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The use of lipid emulsions for sterol supplementation of spat of the Pacific oyster, Crassostrea gigas
Aquaculture 184 Ž2000. 315–326 www.elsevier.nlrlocateraqua-online
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 a
Laboratory of Aquaculture and Artemia Reference Center, UniÕersity of Gent, Rozier 44, 9000 Ghent, Belgium b DRV r A, Laboratoire de Physiologie des InÕertebres, ´ ´ IFREMER Centre de Brest, BP 70, 29280 Plouzane, ´ France c UMR r CNRS 6521, UniÕersite´ de Bretagne Occidentale, BP 809, 29285 Brest, France Accepted 24 September 1999
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 ingestion–absorption 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
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Corresponding author. Tel.: q33-2-98-22-44-02; fax: q33-2-98-22-46-53; e-mail: [email protected]
0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 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 hatchery–nursery. A number of papers on invertebrate nutrition have described the quantitative algal requirements ŽUtting 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 ŽPUFA., 20:5n y 3 and 22:6n y 3, have been demonstrated to be essential for bivalves ŽLangdon 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 ŽLane, 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 ŽTeshima, 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 conditions ŽGladu 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 ŽWikfors 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–digestion 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 ŽICES. — Working Group on Mass Rearing of Juvenile Fish ŽBergen, Norway
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21–23, 1993; ICES 1994., two experimental emulsions 30r0.6rE5 containing either stigmasterol or cholesterol, 5% of the emulsion dry weight ŽDW. were prepared. These emulsions were made by INVE Technologies ŽBaasrode, Belgium. and contained 50% lipid on wet weight ŽWW. basis, water, emulgators, antioxidants, preservatives and liposoluble vitamins ŽA, 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 Žaverage 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 and refilled with 1 mm filtered seawater. A mixed diet Ž50:50 on a DW basis. of T-Isochrysis and Tetraselmis suecica was provided daily to spat at a weight specific ration of 0.75% Žalgal 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 Žnot 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 ŽFW., DW, and organic matter ŽOM. of 10 replicates of five animals were measured. Another three replicates of pooled spats Žfive 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. Ž1957.. The total sterol fraction was first hydrolyzed with sodium methoxide ŽMeONa. for 90 min at room temperature ŽEder 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 Ž12.5 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 ŽCarlo-Erba model 5160 HRGC coupled on mass spectrometer Nermag R10-10H.. Sterol nomenclature Žtrivial 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, 24Ž28.-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-5,24Ž28.-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 times 500 ml of 98:2 chloroform–methanol and put on a silica gel microcolumn Ž30 mm = 5 mm, Kieselgel Merck, 70–230 mesh. for neutral and polar lipid separation. Neutral lipids were eluted with 10 ml chloroform–methanol Ž98:2. 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 Ž14% in methanol. ŽMetcalfe and Schmitz, 1961. and treated according to Marty et al. Ž1992.. The fatty acid methyl esters ŽFAME. were analysed using a Chrompak 9001 gas chromatograph ŽChrompack, Middelburg, Netherlands. equipped with a on-column injector, a DBWAX Ž30 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 ŽCP Sil 8 CB, 30 m = 0.25 mm, 0.25 mm film thickness.. 2.4. Statistics Significant differences Ž P - 0.05. in biological and biochemical measurements between 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 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 Žexpressed as sterol % of the total sterol; Mean, S.D.sstandard deviation; ns 5. Spat at T0
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
T-Isochrysis
Tetraselmis
Emulsion
Mean
S.D.
Mean
S.D.
Mean
S.D.
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
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
– 0.5 – 2.0 3.0 93.9 0.6 – – – – – –
– 0.6 – 0.7 0.4 1.0 0.5 – – – – – –
– – – – 1.9 – – 82.5 14.2 – 1.4 – –
– – – – 0.4 – – 2.1 1.3 – 1.0 – –
40.2
8.0
298.9
17.4
50
50
0.05
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 ŽTable 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 ŽTables 4 and 5.. Table 2 PUFA composition of the spats at the beginning of the experiment, of the algae and of the emulsion Žexpressed as fatty acid % of the total fatty acids; Mean, S.D.sstandard deviation; ns 5. Spat at T0
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
Neutral lipid
Polar lipid
Mean
S.D.
Mean
S.D.
7.9 5.4 6.6 1.0 1.1 6.8 0.7 1.0 1.1 9.4 84.2
1.8 1.3 1.4 0.2 0.2 1.3 0.3 0.3 0.3 1.2 19.6
4.9 2.4 2.6 0.7 3.3 11.2 3.7 6.3 3.3 27.5 136.8
0.7 0.3 0.4 0.2 0.8 2.1 0.8 1.4 0.6 4.9 25.4
T-Isochrysis
Tetraselmis
Mean
S.D.
