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 hatchery–nursery. A number of papers on invertebrate nutrition have described the
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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 Ž
. important. Polyunsaturated fatty acids PUFA , 20:5n y 3 and 22:6 n y 3, have been
Ž 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
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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 Ž
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 ŽBerenberg 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
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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
Ž 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 ingestion–di-
gestion 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
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Ž the Sea ICES — Working Group on Mass Rearing of Juvenile Fish Bergen, Norway
. 21–23, 1993; ICES 1994 , two experimental emulsions 30r0.6rE5 containing either
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stigmasterol or cholesterol, 5 of the emulsion dry weight DW were prepared. These Ž
. emulsions were made by INVE Technologies Baasrode, Belgium and contained 50
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lipid on wet weight WW basis, water, emulgators, antioxidants, preservatives and Ž
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
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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 air–water 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
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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
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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. Ž
. 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, Ž
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. flesh weight FW , DW, and organic matter OM of 10 replicates of five animals were
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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
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Total lipids were extracted according to Folch et al. 1957 . The total sterol fraction Ž
. was first hydrolyzed with sodium methoxide MeONa for 90 min at room temperature
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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
Ž 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
carrier gas, with temperature programmed from 1708C to 2608C at 58C min
y1
and from 2608C to 2808C at 28C min
y1
. The sterols were identified by comparison of their Ž
retention time with standards and verified by GCrMS analysis Carlo-Erba model 5160 .
HRGC coupled on mass spectrometer Nermag R10-10H . Ž
. 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. Cholesterol s Cholesta-5-en-3b-ol; 6. Brassicasterols 24b-methylcholesta-5, 22-dien-3b-ol;
7. desmosterol s Cholesta-5, 24dien-3b-ol; 8. Campesterol s 24a-methylcholesta-5-en-3b-ol;
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9. 24-methylenecholesterols 24b-methylenecholesta-5, 24 28 -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-sitosterol s 24b-ethylcholesta-5-en-3b-ol;
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13. Fucosterol s cholesta-5, 22dien-3b-ol 24b-ethylcholesta-5,24 28 -dien-3b-ol. To separate neutral and polar lipids, the 2:1 chloroform–methanol extracts were
evaporated to dryness under vacuum. The extract was recovered and rinsed with three Ž
times 500 ml of 98:2 chloroform–methanol and put on a silica gel microcolumn 30 .
mm = 5 mm, Kieselgel Merck, 70–230 mesh for neutral and polar lipid separation. Ž
. Neutral lipids were eluted with 10 ml chloroform–methanol 98:2 and polar lipid with
10 ml methanol. The fractions were collected in tapering vials containing C23:0 as internal standard.
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The neutral and polar lipid fractions were transesterified with BF 14 in methanol
3
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Metcalfe and Schmitz, 1961 and treated according to Marty et al. 1992 . The fatty Ž
. acid methyl esters FAME were analysed using a Chrompak 9001 gas chromatograph
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Chrompack, Middelburg, Netherlands equipped with a on-column injector, a DBWAX Ž
. 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
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non-polar column CP Sil 8 CB, 30 m = 0.25 mm, 0.25 mm film thickness . 2.4. Statistics
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Significant differences P - 0.05 in biological and biochemical measurements be- tween dietary treatments were determined by One-way ANOVA. Multiple comparisons
were determined by Newman–Keuls test. Analyses were performed using Statistica computer package.
3. Results
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