Almost total replacement of fish meal by plant protein sources in the diet of a marine teleost, the European seabass, Dicentrarchus labrax

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1 Aquaculture 230 (2004) Almost total replacement of fish meal by plant protein sources in the diet of a marine teleost, the European seabass, Dicentrarchus labrax S.J. Kaushik a, *, D. Covès b, G. Dutto b, D. Blanc a a Fish Nutrition Research Laboratory, Unité Mixte INRA-IFREMER, Unité d Hydrobiolgie, St-Pée-sur-Nivelle, France b Méditerranean Finfish Research Laboratory, Station Expérimentale d Aquaculture IFREMER, Palavas-les-Flots, France Received 1 April 2003; received in revised form 28 May 2003; accepted 28 May 2003 Abstract Five practical diets in which the supply of protein from fish meal was decreased gradually from 100% to about 2% and replaced by plant protein sources were formulated. European seabass weighing about 190 g were fed these diets for 12 weeks at a water temperature of 22 jc. Feed was dispensed using automatic self-feeders and voluntary feed intake (VFI) was closely monitored. We did not find any significant difference among diets in the apparent digestibility coefficients (ADC) of dry matter (80 82%), protein (94 96%), energy (88 92%) or phosphorus (49 58%). Replacement of fish meal by plant protein ingredients did not influence VFI. All groups had very good growth rates (DGI above 1.3%/day) and there were no significant differences in growth rate, feed efficiency or in daily nitrogen gains among groups. There was, however, a slight increase in fat deposition in fish fed diets with plant protein sources. Ammonia nitrogen and soluble phosphorus excretion rates were measured. Nitrogen and phosphorus balance studies indicated that fish meal replacement by plant ingredients led to a slight increase in nitrogen losses (from 83 to 103 g N/kg weight gain) but led to a significant reduction in total phosphorus losses (from 13 to 5 g P/kg weight gain). These results combined with the remarkable acceptability of diets containing high levels of plant protein ingredients with identical growth performances of European seabass show clearly that dietary fish meal levels can be considerably reduced without any adverse consequence in terms of somatic growth or nitrogen utilisation. D 2004 Elsevier B.V. All rights reserved. Keywords: European sea bass; Fish meal replacement; Plant proteins; Growth; Nutrient utilisation * Corresponding author. Tel.: ; fax: address: kaushik@st-pee.inra.fr (S.J. Kaushik) /$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi: /s (03)

2 392 S.J. Kaushik et al. / Aquaculture 230 (2004) Introduction Given the global needs for fish oil and fish meal for aquaculture (FAO, 2002), there is an increasing demand for more insight on the potential of alternative protein sources in aquafeeds (New and Wijkström, 2002). Under intensive farming conditions, in species such as the rainbow trout, development of fish meal free diets has met with some success (Kaushik et al., 1995; Watanabe et al., 1998). When working with marine species, the latter authors, however, observed a number of adverse effects in yellowtail fed non-fish meal diets (Watanabe et al., 1999). This interest for the substitution of fish meal by more sustainable and renewable protein sources applies also for the culture of marine teleosts of interest to the Mediterranean area (Alexis, 1997; Alexis and Nengas, 2001). Although studies were initiated as far back as the late 1970s on the replacement of fish meal by other protein sources in the diets of European seabass (Alliot et al., 1979), only limited knowledge is available today on the nutritional value and the physiological consequences of plant protein use in this species (Dias et al., 1997; da Silva and Oliva-Teles, 1998; Dias, 1999). The aim of this study was to evaluate the possibility of replacing a great portion of fish meal in the diet of European seabass using practical ingredients readily available to the aquafeed industry and to detect possible effects on carcass quality, body composition, excretion and plasma metabolites. 2. Materials and methods 2.1. Diets Five diets were formulated to contain different levels of fish meal incorporation, ranging from 520 to 50 g kg 1 (Table 1). They were formulated so as to meet the nutrient requirements of European seabass (Kaushik, 2001) as well as to be least costly, using the solver macro of Microsoft s Excel software. The diets with fish meal replacement above 50% (diets FM25, FM 12 and FM5) were supplemented with L-lysine and dicalcium phosphate. In the absence of specific data on vitamin, mineral and trace element requirements of European seabass, requirement data for other species were applied (NRC, 1993; Kaushik et al., 1998). All diets contained yttrium oxide as an inert marker for determining apparent digestibility coefficients (ADC). Extruded diets were manufactured by an industrial fish feed manufacturer (Le Gouessant, 29 Lamballe, France) using a twin-screw extruder (Buhler) Rearing facilities The trial was conducted at the experimental facilities of IFREMER (34 Palavas les Flots, France). The rearing system consisted of 15 individual tanks supplied with sand filtered (15 Am) and UV-treated seawater (salinity 31 38x) in a flow-through system (flow rate: 1 m 3 h 1 in each tank). Water temperature was maintained constant (21.8 F 0.1 jc) using titanium heat exchangers and oxygenated to maintain oxygen concentrations above 80% saturation. Each tank was covered and individually equipped with a lamp (75 W)

