Aquaculture 324325 (2012) 111117

Contents lists available at SciVerse ScienceDirect

Aquaculture
journal homepage: www.elsevier.com/locate/aqua-online

Effects of dietary linolenic acid to linoleic acid ratio on growth, tissue fatty acid prole
and immune response of the juvenile grouper Epinephelus malabaricus
Feng-Cheng Wu a, b, Houng-Yung Chen a, c,
a
b
c

Institute of Marine Biology, National Sun Yat-sen University, Kaohsiung 80424, Taiwan
Mariculture Research Center, Fisheries Research Institute, Tainan County 724, Taiwan
Asia-Pacic Ocean Research Center, National Sun Yat-sen University, Kaohsiung 80424, Taiwan

a r t i c l e

i n f o

Article history:
Received 7 January 2011
Received in revised form 22 October 2011
Accepted 28 October 2011
Available online 13 November 2011
Keywords:
Grouper
Epinephelus malabaricus
Fatty acids
Linolenic acid
Linoleic acid
Immune responses

a b s t r a c t
A 12-wk feeding trial was conducted to determine the dietary effects of -linolenic (LN; 18:3n3) and linoleic
acids (LA; 18:2n6) on growth, tissue fatty acids prole and non-specic cellular immune response in kidney of
juvenile grouper Epinephelus malabaricus (initial weight: 11.3 0.6 g). Eight experimental diets and a reference
diet each containing 11.6% lipids were compared. Four experimental diets contained either 1 or 2% diet of pure
LN or LA (N1, N2, L1, and L2). The other 4 diets contained oil mixtures of linseed oil, safower oil, LA, and oleic
acid and had LN:LA ratios of 3.3, 1.4, 0.7 and 0.4 (NL3, NL1.4, NL0.7, and NL0.4). The reference diet contained
a mixture of cod liver oil, linseed oil and safower oil at a ratio of 2:1:1. Weight gain of the grouper
was signicantly increased with increasing dietary LN:LA ratios. The group that fed on the NL3 diet
showed the highest growth. Weight gains of this group equaled those of the reference group. Dietary
inclusion of LN increased the presence of n 3 polyunsaturated fatty acids (PUFAs) in the grouper, except
22:6n 3 and 22:5n 3 in neutral lipid fraction of the liver. 18:2n 6 and 18:3n 6, but not 20:4n 6,
were the predominant n 6 PUFAs in the body of the grouper, and their quantities were closely related
to their quantitative dietary inclusion. Non-specic cellular immune response was signicantly increased
with increasing dietary LN:LA ratios. The grouper that fed on the NL3 diet showed the signicantly highest
head-kidney leucocyte phagocytic and respiratory burst activities among all treatments. Our results suggest
that LN and LA at a dietary level of 2% and a ratio of 3:1 are benecial to weight gain and non-specic cellular
immune responses of the juvenile grouper.
2011 Elsevier B.V. All rights reserved.

1. Introduction
Although sh cannot synthesize de novo n 3 or n 6 series of
polyunsaturated fatty acids (PUFA), they possess varying ability to
desaturate and elongate 18-carbon fatty acids (Greene and
Selivonchick, 1987). The essential fatty acid (EFA) requirements of
sh are closely associated to their metabolism of the precursor fatty
acids. Freshwater sh is capable to convert 18-carbon fatty acids to
longer chain, or more unsaturated fatty acids (Lovell, 1998), and has
a dietary need for linolenic (LN; 18:3n3), linoleic acids (LA;
18:2n6), or both. In contrast, marine sh cannot effectively elongate
and desaturate the 18-carbon fatty acids to highly unsaturated fatty
acids (HUFA) (Cowey et al., 1976; Dhert et al., 1990; Izquierdo et al.,
1989; Wu et al., 2002) and, thus, have a dietary requirement for HUFA.
In the literature there are many studies reporting that EFAs
improve sh growth. The growth of euryhaline milksh was effectively
improved when the diet contained 2% LN (Benitez and Gorriceta, 1983)
Corresponding author at: Institute of Marine Biology, National Sun Yat-sen University,
Kaohsiung 80424, Taiwan.
E-mail address: hychen@mail.nsysu.edu.tw (H.-Y. Chen).
0044-8486/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2011.10.030