Mean
S.D.
6.5 6.5 17.1 0.0 0.1 0.3 – – 2.1 11.1
1.9 0.1 1.0 0.0 0.0 0.0 – – 0.0 0.6
2.9 6.4 3.5 0.0 0.4 3.8 – – – 0.1
1.3 1.6 0.6 0.0 0.0 0.1 – – – 0.0
1.8
0.4
5.8
0.2
Emulsion
5.0 1.1 1.8 0.2 1.1 16.3 – – – 11.8
0.8
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Table 3 Effect of emulsion addition on the WW, FW, DW, % OM content expressed in mg ŽMean, S.D., ns10 of five pooled oysters. Concentration
0%
3%
10%
20%
Mean
S.D.
Mean
S.D.
Mean
S.D.
Mean
S.D.
248.5 375.5 596.1
13.7 66.1 63.1
288.0 426.1 534.1
15.6 53.9 46.7
198.3 345.8 597.6
21.8 31.8 105.0
227.1 333.4 572.3
13.5 41.3 54.4
After 33 days mgr indiÕidu 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
mg r indiÕidu Wet weight, 7 days Wet weight, 19 days Wet weight, 33 days
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 ŽTable 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 ŽMean, S.D., ns 3.. The statistical differences Ž P - 0.05. between treatment are indicated by a letter Control Mean
3% S.D.
Mean
10% S.D.
Mean
20% S.D.
Mean
S.D.
7 Days of supplementation Brassicasterol Campesterol 24-Methylenecholesterol Stigmasterol Cholesterol Total sterol mgrindividu
40.4 14.5 6.9 2.6 21.0 27.0
2.5a 0.8 0.4 0.2 a 0.5a 7.2
40.8 14.1 6.6 3.1 20.7 32.0
1.1a 0.4 0.5 0.1b 0.6 a 8.1
38.7 13.9 6.3 4.0 22.6 25.0
1.5a 0.9 0.1 0.2 c 0.6 b 7.6
35.7 13.8 6.1 4.8 24.5 26.0
0.5 b 0.4 0.4 0.1d 0.9 c 11.5
19 Days of supplementation Brassicasterol Campesterol 24-Methylenecholesterol Stigmasterol Cholesterol Total sterol mgrindividu
49.1 17.2 5.6 1.9 15.0 69.4
0.9 a 0.8 0.1a 0.1a 0.5a 10.3
48.5 16.3 5.2 2.9 17.1 82.5
1.6 a 0.6 0.4 a 0.2 b 0.5 b 11.9
45.4 15.1 4.7 4.5 20.0 60.7
2.0 a 1.1 0.1a 0.0 c 0.4 c 6.5
41.8 15.2 4.5 5.5 22.2 67.5
1.1b 1.2 0.4 b 0.6 d 0.2 d 8.2
33 Days of supplementation Brassicasterol Campesterol 24-Methylenecholesterol Stigmasterol Cholesterol Total sterol mgrindividu
54.0 17.0 5.5 2.2 11.5 90.4
0.4 a 0.4 a 0.3 0.1a 0.4 a 6.8
57.7 13.2 4.6 3.0 12.5 103.6
3.4 a,b 1.5 b 0.7 0.1b 0.9 a 32.9
50.2 13.4 4.0 4.7 16.9 85.6
2.4 a,c 1.0 b 0.3 0.2 c 0.6 b 21.8
48.5 12.9 4.2 5.8 19.2 88.5
2.7 a,c 1.4 b 0.5 0.4 d 1.7 b 10.3
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 ŽMean; S.D.; ns 3.
Control
Polar lipids 3%
Mean
S.D.
Mean
7 Days 20:5ny3 22:6ny3 TFA mgrindividu
5.5 10.7 101.1
0.3 a 0.5 13.0
6.5 11.4 128.9
19 Days 20:5ny3 22:6ny3 TFA mgrindividu
4.6 10.6 286.3
0.3 a 0.3 a 19.2
33 Days 20:5ny3 22:6ny3 TFA mgrindividu
4.3 10.3 490.3
0.2 a 0.4 66.5
10% S.D.
20%
Control
3%
10%
20%
Mean
S.D.
Mean
S.D.
Mean
S.D.
Mean
S.D.
Mean
S.D.
Mean
S.D.
