26 Comparative studies on the nutrition of two species of abalone, Haliotis tuberculata L. and Haliotis discus hannai Ino. IV. Optimum dietary protein level for growth

Aquaculture Aquaculture 136 (1995) 165-180 Comparative studies on the nutrition of two species of abalone, Haliotis tuberculata L and Haliotis discus hannai Ino IV Optimum dietary protein level for growth Kangsen Mai a**, John P Mercer ‘, John Donlon b aShe&h Research Laboratory, University College Galway, Galway, Ireland b Deparmenr of Biochemisrry, University College Galway, Galway, Ireland Accepted 30 May 1995 Abstract A 100 day growth experiment was conducted to identify the optimum dietary protein level for the juveniles of two species of abalone, Haliotis tuberculata and Haliotis discus hannai A mixture of vitamin-free casein and gelatin (4.34: 1) supplemented with crystalline amino acids was used as the protein source to simulate the amino acid profile of abalone body Eight purified diets were formulated to provide graded protein levels ranging approximately from to 50% The weight gain, protein gain, soft body to shell ratio, and carcass levels of protein and lipid of both abalone species were significantly (ANOVA, P < 0.05) affected by the dietary protein level The protein requirements of these abalone were evaluated from weight gain and protein gain respectively, by using the second-order polynomial regression analysis On the basis of weight gain, the optimum protein levels were estimated to be 22.3-32.3%, and 23.3-35.6% for H tuberculata and H discus hannai, respectively According to the protein gain, the statistical analysis indicated that the optimum ranges of protein requirements were 24.0-34.5% and 25.2-36.6% for H tuberculata and H discus hannai, respectively Based on these results, about 35% dietary protein from good quality sources is recommended for the maximum growth of both abalone species; and, if dietary protein is reduced from 35 to 25%, the growth of these abalone may be depressed with 5% likelihood Keywords: Hakotis ruberculata; Halioris discus hnnai; Comparative nutrition; Protein requirements, molluscs Introduction Animals not have an absolute of essential and non-essential protein requirement but require a well-balanced mixture 1983; Wilson, 1989) The most common amino acids (NRC, * Corresponding author at: College of Fisheries, Ocean University of Qingdao, Peoples Republic of China Tel (+86-532) 2864361, Fax (+86-532) 2879091 0044-8486/95/$09.50 1995 Elsevier Science B.V All rights reserved SDr0044-8486(95)01041-6 166 K Mai et al /Aquaculture 136 (1995)165-180 and economical source of amino acid mixture is from natural proteins in feedstuffs Information on the amino acid requirements of an animal provides a basis to evaluate the nutritional value of protein sources and then to select proper protein sources for formulating feeds for the animal Because it is the principal diet component for animal growth, and has the highest cost consideration for commercial feeds, protein has been given priority in nutritional requirement studies (Lim et al., 1979) The minimum amount of dietary protein needed to supply adequate amino acids and produce maximum growth has been determined with semi-purified and purified test diets in about 30 species of fish and crustaceans (NRC, 1983; Tacon and Cowey, 1985; Wilson, 1989) Molluscan nutrition lags far behind that of fish and crustaceans and much less is known of the protein requirements of molluscs largely due to their lower commercial importance in aquaculture (Carefoot, 1982) There have been some publications concerning the protein nutrition of molluscs (e.g Duncan et al., 1985; Nell, 1985; Uki et al., 1985; Utting, 1986; Hawkins et al., 1989; Kreeger and Langdon, 1992; Kreeger, 1993; Mercer et al., 1993) From the viewpoint of quantitative requirements, however, there have been only four reports on the protein requirements of abalone, H discus (Ogino and Kato, 1964)) H discus hunnai (Uki et al., 1986)) H kumtschutkuna (Taylor, 1992) and H midue (Britz et al., 1994), It has been demonstrated that intensive culture of abalone using nutritionally complete diets is feasible; however, information on the nutrient requirements of this animal is far from sufficient to support industrial scale feed manufacture Investigations into the protein requirement of abalone have used either white fish meal (Ogino and Kato, 1964; Uki et al., 1986; Britz et al., 1994) or casein (Uki et al., 1986; Taylor, 1992) as the sole protein source in the test diets Following the results of feeding trials, casein has been considered as the best protein