ICES Journal of Marine Science, 63: 302e310 (2006)
doi:10.1016/j.icesjms.2005.11.002
Diet-induced differences in the essential fatty acid (EFA)
compositions of larval Atlantic cod (Gadus morhua L.)
with reference to possible effects of dietary EFAs on
larval performance
C. J. Cutts, J. Sawanboonchun, C. Mazorra de Quero,
and J. G. Bell
Cutts, C. J., Sawanboonchun, J., Mazorra de Quero, C., and Bell, J. G. 2006. Diet-induced
differences in the essential fatty acid (EFA) compositions of larval Atlantic cod (Gadus
morhua L.) with reference to possible effects of dietary EFAs on larval performance. e
ICES Journal of Marine Science, 63: 302e310.
We studied the performance of cod rearing in which live feed was given under three different essential fatty acid (EFA) enrichment regimes, using commercially available live-feed
enrichments. We assessed the fatty acid profile [docosahexaenoic (DHA), eicosapentaenoic
(EPA), and arachidonic acid (AA)] in larval somatic tissue, relative to its amounts in both
rotifers and Artemia as well as to larval performance. Overall, percentage lipid level of each
experimental diet for the trial was approximately 50%. Further, there were no significant
differences in total fatty acid levels of larvae from each treatment at the end of the trial
(mean ¼ 444.76 mg fatty acid per mg lipid). However, during the rotifer phase, larvae
from each treatment were able to incorporate comparable levels of %DHA, irrespective
of levels in the diet. Despite this, the rotifer diet with more %DHA still promoted better
larval growth than other treatments. Conversely, larvae from two of the treatments did
not exhibit any accumulation of AA, reflecting levels found in the diet instead. However,
between-tank differences in larval %AA showed improved growth during the rotifer period
when larval %AA was high. Low ratios of EPA had no effect. During the Artemia phase,
percentage levels of larval DHA decreased; there was no accumulation of DHA relative
to dietary levels, which in Artemia were significantly lower than in rotifers (6 cf.
20e30%). However, DHA levels in larvae at the end of the experiment correlated positively
with survival. Artemia contained lower levels of AA than rotifers (1.5 cf. 3.0%), yet comparable levels of AA were found in rotifer-fed and Artemia-fed larvae. This also differed
significantly between treatments, and correlated positively with survival.
Ó 2005 International Council for the Exploration of the Sea. Published by Elsevier Ltd. All rights reserved.
Keywords: arachidonic acid, cod (Gadus morhua), docosahexaenoic acid, eicosapentaenoic
acid, growth, live feed, survival.
Received 20 September 2004; accepted 15 November 2005.
C. J. Cutts and C. Mazorra de Quero: Scottish Association for Marine Science, Ardtoe
Marine Laboratory, Acharacle, Argyll PH36 4LD, Scotland, UK. J. Sawanboonchun and
J. G. Bell: Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, Scotland,
UK. Correspondence to C. J. Cutts: tel: þ44 1360 870271; fax: þ44 1360 870381;
e-mail: [email protected].
Introduction
Significant advances have been made in the intensive culture of Atlantic cod (Gadus morhua L.) since Howell
(1984) published feeding protocols using rotifers and Artemia. This renewed interest and advances in protocols are
primarily the result of a reduced supply from the wild fishery, the resulting high market price, and cod’s apparent
suitability for culture (Tilseth, 1990). Protocols for the intensive culture of cod have been dependent on borrowing
techniques from those developed for other species (Brown
et al., 2003). This, however, has not always been effective,
1054-3139/$30.00
and now there is a need for and a move towards research
into cod-specific production protocols.
When rearing larvae immediately after yolk-sac absorption, the correct nutritional parameters in live feed are essential for larval cod performance. However, with species new
to aquaculture, a relatively narrow range of existing commercial proprietary live-feed enrichments must be used for
economic viability and to ascertain the optimal composition
of future products. To date, there have not been any definitive studies aimed at comparing or evaluating the optimal
live food enrichment procedures for cod (Brown et al.,
2003), in terms of either larval performance or dietary fatty
Ó 2005 International Council for the Exploration of the Sea. Published by Elsevier Ltd. All rights reserved.
Diet-induced differences in the EFA compositions of larval Atlantic cod
acid profile. Cod larvae will require long-chain, highly unsaturated fatty acids (HUFA; C 20, double bonds 3), because they may not elongate and/or desaturate 18-carbon
polyunsaturated fatty acids (PUFA; Sargent et al., 1999).
