Ž .
TICA
Microsoft, Statsoft . The homogeneity of the variances of means was checked by Bartlett’s x
2
-test. Arcsine 6 transformation was used prior to statistical analyses of Ž
. percentage data Sokal and Rohlf, 1995 .
3. Results
3.1. Fatty acid composition of the diets Ž
. The major saturated fatty acids SAFA of T. suecica were 16:0, 18:0, the monoun-
Ž .
saturated fatty acids MUFA were dominated by 18:1n y 9 whereas 18:3n y 3, 18:4 n Ž
. y 3 and 20:5n y 3 were the major PUFA Table 1 . The predominant fatty acids in both
Ž .
emulsions were 18:1n y 9, 18:2 n y 6 and 22:6 n y 3 Table 1 . Em2 was prepared to Ž
contain similar amounts of 18:2 n y 6 and 22:6 n y 3 34 of the total fatty acids or 318
y1
. mg g lipid
whereas the 22:6 n y 3 content of Em1 was similar to the 22:6 n y 3 Ž
y1
. content of the liposomes 158 and 153 mg g lipid
, respectively . The fatty acid profile of the liposomes was dominated by 22:6 n y 3 and especially 18:2 n y 6 which compro-
mised 20 and 50, respectively, of the total fatty acids. Compared to the emulsions, the percentage of 18:1n y 9 was approximately three times lower. Except for 22:6 n y 3,
which was present as ethyl esters in the liposomes and the emulsions, fatty acids of the liposomes were supplied as phosphatidylcholine whereas fatty acids of the emulsion
were supplied as triglycerides.
The total amount of lipid supplemented to the algal diet via the liposomes or Ž
. emulsions was similar 50 of the dry weight of the algal diet . However, transesterifi-
cation of 1 g of triglycerides or ethyl esters yields a higher amount of FAME than transesterification of 1 g of phosphatidylcholine. Therefore, the total amount of fatty
Ž .
Ž .
acids mg supplied in via the liposome diet Algae q Lipo was much lower than the Ž
total amount of fatty acids supplied via the emulsion diets Algae q Em1 and Algae q . Ž
. Em2
Table 1 . 3.2. Fatty acid composition of spat fed solely T. suecica
The predominant fatty acids in the neutral lipids of spat fed solely T. suecica were Ž
. 16:0, 18:1n y 9, 18:3n y 3 and 20:5n y 3 Tables 2 and 3 . In the polar lipids, the
Ž .
Ž .
abundance of the nonmethylene-interrupted dienes NMID and DMA mainly 18:0 was compensated by a decrease of the percentage of total MUFA, mainly 18:1n y 9. The
percentage of 22:6 n y 3 was twice as high in the polar lipids than in the neutral lipids Ž
. Tables 2 and 3 .
3.3. Fatty acid composition of emulsion-fed spat The percentage of 18:1n y 9, 18:2 n y 6 and 22:6 n y 3 was significantly higher in
Ž .
the neutral lipids of emulsion-fed C. gigas Algae q Em1 and Algae q Em2 than in C.
Table 2 Ž
. Ž
. Effect of feeding 1 T. suecica Algae , whether or not supplemented with liposomes AlgaeqLipo ,
Ž .
Ž .
Ž emulsion 1 AlgaeqEm1 or emulsion 2 AlgaeqEm2 on the fatty acid composition of the total fatty
. Ž
. acids and DMA of the neutral lipids of C. gigas spat. In each row, mean values ns 3 SD followed by the
Ž .