source for abalone (Uki et al., 1985) At present, no information is available on the quantitative requirements of abalone for essential amino acids (EAA), however, the analyses of amino acid profiles of abalone body indicate that casein is probably limiting in some EAA, especially arginine (Mai et al., 1994) Therefore, the protein requirement of abalone could be overestimated if casein is used as the sole protein source in experimental diets The protein requirements of fish are usually estimated by feeding a balanced diet containing graded levels of high quality protein, which is generally a casein/gelatin mixture supplemented with crystalline amino acids to simulate the amino acid profiles of either fish body or whole hen’s egg protein (Tacon and Cowey, 1985) Hence, this study was designed to identify the optimum level of dietary protein for the growth of the two commercially important abalone species, H tubercuhtu and H discus hunnui by employing the protein source, casein/gelatin mixture supplemented with crystalline amino acids to simulate the EAA pattern of abalone body Materials and methods 2.1 Experimental diets Formulations of the experimental diets and their proximate composition are shown in Table On the basis of essential amino acid ratios (A/E ratios) (Arai, 198 1) , the EAA pattern in the soft-body of these abalone (Mai et al., 1994)) expressed as mean A/E ratios, was used as a reference The mixture of casein and gelatin (4.34: 1) was supplemented with 0 65.00 5.00 18.00 4.00 2.00 0.50 5.00 0.28 5.09 15.8 5653.6 0.4 10.03 5.11 16.3 162.5 15.5 20.15 5.03 17.4 86.1 31.1 25.01 5.16 17.5 70.0 38.6 30.36 5.07 18.7 61.5 46.9 35.32 5.21 18.7 53.0 54.6 28.99 6.68 2.43 26.9 5.00 18.00 4.00 2.00 0.50 5.00 35 39.99 5.03 18.9 47.3 61.8 33.10 7.63 2.77 21.50 5.00 18.00 4.00 2.00 0.50 5.00 40 50.15 5.13 19.7 39.1 78.0 41.39 9.54 3.47 10.6 5.00 18.00 4.00 2.00 0.50 5.00 50 DS” Arg His Ile Leu Lys Met+Cys Phe+Tyr Thr Val 1.ooo 171 38 96 152 133 69 151 88 102 Abalone 0.612 68 51 112 172 139 61 190 76 131 Casein 0.802 308 34 61 129 157 38 102 78 93 Gelatin 0.985 164 43 94 148 124 60 160 91 112 Exp diets ‘All ingredients were purchased from Sigma Chemical, UK.‘Amino acid mix, each 100 g of the mix contained arginine, 71.43g; threonine, 21.35 g and methionine, 7.22 g 3Mineral mix, each 1000 g of diet contained NaCl, 0.4 g; MgSO, 7Hz0, 6.0 g; NaH,PO, 2HZ0, 10.0 g; KH,PO, 12.8 g; Ca( H,PO,) z HzO, 8.0 g; Fe-lactate, 1.O g; Ca-lactate, 1.4 g; ZnSO,.7H,O, 141.2 mg; MnSO,.4H,O, 64.8 mg; CuS04.5Hz0, 12.4 mg; CoC12.6Hz0, 0.40 mg, KIO,, 1.2 mg 4Vitamin mix, each 1000 g of diet contained tbiamin HCI, 120 mg; riboflavin, 100 mg; folic acid, 30 mg; PABA, 400 mg; pyridoxine HCI, 40 mg; niacin, 800 mg; Ca pantothenate, 200 mg; inositol, 4000 mg; biotin, 12 mg; vitamin E, 450 mg; menadione, 80 mg; B,,, 0.18 mg; ascorbic acid, 4000 mg; vitamin A, 100,000 I.U.; vitamin D, 2,000 I.U.; ethoxquin, 400 mg ‘Corn oil and menhaden fish oil ( 1:l) with 0.001% of ethoxquin ?ncluding the AA mix ‘Gross energy aPercentage of protein energy.‘A/E ratio = (Each EAA/Total EAA) X 1000, not including Trp The calculation of A/E ratios for casein and gelatin were based on the amino acid data reported in NRC ( 1983) InDegree of similarity (Mai et al., 1994) Protein(%)6 Lipid( %) GE (kJ/g)’ W/g protein % PEE 30 8.29 16.59 20.70 24.88 1.91 3.82 4.77 5.74 0.70 1.39 1.73 2.08 54.10 43.20 37.80 32.3 5.00 5.00 5.00 5.00 18.00 18.00 18.00 18.00 4.00 4.00 4.00 4.00 2.00 2.00 2.00 2.00 0.50 0.50 0.50 0.50 5.00 5.00 5.00 5.00 25 EAA 20 10 A/E Ratio? diets (% on dry weight basis) Protein levels (%) and proximate analysis of experimental Proximate analysis (means of triplicates) Casein Gelatin AA mix’ Dextrin Cellulose Na alginate Mineral mix’ Vitamin mix4 Choline co/MF@ Ingredients’ Table Ingredient composition 168 K Mai et al /Aquaculture 136 (1995) 165-180 a crystalline amino acid mix including arginine, threonine and methionine to simulate the reference protein The A/E ratios and the degree of similarity (DS) in EAA pattern (Mai et al., 1994) of the protein sources and the experimental diets to that of the reference are also given in Table A mixture of corn oil and menhaden fish oil ( 1: 1) was used as the lipid source Dextrin, the major carbohydrate source, was used to adjust the protein level Eight experimental diets were formulated from the purified ingredients to provide graded protein levels ranging approximately from to 50% Gross energy of experimental diets correspondingly ranged from 15.8 to 19.7 kJ g-’ as determined by bomb calorimetry, and energy/protein ratios (E/P ratios) were from 39.1 