0.5 b 0.9 10.1
8.3 11.8 106.5
0.3 c 0.5 31.6
8.3 12.2 104.5
0.4 c 0.6 24.3
7.1 20.5 162.4
0.2 a 0.5 13.4
7.6 20.7 210.9
0.1a 0.9 8.4
7.9 20.0 174.7
0.4 b 0.6 13.6
8.5 19.9 189.8
0.2 c 0.2 16.0
5.5 11.3 241.2
0.4 a 0.3 a 10.1
7.1 13.0 173.2
0.8 b 0.6 b 27.3
7.7 11.2 324.2
0.5 b 0.4 a 32.2
6.1 20.1 353.4
0.0 a 0.5a 36.0
6.6 19.7 373.5
0.1b 0.3 a 34.0
7.4 21.3 279.5
0.3 c 0.1b 28.6
8.0 20.6 324.2
0.3 d 0.5 a,b 32.2
3.7 11.7 479.5
0.3 a,b 0.5 123
4.3 11.4 479.7
0.1a,c 0.4 25.8
6.0 11.8 647.2
0.2 d 0.7 2.7
6.1 19.4 477.8
0.2 a 0.5 10.2
5.3 19.9 404
0.2 b 0.3 71.2
6.0 19.9 433.6
0.2 a 0.5 29.3
7.1 20.0 458.3
0.2 c 0.7 30.9
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Neutral lipids
321
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to 7 days and at a lesser extent from 19 to 33 days ŽTable 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 ŽTable 4.. After 7 days, only the cholesterol percentage of the groups fed with 10% and 20% of emulsion ŽWWrDW 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 ŽFig. 1a
Fig. 1. Total stigmasterol Ža. and cholesterol Žb. 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 Ž50 mgrspat. by emulsion Ž3% 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 ŽTable 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 ŽSoudant 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 ŽCoutteau 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 ŽDHArEPAs 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 ŽSoudant, 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 Žnot 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 ŽSoudant et al., 1996, 1998.. The low ingestion–accumulation of the emulsion may be due to: Ž1. the physical characteristics
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of emulsion, leading to a possible lower acceptability than algae, Ž2. a lower filtration efficiency due to particle size, Ž3. a control mechanism of the free sterol absorption in the spat, or Ž4. 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 30–40% cholesterol ŽBerenberg 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 ŽSoudant 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 ŽWalton 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 ingestion–absorption of different diets. However, the relationship between the ingestion–accumulation 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 Ž1.25 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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References Berenberg, C.J., Patterson, G.W., 1981. The relationship between dietary phytosterols and the sterols of wild and cultivated oysters. Lipids 16, 276–278. Caers, M., Coutteau, P., Lombeida, P., Sorgeloos, P., 1998. The effect of lipid supplementation on the growth and fatty acid composition of Tapes philippinarum ŽL.. spat. Aquaculture 162, 287–299. Caers, M., Coutteau, P., Sorgeloos, P., 1999. Dietary impact of algal and artificial diets, fed at different feeding rations, on the growth and fatty acid composition of Tapes philippinarum ŽL.. spat. Aquaculture 170, 307–322. Coutteau, P., Castell, J.D., Ackman, R.G., Sorgeloos, P., 1996. The use of lipid emulsions as carriers for essential fatty acids in bivalves: a test case with juvenile Placopecten magellanicus. J. Shellfish Res. 15, 259–264. Eder, K., Reichlmayr-Lais, A.M., Kirchgessne, M., 1992. Simultaneous determination of amounts of major phospholipid classes and their fatty acid composition in erythrocyte membranes using high-performance liquid chromatography and gas chromatography. J. Chromatogr. 598, 33–42. Folch, J., Lees, M., Sloane Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 266, 497–509. Frolov, A.V., Pankov, S.L., 1992. The reproduction strategy of oyster Ostrea edulis L. from the biochemical point of view. Comp. Biochem. Physiol. 