acids. This could have been related to the small size of the liposomes vs. an emulsion. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Fatty acid; Lipid; Emulsion; Liposomes; Oyster; Crassostrea gigas
1. Introduction
Unlike in fish and shrimp lipid nutrition studies, where the use of formulated diets allows the preparation of diets with a specific amount of virtually all important fatty
acids, bivalve studies are mainly performed by feeding different algal diets selected on the basis of their species-dependent fatty acid composition. Although fatty acid composi-
tions of algae can be modified by altering culture conditions, nutritional studies are
Ž compromised by this approach relative to formulated diets
Enright et al., 1986; .
Thompson and Harrison, 1992; Thompson et al., 1993 . Bivalve nutrition studies have Ž
. mainly focussed on n y 3 polyunsaturated fatty acids PUFA whereas the n y 6 PUFA
have received very little attention. Moreover, with a few exceptions, they only report the impact of the diet on the fatty acid composition of the total lipids. However, considering
Ž .
Ž the different function of the polar mainly structural phospholipids and neutral mainly
. storage triglycerides lipids, it is important to consider both lipid fractions.
Compared to emulsions, liposomes have the advantage that they can be used as an encapsulation technique for lipid as well as water-soluble components. We opted to use
the pro-liposome technique since pro-liposomes are a stable product that can easily be transported and the conversion into discrete liposomes and the loading with active
Ž .
ingredients can be carried out locally using simple equipment Arnaud, 1993 . There- fore, the pro-liposome technique holds promise for application in laboratory as well as
commercial hatcheries. Although pro-liposomes have been successfully used to enrich Ž
. Artemia with vitamin C Merchie, 1995 , they have not been used for bivalves.
Ž .
In the present study, lipid emulsions were used to supply n y 6 18:2 n y 6 and Ž
. Ž
. n y 3 PUFA 22:6 n y 3 to an algal diet Tetraselmis suecica containing only 2.6
18:2 n y 6 and no 22:6 n y 3. The fatty acids 18:2 n y 6 and 22:6 n y 3 were supplied as liposomes or emulsions and the partitioning of these fatty acids between the polar and
Ž .
neutral lipids of Crassostrea gigas Thunberg spat were followed. Additionally, the efficiency of emulsions and liposomes as fatty acid carriers for C. gigas spat were
Ž compared. Previous studies indicated that ethyl esters of docosahexaenoic acid DHA,
. 22:6 n y 3 can be used to illustrate the digestion and assimilation of lipid supplements
since 22:6 n y 3, supplied as ethyl esters, was clearly accumulated by larvae, spat and Ž
adults of various bivalve species Coutteau et al., 1994, 1996; Caers et al., 1998, .
Ž .
1999a,c . Therefore, pro-liposomes based on soybean phosphatidylcholine and emul- Ž
. sions based on soybean triglycerides were enriched with 22:6 n y 3 ethyl esters.
2. Materials and methods
2.1. Diets T. suecica was cultured in 4-l bottles in 0.22-mm filtered seawater enriched with
Ž Walne medium at a temperature of 258C under constant illumination Coutteau, 1996;
. Caers et al., 1998 . They were harvested daily in the exponential growth phase and
counted with a haemocytometer. Weekly, samples were removed for lipid analysis. Ž
The experimental ICES emulsions Em1 and Em2 ICES, International Council for the .
Exploration of the Sea prepared by INVE Technologies, Baasrode, Belgium, contained Ž
. 50 lipid soybean oil and 22:6 n y 3 ethyl esters, Itochu-Chemicals, Japan on a wet
Ž .
Ž weight WW basis, water, emulsifiers, antioxidants, preservatives and vitamin E 0.1
. Ž .
of the total lipids ICES, 1997 . The size distribution of the lipid emulsions was
determined with a Malvern Mastersizer S long bed with an automated sample dispersion Ž
. Ž
. unit MSX-17 , 300 RF lens, optic model olive oil in water, 3NAD : Particle R.I.s
Ž .
1.4564, 0.0000 , Dispersant R.I.s 1.3300. The particle size of both emulsions was log-normally distributed with 50 between 1 and 10 mm.
Ž .
Pro-liposomes Lucas Meyer France, Chelles Cedex, France mainly consisted of a mixture of phospholipid, principally unsaturated soybean phosphatidylcholine and small
quantities of charged lipids, dispersed in aqueous ethanol. A mixture of 22:6 n y 3 ethyl esters, vitamin E and ethoxyquin were mixed with the pro-liposome gel. The conversion
into a fully loaded concentrated liposome mixture was done by gradually adding a 3 aqueous NaCl solution. This resulted in liposomes with a lipid content of 9.8 and a
vitamin E and ethoxyquin concentration of 0.1 and 0.88 of the total lipids,
Ž .
respectively. The fatty acid composition of T. suecica n s 4 and the lipid supplements Ž
. n s 3 are given in Table 1.
2.2. Culture conditions and experimental design Ž
. Pacific oyster spat
C. gigas were obtained from Guernsey Sea Farms, UK in
February 1996. The system for rearing spat consisted of 12 aquaria placed in a water bath at 218C. Each 5-l aquarium contained one down-welling silo and one point aeration
to keep the food in suspension. The spat were fed twice a day for 4 weeks, the water Ž
. was renewed three times per week Caers et al., 1998 . T. suecica was fed at a
Ž . Ž
Ž .