to 5653.6 kJ gg’ protein Procedures for food preparation were modified from the method described by Uki and Watanabe ( 1992) Casein, gelatin and some minerals that were in the form of small grains were ground individually using a Pascal Mill and then passed through a mesh with 125 pm pore size After adding water (about 120%, w/w) to the mechanically mixed ingredients containing 18% of sodium alginate, a paste was made by using an electronic mixer The paste was shaped into 0.5 mm thick sheets, which were cut into cm* flakes The flakes were dipped in an aqueous solution of CaCl, (5%, w/v) for one minute The surplus solution was drained naturally, then the flakes were sealed in a sample bag and stored at - 20°C until use 2.2 Animal rearing To maintain the water temperature, a re-circulating system was used This system comprised a glass fibre reservoir tank (3 m3), a high position tank ( 150 1) and two flush trays (220 X 120 X 30 cm) Each flush tray held 24 rearing units constructed from 10 PVC flowerpots with covers, and the bases replaced with 1.0 mm mesh Each rearing unit was stocked with 25 abalone juveniles The seawater in the reservoir tank was pumped into the high position tank, where it was aerated, then delivered to each rearing unit through 1.O cm (i.d.) PVC tubes The flow rate of water through each unit was about 1 min- ‘ The water depth in the flush trays was maintained at about 25 cm and the excess water was returned to the reservoir through the outlets Water temperature during the experiment was maintained at 13-15°C Similar size juveniles of H discus hannai (378.3 f 16.7 mg) and H tubercuzata ( 182.7 + 7.8 mg) were selected from the hatchery produced population, then assigned to the rearing system using a completely randomised design with eight treatments and three replicates per treatment At the same time, 100 juveniles of each species were randomly sampled from the same cohort and stored at - 20°C until subsequent analysis for carcass composition Abalone were fed the appropriate diet every third day at a satiation level with a little leftover The whole system was thoroughly cleaned just before each feeding and refilled with pre-heated and filtered ( 10 pm cartridge) seawater Under such management, good water quality in the system can be maintained (K Mai, unpublished observation) The feeding experiment was run for 100 days 2.3 Sample collection and analysis At the termination of the experiment, 15 abalone from each replicate were frozen for subsequent chemical analysis Growth is expressed as mean weight gain and protein gain 169 K Mai et al /Aquaculture I36 (1995) 165-180 (mg per abalone) The initial and final samples were slightly thawed, and shell and softbody were separated The soft-body to shell ratio (SB/S ratio, w/w) was computed to provide an index of nutritional status for abalone An aliquot of soft-body tissue from each sample was lyophilised to a constant weight to determine the moisture content The rest of each soft-body sample was homogenised with an equivalent volume of distilled water for 10 in an Omni-mixer (Sorvall, New Town, CT), then the homogenate was freeze-dried and ground into fine powder for analyses of protein and total lipid Protein was estimated by a modification of the Lowry procedure (Hartree, 1972) with bovine serum albumin as the calibration standard Extraction of lipid was carried out according to the method of Bligh and Dyer ( 1959) and the lipid levels were determined gravimetrically The levels of protein and lipid in the experimental diets were analysed as described above The gross energy in the diets was estimated with a Gallenkamp ballistic bomb calorimeter The mean gains in body weight and protein were calculated as follows: MWG (mg/abalone) = Wt- Wi MPG(mg/abalone)=SBt.(l-Mr).Pt-SBE’.(l-Mi) Pi where MWG is mean weight gain; Wi, Wt is initial or final mean body weight (mg); MPG is mean protein gain; SE, SBt is initial or final soft-body weight (mg); SBi, t= Ri, t Wi, t/ ( + Ri, t); Ri, Rt is initial or final soft-body to shell ratio (SB/S ratio); Mi, Mt is initial or final moisture level in soft body (%); Pi, Pt is initial or final protein level in soft body (%) 2.4 Statistical analysis All percentage data were square-root arcsine transformed prior to analysis Data from each treatment were subjected to one way ANOVA When overall differences were significant at less than 5%, Tukey test was used to compare the mean values between individual treatments Protein requirements of the juvenile abalone were estimated from weight gain and protein gain using both the broken-line model (Robbins et al., 1979) and the secondorder polynomial regression analysis model (Lovell, 1989) All statistics were calculated using Systat@ package (SYSTAT, 1992) Results 3.1 Survival and weight gain The data of survival and weight gain of abalone fed the experimental diets are shown in Table During the 100 day experimental period the mean survival, ranging from 92.0 to 98.7% for H discus hannai and from 82.7 to 96.0% for H tuberculata, was generally high and not related to dietary protein level, even though both species fed the approximately protein-free diet (Diet 1) showed relatively lower survival The weight gain of both abalone species was significantly affected by dietary protein levels (ANOVA, P < 0.001) The progressive increase in mean weight gain reached a maximum value at a protein level of 2.469 0.064 0.865 0.554 194.7(4.1) 178.1(9.1) 186.9(4.5) 186.0(9.4) 177.3(8.3) 174.8(10.8) 190.1(7.5) 173.6( 10.3) 367.8( 16.9) 358.5(6.6) 388.5(5.7) 404.9( 18.3) 393.8( 16.9) 367.2(6.1) 362.6( 10.0) 383.1(7.2) 54.95 0.000 353.1( 16.3) 643.1(5.5) 874.6(47.9) 972.4(24.1) 917.0(2.9) 874.7(23.0) 806.9(42.6) 872.9( 19.2) H d H t* H d’ 19.389 0.000 239.2(4.0) 380.9(12.4) 499.2( 10.7) 498.9(39.2) 536.7(37.0) 524.0(34.0) 554.9(12.3) 417.8( 12.3) Ht Final weight (mg) Initial weight (mg) 34.53 0.000 359.1(29.7)d 349.1(24.8)’ 364.8(4.9)’ 244.2(7.4)bc 312.3(9.6)cd 313.1(29.6)“’ 44.6(0.39)” 202.7( 13.7)” H t 0.717 0.660 92.0(4.6) 96.0(2.3) 96.0(2.3) 96.0(2.3) 98.7( 1.3) 94.7(3.5) 92.0(2.3) 96.0(2.3) H d 1.76 0.166 82.7(3.5) 96.0(2.3) 94.7( 1.3) 96.0(2.3) 90.7(7.4) 96.0(2.3) 92.0(2.3) 93.3( 1.3) H t Survival (%) different as determined by Tukey test (P > 0.05) 28.17 0.000 - 14.7(0.6)a’ 284.6(7.0)b 486.2(44.3)” 567.5(6X)’ 523.0(7.0)=’ 507.5( 19.9)=’ 444.3(32.7)’ 489.8( 14.8)“’ H d Weight gain (mg/abalone) ‘H d, H discus hannai ‘H t, H tuberculata 3Means in the same column sharing the same superscript letter were not significantly F P 0.3 10.0 20.2 25.0 30.4 35.3 40.0 50.2 ANOVA Protein level( %) Table Effect of dietary protein on growth and survival of the abalone, H ruberculara and H discus hannai (means (s.e), n = 3) % R a R K Mai et al /Aquaculture y = 6.902+30.482x- 0.428x"ZRV = 0.902 y = 171 136 (199.5) 165-180 H dircrcs hami 35.569+ 19.712x - 0.305r'Z R"2 = 0.919 A H.m!xrculata X1=23.3%(H.d) Xmax=35.6%(H.d) Xmax=32.3% (H.I) I 40 I 50 I 60 Dietary protein (%) Fig I, Relationship between weight gain and dietary protein level for H discus hannui and H tubercuhtu a~ described by second-order polynomial regression Y,,,,,, maximum weight gain; Y,, a weight gain below maximum but within the 95% confidence interval; X,,,, a protein level required for Y,,,,,; X,, a protein level associated with Y, 25% for H discus hannai and 40% for H tuberculata The results of Tukey test, however, showed that weight gains of both species at or beyond 20% dietary protein concentration were not significantly different; but 50% dietary protein appeared to significantly depress gain of H tuberculata (Table 2) On the basis of weight gain, the results estimated by the broken-line model showed that the protein requirements of H discus hannai and H tuberculata were 17.3% and 17.4%, respectively (Table 4) However, the second-order polynomial regression analysis revealed that the ranges of dietary protein levels that produced the maximum weight gains (Y,,) and lower gains within the 95% confidence limits of Y,,,,, ( Y, ) were 23.3-35.6% (X,-X,,,) for H discus hannai and 22.3-32.3% for H tuberculata (Fig 1, Table 4) The protein requirements of both abalone species seem to be similar when they were estimated with the same analysis model (Table 4) One remarkable difference in growth performance between these abalone was that feeding on Diet (approximately 0.3% protein), H discus hannai exhibited a negative weight gain, while H tuberculata showed a positive one (Table and Fig 1) 3.2 Carcass composition and protein gain Except for moisture content, the body composition was significantly affected by the dietary treatments (Table 3) Soft body to shell ratios (SB/S ratio, w/w) could be regarded 13.32 0.000 1.91 0.067 ‘Soft-body to shell ratio (w/w) ‘On dry weight basis 3H.t, H tuberdata; H.d, H discus hannai 4Data for initial animals were not subjected to statistical analysis ‘Means in each column sharing the same letter are not significantly 11.97 0.000 F P 1.65(0.03)” 2.02(0.03)b 2.23(0.07)W 2.25(0.04)k 2.32(0.12)W 2.27(0.05)h 2.34(0.04)’ 2.15(0.06)& 1.78(0.03) 10.84 0.000 24.66 0.000 8.6(0.2)’ 8.3(0.1)” 8.2(0.2)“’ 7.9(o.l)k 7.8(0.1)h 7.5(0.l)b 7.4(0.l)b 6.8(o.1)a 6.3(0.2) H t different based on Tukey test (P> 0.05) 2.02 0.058 S.O(O.2)’ 7.8(0.1)’ 7.6(0.1)bc 7.7(O.l)“c 7.3(o.l)ak 7.o(o.l)ab 6.6(0.3)” 6.7(0.3)” 6.2( 0.3) 76.4(0.1) 75.4(0.2) 76.0(0.2) 75.7(0.1) 75.6(0.2) 74.9(0.3) 74.9(0.3) 75.7(0.6) 76.2(0.3) SO.O(O.3) 79.5(0.2) 78.8(0.2) 78.7(0.2) 79.0(0.4) 79.0(0.2) 79.6(0.2) 79.4(0.3) 79.8(0.5) 1.50(o.05)ti 1.83(0.06)b 2.04(0.06)b 2.05(0.06)b 2.07(0.04)b 2.03(0.04)b 1.96(0.06)b l.97(0.06)b 1.81(0.02) H d H t H d H d3 H t3 Lipids (%)’ Moisture (%) 16.24 0.000 40.6(0.6)” 43.6(0.6)ab 44.7(0.8)b 45.5(o.9)b 46.7(0.4)r’=’ 46.5(0.5)““ 48.4(0.7)=“ 49.4(0.8)’ 43.3(0.6) H d Protein (‘70)’ of H tuberculara and H discus hmnai (means (s.e.), n = 3) SB/S Ratios’ 0.3 10.0 20.2 25.0 30.4 35.3 40.0 50.2 Initial animals4 ANOVA Protein level (%) Table Effect of dietary protein on the carcass composition 28.17 0.000 41.5(0.3)” 42.7(0.8)ab 46.0(0.4)bc 46.8(0.4)=’ 49.4(0.5)‘& 50.1( l.o)de 50.2( l.l)* 51.9(0.7)” 45.1(0.8) H t 107.75 0.000 -3.3(0.1)” 17.2(0.4)b 34.0(2.8)= 40.9(0.6)’ 38.4(0.4)cd 36.9( 1.3)cd 32.5(2.2)’ 37.8( l.l)=’ H d 50.55 0.000 1.6(0.1)” 14.9(0.9)b 26.1(0.7)cd 27.0(2.5)Cd 32.8(2.6)de 33.8(2.3)d’ 35.4(0.6)’ 24.3(0.7)’ H t Protein gain (mg/abalone) K Mai et al /Aquaculture Table Comparison of the protein requirements analysis model Statistical model Broken-line analysis Quadratic regression analysis (X, -X,,,) ‘Weight gain 2Protein gain ‘Numbers in parentheses 173 136 (1995) 165-180 of abalone estimated by the broken-line H discus hannai model and the quadratic regression H tuberculata Based on WG’ Based on PC? Based on WG Based on PG 17.3% (2765.8)’ 23.3-35.6% (4905.2) 21.6% (10.24) 25.2-36.6% (20.3) 17.4% (2132.3) 22.3-32.3% (1018.0) 27.3% (17.5) 24.0-34.5% (10.2) are residual mean squares as a good condition index of nutritional status for abalone because it showed a very similar pattern to that of weight gain Statistically, SB/S ratio did not reflect the effect of dietary protein levels on growth performance as sensitively as weight gain The lowest SB/S ratio was always observed for the abalone fed diet (0.3% protein) and higher SB/S values were observed for H discus hannai fed 20-35% protein and for H tuberculata fed 20-40% protein Whole soft-body lipid levels, ranging from 6.6 to 8.0% for H discus hannai and from 6.8 to 8.6% for H tuberculata, steadily decreased with increasing dietary protein Conversely, carcass protein levels, ranging from 40.6 to 49.0% for H discus hannai and from 41.5 to 51.9% for H tuberculata, showed a linear increase when dietary protein increased from 0.3 to 50.2% Based on F values of ANOVA (Table and Table 3), protein gains appeared to be more sensitively affected by dietary protein levels than did weight gains Protein gains ranged from - 3.3 to 40.9 mg per abalone for H discus hannai and from 1.6 to 35.4 mg per abalone for H tuberculata Similarly to weight gains, maximum protein gain was recorded for H discus hannai fed 25% dietary protein and for H tuberculata fed 40% dietary protein Based on protein gain, the protein requirements assessed by the broken-line model were 21.6% and 27.3% for H discus hannai and H tuberculata, respectively (Table 4) The second-order polynomial regression demonstrated that the minimum dietary protein levels giving the maximum protein gains were 36.6% for H discus hannai and 34.5% for H tuberculata, and the lower dietary protein levels, i.e 25.2% and 24.0% for H discus hannai and H tuberculata respectively, could reduce their protein gains with 5% likelihood Discussion Estimates of quantitative nutrient requirements are influenced not only by the criteria used but by the statistical methods chosen to evaluate criterion response to differing dietary nutrient concentrations (Zeitoun et al., 1976) In quantifying protein requirements of fish, the most common criterion used by researchers is growth (Tacon and Cowey, 1985; Lovell, 1989) Some other criteria, such as nitrogen balance, protein efficiency ratio (PER), net K Mai et al /Aquaculture 174 y= y = 136 (1995) 165-180 2.6139+ 2.343 -0.032~A2 ~"2 =0.912 0.171 + 1.865x X1=24.0% (H t) 0.027x”Z RY = 0.924 H.discwhm~i A f/ ~,&rcu,aara Xmax=36.6% (H d) Xmax=34.5% (H L) Dietary protein (%I Fig Relationship between protein gain and dietary protein level for H discus hctnnui and H tuberdata as described by second-order polynomial regression Y,,,, maximum protein gain; Y,, a weight gain below maximum but within 95% confidence interval; X,,,,, a protein level required for Y,,,,,; X, , a protein level associated with Y, protein utilisation (NPU) and feed conversion efficiency (FCE) , are sometimes employed The reliability of the results based on the latter criteria