Peter Coutteau, Gilbert Van Stappen, and Patrick Sorgeloos. Introduction

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1 ICES mar. Sei. Symp., 201: A standard experimental diet for the study of fatty acid requirements of weaning and first ongrowing stages of European sea bass (Dicentrarchus labrax L.): selection of the basal diet Peter Coutteau, Gilbert Van Stappen, and Patrick Sorgeloos Coutteau, P., Van Stappen, G., and Sorgeloos, P A standard experimental diet for the study of fatty acid requirements of weaning and first ongrowing stages of European sea bass (Dicentrarchus labrax L.): selection of the basal diet. - ICES mar. Sei. Symp., 201: A standard experimental diet was developed to study lipid nutrition in marine fish larvae during weaning and first ongrowing. Basal diets were prepared by extrusioncooking, crumbling of the pellets, and sieving to obtain particles in the size ranges i.m and fxm. Three protein sources in the extruded basal diet were evaluated: (1) micronized cod protein granulate (COD); (2) a mixture of casein, gelatin, and albumin (CGA); (3) a mixture of cod fish meal, whey protein, soybean protein concentrate, albumin, hemoglobin meal, and wheat gluten (MIX). A variable lipid fraction was added to the extruded nucleus by coating the particles with an emulsion, coconut oil (COCO), or fish oil emulsion (FO), in a fluidized bed granulator. European sea bass (Dicentrarchus labrax L.) were weaned and consequently reared for 3 weeks on the experimental diets in a recirculation system. A commercial diet and the uncoated COD diet served as control diets. The suitability of the diets was assessed on the basis of the water stability of the particles, survival, growth, resistance to higher salinity stress of the fish, and tissue fatty acid composition. The MIX diet may hold promise as a standard basal diet for lipid nutrition research with marine fish species during weaning and first ongrowing. Furthermore, the MIX basal diet coated with the appropriate lipid fraction may serve as a standard reference diet for comparison among fish species, laboratories, and between experiments. P. Coutteau, G. Van Stappen, and P. Sorgeloos: Laboratory o f Aquaculture and A nem ia Reference Center, University o f Ghent, Rozier 44, B-9000 Ghent, Belgium [tel: (+32) , fax: (+32) ], Introduction Efficient production of fish relies on diets formulated to satisfy the nutritional requirements of each species of fish being cultured. Although all nutrients are important, the lipid and essential fatty acid (EFA) requirements have proven to be most critical and variable between species. The quantitative requirements for highly unsaturated fatty acids (HUFA), primarily eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), have been demonstrated for such species of marine fish as striped jack (Watanabe et al., 1989), red sea bream (Izquierdo et al., 1989; Takeuchi et al., 1992a), yellowtail (Takeuchi et al b), turbot (Witt et al., 1984), and gilthead sea bream (Koven et al., 1989; Mourente et a i, 1993). Nevertheless, major controversies continue to exist over what constitutes the optimal dietary intake of fatty acids, particularly in relation to the balances between various EFAs, such as the (n-6):(n-3) ratio, the DHA:EPA ratio, and the interactions between various lipid classes (Sargent etal., 1993). Furthermore, fatty acid requirements may depend on developmental stage and have been studied primarily in larval stages using enriched live food and in juvenile fish using pelletized diets. On the contrary, few authors have evaluated the fatty acid requirements during the critical period of weaning, i.e., when the larvae are switched from live food to microparticulate diets, partially because of the difficulty in preparing experimental diets with suitable biochemical and physical characteristics. The objective of the present study was the development of a standard diet that yields acceptable growth of European sea bass from weaning onwards and in which the fatty acid composition can be manipulated without