or a combination of 1.0% LN, 0.5% EPA and 0.5% DHA (Borlongan, 1992).
The growth of marine sh red drum was improved when the diet
contained either LN, or LA, or both; but an even better growth was
observed when the sh were fed the diet containing 0.5% or 1.0%
EPA and DHA (Lochmann and Gatlin, 1993). By other hand, excessive
amounts of LA affected adversely the growth of salmonids when their
diet contained the amount of LN that was known to satisfy EFA
requirements (Yu and Sinnhuber, 1976, 1979). Several recent
studies have investigated the effects of dietary LN:LA ratio on nutrient
metabolism, growth and tissue fatty acids prole of several freshwater
shes. Freshwater yellow catsh (Pelteobagrus fulvidraco) showed a
decline in LA content and increase in LN, EPA and DHA contents in
the liver as the dietary LN/LA ratio was increased (Tan et al., 2009).
Activities of enzymes involved in liver intermediary metabolism,
such as lipoprotein lipase, hepatic lipase, pyruvate kinase, succinate
dehydrogenase, malic dehydrogenase and lactate dehydrogenase are
signicantly affected by the dietary LN/LA ratio. Similarly, llets of
freshwater Murray cod (Maccullochella peelii peelii) fed on diet with
higher LN/LA ratios recorded signicantly higher concentrations of
EPA and DHA (Senadheera et al., 2010). LN, EPA and DHA in the body
of Nile tilapia (Oreochromis niloticus) fry increased linearly with the

112

F.-C. Wu, H.-Y. Chen / Aquaculture 324325 (2012) 111117

increasing dietary LN:LA ratio (ElHusseiny et al., 2010). The dietary

roles of and interactions between LN and LA however have been rarely
studied in marine sh.
Development, maintenance, and optimization of the immune system
depend on balanced and adequate nutrition (Kelley, 2001). Dietary
intake of high levels of n3 PUFAs and/or their 18 carbon precursors
affects the immune response of sh (Blazer, 1992). Channel catsh
show an enhanced macrophage bactericidal activity when the sh
were fed a diet high in sh oil or linseed oil (Sheldon and Blazer,
1991) and an elevated antibody production when fed a diet high in
sh oil. Conversely, Atlantic salmon fed with a diet containing high
levels of n 3 PUFAs are immunosuppressed when the sh were
subjected to Yersinia ruckeri challenge (Erdal et al., 1991). Little
information is available regarding the effects of PUFAs on the immunity
of marine sh, which have a rich supply of n3 PUFAs in their natural
diets.
Groupers are economically important food sh in tropical and
subtropical coast waters. Because of a reliable supply of fry, Epinephelus
malabaricus is among the grouper species that are most commonly
cultured in the Asia-Pacic region. The feeding practices and nutritional
requirements of juvenile groupers have recently been reviewed by
Williams (2009). The author indicated that there have been few studies
reported in the literature on essential fatty acid requirements of
groupers. Wu et al. (2002) found that dietary DHA is more potent
than EPA in promoting growth of E. malabaricus. Dietary DHA enhances
the phagocytic functions of leucocytes and T cell proliferation of
juvenile E. malabaricus (Wu et al., 2003). The results of the studies
suggested that a dietary n 3 HUFA level of 11.5% should satisfy
the essential fatty acid requirements of juvenile groupers with
growth rate and immunocompetence optimized when the dietary
DHA content alone is at least 0.75% (Williams, 2009).
The objectives of the present research were to study the possible
dietary effects of LN and LA on juvenile grouper when provided
individually or in combination. In addition to growth performance
and non-cellular immune response, we studied the fatty acid proles
of the sh tissues to understand the metabolic link of the two fatty
acids between diet and sh tissue. The relative importance of LN
and LA in affecting sh growth, tissue fatty acid prole and immune
response was studied.
2. Materials and methods
2.1. Experimental diets
Eight experimental diets and a reference diet were compared. The
diets were derived according to different fatty acids prole from a
basal diet that has been found effective in promoting the growth of
the grouper E. malabaricus (Wu et al., 2002). The basal diet contained
51.2% crude protein, 11.6% crude lipid, and 13.6 MJ/kg digestible
energy (Table 1). All of the diets were of the same composition
as the basal diet except their lipid compositions (Tables 2 and 3). Diets
N1, N2, L1 and L2 contained puried -linolenic acid (LN, 18:3n3)
or linoleic acid (LA, 18:2n6) (Sigma, St. Louis, MO, USA) at 1 or 2%
levels. Diets NL3, NL1.4, NL0.7, and NL0.4 contained mixtures of LA,
linseed oil, and safower seed oil (Table 3) and each had an analyzed
LN:LA ratio of 3.3, 1.4, 0.7 and 0.4, respectively (Table 4). Tristearin
(99% in purity) and oleic acid (95% in purity) were added to adjust the
differences in the fatty acid compositions. The reference diet contained a
natural oil mixture of cod liver oil (ICN Biochemicals, Inc., Aurora, OH,
USA), linseed oil and safower seed oil (Table 3) at a ratio of 2:1:1. The
reference diet thus contained a combination of LN, LA, EPA, DHA, and
other fatty acids (Table 4), which is typical of the fatty acid prole of
commercial grouper feeds. Although HUFAs such as DHA and EPA
were not supplemented in the oil mixtures and the shmeal was
solvent extracted, HUFAs at levels lower than the reference diet
were present in the experimental diets (Table 4). The diets were

