF) increased as dietary CHO : L ratios decreased (Table 2) Whole body and liver lipid increased (P < 0.05) as CHO : L ratios decreased (Table 3).There were no significant differences in whole body and liver crude protein among dietary treatments (Table 3) The highest values of whole body and liver moisture were observed in fish-fed diet with CHO : L ratio of 202.5 (Table 3) Both plasma total cholesterol and triacylglyceride concentrations were significantly affected by CHO : L ratios (Table 4) Cholesterol levels increased linearly as dietary Table Body and tissue proximate composition (g kg)1 of wet weight basis) in juvenile grass carp-fed experimental diets at the end of the growth trial Whole body CHO : L Moisture 202.5 17.1 7.5 4.4 2.8 1.7 767 763 747 748 737 727 ± ± ± ± ± ± Liver Crude protein 5d 6cd 9bc 8bc 9ab 5a 120 113 110 113 108 113 ± ± ± ± ± ± Crude lipid 7b 3ab 3ab 2ab 1a 11ab 78.1 88.4 90.7 91.9 104 106 ± ± ± ± ± ± Moisture 10a 3a 9ab 8abc 10bc 14c 597 481 478 461 458 497 ± ± ± ± ± ± 39b 22a 14a 24a 2a 17a Crude protein Crude lipid 94.1 86.2 89.0 86.8 90.8 88.8 222 300 323 348 364 384 ± ± ± ± ± ± 6 3 ± ± ± ± ± ± 35a 26b 11bc 9cd 7cd 33d Values represents means ± SD of three replicates and values with the same column with different letters were significantly different (P < 0.05) Table Biochemical compositions of plasma from grass carp-fed experimental diets at the end of the growth trial (mmol L)1) CHO : L 202.5 17.1 7.5 4.4 2.8 1.7 Cholesterol Triacylglycerol 6.14 ± 0.19a 3.06 ± 0.11a 6.29 ± 0.15a 3.17 ± 0.06a 7.31 ± 0.21b 3.37 ± 0.13a 7.87 ± 0.06c 4.12 ± 0.30b 8.62 ± 0.23d 4.47 ± 0.20c 9.02 ± 0.12e 5.38 ± 0.22d Values represents means ± SD of three replicates and values with the same column with different letters were significantly different (P < 0.05) Table Liver enzyme activities of grass carp-fed experimental diets at the end of the growth trial CHO : L 202.5 )1 LPS (U mg protein ) ALP(mU mg protein)1) GK (mU mg protein)1) PK (mU mg protein)1) 0.92 108 2.38 99.2 ± ± ± ± 17.1 a 0.06 3.5a 0.14e 0.1e 0.96 114 2.07 89.3 7.5 ± ± ± ± a 0.07 2.4ab 0.11d 2.4d 4.4 1.10 114 1.80 86.2 ± ± ± ± b 0.03 7.2ab 0.14cd 2.4d 1.21 119 1.73 67.0 2.8 ± ± ± ± bc 0.05 2.4b 0.19c 3.1c 1.30 121 1.11 56.1 1.7 ± ± ± ± c 0.07 6.6b 0.18b 1.6b 1.54 136 0.82 33.8 ± ± ± ± 0.09d 4.8c 0.11a 3.2a Values represents means ± SD of three replicates and values with the same column with different letters were significantly different (P < 0.05) ALP, alkaline phosphatase; GK, glucokinase; PK, pyruvate kinase Ó 2009 The Authors Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 327–333 There is little information available concerning the optimal dietary protein level of grass carp Dabrowski (1977) reported that there was a linear relationship in grass carp fry between the percentage of protein in the diet and the increase in fish protein and weight up to optimal levels of 410 and 430 g kg)1 respectively In our previous study (unpublished data) on grass carp, the protein requirement of this species was between 350 and 400 g kg)1 Based on this, the 390 g kg)1 crude protein level in the diets of this study can be considered as the likely optimal dietary protein level for growth in grass carp The results of this study clearly indicate that growth and conversion efficiencies in grass carp are affected by the levels of non-protein energy in the diet Similar results have been reported in other fish species such as in Chinook salmon (Buhler & Halver 1961), plaice (Cowey et al 1975) and walking catfish (Erfanullah & Jafri 1998) However, in channel catfish, diets containing 240 g kg)1 protein with CHO : L ratios ranging from 0.45 to 4.5 have been reported to produce fish with no significant difference in WG or feed conversion (Garling & Wilson 1977), and in tilapia, Tilapia zillii, insignificant effects on growth performance were noted in fish-fed CHO : L ratios ranging from 0.8 to 8.8 in a 240 g kg)1 protein diet (El-Sayed & Garling 1988) In the present study, either high lipid with low carbohydrate