103B, 161–182. Gladu, P.K., Patterson, G.W., Wikfors, G.H., Chitwoods, D.J., Lubsy, W.R., 1991. Sterols of some diatoms. Phytochemistry 30, 2301–2303. Gordon, D.T., Collins, N., 1982. Anatomical distribution of sterols in oysters Ž Crassostrea gigas .. Lipids 17, 811–817. Helm, M.M., Holland, D.L., Utting, S.D., East, J., 1991. Fatty acid composition of early non-feeding larvae of the European flat oyster, Ostrea edulis. J. Mar. Biol. Assoc. U. K. 71, 691–705. Holden, M.J., Patterson, G.W., 1991. Absence of sterol biosynthesis in oyster tissue culture. Lipids 26, 81–82. Lane, A., 1989. The effect of a microencapsulated fatty acid diet on larval production in the European flat oyster Ostrea edulis L. In: de Pauw, N., Jaspers, E., Ackefors, H., Wilkins, N. ŽEds.., Aquaculture: A Biotechnology in Progress, Vol. 2. European Aquaculture Society, Bredene, Belgium, pp. 657–664. Langdon, C.J., Waldock, M.J., 1981. The effect of algal and artificial diets on the growth and fatty acid composition of Crassostrea gigas. J. Mar. Biol. Assoc. U. K. 62, 431–448. Leal, A.M., 1994. Conditioning of Manila Clam Broodstock on Natural and Artificial Diets. PhD Thesis, School of Ocean Sciences, University College of North Wales, Bangor, 343 pp. Lin, D.S., Ilias, A.M., Connor, W.E., Caldwell, R.S., Cory, H.T., Dave, G.D., 1982. Composition and biosynthesis of sterols in selected marine phytoplancton. Lipids 17, 818–824. Marty, Y., Delaunay, F., Moal, J., Samain, J.F., 1992. Change in the fatty acid composition of Pecten maximus ŽL... J. Mar. Biol. Assoc. U. K. 163, 221–234. Metcalfe, L.D., Schmitz, A.A., 1961. The rapid preparation of fatty acid esters for gas chromatography analysis. Anal. Chem. 33, 363–364. Napolitano, G.E., Ackman, R.G., Silva-Serra, M.A., 1993. Incorporation of dietary sterols by the sea scallop Placopecten magellanicus ŽGmelin. fed on microalgae. Mar. Biol. 117, 647–654. Robinson, A., 1992. Dietary supplements for the reproductive conditioning of Crassostrea gigas kumamoto ŽThunberg.: I. Effects on gonadal development, quality of ova and larvae through metamorphosis. J. Shellfish Res. 11, 437–441. Soudant, P., 1995. Les phospholipides et les sterols des geniteurs et des larves de coquille saint Jacques ´ ´ Pecten maximus ŽL... Relations avec la nutrition. These ` de doctorat, Universite´ de Bretagne Occidentale, France, 297 pp. Soudant, P., Marty, Y., Moal, J., Robert, R., Quere, ´ ´ C., Lecoz, J.R., Samain, J.F., 1996. Effect of food fatty acid and sterol quality on Pecten maximus gonad composition and reproduction process. Aquaculture 143, 361–378. Soudant, P., Lecoz, J.R., Marty, Y., Moal, J., Samain, J.F., 1998. Incorporation of microalgae sterols by scallop Pecten maximus ŽL.. larvae. Comp. Biochem. Physiol. 119A, 451–457. Teshima, S., 1983. Sterol metabolism. In: Pruder, G.D., Langdon, C., Conklin, D. ŽEds.., Biological and
326
P. Soudant et al.r Aquaculture 184 (2000) 315–326
Physiological Approaches to Shellfish Nutrition, Vol. 2. Proceedings of the 2nd Conference of Aquaculture Nutrition, Louisiana State University, Baton Rouge, pp. 205–216. Teshima, S., Kanazawa, A., 1974. Biosynthesis of sterols in abalone Haliotis gurneri and mussel Mytilus edulis. Comp. Biochem. Physiol. 43B, 555–561. Teshima, S.I., Patterson, G.W., 1981. Sterol biosynthesis in the oyster, Crassostrea Õirginica. Lipids 16, 234–239. Teshima, S., Kanazawa, A., Miyawaki, H., 1979. Metabolism of b-sitosterol in the mussel and the snail. Comp. Biochem. Physiol. 63B, 323–328. Trider, D.J., Castell, J.D., 1980. Effect of dietary lipids on growth tissue composition and metabolism of the oyster Ž Crassostrea Õirginica.. J. Nutr. 110, 1303–1309. Tsitsa-Tzardis, E., Patterson, G.W., Wickfors, G.H., Gladu, P.K., Harrison, D., 1993. Sterols of Chaetoceros and Skeletonema. Lipids 28, 465–467. Utting, S.D., Millican, P.F., 1995. Techniques for the hatchery conditioning of bivalve broodstocks and the subsequent effect on egg quality and larval quality. In: Lavens, P., Jaspers, E., Roelants, I. ŽEds.., Larvi’95-Fish and Shellfish Larviculture Symposium. European Aquaculture Society, Special Publication No. 24, Gent, Belgium, p. 64. Voogt, P.A., 1975. Investigations on the capacity of synthesizing 3-a sterols in molluscs biosynthesis and composition of sterols in some bivalves ŽAnisomyaria.. Comp. Biochem. Physiol. 50B, 499–504. Walton, M.J., Pennock, J.F., 1972. Some studies on the biosynthesis of ubiquinones, isoprenoic alcohol, squalene and sterols by marine invertebrates. Biochem. J. 127, 471–479. Wikfors, G.H., Gladu, P.K., Patterson, G.W., 1991. In search of the ideal algal diet for oysters: recent progress, with emphasis on sterols. J. Shellfish Res. 10, 292.