Table 1
Ingredient and proximate compositions of the basal diet.1
Ingredient

Quantity
g/kg

Fish meal, solvent-extracted

Corn starch
-starch
Lipid mixture2
Vitamin mixture3
Mineral mixture4
Carboxymethyl-cellulose
-cellulose

651.5
43.5
43.5
100.0
40.0
80.0
30.0
11.5

Proximate composition

% diet5

Crude protein
Crude lipid
Moisture
Ash

51.2 0.8
11.6 0.6
3.6 0.4
9.6 0.6

1
Estimated digestible energy: 13.59 kJ/kg (based on protein:
18.83 kJ/g, carbohydrate: 14.60 kJ/g and lipid: 35.61 kJ/g).
2
Contained various quantities of -linolenic acid, linoleic acid, safower
seed oil, linseed oil, oleic acid, tristearin, or cod liver oil. See Table 2 for
details.
3
Vitamin mixture supplied the following (mg/kg dry diet): thiamin
HCl, 60; riboavin, 200; pyridoxine HCl, 40; nicotinic acid, 800;
Ca-pantothenate, 28; inositol, 4000; biotin, 6; folic acid, 15;
p-aminobenzoic acid, 400; choline chloride, 8000, cyanocobalamin,
0.09; L-ascorbic acid, 2000; -carotene, 12; menadione, 40; vitamin
A acetate, 0.045; and all-rac--tocopherol, 400 (added separately
with oils).
4
Mineral mixture supplied the following (g/kg dry diet): KCl,
5.18; MgSO4.2H2O, 6.85; NaH2PO4.2H2O, 30.805; Ca-lactate, 19.74;
Ferric citrate, 1.845; AlCl3.6H2O, 0.009; ZnSO4.7H2O, 0.178; CuCl,
0.0055; MnSO4.4H2O, 0.04; KI, 0.0085; and CoCl2.6H2O, 0.0525.
5
Mean SEM, n = 27.

prepared and extruded by hand as described previously (Wu et

al., 2002). The resulting pellets were freeze-dried, vacuum packed
in small quantities and stored at 20 C until used.
2.2. Animals and feeding trials
Juvenile grouper that were propagated and raised at the hatchery
of the Mariculture Research Center of the Taiwan Fisheries Research
Institute were used. Before commencing the feeding trial, all sh
were fed on the basal diet without lipid mixture for 4 wk to acclimatize
the sh to the experimental conditions and deplete their lipid reserves.
After acclimation, 270 apparently healthy sh were individually
weighed (mean weight SEM, 11.3 0.6 g) and allocated to 27 outdoor ow-through experimental tanks (290 L) so that the 10 sh in

Table 2
Compositions of the lipid mixtures used in the experimental diets and the reference
diet.1
Diet

Lipid source
LN

LA

Linseed oil

Safower seed oil

Oleic acid

Tristearin

0.0
0.0
0.0
6.5
12.3
18.0
0.0
0.0

0.0
0.0
3.4
3.8
4.5
5.3
0.0
0.0

90.0
80.0
66.5
66.4
66.6
66.7
90.0
80.0

LN/
LA2

% lipid mixture
N1
N2
NL3
NL1.4
NL0.7
NL0.4
L1
L2

10.0
20.0
0.0
0.0
0.0
0.0
0.0
0.0

0.0
0.0
1.3
0.0
0.0
0.0
10.0
20.0

0.0
0.0
28.8
23.3
16.6
10.0
0.0
0.0

3.25
1.42
0.72
0.38

1
The reference diet contained a natural oil mixture of cod liver oil, linseed oil and
safower seed oil at a ratio of 2:1:1.
2
Ratio values were calculated from the results of chromatographic analyses (n = 3).

F.-C. Wu, H.-Y. Chen / Aquaculture 324325 (2012) 111117

Table 3
Major fatty acid compositions of the oils used in the experimental diets and the reference
diet.1
Fatty acid

Linseed oil

113

sh were hand fed once daily at 08:0009:00 to apparent satiation.

Residual feed and fecal materials were removed before feeding.