3 S.J. Kaushik et al. / Aquaculture 230 (2004) Table 1 Ingredient and chemical composition of the practical diets (g/kg) FM52 FM40 FM25 FM12 FM5 Fish meal LT 94 (CP, 70%) Corn gluten meal Wheat gluten Extruded wheat Soybean meal (CP 48%) Rapeseed meal (primor 00) L-Lysine CaHPO 4 2H 2 O (18%P) Fish oil (Scandinavian) Binder Yttrium oxide Mineral premix a Vitamin premix b Dry matter (DM), g/kg Crude protein, g/kg DM Crude fat, g/kg DM Phosphorus, g/kg DM Gross energy, MJ/kg DM Theoretical cost (o/ton) c Both mixtures were manufactured by Unité de préparation des aliments expérimentaux (UPAE), INRA, Jouyen-Josas, France, according to the recommendations of NRC (1993). a Mineral mixture (g or mg/kg diet): calcium carbonate (40% Ca), 2.15 g; magnesium oxide (60% Mg), 1.24 g; ferric citrate, 0.2 g; potassium iodide (75% I), 0.4 mg; zinc sulphate (36% Zn), 0.4 g; copper sulphate (25% Cu), 0.3 g; manganese sulphate (33% Mn), 0.3 g; dibasic calcium phosphate (20% Ca, 18%P), 5 g; cobalt sulphate, 2 mg; sodium selenite (30% Se), 3 mg; KCl, 0.9 g; NaCl, 0.4 g. b Vitamin mixture (IU or mg/kg diet): DL-a tocopherol acetate, 60 IU; sodium menadione bisulphate, 5 mg; retinyl acetate, 15,000 IU; DL-cholecalciferol, 3000 IU; thiamin, 15 mg; riboflavin, 30 mg; pyridoxine, 15 mg; B12, 0.05 mg; nicotinic acid, 175 mg; folic acid, 500 mg; inositol, 1000 mg; biotin, 2.5 mg; calcium panthotenate, 50 mg; choline chloride, 2000 mg. c As per ingredient prices in June about 70 cm above the surface of the water with a photoperiod of 16 h of light (0600 to 2200 h) and 8 h of darkness with twilights of 30 min each at dawn and dusk (Fig. 1). An electronic demand feeding and monitoring system developed by Boujard et al. (1992) was attached to each individual tank. Some adaptations were made as described by Covès et al. (1998) in order to prevent any unintentional triggering of the feeders by fish. This system allows for distribution of feed per demand as well as to closely monitor demands and rewards and to store data directly in a computer Digestibility and growth trial European seabass juveniles (Dicentrarchus labrax), obtained from a commercial farm (Extramer, France), were acclimated to the experimental conditions before the beginning of the trial. Fish were sorted and 15 homogenous groups (coefficient of variation of body weight < 12%) of 50 fish each were randomly allotted to each tank. The five diets