2 i c e s mar. sd. Syrap..201 (1995) FA requirements o f weaning and first ongrowing stages o f European sea bass 131 Table 1. Diet formulation (% of diet). Table 2. Feeding schedule for weaning and ongrowing of Euro pean sea bass on artificial diets (starting density: 400 larvae per Composition COD CGA MIX 301 tank; expressed per tank per day). Extruded basal diet Micronized codfish granulate Casein Gelatin1-8 - Egg white albumin Codfish powder Whey protein concentrate Isolated soybean protein Hemoglobin powder Wheat gluten a-cellulose Native corn starch Glycerol tri-c8/c Emulgator blend Vitamin premix Vitamin C Mineral premix Attractant premix Astaxanthine Butylated hydroxytolueen Butylated hydroxyanisole Total extruded fractiont Coated lipid fraction Oil-free soybean lecithin20 Fish oil21 Coconut oil22 Emulgator blend13 Ethoxyquin2 3 Vitamin E24 Total coated fraction COCO FO (1) Code 7422, Rieber & Son A/S, Norway; (2) Protevit- K4,3; Dena A.G., Belgium; (3) A.S.F., Sanofi Bio-Industrie N.V., Belgium; (4) type HG/LW, Orffa Belgium N.V., Belgium; (5) code 0271, Rieber & Son A/S, Norway; (6) LAC- PRODAN-80, Orffa Belgium N.V., Belgium; (7) SUPRO 500E, Protein Technologies International, Belgium; (8) PRO- SAN 9000, Sanofi Bio-Industrie N.V., Belgium; (9) BIOGLU TEN, Amylum N.V., Belgium; (10) Sigma C8002; (11) SNOW FLAKE 03401, Orffa Belgium N.V., Belgium; (12) MCT2106, Fina Chemicals, Belgium; ( 13) commercial blend, INVE Aquaculture N.V., Belgium; (14) commercial premix, INVE Aquaculture N.V., Belgium; (15) Mg-L-ascorbyl-2-monophosphate. PHOSPHITAN C, Showa Denko K.K., Japan; (16) based on Coves et al. (1991); (17) according to Kanazawa et al. (1989); (18) CAROPHYL PINK, Roche, Belgium; (19) Fédéra, Belgium; (20) EMULPUR N, Lucas Meyer N.V., Belgium. (21) fish oil containing 30% (n-3) HUFA, INVE Aquaculture N.V., Belgium; (22) coconut oil, INVE Aquaculture N.V., Belgium; (23) l,2-dihydro-6-ethoxy-2,2,4-trimethylquinolin. Sigma E8260; (24) dl-a-tocopherol-acetate, Roche, Belgium t Total extruded fraction refers to the weight of extruded basis used for coating. affecting other biochemical and physical characteristics of the diet. Furthermore, it was aimed to use preparation techniques that allow the production of sufficient amounts to avoid batch-to-batch variability of the Fish age A nem ia Artificial diet (g) (d) (l(x)os) xm fraction xm fraction basal diet within and between experiments. Finally, the availability of a standard diet may be a first step in facilitating direct comparisons of results among laboratories, experiments, and species. Materials and methods Experim ental diets The basal diets were prepared at the laboratory facilities of Bühler Ltd., Switzerland as formulated in Table 1. The feed ingredients were mechanically mixed and pelleted in a twin screw cooking-extruder. The pellets were air-dried to a water content of less than 8%, crumbled, and sieved to obtain particles in the ranges (um and jxm. The basal diets were formulated to limit the amount of endogenous lipid and particularly EFAs originating from the protein component. Dietary

3 132 P. Coutteau, G. Van Stappen, and P. Sorgeloos i c e s mar. sd. Symp.. 201(1995) Table 3. Proximate analysis and solubility of the diets. COD-UNCO COD-COCO COD-FO CGA-FO MIX-FO Control Moisture Crude protein Lipid Asha Soluble dry matter _ Available energy (kj/g dry diet)c a Expressed as percent dry weight. b Determined on extruded basal diet, expressed as percent dry weight. c Calculated from physiological fuel values of lipid, protein, and carbohydrate ( = dry weight -protein -lipid -ash). Table 4. Fatty acid composition of total lipids in uncoated and coated diets (mgg 1dry wt). Fatty acid COD UNCOa COD COCO COD FO CGA UNCOa CGA FO MIX UNCOa MIX FO Control 14: _ C 14:1 (n-5) trd : tr :1 (n-5) : :1 (n-7) : : :3 0.3 tr : :1 (n-9) :1 (n-7) : :2 (n-6) : :3 (n-6) : tr :3 (n-3) :1 (n-9) :4 (n-3) :3 (n-6) :4 (n-6) :1(n-9) : :5 (n-3) :4 (n-6) : :1(n-9) :5 (n-3) :6 (n-3) saturates monoenes polyenes (n-3) HUFAb a Uncoated extruded basal diet. b Highly unsaturated fatty acids > 20:3 (n-3). c Not detected. d Trace.

