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Aquaculture 184 2000. 315326 www.elsevier.

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The use of lipid emulsions for sterol supplementation of spat of the Pacific oyster, Crassostrea gigas
Philippe Soudant a , Maica Val Sanles b, Claudie Quere b, Jean Rene Le Coz b, Yanic Marty c , Jeanne Moal b, Jean Francois Samain b,) , Patrick Sorgeloos a
Laboratory of Aquaculture and Artemia Reference Center, Uniersity of Gent, Rozier 44, 9000 Ghent, Belgium b DRV r A, Laboratoire de Physiologie des Inertebres, IFREMER Centre de Brest, BP 70, 29280 Plouzane, France c UMR r CNRS 6521, Uniersite de Bretagne Occidentale, BP 809, 29285 Brest, France Accepted 24 September 1999
a

Abstract To determine the ingestion and absorption of lipid emulsions, spat were fed algae deficient in stigmasterol and cholesterol and an emulsion containing these two sterols. The ingestionabsorption of the emulsion was estimated by measuring incorporation of these two sterols in oyster lipids during the 33-day feeding period. They were supplemented with 0%, 3%, 10% and 20% emulsion wet weight of the algae dry weight. The results showed that after only 7 days of emulsion supplementation, significant differences were observed in the sterol composition. The quantities of stigmasterol and cholesterol absorbed by the spat were time- and dose-dependent. Nevertheless, compared to algal sterols, the absorption rate of the sterols from the emulsion was quite low and decreased with an increasing supply of emulsion. Other emulsion formulations need to be tested to improve the emulsion absorption rate in oyster spat. However, the sterols appeared to be good markers for assessment of lipophilic artificial diet digestion and absorption. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Molluscs; Oyster; Crassostrea gigas; Spat; Microalgae; Artificial diet; Nutrition; Lipids; Sterols; Incorporation and metabolism

Corresponding author. Tel.: q 33-2-98-22-44-02; fax: q 33-2-98-22-46-53; e-mail: jfsamain@ifremer.fr

0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 9 9 . 0 0 3 2 3 - 3

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1. Introduction To produce seed of consistently good quality and at a competitive price is an important priority for the molluscan aquaculture industry. Spat rearing is a critical step. Diet quality often results in variability in growth and quality with unacceptable overcosts for nursery and oyster growers. Sufficient quantities of microalgae are required for feeding, which are costly for the hatcherynursery. A number of papers on invertebrate nutrition have described the quantitative algal requirements Utting and Millican, 1995.. However, after supplying the recommended quantity, large variabilities are still observed for the subsequent success of spat development. This led to the conclusion that the quality of algae is also important. Polyunsaturated fatty acids PUFA., 20:5n y 3 and 22:6n y 3, have been demonstrated to be essential for bivalves Langdon and Waldock, 1981; Helm et al., 1991; Frolov and Pankov, 1992; Marty et al., 1992; Leal, 1994.. It is quite difficult to provide with security a good phytoplanktonic diet to spat. Emulsions have been demonstrated to be a successful way to deliver essential fatty acids Lane, 1989; Robinson, 1992; Coutteau et al., 1996; Caers et al., 1998, 1999.. However, the absorption rates of micronutrients incorporated in emulsions as well as the relative ingestion of the emulsion versus algae are still to be assessed. Likewise, the ability to synthesize or bioconvert sterols de novo is generally low or absent, and varies among bivalve species. This implies that a dietary supply of sterol is necessary for bivalve growth Teshima, 1983; Teshima and Kanazawa, 1974; Teshima and Patterson, 1981; Teshima et al., 1979; Voogt, 1975; Trider and Castell, 1980; Gordon and Collins, 1982; Holden and Patterson, 1991; Napolitano et al., 1993.. The sterol supply by microalgae is highly variable according to the species used Berenberg and Patterson, 1981; Lin et al., 1982; Gladu et al., 1991; Tsitsa-Tzardis et al., 1993.. Sterol composition and associated quantity can also vary with algal culture conditions Gladu et al., 1991.. Consequently, the qualitative and quantitative variability of the sterol composition in microalgae used in nurseries will have consequences on spat phytosterol composition, and will modify growth and survival performance Wikfors et al., 1991.. Lipid emulsions appear to be a potential way to deliver essential sterols as has been demonstrated. The aim of this paper was to estimate ingestion and absorption rates using sterols as markers. To do this, we fed spat with algae, but lacking two sterols, i.e., stigmasterol and cholesterol, that were incorporated as markers in emulsions. The ingestiondigestion of the emulsion was followed by measuring the incorporation of the two sterol markers in oyster spats.

