Marine Biology (2002) 140: 1157–1166
DOI 10.1007/s00227-002-0798-5
M. Caers Æ S.D. Utting Æ P. Coutteau Æ P.F. Millican
P. Sorgeloos
Impact of the supplementation of a docosahexaenoic acid–rich
emulsion on the reproductive output of oyster broodstock,
Crassostrea gigas
Received: 28 February 2001 / Accepted: 7 January 2002 / Published online: 29 March 2002

Springer-Verlag 2002
Abstract Pacific oyster [Crassostrea gigas (Thunberg)]
broodstock were hatchery-conditioned with either a
mixed algal diet of Dunaliella tertiolecta, Tetraselmis
suecica and Rhodomonas sp. [1:1:1, on a dry weight
(DW) basis] (Control), a single diet of D. tertiolecta
(Duna) or a single diet of D. tertiolecta supplemented
with an emulsion rich in eicosapentaenoic (20:5n-3) and
particularly docosahexaenoic acid (22:6n-3) (Du-
na+Em). Oysters were spawned after a conditioning
period of 7 and 8 weeks and larvae were reared for
1 week on an Isochrysis galbana (clone T-Iso) diet. The
percentage recovery of D-larvae from eggs of Control-
fed oysters and Duna+Em-fed oysters was not signifi-
cantly different (80% and 63% after 7 weeks of
conditioning; 87% and 74% after 8 weeks of condi-
tioning, respectively] but was much higher than from
eggs produced by Duna-fed oysters (24% and 41% after
7 and 8 weeks of conditioning, respectively). A reduc-
tion in the percentage recovery of D-larvae as a result of
temperature or salinity stress was most severe in eggs
from Duna-fed oysters. Neither the broodstock diet nor
the conditioning period affected the size of eggs or
D-larvae, larval performance, or lipid content and lipid
Communicated by O. Kinne, Oldendorf/Luhe
M. Caers (&) Æ P. Coutteau Æ P. Sorgeloos
Laboratory of Aquaculture & Artemia Reference Center,
Ghent University, Rozier 44,
9000 Ghent, Belgium
E-mail: marrit.caers@belgacom.net
S.D. Utting Æ P.F. Millican
Centre for Environment,
Fisheries and Aquaculture Science,
Benarth Road, Conwy,
Gwynedd LL32 8UB, United Kingdom
Present address: S.D. Utting
Sea Fish Industry Authority,
PO Box 68, Colwyn Bay LL28 5WR, United Kingdom
Present address: P. Coutteau
INVE Technologies, Oeverstraat 7,
9200 Baasrode, Belgium
class distribution of the eggs. The fatty acid composition
of the eggs was modified by the fatty acid composition of
the algal diet and the lipid emulsion. The supplementa-
tion of the emulsion resulted in a pronounced increase in
the percentage of 22:6n-3 in the polar (PL) and neutral
lipids (NL) of the eggs (4.0–13.4% and 8.5–14.7% in NL
and PL, respectively). The egg lipids (7–8 ng egg–1)
contained about 62% NL and were dominated by tri-
glycerides (TAG), which made up 65% of the NL. The
major phospholipids were phosphatidylethanolamine
(PE), ceramide aminoethylphosphonate and phosphat-
idylinositol (CAEP+PI), phosphatidylserine (PS) and
phosphatidylcholine (PC). Regardless of the broodstock
diet treatment, the PL fraction contained high percent-
ages of non-methylene-interrupted dienes (NMID) and
plasmalogens (detected as dimethyl acetals).
Introduction
Under natural as well as hatchery conditions, the via-
bility and number of eggs produced by bivalves vary
considerably between individuals (Utting and Millican
1998), at different stages of the spawning season (Li-
povsky 1984; LePennec et al. 1998; Robert and Gérard
1999), between different bivalve species (LePennec et al.
1998; Robert and Gérard 1999), among different spaw-
nings of hatchery-conditioned broodstocks (Utting and
Millican 1997, 1998; LePennec et al. 1998; Robert and
Gérard 1999) and between different natural populations
of the same species (Thompson and MacDonald 1991).
These observations stimulated research to unravel the
number of parameters and endogenous regulation
mechanisms that control the process of gametogenesis.
Given that environmental parameters such as tem-
perature, salinity, photoperiod and overall water quality
fall within an acceptable range for a certain species,
broodstock nutrition has been considered as a vital
factor for successful reproduction. Several field studies
revealed that the fecundity of a given population is
strongly related to food availability encountered in its
1158
natural environment (Barber and Blake 1991), whereas
the addition of cultured microalgae usually improved
the fecundity and/or egg quality in hatchery-conditioned
animals (Utting and Millican 1997, 1998).
Successful embryogenesis has been positively corre-
lated with the endogenous lipid reserves laid down in the
eggs during vitellogenesis (Gallager and Mann 1986;
Devauchelle and Mingant 1991). Although information
is still scarce, there is accumulating evidence that the fatty
acid composition of the broodstock diet affects the fatty
acid composition and subsequent viability of the eggs.
Several authors have emphasised the importance of n-3
polyunsaturated fatty acids (PUFA), particularly the
amounts of 20:5n-3 and 22:6n-3, during embryo and
larval development (Whyte et al. 1990a,b; Thompson and
Harrison 1992; Soudant et al. 1996; Jonsson et al. 1999).
The approach used in the aforementioned studies was
to feed broodstock with different algal diets selected on
the basis of their particular fatty acid composition.
However, these diets differed not only in their fatty acid
composition but may also have differed in other pa-
rameters such as palatability, digestibility and bio-
chemical composition (Epifanio 1983; Webb and Chu
1983; Heasman et al. 1996). Earlier work with filter-
feeding organisms such as Daphnia (DeMott and Mül-
ler-Navarra 1997), Artemia (Coutteau and Mourente
1996; Coutteau and Sorgeloos 1997) and bivalves
(Coutteau et al. 1996; Caers et al. 1998, 1999a,b,
2000a,b) illustrated that lipid emulsions are a useful tool
to overcome such potential problems. In the present
study, Dunaliella tertiolecta, an algal diet which was
lacking the long-chain PUFAs 20:5n-3 and 22:6n-3, was
supplemented with an emulsion rich in the latter fatty
acids. Mixed algal diets have been recommended for
successful broodstock conditioning, since they provide a
wide range of nutritional components (lipids, carbohy-
drates, proteins, vitamins and other trace elements)
(Utting and Millican 1997). Therefore a mixed algal diet
of D. tertiolecta, Tetraselmis suecica and Rhodomonas sp.
[1:1:1, on a dry weight (DW) basis] was included as a
control diet (Control) in the present work.
The present study aimed to evaluate the impact of the
fatty acid composition of the parental diet on fecundity,
the lipid and fatty acid content and composition of the
eggs, embryogenesis success and subsequent larval per-
formance. The effect of egg quality on embryo devel-
opment was investigated under normal as well as stress
conditions (temperature and salinity extremes).
Materials and methods
Diets
Rhodomonas sp., Tetraselmis suecica and Dunaliella tertiolecta were
batch cultured in 60-l plastic bags in filtered seawater enriched with
Walne medium (Coutteau 1996). Cultures were harvested daily; cell
densities were determined with a Coulter Counter Model ZM. The
experimental ICES (International Council for the Exploration of
the Sea) emulsion contained 60% lipid (ethyl esters) on a wet
weight (WW) basis, water, emulsifiers, antioxidants, preservatives
and lipid soluble vitamins (A, C, D and E, respectively, 0.18%,
0.08%, 0.013% and 0.32% of WW) (Caers et al. 1999a,b). The
fatty acid composition of the diets is shown in Table 1.
Broodstock conditioning
Oysters [Crassostrea gigas (Thunberg)] were collected in the spring
of 1996 from Tal-y-foel in the Menai Straits, North Wales, UK, and
an initial sample of 25 oysters (shell width=57±8 mm, shell
height=103±22 mm, weight=69±17 g) was used to determine
the shell-free DW (1.63±0.49 g). The experimental rearing system
consisted of three broodstock tanks, each of 150 l, that contained
30 oysters held on a mesh (12 mm aperture) supported off the
bottom (Utting and Spencer 1991). Ambient salinity seawater (31–
33 ppt), heated to 20±1C, flowed to waste through the broodstock
tanks at a rate of 1.0 l min–1. The seawater was pumped from the
Conwy Estuary (5317¢N; 349¢W) into an outdoor concrete storage
tank. It was passed through a sand filter to remove the larger sus-
pended particles, including most of the phytoplankton and some
bacteria, before entering the hatchery (Utting and Doyou 1992).
Algae were held in three 40-l reservoirs above each broodstock
tank and dosed into the tank using a peristaltic pump. The daily
ration, in dry weight of algae, was equivalent to 6% of the initial
shell-free DW of the oysters (Utting and Millican 1997). The
control diet was a mixture of D. tertiolecta, T. suecica and Rho-
domonas sp. (1:1:1 on a DW basis) (Control). Oysters in the re-
maining two tanks were fed D. tertiolecta with (Duna+Em) or
without (Duna) the supplementation of a lipid emulsion at a con-
centration of 50% lipid per total DW of the algal diet (Caers et al.
1999a). Prior to use, the emulsion was diluted in 20 l of seawater,
gently aerated and held at a temperature of 6C. Like the algal
diets, it was continuously supplied via a peristaltic pump during a
period of approximately 8 h day–1.
Spawning and embryonic development
After 6, 7 and 8 weeks of conditioning, ten oysters from each
treatment were selected at random and spawned artificially using
standard techniques (Utting and Spencer 1991). Due to the limited
number of eggs from female oysters that were spawned after
6 weeks, only data for oysters conditioned for 7 and 8 weeks are
reported.
With the exception of the embryo stress tests, all seawater used
for spawning the broodstock and rearing the embryos and larvae
was at ambient salinity (31–33 ppt), filtered through a plate filter
coated with diatomaceous earth, UV-treated and heated to 25C.
Rearing tanks were held in a temperature-controlled room at 25C,
and 2 mg chloramphenicol l–1 and 1 mg EDTA l–1 were added
(Utting and Helm 1985; Utting and Spencer 1991).
For each oyster, eggs were artificially removed with a Pasteur
pipette and concentrated in a known volume of seawater. Then,
three subsamples were taken to measure the number of eggs and the
egg diameter using a Coulter Counter model ZM. For each
broodstock diet treatment, unfertilised eggs from a minimum of
three individual oysters were sampled for lipid analysis. The re-
maining eggs and all the eggs from females not used for lipid
analysis were pooled, concentrated in a known volume of seawater
and counted with a Coulter Counter model ZM (at least two
counts per sample). Pooled sperm, originating from all male oysters
from the same diet treatment, were used for fertilising the eggs. For
each diet treatment, two larval rearing bins (150 l) and five 30-ml
beakers were inoculated at a concentration of 50 eggs ml–1. After
24 h, all the D-larvae were collected from each bin on a 45 lm
sieve. They were concentrated in a known volume of seawater and
counted with a Coulter Counter model ZM. D-larvae from both
150-l vessels per diet treatment were pooled; three subsamples of
100 D-larvae were preserved in formalin for the measurement of
shell length. The remaining larvae were subsequently used in the
larval rearing test.
 1159