4 i c e s mar. sd. Symp., 201 (1995) FA requirements o f weaning and first ongrowing stages o f European sea bass 133 Table 5. Final dry weight and standard length, stress index, and observed mortality of European sea bass (age 81 d) fed various diets. Diet Final dry weight (mg) Specific growth ratet Final body length (mm) Stress index Total observed mortality (%) COD-UNCO ± ± 0.77 b 82 ± ± 8.4 COD-COCO ± ± l.so'5' 88 ± ± 4.2 COD-FO ± 4.35b ± 0.73e 86 ± ± 4.6 CGA-FO ± 8.80a ± ± ± 7.4 MIX-FO ± 6.80h ± 0.79c 90 ± ± 4.2 Control ± 7.90c ± 0.34d 110 ± ± 9.0 ANOVA Table (Fs for each source of variation) Treatment (df = 5) 42.28*** *** 2.37 ns 2.68 ns Block (df = 3) 3.26 ns * 4.08* 2.19 ns Values are means ± standard deviation of four replicate tanks. Dry weight and body length are based on, respectively, four samples and 20 fishes per tank. Values in a column with different superscripts are significantly different (two-way ANOVA without replication, Tukey HSD, p < 0.05). Fish initially averaged mg dry body weight and 15.0mm at an age of 47 days, t Based on initial dry weight of 12.13mg and final dry weight. protein was provided in the micronized cod protein granulate (COD) basal diet by micronized codfish granulate which contained less than 1.5% lipid (dry matter basis). For the casein, gelatin, and albumin mixture (CGA) and the cod fish meal, whey protein, soybean protein concentrate, albumin, hemoglobin meal, and wheat gluten mixture (MIX) basal diets a mixture of various protein sources was used to give a similar essential amino acid composition as the fish meal and a lower content of endogenous fatty acids from marine origin. The slightly lower average purity of the mixed protein sources was partly compensated with the a-cellulose component. A fraction of the dietary lipids was provided in the basal diet as medium chain triglycerides. The remaining lipophilic fraction was added to the extruded u o e 3 s3 o age (days) C0D-UNC0 CGA-FO CONTROL COD-COCO MIX-FO 8 0 COD-FO Figure 1. Cumulative observed mortality as a function of time in European sea bass fed various diets. 9 0 diet by coating the particles with an emulsion in a fluidized bed granulator Glatt, type GPCG1 (see Table 1). Besides soybean lecithin, vitamin E, and ethoxyquin, the coated fraction contained either coconut oil (COD- COCO diet) or fish oil (COD-FO, CGA-FO, and MIX- FO diets). A commercial weaning diet (Lansy A2/W3, INVE Aquaculture N.V., Belgium), which is produced by extrusion (Devresse, pers. comm.), served as a control. Moisture and ash content of the experimental diets were estimated by difference in weight after, respectively, 24 h in a drying oven at 60 C and 6h in a muffling furnace at 600 C. Protein content and total lipid were determined using the Kjeldahl procedure and Soxhlet method, respectively (Williams, 1984). Available energy values of the diets were calculated on the physiological fuel values of 16.74, 33.47, and 6.69 kj/g for protein, lipid, and carbohydrate, respectively (National Research Council, 1981). For the latter, carbohydrate was estimated by subtracting lipid, protein, and ash from the dry weight. Fatty acid analysis was performed according to the ICES Standard Methodology for (n-3) HUFA Analysis (Coutteau and Sorgeloos, 1995). Lipids were extracted with chloroform and methanol (2:1) using a polytron homogenizer and transesterified in acetylchloride/methanol mixture (1:20). Fatty acid methyl esters were separated on a Carlo Erba Mega 5160 HRGC gas chromatograph equipped with a 25 m x 0.32 mm (I.D.) fused silica capillary column with hydrogen as a carrier. Individual fatty acids were identified using known standards and methyl 11,14-eicosadienoate (20:2n-6) was used as internal standard. The solubility of the diets was estimated from the loss of total dry matter after rotating 0.1 g of the diet for 1 h in 20 ml of distilled water, centrifugation for 35min at 3000 rpm, and discarding the supernatans.