These requirements can be met to some extent by commercially available enrichment products, but the relative efficacy
of fatty acid uptake and subsequent effects of such
products on cod larval performance are largely unknown.
The natural plankton on which cod feed has a high content
of different types of fatty acids essential for good growth,
the most important being the n-3 essential long-chain fatty
acids (EFA), since cod may not be able to biosynthesize
these (Klungsoyr et al., 1989). The most important n-3
HUFA are docosahexaenoic acid (DHA; 22:6n-3) and eicosapentaenoic acid (EPA; 20:5n-3), which have been shown
to be essential for larval survival and growth in many marine
fish species (Watanabe, 1983, 1993; Koven et al., 1990; Rodriguez et al., 1997). They are essential because n-3 HUFA
are major components of membrane phospholipids, and
DHA is especially abundant in neural tissue membranes
(Tocher and Harvie, 1988; Bell and Dick, 1991). First-feeding larvae have a very high neurosomatic index, and early
developing larvae have a high requirement for n-3 HUFA,
which must be met by the diet if neural dysfunction is not
to occur (Bell et al., 1995; Sargent et al., 1997).
Much recent work has emphasized the importance of absolute levels and balances of n-3 HUFA to ensure larval development, but it has tended to overlook the dietary
requirement for n-6 HUFA, largely arachidonic acid
(20:4n-6; AA, Bell et al., 1997). Studies on AA have largely
dealt with its competitive interaction with EPA: both are
substrates for the formation of eicosanoids, a group of biologically active molecules that play a wide variety of physiological roles in fish, such as ionic regulation and the
induction of egg shedding in ripe females (Mustafa and Srivastava, 1989; Sargent, 1995; Copeman et al., 2002). Arachidonic acid is the preferred substrate, but EPA-producing
eicosanoids of lower biological activity modulate the efficacy
of AA (Lands, 1989). High levels of AA relative to EPA,
although causing no differences in survival and growth,
caused high levels of malpigmentation in Atlantic halibut
(Hippoglossus hippoglossus; McEvoy et al., 1998) and turbot (Scophthalmus maximus; Estévez et al., 1999). This
may have been the result of excess production of AAderived eicosanoids, which caused fish to experience dietaryinduced stress (Sargent et al., 1999) or an altering of the
neuroendocrine control of metamorphosis (Estévez et al.,
1999). However, AA-supplemented broodstock diet has
improved egg and larval quality in sea bass (Dicentarchus
labrax) and halibut (Sargent et al., 1999; Mazorra et al., 2003).
Similarly, relative levels of n-3 HUFA (DHA to EPA)
have received much attention, as have their absolute levels.
The DHA and EPA competition arises from both molecules
using the same enzymes to esterify fatty acids into phospholipids (Mourente et al., 1991). Since DHA is found at
very high levels in neural tissue, it has been hypothesized
303
that high dietary EPA relative to DHA has a negative impact on larval neural function and on subsequent growth
and survival; this was tested and found to be the case in larval herring (Clupea harengus; Bell et al., 1995).
It is imperative therefore that cod larvae be provided
with live feeds with the correct dietary composition of essential fatty acids (DHA, EPA, and AA). In commercial
production, the prey items e brine shrimp (Artemia salina)
and rotifer (Brachionus plicatilis) e are used predominantly.
Their fatty acid content has been shown to be the main
factor in their dietary value, and it has been demonstrated
(Koven et al., 1990; Czesny et al., 1999; Copeman et al.,
2002) that n-3 HUFA-supplemented diets have a positive
effect on larval growth and survival. However, both prey
items are considered to be suboptimal for larval nutrition,
especially when compared with wild prey such as copepods
(Nanton and Castell, 1999; Han et al., 2000; Cutts, 2003).
Even following enrichment in an emulsion containing large
amounts of HUFA, both rotifers and Artemia are still nutritionally poor. In addition to converting the ethyl esters of
PUFA (the form in which PUFA are frequently present in
commercial enrichment emulsions) to triacylglycerols
(TAG), Artemia can metabolize some of the assimilated
DHA (Fernandez-Reiriz et al., 1993; Rainuzzo et al.,
1994; McEvoy et al., 1996; Navarro et al., 1999), by retroconverting it to EPA (Sargent et al., 1999). This reduces the
levels of DHA available to fish larvae from the prey items.