4 394 S.J. Kaushik et al. / Aquaculture 230 (2004) Fig. 1. Diagram of a tank with photoperiod control, attached demand feeder and fecal trap used for rearing European seabass. were randomly allotted to triplicate tanks and self feeders were filled with the respective diets each day and the feed intake (quantity dispensed minus eventual refusals) monitored daily. At the beginning of the growth trial, a representative sample of six fish was withdrawn and kept frozen ( 20 jc). Growth trial was conducted for 12 weeks (Sept Dec 2000), and every third weeks fish in each tank were bulk-weighed. At the end of the growth study and after an overnight fast, six fish from each tank were sampled and frozen. Fifteen fish per treatment were anesthetised (ethylene glycol monophenyl-ether 1:2500). They were individually weighed and total length measured; blood was collected from the caudal vein with a heparinised syringe. Plasma was recovered after centrifugation and stored at 20jC for total cholesterol and triacylglycerol analysis. Fish were ground and moisture content was determined (110 jc, 24 h) and subsequently freeze-dried before further analyses. For digestibility measurements, fecal matter was collected in a modified version of the decantation chamber described by Cho and Slinger (1979) and directly fitted to the circular rearing tanks (Fig. 1). Almost at the end of the growth trial, faeces were collected over five consecutive days and frozen. ADC of the dietary nutrients and energy were calculated according to Maynard et al. (1979): ADC ð%þ ¼100 1 dietary Y 2O level faecal Y 2 O level faecal nutrient or energy level dietary nutrient or energy level In order to quantify soluble nitrogen (N) and phosphorus (P) losses, outlet water from the same rearing tanks was automatically sampled using a peristaltic pump over three consecutive 24-h cycles and collected daily into bottles containing a small

5 amount of chloroform following procedures used by Kaushik (1980) and Dosdat et al. (1996) Analyses Fecal samples were freeze dried before analyses of dry matter, nitrogen, energy, phosphorus and yttrium oxide. Diets, freeze-dried feces and whole body samples were analysed for dry matter (DM, 110 jc, 24 h), ash (550 jc, 18 h) crude protein (Kjeldahl nitrogen 6.25) and lipids (dichloromethane extraction by Soxlhet method) were performed following AOAC (1984) procedures. Gross energy was determined by calorimetry (IKA Adiabatic Calorimeter C4000A). Yttrium concentrations were determined in diet and fecal samples by atomic absorption spectrophotometry using a nitrous oxide-acetylene flame, after acid digestion (2% nitric acid and 2 g l 1 KCl). Concentrations of ammonia-n and urea-n were analysed by the indophenol blue (Tréguer and Le Corre, 1975) and diacetylmonoxime methods (Aminot and Kérouel, 1982), respectively, using an autoanalyser. Soluble P (PO 4 ) in the water was determined by the ammonium molybdate method (Tréguer and Le Corre, 1975). Based on diet and comparative carcass analysis, daily N, fat and P gains and retention (as % intake) were calculated. Concentrations of plasma triacylglycerols and cholesterol were determined spectrophotometrically using commercial enzymatic kits (BioMérieux and Boehringer Mannheim) Statistical analysis S.J. Kaushik et al. / Aquaculture 230 (2004) Data were analysed using ANOVA using the GLM procedure of SAS (1993). The means were subsequently compared using Duncan s multiple range test (significance level P < 0.05). Data presented are means F standard deviations with superscript letters indicating differences between groups. 3. Results 3.1. Apparent digestibility ADC of dry matter ranged between 80% and 82%, that of protein between 94% and 96% and that of energy between 88% and 92%, little affected by dietary fish meal Table 2 Apparent digestibility coefficients (ADC, %) of the different experimental diets FM52 FM40 FM25 FM12 FM5 Dry matter 82.4 F F F F F 0.3 Protein 94.9 F F F F F 0.3 Energy 91.6 F F F F F 0.5 Phosphorus 49.2 F F F F F 1.5 Values are means F SD. Absence of superscript indicates no significant difference between treatments.