same letter are not significantly different ANOVA, Tukey HSD test, P F 0.05 Fatty acids and DMA
Neutral lipids Algae
AlgaeqLipo AlgaeqEm1
AlgaeqEm2 16:0
15.40.5a 12.70.7b
10.80.2c 9.91.2c
18:0 3.70.3
4.10.4 3.50.1
3.40.6 S SAFA
20.10.7a 17.70.4b
15.50.3c 14.10.6d
16:1ny7 1.50.2a
1.20.2ab 0.60.1c
0.80.2bc 18:1ny9
15.80.7c 10.31.0d
19.90.5a 17.50.1b
18:1ny7 5.40.6a
3.90.4b 3.00.1c
3.00.2c 20:1ny9
3.60.5a 2.90.4a
2.10.0b 1.70.2b
20:1ny7 3.00.4a
2.90.3a 1.20.0b
1.30.1b S MUFA
32.11.6a 24.01.5c
28.30.3b 26.50.9bc
18:2 ny6 3.50.6d
8.80.4c 22.00.3a
18.50.8b 20:2 ny6
0.50.1b 1.00.2b
1.30.0a 1.10.1b
20:4 ny6 1.60.2a
1.70.2a 0.60.0b
0.80.1b S ny6 PUFA
5.70.5d 11.70.7c
24.10.2a 20.60.9b
18:3ny3 8.20.7a
5.10.4b 5.30.1b
4.10.2c 18:4 ny3
3.30.4a 2.30.5a
1.20.1b 0.90.2c
20:5ny3 14.80.8a
12.50.7b 7.20.3d
8.80.5c 22:6 ny3
2.40.2a 12.20.5b
9.30.3c 17.70.6a
S ny3 PUFA 32.41.4a
35.11.9a 24.90.6b
33.40.5a S ny3r ny6
5.80.7a 3.00.1b
1.00.1d 1.60.1c
22:2i 2.00.2a
2.20.1a 0.80.0b
0.90.1b 22:2j
1.80.2a 1.90.2a
0.60.1b 0.50.1b
S NMID 4.50.2a
4.70.1a 2.40.2b
2.20.1b 18:0DMA
tr tr
tr tr
S DMA 3.10.3a
3.50.7a 2.90.2a
1.50.3b
U
Ž .
Trace F 0.5 .
Ž .
gigas fed solely T. suecica Algae . The greatest accumulations of 18:1n y 9, 18:2 n y 6 and 22:6 n y 3 were observed in the neutral lipids of spat fed the emulsion with the
Ž highest concentration of these fatty acids Em1 for 18:1n y 9 and 18:2 n y 6, Em2 for
. 22:6 n y 3 . The same situation was observed in the percentage and absolute concentra-
Ž
y1
. tion mg g DW
of 18:1n y 9 and 18:2 n y 6 and 22:6 n y 3 in the total lipids of C. Ž
. gigas Tables 2–4; Fig. 1A, B, C and D . From the first week onwards, Em 1 and Em2
supplements resulted in a drastic elevation of the percentage of 22:6 n y 3 in the total Ž
. lipids of C. gigas and this remained the case until the end of the experiment Fig. 1A .
For treatments consisting of either emulsion, a 50 reduction of 20:5n y 3 was observed in the total lipids of C. gigas during the first week after which it remained
Ž .
quite constant at approximately 8 Fig. 1B . As with 22:6 n y 3, a profound increase in 18:1n y 9 and 18:2 n y 6 occurred in the first week. However, a parallel decrease in the
Ž proportions of both fatty acids took place during the last 3 weeks of the experiment Fig.
. 1C and D . When T. suecica was supplemented with equal amounts of 18:2 n y 6 and
Table 3 Ž
. Ž
. Effect of feeding 1 T. suecica Algae , whether or not supplemented with liposomes AlgaeqLipo ,
Ž .
Ž .
Ž emulsion 1 AlgaeqEm1 or emulsion 2 AlgaeqEm2 on the fatty acid composition expressed as of the
. Ž
. total fatty acids and DMA of the polar lipids of C. gigas spat. In each row, mean valuesSD ns 3
Ž .
