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
Growth of 5–19 day old seabream larvae at the end of the screening study was Ž
. independent of dietary AA level and not
P 0.05 different among the rotifer
Ž .
treatments data not shown . However, the results indicated that larvae fed rotifers Ž
y1
. enriched on the 12.5 AA-PL 1.8 mg AA g
DW replacement of the DHA-PL Ž
. consistently gave the lowest, although non-significant P 0.05 , accumulated mortality
throughout the 7 days, following transfer than larvae fed the 0 AA-PL replacement or Ž
y1
. Ž
y1
. Ž
DHA-PL 0.8 mg AA g DW , 25 AA-PL 3.0 mg AA g
DW , 50 AA-PL 5.3
y1
. Ž
y1
. Ž
. mg AA g
DW and the ALGA 1.5 mg AA g DW rotifers Fig. 1 .
Ž .
In general, the selected fatty acids and fatty acid classes in rotifers Table 3 and Ž
. Artemia Table 4 from the main study on early and late larval development reflected the
Ž .
fatty acid content of the enrichment preparations they were fed Table 2 . In rotifers, the
Ž
y1
. Fig. 1. Accumulated larval mortality, as a function of rotifer treatment mg AA g
DW , in the screening study after transfer to aquaria and start of Artemia feeding.
Ž
y1
. AA levels were highest in the AADHA 2.7 mg g
DW , compared to the DHA-PL Ž
y1
. and ALGA treatments 0.8 and 1.3 mg g
DW, respectively , while DHA levels did
Table 3 Ž
y1
. Selected fatty acids and fatty acid groups mg g
DW found in rotifers Rotifer treatments
Fatty acids AADHA
DHA-PL ALGA
Saturates 10.90.3
11.40.3 16.73.2
Monounsaturates 25.13.2
25.30.2 9.40.1
ny3 Polyunsaturates 19.11.7
22.31.3 17.30.6
ny6 Polyunsaturates 7.40.8
5.20.1 6.60.3
ny3r ny6 Polyunsaturates 2.60.5
4.30.2 2.60.1
Ž .
DHA 22:6 ny3 14.71.6
16.01.6 13.40.7
Ž .
EPA 20:5ny3 2.90.1
4.20.6 2.50.1
Ž .
AA 20:4 ny6 2.70.3
0.80.1 1.30.0
Ž .
DPA 22:5ny6 0.60.6
0.10.1 4.40.3
DHArEPA 5.10.4
3.90.9 5.40.5
DHArAA 5.61.2
20.95.0 10.10.4
AArEPA 0.90.1
0.20.0 0.50.0
Table 4 Ž
. Ž
. Artemia enriched on the arachidonic acid AADHA , docosahexaenoic acid DHA-PL and the AlgaMac
Ž .
ALGA enrichments Artemia treatments
Fatty acids AADHA
DHA-PL ALGA
Saturates 15.50.9
19.31.4 20.20.8
Monounsaturates 36.22.4
47.24.6 20.10.6
Polyunsaturates 10.60.6
12.81.1 7.70.2
ny3 Polyunsaturates 40.13.3
51.63.8 30.81.1
ny6 Polyunsaturates 10.81.2
8.90.8 7.70.3
ny3r ny6 3.80.3
5.80.2 4.00.1
Ž .
DHA 22:6 ny3 15.32.9
22.92.2 8.90.5
Ž .
EPA 20:5ny3 5.00.3
7.40.7 4.20.1
Ž .
AA 20:4 ny6 4.60.9
0.90.1 1.90.1
Ž .
DPA 22:5ny6 0.00.0
0.10.1 2.90.2
DHArEPA 3.00.4
3.20.3 2.10.1
DHArAA 3.70.8
25.83.8 4.60.1
AArEPA 0.90.2
0.10.0 0.50.0
not vary markedly. This resulted in the AADHA treatment rotifers having significantly Ž
. Ž
. Ž
. Ž
. P - 0.05 lower DHArAA ratios 5.6 than the DHA-PL 20.9 and ALGA 10.1
Ž
y1
. treatments. Noteworthy is the elevated level of 22:5n y 6 4.4 mg g
DW in the ALGA rotifers, reflecting the relatively high levels of this fatty acid in this enrichment
Ž .