Ž .
. weight-specific feeding ration FR
dry weight DW per wet weight WW per day
y1 y1
Ž .
of 1 DW WW day
. The algal diet was fed alone Algae or supplemented with Ž
. Ž
. Ž
. liposomes Algae q Lipo , emulsion 1 Algae q Em1 or emulsion 2 Algae q Em2 at a
Ž .
concentration of 50 lipid expressed as percentage of total algal DW . All treatments were run in triplicate aquaria. The spat were stocked at an initial concentration of 0.75 g
WW per silo and every week were rinsed with tap water, blotted dry with paper towel, Ž
. weighed and restocked at 0.75 g per silo WW . The amount of food was adjusted daily
i
Ž .
to keep the FR constant as previously described in Caers et al. 1999c . 2.3. Biochemical analysis
Before sampling, spat were starved for 24 h to empty their guts and the number of oysters in each silo was counted. The total weight of the freeze-dried oysters of each silo
was determined after which the oysters were pulverised in a mortar. For each silo, three Ž
. Ž
subsamples of the freeze-dried powder were used for DW 24 h at 608C and ash 24 h
Table 1 Ž
. Ž
. Ž
. Fatty acid composition Tetraselmis suecica Algae , liposomes Lipo and emulsions 1 Em1 and emulsion 2
Ž . Ž
y1
. Em2
, expressed as of the total fatty acids; mg g , expressed as mg fatty acids per g lipid . Data
Ž .
Ž .
Ž represent mean valuesSD standard deviation of four T. suecica and three replicates liposomes and
. emulsions
Ž . Fatty acid
Algae Lipo
Em1 Em2
y1 y1
y1
mg g mg g
mg g 16:0
23.81.1 10.90.2
82.82.1 9.20.5
92.911.7 7.00.1
65.61.0 18:0
3.50.1 2.70.0
20.70.5 3.40.1
33.71.4 2.70.0
25.20.2 S SAFA
28.01.1 14.00.2
1062.4 13.40.3
135.313.4 10.40.1
97.71.3 16:1ny7
2.00.1 tr
1.10.1 tr
1.10.2 tr
tr 18:1ny9
11.70.6 6.80.2
51.71.4 18.10.2
182.212.5 14.50.1
135.91.2 18:1ny7
3.40.2 1.20.0
9.40.3 1.40.4
14.85.1 0.90.1
8.80.8 20:1ny9
0.80.1 tr
tr tr
2.00.3 tr
1.20.1
U
20:1ny7 nd
nd nd
tr tr
tr tr
S MUFA 18.80.5
8.40.3 63.72.1
20.40.2 205.217.6
16.10.1 150.71.1
18:2 ny6 2.60.6
49.90.7 379.89.7
44.10.8 444.141.3
33.80.0 3170.9
UU
20:2 ny6 tr
0.50.3 3.72.4
tr tr
tr tr
20:4 ny6 0.80.1
nd nd
nd nd
nd nd
S ny6 PUFA 4.10.3
50.50.4 383.99.3
44.20.8 445.141.3
33.90.0 317.80.9
18:3ny3 15.40.6
5.80.0 44.20.9
5.30.0 53.14.0
4.00.1 37.81.0
18:4 ny3 8.60.5
tr tr
tr tr
tr tr
20:5ny3 6.80.3
1.00.1 7.90.7
0.70.0 7.00.2
1.40.0 13.10.1
22:6 ny3 nd
20.10.3 152.61.4
15.71.2 157.80.1
33.90.1 318.51.6
S ny3 PUFA 42.51.1
27.00.2 205.32.2
21.71.2 218.03.9
39.40.1 369.439.4
S ny3r ny6 5.60.4
0.50.0 0.50.0
0.50.0 0.50.0
1.20.0 1.20.0
U
Not detected.
UU
Ž
y1
. Trace F 0.5 or 1 mg g
.
. at 4508C determinations, the rest of the powder was used for lipid extraction. The ash
Ž .
free dry weight AFDW defines the organic matter content of the spat and was calculated as the difference between DW and ash and expressed as percentage of the
DW. Total lipids of freeze-dried and pulverised samples of the diets and oysters were
Ž .
extracted with chloroform and methanol 2:1, vrv and determined gravimetrically. Ž
. Ž
. Fatty acid methyl esters FAME and dimethyl acetals DMA of total lipid were
Ž .
prepared by transmethylation with a mixture of sulfuric acid and methanol 1:100, vrv for 16 h at 508C, using 22:4 n y 6 as the internal standard. Neutral and polar lipids were
Ž .
separated on a silicic acid column according to the method of Marty et al. 1992 . The Ž
fatty acid and DMA compositions were determined as previously described Caers et al., .
1998, 1999a,c . 2.4. Statistical analysis
Ž .
Statistical analysis included one way analysis of variance ANOVA and Tukey’s Ž
. honest significant difference test Tukey HSD test using the software-program
STATIS
-
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