Embryo stress test
Embryo development was measured at 25C, 30C and 35C and
salinities of 20, 30 and 40 ppt (see Table 3). For each salinity–
temperature combination and for each of the three broodstock diet
treatments, five 30-ml beakers were inoculated with 50 eggs ml–1.
The beakers were placed in a thermoregulated water bath of the
appropriate temperature. Before the larvae were inoculated, salin-
ity was adjusted from ambient by the addition of NaCl or distilled
water. Twenty-four hours after fertilisation, larvae were killed with
a few drops of formalin and the number of normal D-larvae (i.e.
perfect D-shape) was counted under the microscope. D-larva yield
or percentage D-larva recovery was defined as the percentage of
eggs that developed into normal D-larvae within 24 h.
Larval rearing test
Triplicate samples of D-larvae per broodstock diet treatment were
inoculated into 2.5-l glass beakers containing 2 l seawater at an
initial concentration of 10 larvae ml–1. The water was renewed at
2-day intervals, and I. galbana (clone T-Iso) was added at a
concentration of 100 cells ll–1. After 7 days, the size and number of
larvae were determined. A minimum of 100 larvae were measured
per beaker.
Lipid and fatty acid analysis
Lipid analysis was carried out in triplicate on each sample
(one sample per oyster). Total lipids were extracted with
chloroform:methanol (2:1, v/v) as the solvent mixture and deter-
mined gravimetrically. Lipid classes were analysed using high-per-
formance thin-layer liquid chromatography (HPTLC) on HPTLC
plates (10·10 cm, silicagel 60, Merck, Germany). Lipids were
separated in a one-dimensional double development using methyl
acetate:isopropanol:chloroform:methanol:0.25% aqueous KCl (25:
25:25:10:9, v/v) and hexane:diethyl ether:acetic acid (80:20:2, v/v)
as a developing solvent mixture for polar (PL) and neutral lipid
(NL) classes, respectively (Henderson and Tocher 1992). For
quantification of the lipid classes, plates were sprayed with 3% (w/
v) cupric sulphate in 8% (v/v) phosphoric acid, charred at 160C
for 20 min and quantified with a Sharp JX-325 scanner supported
with ImageMaster software. Identification was done by using spe-
cific stain reagents, lipid standards and two-dimensional develop-
ment systems (Henderson and Tocher 1992). Since plasmalogen
and phosphonolipids are not separated from their corresponding
diacylphospholipid analogues, they were quantified as one group.
For clarity, only the name of the diacylphospholipids is used in the
presentation of the results and the discussion. Similarly, the frac-
tion named TAG includes triglycerides as well as alkyl and alkenyl
forms, since they were not separated by the applied neutral solvent
system.
Neutral and polar lipids were separated on a silicic acid
column according to the method of Marty et al. (1992). Fatty
acid methyl esters (FAME) were prepared by transmethylation
with a mixture of sulphuric acid and methanol (1:100, v/v) for
16 h at 50C, using 22:4n-6 as internal standard. FAME were
extracted with hexane:diethylether (1:1, v/v), washed with
KHCO3 and dissolved in iso-octane. During acid-catalysed
transmethylation, FAME are formed simultaneously with dim-
ethyl acetals (DMA) which originate from the 1-alkenyl chains of
plasmalogens. For the calculation of the percentage (expressed as
a percent of total fatty acids) of individual DMA and FAME,