Ž .
preparation Table 1 . Similarly, Artemia nauplii Table 4 fed the AADHA treatment Ž
y1
. demonstrated the highest levels of AA 4.6 mg g
DW compared to the DHA and Ž
y1
. ALGA nauplii 0.9 and 1.9 mg g
DW, respectively . On the other hand, the DHA Ž
y1
. levels in the AADHA and DHA-PL nauplii 15.3 and 22.9 mg g
DW, respectively Ž
y1
. were considerably higher than the ALGA nauplii 8.9 mg g
DW . This resulted in the Ž
. DHArAA ratios in the AADHA nauplii 3.7 being lower but similar to the ALGA
Ž .
nauplii 4.6 , while both these treatment nauplii were much smaller than the ratio in the Ž
. DHA-PL nauplii 25.8 . As in rotifers, the level of 22:5n y 6 in the ALGA nauplii was
Ž
y1
. notably higher 2.9 mg g
DW than the trace levels in the AADHA and DHA-PL Ž
y1
. nauplii 0.0 and 0.1 mg g
DW, respectively . The absence or presence of dietary AA in rotifers or Artemia had no discernible
Ž .
effect on larval growth Fig. 2 . On the other hand, an effect of dietary AA fed prior to handling stress on accumulated mortality following handling stress was very clear
Ž .
P - 0.05 . Larvae fed the AADHA rotifers in the V-tanks prior to transfer to the aquaria showed a markedly and consistently reduced daily accumulated mortality after
Ž .
transfer than the DHA-PL and ALGA larvae Fig. 3a . This trend was verified by the Ž
. Ž
. Ž
. higher P - 0.05 survival Fig. 3b at the end of the experiment 35 days post-hatching
Ž .
in larvae fed the AADHA rotifers 42.9 compared to larvae fed the ALGA rotifers Ž
. Ž
. 31 . The ALGA rotifer fed larvae, in turn, survived better P - 0.05 than larvae fed
Ž .
the DHA-PL rotifers 17.5 . In contrast, the absence or presence of dietary AA in Artemia had no effect on accumulated mortality after the handling stress, regardless if
Ž . Fig. 2. a The effect of the AADHA, DHA-PL and ALGA rotifer treatments on larval dry weight prior
Ž . transfer to aquaria and b the effect of AADHA, DHA-PL and ALGA Artemia treatments on larval dry
weight at end of feeding trial in the aquaria.
Ž .
Ž .
the fish were fed AADHA Fig. 4a or DHA-PL Fig. 4b rotifers prior transfer to the aquaria.
Ž . Fig. 3. a Larval accumulated mortality occurring in the aquaria as a function of the rotifer treatments fed
Ž . Ž .
prior to transfer and b larval survival at the end of Artemia feeding in the aquaria. Survival values Ž
. having the same letters were not significantly P - 0.05 different.
The selected fatty acids and fatty acid classes of day 19 and day 35 larvae are shown Ž
. in Tables 5–7. The fatty acid composition of day 19 larvae Table 5 showed similar
Fig. 4. Accumulated mortality occurring in the aquaria as a function of the Artemia treatments in larvae fed Ž .
Ž . the a AADHA or b DHA-PL rotifers prior transfer to the aquaria.
levels of DHA, while those fed the AADHA rotifers demonstrated the highest levels of Ž
y1
. AA 2.7 mg g
DW , compared to the same age larvae fed the DHA-PL and ALGA
Table 5 Selected fatty acids and fatty acid groups in day 19 larvae fed the DHA-PL, AADHA and ALGA rotifer
treatments Rotifer treatments
Fatty acids AADHA
DHA-PL ALGA
Saturates 15.21.5
17.42.6 14.00.9
Monounsaturates 10.50.9
13.91.5 8.70.7
Polyunsaturates 5.71.2
5.20.5 3.50.3
ny3 Polyunsaturates 17.10.8
20.31.5 17.31.6
ny6 Polyunsaturates 5.00.2
4.30.4 5.80.5
Ž .
