1. Introduction
Ž .
The long-chain highly unsaturated fatty acids HUFAs , particularly eicosapentaenoic Ž
. Ž
. acid EPA, 20:5n y 3 and docosahexaenoic acid DHA, 22:6 n y 3 , are important in
Ž .
the nutrition of young marine fish Kanazawa, 1985; Watanabe et al., 1989 . Various investigators have used DHArEPA ratios as an index of the optimal level required for
Ž normal growth and development in fish larvae Koven et al., 1993; Mourente et al.,
1993; Rainuzzo et al., 1994; Reitan et al., 1994; Tocher et al., 1997; Rodriguez et al., .
1998 . This is based on the proposition that optimum DHA and EPA levels are Ž
. determined not on total amounts per se as excess can be harmful but rather on the
Ž .
Ž relative proportions of these essential fatty acids EFAs in the diet Watanabe and
. Kiron, 1994; Rodriguez et al., 1997, 1998 . Apart from DHA and EPA, arachidonic acid
Ž .
Ž ARA, 20:4 n y 6 has also been recognized as essential for marine fish Castell et al.,
. 1994 . ARA is the main precursor of eicosanoids responsible for osmoregulation,
Ž cardiovascular functions, neural control and reproduction
Mustafa and Srivastava, .
Ž .
1989 . Sargent et al. 1997, 1999 have suggested that desirable ratios of 22:6 n y 3r20:5n y 3r20:4 n y 6 can be useful in determining optimal requirements in fish
larval nutrition. Ž
. Opercular abnormalities in fish affect its morphology Koumoundouros et al., 1997
Ž .
and biological performance Andrades et al., 1996; Sumagaysay et al., 1999 . Shortened operculum and distortion of the support cartilage have been described to be signs of
Ž .
nutritional deficiency e.g., vitamin C in fish caused by impaired collagen formation Ž
. and support cartilage formation Halver et al., 1975 . Some researchers have theorized
Ž .
that opercular deformities e.g., milkfish are caused by mechanical stress, especially Ž
during egg collection, transport or routine hatchery operations Toledo et al., 1996; .
Ž .
Hilomen-Garcia, 1997, 1998 . Toledo et al. 1996 even recommended transporting milkfish eggs at C-shaped embryonic stage to improve viability. Other causes such as
Ž .
genetic variations or factors, however, cannot be discounted Sargent, 1995 . Recently, Ž
. Gapasin et al.
1998 observed that the incidence of opercular deformities among hatchery-reared milkfish larvae could be alleviated by feeding them live food supple-
mented with EFAs and vitamin C. The DHArEPA ratios reported in that study ranged from 0.33–0.74 for HUFA-enriched rotifers and Artemia nauplii while values for the
unenriched live food were very low ranging from 0.01–0.04.
This follow-up study was therefore conducted to determine whether increasing the Ž
. DHA levels and the corresponding DHArEPA ratio to a value of at least G 1.0 in live
Ž .
food organisms using commercially available enrichers could further improve milkfish larvae performance.
2. Materials and methods
2.1. Milkfish eggs Ž
. Natural spawned eggs used for trial 1 came from milkfish Chanos chanos spawners
reared at SEAFDECrAQD Tigbauan Main Station in a 10 = 10 = 10 m concrete tank
whereas eggs for trial 2 came from broodstock maintained at SEAFDECrAQD Igang Marine Substation in a 10-m diameter = 3-m deep sea cage. Eggs were incubated in
500-l capacity circular, flat-bottomed, fiberglass tanks following standard procedures Ž
. Gapasin and Marte, 1990; Gapasin et al., 1998 .
2.2. LiÕe food enrichment Ž
. Ž
Rotifers Brachionus rotundiformis, S-type were first cultured in baker’s yeast Red .
Ž Star brand for 3 days and then enriched with DHA Protein Selco INVE Aquaculture,
. Ž
. Dendermonde, Belgium on the 4th day as described by Lavens et al. 1994 with
modification. Rotifers cultured extensively in Chlorella sp. served as a control. Ž
. Artemia nauplii were hatched following standard practice Sorgeloos et al., 1986 and
Ž .
enriched thereafter with DHA Selco INVE Aquaculture, Dendermonde, Belgium
Ž .
following the procedures of Leger et al. 1987 . Newly hatched Artemia served as a control.