Table 1. Fatty acid composi-

Fatty acid composition
tion (expressed as % of the total
fatty acids and mg g–1 dry wei- (%) (mg g–1 dry weight)
ght), total fatty acid content
(TFA) and total lipid content Control Duna Em Control Duna Em
(TL) of the diets (Control, Du-
naliella tertiolecta:Rhodomon- 14:0 4.0±0.2 1.1±0.1 tr 3.4±0.3 1.1±0.1 3.5±0.1
as sp.:Tetraselmis suecica, 1:1:1 16:0 15.4±0.1 16.7±0.2 9.1±0.5 13.1±0.6 17.0±0.9 76.7±2.8
on a dry weight basis; Duna, 18:0 0.5±0.1 0.9±0.3 6.7±0.5 0.4±0.1 1.0±0.3 56.3±3.2
D. tertiolecta; Em, lipid emul-
sion) used in broodstock con- SSAFA 21.0±0.1 21.1±0.6 17.3±1.3 17.9±0.7 21.4±1.5 146.6±8.2
ditioning trials with Crassostrea 16:1n-7 1.9±0.0 1.8±0.3 0.7±0.1 1.6±0.1 1.8±0.2 5.9±0.5
gigas. Data represent the aver- 18:1n-9 5.7±0.1 7.0±0.9 5.1±0.2 4.9±0.2 7.1±1.0 42.9±0.4
age of three replicate samples 18:1n-7 2.1±0.0 4.6±0.9 tr 1.8±0.1 4.6±0.8 4.0±0.3
(±SD) (SAFA, MUFA, PUFA 20:1n-9 tr n.d. tr tr n.d. 1.7±0.1
saturated, monounsaturated, 20:1n-7 n.d. n.d. n.d. n.d. n.d. n.d.
polyunsaturated fatty acids, SMUFA 13.4±0.2 14.9±1.2 6.8±0.2 11.4±0.4 15.1±0.8 56.7±1.0
respectively; tr trace, £ 0.5%; 18:2n-6 10.2±0.1 5.9±0.3 5.7±0.1 8.7±0.4 6.0±0.4 47.9±0.4
n.d. not determined) 20:2n-6 1.1±0.0 tr n.d. 1.0±0.1 tr n.d.
20:3n-6 n.d. n.d. n.d. n.d. n.d. n.d.
20:4n-6 1.1±0.0 n.d. 3.8±0.1 1.0±0.0 n.d. 32.1±1.5
22:5n-6 tr tr 2.5±0.1 tr tr 20.9±0.9
Sn-6 PUFA 16.5±0.2 11.9±0.2 12.5±0.1 14.1±0.7 12.1±0.7 104.8±3.0
16:4n-3 9.5±0.2 13.6±0.7 n.d. 8.1±0.4 13.8±0.3 n.d.
18:3n-3 19.5±0.3 29.5±1.2 0.9±0.1 16.6±0.6 30.0±1.9 7.3±0.3
18:4n-3 8.4±0.3 1.2±0.1 0.9±0.0 7.1±0.4 1.2±0.1 7.4±0.2
20:3n-3 tr n.d. n.d. tr n.d. n.d.
20:4n-3 tr n.d. tr tr n.d. 3.1±0.2
20:5n-3 4.4±0.3 n.d. 12.1±0.4 3.7±0.2 n.d. 101.9±5.1
21:5n-3 tr n.d. tr tr n.d. 2.1±0.2
22:5n-3 tr n.d. 2.0±0.0 tr n.d. 16.6±0.3
22:6n-3 2.5±0.2 tr 45.3±1.3 2.1±0.0 tr 380.4±18.6
Sn-3 PUFA 45.5±0.3 46.6±1.2 62.8±1.8 38.8±1.5 47.3±2.0 521.2±24.8
n-3/n-6 2.8±0.0 3.9±0.1 5.0±0.1
TFA 85.2±3.5 101.5±4.2 838.9±16.8
TL 182.8±2.2 180.1±10.2
1160