5 134 P. Coutteau, G. Van Stappen, and P. Sorgeloos ic e s mar. sd.symp., 201(1995) Table 6. Fatty acid composition of total lipids in fish at age of 81 d (area %). Fatty acid COD-UNCO COD-COCO COD-FO CGA-FO MIX-FO Control 14: :1 (n-5) : :1 (n-5) : :1 (n-7) : : : : :1 (n-9) :1 (n-7) : :2 (n-6) _ b 19: _ 18:3 (n-6) : (n-3) :1 (n-9) :4 (n-3) :3 (n-6) :4 (n-6) :1 (n-9) : :5 (n-3) :4 (n-6) : :1 (n-9) :5 (n-3) :6 (n-3) saturates monoenes polyenes (n-3) HUFA a Highly unsaturated fatty acids a 20:3 (n-3). b Not detected. C ulture system and experim ental design Juvenile 44-d-old sea bass were obtained from the hatchery of Sepia International at Gravelines, France, and acclimated for 3 days to the experimental conditions. During this period they were fed ad libitum 48-h-old Artemia nauplii (EG type, INVE Aquaculture N.V., Belgium), which had been enriched for 24h with a commercial emulsion (Super Selco, INVE Aquaculture N.V., Belgium). The experiment was set up as a randomized complete block design with four blocks each containing one replicate of six treatments. Each block consisted of an independent recirculating system with six 30 liter dark grey rectangular tanks, a mechanical filter, a biological filter, a carbon filter, and a water reservoir equipped with heaters and aeration. Filtered water from the reservoir (1001) was circulated by an electric pump through a constant head tube which delivered water to the rearing units. Retention time of the recirculated water in the rearing units was stepwise shortened in the course of the experiment from 90 to 30 min. Daily, 20% of the volume of the water reservoir was replaced by natural sea water (33-36 ppt). Temperature was kept at 21 ± 1 C; NH4+ and N 0 2_ concentration never exceeded 0.5 and 0.4ppm, respectively. The 47-day-old fish were individually counted and equally distributed to the 24 tanks, resulting in a density of 13 fish I -1. Illumination was provided by dimmed TL lamps ( lux at the water surface) for 12hd_l. Weaning was spread over 11 days, starting from day 48 onwards, by gradually decreasing the amount of enriched Artemia nauplii fed twice daily and increasing the ration of formulated feeds fed at a frequency of nine feed distributions d-1. After weaning, the fish were fed the formulated diets for an additional 3 weeks in accordance with the feeding schedule in Table 2. Daily, excess feed, faeces, and dead fishes were siphoned, and mortality was recorded.

6 ICES mar. Sei. Symp., 201 (1995) FA requirements o f weaning and first ongrowing stages o f European sea bass < O COD-UNCO V COD-COCO COD-FO A CGA-FO O MIX-FO 0 CONTROL d ie ta ry E(n-3)H U FA (area% ) 5 0 <0 u «< Cx* D X CO I >. -o o JO _ B o COD-UNCO v COD-COCO o COD-FO * CGA-FO MIX-FO - CONTROL / / DHA - o, / O - & s' V s ^ EPA DPA r. i i.. i i i d ie ta ry (n-3)h U FA (area% ) Figure 2. Proportion of (n-3) HUFA in total lipids of European sea bass (age 81 d) in relation to dietary (n-3) HUFA concentration (regression equations are given in parentheses). A: S(n-3) HUFA [y = 0.764x r2 = 0.95], B: 20:5 (n-3) [EPA: y = 0.475x ,? = 0.80], 22:5 (n-3) [DPA: y = 0.503x , r2 = 0.91], and 22:6 (n-3) [DHA: y = 0.810x , r2 = 0.94], 3 0 Sample collection and analysis At the end of the feeding experiment, 40 fish per culture tank were sampled randomly for the estimation of individual standard length and dry weight. The fish were starved for 15 h prior to sampling. Fish samples of approximately 1 g wet weight were frozen and served for fatty acid analysis of total lipids. A separate sample of 10 fish per tank was used to evaluate the overall physiological condition in a stress test. For the latter, the fish were immersed in sea water of a salinity of 72 g I-1 and mortality was monitored every 3min during maximum 1 h. Sensitivity was expressed by the index calculated from addition of the cumulative mortalities in the 3 min time intervals (Dhert eta/., 1992). Statistical analysis Biological data, obtained in the randomized complete blocks design, were analysed by two-way analysis of variance without replication, followed when pertinent for the treatment effect by Tukey s multiple range test (Sokal and Rohlf, 1981). Linear regression analyses were performed between fatty acid proportions of total lipids in fish and diets. Results The proximate analysis of the diets showed a considerable variation of crude protein and lipid content among the experimental diets (Table 3). The accidental omission of the medium chain triglyceride component in the CGA basal diet (containing only 0.3% lipid versus 3.3% for the COD and MIX diet) resulted in the low lipid content of the CG A -FO coated diet. The lower lipid level in the COD-COCO diet was probably due to the loss of a fraction of the coconut oil emulsion by adhesion to the walls of the fluidized bed granulator. The loss of total dry matter was highest for the CGA basal diet, and was limited to 20% and 30% for the COD and MIX diet, respectively. The COD basal diet still contained a relatively high level of endogenous (n-3) HUFA, i.e., 0.44% (on dry weight basis) (Table 4). Coating the COD diet with coconut oil emulsion resulted in increased proportions of the saturated fatty acids and oleic acid, and a slight dilution of the (n-3) HUFA to 0.39%, whereas a fish oil coating primarily increased the proportion of 20:5(n-3) and the (n-3) HUFA content. The fish oil coating increased the background (n-3) HUFA level of the COD and the MIX basal diets with %. Weaning and consequent feeding of the uncoated COD diet strongly reduced growth compared to the control treatment fed the commercial diet (Table 5). The addition of the lipid fraction based on coconut oil did not significantly improve growth, whereas this was the case when fish oil coating was applied. The CGA nucleus yielded significantly lower growth than the COD and MIX based diets. Best growth was observed in the group fed the MIX-FO experimental diet, although this was still inferior to the performance of the fish fed the commercial diet. Although not significant, the stress test

7 136 P. Coutteau, G. Van Stappen, and P. Sorgeloos indicated a slightly higher sensitivity of the fast growing fish fed the commercial diet compared to those fed the experimental diets. The total observed mortality, which was not significantly different among treatments (Table 5), showed a loss of 13-26% of the fish population during and just after finishing weaning (Fig. 1). With the exception of the treatment fed the uncoated diet, observed mortality was negligible during the last 2 weeks of the experiment. The accuracy of estimates of daily mortality remains limited in weaning experiments since the observation of the number of dead fishes does not account for cannibalism and desintegration of fishes prior to counting. Nevertheless, it is interesting to note that over 10% less mortality was observed in the treatment fed the CO D -FO diet compared to all other treatments. The fatty acid composition of total lipid in the fish tissue largely reflected the fatty acid composition of dietary lipids, as illustrated by the concentrations of 16:0, 18:1 (n-9), 20:5(n-3), 22:5(n-3), and 22:6(n-3) in fish fed the COD diet coated with either coconut oil or fish oil (Table 6). Notwithstanding the differences in overall composition between the six diets tested, the proportion of (n-3) HUFA in the fish lipids was correlated with the (n-3) HUFA level in the diet (Fig. 2). The higher concentration of 18:2(n-6) and particularly 14:0 in the COD-COCO diet did not result in a proportional increase of these fatty acids in the fish lipids. Discussion Despite its low contents of endogenous essential fatty acids, the use of the CGA basal diet was rejected for future formulations because of its high solubility, which may in turn have caused its low nutritional value for juvenile sea bass. Because of the relatively low background level of (n-3) HUFA (< 0.2% on dry weight basis) and the low leaching rate (30% loss of total dry matter after 1 h exposure), the MIX basal diet was retained for future formulations oof the standard diet. Furthermore, feeding the MIX basal diet after coating with a fish oil-based lipophilic fraction yielded acceptable growth of sea bass, i.e., over 85% of the standard growth rate observed for the control group