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;
Ž .
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;
Ž .
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.
Ž .
The neutral and polar lipid fractions were transesterified with BF 14 in methanol
3
Ž .
Ž .
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
Ž .
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
Ž .
non-polar column CP Sil 8 CB, 30 m = 0.25 mm, 0.25 mm film thickness . 2.4. Statistics
Ž .
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
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.
Table 1 Ž
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
T-Isochrysis Tetraselmis
Emulsion Mean
S.D. Mean
S.D. Mean
S.D. Norcholesterol
2.4 0.1
– –
– –
c-Dehidrocholesterol 0.7
0.1 0.5
0.6 –
– t-Dehidrocholesterol
3.7 0.1
– –
– –
Dihydrocholesterol 1.3
0.2 2.0
0.7 –
– Cholesterol
25.0 0.5
3.0 0.4
1.9 0.4
50 Brassicasterol
34.0 1.7
93.9 1.0
– –
Desmosterol 2.1
0.2 0.6
0.5 –
– Campesterol
11.9 1.1
– –
82.5 2.1
24-Methylenecholesterol 8.6
0.5 –
– 14.2
1.3 Stigmasterol
2.6 0.4
– –
– –
50 Methylporiferasterol
0.8 0.1
– –
1.4 1.0
Betasitosterol 3.5
0.4 –
– –
– Fucosterol
0.7 0.1
– –
– –
Total sterol in spat mgrindividu 17.1
3.8 Total sterol in algae fgrcell
40.2 8.0
298.9 17.4
Total sterol in emulsion grg DW 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
Ž .
treatments along the experiment Table 3 . The sterol supplementation did not change the total sterol content of the spat as well
Ž .
as the total fatty acid content measured in the neutral and polar lipids Tables 4 and 5 .
Table 2 Ž
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
T-Isochrysis Tetraselmis
Emulsion Neutral lipid
Polar lipid Mean
S.D. Mean
S.D. Mean
S.D. Mean
S.D. 18:2 ny6
7.9 1.8
4.9 0.7
6.5 1.9
2.9 1.3
5.0 18:3ny3
5.4 1.3
2.4 0.3
6.5 0.1
6.4 1.6
1.1 18:4 ny3
6.6 1.4
2.6 0.4
17.1 1.0
3.5 0.6
1.8 20:2 ny6
1.0 0.2
0.7 0.2
0.0 0.0
0.0 0.0
0.2 20:4 ny6
1.1 0.2
3.3 0.8
0.1 0.0
0.4 0.0
1.1 20:5ny3
6.8 1.3
11.2 2.1
0.3 0.0
3.8 0.1
16.3 22:2D7,15
0.7 0.3
3.7 0.8
– –
– –
– 22:2D7,13
1.0 0.3
6.3 1.4
– –
– –
– 22:5ny6
1.1 0.3
3.3 0.6
2.1 0.0
– –
– 22:6 ny3
9.4 1.2
27.5 4.9
11.1 0.6
0.1 0.0
11.8 Total FA in spat mgrindividu
84.2 19.6
136.8 25.4
Total FA in algae pgrcell 1.8
0.4 5.8
0.2 Total FA in emulsion grg DW
0.8
Table 3 Ž
Effect of emulsion addition on the WW, FW, DW, OM content expressed in mg Mean, S.D., ns10 of five .
pooled oysters Concentration
3 10
20 Mean
S.D. Mean
S.D. Mean
S.D. Mean
S.D. mg r indiÕidu
Wet weight, 7 days 248.5
13.7 288.0
15.6 198.3
21.8 227.1
13.5 Wet weight, 19 days
375.5 66.1
426.1 53.9
345.8 31.8
333.4 41.3
Wet weight, 33 days 596.1
63.1 534.1
46.7 597.6
105.0 572.3
54.4 After 33 days mg r indiÕidu
Flesh weight 85.9
15.8 84.1
1.4 84.2
25.5 77.1
11.3 Dry weight
19.0 3.0
19.2 1.0
18.1 5.5
17.9 1.7
Organic matter 15.9
2.6 15.8
0.9 15.0
4.8 14.9
1.5
The percentage of cholesterol decreased over time in all treatments while brassicast- erol and campesterol increased. However, the cholesterol decrease was gradually
Ž .
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 Ž
Effect of sterol addition on the total sterol composition of the spat after 7, 19, 33 days of feeding Mean, S.D., .