Safower seed oil

Cod liver oil

2.3. Sampling and preparation of head-kidney leucocytes

1.5
0.3
2.3
13.5
76.1
0.3

6.00

2.8
10.7
8.9
3.7
23.9
1.5
0.9
2.6
1.0
8.0
14.3
21.70

The experiment was conducted in accordance with the guidelines

of the University regarding research on experimental animals. The
experimental sh were not fed the day the trial was terminated.
All sh were anesthetized by adding 0.4 g/L benzocaine (Sigma) to
each tank. Fish number was counted to obtain survival rate before
weighing and sample collections. Each sh was individually
weighed and mean weight gain (100 (nal weight initial
weight) / initial weight) of each tank was calculated. Three sh
from each tank (i.e., nine sh/treatment) were randomly sampled.
The liver and a ventral muscle strip posterior to the dorsal n of
the sampled sh were excised and weighed. Liver somatic index
(100 liver weight / body weight) was calculated. The liver and the
muscle from the sh of a same tank were pooled, respectively. The
pooled samples were minced before lipid extraction. Head-kidney
from all sampled sh (9 replicates in each treatment) was also
excised and processed individually for the leucocyte preparation
and immunological assay. The head-kidney leucocyte suspension
was prepared following the methods of Braun-Nesje et al.
(1981) and Lemaire-Gony et al. (1995), with modied (Solem et
al., 1995) Percoll densities (37%51%). Cell viability was checked in
Neubauer hemocytometers by the trypan blue exclusion method,
and was greater than 95% for all assays.

% lipid
b 0.1
1.7
0.1
0.7
22.3
13.5
52.3

9.30

14:0
16:0
16:1
18:0
18:1
18:2(-6)
18:3(-3)
18:4(-3)
20:4(-6)
20:5(-3)
22:6(-3)
Others

1
LN (-linolenic acid, 99% purity); LA (linoleic acid, 99% purity); tristearin (99%
purity); oleic acid (95% purity); All lipids were purchased from Sigma, St. Louis,
MO.

each tank had approximately equal biomass. Each dietary treatment

was randomly assigned to triplicate tanks. Seawater was replaced at a
ow rate of 600 mL/min and extra aeration was provided. During the
12-wk feeding trial, the water temperature ranged between 19 and
26 C and the salinity ranged between 33 and 35 parts/thousand. The

2.4. Lipid extraction and analysis

Table 4
Fatty acid compositions of the experimental diets.1,2
Fatty acid

Diet
N1

N2

NL3

NL1.4

NL0.7

NL0.4

L1

L2

Reference

0.5
0.3
10.7
0.1
38.5
4.8
13.9
19.8
0.1
0.1
1.0
tr
0.1
tr
1.2
4.1
0.1
0.2
tr
0.1
3.5
0.1
0.2
50.0
6.6
42.8
27.7
15.1
9.0
1.4

0.5
Tr
11.2
0.3
37.3
3.9
21.1
15.2
0.3
tr
0.8
tr
0.3
tr
0.9
4.3
tr
0.1
0.2
0.1
3.0
tr
0.2
49.0
5.3
45.4
23.2
22.2
8.8
0.7

0.9
tr
11.3
0.1
39.1
4.1
26.0
7.9
0.2
0.3
1.0
0.2
0.1
0.1
1.2
3.5
tr
0.1
0.1
0.3
3.1
0.1
0.1
51.7
5.4
42.7
15.1
27.6
8.6
0.4

1.0
0.3
11.6
0.4
50.8
1.8
18.7
4.1
0.1
0.2
0.5
0.3
0.2
0.2
1.3
3.9
0.2
0.3
0.1
0.2
3.0
tr
tr
63.8
3.3
32.1
11.5
20.6
9.2
0.2

0.3
0.2
10.3
0.5
39.8
2.6
31.3
3.3
0.2
0.1
0.4
0.1
0.1
0.4
1.2
4.5
0.2
tr
0.1
0.3
3.3
0.1
0.2
50.8
3.9
44.8
11.7
33.1
10.0
0.1

3.3
0.1
8.2
10.3
19.8
25.9
4.2
8.5
0.8
0.1
1.2
0.3
0.4
tr
1.3
7.0
0.3
1.2
0.1
0.5
5.4
tr
1.2
31.7
39.9
28.1
22.2
5.9
14.7
2.0

% total fatty acids

14:0
14:1
16:0
16:1
18:0
18:1n 9
18:2n 6
18:3n 3
18:4n 3
20:0
20:1n 9
20:2n 6
20:3n 3
20:3n 6
20:4n 6
20:5n 3
22:0
22:1n 9
22:4n 6
22:5n 3
22:6n 3
24:0
24:1n 9
SAT
MONO
PUFA
n3 PUFA
n 6 PUFA
HUFA
LN:LA ratio
1

0.3
0.2
10.9
0.4
55.4
1.4
2.9
15.2
0.3
0.4
0.7
0.2
0.2
tr
1.3
4.9
0.3
0.1
tr
0.3
3.4
0.1
0.3
67.4
3.1
28.9
24.5
4.4
10.3
5.2

0.5
0.1
10.7
tr
40.1
1.1
2.6
35.2
0.2
0.2
1.2
0.1
0.2
tr
0.9
3.2
0.1
0.3
tr
0.2
2.5
tr
0.2
51.8
2.9
45.1
41.5
3.6
7.1
13.5

0.6
tr
10.2
0.1
40.1
4.2
8.1
26.3
0.2
0.2
0.9
tr
0.1
0.1
1.0
3.8
0.1
tr
0.1
0.3
3.2
tr
0.1
51.2
5.3
43.2
33.9
9.3
7.7
3.3

Values are means of three replicates.