Enrichment of live feed can be done by feeding with cultured algae (Gatesoupe, 1991), but microalgae can also vary
in fatty acid composition according to species and culture
technique (Volkman et al., 1989). Commercial enrichment
formulations, which are rich in HUFA, have been developed and are convenient for commercial operations. These
products include AlgaMac 2000 (Aquafauna Biomarine),
a spray-dried single cell protist, and DHA Protein Selco,
Protein Selco, and Super Selco (INVE Aquaculture), which
are lipid emulsions containing marine oils, vitamins, antioxidants, and emulsifiers. Enrichment of Artemia with Super Selco and AlgaMac improved growth and survival in
halibut larvae but they were inferior to wild zooplankton
in reducing malpigmentation (Naess et al., 1995; Gara
et al., 1998). In yellowtail flounder (Limanda ferruginea),
rotifers enriched with high DHA emulsions gave significantly higher growth and survival (Copeman et al., 2002).
Since there have been no definitive studies so far to determine the optimal live-feed requirements of cod larvae
(Brown et al., 2003), using commercial enrichments or otherwise, this study aims, through both regressional and variance analyses, to define the EFA characteristics of several
commercial enrichments that maximize larval performance.
EFA levels in cod larvae were compared in detail with EFA
levels in their corresponding dietary treatments and were related to performance. This allowed us to determine which
enrichments were most effective in yielding larval EFA levels appropriate to good performance. A standard reference
enrichment diet of AlgaMac 2000 plus Super Selco was
200
200
3
3
65
65
n/a
n/a
30
30
DC DHA Selco
DC Selco
75
88
12
3
29
42
5
5
DHA Protein Selco
Protein Selco
29
24
n/a
400
20
1
38
67
20
n/a
4
30
AlgaMac 2000
Super Selco
n/a
400
20
1
38
67
20
n/a
II
III
In addition to dietary samples, larval samples were taken at
days 10, 24, and 51 post-hatch. Live feed or larval samples
4
30
Lipid analysis
AlgaMac 2000
Super Selco
Twelve 100-l tanks were each stocked with 3000 disinfected stage V (sensu Thompson and Riley, 1981) cod
eggs. Routine husbandry regimes were established immediately post-hatch (Shields et al., 2003), with the exception of
the live-feeding regime. Samples of cod larvae at hatch
were taken for dry weight analysis in order to estimate
growth. Growth was calculated as specific growth rate
(SGR; % d1 ¼ [(ln (final dry weight) ln (initial dry
weight))/days] 100). The mean temperature throughout
the experiment was 10.5(C (range 9e11(C), and mean
salinity was 33.8 (range 33.7e34.3).
Three live-feeding regimes were administered. Rotifers
were fed until 25 days post-hatch, with Artemia being
added thereafter, following the method of Shields et al.
(2003). The regimes differed in terms of live-feed enrichment: there were three different treatments, each with four
tanks (see Table 1 for details of live-feed enrichments).
Treatment I was the standard Ardtoe treatment (control),
and was fed with rotifers enriched with AlgaMac 2000 until
day 10, rotifers enriched with Super Selco (50% of prey
items) and AlgaMac 2000 (50% of prey items), until day
25, and Artemia enriched with Super Selco (50%) and
AlgaMac 2000 (50%) thereafter (Table 1).
Ten larval cod from each tank were sampled for length,
dry weight, feeding status, and swimbladder inflation at 10,
24, and 51 (termination of the experiment) days post-hatch.
Furthermore, the tank bottoms were also thoroughly siphoned at regular intervals in order to estimate mortality
in each tank: subsamples of detritus collected from the
tank bottoms were examined under a microscope, mortalities counted, and total mortality assessed. Overall survival
was assessed at day 51 by the removal and counting of cod
juveniles from each tank.
I
Husbandry protocol
Artemia
enrichment
The experiment was carried out at Ardtoe Marine Laboratory,
Seafish Industry Authority [now part of SAMS (Scottish
Association of Marine Science)]. Fertilized Atlantic cod
eggs were obtained from a natural spawning population
maintained under ambient temperature and photoperiod conditions. The broodstock population was held in a 10-m diameter circular tank (volume 150 000 l) and fed with a moist
diet containing low temperature fishmeal and squid ad lib.