‘‘total fatty acids’’ refer to the sum of all DMA and FAME
present in the sample.
The FAME and DMA were analysed with a Chrom-
pack CP9001 Gas, equipped with an autosampler. Injection was
done on-column into a very polar 50 m capillary column, BPX70,
with a diameter of 0.32 mm and a layer thickness of 25 lm, con-
nected to a 2.5 m methyl-deactivated precolumn. The carrier gas
was H2, and the detection mode FID (flame ionisation detection).
The oven temperature was set to raise the initial temperature from
85C to 150C at a rate of 20C min)1, from 150C to 152C at
0.1C min)1, from 152C to 174C at 0.7C min)1, from 174C to
180C at 10C min)1 and to stay at 180C for 2 min. Individual
FAME and DMA were identified by reference to authentic stan-
dards (Nu-Check Prep., USA) and well-characterised shellfish and
fish oil samples, and injection into a gaschromatograph (Carlo
Erba) equipped with a non-polar column. Integrations and calcu-
lations were done with the software program ‘‘Maestro’’ (Chrom-
pack).
Dunaliella tertiolecta (Duna) of 20:5n-3 and 22:6n-3 and
any other n-3 or n-6 polyunsaturated fatty acids (PUFA)
with 20 or more carbon atoms. Although the percentage
of total n-3 PUFA was similar in both algal diets, the
Control diet contained less 18:3n-3 (19.5% and 29.5% in
Control and Duna, respectively) but more 18:4n-3 (8.4%
and 1.2% in Control and Duna, respectively) than the
Duna diet. The higher n-6 PUFA level of the Control diet
as compared to the Duna diet was mainly attributed to a
higher level of 18:2n-6. Although the total lipid content
(TL) of both algal diets was similar, the Duna diet showed
a higher total fatty acid content (TFA). The fatty acid
profile of the emulsion was dominated by the abundance
of 20:5n-3 and especially 22:6n-3, which accounted for
12.1% and 45.3%, respectively, of the total fatty acids.

Statistical analysis
Statistical analysis included one-way analysis of variance (ANO-
VA) and Tukey’s honest significant difference test (Tukey HSD-
test) using the software program STATISTICA (Statsoft, Tulsa,
Okla.). Homogeneity of the variances of means was checked by the
univariate test (Cohran’s C, Hartley, Barlett). Arcsine–square-root
transformation was used prior to statistical analyses of percentage
data (Sokal and Rohlf 1995).
Results
Diets
The percentage total saturated (SAFA) and monounsat-
urated (MUFA) fatty acids was the same for both algal
diets, although some minor differences between the indi-
vidual fatty acids were found (e.g. 14:0 and 18:1n-7)
(Table 1). The main difference between the fatty acid
composition of the two algal diets was the absence in
Biological parameters and stress test
Irrespective of the conditioning period, the percentage
recovery of D-larvae from eggs of broodstock fed solely
D. tertiolecta (Duna) was significantly lower than the
yield from eggs of broodstock fed the Control diet
(Table 2). Supplementation of D. tertiolecta with lipids
(Duna+Em) resulted in a significant increase in the
percentage recovery of D-larvae to a level which was
similar to that found with the Control diet (Table 2).
When embryogenesis occurred under superoptimal
temperature (30C) or salinity (20 ppt) conditions, the
percentage D-larva recovery was similarly dependent on
broodstock diet treatment (Table 3). The effect of in-
creasing the temperature from 25C to 30C on the
percentage D-larva recovery was similar to the effect of
decreasing the salinity from 30 to 20 ppt. Embryonic
development was virtually absent at 35C or at a salinity
of 40 ppt. Neither the broodstock diet nor the length of

Table 2. Crassostrea gigas.

Parameters Control Duna Duna+Em
Effect of the different brood-
stock diets (Control, Dunaliella
7 weeks of conditioning
tertiolecta:Rhodomonas sp.:
Number of female oysters/total 4/10 4/10 5/10
Tetraselmis suecica, 1:1:1 on a
number of oysters
dry weight basis; Duna,
Eggs per oyster (106)a 9.2±5.2 10.6±8.4 9.7±7.7
D. tertiolecta only; Duna+Em,
Egg diameter (lm) 51.9±1.3 53.1±0.5 52.5±0.7
D. tertiolecta with lipid emul-
Egg lipid content (ng egg–1) 7.4±1.0 7.8±0.7 8.1±1.7
sion) on the fecundity, egg
Size D-larvae (lm) 68.8±0.8 69.1±0.8 69.0±0.9
characteristics and larval
Percentage D-larvae recovery (n=5) 80.0±8.0a 24.4±2.6b 63.2±7.0a
performance of C. gigas. Mean
Larval growth (day 1–7) (lm day–1) 10.3±0.9 10.4±1.3 10.9±1.1
values within the same row
Larval survival (day 1–7) 67±9 67±8 67±11
followed by a different letter are
8 weeks of conditioning
significantly different (Tukey’s
Number of female oysters/total 5/10 5/10 7/10
HSD-test, P<0.05). Data rep-
number of oysters
resent the average±SD (n=3
Eggs per oyster (106)a 24.4±4.2 12.6±8.8 24.5±14.4
unless otherwise indicated)
Egg diameter (lm) 52.4±0.4 53.5±2.7 52.7±0.6
(DW dry weight)
Egg lipid content (ng egg–1) 7.3±1.1 7.1±0.9 7.6±0.9
Size D-larvae (lm) 68.4±0.5 68.3±0.4 69.2±0.7
Percentage D-larva recovery (n=5) 87.5±12.2a 41.1±3.0b 74.2±12.3a
Larval growth (day1–7) (lm day–1) 9.5±0.2 8.8±0.3 9.6±0.5
Larval survival (day 1–7) 71±15 63±9 70±11
a
Number of replicates used to calculate the SD is similar to the number of female oysters that produced
eggs