LN: -linolenic acid; LA: linoleic acid; SAT: sum of saturated fatty acids;
MONO: sum of monounsaturated fatty acids; PUFA: sum of polyunsaturated fatty
acids (2 or more insaturations); n3 PUFA: sum of n 3 polyunsaturated fatty
acids; n6 PUFA: sum of n 6 polyunsaturated fatty acids; HUFA: sum of highly
unsaturated fatty acids (3 or more insaturations); tr: trace amount (b0.1).
2

Total lipids were extracted from the experimental diets, reference

diet and tissue samples by the modied Folch method (Ways and
Hanahan, 1964). The extracted crude lipid was separated into polar
and neutral lipid fractions using silica cartridges (Sep-Pak, Waters
Associates, Milford, MA) to reect their roles in physiological functions
(membrane) and storage, respectively. Both fractions were transmethylated with BF3 in methanol to yield methyl esters (Metacalfe and
Schmitz, 1961). Fatty acid methyl esters (FAMEs) were analyzed by
the 5890A gasliquid chromatograph (Hewlett-Packard, Palo Alto, CA)
equipped with a ame ionization detector. A 30-m 0.25-mm ID
fused silica capillary column (Supelcowax-10, Supelco, Inc., Bellefonte,
PA) was used. The ow rate of carrier gas helium was 20 cm/s. The
thermal gradient program was initially 175 C for 15 min, followed
by increments of 2 C min 1 to 220 C, and nally 220 C for 45 min.
Injector and detector temperatures were 235 and 250 C, respectively.
Individual FAME was identied, integrated and quantied with the HP
ChemStation 1201A PC software package through comparison to
known standards (Nu-Chek 461, Nu-Chek Prep, Elysian, MN; Supelco
37 and PUFA No. 3, Supelco). Fatty acids were quantied relative to an
internal standard, nonadecanoate (19:0, Sigma), and expressed as the
percentage of total fatty acids.
2.5. Phagocytic and respiratory burst activities assay
Phagocytic activity of head-kidney leucocytes was assayed at 27 C
using the colorimetric method (Gebran et al., 1992; Solem et al.,
1995). Phagocytosis activity was calculated by subtracting OD620
values of the control wells from the values of the experimental
wells. The cell number in each well was determined (Secombes,
1990) and was approximately 7 10 4 cells/well. The respiratory
burst activity was assayed following the methods of Pick and Mizel
(1981) and Lemaire-Gony et al. (1995) with some modications to
optimize for the grouper leucocytes (Wu et al., 2003). The respiratory
burst activity was calculated as the difference between the basal and
stimulated cell activities and expressed as OD620 per 2 10 7 cells.

114

F.-C. Wu, H.-Y. Chen / Aquaculture 324325 (2012) 111117

2.6. Statistical analysis

Weight gain, percent of survival, liver somatic index, and immunoassay results are expressed as mean SEM. All data was analyzed by
one-way ANOVA and Tukey's test using the SAS statistical procedures
(SAS Institute, Cary, NC). Differences were regarded as signicant
when P b 0.05. Data of major fatty acids composition were found to
be nonhomogeneous using the Bartlett's test and were arc-sine transformed before ANOVA. Linear correlations were conducted to assess
the incorporation of dietary fatty acids into body tissues. Only the
correlations between dietary and tissue fatty acids that are signicant
are presented.

highest concentration of DHA and 18:1n 9 (Table 4) did not result

in high tissue levels except the DHA concentration in liver neutral
lipid fraction.
No signicant correlations exist between the dietary LN levels and
tissue DHA concentrations. Correlations between the dietary LN levels
and tissue n 3 PUFA levels are highly signicant (P b 0.01) except
22:5n3 in liver polar lipid fraction (Table 7). The levels of tissue
18:2n 6 and 18:3n 6, the only two n 6 PUFAs, were closely
reective of dietary LA levels. Dietary LN levels affected markedly
tissue levels of LN, 18:4n 3, 20:3n 3, 20:4n 3, 20:5n 3 and
22:5n 3, but not 22:5n 3 levels in liver polar lipid faction and
tissue DHA. Increased levels of dietary LN resulted in a signicant
accumulation of 20:3n 3 in liver (Table 7).