2.3. LarÕal culture Newly hatched milkfish larvae were estimated volumetrically and stocked in 5-ton
circular, concrete larval rearing tanks at a density of 30 larvaerl. Water management Ž
. and feeding scheme followed previous protocol outlined in Gapasin et al. 1998 . The
Table 1 Ž
. Certain fatty acids
in the total lipids dry weight basis from Brachionus rotundiformis fed Chlorella sp. Ž .
Ž . A and rotifers enriched with a commercial diet, DHA Protein Selco B
Fatty acid A
B
b a
14:0 0.180.02
0.260.03
b a
16:0 1.610.16
2.010.15 18:0
0.600.12 0.700.08
b a
18:1ny9 1.870.09
2.260.04
b a
18:2 ny6 0.450.08
0.820.10
b a
18:3ny3 1.010.06
1.450.05
b a
18:4 ny6 0.020.00
0.700.03
b a
20:1ny9 0.100.00
0.250.01
b a
20:4 ny6 0.260.04
0.581.09
b a
20:5ny3 0.450.03
0.850.01 22:4 ny6
0.240.03 0.260.05
22:4 ny3 –
0.150.03 22:5ny6
– 0.090.01
b a
22:5ny3 0.050.00
0.470.03
b a
22:6 ny3 0.030.00
1.280.01
b a
Sny6 0.970.12
2.450.16
b a
Sny3 1.540.16
4.200.05
b a
DHArEPA 0.070.00
1.500.04 Sny3r Sny6
1.590.07 1.710.06
Ž .
Means S.E.M.
within rows not bearing the same letter superscripts are significantly different Ž
. P - 0.05 .
Ž .
Ž . Ž
. treatments with three replicates were as follows: a Trt 1 control — larvae fed
Ž . Chlorella-cultured rotifers and newly hatched Artemia nauplii and b Trt 2 — larvae
fed rotifers and Artemia nauplii enriched with DHA Protein Selco and DHA Selco, Ž
. respectively. Fish larvae n s 20–25 were sampled for total length every week from
Ž .
each replicate tank. Percent survival surviving fishrinitial stock = 100 were deter- Ž
. mined at harvest day 25 . Two larviculture trials were conducted.
Ž Harvested 25-day old larvae were packed in separate plastic bags according to
. Ž
treatments , transported by land and stocked in 27 = 38 m earthen nursery ponds three .
2
replicates at a density of 15–20 larvaerm . Prior to stocking, ponds were fertilized and Ž
. prepared following standard practice Lijauco et al., 1979 . Fish were reared extensively
Ž .
on natural food present in ponds for 60 days after which the young juveniles or Ž
. fingerlings n s 100 were randomly sampled. Live fish were individually examined for
Ž .
opercular deformities following procedures in Gapasin et al. 1998 . Of the 100
Ž .
Ž .
fingerlings per replicate pond checked for abnormalities, half of these n s 50 were sampled for total length measurements. The rest of the fish stock were seined and
Ž .
counted to determine survival done only for trial 2 . 2.4. Lipid extraction and FAME analysis
Ž .
Random samples of rotifers Chlorella-cultured or DHA Protein Selco-enriched , Ž
. Ž
Artemia nauplii newly hatched or DHA Selco-enriched and 25-day old fish larvae fed
Table 2 Ž
. Ž .
Certain fatty acids in the total lipids dry weight basis from newly hatched Artemia nauplii A and
Ž . Artemia metanauplii enriched with a commercial diet, DHA Selco B
Fatty acid A
B 14:0
0.060.01 0.080.01
b a
16:0 0.310.03
0.470.05 18:0
0.210.06 0.280.01
18:1ny9 0.520.06
0.660.07
b a
18:2 ny6 0.130.02
0.260.05
b a
18:3ny3 0.470.01
0.780.05
b a
18:4 ny6 0.140.02
0.280.04 20:1ny9
– 0.100.02
b a
20:4 ny6 0.050.00
0.190.04
b a
20:5ny3 0.030.00
0.480.05
b a
22:4 ny6 0.020.00
0.140.04 22:4 ny3
– 0.110.02
22:5ny6 –
0.020.00 22:5ny3
– 0.080.04
22:6 ny3 –
0.610.05
b a
Sny6 0.330.04
0.890.02
b a
Sny3 0.510.01
2.060.09 DHArEPA
– 1.270.04
b a
Sny3r Sny6 1.540.07
2.310.05 Ž
. Means
S.E.M. within rows not bearing the same letter superscripts are significantly different
Ž .
P - 0.05 .