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 a
Laboratory of Aquaculture and Artemia Reference Center, UniÕersity of Gent, Rozier 44, 9000 Ghent, Belgium b DRV r A, Laboratoire de Physiologie des InÕertebres, ´ ´ IFREMER Centre de Brest, BP 70, 29280 Plouzane, ´ France c UMR r CNRS 6521, UniÕersite´ de Bretagne Occidentale, BP 809, 29285 Brest, France Accepted 24 September 1999
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 ingestion–absorption 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
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Corresponding author. Tel.: q33-2-98-22-44-02; fax: q33-2-98-22-46-53; e-mail: [email protected]
0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 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 hatchery–nursery. A number of papers on invertebrate nutrition have described the quantitative algal requirements ŽUtting 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 ŽPUFA., 20:5n y 3 and 22:6n y 3, have been demonstrated to be essential for bivalves ŽLangdon 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 ŽLane, 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 ŽTeshima, 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 conditions ŽGladu 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 ŽWikfors 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–digestion 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 ŽICES. — Working Group on Mass Rearing of Juvenile Fish ŽBergen, Norway
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21–23, 1993; ICES 1994., two experimental emulsions 30r0.6rE5 containing either stigmasterol or cholesterol, 5% of the emulsion dry weight ŽDW. were prepared. These emulsions were made by INVE Technologies ŽBaasrode, Belgium. and contained 50% lipid on wet weight ŽWW. basis, water, emulgators, antioxidants, preservatives and liposoluble vitamins ŽA, 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 Žaverage 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 and refilled with 1 mm filtered seawater. A mixed diet Ž50:50 on a DW basis. of T-Isochrysis and Tetraselmis suecica was provided daily to spat at a weight specific ration of 0.75% Žalgal 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 Žnot 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 ŽFW., DW, and organic matter ŽOM. of 10 replicates of five animals were measured. Another three replicates of pooled spats Žfive 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. Ž1957.. The total sterol fraction was first hydrolyzed with sodium methoxide ŽMeONa. for 90 min at room temperature ŽEder 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 Ž12.5 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 ŽCarlo-Erba model 5160 HRGC coupled on mass spectrometer Nermag R10-10H.. Sterol nomenclature Žtrivial 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, 24Ž28.-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-5,24Ž28.-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 times 500 ml of 98:2 chloroform–methanol and put on a silica gel microcolumn Ž30 mm = 5 mm, Kieselgel Merck, 70–230 mesh. for neutral and polar lipid separation. Neutral lipids were eluted with 10 ml chloroform–methanol Ž98:2. 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 Ž14% in methanol. ŽMetcalfe and Schmitz, 1961. and treated according to Marty et al. Ž1992.. The fatty acid methyl esters ŽFAME. were analysed using a Chrompak 9001 gas chromatograph ŽChrompack, Middelburg, Netherlands. equipped with a on-column injector, a DBWAX Ž30 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 ŽCP Sil 8 CB, 30 m = 0.25 mm, 0.25 mm film thickness.. 2.4. Statistics Significant differences Ž P - 0.05. in biological and biochemical measurements between 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 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 Žexpressed as sterol % of the total sterol; Mean, S.D.sstandard deviation; ns 5. Spat at T0
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
T-Isochrysis
Tetraselmis
Emulsion
Mean
S.D.
Mean
S.D.
Mean
S.D.
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
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
– 0.5 – 2.0 3.0 93.9 0.6 – – – – – –
– 0.6 – 0.7 0.4 1.0 0.5 – – – – – –
– – – – 1.9 – – 82.5 14.2 – 1.4 – –
– – – – 0.4 – – 2.1 1.3 – 1.0 – –
40.2
8.0
298.9
17.4
50
50
0.05
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 ŽTable 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 ŽTables 4 and 5.. Table 2 PUFA composition of the spats at the beginning of the experiment, of the algae and of the emulsion Žexpressed as fatty acid % of the total fatty acids; Mean, S.D.sstandard deviation; ns 5. Spat at T0
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
Neutral lipid
Polar lipid
Mean
S.D.
Mean
S.D.
7.9 5.4 6.6 1.0 1.1 6.8 0.7 1.0 1.1 9.4 84.2
1.8 1.3 1.4 0.2 0.2 1.3 0.3 0.3 0.3 1.2 19.6
4.9 2.4 2.6 0.7 3.3 11.2 3.7 6.3 3.3 27.5 136.8
0.7 0.3 0.4 0.2 0.8 2.1 0.8 1.4 0.6 4.9 25.4
T-Isochrysis
Tetraselmis
Mean
S.D.