6 396 S.J. Kaushik et al. / Aquaculture 230 (2004) replacement levels (Table 2). Availability of phosphorus was lower in all diets (49% to 58%) but given the variability, there was no significant difference among diets Growth performance and nutrient utilisation At the end of the 12 weeks of the growth trial, all groups of European seabass had reached mean individual body weights ranging from 313 to 333 g (Table 3) with no significant differences among groups. Analysis of length weight relationships did not show any differences either between groups described by a common relation of y = 0.027X with an R 2 of 0.682, where X and Y represent total length (cm) and body weight (g), respectively. Over the 12-week period of the growth trial, the daily mean voluntary feed intake (VFI) was slightly higher in fish fed FM40 (10 g/kg/day) than in the other groups (9.1 to 9.4 g/kg/day) which did not differ among themselves. The values of daily growth index (DGI) were high for seabass of this size, grown at 22 jc, reflecting the good rearing conditions and physico-chemical quality of water, with no significant differences among treatments (Table 3). While there was no difference between groups in feed efficiency (FE = wet weight gain/dry feed intake), the protein efficiency ratios (PER = wet weight gain/crude protein intake) varied and the lowest value was observed in seabass fed diet FM5. Whole body protein or phosphorus contents did not vary among groups but body fat content increased with increasing level of fish meal replacement (Table 4) Nitrogen and phosphorus losses Measurements of total ammonia-n rates showed that ammonia-n excretion rates were lower in FM52-fed fish than in all other groups (Table 5), reflecting possible amino acid imbalance or more probably an excess supply in the latter groups, which, however, did not affect N-gains differently. Urea N excretion rates differed between treatments ranging between 35 and 50 mg urea-n/kg BW/day. The relative proportion of urea-n to ammonia + urea N decreased with decreasing levels of dietary fish meal levels. Daily P Table 3 Growth performance and nutrient utilisation in European seabass (initial body weight: 190 F 2 g) fed diets with graded levels of fish meal replacement over 12 weeks at 22 jc Diet FM52 FM40 FM25 FM12 FM5 Final body weight, g F F F F F 11.6 Final total length, cm 28.9 F F F F F 1.0 Voluntary feed intake, g/kg BW/day 9.25 F 0.09 b F 0.20 a 9.17 F 0.52 b 9.42 F 0.40 b 9.07 F 0.08 b Daily growth index (DGI) F 0.11 ab 1.48 F 0.12 a 1.34 F 0.09 ab 1.38 F 0.09 ab 1.25 F 0.07 b Feed efficiency (FE) F F F F F 0.02 Protein efficiency ratio (PER) F 0.09 a 1.41 F 0.07 ab 1.36 F 0.02 bc 1.46 F 0.03 ab 1.28 F 0.05 c Values are mean F SD. Within a row, means with different superscript letters differ significantly (P < 0.05). Absence of superscript indicates no significant difference between treatments. 1 DGI: (FBW 1/3 IBW 1/3 )/84 days) FE, Weight gain/dry feed intake. 3 PER, Wet weight gain/crude protein intake.

7 S.J. Kaushik et al. / Aquaculture 230 (2004) Table 4 Whole body composition, nutrient retention and gain in European seabass (IBW: 190 F 2 g) fed diets with graded levels of fish meal replacement over 12 weeks at 22 jc Diet FM52 FM40 FM25 FM12 FM5 HSI 1, % 2.1 F F F F F 0.6 VSI 2, % 11.0 F F F F F 1.5 GSI 3, % 0.52 F F F F F 0.75 Final body composition 4 Moisture, % 62.3 F 1.9 ab 60.5 F 0.4 b 60.2 F 0.5 b 60.8 F 0.1 ab 59.6 F 0.2 b Protein, % 16.1 F F F F F 0.5 Fat, % 18.4 F 1.0 c 19.8 F 0.3 b 19.9 F 0.5 b 20.0 F 0.2 b 21.8 F 1.0 a Phosphorus, % 0.6 F F F F F 0.1 Ash, % 3.1 F F F F F 0.7 Energy, kj/g 10.8 F 0.7 c 11.5 F 0.2 ab 11.6 F 0.3 ab 11.4 F 0.2 bc 12.1 F 0.2 a Retention, % of intake Dry matter 26.8 F F F F F 1.2 Protein 22.3 F F F F F 2.2 Energy 34.2 F 6.0 b 38.2 F 1.2 ab 39.5 F 1.5 ab 36.8 F 0.8 ab 40.9 F 1.6 a Phosphorus 23.1 F 7.0 c 25.8 F 3.5 bc 36.1 F 11.9 abc 46.5 F 7.8 a 44.2 F 16.0 ab Daily nutrient gain (mg or g/kg/d) Nitrogen (mg) F F F F F 17.0 Fat (g) 1.5 F 0.2 b 1.8 F 0.1 ab 1.7 F 0.1 ab 1.8 F 0.1 ab 1.9 F 0.2 a Phosphorus (mg) 25.1 F F F F F 8.5 Values are mean F SD. Within a row, means with different superscript letters differ significantly (P < 0.05). Absence of superscript indicates no significant difference between treatments. 1 HSI = hepato-somatic index, 100 weight of the liver/whole body weight. 2 VSI = viscero-somatic index, 100 weight of digestive tract/whole body weight. 3 GSI = gonado-somatic index, 100 weight of gonads/whole body weight. 4 Initial body composition was: moisture 63.6, protein 17.1, fats 14.7, phosphorus 0.78, ash 4.7, and energy 9.9. Table 5 Daily mean NH 4 N and PO 4 P excretion rates in European seabass fed the different diets Dietary Treatment FM52 FM40 FM25 FM12 FM5 Ammonia-N excretion, F F F F F 50.9 mg N/kg/d Urea N-excretion, 37.1 F F F F F 5.1 mg N/kg/d Urea N/[Ammonia F F F F F 0.4 Urea N, % [Ammonia + urea] N 44.2 F 0.7 b 48.0 F 1.8 b 52.7 F 3.1 b 45.7 F 3.1 b 53.3 F 2.3 a excreted as % of digestible N intake PO 4 P excreted, 23.1 F F F F F 0.9 mg P/kg/d PO 4 P excretion as % of available P intake 41.8 F 6.7 a 40.3 F 5.4 a 23.6 F 4.8 b 6.6 F 2.1 c 0 Values are mean F SD. Within a row, means with different superscript letters differ significantly (P < 0.05). Absence of superscript indicates no significant difference between treatments.

8 398 S.J. Kaushik et al. / Aquaculture 230 (2004) Fig. 2. Nitrogen budget in European seabass (mg N/kg BW/day) based on digestibility, ammonia + urea N excretion and comparative carcass analyses. The fraction corresponding to Other N represents that not accounted for by measurements of fecal and metabolic losses. gain did not differ among groups, P retention values increased with increasing levels of plant protein incorporation. Based on data on VFI, digestibility and on measured soluble nutrient losses, combined with comparative carcass analysis, total nitrogen and phoshorus fluxes were calculated and reported in Figs. 2 and 3. Fig. 3. Phosphorus budget in European seabass (mg P/kg BW/day) based on digestibility, total soluble PO 4 excretion and comparative carcass analyses.

9 S.J. Kaushik et al. / Aquaculture 230 (2004) Fig. 4. Concentrations of plasma triacylglycerols in European seabass fed the different diets. Values are mean F SD. Means with different superscript letters differ significantly ( P < 0.05) Other metabolic consequences Very few fish (less than 3%) were found to be mature and the gonadosomatic index varied between 0.5% and 1.4% with no differences between sexes. We did not find any differences between groups in the liver to body weight ratio (HSI) or in the viscerosomatic index (VSI; Table 4). Concentrations of plasma triacylglycerols ranged between 100 and 400 mg/dl (Fig. 4) with much variability among treatments. Plasma cholesterol levels decreased with increasing levels of fish meal replacement, the lowest level being found in fish fed diet FM5 (Fig. 5). Fig. 5. Plasma cholesterol concentrations in European seabass fed the different diets. Values are mean F SD. Means with different superscript letters differ significantly ( P < 0.05).

10 400 S.J. Kaushik et al. / Aquaculture 230 (2004) Discussion Some earlier studies with rainbow trout as well as European seabass have suggested that the major problem connected with poor growth of fish fed fish meal-free, plantprotein-based diets is caused by poor feed intake (Gomes et al., 1995; Dias, 1999). In European seabass fed diets containing very high levels of single protein sources such as soy protein concentrate or corn gluten meal, there was a decrease in VFI, which was improved by supplementation with an attractant mix (Dias et al., 1997). But other data indicate that when the same protein sources replaced about 60% of fish meal, adequately supplemented with limiting amino acids such as lysine or methionine, there was no need for an attractant mix such as squid extract (Tibaldi et al., 1999). It is thus of interest to note that the diets used here and made commercially did not lead to any reduction in voluntary feed intake. The growth rates observed here were higher than those reported earlier by Dias (1999) for seabass grown at 18 jc and even higher than those reported by Ballestrazzi et al. (1994) for fish reared at similar temperature levels. Earlier data have shown that partial replacement of fish meal by plant protein sources (Tibaldi et al., 1999; Tulli et al., 1999) or single cell proteins (Oliva-Teles and Goncalves, 2001) is feasible in European seabass. Dias (1999) observed that inclusion of corn gluten meal or soy protein concentrates as the sole protein source led to significant growth reduction. This is the first ever demonstration of an almost total replacement of fish meal and soybean meal by a mixture of other plant protein sources in European seabass. Diets were formulated to be least costly and it is apparent that considerable reduction in fish meal levels can be achieved without comprising fish performance under similar economic terms. We observed, however, a significant increase in fat content with increasing levels of fish meal replacement. This consequently resulted in a similar increase in whole body energy content. The high fat and energy retention values in this group clearly suggest that there was increased lipogenesis with increasing levels of fish meal replacement, without any effect on nitrogen utilisation. Indeed, daily N gains or N retention expressed as a percentage of unit N intake did not vary among groups. The HSI values of above 2 as found here are common in European seabass (Ballestrazzi et al., 1998; Dias, 1999), where hepatic fat deposition indeed is very high (Dias, 1999). There is evidence that replacement of fish meal by plant protein sources such as corn gluten meal or soy protein concentrates affects hepatic lipogenic enzyme activities variably in seabass (Dias, 1999): while the activity of malic enzyme decreased, that of fatty acid synthetase increased significantly with high levels of corn gluten meal in the diet. The data on the relative proportion of urea-n to ammonia + urea N which decreased with decreasing levels of dietary fish meal levels have to be treated with caution, since the daily excretion rates measured here in well growing fish were relatively lower than what has been found earlier (Dosdat et al., 1996). Expressed per unit digestible N intake, measured (ammonia + urea) N excretion rates ranged between 43% and 53%, higher than values reported earlier by Robaina et al. (1999), but close to other data (Ballestrazzi et al., 1994). Reactive phosphorus losses decreased with increasing levels of fish meal replacement, with practically no measurable soluble phosphorus in the effluents in fish fed diet FM5. The sensitivity of the analytical method, albeit low, was not sufficient to detect