2. Materials and methods 2.1. Emulsion Following recommendation three of the International Council for the Exploration of the Sea ICES. Working Group on Mass Rearing of Juvenile Fish Bergen, Norway

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2123, 1993; ICES 1994., two experimental emulsions 30r0.6rE5 containing either stigmasterol or cholesterol, 5% of the emulsion dry weight DW. were prepared. These emulsions were made by INVE Technologies Baasrode, Belgium. and contained 50% lipid on wet weight WW. basis, water, emulgators, antioxidants, preservatives and liposoluble vitamins A, C, D and E, respectively, 0.18%, 0.08%, 0.013% and 0.32% of WW.. The emulsion DW was 70% of the emulsion WW. 2.2. Spat culture Crassostrea gigas juveniles were provided by a French commercial hatchery. The spats average initial weight 0.1 g., were acclimatized to experimental conditions for 10 days prior to the experiment in a 20-l tank equipped by airwater lift at the density of 200 spatsrtank. Spats reached an average weight of 0.22 g after the acclimatization period. The spat was daily washed with a jet of fresh water, and the tanks were cleaned and refilled with 1 mm filtered seawater. A mixed diet 50:50 on a DW basis. of T-Isochrysis and Tetraselmis suecica was provided daily to spat at a weight specific ration of 0.75% algal DW per WW of spat.. The algae of the diet were selected on the basis of their known nutritional quality and their absence of two sterols, namely cholesterol and stigmasterol, which were supplied by the emulsions. Preliminary experiments not reported here. were performed to determine the best protocol of emulsion supplementation to prevent algal filtration perturbation. For the present experiment, spats were fed algal diet supplemented with 0%, 3%, 10%, and 20% WW emulsion per algal DW. The emulsion contained a 1:1 mixture of the two previously prepared emulsions. Algae were provided continuously using a reservoir while emulsion was added twice a day. After 7, 19 and 33 days, the spats were harvested, rinsed, and weighed. The WW, flesh weight FW., DW, and organic matter OM. of 10 replicates of five animals were measured. Another three replicates of pooled spats five animalrreplicate. were sampled for lipid analysis. The animals sampled for lipid analysis were starved for 48 h to avoid artifacts of sterols from microalgae and emulsion which could be present in the digestive tract at the time of the sampling. The total cells of T-Isochrysis and T. suecica provided to each tank during the 33 days of experiment were 2.42 = 10 11 and 1.8 = 10 10 cells, respectively. The total amounts of WW emulsion added in the 3%, 10% and 20% tanks for the experiment were 0.22, 0.67 and 1.35 g, respectively. 2.3. Lipid analysis Total lipids were extracted according to Folch et al. 1957.. The total sterol fraction was first hydrolyzed with sodium methoxide MeONa. for 90 min at room temperature Eder et al., 1992.. The sterols were extracted in hexane and injected directly into the GC. Sterol composition was analyzed in a Chrompak CP 9002 gas chromatograph equipped with a Restek Rt = 65 fused silica capillary column 12.5 m = 0.25 mm, 0.25 mm film thickness. using an on-column injection system. Hydrogen was used as the