Table 1. Live-feed enrichments for three experimental treatments. Proximate analysis refers to manufacturer’s specifications of enrichment.
Source of experimental animals
Total n-3 HUFA
% Moisture % Protein % Lipid % Ash (mg g1 dry weight)
Material and methods
Rotifer
enrichment
used, because these products are ubiquitous live-feed enrichments in cold-water larviculture and constitute current
Ardtoe best practice; the diet was compared with larval
performance under two other dietary regimes.
Total n-3 HUFA
% Moisture % Protein % Lipid % Ash (mg g1 dry weight)
C. J. Cutts et al.
Treatment
304
Diet-induced differences in the EFA compositions of larval Atlantic cod
of at least 100 mg were collected and stored in glass vials
containing 5 ml of a 2:1 (vol/vol) mixture of chloroform:methanol with 0.01% (wt/vol) butylated hydroxytoluene
(C:M þ BHT) as antioxidant. Lipids were extracted in at
least ten volumes of C:M þ BHT, essentially by the method
of Folch et al. (1957). Total lipids were quantified gravimetrically after evaporation of the solvent under a stream
of nitrogen and overnight vacuum desiccation. Total lipids
were then stored in C:M þ BHT at a concentration of
10 mg total lipid per ml at 20(C until later analysis.
Fatty acid methyl esters from individual samples were
prepared by the addition of methylating solution [1% (vol/
vol) sulphuric acid in methanol] and toluene (2:1, vol/vol).
Following acid neutralization with 2% (wt/vol) KHCO3,
methyl esters were extracted twice using 5 ml isohexane:
diethyl ether (1:1, vol/vol), purified on TLC plates, and
analysed by gaseliquid chromatography (Carlo Erba Vega
8160, Milan, Italy) using a 30-m 0.32-mm id capillary
column (CP Wax 52 CB Chrompak, London, UK). Hydrogen was used as a carrier gas and temperature programming
was from 50(C to 150(C at 40(C min1 and then to 225(C
at 2(C min1. Individual methyl esters were identified by
comparison with known standards and by reference to
published data (Ackman, 1980). Data were collected and
processed using the Chromcard for Windows computer
package (Thermoquest Italia, Milan, Italy).
Performance data between experimental treatments were
assessed by one-way ANOVA, and data between individual
tanks were assessed by non-parametric Spearman’s R tests.
Statistical significance was taken as p < 0.05.
Results
There were no differences in survival between treatments at
the termination of the experiment at day 51 post-hatch
[mean survival (s.d.) ¼ 5.81 2.39%; one-way ANOVA:
F2,9 ¼ 0.408, p ¼ 0.677]. In addition, there were no significant differences in swimbladder development at day 10
(mean ¼ 39.9 21.8%; F2,9 ¼ 1.67, p ¼ 0.242) or day 24
(mean ¼ 80.6 14.8%; F2,9 ¼ 2.44, p ¼ 0.143). However,
larvae fed on the control diet grew significantly faster during the rotifer feeding stage (up to 25 days post-hatch) than
305
those from the experimental treatments (F2,9 ¼ 6.38,
p < 0.05; Table 2). Overall mean specific growth rate
(SGR) did not differ significantly between treatments
(mean ¼ 6.99 0.70%, F2,9 ¼ 2.95, p ¼ 0.103), since there
were no significant differences in growth during the
Artemia feeding stage (mean ¼ 13.1 1.45%, F2,9 ¼ 0.386,
p ¼ 0.691).
Furthermore, there were no significant differences in fatty
acid content of lipids (mg fatty acid per mg lipid) between
larvae from the three treatments [means ¼ 446.9 39.1
(Treatment I); 453.3 40.1 (Treatment II); 434.1 54.8
(Treatment III) mg mg1; F2,9 ¼ 0.188, p ¼ 0.832] at the
end of the trial, despite large differences in the composition
of the enrichments (Table 1).
Although there were few significant differences between
dietary treatments with regard to larval performance, the
mean biochemical characteristics of surviving larvae (i.e.
essential fatty acid content) from tanks across all treatments
could be compared with the performance parameters of
those tanks using non-parametric regression analyses. However, there were no significant correlations between mean
larval fatty acid content of lipids (mg fatty acid per mg lipid)
and survival or growth. Moreover, taking the trial as
a whole (rotifer and Artemia phases), the percentage lipid
values for the enrichments within each treatment did not
differ greatly from each other (Treatment I ¼ 50%, Treatment II ¼ 47%, Treatment III ¼ 53.5%). Therefore, percentage values of particular EFAs within the total fatty
acid content of both diets and larvae were assessed. For example, percentage survival at day 51 post-hatch correlated
strongly with DHA content of the surviving larvae (measured as a percentage of total fatty acid content; Spearman’s R ¼ 0.669, p < 0.05; Figure 1a). There was no such
correlation for percentage EPA, although there was a significant relationship between the DHA/EPA ratio of surviving
larvae and percentage survival (mean ¼ 3.91, range ¼
2.22e5.32; Spearman’s R ¼ 0.573, p ¼ 0.05), as a result
of increasing amounts of DHA promoting survival. Furthermore, there was a significant positive correlation between
levels of %AA and survival at day 51 (Spearman’s
R ¼ 0.605, p < 0.05; Figure 1b), although this relationship
was weaker than that of DHA.
Table 2. Mean dry weight (mg) and specific growth rate (SGR; % d1) at three different periods of the trial for three experimental
treatments, and overall mean SGR and percentage survival at the end of the trial. Errors denote standard deviation.
Days 1e10
Days 10e25
Treatment
Dry weight
(mg)
I
II
III
0.21 0.03 6.13 1.73 0.28 0.01 3.93 0.14 5.03 0.91
0.16 0.02 3.68 1.33 0.22 0.06 2.77 1.17 3.22 0.68
0.17 0.01 4.33 0.85 0.23 0.03 2.95 0.62 3.64 0.62
SGR
Dry weight
(mg)
Days 1e25
*Denotes statistical significance at p < 0.05.
SGR
SGR*
Days 25e51
Dry weight
(mg)
8.8 3.2
7.8 3.6
9.1 3.5
SGR
Days 1e51
Mean
SGR
Mean %
survival
12.55 1.25 7.54 0.94 5.52 1.86
13.07 1.54 6.50 0.28 6.72 3.30
13.51 1.79 6.93 0.35 5.18 2.20
C. J. Cutts et al.
Percentage survival
at day 51
12
a
Treatment I
8
Treatment II
Treatment III
4
0
2
4
6
8
10
Mean specific growth rate
(% d-1) up to day 10
306
4
2
0
20
Treatment II
Treatment III
4
0
3.5
Mean specific growth rate
(% d-1) up to day 10
Percentage survival
at day 51
8
3
Treatment III
25
30
35
DHA content of cod larvae at day 10
(% of total fatty acids)
Treatment I
2.5
Treatment II
6
12
b
2
Treatment I
8
DHA content of larvae at day 51
(% of total fatty acids)
12
a
10
10
Treatment II
8
Treatment III
6
4
2
0
4
5
6
AA content of cod larvae at day 10
(% of total fatty acids)
AA content of larvae at day 51
(% of total fatty acids)
Percentage DHA of total
fatty acids at day 10
Figure 2. The relationship between mean specific growth rate (%
d1) from day 1 to day 10 post-hatch and (a) mean DHA content
of cod larvae at day 10 (Spearman’s R ¼ 0.736, p < 0.01) and (b)
mean AA content of larvae at day 10 (R ¼ 0.675, p < 0.05). Both
EFA levels are expressed as a percentage of total fatty acids.
35
a
30
Treatment I
Treatment II
Treatment III
25
20
15
Diet
Percentage AA of total
fatty acids at day 10
In addition, specific growth rate also correlated positively
with DHA and AA levels (sampled at day 10) during the
first 10 days of feeding (DHA vs. SGR: Spearman’s
R ¼ 0.736, p < 0.01; AA vs. SGR: Spearman’s R ¼ 0.675,
p < 0.05; Figure 2).
Larvae fed on the control diet grew significantly faster
during the rotifer phase of feeding (Table 2). We found
that the control diet had significantly higher levels of
DHA than the other two diets (F2,9 ¼ 8.65, p < 0.01;
Figure 3a), but there was no significant difference in AA
levels (F2,9 ¼ 0.482, p ¼ 0.704; Figure 3b). Furthermore,
larvae fed on the control diet and sampled at day 10 had
higher levels of DHA than those from the other two treatments (F2,9 ¼ 4.03, p ¼ 0.05; Figure 3a), although this result was only weakly significant. In addition, although
there were no significant differences in AA levels among diets, larvae fed on the control diet had significantly higher
levels of AA by day 10 than those from the other two treatments (F2,9 ¼ 16.0, p < 0.005; Figure 3b).