or low lipid with high carbohydrate reduced WG, PER and PPV in fish, indicating that fish-fed diets with similar energy content, supplied by different energy sources, result in differential utilization of lipid and carbohydrate energy It appears that utilization of dietary lipid and carbohydrate is moderate in grass carp Similar results have also been observed in other species such as Clarias catfish (Jantrarotai et al 1994) and walking catfish (Erfanullah & Jafri 1998) But grass carp fed 100 g kg)1 lipid and 175 g kg)1 carbohydrate in the diet performed much better based on growth and feed utilization than that fed lipid-free and 400 g kg)1 carbohydrate in the diet However, decreasing carbohydrate in the diet to 355 g kg)1 resulted in a significant increase in growth of the fish Therefore, it appears that grass carp cannot tolerate up to 400 g kg)1 carbohydrate in the diet, and are either a little more capable of utilizing lipid or less tolerant to high carbohydrate intake Low growth and poor conversion efficiencies with high carbohydrate–low lipid diet could perhaps be attributed to a deficiency of essential fatty acids in diet As the diet with a CHO : L ratio of 202.5 contained only g kg)1 dietary lipid, it may not have met the needs of the fish for essential fatty acids, thus resulting in reduced growth and poor feed efficiency In T zillii, low lipid (17 g kg)1) and high carbohydrate (410 g kg)1) in a 300 g kg)1 CP diet similarly produced low growth rates and feed utilization (El-Sayed & Garling 1988) Crude fibre content (165 g kg)1), as cellulose, in diet is unlikely to be the cause affecting performance of grass carp Das & Tripathi (1991) reported that the presence of cellulose activity in grass carp suggested the necessity of providing cellulose in the diet, and Stanley (1974) reported that grass carp were highly efficient in converting assimilated food into fish biomass giving it a rapid growth rate despite wasteful digestion In other studies, cellulose was used at levels up to 400 g kg)1 for channel catfish and T zillii without affecting growth rates (Garling & Wilson 1977; El-Sayed & Garling 1988) Growth maxima were observed when the dietary carbohydrate and lipid levels were 274.7 and 58.7 g kg)1, evaluated by a second-order polynomial regression model This carbohydrate level is similar to catfish (Erfanullah & Jafri 1998) and the lipid level is much lower than for both catfish (Erfanullah & Jafri 1998) and yellowfin seabream (Hu et al 2007) Martino et al (2002) reported that dietary lipid level is usually associated with an increase in viscera lipid deposition This phenomenon was also supported by the results of VSI, HSI, IPF and liver lipid content in the present experiment HSI and IPF values increased as dietary lipid levels increased; this agrees with the result of hybrid Clarias catfish (Jantrarotai et al 1994) and hybrid striped bass (Gaylord & Gatlin 2000) As reported for many fish species, the increase in dietary lipid levels generally leads to an increase in fat deposited in the carcass (NRC 1993; Helland & GrisdaleHelland 1998; Company et al 1999) The positive correlation between whole-body lipid content and dietary lipid level in the present study followed similar observations as reported for other fish species such as T zillii (El-Sayed & Garling 1988), hybrid Clarias catfish (Jantrarotai et al 1994), hybrid tilapia (Chou & Shiau 1996) and walking catfish (Erfanullah & Jafri 1998) Increased carbohydrate failed to produce undesirable fat accumulation in the body and liver of the fish, similarly to findings, in turbot fed increasing percentages of starch, which also resulted in reduced lipid deposition (Nijhof & Bult 1994) There were almost no significant differences among dietary treatments in whole body and liver protein But Catacutan & Coloso (1997) observed decrease in body protein in Asian seabass fed a high lipid diet, and Ellis & Reigh (1991) reported that the percentage decrease was due to a proportional increase in tissue