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carrier gas, with temperature programmed from 1708C to 2608C at 58C miny1 and from 2608C to 2808C at 28C miny1 . The sterols were identified by comparison of their retention time with standards and verified by GCrMS analysis Carlo-Erba model 5160 HRGC coupled on mass spectrometer Nermag R10-10H.. Sterol nomenclature trivial name and systematic name.: 1. Norcholesterols 22-trans-24-5,22-dien-3b-ol; 2. c-dehydrocholesterols 22-cis-cholesta-5,22dien-3b-ol; 3. t-dehydrocholesterols 22-trans-cholesta-5,22dien-3b-ol; 4. Dihydrocholesterols Cholesta-3b-ol 5. Cholesterols Cholesta-5-en-3b-ol; 6. Brassicasterols 24b-methylcholesta-5, 22-dien-3b-ol; 7. desmosterols Cholesta-5, 24dien-3b-ol; 8. Campesterols 24a-methylcholesta-5-en-3b-ol; 9. 24-methylenecholesterols 24b-methylenecholesta-5, 2428.-dien-3b-ol; 10. Stigmasterols 24b-ethylcholesta-5, 22-dien-3b-ol; 11. 4a-methyl poriferasterols 4-a-methyl-24-a-ethylcholesta-22-en-3b-ol; 12. b-sitosterols 24b-ethylcholesta-5-en-3b-ol; 13. Fucosterols cholesta-5, 22dien-3b-ol 24b-ethylcholesta-5,2428.-dien-3b-ol. To separate neutral and polar lipids, the 2:1 chloroformmethanol extracts were evaporated to dryness under vacuum. The extract was recovered and rinsed with three times 500 ml of 98:2 chloroformmethanol and put on a silica gel microcolumn 30 mm = 5 mm, Kieselgel Merck, 70230 mesh. for neutral and polar lipid separation. Neutral lipids were eluted with 10 ml chloroformmethanol 98:2. and polar lipid with 10 ml methanol. The fractions were collected in tapering vials containing C23:0 as internal standard. The neutral and polar lipid fractions were transesterified with BF3 14% in methanol. Metcalfe and Schmitz, 1961. and treated according to Marty et al. 1992.. The fatty acid methyl esters FAME. were analysed using a Chrompak 9001 gas chromatograph Chrompack, Middelburg, Netherlands. equipped with a on-column injector, a DBWAX 30 m = 0.35 mm, 0.25 mm film thickness. capillary column, and flame ionization detector. Hydrogen was used as the carrier gas. The fatty acids were identified by comparing their retention times with those of standards and were confirmed using a non-polar column CP Sil 8 CB, 30 m = 0.25 mm, 0.25 mm film thickness.. 2.4. Statistics Significant differences P - 0.05. in biological and biochemical measurements between dietary treatments were determined by One-way ANOVA. Multiple comparisons were determined by NewmanKeuls test. Analyses were performed using Statistica computer package. 3. Results The sterol and PUFA composition of the diets and of the initial spats after 10 days of acclimatization are presented in Tables 1 and 2.

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Table 1 Sterol composition of the spat at the beginning of the experiment, of the algae, and of the emulsion expressed as sterol % of the total sterol; Mean, S.D.sstandard deviation; ns 5. Spat at T0 Mean Norcholesterol c-Dehidrocholesterol t-Dehidrocholesterol Dihydrocholesterol Cholesterol Brassicasterol Desmosterol Campesterol 24-Methylenecholesterol Stigmasterol Methylporiferasterol Betasitosterol Fucosterol Total sterol in spat mgrindividu Total sterol in algae fgrcell Total sterol in emulsion grg DW 2.4 0.7 3.7 1.3 25.0 34.0 2.1 11.9 8.6 2.6 0.8 3.5 0.7 17.1 S.D. 0.1 0.1 0.1 0.2 0.5 1.7 0.2 1.1 0.5 0.4 0.1 0.4 0.1 3.8 T-Isochrysis Mean 0.5 2.0 3.0 93.9 0.6 40.2 S.D. 0.6 0.7 0.4 1.0 0.5 8.0 Tetraselmis Mean 1.9 82.5 14.2 1.4 298.9 S.D. 0.4 2.1 1.3 1.0 17.4 0.05 Emulsion

50

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Spat exhibited growth during the experiment but no significant difference was observed in the WW, FW, DW, and OM quantities of spat between all the dietary treatments along the experiment Table 3.. The sterol supplementation did not change the total sterol content of the spat as well as the total fatty acid content measured in the neutral and polar lipids Tables 4 and 5..
Table 2 PUFA composition of the spats at the beginning of the experiment, of the algae and of the emulsion expressed as fatty acid % of the total fatty acids; Mean, S.D.sstandard deviation; ns 5. Spat at T0 Neutral lipid Mean 18:2 ny6 18:3ny3 18:4ny3 20:2 ny6 20:4ny6 20:5ny3 22:2D7,15 22:2D7,13 22:5ny6 22:6ny3 Total FA in spat mgrindividu Total FA in algae pgrcell Total FA in emulsion grg DW 7.9 5.4 6.6 1.0 1.1 6.8 0.7 1.0 1.1 9.4 84.2 S.D. 1.8 1.3 1.4 0.2 0.2 1.3 0.3 0.3 0.3 1.2 19.6 Polar lipid Mean 4.9 2.4 2.6 0.7 3.3 11.2 3.7 6.3 3.3 27.5 136.8 S.D. 0.7 0.3 0.4 0.2 0.8 2.1 0.8 1.4 0.6 4.9 25.4 6.5 6.5 17.1 0.0 0.1 0.3 2.1 11.1 1.8 1.9 0.1 1.0 0.0 0.0 0.0 0.0 0.6 0.4 2.9 6.4 3.5 0.0 0.4 3.8 0.1 5.8 1.3 1.6 0.6 0.0 0.0 0.1 0.0 0.2 0.8 5.0 1.1 1.8 0.2 1.1 16.3 11.8 T-Isochrysis Mean S.D. Tetraselmis Mean S.D. Emulsion