followed by the same letter are not significantly different ANOVA, Tukey HSD test, P F 0.05 Fatty acids and DMA
Polar lipids Algae
AlgaeqLipo AlgaeqEm1
AlgaeqEm2 16:0
16.90.7a 16.20.5ab
15.30.4b 15.60.6ab
18:0 3.80.4
4.00.2 4.50.1
4.40.5 S SAFA
22.51.2 21.50.4
21.60.4 22.00.5
16:1ny7 1.20.3a
1.00.1ab 0.60.2b
1.00.1ab 18:1ny9
4.30.4ab 3.50.3b
5.40.3a 4.84.8a
18:1ny7 4.20.2a
4.30.6a 3.00.2b
3.00.5b 20:1ny9
3.00.3a 1.30.2c
2.10.1b 2.00.1b
20:1ny7 3.10.4a
2.20.2b 1.40.1c
1.40.2c S MUFA
17.40.7a 13.11.0b
14.70.7ab 13.91.6b
18:2 ny6 1.70.3d
3.80.2c 7.20.3a
4.70.4b 20:2 ny6
0.10.1b 0.00.0b
0.80.1a 0.90.1a
20:4 ny6 2.00.2a
1.70.2a 1.20.0b
1.10.1b S ny6 PUFA
4.30.6c 6.00.4b
9.30.3a 6.90.4b
18:3ny3 4.10.4a
2.60.2b 2.20.1b
1.50.2c 18:4 ny3
2.40.3a 2.10.2a
0.60.1b 0.60.0b
20:5ny3 16.70.6a
11.80.5b 9.60.3c
8.20.8d 22:6ny3
4.80.7c 14.20.8b
16.50.3b 21.51.5a
S ny3 PUFA 31.50.6b
32.70.4ab 31.70.5b
34.01.4a S ny3r ny6
7.50.9a 5.40.4b
3.40.2c 4.90.5b
22:2i 4.60.2a
3.60.2b 3.00.1c
2.80.2c 22:2j
3.30.3a 2.80.2a
1.60.1b 1.50.1b
S NMID 8.40.4a
7.80.6a 5.10.2b
5.00.2b 18:0DMA
11.40.4b 13.10.4a
13.40.4a 12.60.9ab
S DMA 13.50.5a
15.50.5b 15.20.5b
14.70.6ab
Ž .
Ž .
Ž 22:6 n y 3 Em2 , a similar increase 15 of both fatty acids compared to spat fed
. solely T. suecica was observed in the neutral lipids of C. gigas whereas a clear
Ž .
preferential incorporation 17 vs. 3 of 22:6 n y 3 compared to 18:2 n y 6 occurred Ž
. in the polar lipid fraction Tables 2 and 3 . Supplementing similar amounts of 18:1n y 9
Ž .
Ž . Ž
. 18 and 22:6 n y 3 16
Em1 resulted in a significant increase the percentage of Ž
22:6 n y 3 in the polar and neutral lipids of Algae q Em1-fed spat compared to spat fed .
solely T. suecica while a significant augmentation of the percentage of 18:1n y 9 only Ž
. took place in the neutral lipid fraction Tables 2 and 3 .
When the fatty acid composition is expressed as percentage of the total fatty acids Ž
and DMA, a strong increase of one fatty acid such as the percentage of 18:1n y 9, .
18:2 n y 6 and especially 22:6 n y 3 in emulsion-fed oysters inevitably results in a reduction of the percentage of other fatty acids. This explains why, e.g., the percentage
Ž of 16:0 in total lipids of emulsion-fed oysters decreased 15.4, 12.4 and 12.1 in
. Algae, Algae q Em1 and Algae q Em2-fed oysters, respectively
even though the Ž
y1
. Ž
. concentration mg g DW
of 16:0 increased Table 4 . The supplementation of Em1
Table 4 Ž
. Ž
. Effect of feeding 1 T. suecica Algae , whether or not supplemented with liposomes AlgaeqLipo ,
Ž .
Ž .
Ž emulsion 1 AlgaeqEm1 or emulsion 2 AlgaeqEm2 on the fatty acid composition expressed as mg per g
. Ž
. dry weight of the total lipids of C. gigas spat. In each row, mean valuesSD ns 3 followed by the same
Ž .
letter are not significantly different ANOVA, Tukey HSD test, P F 0.05 Fatty acids and DMA
Total lipids Algae
AlgaeqLipo AlgaeqEm1
AlgaeqEm2 16:0
24514d 31615c
4875a 41517b
18:0 724c
846c 1524a
1247b S SAFA
35212d 4219c
7024a 59530b
16:1ny7 173c
224abc 273a
243abc 18:1ny9
1082d 13615c
5177a 38012b
18:1ny7 775c
928b 1141a
1064ab 20:1ny9
552b 412c
793a 718a
20:1ny7 323c
294c 544a
435b S MUFA
3593.4c 38024c
87725a 69132b
18:2 ny6 424d
13410c 5995a
38115b 20:2 ny6
71b 142b
386a 395a
20:4ny6 351ab
403a 313b
334ab S ny6 PUFA
954d 19710c
67118a 46017b
18:3ny3 857c
819c 1625a
1074b 18:4ny3
345ab 332ab
422a 31.75b
20:5ny3 26019b
26720b 31520a
29820ab 22:6 ny3
445d 28323c
55243b 72618a
S ny3 PUFA 48013c
71345b 115132a
122830a S ny3r ny6
50a 40b
20d 30c
22:2i 6310
645 626
622 22:2j
484ab 577a
5010ab 372b
S NMID 1769a
14412bc 1503b
1235c 18:0DMA
1128.6c 20011b
24121a 2086ab
S DMA 1148c
21511b 29830a
2584a
Ž or Em2 also significantly reduced the percentage of 20:4 n y 6 from 2.2 to 0.8 and
. Ž
. 1.0, respectively and 20:5n y 3 from 16.3 to 8.1 and 8.7, respectively in the
total lipids of C. gigas while the concentration of the latter two fatty acids was not Ž
. affected Table 4 . Similarly, the five-fold increase of the 20:2 n y 6 tissue concentration
Ž .
in emulsion-fed oysters Table 4 was limited to a two-fold increase in the percentage of Ž
. 20:2 n y 6 from 0.5 to 1.0 and 1.2, respectively .
3.4. Fatty acid composition of liposome-fed spat Although the concentration of 22:6 n y 3 was similar in Em1 and the liposomes
Ž
y1
. 152.6 and 157.8 mg g
, the 22:6 n y 3 concentration was twice as high in Algae q Ž
y1
. Ž
y1
. Em1-fed oysters 552 mg g
than in Algae q Lipo-fed oysters 283 mg g . The
supplementation of liposomes caused a significant increase of the percentage of 18:2 n y 6 in the polar and especially the neutral lipids compared to oysters fed only algae but it
Ž .
Ž . Ž .
Fig. 1. Evolution of the percentage of the total fatty acids and DMA of 22:6 ny3 A , 20:5ny3 B , Ž .
Ž . Ž
. 18:1ny9 C and 18:2 ny6 D in the total lipids of C. gigas spat fed T. suecica Algae , whether or not
Ž .
Ž .
Ž .
supplemented with liposomes AlgaeqLipo , emulsion 1 AlgaeqEm1 or emulsion 2 AlgaeqEm2 . Data represent the mean of three replicates, SD never exceeded 10 of the mean and were omitted for clarity.
did not clearly reflect the abundance of 18:2 n y 6 in the liposomes. Feeding Em1 Ž
. 44.1 18:2 n y 6 , resulted in a much higher percentage of 18:2 n y 6 in the polar
Ž .
Ž .
Ž .
7.2 and neutral 22.0 lipids of C. gigas than feeding liposomes 49.9 18:2 n y 6 Ž
. 3.8 and 8.8 18:2 n y 6 in the polar and neutral lipids, respectively . Similarly,
Ž
y1
. despite the higher 18:2 n y 6 concentration in the liposomes 380 mg g
than in Em2 Ž
y1
.
y1
317 mg g , spat fed the latter supplement contained 599 mg g
of 18:2 n y 6 compared to 134 mg g
y1
in Algae q Lipo-fed oysters. 3.5. Growth and surÕiÕal
No mortality was observed during the experimental period of 4 weeks. The supple- mentation of liposomes caused a significant increase in AFDW per oyster but DW and
Ž .
ash content were similar as for oysters fed only T. suecica Table 5 . The impact of both emulsions on oyster growth and ash content was similar. Both lipid emulsions resulted
in a significantly higher DW and AFDW per oyster whereas the ash content was
Table 5 Ž
. Ž
. Effect of algal and lipid supplemented diets on the dry weight DW , ash free dry weight AFDW and ash
Ž .
Ž .
content of the DW of C. gigas spat after 4 weeks. Values meanSD from three replicates indicated Ž
. with the same letter in one row are not significantly different ANOVA, Tukey HSD test, P F 0.05
Initial Algae
AlgaeqLipo AlgaeqEm1
AlgaeqEm2 Ž
. DWroyster mg
4.30.2 11.90.9b
12.70.9b 18.21.8a
17.21.0a Ž
. AFDWroyster mg
26510 69422c
85156b 136254a
128583a Ž .
AshrDW 94.60.2
94.50.3a 93.90.1a
92.90.5b 93.00.1b
significantly lower than in spat fed T. suecica whether or not supplemented with Ž
. liposomes Table 5 .
4. Discussion