Figure 3a shows that larvae from all dietary treatments
were capable of accumulating DHA by day 10 at significantly greater amounts than DHA present in the diet
[F1,6 ¼ 20.3 (Treatment I), 27.1 (Treatment II), and 99.0
(Treatment III); p < 0.01, 0.005, and 0.001, respectively].
Treatment I
3
4
Figure 1. The relationship between percentage survival at day 51
post-hatch and (a) mean docosahexaenoic acid (DHA) content of
surviving cod larvae (Spearman’s R ¼ 0.669, p < 0.05) and (b)
mean arachidonic acid (AA) content of surviving cod larvae
(R ¼ 0.605, p < 0.05). Both EFA levels are expressed as a percentage of total fatty acids.
b
5
Larvae
b
Treatment I
Treatment II
Treatment III
4
3
2
Diet
Larvae
Figure 3. Levels of essential fatty acids (expressed as a percentage
of total fatty acids) in three rotifer diets enriched with different
products and in the cod larvae fed on the diets (sampled at 10
days post-hatch) for (a) DHA and (b) AA. Error bars denote standard deviation. See text for statistical analysis.
Diet-induced differences in the EFA compositions of larval Atlantic cod
11
a
Treatment I
Treatment II
Treatment III
9
7
5
p ¼ 0.149), whereas there was a significant difference in
percentage levels among larvae fed on different treatments
(F2,9 ¼ 4.68, p < 0.05). This was most pronounced between
Treatment I (reference diet) and Treatment III. However,
none of the differences between DHA content of the diet
and the larvae fed on the diet were significantly different
(F1,9 ¼ 0.628, 1.81, and 0.024; p ¼ 0.451, 0.236, and
0.884 for Treatments I to III, respectively).
AA concentration was also lower than during the rotifer
phase, with the reference diet exhibiting higher levels than
Treatment III, although this was not quite significant
(F3,11 ¼ 2.85, p ¼ 0.069). However, cod larvae had higher
levels of AA compared with their diet by day 50 post-hatch
(Figure 4b). These levels were significantly higher for each
treatment (F1,9 ¼ 112.4, 63.2, and 125.3; p < 0.0001, 0.005,
and 0.0001 for the Treatments I to III, respectively). In addition, AA levels in larvae fed on Treatments I and III diets
were significantly higher than AA levels in larvae fed on
Treatment II (F2,9 ¼ 14.6, p < 0.005).
Figure 5 shows the changing levels of DHA and AA over
the 51 days of the trial. There were marked changes in larval DHA levels during the trial (Figure 5a). In comparison
with day 50, cod DHA levels were approximately six times
higher at days 10 and 22 when the larvae were still fed rotifers. These high levels were largely the result of higher
levels of DHA in all three rotifer diets and greater incorporation of DHA by the larvae. By day 50, DHA levels in the
larvae had dropped to levels similar to those in the Artemia
diet, with much reduced DHA concentration.
AA levels appeared to be far more conservative than
DHA levels throughout the trial (Figure 5b), reflecting the
Percentage DHA of
total fatty acids
Percentage DHA of total
fatty acids at day 50
Although larvae from the standard reference diet had a significantly higher %DHA than larvae from the other two
treatments, this was only just significant, and larvae from
Treatments II and III were able to incorporate similar
amounts from diets with much less DHA.
Conversely, Figure 3b shows that (in addition to no significant difference in AA content between the diets but a significant difference in %AA content between larvae fed on
the three treatments) larvae from Treatments II and III
did not show significant selective incorporation of AA (differences between dietary and larval levels: F1,6 ¼ 5.37 and
1.83, p ¼ 0.07 and 0.234 for Treatments II and III, respectively). However, by directly comparing %AA of total fatty
acid levels of both larvae and diet, larvae fed on the control
diet (Treatment I) exhibited significantly higher %AA levels than reflected in their diet (F1,6 ¼ 25.2, p < 0.005), suggesting a selective uptake and deposition of AA.
Figure 1 showed the relation of both DHA and AA levels
in larvae at the end of the experiment with larval survival in
individual tanks. Therefore, it is important to study any relationship between larval EFA levels and the corresponding
dietary EFA levels in the Artemia feeding phase of the experiment. Figure 4 shows that the %DHA of total fatty acids in
50-day-old larvae had been considerably reduced since day
10 post-hatch (mean ¼ 6.18 2.07 cf. 28.98 2.00%),
whereas %AA remained similar (mean ¼ 3.08 0.43 cf.