the conditioning period affected the size of eggs or
D-larvae (Table 2).
After 8 weeks of conditioning, fecundity of oysters
fed the Control or Duna+Em diet was twice as high as
for oysters fed the Duna diet, but no diet-related dif-
ferences were observed after a conditioning period of
7 weeks (Table 2). An increase of the conditioning pe-
riod from 7 to 8 weeks had no effect on the fecundity of
Duna-fed oysters, but D-larva recovery was twice as
high. On the contrary, fecundity of oysters fed the
Control or Duna+Em diet was twice as high after 8
compared to after 7 weeks, but the percentage D-larva
recovery remained similar (Table 2).
Neither the broodstock diet nor the length of the
conditioning period had a lasting effect on subsequent
larval development under standard hatchery conditions,
as size and survival of 1-week-old larvae were similar
regardless of the broodstock diet or the length of con-
ditioning.
Lipid composition and content of the eggs
The NLs were around 65% of the total egg lipids and
were dominated by TAG (65% of the NL) and SE
(15%). The remaining 20% of the NLs included FFA,
FS+DAG and MAG+Pig (Table 4, see table also for
lipid class abbreviations). Quantitatively, the most im-
portant PL classes were PC and PE+UN, each repre-
senting about 30% of the PL. The level of PI+CAEP,
which accounted for a quarter of the total PL, was twice
as high as the PS level. Except for GLY, a minor gly-
colipid which represented only 2% of the PL, neither the
broodstock diet nor the length of the conditioning pe-
riod had an effect on the relative lipid class distribution
or lipid content of the oyster eggs (Table 4).
Comparison of the fatty acid composition of the polar
versus neutral lipids of the eggs
The fatty acid composition of the neutral and polar lipid
fraction of eggs originating from oyster broodstock fed
during 8 weeks on three different diets is shown in
Table 5. The total percentages of SAFA and MUFA
Table 3. Crassostrea gigas. Effect of salinity (in ppt) and temper-
ature (C) stress on the percentage D-larva recovery from eggs
originating from C. gigas broodstock (experiment 2) fed three
different diets (Control, Dunaliella tertiolecta:Rhodomon-
as sp.:Tetraselmis suecica, 1:1:1 on a dry weight basis; Duna,
Diet D-larva recovery (%) at:
25C
20 ppt 30 ppt 40 ppt
Control 53.8±5.4a 87.5±12.2a 0 47.3±2.0a
Duna 11.9±0.9b 41.1±3.0b 0 12.3±1.6b
Duna+Em 45.4±3.0a 74.2±12.3a 0 39.2±5.8a
1161
were much lower in the PL than in the NL. Since the
lower percentage of 18:2n-6 in the PL was mainly com-
pensated by an increase of the percentage of 20:4n-6, the
percentage of total n-6 PUFA was not different from the
level in the NL. Similarly, no differences were found
between the total n-3 PUFA of both lipid fractions, but
there was a reduction in the 18:3n-3 level in the PL
compared to the NL in each treatment. Another feature
of the PL was the abundance of non-methylene-inter-
rupted dienes (NMID) and plasmalogens (detected as
dimethylacetals).
Impact of the algal diets on the fatty acid composition
of the eggs
Despite the high algal 16:4n-3 supply (Table 1), this fatty
acid was not accumulated in the NL or PL of the eggs
(Table 5). By comparison, the abundance of 18:3n-3 in
the Control (19.5%) and especially the Duna (29.5%)
diet was clearly reflected in the fatty acid profile (PL and
NL) of the eggs. Eggs from oysters fed the algal diet
most rich in 18:4n-3 and 18:2n-6 (Control) showed the
highest level of these fatty acids in the NL, but the effect
was less pronounced than for 18:3n-3, and no dietary
effects were observed in the PL fraction. Although 20:5n-
3 was absent in D. tertiolecta, its level in the PL fraction
of the eggs was similar to that of the Control treatment
(4.4% 20:5n-3 in the diet). However, the percentage of
20:5n-3 was nearly twice as high in the NL of eggs from
Control-fed broodstock (9.4%) in comparison to eggs
from Duna-fed broodstock (5.3%). The lack of 22:6n-3
in D. tertiolecta resulted in lower percentages of 22:6n-3
in the NL (4.0% versus 6.8%) and the PL (8.5% versus
11.3%) of the eggs compared to the eggs spawned by
oysters fed the Control diet.
Impact of lipid supplementation on the fatty acid
composition of the eggs
The high level of 22:6n-3 in the lipid emulsion (45.3%)
was clearly reflected in the fatty acid composition of the
eggs, since its level increased from 4.0% to 13.4% and
from 8.5% to 14.7% in the NL and PL, respectively.
D. tertiolecta only; Duna+Em, D. tertiolecta with lipid emulsion).
Mean values within the same column followed by a different letter
are significantly different (Tukey’s HSD-test, P<0.05). Data
represent the average of five replicate samples (±SD)
30C 35C
30 ppt 40 ppt 30 ppt 40 ppt
0 1.1±0.4 0
0 1.3±0.4 0
0 1.2±0.2 0
1162