11 S.J. Kaushik et al. / Aquaculture 230 (2004) differences between inlet and outlet water. Indeed, measurements of low levels of soluble P in fish farm effluent water is difficult due to the low concentrations generally found. Data of Ballestrazzi et al. (1994) also provide evidence that seabass fed diets containing corn gluten meal had lower reactive phosphate concentration in the effluent water than in those fed herring meal based diets. It is clear from Fig. 2 that while fecal N losses are relatively small and that daily N gains are similar among groups (Tables 4 and 6), metabolic N losses are high. We also observed that measurement of ammonia-n + urea N as measured here does not, at least in our case, reflect total metabolic losses, confirming earlier suggestions that in fish, N balance measurement based on comparative carcass analysis is more reliable than measurement of excretory rates (Cho and Kaushik, 1990). Earlier data have shown that phosphorus availability was reduced in European seabass fed soy protein concentrates (Dias, 1999). Given that the raw materials used here did not contain detectable levels of phytic acid, addition of phytase, known to improve phytic P availability even in European seabass (Oliva-Teles et al., 1998), was not found necessary. Although daily P gain did not differ among groups, P retention values increased with increasing levels of plant protein incorporation, reflecting that the inorganic phosphorus supplementation was efficient. The data on phosphorus budget (Fig. 3) show that quite a significant portion of soluble P was not accounted for by measured soluble PO 4 in effluents from fish tanks. It is, however, interesting to note that total P losses were reduced in fish fed plant-protein-rich diets (Table 6). The decrease in plasma cholesterol levels in fish fed diets with plant proteins (Fig. 5) has already been reported in rainbow trout (Kaushik et al., 1995) and in European seabass fed different plant protein sources (Dias, 1999; Tulli et al., 1999) in replacement of fish meal. In terrestrial animals, plant products are generally considered to have a hypocholesteromic effect (de Schrijver, 1990), mainly due to the relatively high levels of estrogeno-mimetic isoflavones (Setchell and Cassidy, 1999). Given that the diets used here contained small amounts of soy bean meal, it appears that the hypocholesterolemic effects seen in such studies have more to do with the withdrawal of fish meal rather than with a direct effect of plant proteins. Besides, measurements of phytoestrogens in several plant products do show that there was considerable variability in the flavonoid contents of soybean products and that other plant products used here contain very low or no genistein Table 6 Nitrogen and phosphorus budget per unit body weight gain in European seabass fed the different diets Dietary treatment FM52 FM40 FM25 FM12 FM5 Nitrogen, g N/kg BW gain Intake F F F F F 4.8 Gain 23.6 F F F F F 2.0 Losses 82.8 F F F F F 6.3 Phosphorus, g P/kg BW gain Intake 17.1 F F F F F 0.3 Gain 3.9 F F F F F 1.4 Losses 13.2 F F F F F 1.5 Values are mean F SD. Absence of superscript indicates no significant difference between treatments.