Mean
S.D.
6.5 6.5 17.1 0.0 0.1 0.3 – – 2.1 11.1
1.9 0.1 1.0 0.0 0.0 0.0 – – 0.0 0.6
2.9 6.4 3.5 0.0 0.4 3.8 – – – 0.1
1.3 1.6 0.6 0.0 0.0 0.1 – – – 0.0
1.8
0.4
5.8
0.2
Emulsion
5.0 1.1 1.8 0.2 1.1 16.3 – – – 11.8
0.8
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Table 3 Effect of emulsion addition on the WW, FW, DW, % OM content expressed in mg ŽMean, S.D., ns10 of five pooled oysters. Concentration
0%
3%
10%
20%
Mean
S.D.
Mean
S.D.
Mean
S.D.
Mean
S.D.
248.5 375.5 596.1
13.7 66.1 63.1
288.0 426.1 534.1
15.6 53.9 46.7
198.3 345.8 597.6
21.8 31.8 105.0
227.1 333.4 572.3
13.5 41.3 54.4
After 33 days mgr indiÕidu 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
mg r indiÕidu Wet weight, 7 days Wet weight, 19 days Wet weight, 33 days
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 ŽTable 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 ŽMean, S.D., ns 3.. The statistical differences Ž P - 0.05. between treatment are indicated by a letter Control Mean
3% S.D.
Mean
10% S.D.
Mean
20% S.D.
Mean
S.D.
7 Days of supplementation Brassicasterol Campesterol 24-Methylenecholesterol Stigmasterol Cholesterol Total sterol mgrindividu
40.4 14.5 6.9 2.6 21.0 27.0
2.5a 0.8 0.4 0.2 a 0.5a 7.2
40.8 14.1 6.6 3.1 20.7 32.0
1.1a 0.4 0.5 0.1b 0.6 a 8.1
38.7 13.9 6.3 4.0 22.6 25.0
1.5a 0.9 0.1 0.2 c 0.6 b 7.6
35.7 13.8 6.1 4.8 24.5 26.0
0.5 b 0.4 0.4 0.1d 0.9 c 11.5
19 Days of supplementation Brassicasterol Campesterol 24-Methylenecholesterol Stigmasterol Cholesterol Total sterol mgrindividu
49.1 17.2 5.6 1.9 15.0 69.4
0.9 a 0.8 0.1a 0.1a 0.5a 10.3
48.5 16.3 5.2 2.9 17.1 82.5
1.6 a 0.6 0.4 a 0.2 b 0.5 b 11.9
45.4 15.1 4.7 4.5 20.0 60.7
2.0 a 1.1 0.1a 0.0 c 0.4 c 6.5
41.8 15.2 4.5 5.5 22.2 67.5
1.1b 1.2 0.4 b 0.6 d 0.2 d 8.2
33 Days of supplementation Brassicasterol Campesterol 24-Methylenecholesterol Stigmasterol Cholesterol Total sterol mgrindividu
54.0 17.0 5.5 2.2 11.5 90.4
0.4 a 0.4 a 0.3 0.1a 0.4 a 6.8
57.7 13.2 4.6 3.0 12.5 103.6
3.4 a,b 1.5 b 0.7 0.1b 0.9 a 32.9
50.2 13.4 4.0 4.7 16.9 85.6
2.4 a,c 1.0 b 0.3 0.2 c 0.6 b 21.8
48.5 12.9 4.2 5.8 19.2 88.5
2.7 a,c 1.4 b 0.5 0.4 d 1.7 b 10.3
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 ŽMean; S.D.; ns 3.
Control
Polar lipids 3%
Mean
S.D.
Mean
7 Days 20:5ny3 22:6ny3 TFA mgrindividu
5.5 10.7 101.1
0.3 a 0.5 13.0
6.5 11.4 128.9
19 Days 20:5ny3 22:6ny3 TFA mgrindividu
4.6 10.6 286.3
0.3 a 0.3 a 19.2
33 Days 20:5ny3 22:6ny3 TFA mgrindividu
4.3 10.3 490.3
0.2 a 0.4 66.5
10% S.D.
20%
Control
3%
10%
20%
Mean
S.D.
Mean
S.D.
Mean
S.D.
Mean
S.D.
Mean
S.D.
Mean
S.D.