4.01 0.57%). This coincides with reduced concentration
of DHA in the Artemia (circa 6% of total fatty acids) with
respect to that found in the rotifers (20e30%).
Figure 4a shows that %DHA levels did not differ significantly among experimental Artemia diets (F3,11 ¼ 2.35,
3
Diet
307
40
a
Treatment I
Treatment III
30
Treatment II
20
10
0
10
Larvae
22
50
4
b
Treatment I
Treatment II
Treatment III
3
2
1
Diet
Larvae
Figure 4. Levels of essential fatty acids (expressed as a percentage
of total fatty acids) in three Artemia diet treatments and in the cod
larvae fed on the diets (sampled at 50 days post-hatch) for (a) DHA
and (b) AA. Error bars denote standard deviation. See text for statistical analysis.
Percentage AA of
total fatty acids
Percentage AA of total
fatty acids at day 50
Days post-hatch
6
5
b
Treatment I
Treatment III
Treatment II
4
3
2
1
0
10
22
50
Days post-hatch
Figure 5. Levels of (a) DHA and (b) AA (expressed as a percentage
of total fatty acids) in larval cod somatic tissue under three different
dietary treatments at 10, 22, and 50 days post-hatch.
308
C. J. Cutts et al.
lesser change of content in the two prey items. By day 10
post-hatch, all larvae, with the exception of control fish, reflected their diet with regard to %AA (control fish were able
to accumulate AA at levels nearly twice those of their diet;
Figure 4b). Despite the %AA in the Artemia diets being
substantially lower than that in the rotifer diets (1.5 cf.
3.0% on average; Figures 3b and 4b), by the end of the trial,
fish from each group were all exhibiting selective incorporation of AA, since its levels were approximately similar
throughout the trial.
Discussion
Larvae fed on the control treatment grew faster than those
from the other two dietary treatments, but only during the
rotifer feeding phase (hatch to 25 days post-hatch). However,
because of similar growth rates between treatments during the Artemia feeding phase, there were no significant
differences in overall growth.
Percentage levels of certain essential fatty acids appeared
to vary far more with performance parameters than either
percentage lipid in the enrichment diets or total fatty acid
content in the larvae themselves, so they are discussed
here in detail. During the rotifer feeding phase, it is therefore
worthwhile to note that growth rate correlated positively
with both larval DHA and AA levels when measured at 10
days post-hatch. As noted before, it is hard to disassociate
cause and effect with these two correlations: the larvae
may grow faster as a result of elevated levels of both DHA
and AA, or they may have elevated levels as a result of being
bigger and incorporating DHA and AA at a greater rate.
However, larvae in the control treatment grew faster during
the first 25 days, and the control diet had significantly higher
levels of DHA, but not AA. Furthermore, this was reflected
in the DHA levels of control larvae at day 10; they had
significantly more %DHA than the two experimental treatments. Although larvae fed on Treatment I had significantly
more DHA than those from the other two treatments, larvae
fed on Treatments II and III were able to incorporate comparable levels using diets with significantly less %DHA than
the standard reference diet. However, there was the slight
trend of enhanced %DHA levels in the control diet promoting %DHA levels in the faster growing control larvae during
the rotifer phase. Therefore, providing adequately high
levels of DHA to cod larvae may well promote growth, especially when supplied in the early feeding phase.
As with DHA, there was also a positive correlation between growth and %AA levels at day 10, albeit weaker.
However, unlike DHA levels in the three different rotifer
diets, AA levels did not differ significantly among dietary
treatments. Despite this, larvae from the control group
showed significant selective incorporation of AA, exhibiting levels significantly higher than both their diet and larvae from the two experimental treatments. Conversely,
larvae from the two experimental treatments did not exhibit
significant incorporation of AA over and above levels in
their diet. Since Treatment I larvae grew faster and showed
higher levels of both DHA and AA, and the reference diet
contained significantly more DHA (but not AA), higher levels of DHA and AA in the diet may improve cod larval
growth during this period. However, other factors not measured in this study, such as dietary total energy content and
digestibility, will also influence growth.