fed a commercial weaning diet under the same culture conditions. The latter growth rate was comparable with data reported for weaning and consequent ongrowing of sea bass juveniles using the commercial diet Sevbar (SGR for the period of days after hatching; Person-Le Ruyet et al., 1993). The significant effects of the composition of the coated lipophilic fraction on the performance and fatty acid composition of the fish demonstrated that a standard diet, consisting of an extruded basal diet to which a lipid fraction is added by coating, could be used to study ICES mar. Sei. Symp., 201 (1995) lipid requirements of the European sea bass from weaning onwards. Changes of fatty acid composition of fish body lipids according to dietary fatty acid composition have been observed for various other fish species, including juvenile sunshine bass (Nematipour and Gatlin, 1993) and red drum (Lochmann and Gatlin, 1993). Kalogeropoulos etal. (1992) found that the (n-3) HUFA content of liver phospholipids levelled off in gilthead sea bream (Sparus aurata) (lg) fed diets containing more than 0.9% EPA + DHA. The maximum concentration of EPA and DHA observed by the latter authors was 43.4% of the total phospholipid fatty acids, which was approximated in the present study in the total lipids of the fish fed the commercial diet containing 4.9% (n-3) HUFA. The higher sensitivity to salinity stress of the latter fish needs further experimentation. Possibly, saturating concentrations of (n-3) HUFA may have negatively affected membrane structure and function. The establishment of a standard reference diet has been considered by various working groups on the standardization of nutrition research as one of the most important steps in facilitating direct comparison of results among laboratories, experiments, and species (Castell et al., 1989) and was recently recommended by the ICES Working Group on Mass Rearing of Juvenile Marine Fish (ICES, 1993). The use of extrusion technology allows the production of relatively large batches of the present standard diet and the addition of a variable lipid fraction through coating allows the possibility of preparing diets differing in lipophilic compounds. The combined technique of extrusion/coating was recently compared with the preparation of diets by extrusion only (Coutteau et al., 1995). The latter study showed a similar response of the tissue fatty acid composition to increasing dietary (n-3) HUFA contents for sea bass offered the lipid fraction containing the essential fatty acids either as part of the extruded diet or coated on an extruded basal diet. In addition, growth was not significantly different for fish fed either extruded/coated or extruded diets with an identical formulation. The combined technique of cooking-extrusion of a basal diet followed by coating of a lipid emulsion, which allows the preparation of diets that differ only with regard to their lipid and fatty acid composition, may hold promise to prepare diets for studying quantitative fatty acid requirements of weaning and first ongrowing stages of marine fish. Furthermore, the MIX basal diet coated with the appropriate lipid fraction may be used as a standard reference diet for comparison among fish species, laboratories, and between experiments. Acknowledgements This study was supported by the Belgian National Fund for Scientific Research (PC and PS are, respectively,

8 i c e s mar. sd. Symp., 201 (1995) FA requirements o f weaning and first ongrowing stages o f European sea bass 137 Senior Research Assistant and Research Director with the BNFSR), the Belgian Ministry for Science Policy (OOA Programme), and INVE Aquaculture N.V.