depends strongly upon the accuracy in determining the feed consumed The aquatic environment makes it difficult to obtain accurate data on feed intake of aquatic animals Therefore, growth in terms of weight gain is generally sensitive to the dietary protein content, and sometimes shows a more clear-cut protein requirement level than PER, FCE, etc (Moore et al., 1988) Because abalone eat very slowly, using radula to scrape off particles of feed and send them into the mouth rather than swallowing feed pellets directly as most fish do, great difficulties are encountered in obtaining reliable data on their feed intake, particularly on artificial feeds As a result, the protein requirements of the juveniles of H discus hannai and H tuberculata were evaluated by their growth data in the present study Weight gain is a reliable indicator for growth as long as the experimental variable is not expected to affect composition of gain in the animal (Lovell, 1989) In this study, dietary protein significantly affected the carcass composition of both abalone species (Table 3) Hence, it is generally believed that protein gain could be a more reliable measure for true growth than weight gain, and thus more reliable estimates of protein requirements may be yielded from the protein gain data In the current study, however, protein gain was still highly correlated with weight gain in both abalone species (r = 0.99)) and very similar estimates of protein requirements were obtained from these two types of data by the second-order polynomial regression analysis (Fig and Fig 2) K Mai et al /Aquaculture 136 (1995) 165-180 175 Different statistical methods, such as ANOVA and Duncan’s multiple range test, the broken-line model and the second-order polynomial regression analysis, have been used to determine the protein requirements of several species of fish (e.g Delong et al., 1958; Zeitoun et al., 1976; Cowey, 1979; Moore et al., 1988; Santiago and Reyes, 1991; Khan et al., 1993) The broken-line model and the quadratic regression model employed in the current study produced quite different estimates (Table 4) Comparing the residual mean squares yielded from the two mathematical models (Table 4), the broken-line model appeared to be more suitable for the data of H discus hannai, whereas the second-order polynomial regression analysis method seemed to be more appropriate for those of H tuberculatu It may not be preferable to compare the protein requirements, which were not assessed by the same mathematical model, between the abalone species The observed data (Table and Table 3, Fig and Fig 2) showed that maximum growth of H discus ham& was reached around 25-30% of dietary protein, and the fastest growth of H tuberculata was attained around 3&40% protein Hence, the broken-line model appeared to underestimate the protein requirements of these abalone The polynomial regression analysis shows the advantage of being more accurate than other methods when the relationship between dietary nutrient and growth data is curvilinear Additionally, this method can yield nutrient requirements for maximum and less than maximum rates of growth for both physiological and economical considerations (Lovell, 1989) It is believed that the second-order polynomial regression analysis gave better estimates in the present study That is to say, on the basis of protein gain data, the optimum dietary protein levels were 25.2-36.6% for H discus hannai and 24.0-34.5% for H tuberculata Statistically, H discus hannai seems to have a slightly higher protein requirement than H tuberculata Conversely, the observed data indicate that H tuberculatu may have a slightly higher requirement for protein than H discus hannai (Table and Table 3) However, it may be premature to draw any conclusion on the different protein requirements of these two species because their initial size in this experiment was different (Table 2) For the sake of simplification, it could be concluded that about 35% dietary protein may produce maximum growth, in terms of protein gain, for the juveniles of both H discus hannai and H tuberculata, and when dietary protein is reduced from 35 to 25% the growth of these abalone may be depressed with 5% likelihood The optimum level (25-35%) of dietary protein for these abalone is lower than those for most species of fish and crustaceans (NRC, 1983; Tacon and Cowey, 1985; Wilson, 1989) This result looks similar to those recommended by Ogino and Kato ( 1964) for H discus (20%)) by Uki et al ( 1986) for H discus hannai (2&30%), and by Taylor ( 1992) for H karntschatkana (30%), but much lower than that of H midue (47%) (Britz et al., 1994) However, it is difficult to compare these values directly Among the reported data for abalone, the protein requirements of H discus and H midae were evaluated by using fish meal as a sole protein source (Ogino and Kato, 1964; Britz et al., 1994) In fact, Ogino and Kato ( 1964) obtained very different results in their three experiments In their last two experiments, maximum growth rates of H discus were reached at the highest protein levels tested (42.7% and 44.3%) For H kamtschatkuna, the range of dietary protein level tested was that which resulted from O-30% casein used as the sole protein source, and of these, only 20%- and 30%-casein diets yielded positive growth (Taylor, 1992) Protein levels above 30% have not been investigated for this abalone Uki et al (1986) evaluated the protein requirement of H discus hannai using both casein and fish meal as the sole protein 176 K Mai et al /Aquaculture 136 (1995) 165-180 54 - 52 - H discushannai A H tuberculnta 50 48 - +P B 46 44 - $0 f P I 42 25 I I 50 I , 75 100 125 , I , 150 175 150 175 200 8.5 8.0 - 7.5 - @G 7.0- P P 6.5 6.0 @ I I I 25 50 75 E/F’ ratio I , 100 (kJ/G I 125 , , ml protein) Fig Relationship between dietary energy/protein ratio and carcass lipid and protein in H discus hannai and H tuberculata Data from Diet are not depicted to simplify the graph Error bars, f s.e sources, based on FCE and NPU instead of growth Actually, weight and protein gains of H discus hunnai increased up to the highest protein level (43%) when fish meal was used as the sole protein source, and maximum growth was reached at 50% of dietary protein when casein was used as the sole protein source (Uki et al., 1986; see Uki and Watanabe, 1992 for a review) From this standpoint, our estimate is much lower than theirs This difference could probably be attributed to the simulation of the EAA pattern of abalone body by using a mixture of casein and gelatin supplemented with crystalline amino acids as protein sources in the current study It can be seen from Table that the simulation increased the DS (degree of similarity) value of EAA pattern from 0.612 of casein and 0.802 of gelatin to 0.985 of the experimental diets However, it is necessary to further investigate the utilisation of gelatin by these abalone and the supplemental effectiveness of crystalline amino acids, because it has been documented that the utilisation of gelatin or crystalline amino acids by some species of fish and crustaceans can be very poor (Wilson et al., 1977; Cowey and Sargent, 1979; Pieper and Pfeffer, 1980; Deshimaru, 1982; Mai et al., 1988; Lim, 1993) However, it has been demonstrated that over 50% of crystalline amino acids diffuse from agar gel in just a few hours (Carefoot, 1982) Leaching could be a major problem, even though the procedure of diet preparation in the current study produces K Mai et al /Aquaculttwe 111 I36 (1995) 165-180 45 25 50 7s E/P ratios 100 H discus hannar A H tuberculata 125 150 17s 2M) (kJ/g protein) Fig Relationship between dietary energy/protein ratio and protein gain of H discus hnnai and H tuberculatu Data from Diet are not depicted to simplify the graph Error bars, f se good water stability of feed pellets (Mai et al., 1995) Therefore, the contribution of the added crystalline amino acids largely depends on the first day intake in each day feeding The notably different performance between H discus hannai and H tuberculata when both fed on Diet (without protein sources added but 0.3% protein estimated) might result from their differences in initial size, energy requirement, appetite for the ‘non-protein’ diet, etc The mean initial body weight of H discus hannai was about twice as big as H tuberculutu (Table 2) As a result, H discus hannui certainly required a higher absolute amount of energy and protein for maintenance than H tuberculata Based on visual inspection of uneaten feed remaining in the rearing units, the approximately protein-free diet (Diet 1) apparently depressed the appetite of both abalone species Judging from the negative gains in both body weight and protein of H discus hannai fed diet (Table and Table 3) and the lowest gross energy level in this diet (Table l), these larger abalone obviously did not ingest sufficient protein and/or energy to meet their maintenance requirements In the smaller H tubercuhta, however, the consumed protein and energy were not only sufficient to meet the maintenance requirements, but also produced observable growth in weight and shell If the ingested nutrients came completely from the artificial feed (Diet 1), H tuberculutu appeared to