0.5 b 0.9 10.1
8.3 11.8 106.5
0.3 c 0.5 31.6
8.3 12.2 104.5
0.4 c 0.6 24.3
7.1 20.5 162.4
0.2 a 0.5 13.4
7.6 20.7 210.9
0.1a 0.9 8.4
7.9 20.0 174.7
0.4 b 0.6 13.6
8.5 19.9 189.8
0.2 c 0.2 16.0
5.5 11.3 241.2
0.4 a 0.3 a 10.1
7.1 13.0 173.2
0.8 b 0.6 b 27.3
7.7 11.2 324.2
0.5 b 0.4 a 32.2
6.1 20.1 353.4
0.0 a 0.5a 36.0
6.6 19.7 373.5
0.1b 0.3 a 34.0
7.4 21.3 279.5
0.3 c 0.1b 28.6
8.0 20.6 324.2
0.3 d 0.5 a,b 32.2
3.7 11.7 479.5
0.3 a,b 0.5 123
4.3 11.4 479.7
0.1a,c 0.4 25.8
6.0 11.8 647.2
0.2 d 0.7 2.7
6.1 19.4 477.8
0.2 a 0.5 10.2
5.3 19.9 404
0.2 b 0.3 71.2
6.0 19.9 433.6
0.2 a 0.5 29.3
7.1 20.0 458.3
0.2 c 0.7 30.9
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Neutral lipids
321
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to 7 days and at a lesser extent from 19 to 33 days ŽTable 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 ŽTable 4.. After 7 days, only the cholesterol percentage of the groups fed with 10% and 20% of emulsion ŽWWrDW 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 ŽFig. 1a
Fig. 1. Total stigmasterol Ža. and cholesterol Žb. 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 Ž50 mgrspat. by emulsion Ž3% 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 ŽTable 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 ŽSoudant 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 ŽCoutteau 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 ŽDHArEPAs 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 ŽSoudant, 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 Žnot 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 ŽSoudant et al., 1996, 1998.. The low ingestion–accumulation of the emulsion may be due to: Ž1. the physical characteristics
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of emulsion, leading to a possible lower acceptability than algae, Ž2. a lower filtration efficiency due to particle size, Ž3. a control mechanism of the free sterol absorption in the spat, or Ž4. 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 30–40% cholesterol ŽBerenberg 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 ŽSoudant 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 ŽWalton 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 ingestion–absorption of different diets. However, the relationship between the ingestion–accumulation 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 Ž1.25 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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References Berenberg, C.J., Patterson, G.W., 1981. The relationship between dietary phytosterols and the sterols of wild and cultivated oysters. Lipids 16, 276–278. Caers, M., Coutteau, P., Lombeida, P., Sorgeloos, P., 1998. The effect of lipid supplementation on the growth and fatty acid composition of Tapes philippinarum ŽL.. spat. Aquaculture 162, 287–299. Caers, M., Coutteau, P., Sorgeloos, P., 1999. Dietary impact of algal and artificial diets, fed at different feeding rations, on the growth and fatty acid composition of Tapes philippinarum ŽL.. spat. Aquaculture 170, 307–322. Coutteau, P., Castell, J.D., Ackman, R.G., Sorgeloos, P., 1996. The use of lipid emulsions as carriers for essential fatty acids in bivalves: a test case with juvenile Placopecten magellanicus. J. Shellfish Res. 15, 259–264. Eder, K., Reichlmayr-Lais, A.M., Kirchgessne, M., 1992. Simultaneous determination of amounts of major phospholipid classes and their fatty acid composition in erythrocyte membranes using high-performance liquid chromatography and gas chromatography. J. Chromatogr. 598, 33–42. Folch, J., Lees, M., Sloane Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 266, 497–509. Frolov, A.V., Pankov, S.L., 1992. The reproduction strategy of oyster Ostrea edulis L. from the biochemical point of view. Comp. Biochem. Physiol. 