Marine lipids
water under continuous aeration. The sea water inlets of the raceways
were located at the opposite end of the drain outlets. Oysters placed
in the raceway R 1 were not fed with Skeletonema costatum and were therefore used as controls. Oysters placed in the raceway R2 were fed with
Skeletonema costatum at a daily rate of 2x10 9 cells per oyster. Temperature, salinity and oxygen content of the sea water were measured daily or every second day in each raceway. These parameters were kept
relatively constant during the experimental period (respectively 12.0
± 0.6°C, 32.0 ± 1.1 %o and 9.9 ± 4.4%). Skeletonema costatum content in
each raceway was estimated twice per week by measuring chlorophyll a
and phaeopigment levels as described by Baud et al. (1990). As expected,
these values were approximately twenty time higher in raceway R2
than in the control raceway (Rl) respectively 39.2 ± 10.4 µg.l -1 versus 1.8 ± 2.2 µg.l -1 of chlorophyll a and 24.1 ± 4.0 µg.l -1 versus 2.3
± 2.5 µg.l -1 of phaeopigment. Oysters were sampled for chemical
analysis at time zero (W0) and after one (Wl), two (W2), three (W3),
four (W4) and six (W6) weeks of the experiment. Following sampling,
but before analysis, oysters were starved for 24 h to clear the gut of
residual food.
Chemical analysis
Glycogen content was determined as described by Dubois et al. (1956).
The analyses were conducted using a sample size of 30 freeze-dried
oysters.
Lipid content: flesh of 12 fresh oysters was grounded using an UltraTurrax homogenizer. Lipid extraction was carried out in triplicate on
10 g of the mixture according to the procedure of Folch et al. (1957).
The extraction solution of chloroform-methanol (2:1, v/v) containecl
0.005% BHT (butyl hydroxy toluene) to prevent lipid oxidation. The
total lipid content of the oysters was estimated by gravimetry after the
solvent was evaporated under vacuum. A similar procedure was used
for lipid analysis of Skeletonema costatum.
Fatty acids composition: total lipid extracts were transmethylated using
boron trifluoride-methanol (14%) as described by Morrison & Smith
(1964). The resulting fatty acid methyl esters (FAMEs) were separated by
gas-liquid chromatography (GLC) using a HP 5890 series II chromatograph equipped with a split injector and a flame ionisation detector
(FID). The capillary column was 30 m length, 0.32 pm i.d. and 0.25 pm
film thickness. Helium was used as carrier gas with the head pressure
set at 15 pounds per square inch (psi). The temperatures of the injector
and the detector were set at 250°C and 280°C, respectively. The oven
temperature program involved an initial hold at 150°C for three minutes,
followed by an increase from 150°C to 180 °C at rate of 10°C/min, then
held at 180°C for seven minutes, followed by another increase to 215°C
at rate of 5 C min, before a final hold at 215°C for 15 minutes.
114
water under continuous aeration. The sea water inlets of the raceways
were located at the opposite end of the drain outlets. Oysters placed
in the raceway R 1 were not fed with Skeletonema costatum and were therefore used as controls. Oysters placed in the raceway R2 were fed with
Skeletonema costatum at a daily rate of 2x10 9 cells per oyster. Temperature, salinity and oxygen content of the sea water were measured daily or every second day in each raceway. These parameters were kept
relatively constant during the experimental period (respectively 12.0
± 0.6°C, 32.0 ± 1.1 %o and 9.9 ± 4.4%). Skeletonema costatum content in
each raceway was estimated twice per week by measuring chlorophyll a
and phaeopigment levels as described by Baud et al. (1990). As expected,
these values were approximately twenty time higher in raceway R2
than in the control raceway (Rl) respectively 39.2 ± 10.4 µg.l -1 versus 1.8 ± 2.2 µg.l -1 of chlorophyll a and 24.1 ± 4.0 µg.l -1 versus 2.3
± 2.5 µg.l -1 of phaeopigment. Oysters were sampled for chemical
analysis at time zero (W0) and after one (Wl), two (W2), three (W3),
four (W4) and six (W6) weeks of the experiment. Following sampling,
but before analysis, oysters were starved for 24 h to clear the gut of
residual food.
Chemical analysis
Glycogen content was determined as described by Dubois et al. (1956).
The analyses were conducted using a sample size of 30 freeze-dried
oysters.
Lipid content: flesh of 12 fresh oysters was grounded using an UltraTurrax homogenizer. Lipid extraction was carried out in triplicate on
10 g of the mixture according to the procedure of Folch et al. (1957).
The extraction solution of chloroform-methanol (2:1, v/v) containecl
0.005% BHT (butyl hydroxy toluene) to prevent lipid oxidation. The
total lipid content of the oysters was estimated by gravimetry after the
solvent was evaporated under vacuum. A similar procedure was used
for lipid analysis of Skeletonema costatum.
Fatty acids composition: total lipid extracts were transmethylated using
boron trifluoride-methanol (14%) as described by Morrison & Smith
(1964). The resulting fatty acid methyl esters (FAMEs) were separated by
gas-liquid chromatography (GLC) using a HP 5890 series II chromatograph equipped with a split injector and a flame ionisation detector
(FID). The capillary column was 30 m length, 0.32 pm i.d. and 0.25 pm
film thickness. Helium was used as carrier gas with the head pressure
set at 15 pounds per square inch (psi). The temperatures of the injector
and the detector were set at 250°C and 280°C, respectively. The oven
temperature program involved an initial hold at 150°C for three minutes,
followed by an increase from 150°C to 180 °C at rate of 10°C/min, then
held at 180°C for seven minutes, followed by another increase to 215°C
at rate of 5 C min, before a final hold at 215°C for 15 minutes.
114