consumed relatively more feed (% body weight) than H discus hannai, indicating that the European abalone might have better appetite to the unpalatable diet, and/ or have a higher energy requirement than the Japanese abalone It is still unknown whether these differences are size-dependent or species-dependent Though the quantity of benthic microalgae growing on the inside surfaces of the rearing units was limited, it might be a supplemental source of nutrients particularly for smaller abalone, which require smaller absolute amounts of nutrients for maintenance and are better grazers of microalgae than larger ones Although the moisture content of the abalone was not significantly affected by dietary protein level, it tended to be lower when fed the diets with optimum protein levels (Table 2) relatively 178 K Mai et al /Aquaculture 136 (1995) 165-180 Carcass lipid levels significantly decreased with increasing dietary protein, meanwhile, carcass protein concentrations were significantly elevated (Table 2) Similar observations have also been reported in some fish species (Cowey et al., 1972; Wee and Tacon, 1982; Santiago and Reyes, 1991; Khan et al., 1993) Nevertheless, some studies illustrate the lack of significant influence of dietary protein on body protein of fish and conclude that whole body protein is influenced to a greater degree by dietary lipid than dietary protein (Millikin, 1982; Daniels and Robinson, 1986; Brown et al., 1992) This conclusion is largely true In the present study, however, all diets contained approximately 5% lipid (Table 1)) thus, the significant difference in carcass protein content undoubtedly resulted from the progressive substitution of dietary protein Millikin (1982) suggested that a shorter experimental duration may account for a lack of differences in whole-body protein concentration of striped bass In our opinion, the manifestation of the effect of dietary protein on carcass protein content depends largely on the range of dietary protein levels, or of energy/protein ratios (E/P ratios) tested; the wider the range, the more significant the effect In the present study, the experimental diets contained a wide range of protein levels (O-50%), particularly including the extreme end of low levels (Diets and 2) The corresponding range of E/P ratios was from 39.1 to 5653.6 kJ g- ’ protein, and the percentage of protein energy from 0.4 to 78.0% (Table 1) Apparently, the non-protein energy in low protein diets can easily be converted to lipid and then deposited in animal tissues Subsequently, the deposited lipid dilutes the whole-body protein (Page and Andrews, 1973) However, low protein diets can possibly not provide sufficient EAA for protein biosynthesis, and thus lead to lower body protein The relationship between E/P ratios and the carcass levels of protein and lipid (Fig 3) illustrates that E/P ratio is the dominant factor influencing the carcass composition of these abalone It can be seen from Fig and Fig that the optimum gross energy/protein ratios seem to be around 50-60 kJ g-’ protein in casein/gelatin-based diets for feeding abalone to produce maximum protein gain and higher carcass protein but lower carcass lipid As digestible energy in feedstuffs varies considerably with sources, digestible energy, instead of gross energy, is preferably used to express optimum E/P ratio At present, however, there is no information available on digestible energy of feed ingredients for abalone Further experimentation is required to obtain more reliable optimum E/P ratio for abalone by using varying levels of both dietary protein and digestible energy Acknowledgements The authors would like to thank P Crawford, University of Dublin, for use of the Gallenkamp ballistic bomb calorimeter and B Mannion for the preparation of the manuscript Special thanks are due to D Brown and Y Mgaya for their valuable comments on the manuscript Part of the work was supported by Taighde MaraTeo Kangsen Mai received a postgraduate fellowship from University College Galway References Arai, S., 1981 A purified test diet for coho salmon, Oncorhynchus 550 kisutch, fry Bull Jpn Sot Sci Fish., 47: 547- K Mai et al /Aquaculture 136 (1995) 165-180 179 Bligh, E.G and Dyer, W.J., 1959 A rapid method of total lipid extraction and purification Can J Biochem., 37: 911-917 Britz, P.J., Hecht, T., Knauer, J and Dixon, M.G., 1994 The development of an artificial feed for abalone farming S Afr J Sci., 90: 7-8 Brown, M.L., Nematipour, G.R and Gatlin, D.M., 1992 Dietary protein requirement ofjuvenile sunshine bass at 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