Nevertheless, the standard reference diet fed during the
rotifer feeding phase seemed to promote growth and also
yielded enhanced levels of larval %DHA, although larvae
from all treatments were able to incorporate DHA at significant rates. In addition, although AA levels did not differ
among diets, the ability to incorporate AA was markedly
higher in larvae fed on the reference diet.
The dynamics of EFA supplementation and uptake were
markedly different between the two prey items. Percentage
levels of larval somatic DHA had dropped markedly during
the Artemia phase, from 28.98% to 6.18%. This was a reflection of much lower DHA levels in Artemia from all dietary
treatments, with none of the larvae from each treatment expressing selective incorporation of DHA. Thus, Artemia enriched in the conditions of this experiment seemed unable to
meet the requirements of larvae for DHA. However, larvae
in Treatment III did show significantly more DHA than control larvae, although this was the result of low levels in the
reference group rather than specific incorporation of DHA
in Treatment III. There was a marked dietary difference between the rotifer and the Artemia phases, substantially owing
to differences in the final concentration of EFAs in the two
prey items. The poor DHA enrichment capability of Artemia
has been explained by their naturally high lipid content prior
to enrichment and the presence of nutritionally poor fatty
acids; a relatively high EPA content in unenriched nauplii;
and the rapid retroconversion of DHA to EPA following enrichment (Navarro et al., 1999; Bell et al., 2003). Furthermore, there is evidence that early developing marine fish
larvae have an absolute requirement for pre-formed phospholipids, since they cannot synthesize phospholipids de
novo in sufficient quantities to meet requirements (Teshima
et al., 1987). In addition, phospholipids derived from rotifers
(and copepods; Shields et al., 1999) can be incorporated directly into the cell membranes of larvae while still retaining
a profile of favourable HUFA, especially DHA. Conversely,
the concentration of DHA in the polar lipid fraction of
Artemia is very low (Bell et al., 2003).
However, larvae from each group retained the ability to
incorporate AA from lower levels in the Artemia. This was
done to such an extent that larval AA levels at the end of
the experiment were comparable with levels during the rotifer
feeding phase. Moreover, larvae fed on Treatments I and III
accumulated AA to a greater extent than those fed on Treatment II. Therefore, in contrast to their ability to accumulate
DHA, larval cod seem able to continue accumulating AA at
levels beyond its presence in Artemia. The inefficiency of using Artemia as a conduit for transferring EFAs from enrichment emulsions to larval fish is well known: Artemia
Diet-induced differences in the EFA compositions of larval Atlantic cod
catabolize lipids, converting surplus phospholipids into triacylglycerols, which are less easily assimilated by fish larvae.
Furthermore, Artemia store a large proportion of their EFAs
in their neutral lipids as opposed to polar membrane lipids.
This makes them less digestible, since it is polar lipids that
form lipid emulsions in the larval gut, facilitating lipid digestion (Sargent et al., 1999). This is a well known problem with
regard to DHA, but this study suggests that it is less so for
AA, which may reflect the distribution of DHA and AA
among different lipid classes (Sargent et al., 2002). This
preferential storage of AA has also been noted in gilthead
sea bream larvae (Rodriguez et al., 1998).
In addition to the significant effects of dietary and larval
EFAs on larval growth, mean larval levels of both DHA
and AA also correlated positively with individual tank survival at the end of the trial. This again demonstrates the importance of ensuring high levels of DHA and AA in cod
larvae for generally good performance.
Despite the inefficiency of Artemia as a live food, and acknowledging the importance of both DHA and AA, this
study suggests that, purely in terms of larval EFA composition, Treatment III is the highest performing diet during
the Artemia phase. This recommendation is based on the
finding that fish from this treatment exhibited higher
%DHA and comparable %AA to Treatment I fish, although
AA levels in their diet were almost significantly lower. The
study also highlights the relative efficacy of different commercially available live-feed enrichments, both in the relative amounts of EFAs in the live feed and their degree of
uptake by larval cod.
Acknowledgements
We thank Peter Smith and Les Ford for egg collection and
maintenance; Jon Sherwood, Kay Robins, and Hazel
Cameron for live feed and husbandry assistance; and James
Dick, Elizabeth MacKinlay, and Fiona McGhee for assistance with lipid analysis. The study was carried out as partial fulfilment of J.S.’s Master’s thesis, and was funded, in
part, by a grant awarded to Seafish Aquaculture by Highlands and Islands Enterprise, Scotland.
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