ICES Journal of Marine Science, 63: 285e295 (2006)

doi:10.1016/j.icesjms.2005.10.011

Effect of enriched rotifers on growth, survival, and

composition of larval Atlantic cod (Gadus morhua)
Heum Gi Park, Velmurugu Puvanendran, Anne Kellett,
Christopher C. Parrish, and Joseph A. Brown

Downloaded from https://academic.oup.com/icesjms/article/63/2/285/640037 by guest on 22 September 2021

Park, H. G., Puvanendran, V., Kellett, A., Parrish, C. C., and Brown, J. A. 2006. Effect of
enriched rotifers on growth, survival, and composition of larval Atlantic cod (Gadus
morhua). e ICES Journal of Marine Science, 63: 285e295.

Recently, the nutritional requirements of marine finfish larvae have received considerable
attention, and studies have shown that docosahexaenoic acid (DHA) affects the growth
and survival of marine finfish larvae. We investigated the effects of different rotifer diets
containing variable amounts of DHA on the growth and survival of larval Atlantic cod
(Gadus morhua L.). Four different commercial rotifer enrichment formulations were
used: spray-dried whole cells composed of Crypthecodinium sp. (ED1), spray-dried whole
cells of Schizochytrium sp. (ED2), an oil emulsion (ED3) and ED1, and dried Chlorella at
a 7:3 ratio by weight (ED4). The resultant rotifers contained a similar concentration of DHA
(1.1e1.6% DW), but the level of DHA differed in proportion to EPA for each enrichment,
and was designated ER1e4. Twelve 30-l aquaria were used with three replicates per treat-
ment. Larvae were fed with rotifers from 3 to 43 days post-hatch (dph) at 4000 prey l
1. At
the end of the experiment, no significant differences were found in body length and dry
weight between the larvae reared on ER1 and ER2. However, larvae reared on ER3 were
significantly smaller (both in length and weight) than larvae reared on ER1 and ER2. Larval
survival on the ER2 treatment at 43 dph was significantly higher than on the other three
treatments. Our results showed a positive effect of rotifer DHA proportions on growth
and survival of cod larvae, and demonstrated that Atlantic cod larvae require a high ratio
of dietary DHA to EPA.
Ó 2005 International Council for the Exploration of the Sea. Published by Elsevier Ltd. All rights reserved.
Keywords: Atlantic cod larvae, DHA proportion, DHA/EPA ratio, enrichments, rotifer.
Received 13 June 2004; accepted 6 October 2005.
H. G. Park: Faculty of Marine Bioscience and Technology, Kangnung National University,
Kangnung 210-702, South Korea. V. Puvanendran, A. Kellett, C. C. Parrish, and J. A.
Brown: Ocean Sciences Centre, Memorial University, St. John’s, Newfoundland, Canada,
A1C 5S7. Correspondence to V. Puvanendran: tel: þ1 709 737 3026; fax: þ1 709 737
3220; e-mail: puvy@mun.ca.

Introduction Artemia, are naturally poor in these fatty acids, so enrich-

Lipids and amino acids are major sources of metabolic en-
ergy during the embryonic and pre-feeding larval stages in
fish. At hatch, yolk-sac larvae have high levels of these en-
ergy sources, but they are dramatically reduced during the
endogenous feeding stage (Evans et al., 2000). Thus,
start-feeding larvae require a live feed that provides suffi-
cient levels of these energy sources. Studies have shown
that essential fatty acids (EFA), such as docosahexaenoic
acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-
3), and arachidonic acid (ARA, 20:4n-6) are also important
in larval fish nutrition (Takeuchi, 1997; McEvoy et al.,
1998; Estevez et al., 1999; Sargent et al., 1999). These fatty
acids, as components of phospholipids (PL), are critical
structural and physiological components of the cell mem-
branes of most tissues. However, the live feeds commonly
used for the first-feeding larval stages, such as rotifers and
ment of live foods with lipids rich in EFA is necessary to
achieve better growth and survival through metamorphosis
(Rainuzzo et al., 1997).
Recently, absolute and relative levels of DHA, EPA, and
ARA in the diets of marine fish larvae have received con-
siderable attention (Sargent et al., 1999; Harel et al.,
2002; Bell and Sargent, 2003). DHA, which has a competi-
tive relationship with EPA, is particularly important for
normal neural development and function, including that
of retina and brain (Sargent et al., 1999). Studies have
shown that the DHA requirement in the diet differs among
fish species, especially in cold-water fish species such as
yellowtail flounder (Limanda ferruginea) and Atlantic hal-
ibut (Hippoglossus hippoglossus), which require high levels
of dietary DHA (McEvoy et al., 1998; Copeman et al.,
2002). However, Planas and Cunha (1999) reported that
turbot larvae (Scophthalmus maximus) require lower levels

1054-3139/$30.00 Ó 2005 International Council for the Exploration of the Sea. Published by Elsevier Ltd. All rights reserved.
286 H. G. Park et al.

Table 1. Lipid content and lipid class composition of the four experimental enrichment products.

ED1 ED2 ED3 Chlorella

Total lipid (mg g
1 DW) 141.5  4.1b 289.7  5.3c 658.7  24.3d 58.4  3.5a
Lipid class (% total lipid)
Hydrocarbons 3.4  1.1b 1.8  0.5ab 0.7  0.2a 6.5  0.9c
Triacylglycerols 59.8  1.0b 66.9  0.7c 52.2  1.4d 0.0  0.0a
Free fatty acids 5.7  0.6c 17.1  0.3d 2.7  0.1b 0.0  0.0a
Alcohols 1.7  0.9b 0.2  0.2ab 0.0  0.0a 0.0  0.0a
Sterols 4.6  3.2a 2.2  1.4a 0.7  0.4a 0.0  0.0a

Downloaded from https://academic.oup.com/icesjms/article/63/2/285/640037 by guest on 22 September 2021

Acetone mobile polar lipids 12.2  1.8b 5.3  1.2a 6.2  1.7ab 17.7  0.8c
Phospholipids 12.7  1.6b 6.0  1.0a 4.2  0.4a 75.8  1.6c

Values (mean  s.e. of three replicates) in the same row not sharing a common superscript are significantly different ( p < 0.05).

of DHA in their diet for better growth and survival. Other

specific benefits of feeding DHA-enriched diets to fish
larvae include successful metamorphosis, reduced pig-
mentation problems, enhanced vision capabilities, im- Table 2. Fatty acid composition of the four experimental enrich-
proved neural development and stress resistance ment products.
(Watanabe, 1993).
Atlantic cod (Gadus morhua L.) is emerging as an alter- Fatty
native aquaculture species in the North Atlantic region acids (%) ED1 ED2 ED3 Chlorella
(Brown et al., 2003). In order to develop this species as a
new candidate for aquaculture at a commercial scale, a con- 14:0 14.7  0.2b 18.0  0.4d 16.8  0.0c 1.4  0.0a
sistent production of juvenile fish must be achieved. Under- 16:0 17.4  0.9a 17.7  0.4a 39.9  0.5c 20.2  0.2b
standing the nutritional requirements of early larval cod, 18:0
P 1.3  0.1b 0.5  0.0a 1.1  0.0b 0.4  0.0a
SFA) 33.8  1.4a 36.4  0.9b 58.9  0.5d 43.5  0.1c
especially of EFA such as DHA and EPA, is important for
16:1 (n-7) 2.6  0.1c 2.0  0.0b 3.7  0.0d 0.2  0.2a
successful mass production. This study investigated the
18:1 (n-9) 10.0  0.1c 9.8  0.4c 0.0  0.0a 1.7  0.0b
effect of DHA level and DHA/EPA ratio in four rotifer- 18:1 (n-7) 0.4  0.0b 0.0  0.0a 2.4  0.0c 0.4  0.0b
enriched diets on growth and survival of Atlantic cod larvae. 20:1 (n-9) 0.0  0.0a 0.0  0.0a 0.0  0.0a 0.1  0.1a
P
MUFAy 3.5  0.0d 12.0  0.5c 6.2  0.0b 3.9  0.2a
18:2 (n-6) 6.4  0.7b 0.1  0.0a 0.1  0.0a 49.8  0.0c
Material and methods 20:3 (n-6) 0.1  0.1a 0.1  0.0a 0.2  0.0a 0.2  0.1a
Experimental enrichments 20:4 (n-6) 0.0  0.0a 0.0  0.0a 0.6  0.0b 0.0  0.0a
(ARA)
Four different commercial enrichment formulations were 20:4 (n-3) 0.2  0.1ab 0.0  0.0a 0.4  0.0c 0.3  0.1bc
used: spray-dried whole cells composed of Crypthecodi- 20:5 (n-3) 0.5  0.0b 0.0  0.0a 0.7  0.0c 0.0  0.0a
nium sp. (ED1), spray-dried whole cells of Schizochytrium (EPA)
sp. (ED2), an oil emulsion (ED3) and ED1, and dried 22:5 (n-3) 0.5  0.2b 0.3  0.0ab 0.1  0.0a 0.0  0.0a
Chlorella at a 7:3 ratio by weight (ED4). Three of these en- 22:6 (n-3) 43.8  2.2c 51.1  1.5d 24.2  0.3b 0.1  0.0a
richments, ED1, ED2, and ED3, were high (52.2e66.9%) (DHA)
P
in triacylglycerol (TAG) and relatively low (4.2e12.7%) PUFAz 52.7  1.3a 51.6  1.4a 34.9  0.5b 52.6  0.3a
P
in PL (Table 1). The Chlorella, however, was high in phos- P(n-3) 46.2  2.2c 51.4  1.4d 25.8  0.4b 2.4  0.1a
pholipids (75.8%) and contained no TAG. The fatty acids in (n-6) 6.5  0.9b 0.2  0.0a 9.2  0.1c 50.1  0.1d
(n-3) PUFA 45.0  2.3c 51.4  1.5d 25.6  0.3b 0.5  0.1a
ED1, ED2, and ED3 were composed of 43.8%, 51.1%, and
(n-3)/(n-6) 7.2  1.3a 283.8  15.3b 2.8  0.0a 0.0  0.0a
24.2% DHA in total fatty acid, respectively, and they had DHA/EPA 95.7  1.3c 0.0  0.0a 33.6  0.6b 0.0  0.0a
less than 0.7% of EPA (Table 2). The Chlorella contained
very low proportions of DHA and no EPA. Values (mean  s.e. of three replicates) in the same row not sharing
a common superscript are significantly different ( p < 0.05).
)Includes ai-15:0, 15:0, ai-17:0, i-17:0, 17:0, 19:0, 20:0, 22:0, 24:0
Rotifer enrichment
at <0.28% each.
Rotifers were cultured with baker’s yeast and Culture yIncludes 16:1n-9, 18:1n-11, 18:1n-6, 18:1n-5, 20:1n-7, 22:1n-9,
SelcoÒ (INVE, Dendermond, Belgium), and were enriched 22:1n-7, 24:1 at <1.60% each.
with ED1, ED2, ED3, and ED4 in 10-l vessels at a density zIncludes 16:2n-4, 16:3n-4, 16:4n-3, 18:2n-4, 18:3n-6, 18:3n-3,
of 4  105 rotifers 1
1. The resultant rotifers were designated 18:4n-3, 20:2n-6, 21:5n-3, 22:5n-6 at <1.96% each.
 Effect of enriched rotifers on growth, survival, and composition of larval Atlantic cod (Gadus morhua)
as enriched rotifer 1 (ER1), enriched rotifer 2 (ER2), en-
riched rotifer 3 (ER3), and enriched rotifer 4 (ER4). Prelim-
inary enrichment trials showed better results in 24-h
enrichment than with 8- and 16-h enrichments. Each batch
of rotifers was enriched at 0.08 g of enrichment material l
1
of rotifer culture for 24 h. Enrichment diets were divided
into three portions and added at times: 0:00, 6:00, and
12:00. Water temperature and salinity for enrichment
were 22(C and 31 psu, respectively. Once a week, from
weeks 1 to 4, samples of 24-h enriched rotifers were taken
from each enrichment vessel for lipid analysis.
Larviculture
Larviculture was conducted following the guidelines of
Puvanendran and Brown (1999, 2002). Spontaneously
spawned, fertilized eggs were collected from captive brood-
stock maintained at the Ocean Sciences Centre, Memorial
University. Eggs were incubated in a 250-l flow-through
silo system (2e3 l min
1) with aeration. Water temperature
was maintained at 5e6(C. Lights remained on around the
clock at an intensity of 300e400 lux. Dead eggs were si-
phoned out daily. Once completely hatched, 1500 larvae
(50 larvae l
1) were transferred to each of the twelve 30-l
rectangular glass aquaria (three replicates per treatment)
that were randomly placed in a thermo-regulated water
bath. This was considered day 1 of the experiment. The sides
of the aquaria were covered with opaque black plastic.
The water temperature in the experimental tanks was
maintained at 11(C and monitored twice daily. A flow-
through saltwater system (31 psu) was provided at an initial
flow of 150 ml min
1 (7.2 exchanges d
1), and gradually
increased to 350 ml min
1 (16.8 exchanges d
1). Dissolved
oxygen was monitored weekly, and remained at around
10.2 mg l
1. Light was provided around the clock at
2000 lux (Puvanendran and Brown, 2002).
Rotifer concentration (4000 prey l
1) was chosen from
a previous study (Puvanendran and Brown, 1999), and lar-
vae were fed four times a day. Each experimental tank was
aerated, which ensured a homogenous distribution of prey
within the tank. To maintain the desired prey concentration
within each experimental tank, just before feeding, a 10-ml
water aliquot was sampled from each tank. The number of
prey in each sample was counted, and prey concentrations
were adjusted as needed.
Data collection
Five larvae (15 per treatment) were randomly sampled for
morphometric measurements from each experimental tank
on 1, 8, 15, 22, and 29 dph. On days 36 and 43, ten larvae
were sampled from each experimental tank (30 per treat-
ment). As greater size variation among larvae was observed
after the fourth week, the number of larvae sampled from
each tank was increased at day 36 to reduce the effect of
size variation between treatments. Each larva was photo-
graphed, and later, larval length was measured using an
287
image analysis software (Matrox Inspector, Matrox Elec-
tronic Systems Ltd, Quebec, Canada). Once photographed,
larvae were washed with distilled water to remove salt, and
five larvae were placed on a 1.0-cm2 pre-weighed alumini-
um foil. This method resulted in three samples (six samples
at 36 and 43 dph) of five larvae being analysed for dry
weight tank
1 week
1. The foils were dried at 60(C for
48 h. Foils were then stored in a desiccator and weighed
again using a microbalance (UMT2, Mettler Toledo, Swit-
zerland). Survival at 43 dph was determined by counting all
Downloaded from https://academic.oup.com/icesjms/article/63/2/285/640037 by guest on 22 September 2021
larvae from each tank.
Lipid samples and lipid analysis
Triplicate samples, consisting of approximately 10 mg dry
weight of larvae, were taken from each tank at hatching
and at 22 and 43 dph. Samples were placed directly in chlo-
roform and stored under nitrogen at
20(C until extrac-
tion. Lipids were extracted in chloroform/methanol
according to Parrish (1999), using a modified Folch proce-
dure (Folch et al., 1957). Lipid classes were determined us-
ing thin layer chromatography with flame ionization
detection (TLC/FID) with a MARK V Iatroscan (Iatron
Laboratories, Tokyo, Japan), as described by Parrish
(1987). Extracts were spotted on silica gel-coated Chromar-
ods, and a three-stage development system was used to sep-
arate lipid classes.
The first separation consisted of a 25-min and a 20-min
development in 99:1:0.05 hexane/diethyl ether/formic
acid. The second separation consisted of a 40-min develop-
ment in 80:20:1 hexane/diethyl ether/formic acid. The last
separation consisted of two 15-min developments in
100% acetone followed by two 10-min developments in
5:4:1 chloroform/methanol/water. After each separation,
the rods were scanned, and the three chromatograms were
combined using T-Data Scan software (RSS, Bemis, Ten-
nessee, USA). The signal detected in millivolts was quanti-
fied using lipid standards (Sigma).
Fatty acid methyl esters (FAME) were prepared by trans-
esterification with 10% boron triflorate (BF3) in methanol at
85(C for 1.5 h (Morrison and Smith, 1964; Budge, 1999).
A Varian model 3400 Gas Chromatograph (GC) equipped
with a Varian 8100 Auto-Sampler was used for fatty acid
analysis (Varian, California, USA). An Omegawax 320 col-
umn, 30 m long, 0.32 mm i.d., 0.25 mm film thickness (Su-
pelco, Bellefonte, Pennsylvania, USA) was used for
separations. Hydrogen was used as the carrier gas, and
the flow rate was set at 2 ml min
1. The column tempera-
ture profile was as follows: 65(C for 0.5 min, hold at
195(C for 15 min after ramping at 40(C min
1, and hold
at 220(C for 0.75 min after ramping at 2(C min
1. The in-
jector temperature was increased from 150(C to 250(C at
200(C min
1. Peaks were detected by flame ionization
with the detector held at 260(C. Fatty acid peaks were in-
tegrated using Varian Star Chromatography Software (ver-
sion 4.02), and identification was made with reference to
288 H. G. Park et al.

known standards (PUFA 1, 3 and 37 Component FAME
Mix, Supelco Canada, Ontario, Canada).
Statistical analysis
All data were tested for normality to satisfy the assumptions
of ANOVA. Two-way ANOVAs with the Bonferroni mul-
tiple comparison test were used to determine the statistical
significance of age and treatment on dry weight and stan-
dard length of cod larvae. One-way ANOVAs with the
Duncan multiple comparison test were used to compare dif-
were 23.4% DHA in the ER1 treatment and 3.5% EPA in
the ER3 treatment (Table 4). However, no significant differ-
ences were found among enriched rotifers from all four
treatments when considering the DHA content as a propor-
tion of total lipid dry weight. Levels of n-3 polyunsaturated
fatty acids (PUFA) were significantly higher (F ¼ 89.2,
d.f. ¼ 4, p < 0.0001) in the ER1 treatment (28.2%) than
in the other three treatments. Rotifers from ER1 and ER2
had significantly higher DHA/EPA ratios than rotifers
from ER3 and ER4. Contents of DHA per dietary dry

Downloaded from https://academic.oup.com/icesjms/article/63/2/285/640037 by guest on 22 September 2021

ferences in survival of larvae and lipid class, and fatty acid weight (DW) were significantly higher (F ¼ 19.9, d.f. ¼ 4,
composition of rotifer and larvae between treatments. Dif- p < 0.0001) in all treatments, compared with the initial
ferences were considered significant at the p < 0.05 level. content.

Results Growth and survival of larvae

Rotifer enrichment
The total protein content of enriched rotifers in all treat-
ments was significantly lower (F ¼ 3.38, d.f. ¼ 4,
p < 0.037) than in the initial rotifers, and in ER4, it was sig-
nificantly higher than in the ER2 and ER3 treatments
(Table 3). The total lipid content of rotifers increased in
all treatments after 24-h enrichment, but it was significantly
higher (F ¼ 12.6, d.f. ¼ 4, p < 0.0001) in the ER2 and ER3
treatments (98.7 mg g
1 DW) than in the ER1 and ER4
treatments. The protein/lipid ratio of the enriched rotifers
in all groups was lower than in the initial rotifers. However,
ER4 showed a significantly higher ratio than ER2 and ER3.
TAG content of the ER2 and ER3 treatments was signifi-
cantly higher than in the initial rotifers (25.8  5.2%),
while the PL level was significantly lower than in the initial
sample (59.0  8.1%). Meanwhile, no significant differen-
ces were found in TAG and PL contents among the ER1
and ER4 treatments and the initial rotifer samples.
All treatments resulted in higher levels of DHA than
EPA, and the highest levels of DHA (F ¼ 174.4, d.f. ¼ 4,
p < 0.0001) and EPA (F ¼ 10.3, d.f. ¼ 4, p < 0.0001)
The effects of enrichment (F ¼ 38.3, d.f. ¼ 3, p < 0.0001)
and age (F ¼ 2821, d.f. ¼ 6, p < 0.0001) on standard length
of cod larvae were significant. From 29 dph to the end of
the experiment, larvae reared on ER1 and ER2 (except
for 22 dph) were significantly larger than larvae reared on
ER3 (Figure 1a). Throughout the experiment, except for
22 dph, no significant difference was found in larval stan-
dard length between the ER1 and ER2 treatments. Except
for 22 and 43 dph, no significant difference was found in
larval standard length between the ER3 and ER4
treatments.
Enrichment (F ¼ 13.1, d.f. ¼ 3, p < 0.0001) and age
(F ¼ 1572, d.f. ¼ 6, p < 0.0001) also had a significant effect
on the dry weight of larval cod. Initially (15, 22, 29, and
36 dph), larvae reared on the ER2 treatment were signifi-
cantly larger than those reared on the ER3 treatment, but
no significant difference ( p ¼ 0.056) was found at the end
of the experiment (Figure 1b). In general, no significant dif-
ference was found in the dry weight among the ER1, ER2,
and ER4 treatments at the beginning and end of the exper-
iment. However, from 29 to 43 dph, larvae reared on the

Table 3. Lipid content and lipid class composition of rotifers enriched for 24 h using four different enrichment products.

Initial ER1 ER2 ER3 ER4

Total protein (mg g
1 DW) 386.5  4.5c 352.4  4.7ab 340.5  5.2a 341.1  6.4a 360.4  5.4b
Total lipid (mg g
1 DW) 68.7  4.5a 75.3  7.3ab 98.7  4.9c 92.1  11.8bc 72.8  4.2ab
Protein/lipid 5.7  0.5c 4.8  0.4bc 3.5  0.1a 3.9  0.4ab 5.0  0.4c
Lipid class (% total lipid)
Hydrocarbons 5.0  2.4b 0.3  0.3a 4.6  2.0ab 1.3  0.5ab 0.1  0.1a
Triacylglycerols 25.8  5.2a 32.1  1.1ab 40.2  1.8b 50.4  3.3c 27.2  0.9a
Free fatty acids 2.1  0.9ab 0.6  0.4a 10.8  1.0c 5.2  1.5b 1.7  1.7ab
Alcohols 0.9  0.4b 0.0  0.0a 0.0  0.0a 0.0  0.0a 0.0  0.0a
Sterols 3.5  0.7a 4.3  0.6a 3.7  0.7a 2.7  0.6a 5.2  1.2a
Acetone mobile polar lipids 3.8  1.3ab 2.0  0.3a 2.1  0.7a 6.2  1.0b 2.4  0.8a
Phospholipids 59.0  8.1b 60.7  1.6b 38.6  3.6a 34.1  1.4a 63.3  2.1b

Values (mean  s.e. of three replicates) in the same row not sharing a common superscript are significantly different ( p < 0.05).
 Effect of enriched rotifers on growth, survival, and composition of larval Atlantic cod (Gadus morhua) 289

Table 4. Fatty acid composition of rotifers enriched for 24 h using four different enrichment products.

Fatty acids (%) Initial ER1 ER2 ER3 ER4

14:0 1.8  0.1a 5.0  0.2c 8.0  0.3d 2.4  0.0b 4.3  0.2c
16:0 7.3  0.3a 11.2  0.3c 24.6  1.4b 13.1  0.2b 11.0  0.5b
17:0 0.4  0.0b 0.3  0.0a 0.3  0.0a 0.8  0.0c 0.3  0.0a
18:0 3.7  0.2b 2.5  0.1a 2.3  0.1a 3.5  0.1b 2.6  0.1a
P
SFA) 16.7  0.2a 21.2  0.3b 37.0  1.6c 22.9  0.2b 22.4  0.3b
16:1 (n-7) 20.4  0.7d 11.7  0.1bc 8.7  0.1a 11.0  0.4b 12.3  0.1c
18:1 (n-9) 20.9  0.6d 16.7  0.3b 7.6  0.2a 19.4  0.3c 16.2  0.2b

Downloaded from https://academic.oup.com/icesjms/article/63/2/285/640037 by guest on 22 September 2021

18:1 (n-7) 5.1  0.1c 3.7  0.1a 4.3  0.1b 4.2  0.1b 4.0  0.1ab
20:1
P (n-9) 3.2  0.1e 2.3  0.1d 1.4  0.0b 1.9  0.1c 0.0  0.0a
MUFAy 60.5  1.3c 42.0  0.4b 27.6  0.4a 42.0  1.0b 42.0  0.5b
18:2 (n-6) 7.7  0.4d 3.6  0.3b 2.3  0.2a 6.5  0.1c 9.4  0.4e
20:3 (n-6) 1.4  0.1c 0.7  0.1b 0.6  0.0ab 0.6  0.0a 0.7  0.0b
20:4 (n-6) (ARA) 1.6  0.1b 1.0  0.1a 1.8  0.1b 1.7  0.1b 0.9  0.1a
20:4 (n-3) 1.6  0.3b 0.6  0.1a 0.7  0.1a 0.5  0.2a 0.3  0.1a
20:5 (n-3) (EPA) 2.0  0.4a 2.4  0.0a 2.0  0.1a 3.5  0.2b 2.1  0.1a
22:5 (n-3) 0.6  0.2a 0.8  0.0a 0.6  0.0a 1.1  0.1b 0.6  0.0a
22:6
P (n-3) (DHA) 1.0  0.1a 23.4  0.6d 18.3  0.8c 13.8  1.0b 16.8  0.4c
PUFAz 22.8  1.3a 36.7  0.5b 35.4  1.3b 35.1  1.2b 35.6  0.5b
P
P(n-3) 5.7  0.8a 27.7  0.6c 22.0  0.9b 20.4  1.3b 20.3  0.5b
(n-6) 11.1  0.4c 5.6  0.4a 10.0  0.3b 9.5  0.1b 11.8  0.4c
(n-3) PUFA 6.9  0.7a 28.2  0.5c 22.2  0.9b 19.7  1.3b 20.7  0.5b
(n-3)/(n-6) 0.5  0.1a 5.1  0.6c 2.2  0.0b 2.1  0.2b 1.7  0.1b
EPA %x 0.1  0.0a 0.2  0.0a 0.2  0.0a 0.3  0.0b 0.1  0.0a
DHA % DWx 0.1  0.0a 1.6  0.2b 1.6  0.1b 1.2  0.2b 1.1  0.0b
DHA/EPA 0.5  0.1a 9.7  0.2d 9.2  0.4d 4.0  0.1b 8.1  0.5c

Values (mean  s.e. of three replicates) in the same row not sharing a common superscript are significantly different ( p < 0.05).
)Includes ai-15:0, i-15:0, 15:0, ai-16:0, i-16:0, ai-17:0, i-17:0, 18:0, 19:0, 20:0, 22:0, 23:0, 24:0 at <1.9% each.
yIncludes 16:1n-9, 17:1, 18:1n-11, 18:1n-6, 18:1n-5, 20:1n-11, 20:1n-7, 22:1n-11, 22:1n-9, 22:1n-7, 24:1 at <3.5% each.
zIncludes 16:2n-4, 16:3n-4, 16:4n-3, 16:4n-1, 18:2n-4, 18:3n-6, 18:3n-4, 18:3n-3, 18:4n-3, 20:2a, 20:2b, 20:2n-6, 20:3n-3, 21:5n-3, 22:5n-
6, 22:2NIMDb at <1.4% each.
xDHA and EPA contents as a proportion of dry weight (%) ¼ total lipid per dry weight (%)  area (g g
1)  0.892 (Yoshimatsu et al.,
1997).

ER1 treatment were significantly larger than larvae from
the ER3 treatment.
The survival rate of larval cod in the ER2 treatment was
higher than in the other treatments, although no significant
difference was found (F ¼ 2.21, d.f. ¼ 3, p > 0.165) among
treatments (Figure 2). Larval survival in one of the tanks in
the ER2 treatment was lower (20%) than in the others
(34%). Cochran’s test for variance outliers (Kanji, 1994)
was used to determine outliers in the data, and a significant
critical value ( p < 0.05) was found for the ER2 survival
data. When the data were analysed after removing this out-
lier, larval survival in the ER2 treatment was significantly
higher (F ¼ 6.56, d.f. ¼ 3, p < 0.019), while no significant
difference was found among the other three treatments.
Larval lipids
The total lipid content of cod larvae at 3 and 6 weeks was
not significantly different (96.6e123.8 mg g
1 DW) among
all treatments (Table 5, Figure 3a). The major lipid class of
cod larvae was PL in all treatments, and the PL content in
the ER3 treatment was significantly lower (F ¼ 41.1,
d.f. ¼ 3, p < 0.0001) than in ER1 and ER4 at 43 dph
(Figure 3b). Concomitantly, the TAG level in the
ER3 treatment was significantly higher than in all other
treatments at 43 dph (Figure 3c; F ¼ 25.3, d.f. ¼ 3,
p < 0.019).
The percentage of ARA in larvae from all treatments at 22
and 43 dph was higher than the initial value (1.3  0.0%).
The ARA level in larvae reared in ER2 was significantly
higher (F ¼ 793, d.f. ¼ 3, p < 0.0001 and F ¼ 89.7,
d.f. ¼ 3, p < 0.0001 at 22 and 43 dph, respectively) than in
the other three treatments (Table 6, Figure 4). The level
of EPA in all treatments, at 22 and 43 dph, was lower
than initially (16.7  0.1%), and it was significantly lower
(F ¼ 351, d.f. ¼ 3, p < 0.0001 and F ¼ 593, d.f. ¼ 3,
p < 0.0001 at 22 and 43 dph, respectively) in the ER2
treatment.
DHA levels of 22- and 43-dph cod larvae fed with roti-
fers from ER1 and ER2 treatments were similar to those
of 1-dph cod larvae (initial: 32.7  0.1%), while DHA
290
Figure 1. Growth of Atlantic cod larvae fed the different enriched rotifers during the first 43 days post-hatch. Data are mean  s.e. (a)
Standard length (n ¼ 15), (b) dry weight (n ¼ 3).
levels in the ER3 and ER4 treatments were lower than the
initial values. DHA levels in the ER1 and ER2 treatments
were significantly higher than in ER3 and ER4, but not sig-
nificantly different between each other. The ratio of DHA/
EPA was highest at 22 dph (10.4) and 43 dph (12.5) in
the ER2 treatment, and lowest at 22 dph (5.4) and 43 dph
(5.6) in the ER3 treatment.
Discussion
Several studies have suggested that PL rather than TAG
are the preferred vehicle for delivering PUFA to marine
fish larvae (Kanazawa, 1997; Sargent et al., 1997; Harel
H. G. Park et al.
Downloaded from https://academic.oup.com/icesjms/article/63/2/285/640037 by guest on 22 September 2021
et al., 1999). In our study, rotifers from ER1 had signif-
icantly higher levels of PL than rotifers from ER2 and
ER3 treatments, while rotifers from the ER3 treatment
had a significantly higher level of TAG than the remain-
ing treatments. The lack of a significant difference in
growth between cod larvae reared in the ER1 and ER2
treatments suggests that PL levels in the diet may not
have a dominant influence on the growth of cod larvae.
Nonetheless, our results agree with other studies, which
indicate that TAG may not be a preferred vehicle for de-
livering PUFA, which has an important role in the growth
of marine finfish larvae (Kanazawa, 1997; Sargent et al.,
1997; Harel et al., 1999).
 Effect of enriched rotifers on growth, survival, and composition of larval Atlantic cod (Gadus morhua) 291

Downloaded from https://academic.oup.com/icesjms/article/63/2/285/640037 by guest on 22 September 2021

Figure 2. Survival of Atlantic cod larvae fed four different enriched
rotifers at 43 dph.)Represents a significant difference ( p < 0.05)
among treatments (ANOVA, Duncan’s multiple comparison).

A relationship between DHA levels and DHA/EPA ratios

of rotifers, and the growth and survival of cod larvae was
found in the present study. Takeuchi et al. (1994) investi-
gated the effect of DHA levels of rotifers on the growth,
survival rate, and abnormalities of larval cod (Gadus mac-
rocephalus), and suggested that the appropriate level of
DHA that should be contained in the rotifers was around
1% DW. In their study, any amount higher than 1% DHA
resulted in a high percentage of abnormal fish, together
with high mortality. However, in our study, larval cod fed
Figure 3. Lipid content of Atlantic cod larvae fed the different en-
ER2 containing 1.6% DW of DHA had higher growth
riched rotifers at 1, 22 and 43 days post-hatch. Data are mean  s.e.
and survival than larvae fed with ER3 and ER4 with (n ¼ 3). (a) Lipid (mg g
1 DW), (b) PL (% total lipids), (c) TAG
1.1e1.2% DW of DHA. Sargent et al. (1999) suggested (% total lipids). )Represents a significant difference ( p < 0.05)
that species-specific requirements for DHA exist among among dietary groups (ANOVA, Duncan’s multiple comparison).
marine finfish larvae. Several other studies suggested that
much higher levels of DHA (or n-3 highly unsaturated fatty
acids e HUFA) could reduce larval survival (Planas and

Table 5. Lipid content and lipid class composition of cod larvae fed four types of differently enriched rotifers for the 43 dph.

Initial ER1 ER2 ER3 ER4

Total lipid (mg g
1 DW) 112.9  6.1 115.2  9.6 96.6  3.7 123.8  19.7 111.1  12.8
Lipid class (% total lipid)
Hydrocarbons 0.1  0.1 1.7  0.5 3.0  0.3 3.6  1.0 2.6  0.8
Triacylglycerols 12.9  4.1 2.1  0.2a 6.1  0.4b 13.3  1.0c 3.6  1.5ab
Free fatty acids 6.2  3.1 0.8  0.2b 0.2  0.2ab 0.0  0.0a 0.2  0.2ab
Alcohols 2.0  1.9 0.0  0.0 0.0  0.0 0.0  0.0 0.0  0.0
Sterols 12.5  0.4 20.5  1.1ab 18.6  0.8ab 16.0  1.5a 21.0  1.9b
Acetone mobile polar lipids 0.2  0.2 0.0  0.0a 0.6  0.6a 1.9  0.5b 0.0  0.0a
Phospholipids 66.0  7.6 75.0  1.8b 71.6  1.4ab 65.2  0.2a 72.5  3.5b

Values (mean  s.e. of three replicates) in the same row not sharing a common superscript are significantly different ( p < 0.05).
292 H. G. Park et al.

Table 6. Fatty acid composition of the 43-dph-old cod larvae fed on rotifers enriched with four different enrichment products.

Fatty acids (%) Initial ER1 ER2 ER3 ER4

14:0 1.0  0.0 1.2  0.0b 1.4  0.1c 1.0  0.1a 1.0  0.0a
16:0 19.2  0.2 14.1  0.0b 15.6  0.1d 11.4  0.2a 14.7  0.2c
17:0 0.2  0.0 0.5  0.0b 0.4  0.0a 0.7  0.0d 0.5  0.0c
18:0 5.0  0.1 7.1  0.1b 7.0  0.2b 6.1  0.1a 7.1  0.1b
P
SFA) 26.4  0.2 24.1  0.1b 25.4  0.2d 21.3  0.2a 24.8  0.1c
16:1 (n-7) 3.6  0.0 5.9  0.1b 4.5  0.2a 6.8  0.2d 5.9  0.2b
18:1 (n-9) 8.8  0.1 13.2  0.0b 9.0  0.0a 13.3  0.3b 12.8  0.2b

Downloaded from https://academic.oup.com/icesjms/article/63/2/285/640037 by guest on 22 September 2021

18:1
P (n-7) 3.1  0.0 4.6  0.0ab 4.7  0.0b 4.6  0.0a 4.7  0.1b
MUFAy 21.0  0.2 28.7  0.1b 22.0  0.3a 29.6  0.7b 28.3  0.4b
18:2 (n-6) 0.5  0.0 2.5  0.0b 1.4  0.0a 4.2  0.1c 6.2  0.2d
20:3 (n-6) 0.0  0.0 0.6  0.0b 0.4  0.0a 0.5  0.0a 0.9  0.0c
20:4 (n-6) (ARA) 1.3  0.0 2.2  0.0a 4.7  0.1d 4.2  0.0c 2.4  0.0b
20:4 (n-3) 0.1  0.0 0.2  0.1 0.1  0.1 0.1  0.1 0.3  0.1
20:5 (n-3) (EPA) 16.7  0.1 4.2  0.1b 2.7  0.0a 4.9  0.0d 4.6  0.0c
22:5 (n-3) 0.0  0.0 1.1  0.0b 0.8  0.0a 2.0  0.0d 1.2  0.0c
22:6
P (n-3) (DHA) 32.7  0.1 34.2  0.4c 33.7  0.2c 27.4  0.5a 28.8  0.3b
PPUFAz 52.7  0.1 47.1  0.2a 52.5  0.1c 49.0  0.6b 46.9  0.3a
(n-3) 49.8  0.1 40.0  0.3c 37.4  0.3b 35.0  0.5a 35.2  0.4a
P
(n-6) 2.2  0.0 6.1  0.1a 14.5  0.2d 11.3  0.1c 10.6  0.1b
(n-3) PUFA 49.5  0.1 39.8  0.3c 37.3  0.3b 34.4  0.5a 35.0  0.4a
(n-3)/(n-6) 22.5  0.5 6.5  0.2c 2.6  0.0a 3.1  0.1b 3.3  0.1b
DHA/EPA 2.0  0.0 8.1  0.2c 12.5  0.1d 5.6  0.1a 6.3  0.1b

Values (mean  s.e of three replicates) in the same row not sharing a common superscript are significantly different ( p < 0.05).
)Includes ai-15:0, i-15:0, 15:0, ai-16:0, i-16:0, ai-17:0, i-17:0, 18:0, 19:0, 20:0, 22:0, 23:0, 24:0 at <0.5% each.
yIncludes 16:1n-9, 17:1, 18:1n-11, 18:1n-6, 18:1n-5, 20:1n-11, 20:1n-7, 22:1n-11, 22:1n-9, 22:1n-7, 24:1 at <3.6% each.
zIncludes 16:2n-4, 16:3n-4, 16:4n-3, 16:4n-1, 18:2n-4, 18:3n-6, 18:3n-4, 18:3n-3, 18:4n-3, 20:2a, 20:2b, 20:2n-6, 20:3n-3, 21:5n-3, 22:5n-
6, 22:2NIMDb at <1.6% each.

Cunha, 1999). Izquierdo et al. (1992) showed that, in larval
Japanese flounder (Paralichthys olivaceus), lower (or
higher) DHA content (1.5%) of Artemia did not affect sur-
vival, but larvae were significantly larger when fed Artemia
containing a higher percentage of DHA (up to 3.5%). How-
ever, Salhi et al. (1994), in their study with gilthead sea
bream (Sparus aurata), showed that larvae fed with a lower
DHA microdiet (>0.5%) had a significantly lower survival
than larvae fed with a higher DHA microdiet (1.2e1.3%).
They suggested that the growth of larvae was affected by
a combination of DHA content and total dietary lipid. In
our study, however, the ER2 (1.6% DW of DHA) treatment
gave a significantly higher survival than the ER3 (1.2%
DW of DHA) and ER4 (1.1% DW of DHA) treatments.
Thus, it seems that the requirement of dietary DHA levels
of marine finfish larvae is species dependent.
Rodriguez et al. (1997) reported that a higher DHA/EPA
ratio (1.4:1e0.3:1) during the rotifer stage improved the
growth and survival of gilthead sea bream. Copeman et al.
(2002) found that yellowtail flounder fed high DHA/EPA
(8:1) had a higher growth and survival than those fed
a DHA/EPA ratio of 1.9:1. However, there was no significant
difference in the growth of Japanese flounder and turbot larvae
when they were fed with different dietary ratios of DHA and
EPA (Estevez et al., 1999; Furuita et al., 1999). Harel et al.
(2002) investigated the effect of commercial enrichment ma-
terials on early development of three larval fish. They reported
no significant difference in growth between striped bass
(Morone saxatilis) and gilthead sea bream larvae fed with Ar-
temia enriched with Algamac 2000Ò or PL-Cr (DHA-rich
phospholipid extract of Crypthecodinium sp.). However, the
growth of halibut larvae fed Artemia enriched with DHA
SelcoÒ was lower than the growth of larvae fed with PL-Cr.
Our studies also showed that cod larvae fed high DHA diets
(ER2) showed better growth and survival than those fed low
DHA diets (ER3 and ER4). On the other hand, cod larvae
fed ER1, which had an equivalent level of DHA compared
with ER2, had a lower survival than those fed ER2 treatments.
All these studies, including our own, suggest the existence of
species-specific requirements for the DHA/EPA ratio for
growth and survival of marine finfish larvae.
The lipid composition of eggs/yolk has been suggested as
an indicator for determining the nutritional requirements of
first-feeding larvae. Typically, a dietary DHA/EPA ratio of
2:1 is found in marine species, and has been suggested as
adequate for larval feeding (Tocher and Sargent, 1984; Sar-
gent et al., 1999). However, in our experiment, growth and
survival of larval cod improved with increasing ratio. Sim-
ilar to other experiments (Tocher and Sargent, 1984), newly
hatched cod larvae in our experiment had a DHA/EPA ratio
 Effect of enriched rotifers on growth, survival, and composition of larval Atlantic cod (Gadus morhua) 293

Downloaded from https://academic.oup.com/icesjms/article/63/2/285/640037 by guest on 22 September 2021

Figure 4. Fatty acid content of Atlantic cod larvae fed the different enriched rotifers at 1, 22 and 43 days post-hatch. Data are mean  SEM
(n ¼ 3). (a) ARA (% total lipids), (b) EPA (% total lipids), (c) DHA (% total lipids), and (d) DHA/EPA. Different letters represent a sig-
nificant difference ( p < 0.05) among dietary groups (ANOVA, Duncan’s multiple comparison).

of 2:1. DHA/EPA ratio of larval cod increased as the larvae
grew, irrespective of the rotifer enrichment. However, in-
crease in the DHA/EPA ratio was significantly higher in
the ER1 and ER2 treatments, which yielded better growth
than the other two treatments. From our results, it seems
that larval cod require a higher DHA:EPA ratio than
some other marine finfish species. Copeman et al. (2002)
suggested that larval yellowtail flounder require higher di-
etary DHA levels for better growth. Thus, our results indi-
cate that the presence of high DHA and lower EPA levels in
the diet may be important for better growth of cold-water
marine finfish larvae.
It has been suggested that the ideal diet for fish larvae
should provide a similar lipid class composition as the
yolk of eggs or yolk-sac larvae (Sargent et al., 1999). In
our study, lipid class constituents of newly hatched cod
larvae were 12.9% TAG and 66.0% PL. The lipid class
of larval cod reared on the ER3 treatment was similar at
43 dph to that of newly hatched larvae. The lipids of cod
larvae reared on the remaining treatments had lower TAG
and higher PL levels than those of larvae from the ER3
treatment and the newly hatched larvae. Therefore, our
study suggests that increased PL levels and decreased
TAG levels of older cod larvae, compared with newly
hatched larvae, could be an indicator of better larval
condition.
Watanabe (1993) suggested that the DHA content of At-
lantic cod larvae could be reduced rapidly during larval de-
velopment after hatching. In our study, the DHA levels of
Atlantic cod larvae in ER1 and ER2 treatments did not
change compared with the initial value. Meanwhile, the
DHA levels of larval cod fed with ER3 and ER4 were lower
than the initial value. This suggests that the DHA levels of
cod larvae should be kept close to the initial levels for bet-
ter larval growth, and that this can be accomplished by
feeding diets with a relatively high DHA level and high
DHA/EPA ratio. Copeman et al. (2002) found that supple-
menting diets with high EPA levels was not effective for the
growth of yellowtail flounder. Similarly, in our experiment,
EPA levels of larvae at 22 and 43 dph were very low in all
treatments, compared with initial levels (newly hatched
larvae), and had no effect on the growth of cod larvae.
Recently, studies have indicated that arachidonic acid
(ARA) levels in marine fish larvae may be important for
stress tolerance, pigmentation, growth, and survival (Bell
and Sargent, 2003). In particular, the competitive interac-
tions between EPA and ARA are important in the formation
of eicosanoids (Harel and Place, 2003). In our study, ARA
levels in larvae were higher in all treatments than in their
respective diets. Thus, larval cod appear to have the ability
to selectively incorporate dietary ARA into their body tis-
sues. Similarly, Copeman et al. (2002) found that yellowtail
294 H. G. Park et al.
flounder larvae have the ability to increase the dietary ARA
levels in the body tissue in spite of lower dietary ARA lev-
els (as low as 2.2% of total fatty acid). Zheng et al. (1996)
reported that prey enriched with higher ARA provided no
improvement in survival for Pacific cod larvae. Similarly,
in our studies, ARA levels in rotifers did not affect growth
and survival of Atlantic cod. However, studies have shown
that dietary ARA levels are important for improved growth
and survival in gilthead sea bream (Bessonart et al., 1999;
Koven et al., 2001). Sargent et al. (1999) suggested that
both the concentration and ratio, not only between DHA
and EPA, but also between EPA and ARA, are important
in larval marine fish nutrition. Thus, it appears that the
ARA levels in diet have a species-dependent effect on
cold-water marine fish.
Chemical composition of the enrichment diets used in
our experiment differed not only in essential fatty acids,
but also in phospholipids, proteins, and micronutrients. Al-
though our results showed that DHA, EPA, and DHA:EPA
ratio had significant effects on the growth, survival, and
composition of larval cod, differences in other nutrients
could have also affected our results. Unfortunately, it is dif-
ficult to control all the nutrients in studies involving com-
mercial enrichments and live prey, and could only be
achieved using formulated inert diets.
In conclusion, high dietary levels of DHA, relative to
EPA, with ratios up to 10:1 in rotifers promoted growth in
Atlantic cod. ER1 and ER2 had higher levels of DHA com-
pared with ER3, and larvae fed with these two rotifers (ER1
and ER2) had a better growth than larvae fed with ER3. En-
richment of rotifers with a DHA-rich diet appears effective in
improving the nutritional value of rotifers for the improve-
ment of growth and survival of Atlantic cod larvae.
Acknowledgements
We thank Danny Boyce, Cathy Williams, and other ARDF
personnel for their help. This work was supported by the
Postdoctoral Fellowship Programme of Korea Science &
Engineering Foundation (KOSEF) to HGP and the Natural
Sciences and Engineering Research Council of Canada
(NSERC) strategic project (JAB and VP). We thank New-
foundland Aqua Ventures and Department of Fisheries
and Oceans for providing cod eggs.
References
Bell, J. G., and Sargent, J. R. 2003. Arachidonic acid in aquaculture
feeds: current status and future opportunities. Aquaculture, 218:
491e499.
Bessonart, M., Izquierdo, M. S., Salhi, M., Hernández-Cruz, C. M.,
González, M. M., and Fernández-Palacios, H. 1999. Effect of di-
etary arachidonic acid levels on growth and survival of gilthead
sea bream (Sparus aurata L.) larvae. Aquaculture, 179:
265e275.
Brown, J. A., Minkoff, G., and Puvanendran, V. 2003. Larviculture
of Atlantic cod (Gadus morhua): progress, protocol and prob-
lems. Aquaculture, 227: 357e372.
Budge, S. M. 1999. Fatty acid biomarkers in a cold water marine
environment. PhD thesis, Memorial University of Newfound-
land, St. John’s, Newfoundland, Canada. 197 pp.
Copeman, L. A., Parrish, C. C., Brown, J. A., and Harel, M. 2002.
Effects of docosahexaenoic, eicosapentaenoic and arachidonic
acids on the early growth, survival, lipid composition and pig-
mentation of yellowtail flounder (Limanda ferruginea): a live
food enrichment experiment. Aquaculture, 210: 285e304.
Evans, R. P., Zhu, P., Parrish, C. C., and Brown, J. A. 2000. Lipid
and amino acid metabolism during early development of marine
fish. In Seafood in Health and Nutrition-Transformation in Fish-
eries and Aquaculture: Global Perspectives, pp. 477e493. Ed. by
F. Shahidi. ScienceTech Publishing Company, St. John’s,
Downloaded from https://academic.oup.com/icesjms/article/63/2/285/640037 by guest on 22 September 2021
Newfoundland.
Estevez, A., McEvoy, L. A., Bell, J. G., and Sargent, J. R. 1999.
Growth, survival, lipid composition and pigmentation of turbot
(Scophthalmus maximus) larvae fed live-prey enriched in arach-
idonic and eicosapentaenoic acids. Aquaculture, 180: 321e343.
Folch, J., Lees, M., and Sloane-Stanley, G. H. 1957. A simple
method for the isolation and purification of total lipids from an-
imal tissues. Journal of Biological Chemistry, 22: 497e509.
Furuita, H., Konishi, K., and Takeuchi, T. 1999. Effect of different
levels of eicosapentaenoic and docosahexaenoic acid in Artemia
nauplii on growth, survival and salinity tolerance of larvae of the
Japanese flounder, Paralichthys olivaceus. Aquaculture, 170:
59e69.
Harel, M., Ozkizilcik, S., Lund, E., Behrens, P., and Place, A. R.
1999. Enhanced absorption of docosahexaenoic acid (DHA,
22:6n-3) in Artemia nauplii using a dietary combination of
DHA-rich phospholipids and DHA-sodium salts. Comparative
Biochemistry and Physiology Part B, 124: 169e176.
Harel, M., Koven, W., Lein, I., Bar, Y., Behrens, P., Stubblefield,
J., Zohar, Y., and Place, A. R. 2002. Advanced DHA, EPA and
ArA enrichment materials for marine aquaculture using single
cell heterotrophs. Aquaculture, 213: 347e362.
Harel, M., and Place, A. R. 2003. Tissue essential fatty acid com-
position and competitive response to dietary manipulations in
white bass (Morone chrysops), striped bass (M. saxatilis) and hy-
brid striped bass (M. chrysops  M. saxatilis) Comparative.
Biochemistry and Physiology Part B, 135: 83e94.
Izquierdo, M. S., Arakawa, T., Takeuchi, T., Haroun, R., and
Watanabe, T. 1992. Effect of n-3 HUFA levels in Artemia on
growth of larval Japanese flounder (Paralichthys olivaceus).
Aquaculture, 105(1): 73e82.
Kanazawa, A. 1997. Effects of docosahexaenoic acid and phospho-
lipids on stress tolerance of fish. Aquaculture, 155: 129e134.
Kanji, G. K. 1994. 100 Statistical Tests. Sage Publications,
London. 216 pp.
Koven, W., Barr, Y., Lutzky, S., Ben-Atia, I., Weiss, R., Harel, M.,
Behrens, P., and Tandler, A. 2001. The effect of dietary
arachidonic acid (20:4n-6) on growth, survival and resistance
to handling stress in gilthead sea bream (Sparus aurata) larvae.
Aquaculture, 193: 107e122.
McEvoy, L. A., Naess, T., Bell, J. G., and Lie, Ø. 1998. Lipid and
fatty acid composition of normal and malpigmented Atlantic hal-
ibut (Hippoglossus hippoglossus) fed enriched Artemia: a com-
parison with fry fed wild copepods. Aquaculture, 163: 237e250.
Morrison, W. R., and Smith, L. M. 1964. Preparation of fatty acid
methyl esters and dimethylacetals from lipids with boron fluo-
ride methanol. Journal of Lipid Research, 5: 600e608.
Parrish, C. C. 1987. Separation of aquatic lipid classes by Chro-
marod thin-layer chromatography with measurement by Iatro-
scan flame ionization detection. Canadian Journal of Fisheries
and Aquatic Sciences, 44: 722e731.
Parrish, C. C. 1999. Determination of total lipid, lipid classes and fat-
ty acids in aquatic samples. In Lipids in Freshwater Ecosystems,
pp. 4e12. Ed. by M. T. Arts, and B. C. Wainman. Springer-Verlag,
New York.
 Effect of enriched rotifers on growth, survival, and composition of larval Atlantic cod (Gadus morhua)
Planas, M., and Cunha, I. 1999. Larviculture of marine fish: prob-
lems and perspectives. Aquaculture, 177: 171e190.
Puvanendran, V., and Brown, J. A. 1999. Foraging, growth and sur-
vival of Atlantic cod larvae reared in different prey concentra-
tions. Aquaculture, 175: 77e92.
Puvanendran, V., and Brown, J. A. 2002. Foraging, growth and sur-
vival of Atlantic cod larvae reared in different light intensities
and photoperiods. Aquaculture, 214: 131e151.
Rainuzzo, J. R., Reitan, K. I., and Olsen, Y. 1997. The significance
of lipids at early stages of marine fish: a review. Aquaculture,
155: 103e115.
Rodriguez, C., Perez, J. A., Diaz, M., Izquierdo, M. S., Fernandez-
Palacios, H., and Lorenzo, A. 1997. Influence of EPA/DHA ratio
in rotifers on gilthead sea bream (Sparus aurata) larval develop-
ment. Aquaculture, 150: 77e89.
Salhi, M., Izquierdo, M. S., Hernandez-Cruz, C. M., Gonzalez, M.,
and Fernandez-Palacios, H. 1994. Effect of lipid and n-3 HUFA
levels in microdiets on growth, survival and fatty acid composi-
tion of larval gilthead sea bream (Sparus aurata). Aquaculture,
124: 275e282.
Sargent, J., McEvoy, L. A., and Bell, J. G. 1997. Requirements,
presentation and sources of polyunsaturated fatty acids in marine
fish larval feeds. Aquaculture, 155: 117e127.
295
Sargent, J., Bell, J. G., McEvoy, L. A., Tocher, D., and Estevez, A.
1999. Recent developments in the essential fatty acid nutrition of
fish. Aquaculture, 177: 191e199.
Takeuchi, T., Zheng, F., Takeuchi, T., Yosheda, M., Hirokawa, J., and
Watanabe, T. 1994. Nutritive value of DHA-enriched rotifer for
larval cod. Nippon Suisan Gakkaishi, 60: 641e652 (In Japanese).
Takeuchi, T. 1997. Essential fatty acid requirements of aquatic
animals with emphasis on fish larvae and fingerlings. Fisheries
Science, 5: 1e25.
Tocher, D. R., and Sargent, J. R. 1984. Analysis of lipids and fatty
acids in ripe roes of some Northwest European marine fish
Lipids, 7: 492e499.
Watanabe, T. 1993. Importance of docosahexaenoic acid in marine lar-
Downloaded from https://academic.oup.com/icesjms/article/63/2/285/640037 by guest on 22 September 2021
val fish. Journal of the World Aquaculture Society, 24: 152e161.
Yoshimatsu, T., Imoto, H., Hayashi, M., Toda, K., and Yoshimura, K.
1997. Preliminary results in improving essential fatty acids enrich-
ment of rotifer cultured in high density. Hydrobiologia, 358:
153e157.
Zheng, F., Takeuchi, T., Yosheda, K., Kobayashi, M., Hirokawa, J.,
and Watanabe, T. 1996. Requirement of larval cod for arachi-
donic acid, eicosapentaenoic acid and docosahexaenoic acid
using enriched Artemia nauplii. Nippon Suisan Gakkaishi, 62:
669e676 (In Japanese).