Although the emulsion also contained 12.1% 20:5n-3, its
level in the PL of the eggs was not affected, while the
percentage 20:5n-3 increased from 5.3% to 7.1% in the
NL fraction. Similarly, an increase in the percentage of
18:1n-9 and 18:2n-6 in eggs from oysters fed Duna+Em
as compared to eggs from oysters fed the non-supple-
mented Duna diet was noted in the NL but not in the PL
fraction of the eggs. Some PUFAs such as 20:4n-6,
22:5n-6 and 22:5n-3, representing <4% of the total fatty
acids of the emulsion, were found in higher amounts in
the NL as well as in the PL of the eggs from oysters fed
the lipid-supplemented diet (Duna+Em) as compared
to oysters fed solely D. tertiolecta (Duna).
Discussion
After 8 weeks of conditioning, fecundity of Duna+Em-
fed oysters was much higher than that of Duna-fed
broodstock. No such effect was seen in oysters spawned
1 week earlier, but the number of eggs produced in
week 7 indicated that oysters were still not fully condi-
tioned at that stage. The supplementation of emulsified
or encapsulated lipids also tended to enhance matura-
tion/fecundity in Argopecten purpuratus and Ostrea ed-
ulis broodstock (Lane 1989; Caers et al. 1999a; Martı́nez
et al. 2000), but hardly affected fecundity in Crassostrea
virginica (Robinson 1992a). This is not surprising since
data on the value of different algal species for brood-
stock conditioning can also be contradictory. For ex-
ample, the composition of the algal diet affected the
number of newly released O. edulis and Ostrea chilensis
larvae (Millican and Helm 1994; Wilson et al. 1996;
Berntsson et al. 1997), but in other trials had no clear
impact on the fecundity of clams and oysters (Laing and
Lopez-Alvarado 1994; Utting and Millican 1998). Many
more data are needed to prove conclusively that specific
dietary components can promote fecundity, especially
since studies are hampered by the high variability in
fecundity between individuals and the often limited
number of spawners per diet treatment.
Evidence from the present study and from earlier
work (Laing and Lopez-Alvarado 1994; Soudant et al.
1996) showed that egg lipid content was not related to
the broodstock diet. This supports the hypothesis of
Utting and Millican (1997) that bivalves adjust their
fecundity in order to maintain a consistent lipid level in
the eggs. However, Gallager and Mann (1986) observed
large variations in the lipid contents of clam and oyster
eggs of different batches, and the maturation diet has
been shown to influence the lipid level in O. chilensis
(Wilson et al. 1996) and A. purpuratus (Caers et al.
1999a).
The average lipid content per egg (7–8 ng egg–1;
present study) falls within the range reported by Gal-
lager et al. (1986) for C. virginica eggs (4.3–9.2 ng egg–1),
but was considerably higher than the 2.5 ng lipid per
C. virginica egg reported by Lee and Heffernan (1991).
The percentage of TAG, approximately 40% of the TL,
was comparable with the data reported for Mercenaria
mercenaria and C. virginica (Gallager et al. 1986), but
the percentages of sterol (9% of TL) and SE+DAG
(5% of TL) were more than double those in the latter
two species. It has been shown that TAG, which are
located in lipid droplets dispersed throughout the eggs,
are the predominant fuel during embryogenesis (Gal-

Table 4. Crassostrea gigas. Impact of the broodstock diet on the lipid class composition of the neutral and polar lipids of
(Control, Dunaliella tertiolecta:Rhodomonas sp.:Tetraselmis sueci- C. gigas eggs. Data represent the average of three replicate samples
ca, 1:1:1 on a dry weight basis; Duna, D. tertiolecta only; (±SD) (TL total lipid)
Duna+Em, D. tertiolecta with lipid emulsion), fed during 8 weeks,

Lipid class Abbrev. 7 weeks of conditioning 8 weeks of conditioning

Control Duna Duna+Em Control Duna Duna+Em

NL/PL 1.7±0.1 1.7±0.1 1.7±0.1 1.7±0.2 1.9±0.1 1.8±0.1

Neutral lipid (% of TL) NL 62.6±1.1 63.2±1.6 62.9±1.0 63.0±2.4 65.0±1.2 64.1±1.4
Sterolesters SE 15.3±2.1 15.7±1.3 15.4±1.1 15.5±0.7 15.7±2.0 15.7±1.0
Triglycerides TAG 64.9±2.9 64.6±2.0 64.2±1.9 65.6±0.9 64.0±3.1 66.0±0.7
Free fatty acids FFA 4.4±0.4 4.8±1.1 4.7±1.0 4.1±0.6 4.7±0.8 4.1±0.3
Free sterols+ FS+DAG 8.0±0.6 7.8±0.6 8.7±1.0 8.7±0.3 8.9±1.1 7.5±0.4
diglycerides
Monoglycerides+ MAG+Pig 7.3±0.5 7.1±0.3 7.0±0.3 6.1±0.6 6.7±0.4 6.7±0.5
pigments
Polar lipid (% of TL) PL 37.4±1.1 36.8±1.6 37.1±1.0 37.0±2.4 35.0±1.2 35.9±1.4
Glycophospholipids GLY 2.1±0.3 2.1±0.2 2.0±0.2 1.6±0.1 2.1±0.1 1.8±0.3
Phosphatidylethanol- PE+UN 32.4±1.6 31.9±1.6 31.0±2.0 32.6±1.6 32.3±1.4 32.8±1.5
amine+unknown
phospholipid
Phosphatidylinositol+ PI+CAEP 24.1±1.6 25.8±1.7 25.1±1.6 24.6±1.1 24.6±2.2 24.5±1.4
ceramide
aminoethanolphosphate
Phosphatidylserine PS 12.0±0.8 10.0±1.2 10.6±2.2 12.0±1.2 10.2±1.4 11.4±1.1
Phosphatidylcholine PC 29.5±2.4 30.2±3.1 31.2±3.3 29.2±0.9 30.8±1.2 29.5±0.8
 1163

lager and Mann 1986; Gallager et al. 1986; Whyte et al.
1990a; Lee and Heffernan 1991).
The observation that PC and PE were the quantita-
tively most important polar lipid classes in the oyster
eggs is consistent with the findings of Soudant et al.
(1998) for Pecten maximus eggs. However, the latter
authors did not report the presence of CAEP. Although
this sphingophophonolipid is a common lipid class in a
wide range of bivalve species, it is often not reported,
most likely due to the absence of adequate analytical
techniques (Vaskovsky 1989). The functional role of
CAEP remains to be determined, but Vaskovsky (1989)
speculated that it might replace sphingomyelin, a
sphingolipid that has been found in many marine in-
vertebrates but not in bivalves.
Egg size was not affected by the diet or the length of
the conditioning period, supporting the conclusions of
Bayne and Newell (1983) and Devauchelle and Mingant
(1991) that egg size is a species-specific characteristic. On
the contrary, others have observed that environmental
conditions can affect the size and subsequently the via-
bility of eggs (Kraeuter et al. 1982; Bayne 1985; Gallager
and Mann 1986; Wilson et al. 1996). In the present trial,
the size of the D-larvae was the same for all treatments
and was within the range reported for wild (His and
Maurer 1988) as well as hatchery-produced larvae (Ut-
ting and Spencer 1991; Robert and Gérard 1999). The
D-larva yield obtained with eggs from oysters fed the
Control or Duna+Em diet always exceeded 60%, which
indicated normal egg quality and compared with data
reported for C. gigas conditioned under standard
hatchery conditions (Utting and Helm 1985; Robert and
Gérard 1999). Larval survival in the present study
agreed well with data provided by the latter authors,
while growth rate was higher.
Although the percentage D-larva recovery from
Duna-fed C. gigas was nearly half that of Control- or
Duna+Em-fed oysters, subsequent larval performance
in terms of survival and growth was similar. This agreed
with data collected by Lipovsky (1984) and Devauchelle
and Mingant (1991) that embryogenesis success is no
indication of subsequent larval performance. However,
in M. mercenaria, high D-larva yields were linked with
high larval survival and spat yields (Kraeuter et al. 1982;
Gallager and Mann 1986). In the larviparous species
O. edulis and O. chilensis, broodstock nutrition had a

Table 5. Crassostrea gigas.