- S.A., Belgium. The authors thank Bart Van Houcke, Geert Van de Wiele, Anita De Haese, Armando Garcia Ortega, and Andres Pedrazolli for their technical support. Special thanks are extended to the Bühler company, Switzerland for providing the facilities and the technical assistance for preparing the extruded diets. References Castell, J. D., Kean, I.V., D'Abramo, L. R., and Conklin, D. E Evaluation of two formulations for use as a standard reference diet for Crustacean Nutrition Research. J. World Aquacult. Soc., 20: Coutteau, P., and Sorgeloos, P Intercalibration exercise for quantitative analysis of fatty acids in marine samples. ICES Coop. Res. Rep. (In press). Coutteau, P., Van Stappen, G., and Sorgeloos, P A standard experimental diet for the study of fatty acid requirements of weaning and first ongrowing stages of the European sea bass Dicentrarchus labrax L.: comparison of extruded and extruded/coated diets. Arch. Anim. Nutr. (In press). Coves, D., Dewavrin, G., Breuil, G., and Devauchelle, N Culture of sea bass (Dicentrarchus labrax L.). In Handbook of mariculture, vol. II. Finfish aquaculture, pp Ed. by J. P. McVey. CRC Press, Inc., Boca Raton, Florida. Dhert, P., Lavens, P., and Sorgeloos, P A simple test for quality evaluation of cultured fry of marine fish. Med. Fac. Landbouww. Univ. Ghent, 57: ICES, Report of the Working Group on Mass Rearing of Juvenile Marine Fish. ICES CM 1993/F: 8. 8 pp. Izquierdo, M. S., Watanabe, T., Takeuchi, T., Arakawa, T., and Kitajima, C Requirements of larva! red sea bream Pagrus major for essential fatty acids. Nippon Suisan Gakkaishi, 55: Kalogeropoulos, N., Alexis, M. N., and Henderson, J. R Effects of dietary soybean and cod-liver oil levels on growth and body composition of gilthead bream (Sparus aurata). Aquaculture, 104: Kanazawa, A., Koshio, S., andteshima, S Growth and survival of larval red sea bream Pagrus major and Japanese flounder Paralichthys olivaceus fed microbound diets. J. World Aquacult. Soc., 20: Koven, W. M., Kissil, G. W., andtandler, A Lipid and n-3 requirement of Sparus aurata larvae during starvation and feeding. Aquaculture, 79: Lochmann, R. T., and Gatlin, D. M Evaluation of different types and levels of triglycerides, singly and in combination with different levels of n-3 highly unsaturated fatty acid ethyl esters in diets of juvenile red drum, Sciaenops ocellatus. Aquaculture, 114: Mourente, G., Rodriguez, A., Tocher, D. R., and Sargent, J. R Effects of dietary docosahexaenoic acid (DHA; 22:6n-3) on lipid and fatty acid compositions and growth in gilthead sea bream (Sparus aurata L.) larvae during first feeding. Aquaculture, 112: National Research Council Nutrient requirements of domestic animals. Number 216. Nutrient requirements of Coldwater Fishes. National Academy Press, Washington, DC. 63 pp. Nematipour, G. R., and Gatlin, D. M Effects of different kinds of dietary lipid on growth and fatty acid composition of juvenile sunshine bass, Morone chrysops 9 x M. saxatilis cf. Aquaculture, 114: Person-Le Ruyet, J., Fischer, C., and Thebaud, L Sea bass (Dicentrarchus labrax) weaning and ongrowing onto Sevbar. In Fish nutrition in practice, Biarritz (France), June, 24-27, 1991, pp Ed. by S. J. Kaushik and P. Luquet. INRA, Paris 1993 (Les Colloques, no. 61). Sargent, J. R., Bell, J. G., Bell, M. V., Henderson, R. J., and Tocher, D. R The metabolism of phospholipids and polyunsaturated fatty acids in fish. In Aquaculture: fundamental and applied research, pp Ed. by B. Lahlou and P. Vitiello. American Geophysical Union. Sokal, R. R., and Rohlf, F. J Biometry. W. H. Freeman and Company, San Francisco, California. 859 pp. Takeuchi, T., Shiina, Y., and Watanabe, T. 1992a. Suitable levels of n-3 highly unsaturated fatty acids in diet for fingerlings of red sea bream. Nippon Suisan Gakkaishi, 58: Takeuchi, T., Shiina, Y., Watanabe, T., Sekiya, S., and Imaizumi, K. 1992b. Suitable levels of n-3 highly unsaturated fatty acids in diet for fingerlings of yellowtail. Nippon Suisan Gakkaishi, 58: Watanabe, T., Takeuchi, T., Arakawa, T., Imaizumi, K., Sekiya, S., and Kitajima, C Requirements of juvenile striped jack (Longirostris delicatissimus) for n-3 highly unsaturated fatty acids. Nippon Suisan Gakkaishi, 55: Williams, S Official methods of analysis of the association of official analytical chemists. Association of Official Analytical Chemists, Inc., Arlington, Virginia, USA. 572 p p. Witt, U., Quantz, G., Kuhlman, D., and Kattner, G Survival and growth of turbot larvae Scophthalmus maximus L. reared on different food organisms with special regard to long-chain polyunsaturated fatty acids. Aquacult. Eng., 3: