Ž .
Ostrowski and Divakaran, 1990; Castell et al., 1994; Bessonart et al., 1999 . Workers have reported that AA was preferentially retained in various species together with DHA
during starvation, suggesting a metabolic priority for conservation of certain fatty acids Ž
. Ostrowski and Divakaran, 1990; Rainuzzo et al., 1994; Izquierdo, 1996 . In turbot,
Ž dietary deficiencies in AA have resulted in high mortality and obvious pathology Bell
. Ž
. et al., 1985a , while Castell et al. 1994 reported a positive effect of AA on survival
Ž .
from levels ranging from 0.5–1.0. Bessonart et al. 1999 found that AA was more effective in improving survival of gilthead seabream larvae if provided in the presence
of a high dietary DHArEPA ratio. On the other hand, AA supplementation may also have adverse affects. Other studies on turbot and halibut found an increased incidence of
malpigmentation correlated with brain AA, whereas higher EPArAA dietary levels
Ž .
improved pigmentation McEvoy et al., 1998 . The above findings suggest that AA contributes to survival in larvae of a variety of
marine teleosts. However, the question remains whether AA is equally effective throughout larval development. Moreover, due to the possible effect of AA-based
eicosanoids on cortisol production during stress, the dietary AA effect on survival may be related to stress resistance. Consequently, the aim of the present study on the larvae
of the gilthead seabream was twofold. Firstly, to test the effect of dietary AA on growth and survival in early and late larval development. Secondly, to determine the influence
of dietary AA on larval growth and survival, prior to andror following handling stress.
2. Methods and materials
2.1. Screening study to determine optimal dietary AA leÕel during early larÕal rearing The determination of an optimal dietary AA level in early seabream larvae was
carried out in a screening study. In this experiment, five rotifer enrichment treatments, Ž
varying in their DHArAA ratios, were fed to 5–19 day old gilthead seabream Sparus .
aurata larvae in conical V-tanks. Following the handling stress of transfer to glass aquaria, all the larvae, from day 19 to day 35 post-hatching, were fed Artemia nauplii
Ž enriched on the commercial preparation AlgaMac 2000 Biomarine, Hawthorne, Califor-
. nia, USA . During this period, growth and accumulated mortality of larvae fed the
different rotifer treatments before transfer were monitored. The control treatment rotifers were fed a spray dried, lipid extracted algal meal mixed
Ž .
Ž .
with a phospholipid containing 35.9 of total fatty acids TFA as DHA DHA-PL . These ingredients were residual products from an industrial oil extraction process of the
Ž heterotrophically grown dinoflagellate Crypthecodinium sp.
Martek Biosciences, .
Columbia, Maryland, USA . The other rotifer treatments consisted of replacing 12.5, Ž
. 25 and 50 of the DHA-rich meal with a meal mixed with an AA-rich 52.5 TFA
Ž .
phospholipid AA-PL . This phospholipid and meal were residual products remaining from an industrial oil extraction process of the heterotrophically grown fungus
Ž .
Mortierella alpina Martek Biosciences, Columbia, Maryland, USA . A fifth treatment
Ž was a commercial enrichment preparation, AlgaMac 2000 Bio-Marine, Hawthorne,
. Ž
. California, USA . The component and selected fatty acid composition TFA of the
enrichment preparations and of the rotifers that consumed the different treatments are shown in Tables 1 and 2, respectively.
Ž .
The experimental system consisted of 25 conical V-tanks 400 l supplied with Ž
. continuously filtered seawater 25‰ at a flow rate of one tank exchange per day, where
the temperature was gradually raised from 198C to 218C. The tanks were stocked with Ž
y1
. 2-day-old gilthead seabream larvae 100 larvae l
, originating from spawns of locally maintained broodstock. The five rotifer treatments were tested in five tank replicates per
Ž treatment, where larvae from day 3 to day 19 were fed twice daily rotifers 7.5 rotifers
y1 y1
. ml
tank that were previously enriched for 18 h with Nannochloropsis sp.
Ž
6 y1
. 30 = 10
cells ml , followed by their respective enrichment treatments. Based on
Ž .
routine rearing procedures for gilthead seabream Tandler et al., 1987 , Nannochloropsis sp. was also added to the larval rearing tanks to reach a tank concentration of 0.5 = 10
6
cells ml
y1
. Rotifer enrichments were carried out in V-tanks containing 600 l of well-aerated
Ž .
seawater 25‰ thermostatically controlled at 288C and stocked with approximately 300–500 rotifers ml
y1
. Each of the five rotifer-enrichment tanks received a 100 mg l
y1
feeding of enrichment preparation at the start of feeding and after 4 h during the 8 h enrichment period. At the end of rotifer enrichment, the rotifers were washed well with
clean filtered seawater, dead rotifers removed and the surviving zooplankters counted before feeding to the larvae. Larvae were fed according to this protocol until they
reached 19 days post-hatching and then sampled for dry weight determination.
The second stage of the study commenced with the transfer and stocking of 20-day-old larvae from each of the DHArAA treatments into five 27 l glass aquaria
Table 1 Components and selected fatty acids of the enrichment preparations and the commercial product AlgaMac
2000 Components
DHA-PL ARA-PL
AlgaMac q algal meal
qalgal meal 2000
Proximate analysis Ž
. Total fat dry wt
48.2 29.4
32.0 Ž
. Total carbohydrate dry wt
27.5 40.0
13.0 Ž
. Protein dry wt
6.9 8.0
39.0 Ž .
Moisture 3.0
5.0 3.0
Ž .
Fiber dry wt 0.4
3.0 0.0
Ž .