3. Results
3.3. Leucocyte phagocytic and respiratory burst activities

3.1. Growth performance

Fish mortality during the feeding trial was low. Signicant differences
were found in sh growth and liver somatic index (Table 5). Weight gain
of the grouper was signicantly increased with increasing dietary LN to
LA ratios (Table 5). Grouper fed the diet with the highest LN:LA ratio
(NL3) had the signicantly greatest weight gain than those sh fed on
the diet contained only LA (L1 and L2) or on the diet with the lowest
LN:LA ratio (NL0.4), or the diet containing only 1% LN (N1). The sh fed
on the diet L1 had the lowest weight gain. Despite that the reference
diet contained more DHA and EPA than the experimental diets; the
sh fed on the reference diet did not grow signicantly better than the
sh fed on any of the experimental diets except the diet L1 (Table 5).
The group fed the diet L2 that contained the highest amount of LA
shows the greatest liver somatic index, which was signicantly higher
than the reference diet group.
3.2. Fatty acid prole
Because the patterns and trends of fatty acid compositions of the
liver and the muscle are very similar, only the liver fatty acid compositions are presented (Table 6). The sh fed on any of the experimental
diets containing LN showed a total n 3 PUFA level in the liver higher
than the sh fed on the LN-free diets (L1 and L2). As the dietary LN
level was increased, such as among the NL diets or the L1 and L2
diets (Table 4), the total n 6 PUFA levels in the liver polar lipid
fractions (Table 6) increased signicantly. Level of total n 6 HUFAs
in liver polar lipid fraction of the sh fed on the reference diet were
signicantly different from the other experimental groups, but not
its neutral lipid fraction (Table 6). The reference diet that had the

Table 5
Weight gain, percent of survival and liver somatic index (LSI) of the juvenile groupers
fed for 12 weeks on diets differing in linolenic acid and linoleic acid compositions and
ratios.1
Diet2

N1
N2
NL3
NL1.4
NL0.7
NL0.4
L1
L2
Reference

Weight gain3

Survival

LSI4

% initial weight

187.5 5.7bc
205.5 15.4ab
219.2 22.3a
209.1 7.7ab
197.0 17.4abc
185.6 23.7bc
168.1 6.9c
189.2 19.6bc
202.5 22.6ab

96.7 5.8
93.3 11.5
96.7 5.8
93.3 11.5
93.3 5.8
96.7 5.8
90.0 10.0
100.0
93.3 11.5

2.68 0.52ab
2.53 0.37ab
2.60 0.09ab
2.69 0.15ab
2.76 0.33ab
2.62 0.14ab
2.58 0.19ab
3.06 0.25a
2.29 0.16b

1
Values are means SEM, n = 3 replicates of 10 sh initially. Means in a same column
without a common superscript letter differ signicantly (P b 0.05).
2
Diet nomenclature: N (-linolenic acid); L (linoleic acid); NL (N:L); Subscript numbers
indicate amount or ratio of fatty acids.
3
Weight gain= 100 (Final weight Initial weight) /Initial weight.
4
LSI = 100 Liver weight/body weight.

Signicant differences (P b 0.05) among dietary treatments were

observed in phagocytic and respiratory burst activities of grouper headkidney leucocytes (Table 8). The highest phagocytic and respiratory
burst activities were found in sh fed the diet with the highest LN:LA
ratio (NL3, 3:1) and are signicantly greater than other dietary groups.
Grouper fed on the diet N1 showed phagocytic and respiratory burst
activities signicantly higher than those of sh fed the diet N2, L1, L2,
or reference diet. The group fed the diet NL0.7 exhibited signicantly
higher phagocytic activity than those of sh fed the diets NL0.4, NL1.4,
L1, or reference diet. The lowest phagocytic activity was found in the
group fed the diet L2, which was not different from the reference diet
group. The lowest respiratory burst activity, however, was observed in
the sh fed the diet N2, which is signicantly lower from the sh fed on
the reference diet. No signicant correlations were established between
leucocyte phagocytic or respiratory burst activities and tissue concentration of major fatty acids.
4. Discussion
A dietary n 3 HUFA specication of 11.5% has been suggested to
satisfy the essential fatty acid requirements of juvenile groupers; and
growth rate and immunocompetence could be optimized when the
dietary DHA content alone is at least 0.75% (Williams, 2009). In the
present study, the grouper that fed on the diet with the highest LN:
LA (NL3, 3:1) and a relatively low HUFA level (7.7% of lipid) had the
best growth performance, although not signicantly different from
the sh fed on the reference diet with a LN:LA ratio of 2:1 and a
high HUFA level (14.7% of lipid). The sh fed on the diet L1 had the
lowest growth. No overt symptoms of EFA deciency or related detrimental responses were observed during the course of the feeding trial
independently of dietary treatment. In a previous study (Wu et al.,
2002), we have showed that the grouper fed on diet containing 1%
diet of LN and LA in a ratio 3:1 and a trace amount of DHA and EPA
achieved a better growth than the sh fed on the reference diet, but a
lower growth than the sh fed on the diet containing lipid with DHA:
EPA in a high ratio of 3:1. The milksh (Chanos chanos), also a tropical
sh, also shows an EFA requirement for LN (Borlongan, 1992). Although
LN at a dietary level of 2% diet was shown to enhance growth of
milksh, combination of 1% LN, 0.5% DHA, and 0.5% EPA promotes
even more growth. The present result showed that the grouper fed
the diet containing LN alone had a better growth than the LA alone
group. It is, thus, likely that LN and LA are both essential for the
growth of the grouper, and LN is required preferentially than is LA.
Like in mammals (Brenner and Peluffo, 1966), there seemingly exists
in the grouper a substrate preference of 6 fatty acid desaturase for
18:3n3 than for 18:2n6 although competition exists between
the two substrates. The substrate afnity and biosynthesis of HUFA
from precursor fatty acids in sh has been documented (Henderson
and Sargent, 1985; Sargent et al., 1989). The dietary requirements