12 402 S.J. Kaushik et al. / Aquaculture 230 (2004) or daidzein (Bennetau-Pelissero et al., in press). In an earlier study with rainbow trout (Kaushik et al., 1995), we also observed that casein had a stronger hypocholesterolemic effect than soy by-products. From a comparative perspective, it will be of interest to gather more insight on the specific effects of plant proteins on lipid and cholesterol synthesis and metabolism, in as much as these factors might affect, among others, flesh quality. 5. Conclusions Data obtained here show that development of diets based mainly on commonly available practical ingredients of plant origin with very low levels of fish meal incorporation and appropriate supplementation of essential amino acids does not affect growth or nitrogen utilisation in European seabass. The low levels of phosphorus in these diets can be easily supplemented with inorganic phosphorus to maintain body phosphorus status while having a significant reduction in phosphorus discharges. Acknowledgements We express our thanks to Le Gouessant Aquaculture, 29 Lamballe, France, for the manufacture of extruded diets used in this study. Recognition is given to all the technical staff of Ifremer, Palavas and Inra, St-Pée sur Nivelle for their assistance in conducting growth trials, samplings and chemical analyses. Special thanks are due to A. Charrier, (IFREMER) for her help in excretion measurements and to C. Vachot (INRA) for analysis of plasma metabolites. This study is a part of the Eureka project A! 1960: Aqua-Maki 2. References Alexis, M.N., Fish meal and fish oil replacers in Mediterranean marine fish diets. In: Tacon, A., Basurco, B. (Eds.), International Cent. for Advanced Mediterranean Agronomic Studies, CIHEAM, Zaragoza (Spain). Cah. Options Mediterr., vol. 22, pp Alexis, M.N., Nengas, I., Current State of Knowledge Concerning the Use of Soy Products in Diets for Feeding Sea Bass and Seabream. Needs for Future Research Publ. American Soybean Assn., Brussels, Belgium. No. 5, 32 pp. Alliot, E., Pastoureaud, A., Pelaez-Hudlet, J., Métailler, R., Utilisation des farines végétales et des levures cultivées sur les alcanes pour l alimentation du bar (Dicentrarchus labrax L.). In: Tiews, K., Halver, J.E. (Eds.), Finfish Nutrition and Fishfeed Technology, vol. 2. Heenemann, Berlin, pp Aminot, A., Kérouel, R., Dosage automatique de l urée dans l eau de mer: une méthode très sensible à la diacétylmonoxyme. Can. J. Fish. Aquat. Sci. 39, AOAC (Association of Official Analytical Chemists), Official Methods of Analysis, 12th ed. Association of Official Analytical Chemists, Washington, DC pp. Ballestrazzi, R., Lanari, D., D Agaro, E., Mion, A., The effect of dietary protein level and source on growth, body composition, total ammonia and reactive phosphate excretion of growing sea bass (Dicentrarchus labrax). Aquaculture 127, Ballestrazzi, R., Lanari, D., D Agaro, E., Performance, nutrient retention efficiency, total ammonia and reactive phosphorus excretion of growing European sea-bass (Dicentrarchus labrax, L.) as affected by diet processing and feeding level. Aquaculture 161,

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14 404 S.J. Kaushik et al. / Aquaculture 230 (2004) Satoh, S., Bani, P., Calamari, L. (Eds.), Recent Progress in Animal Production Science: I. Proc. A.S.P.A. XIII Congress, Piacenza, Italy, June Assn. Sci. Anim. Production, Italy, pp Tréguer, P., Le Corre, P., Manuel d analyse des sels nutritifs dans l eau de mer (Utilisation de l autoanalyseur Technicon), 2nd ed. Laboratoire d Océanographie Chimique, Université de Bretagne Occidentale, France. 110 pp. Tulli, F., Tibaldi, E., Comin, A., Dietary protein sources differently affect plasma lipid levels and body fat deposition in juvenile sea bass. In: Piva, G., Bertoni, G., Masoero, F., Bani, P., Calamari, L. (Eds.), Recent Progress in Animal Production Science: I. Proc. A.S.P.A. XIII Congress, Piacenza, Italy, June Assn. Sci. Anim. Production, Italy, pp Watanabe, T., Verakunpiriya, V., Watanabe, K., Viswanath, K., Satoh, S., Feeding of rainbow trout with non-fish meal diets. Fish. Sci. 63, Watanabe, T., Aoki, H., Shimamoto, K., Hadzuma, M., Maita, M., Yamagata, Y., Viswanath, K., Satoh, S., A trial to culture yellowtail with non-fish meal diets. Fish. Sci. 64,