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Table 3 Effect of emulsion addition on the WW, FW, DW, % OM content expressed in mg Mean, S.D., ns10 of five pooled oysters. Concentration 0% Mean mg r indiidu Wet weight, 7 days Wet weight, 19 days Wet weight, 33 days 248.5 375.5 596.1 S.D. 13.7 66.1 63.1 3% Mean 288.0 426.1 534.1 S.D. 15.6 53.9 46.7 10% Mean 198.3 345.8 597.6 S.D. 21.8 31.8 105.0 20% Mean 227.1 333.4 572.3 S.D. 13.5 41.3 54.4

After 33 days mgr indiidu Flesh weight 85.9 Dry weight 19.0 Organic matter 15.9

15.8 3.0 2.6

84.1 19.2 15.8

1.4 1.0 0.9

84.2 18.1 15.0

25.5 5.5 4.8

77.1 17.9 14.9

11.3 1.7 1.5

The percentage of cholesterol decreased over time in all treatments while brassicasterol and campesterol increased. However, the cholesterol decrease was gradually compensated by the increasing supply of cholesterol provided by the emulsion Table 4.. The percentage of stigmasterol in spats treated with emulsion increased strongly from 0
Table 4 Effect of sterol addition on the total sterol composition of the spat after 7, 19, 33 days of feeding Mean, S.D., ns 3.. The statistical differences P - 0.05. between treatment are indicated by a letter Control Mean 7 Days of supplementation Brassicasterol Campesterol 24-Methylenecholesterol Stigmasterol Cholesterol Total sterol mgrindividu 19 Days of supplementation Brassicasterol Campesterol 24-Methylenecholesterol Stigmasterol Cholesterol Total sterol mgrindividu 33 Days of supplementation Brassicasterol Campesterol 24-Methylenecholesterol Stigmasterol Cholesterol Total sterol mgrindividu 40.4 14.5 6.9 2.6 21.0 27.0 S.D. 2.5a 0.8 0.4 0.2 a 0.5a 7.2 0.9 a 0.8 0.1a 0.1a 0.5a 10.3 0.4 a 0.4 a 0.3 0.1a 0.4 a 6.8 3% Mean 40.8 14.1 6.6 3.1 20.7 32.0 S.D. 1.1a 0.4 0.5 0.1b 0.6 a 8.1 1.6 a 0.6 0.4 a 0.2 b 0.5 b 11.9 3.4 a,b 1.5 b 0.7 0.1b 0.9 a 32.9 10% Mean 38.7 13.9 6.3 4.0 22.6 25.0 S.D. 1.5a 0.9 0.1 0.2 c 0.6 b 7.6 2.0 a 1.1 0.1a 0.0 c 0.4 c 6.5 2.4 a,c 1.0 b 0.3 0.2 c 0.6 b 21.8 20% Mean 35.7 13.8 6.1 4.8 24.5 26.0 S.D. 0.5 b 0.4 0.4 0.1d 0.9 c 11.5 1.1b 1.2 0.4 b 0.6 d 0.2 d 8.2 2.7 a,c 1.4 b 0.5 0.4 d 1.7 b 10.3