F.-C. Wu, H.-Y. Chen / Aquaculture 324325 (2012) 111117

115

Table 6
Major fatty acid composition of polar and neutral fractions of lipids extracted from the liver of the juvenile groupers fed for 12 weeks on diets differing in linolenic acid and linoleic
acid compositions and ratios.1
Fatty acid2

Diet
N1

N2

NL3

NL1.4

NL0.7

NL0.4

L1

L2

Reference

% total fatty acids

Polar lipid
16:0
18:0
18:1n9
18:2n6
18:3n3
18:3n6
18:4n3
20:1n9
20:2n6
20:3n3
20:3n6
20:4n 3
20:4n6
20:5n3
22:5n3
22:6n3
SAT
MONO
n 3 PUFA
n 6 PUFA
n 3HUFA
n 6HUFA

22.2cd
19.3abc
11.8c
1.9fg
11.4c
trf
4.4bc
2.6c
trb
3.0c
tr
2.4b
0.2bcd
5.0bcd
2.4cd
6.2a
47.8bc
15.1c
34.8cd
2.2f
19.0c
0.3b

15.4g
12.4e
11.0cd
2.2fg
15.8a
0.1f
6.2a
4.2b
trb
5.8a
tr
3.8a
0.1cd
6.0ab
3.5ab
5.7ab
34.3f
16.5d
46.7a
2.5f
24.7a
0.3b

19.0ef
15.7d
8.9d
4.1de
14.1ab
0.4de
5.2ab
2.4c
trb
4.3b
tr
3.2ab
0.2bcd
5.5abc
2.9abc
6.4a
42.0de
11.7c
41.5b
4.8e
22.3b
0.3b

17.5fg
17.9abcd
12.3c
5.8cd
11.7bc
0.6cd
4.8b
2.7c
trb
3.2c
tr
2.8b
0.1cd
5.2bc
2.5bcd
6.5a
41.0e
15.5c
36.7c
6.8cd
20.2c
0.4b

20.9de
18.4abcd
12.9c
7.0c
10.1c
1.5ab
3.4cd
3.3bc
trb
1.4d
tr
1.5c
0.2bc
4.5cde
2.5bcd
6.4a
44.9cd
16.7c
29.7e
8.8c
16.1d
0.3b

20.2de
20.4ab
11.2cd
13.5a
7.5d
2.4a
2.5d
2.7bc
0.1b
0.5e
0.1
1.2c
0.2bc
4.3cde
2.2cde
5.8ab
45.1cd
14.5cd
24.0f
16.4a
14.0e
0.5b

24.1bc
21.2a
20.0ab
9.7b
3.0e
1.5ab
0.2e
2.7bc
trb
0.3e
tr
trd
0.3ab
4.6cde
1.9de
4.2c
48.8ab
25.3b
14.2h
11.7 b
11.0f
0.5b

28.1a
19.9ab
12.1c
14.6a
3.7e
2.4a
0.1e
2.5c
trb
0.3e
Tr
trd
0.3ab
3.6e
2.0cde
4.9bc
51.0ab
16.9c
14.6gh
17.5a
10.8f
0.4b

21.8cd
16.6cd
18.1b
3.2ef
6.3d
1.2bc
3.1d
2.2c
0.2a
3.0c
tr
3.1ab
0.6a
6.9a
3.8c
6.6a
40.4e
21.4b
32.7d
5.5de
23.4ab
1.1a