Impact of the broodstock diet Neutral lipid Polar lipid
(Control, Dunaliella tertiolec-
Control Duna Duna+Em Control Duna Duna+Em
ta:Rhodomonas sp.:Tetraselmis
suecica, 1:1:1 on a dry weight
14:0 4.8±0.3 4.0±0.5 3.2±0.2 0.5±0.0 0.8±0.1 0.5±0.1
basis; Duna, D. tertiolecta only; 16:0 24.8±0.7 27.2±1.3 23.2±0.6 12.9±1.3 12.8±0.7 12.9±0.5
Duna+Em, D. tertiolecta with
18:0 4.6±0.4 5.6±0.2 7.2±0.4 5.1±0.2 5.5±0.4 6.3±0.4
lipid emulsion), fed during
8 weeks, on the fatty acid com- S SAFA 39.1±1.0 41.4±0.3 37.3±0.5 23.2±1.1 23.5±1.8 23.5±1.1
position of the neutral and 16:1n-7 4.3±0.5 4.2±0.4 2.2±0.4 1.3±0.1 1.3±0.1 0.7±0.1
polar lipid fraction of C. gigas 18:1n-9 5.5±0.4 5.6±0.3 6.8±0.3 3.0±0.2 2.8±0.2 2.0±0.1
eggs. Data represent the aver- 18:1n-7 5.2±0.5 3.9±0.4 2.9±0.4 2.5±0.1 2.0±0.2 1.1±0.3
age of three replicate samples 20:1n-9 1.3±0.2 1.7±0.4 1.4±0.1 3.7±0.3 4.0±0.2 3.5±0.3
(±SD) (SAFA, MUFA, PUFA 20:1n-7 0.3±0.2 0.3±0.1 0.4±0.1 0.6±0.2 1.0±0.1 1.0±0.1
saturated, monounsaturated,
S MUFA 18.8±1.0 18.2±1.1 16.2±0.3 12.2±0.2 12.9±0.4 9.5±0.2
polyunsaturated fatty acids, 18:2n-6 5.6±0.4 3.8±0.4 5.2±0.5 2.4±0.1 2.3±0.4 1.7±0.2
respectively; tr trace, £ 0.5%;
20:2n-6 0.6±0.1 0.5±0.1 0.7±0.1 0.7±0.2 0.8±0.1 0.6±0.1
n.d. not determined) 20:3n-6 0.4±0.0 0.6±0.1 0.6±0.1 0.2±0.0 0.9±0.2 0.5±0.3
20:4n-6 1.5±0.1 0.9±0.1 2.1±0.4 3.0±0.2 2.6±0.3 4.1±0.2
22:5n-6 0.2±0.0 0.1±0.0 0.7±0.1 0.6±0.1 0.3±0.1 1.0±0.1
16:4n-3 tr tr tr n.d. n.d. n.d.
18:3n-3 4.5±0.3 13.6±0.9 8.6±0.6 2.2±0.1 6.0±0.1 2.7±0.3
18:4n-3 4.1±0.2 2.3±0.1 1.8±0.3 2.4±0.1 2.3±0.3 1.4±0.2
20:3n-3 0.4±0.1 0.9±0.3 0.5±0.0 0.4±0.1 0.6±0.1 0.4±0.0
20:4n-3 0.6±0.1 0.3±0.0 0.3±0.0 0.4±0.1 0.3±0.1 0.1±0.0
20:5n-3 9.4±0.6 5.3±0.4 7.1±0.7 11.1±0.5 9.5±0.7 9.6±0.9
21:5n-3 0.7±0.2 0.4±0.0 0.3±0.1 0.5±0.1 0.3±0.1 0.2±0.0
22:5n-3 0.6±0.2 0.3±0.0 0.8±0.1 1.3±0.1 0.9±0.2 1.9±0.1
22:6n-3 6.8±0.5 4.0±0.5 13.4±0.6 11.3±0.5 8.5±0.6 14.7±0.8
Sn-3 PUFA 27.3±0.8 27.7±0.8 33.0±0.8 29.9±0.8 28.6±1.2 31.1±1.2
n-3/n-6 2.9±0.2 3.7±0.2 3.2±0.2 3.7±0.3 3.7±0.4 3.3±0.1
22:6n-3/20:5n-3 0.7±0.1 0.7±0.1 1.9±0.2 1.0±0.1 0.9±0.1 1.5±0.1
20NMIDi 1.2±0.1 0.6±0.1 0.3±0.1 3.0±0.1 3.6±0.5 2.9±0.2
20NMIDj 0.5±0.1 0.2±0.1 0.1±0.0 0.4±0.0 0.4±0.1 0.3±0.1
22NMIDi 0.4±0.0 0.2±0.1 0.3±0.1 1.8±0.1 1.7±0.2 2.0±0.1
22NMIDj 1.5±0.2 2.7±0.7 0.8±0.1 5.0±0.0 4.4±0.5 3.6±0.1
S NMID 3.6±0.1 3.6±0.4 1.4±0.1 10.3±0.2 10.2±0.9 8.8±0.2
18:0DMA 0.3±0.1 0.3±0.1 0.5±0.2 12.4±0.6 13.3±0.8 14.9±0.1
S DMA 0.6±0.1 0.6±0.1 0.8±0.2 15.6±0.9 16.1±1.0 17.0±0.3
1164
lasting impact on growth of newly released larvae (Helm
et al. 1973; Millican and Helm 1994; Berntsson et al.
1997) and on the yield and growth of spat (Helm et al.
1973; Wilson et al. 1996).
The abundance of plasmalogen (detected as DMA) in
the polar lipids of C. gigas eggs and the predominance of
18:0 DMA, regardless of the broodstock diet, was
comparable with recent information on the composition
of scallop eggs (Caers et al. 1999a). The exact functions
of plasmalogen and the importance of the fatty chain
composition remain to be elucidated, but Chapelle
(1987) speculated that they probably play a critical role
in membrane integrity. Although NMIDs have been
found in numerous bivalves, their presence in oyster or
clam eggs has not previously been reported (Joseph
1989; Robinson 1992b; Utting and Doyou 1992).
NMIDs, which can be synthesised de novo (Zhukova
1991), are thought to play a crucial structural/metabolic
role (Joseph 1989).
Dietary 16:4n-3 did not accumulate in the eggs, 18:1n-9,
18:2n-6 and 18:3n-3 were mainly incorporated in the NL,
while 22:6n-3 was preferentially retained from the diet
and distributed among the PL and NL fractions of the
eggs. It confirms the fatty acid–specific accumulation
and the distribution of dietary fatty acids among the PL
and NL fractions in the tissue of oyster spat (Caers et al.
2000b). It is well established that the NLs in larva, ju-
venile as well as adult bivalves resemble the fatty acid
composition of the diet more closely than the mem-
brane-associated PLs. Nevertheless, some fatty acids did
not accumulate to the extent that they were provided in
the diet (18:3n-3 versus 18:4n-3), while others were
quickly metabolised (16:4n-3); this indicates that bival-
ves can at least partially control the fatty acid compo-
sition of the eggs.
The data presented show that the supplementation of
a 22:6n-3-deficient algal diet (Dunaliella tertiolecta) with
a 22:6n-3-rich lipid emulsion greatly enhanced the per-