Ž .
ns 3 . The statistical differences P -0.05 between treatment are indicated by a letter Control
3 10
20 Mean
S.D. Mean
S.D. Mean
S.D. Mean
S.D. 7 Days of supplementation
a a
a b
Brassicasterol 40.4
2.5 40.8
1.1 38.7
1.5 35.7
0.5 Campesterol
14.5 0.8
14.1 0.4
13.9 0.9
13.8 0.4
24-Methylenecholesterol 6.9
0.4 6.6
0.5 6.3
0.1 6.1
0.4
a b
c d
Stigmasterol 2.6
0.2 3.1
0.1 4.0
0.2 4.8
0.1
a a
b c
Cholesterol 21.0
0.5 20.7
0.6 22.6
0.6 24.5
0.9 Total sterol mgrindividu
27.0 7.2
32.0 8.1
25.0 7.6
26.0 11.5
19 Days of supplementation
a a
a b
Brassicasterol 49.1
0.9 48.5
1.6 45.4
2.0 41.8
1.1 Campesterol
17.2 0.8
16.3 0.6
15.1 1.1
15.2 1.2
a a
a b
24-Methylenecholesterol 5.6
0.1 5.2
0.4 4.7
0.1 4.5
0.4
a b
c d
Stigmasterol 1.9
0.1 2.9
0.2 4.5
0.0 5.5
0.6
a b
c d
Cholesterol 15.0
0.5 17.1
0.5 20.0
0.4 22.2
0.2 Total sterol mgrindividu
69.4 10.3
82.5 11.9
60.7 6.5
67.5 8.2
33 Days of supplementation
a a,b
a,c a,c
Brassicasterol 54.0
0.4 57.7
3.4 50.2
2.4 48.5
2.7 Campesterol
17.0 0.4
a
13.2 1.5
b
13.4 1.0
b
12.9 1.4
b
24-Methylenecholesterol 5.5
0.3 4.6
0.7 4.0
0.3 4.2
0.5
a b
c d
Stigmasterol 2.2
0.1 3.0
0.1 4.7
0.2 5.8
0.4
a a
b b
Cholesterol 11.5
0.4 12.5
0.9 16.9
0.6 19.2
1.7 Total sterol mgrindividu
90.4 6.8
103.6 32.9
85.6 21.8
88.5 10.3
P. Soudant
et al.
r Aquaculture
184 2000
315 –
326
321 Table 5
Ž .
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 Neutral lipids
Polar lipids Control
3 10
20 Control
3 10
20 Mean
S.D. Mean
S.D. Mean
S.D. Mean
S.D. Mean
S.D. Mean
S.D. Mean
S.D. Mean
S.D. 7 Days
a b
c c
a a
b c
20:5ny3 5.5
0.3 6.5
0.5 8.3
0.3 8.3
0.4 7.1
0.2 7.6
0.1 7.9
0.4 8.5
0.2 22:6 ny3
10.7 0.5
11.4 0.9
11.8 0.5
12.2 0.6
20.5 0.5
20.7 0.9
20.0 0.6
19.9 0.2
TFA mgrindividu 101.1
13.0 128.9
10.1 106.5
31.6 104.5
24.3 162.4
13.4 210.9
8.4 174.7
13.6 189.8
16.0 19 Days
a a
b b
a b
c d
20:5ny3 4.6
0.3 5.5
0.4 7.1
0.8 7.7
0.5 6.1
0.0 6.6
0.1 7.4
0.3 8.0
0.3
a a
b a
a a
b a,b
22:6 ny3 10.6
0.3 11.3
0.3 13.0
0.6 11.2
0.4 20.1
0.5 19.7
0.3 21.3
0.1 20.6
0.5 TFA mgrindividu
286.3 19.2
241.2 10.1
173.2 27.3
324.2 32.2
353.4 36.0
373.5 34.0
279.5 28.6
324.2 32.2
33 Days
a a,b
a,c d
a b
a c
20:5ny3 4.3
0.2 3.7
0.3 4.3
0.1 6.0
0.2 6.1
0.2 5.3
0.2 6.0
0.2 7.1
0.2 22:6 ny3
10.3 0.4
11.7 0.5
11.4 0.4
11.8 0.7
19.4 0.5
19.9 0.3
19.9 0.5
20.0 0.7
TFA mgrindividu 490.3
66.5 479.5
123 479.7
25.8 647.2
2.7 477.8
10.2 404
71.2 433.6
29.3 458.3
30.9
Ž .
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, Ž
. 19 and 33 days of feeding Table 4 . After 7 days, only the cholesterol percentage of the
Ž .
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 Ž
as a polynomial function of the total sterol added with the emulsion per spat Fig. 1a
Ž . Ž .
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.
. 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
Ž .
Ž 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 22:6 n 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 22:6 n y 3 was only noted after
Ž .
19 days of feeding in the group fed with 10 emulsion supplement Table 5 .
4. Discussion