lipid level Whole body and liver moisture increased with increasing CHO : L Ó 2009 The Authors Journal compilation Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 327–333 ratios in our study, which was similar to the result of yellowfin seabream (Hu et al 2007) Tissue cholesterol concentrations are known to vary depending upon the nutritional status of fish (Kennish et al 1992; Kaushik et al 1995) In the present study, plasma cholesterol and triacylglyceride concentrations observed indicated a more active lipid transport, in response to the higher dietary lipid level Similar results were also observed by Regost et al (2001) and Du et al (2005) Adaptation of hepatic enzymes to increased dietary carbohydrate levels have been reported consistently for herbivorous and omnivorous species such as carp (Shimeno et al 1981) and channel catfish (Likimani & Wilson 1982) In contrast, Dias et al (2004) reported that the activities of hepatic GK and PK were not affected by the extruded starch level of the diets in sole juveniles In the present study, the activities of hepatic PK and GK were significantly increased with the increasing carbohydrate levels in the diets It is important to note that the level of GK activity was extremely low when compared with activities found in rainbow trout or gilthead seabream fed with almost the same level of digestible carbohydrates (Panserat et al 2000); the reason for this result can be either a low induction of the enzyme in carp (Panserat et al 2000) or a too long sampling delay after the meal (24 h in the present trial) For instance, hepatic GK activity in gilthead seabream was high when assayed at h after a meal, while at 24 h it was extremely low (Panserat et al 2000) Positive correlation between lipase activity and lipid digestibility has been reported in mahseer (Tor khudree) (Bazaz & Keshavanath 1993), rohu (Labeo rohita) (Gangadhar et al 1997) and European sea bass (Dicentrarchus labrax) (Peres & Olı´ va-Teles 1999), in agreement with our finding, indicating a higher activity of lipase, in response to the higher dietary lipid level In the present study, the value of ALP activity was the highest in grass carp fed 100 g kg)1 lipid and 175 g kg)1 carbohydrate ALP is considered to be involved in the absorption of nutrients such as lipid, glucose, calcium and inorganic phosphate (Dupuis et al 1991; Mahmood et al 1994), and its activity has been related to food intake (Fraisse et al 1981) Ribeiro et al (1999) has found a trend of higher ALP activity in Senegalese sole that were seen to feed more actively Growth maxima were observed when the dietary carbohydrate and lipid levels were 274.7 and 58.7 g kg)1, corresponding to a CHO : L ratio of 4.68 The utilization of dietary lipid and carbohydrate was comparatively moderate in grass carp, but the fish were a little more capable of utilizing lipid when compared with high carbohydrate intake AOAC (1995) Official Methods of Analysis Association of Official Analytical Chemists, Arlington, 1141 pp Bautista, M.N (1986) The response of Penaeus monodon juveniles to varying protein/energy ratios in test diets Aquaculture, 53, 229– 242 Bazaz, M.M & Keshavanath, P (1993) Effect of feeding different levels of sardine oil on growth, muscle composition and digestive enzyme activities of mahseer, Tor Khudree Aquaculture, 115, 111– 119 Bergot, F (1979) Carbohydrate in rainbow trout diets: effects of the level and source of carbohydrate and number of meals on growth and body composition Aquaculture, 18, 157–161 Bessey, O.A., Lowry, O.H & Brock, M.J (1946) A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum J Biol Chem., 164, 321–329 Buhler, D.R & Halver, J.E (1961) Nutrition of salmonid fishes: IX Carbohydrate requirements of Chinook salmon J Nutr., 74, 307– 318 Capuzzo, J.M & Lancaster, B.A (1979) The effects of dietary carbohydrate levels on protein utilization in the American lobster (Homarus americanus) Proc World Maric Soc., 10, 689–700; 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