DHA 22:6 ny3 11.70.6
14.31.3 12.91.1
Ž .
EPA 20:5ny3 3.50.2
3.80.1 2.70.3
Ž .
AA 20:4 ny6 2.70.1
1.80.1 2.10.2
Ž .
DPA 22:5ny6 0.20.1
0.10.1 2.60.2
DHArEPA 3.40.2
3.70.2 5.00.3
DHArAA 4.40.1
7.80.3 6.10.1
AArEPA 0.80.0
0.50.0 0.80.0
Ž
y1
. Ž
rotifers 1.8 and 2.1 mg g DW, respectively . The DHArAA ratios in day 19 Table
. Ž
. 5 and day 35 larvae Tables 6 and 7 consistently followed the treatment pattern of
DHA-PL ALGA AADHA, which agreed with the pattern of decreasing accumula- tive mortality. On the other hand, the AA levels of ALGA rotifer and ALGA Artemia
fed larvae were always higher than expected considering that AA was absent in both the
Ž .
DHA-PL and ALGA enrichment preparations Tables 5, 6 and 7 . In fact, the AA levels of larvae fed first the AA rotifers and then the ALGA Artemia were virtually identical
Table 6 Selected fatty acids and fatty acid groups in day 35 larvae fed the AADHA rotifer treatment prior to transfer
followed by the Artemia treatments following transfer Artemia treatments
Fatty acids AADHA
DHA-PL ALGA
Saturates 10.71.6
12.70.8 15.91.9
Monounsaturates 13.01.8
16.01.1 14.51.8
Polyunsaturates 4.80.7
5.40.4 6.00.7
ny3 Polyunsaturates 15.02.2
19.71.4 18.92.6
ny6 Polyunsaturates 4.90.7
3.60.2 6.80.9
Ž .
DHA 22:6 ny3 7.61.1
10.70.7 7.91.1
Ž .
EPA 20:5ny3 2.20.3
3.50.3 3.50.5
Ž .
AA 20:4 ny6 2.80.4
1.00.1 2.70.4
Ž .
DPA 22:5ny6 0.00.0
0.00.0 2.00.3
DHArEPA 3.40.2
3.100.0 2.30.0
DHArAA 2.70.1
10.30.2 2.90.0
AArEPA 1.30.0
0.30.0 0.80.0
Table 7 Selected fatty acids and fatty acid groups in day 35 larvae fed the DHA-PL rotifer treatment prior to transfer
followed by the Artemia treatments following transfer Artemia treatments
Fatty acids AADHA
DHA-PL ALGA
Saturates 15.20.8
14.51.0 16.71.7
Monounsaturates 19.01.0
20.41.5 16.11.6
Polyunsaturates 6.60.3
6.90.5 6.70.6
ny3 Polyunsaturates 21.01.2
21.02.8 20.01.1
ny6 Polyunsaturates 7.60.5
4.40.3 7.00.4
Ž .
DHA 22:6 ny3 10.00.4
8.92.7 8.00.2
Ž .
EPA 20:5ny3 3.40.3
4.70.3 3.80.4
Ž .
AA 20:4 ny6 4.20.1
1.10.1 2.90.2
Ž .
DPA 22:5ny6 0.30.3
0.00.0 1.90.1
DHArEPA 3.00.1
1.90.6 2.10.2
DHArAA 2.40.0
7.52.2 2.80.2
AArEPA 1.20.1
0.30.0 0.80.0
Ž
y1
to those of larvae fed both AADHA rotifers and Artemia 2.7 and 2.8 mg g DW,
. respectively .
4. Discussion