49.1 17.2 5.6 1.9 15.0 69.4

48.5 16.3 5.2 2.9 17.1 82.5

45.4 15.1 4.7 4.5 20.0 60.7

41.8 15.2 4.5 5.5 22.2 67.5

54.0 17.0 5.5 2.2 11.5 90.4

57.7 13.2 4.6 3.0 12.5 103.6

50.2 13.4 4.0 4.7 16.9 85.6

48.5 12.9 4.2 5.8 19.2 88.5

Table 5 PUFA composition of neutral and polar lipids from the spat supplemented with four levels of emulsion after 7, 19 and 33 days of feeding Mean; S.D.; ns 3. P. Soudant et al.r Aquaculture 184 (2000) 315326 Neutral lipids Control Mean 7 Days 20:5ny3 22:6ny3 TFA mgrindividu 19 Days 20:5ny3 22:6ny3 TFA mgrindividu 33 Days 20:5ny3 22:6ny3 TFA mgrindividu 5.5 10.7 101.1 S.D. 0.3 a 0.5 13.0 0.3 a 0.3 a 19.2 0.2 a 0.4 66.5 3% Mean 6.5 11.4 128.9 S.D. 0.5 b 0.9 10.1 0.4 a 0.3 a 10.1 0.3 a,b 0.5 123 10% Mean 8.3 11.8 106.5 S.D. 0.3 c 0.5 31.6 0.8 b 0.6 b 27.3 0.1a,c 0.4 25.8 20% Mean 8.3 12.2 104.5 S.D. 0.4 c 0.6 24.3 0.5 b 0.4 a 32.2 0.2 d 0.7 2.7 Polar lipids Control Mean 7.1 20.5 162.4 S.D. 0.2 a 0.5 13.4 0.0 a 0.5a 36.0 0.2 a 0.5 10.2 3% Mean 7.6 20.7 210.9 S.D. 0.1a 0.9 8.4 0.1b 0.3 a 34.0 0.2 b 0.3 71.2 10% Mean 7.9 20.0 174.7 S.D. 0.4 b 0.6 13.6 0.3 c 0.1b 28.6 0.2 a 0.5 29.3 20% Mean 8.5 19.9 189.8 S.D. 0.2 c 0.2 16.0 0.3 d 0.5 a,b 32.2 0.2 c 0.7 30.9

4.6 10.6 286.3

5.5 11.3 241.2

7.1 13.0 173.2

7.7 11.2 324.2

6.1 20.1 353.4

6.6 19.7 373.5

7.4 21.3 279.5

8.0 20.6 324.2

4.3 10.3 490.3

3.7 11.7 479.5

4.3 11.4 479.7

6.0 11.8 647.2

6.1 19.4 477.8

5.3 19.9 404

6.0 19.9 433.6

7.1 20.0 458.3

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to 7 days and at a lesser extent from 19 to 33 days Table 4.. In the control, the level of stigmasterol was relatively unchanged. The percentage of stigmasterol was statistically different among all treatments after 7, 19 and 33 days of feeding Table 4.. After 7 days, only the cholesterol percentage of the groups fed with 10% and 20% of emulsion WWrDW of microalgae. was significantly different from the control. After 19 days of emulsion supplementation, the percentages of cholesterol differed significantly among all groups. After 33 days, the cholesterol percentages of groups fed with 10% and 20% of emulsion were significantly higher than the control and the group fed 3% emulsion. The total accumulation during the experiment of each sterol by spat from algae and emulsion for each treatment was calculated. The dietary ration of the major algal sterols: brassicasterol, campesterol and 24-methylenecholesterol were, respectively, 54, 26 and 4.5 mgrspat. The accumulation rates of these algae sterols in all treatments were estimated from 68% to 90% for the brassicasterol, from 35% to 51% for the campesterol and from 42% to 75% for the 24-methylenecholesterol. Total cholesterol and stigmasterol gained by spat during 33 days of feeding increased as a polynomial function of the total sterol added with the emulsion per spat Fig. 1a

Fig. 1. Total stigmasterol a. and cholesterol b. accumulated by spat after 33 days of feeding with 0, 3, 10 and 20% of emulsion per algae DW as a function of the total stigmasterol and cholesterol added.

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and b.. The accumulation rates of emulsion sterols by spats were 4.7%, 3.0%, and 2.4% for cholesterol and 2.2%, 1.5%, and 1.0% for stigmasterol after 30 days of feeding with 3%, 10% and 20% of emulsion, respectively. The accumulation of brassicasterol provided at 54 mgrspat by algae was much higher than the accumulations of cholesterol and stigmasterol provided at a similar range 50 mgrspat. by emulsion 3% emulsion treatment.. Supplementation with lipid emulsion affected the level of 20:5n y 3 and to some extent the level of 22:6n y 3. The percentage of 20:5n y 3 in both neutral and polar lipids was significantly increased in the group supplemented with emulsion after 7, 19 and 33 days of feeding. However, significant increase in 22:6n y 3 was only noted after 19 days of feeding in the group fed with 10% emulsion supplement Table 5..