Ash dry wt 9.6
4.0 12.0
Selected Fatty acids of TFA Ž
. DHA 22:6 ny3
35.9 2.4
24.0 Ž
. AA 20:4 ny6
0.0 52.5
0.0 Ž
. EPA 20:5ny3
0.0 0.2
0.6 Ž
. DPA 22:5ny6
0.0 0.0
12.9
Table 2 Ž
y1
. The values mg g
, SEM of selected fatty acids and fatty acid ratios of the rotifers fed the enrichment treatments
AlgaMac DHA-PL
12.5 AA 25 AA
50 AA Ž
. DHA 22:6 ny3
7.72.0 6.51.4
5.70.1 6.22.2
4.20.2 Ž
. EPA 20:5ny3
3.40.8 2.30.4
3.01.0 2.20.8
2.51.0 Ž
. DPA 22:5ny6
2.20.6 0.00.0
0.10.1 0.00.0
0.00.0 Ž
. AA 20:4 ny6
1.50.3 0.80.3
1.70.2 3.01.0
5.30.6 DHArEPA
2.30.6 3.00.6
2.40.7 2.80.1
2.30.7 DHArAA
5.21.4 9.11.6
3.40.4 2.10.1
0.80.1 EPArAA
2.20.0 3.40.9
1.70.3 0.70.0
0.50.2
Ž
y1
. 200 larvae aquarium
. The aquaria were supplied with filtered seawater whose temperature was gradually increased from 218C to 248C during the feeding trial. From
day 20 to day 34, the larvae from the 25 glass aquaria were fed Artemia nauplii twice Ž
y1
. daily 1 nauplii ml
that were enriched on AlgaMac 2000. Dead larvae from all the aquaria were collected each day and recorded for the first 7 days following transfer.
2.2. The dietary AA effect on growth and surÕiÕal in early and late larÕal seabream deÕelopment
Following the determination of the optimal level of AA for early larval performance, a study was conducted to determine the effect of dietary AA on growth and survival in
early and late larval seabream development. In this experiment, the treatment that gave the best larval performance in the screening trial, the 12.5 AA-PL supplement
Ž .
AADHA , was compared with the high DHA but AA-deficient treatment DHA-PL and Ž
. the commercial product AlgaMac 2000 ALGA . The experimental tank system was
identical and similarly stocked with larvae as described in the screening study. The three rotifer treatments were prepared, as detailed earlier, and tested on 3–19 day old larvae in
eight tank replicates per treatment. At the end of the experiment, 19-day-old larvae were sampled for fatty acid analysis and dry weight determination.
The following day, 20-day-old larvae from each of the DHA-PL and AADHA Ž
treatments were transferred and stocked into sets of 12 glass aquaria 27 l, 300 larvae
y1
. aquarium
supplied with filtered and temperature controlled seawater, as outlined in the screening study. Each set of 12 aquaria per treatment was subdivided into three
Ž groups of four so that the three Artemia enrichment treatments DHA-PL, AADHA and
. ALGA could be tested in replicates of four on larvae that were previously fed DHA-PL
or AADHA enriched rotifers. The commercial control larvae from the ALGA treatment Ž
y1
. were stocked 300 larvae aquarium
in three 27 l glass aquaria and were fed ALGA enriched Artemia.
The Artemia nauplii enrichments were carried out in 600 l V-tanks supplied with Ž
. well-aerated filtered seawater 25‰ thermostatically controlled at 288C and stocked
with approximately 200 nauplii ml
y1
. The Artemia were then fed a ration of enrichment
Ž
y1
. preparation 300 mg l
at the onset of feeding and after 8 h during the 16 h enrichment period. At the end of enrichment, the Artemia were washed well with clean
seawater, dead nauplii removed and the remaining Artemia counted. From day 20 to day 34, the larvae from the 27 glass aquaria were fed their respective Artemia
Ž
y1
. treatments twice daily 1 nauplii ml
. Dead larvae from all the aquaria were collected each day and recorded.
During the course of the experiment, Artemia and rotifer samples were taken on three separate occasions for lipid and fatty acid analyses. At the completion of the
experiment, the larvae from each of the aquaria were counted and samples were taken for dry weight determination and for lipid and fatty acid analyses. The average larval dry
weight per treatment was determined by washing a sample from each of the aquaria, first with fresh water, and then, distilled water. This was followed by averaging 50
Ž .
individually weighed larvae Sartorius BP 210S 0.1 mg that had previously been oven dried for 48 h at 608C.
The rotifer, Artemia and larval samples were lyophilized and then lipid extracted Ž
. Ž
Folch et al. 1957 . Total lipid levels were determined gravimetrically Sartorius BP .
210S 0.1 mg . The lipid samples were transmethylated to their corresponding fatty Ž
. acid methyl esters FAME by acidified methylation overnight at 508C in 1 H SO in
2 4
Ž .
methanol vrv . After purification on 20 = 20 cm TLC plates pre-coated with silica gel Ž
. Ž
G60 E. Merk, Darmstadt, FRG using the solvent system hexanerdiethyl ether 1:1, .
Ž
y1
. vrv , the resulting FAME were concentrated in hexane 2 mg FAME ml
hexane . The samples were injected into an on-column Chrompack CP9001 gas chromatograph
equipped with a Chrompack WCOT fused silica 30 M = 0.32 mm capillary column that used hydrogen as a carrier gas. FAME were identified by known purified standards and
Ž .
quantified using a response factor to an internal standard heptadecanoic acid, 19:0 . Following testing for homogeneity of variance, the growth, cumulative mortality and
survival data were analyzed using the statistical software package SPSS for Windows Ž
. Version 6.1.3 SPSS, Chicago, IL, USA to perform one-way analysis of variance
Ž .
ANOVA and the Duncan’s multiple range test.
3. Results