. unenriched or DHA-enriched live food were periodically taken and placed in coded-
Ž plastic vials. For reference and comparison, newly spawned milkfish eggs
from .
broodstock maintained at SEAFDECrAQD Igang Marine Substation and wild milkfish Ž
fry approximately 3-week old postlarvae seined by fry collectors along Tigbauan
. Ž
. coastal waters were also collected. All samples were kept in deep freeze y708C until
Ž .
analyzed for total lipids and fatty acid methyl esters FAME . Ž
Total lipids were extracted from triplicated pooled samples except for the eggs and .
wild milkfish fry which had only one pooled replicate sample after homogenization in Ž
. Ž
. chloroformrmethanol 2:1, vrv with 0.05 butylated hydroxytoluene BHT following
Ž .
the method of Folch et al. 1957 . Extracted lipid fractions were saponified with 0.5 N KOH in methanol and fatty acids were esterified in 14 boron triflouride–methanol
Ž .
complex Metcalfe et al., 1966 . FAMEs were resuspended in isooctane, flushed with Ž
. nitrogen in glass vials and stored in deep freeze y708C before injection into the
chromatograph. Fatty acid compositions were analyzed using a Shimadzu gas–liquid chromatograph
Ž .
Ž GC-4PTF, Japan equipped with flame ionization detector initial temperature: 1508C
. for 5 min, program rate: 108Crmin, final temperature: 2008C , using a 30 m = 0.32
Ž .
mm = 0.2 m film thickness SPB-PUFA capillary column Supelco, USA . The chro- Ž
matograph was linked to a Shimadzu integratorrrecorder Chromatopac C-R7A Plus,
Table 3 Ž
. Certain fatty acids
in the total lipids dry weight basis from whole body tissues of 25-day old milkfish Ž .
larvae fed Chlorella-cultured rotifersrnewly hatched Artemia sp. A , larvae fed DHA Protein Selco-enriched Ž .
Ž . Ž .
rotifersrDHA Selco-enriched Artemia sp. B , wild milkfish fry C and newly spawned milkfish eggs D Fatty acid
A B
C D
b a
14:0 0.030.00
0.190.05 0.14
0.08
b a
16:0 0.700.01
0.900.03 1.21
2.77 18:0
0.370.02 0.450.05
0.38 0.83
b a
18:1ny9 0.400.01
0.600.05 0.24
2.18
b a
18:2 ny6 0.040.00
0.210.01 0.13
0.65
b a
18:3ny3 0.190.02
0.400.05 0.09
0.05 18:4 ny6
0.010.00 0.020.01
0.09 0.09
20:1ny9 0.140.02
0.120.01 0.01
0.12 20:4 ny6
0.130.01 0.160.02
0.12 0.36
b a
20:5ny3 0.300.02
0.390.02 0.42
0.29 22:4 ny6
0.030.00 0.030.00
0.01 0.02
22:4 ny3 0.140.02
0.120.01 0.01
0.01 22:5ny6
0.070.03 0.110.02
0.10 0.03
b a
22:5ny3 0.060.00
0.090.01 0.04
0.13
b a
22:6 ny3 0.160.01
0.600.04 0.96
1.99
b a
Sny6 0.290.01
0.530.01 0.45
1.16
b a
Sny3 0.850.01
1.580.02 1.52
2.47
b a
DHArEPA 0.530.01
1.540.09 2.28
6.75 Sny3r Sny6
2.930.11 2.980.13
3.41 2.14
Ž .
Means S.E.M.
within rows not bearing the same letter superscripts are significantly different Ž
. P - 0.05 .
Ž Fig. 1. Growth of 25-day-old milkfish fed Chlorella-cultured rotifers and newly hatched Artemia nauplii Trt
. Ž
. 1, control and DHA-enriched rotifers and Artemia metanauplii Trt 2, DHA-treated . Each data point
Ž .
represents mean total lengthS.E.M. ns 3, 20–25 fish per replicate sample .
. Japan . Individual fatty acids were identified by comparing the retention times with
Ž .
commercially available standards Sigma-Aldrich, USA . 2.5. Statistical analysis
Ž Total length, percent survival and deformities log- or arcsine-transformed where
. appropriate , individual fatty acid levels, total n y 3, total n y 6, DHArEPA and total
n y 3rn y 6 ratios between the two treatments were subjected to Student’s t-test Ž
. Ž
. Ž
analysis P s 0.05 using the Statistical Analysis System SAS software program SAS .
Institute, 1988 for PCs.
3. Results
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