centage of 22:6n-3 in the PL and particularly in the NL
of oyster eggs, whereas the size, lipid content and lipid
class composition of the eggs were not affected. This
may suggest that the higher D-larva yield of eggs from
Duna+Em- than from Duna-fed oysters was partially
associated with the 22:6n-3 content of the eggs. It is
consistent with some of the results obtained by authors
who used algal diets with a distinctly different fatty acid
composition (Soudant et al. 1996). However, apart from
the fatty acid composition, a number of parameters
specific to algal species may interfere (e.g. palatability,
gross biochemical composition) and affect the results.
This was clearly shown by Utting and Doyou (1992) and
Utting and Millican (1998).
Irrespective of the diet, oyster broodstock (present
study) were able to maintain minimum 22:6n-3 levels in
the eggs. This agrees with data obtained with clam and
scallop broodstock (Soudant et al. 1996; Utting and
Millican 1998). The contribution of PUFA, originating
from dissolved fatty acids, picoplankton or de novo syn-
thesis, to the high accumulation of PUFA in the eggs is
most likely of minor quantitative importance (Utting and
Doyou 1992). Consistent with the findings and conclu-
sions of Utting and Doyou (1992), the presence of 20:5n-3
and 22:6n-3 in eggs from Duna-fed oysters is assumed to
result from the transfer of lipids from reserves that were
built up prior to the initiation of our experiment. How-
ever, the poor D-larva yield in eggs from D. tertiolecta–fed
oysters may indicate that the 22:6n-3 level in the eggs was
too low to guarantee the successful embryonic develop-
ment observed in the eggs from oysters fed the lipid-sup-
plemented D. tertiolecta. The fact that 22:6n-3 is
selectively retained while 20:5n-3 is largely catabolised
during the process of embryogenesis has been associated
with their structural and energy role, respectively (Whyte
et al. 1990a). However, the 2.5% of 22:6n-3 in the mixed
algal diet appeared to be sufficient to obtain the same
percentage of D-larva recovery as in eggs from oysters fed
the lipid-supplemented D. tertiolecta diet. These findings
may support the idea of the existence of a 22:6n-3
threshold level, beyond which more 22:6n-3 will not
further improve embryonic development. Similar
conclusions have been formulated for the essential fatty
acid requirements of larvae (Thompson and Harrison
1992) and spat (Caers et al. 1998). In addition, it should be
noted that, apart from the possibility that the lipid
emulsion did not fully meet the fatty acid requirements of
oyster broodstock (e.g. with respect to the optimal
20:5n-3/22:6n-3 ratio), D. tertiolecta may be deficient in
other important nutritional components.
The impact of the parental diet on the viability of the
eggs was even more profound when embryogenesis oc-
curred under conditions of temperature or salinity stress.
When eggs were left to develop at 30C or 20 ppt, D-larva
recovery rates were reduced by <50% in eggs from
oysters fed the mixed or lipid-supplemented diet, whereas
a near fourfold decrease was noted in eggs from oysters
conditioned on a diet of D. tertiolecta only. Interesting in
this context are the findings of Utting and Doyou (1992),
i.e. the broodstock diet-dependent consumption of egg
lipids observed during embryogenesis of Tapes philipp-
inarum was much more pronounced under stressful con-
ditions such as the induction of triploidy (76% and 22%
of the initial and similar egg lipid content for D. tertio-
lecta– and S. costatum–conditioned clams, respectively)
than under normal conditions. The foregoing indicates
that one must not only focus on the lipid content of the
eggs, but also judge their quality, which in turn is most
likely determined by their component fatty acids.
The increase in 22:6n-3 in the eggs of oysters fed lipid-
supplemented D. tertiolecta as compared to eggs
spawned by oysters fed solely D. tertiolecta clearly
showed that emulsions provide the tools to manipulate
the fatty acid composition of the eggs. The observation
that emulsified fatty acids were at least partially allo-
cated to the eggs confirms the results of earlier work
with A. purpuratus broodstock (Caers et al. 1999a)
and illustrates their potential application in batch
(A. purpuratus) as well as flow-through (present study)
conditioning systems.

Acknowledgements This work was supported by a grant from the
Flemish government (IWT) to M.C.
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