103B, 161–182. Gladu, P.K., Patterson, G.W., Wikfors, G.H., Chitwoods, D.J., Lubsy, W.R., 1991. Sterols of some diatoms. Phytochemistry 30, 2301–2303. Gordon, D.T., Collins, N., 1982. Anatomical distribution of sterols in oysters Ž Crassostrea gigas .. Lipids 17, 811–817. Helm, M.M., Holland, D.L., Utting, S.D., East, J., 1991. Fatty acid composition of early non-feeding larvae of the European flat oyster, Ostrea edulis. J. Mar. Biol. Assoc. U. K. 71, 691–705. Holden, M.J., Patterson, G.W., 1991. Absence of sterol biosynthesis in oyster tissue culture. Lipids 26, 81–82. Lane, A., 1989. The effect of a microencapsulated fatty acid diet on larval production in the European flat oyster Ostrea edulis L. In: de Pauw, N., Jaspers, E., Ackefors, H., Wilkins, N. ŽEds.., Aquaculture: A Biotechnology in Progress, Vol. 2. European Aquaculture Society, Bredene, Belgium, pp. 657–664. Langdon, C.J., Waldock, M.J., 1981. The effect of algal and artificial diets on the growth and fatty acid composition of Crassostrea gigas. J. Mar. Biol. Assoc. U. K. 62, 431–448. Leal, A.M., 1994. Conditioning of Manila Clam Broodstock on Natural and Artificial Diets. PhD Thesis, School of Ocean Sciences, University College of North Wales, Bangor, 343 pp. Lin, D.S., Ilias, A.M., Connor, W.E., Caldwell, R.S., Cory, H.T., Dave, G.D., 1982. Composition and biosynthesis of sterols in selected marine phytoplancton. Lipids 17, 818–824. Marty, Y., Delaunay, F., Moal, J., Samain, J.F., 1992. Change in the fatty acid composition of Pecten maximus ŽL... J. Mar. Biol. Assoc. U. K. 163, 221–234. Metcalfe, L.D., Schmitz, A.A., 1961. The rapid preparation of fatty acid esters for gas chromatography analysis. Anal. Chem. 33, 363–364. Napolitano, G.E., Ackman, R.G., Silva-Serra, M.A., 1993. Incorporation of dietary sterols by the sea scallop Placopecten magellanicus ŽGmelin. fed on microalgae. Mar. Biol. 117, 647–654. Robinson, A., 1992. Dietary supplements for the reproductive conditioning of Crassostrea gigas kumamoto ŽThunberg.: I. Effects on gonadal development, quality of ova and larvae through metamorphosis. J. Shellfish Res. 11, 437–441. Soudant, P., 1995. Les phospholipides et les sterols des geniteurs et des larves de coquille saint Jacques ´ ´ Pecten maximus ŽL... Relations avec la nutrition. These ` de doctorat, Universite´ de Bretagne Occidentale, France, 297 pp. Soudant, P., Marty, Y., Moal, J., Robert, R., Quere, ´ ´ C., Lecoz, J.R., Samain, J.F., 1996. Effect of food fatty acid and sterol quality on Pecten maximus gonad composition and reproduction process. Aquaculture 143, 361–378. Soudant, P., Lecoz, J.R., Marty, Y., Moal, J., Samain, J.F., 1998. Incorporation of microalgae sterols by scallop Pecten maximus ŽL.. larvae. Comp. Biochem. Physiol. 119A, 451–457. Teshima, S., 1983. Sterol metabolism. In: Pruder, G.D., Langdon, C., Conklin, D. ŽEds.., Biological and
326
P. Soudant et al.r Aquaculture 184 (2000) 315–326
Physiological Approaches to Shellfish Nutrition, Vol. 2. Proceedings of the 2nd Conference of Aquaculture Nutrition, Louisiana State University, Baton Rouge, pp. 205–216. Teshima, S., Kanazawa, A., 1974. Biosynthesis of sterols in abalone Haliotis gurneri and mussel Mytilus edulis. Comp. Biochem. Physiol. 43B, 555–561. Teshima, S.I., Patterson, G.W., 1981. Sterol biosynthesis in the oyster, Crassostrea Õirginica. Lipids 16, 234–239. Teshima, S., Kanazawa, A., Miyawaki, H., 1979. Metabolism of b-sitosterol in the mussel and the snail. Comp. Biochem. Physiol. 63B, 323–328. Trider, D.J., Castell, J.D., 1980. Effect of dietary lipids on growth tissue composition and metabolism of the oyster Ž Crassostrea Õirginica.. J. Nutr. 110, 1303–1309. Tsitsa-Tzardis, E., Patterson, G.W., Wickfors, G.H., Gladu, P.K., Harrison, D., 1993. Sterols of Chaetoceros and Skeletonema. Lipids 28, 465–467. Utting, S.D., Millican, P.F., 1995. Techniques for the hatchery conditioning of bivalve broodstocks and the subsequent effect on egg quality and larval quality. In: Lavens, P., Jaspers, E., Roelants, I. ŽEds.., Larvi’95-Fish and Shellfish Larviculture Symposium. European Aquaculture Society, Special Publication No. 24, Gent, Belgium, p. 64. Voogt, P.A., 1975. Investigations on the capacity of synthesizing 3-a sterols in molluscs biosynthesis and composition of sterols in some bivalves ŽAnisomyaria.. Comp. Biochem. Physiol. 50B, 499–504. Walton, M.J., Pennock, J.F., 1972. Some studies on the biosynthesis of ubiquinones, isoprenoic alcohol, squalene and sterols by marine invertebrates. Biochem. J. 127, 471–479. Wikfors, G.H., Gladu, P.K., Patterson, G.W., 1991. In search of the ideal algal diet for oysters: recent progress, with emphasis on sterols. J. Shellfish Res. 10, 292.