4. Discussion Brassicasterol and campesterol, the two major algal sterols, increased in all treatments while cholesterol decreased, confirming that bivalves are quite sensitive to algal diet composition Soudant et al., 1998.. Good spat growth obtained in the experiment suggests that the algal mixture provided necessary nutrients despite lacking cholesterol, once considered essential. Individual WW, FW, DW and OM contents showed that the emulsion had no effect on the growth of the reared spat. The relative sterol composition of the spat was significantly influenced by the artificial diet composition after 7 days of supplementation. The relative differences of sterol composition were maintained during the experiment. These changes in sterol composition of the spats fed with emulsion showed that sterols incorporated in emulsion were ingested, digested and accumulated by spat. These results confirmed that emulsions can be used to deliver essential lipids Coutteau et al., 1996; Caers et al., 1998, 1999.. Essential PUFA composition of spats was affected but to a lesser extent than the sterols. Only 20:5n y 3, along with 22:6n y 3, the major PUFA in the emulsion DHArEPAs 0.6., was clearly more accumulated in neutral lipids and polar lipids according to the amount of added emulsion. The presence of 20:5n y 3 and 22:6n y 3 in significant amounts in the dietary algae, and in the initial spats, could explain the lesser effect on essential PUFA contents compared to the sterol contents. Essential PUFAs are more regulated than sterols Soudant, 1995. but saturation processes for PUFA incorporation could also occur in bivalves, especially in the membrane lipids. The polynomial relationship between sterol added via emulsion and accumulated sterol amounts in the spat indicated the ability of the oysters to accumulate sterols from the emulsion until saturation not reached in that experiment.. An increasing percentage of emulsion in the diets resulted in a decreasing accumulation rate of sterols from emulsion. Emulsion sterol accumulation rates were much lower than algal sterols when supplied in equivalent amounts. This indicated a very low transfer efficiency between the artificial diet and the spat because these sterols are usually assimilated at similar rates when they are only provided by microalgae Soudant et al., 1996, 1998.. The low ingestionaccumulation of the emulsion may be due to: 1. the physical characteristics

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of emulsion, leading to a possible lower acceptability than algae, 2. a lower filtration efficiency due to particle size, 3. a control mechanism of the free sterol absorption in the spat, or 4. loss due to possible emulsion adhesion to the tank. A saturation process of cholesterol incorporation can be eliminated. The highest percentage of cholesterol in total sterols of spat was 25% in this experiment while oysters in the field may contain 3040% cholesterol Berenberg and Patterson, 1981; Wikfors et al., 1991.. Cholesterol accumulation by the spat was three times higher than stigmasterol, demonstrating selective incorporation of cholesterol. Such a preferential incorporation of cholesterol over other sterols has been previously observed in Pecten maximus Soudant et al., 1998.. Bioconversions of algal brassicasterol, campesterol or 24-methylenecholesterol in cholesterol could have also occurred. This may allow a sufficient level of cholesterol for cellular regulation by the spats. Studies on biotransformation and de novo synthesis of sterols have shown contradictory results Walton and Pennock, 1972; Teshima and Kanazawa, 1974; Voogt, 1975; Holden and Patterson, 1991.. Some bivalve species have the ability of de novo synthesis andror modifying dietary sterols. But in most cases, synthetic ability was limited. The substitution of cholesterol with other algal sterols such as brassicasterol or campesterol did not appear to result in physiological damage during spat development. This observation suggests that the main function of phytosterols at this juvenile stage is structural. Nevertheless, possible perturbations of the early sexual maturation of spats or of the physiological processes involving steroids could not be excluded at this point in our study. In conclusion, sterols appeared to be good markers for assessment of ingestionabsorption of different diets. However, the relationship between the ingestionaccumulation of these sterols and the other dietary components remains to be assessed. A concentration over 10% WW emulsion per algal DW appeared to give substantial response in terms of nutrition application. However, it has been observed in preliminary experiments that emulsion concentrations over 1.6 mg WWrl resulted in perturbation of algal filtration. This is why we supplied the 20% dose twice a day 1.25 mg WWrl at the time of the addition.. For higher concentrations, a continuous distribution of the emulsion may provide a compromise between the low emulsion accumulation rate and a sufficient supply of sterols for the spats. Increasing the emulsion sterol concentration, if technically feasible, should also be considered as a way to improve the yield of sterol transfer from artificial diet to oysters.

Acknowledgements This material is based upon work supported by the European Commission under grant FAIR CT.96.1852, Breeding improvement of C. gigas by nutritional and gametogenesis control, and under Training Mobility Research grant FAIR GT96 0762, Contribution to study essential requirements for C. gigas broodstock using artificial complementation. The authors would like to thank Pr. F.L.E. Chu and V. Encomio for the constructive review of the manuscript.

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