Neutral lipid
16:0
18:0
18:1n9
18:2n6
18:3n3
18:3n6
18:4n3
20:1n9
20:2n6
20:3n3
20:3n6
20:4n3
20:4n6
20:5n 3
22:5n3
22:6n3
SAT
MONO
n 3 PUFA
n 6 PUFA
n 3 HUFA
n 6 HUFA

20.0
19.9bc
18.4bc
3.6fg
8.5c
0.1ef
2.7bc
3.7bc
0.2a
3.8ab
0.2a
0.7d
tr
5.2ab
3.0a
4.5b
43.7ab
23.7de
28.4cd
4.2de
17.2b
0.5

18.3
18.1c
16.6d
3.0g
13.6a
trf
4.6a
2.7cd
0.1ab
5.1a
trb
2.3a
tr
5.1ab
3.6a
3.9b
38.0c
20.4f
38.2a
3.3e
20.0a
0.3

18.2
21.5abc
17.1d
4.2fg
11.1b
0.1ef
3.8ab
2.5cd
trab
4.5a
trb
1.9b
tr
4.6bc
3.6a
4.0b
41.4bc
20.6f
33.5b
4.5de
18.6ab
0.2

19.2
22.3ab
17.3cd
4.7ef
10.3b
0.1ef
3.3ab
2.1d
0.1ab
4.1ab
trb
1.8bc
0.1
4.2bcd
3.5a
4.2b
43.2ab
20.4f
31.4bc
5.0d
17.7ab
0.2

19.5
21.9ab
18.6bc
7.1cd
8.5c
0.2de
2.7bc
2.0d
0.1ab
3.6ab
trb
1.4c
0.1
3.6cde
3.5a
4.1b
43.1ab
21.5ef
27.5d
7.5c
16.3b
0.2

20.1
20.8abc
20.1b
9.3b
7.2c
0.5cd
2.0c
3.9b
trab
1.5c
trb
1.3c
0.2
3.3de
2.6a
4.2b
43.0ab
25.0d
22.0e
10.0b
12.9c
0.2

19.6
23.5a
24.4a
8.3bc
4.0d
1.6b
0.4d
4.1b
0.1ab
0.1d
trb
0.1e
0.1
2.9e
2.0a
4.1b
46.9a
29.3b
13.5f
10.3b
9.1d
0.4

20.4
20.5abc
21.0b
12.8a
4.6d
2.4a
0.4d
4.1b
trb
0.1d
trb
tre
0.2
3.0e
1.7b
4.7b
43.9ab
26.1cd
14.7f
15.5a
9.6d
0.3

19.1
11.7d
25.6a
5.7de
8.1c
0.8c
3.1b
2.6cd
0.1ab
2.9b
trb
1.7bc
0.2
5.8a
2.8a
7.3a
32.5d
29.0bc
31.6bc
6.9c
20.5a
0.4

Means (n = 3) in a same row without common superscript letter differ signicantly (P b 0.05). Original data were arc sine transformed before ANOVA.
SAT: sum of saturated fatty acids; MONO: sum of monounsaturated fatty acids; n 3 PUFA: sum of n 3 polyunsaturated fatty acids (2 or more insaturations); n 6 PUFA
: sum of n 6 polyunsaturated fatty acids; n 3 HUFA : sum of n 3 highly unsaturated fatty acids (3 or more insaturations); n 6 HUFA : sum of n 6 highly unsaturated fatty
acids; tr: trace amount (b 0.1).
2

for LN and LA in combinations are ubiquitous in carnivorous tropical

marine sh.
The present results show that grouper could have the ability to
convert dietary LN to 20-C n3 fatty acids, but are unable to transform
LA to 20:4n6. Like in other sh (Stowell and Gatlin, 1992; Xu et al.,
1993), fatty acid compositions of grouper (Wu et al., 2002) are closely
associated with diet. Dietary LN and grouper tissue n3 PUFAs are
highly correlated except for DHA. The incorporation into liver and
muscle of 18:3n3, 18:4n3, 20:3n3, 20:4n3, 20:5n3 and
22:5n3, with the exceptions of 22:5n3 in the neutral fraction
of the liver, was proportional to dietary LN levels, implicating that
the grouper might possess some capacity to convert LN to 20:5n3
or 22:5n3 via chain elongation. LN might be converted to 18:4n3
and 20:4n3, and then desaturated to 20:5n3, or even elongated
to 22:5n3. The grouper seem to be more capable of bioconverting

18:3n3 to 20:5n3 than are the turbot (Owen et al., 1975), another
marine nsh. Its capability to synthesize 22:6n3 from 20:5n3 via
24:6n3 by peroxisomal -oxidation pathway (Buzzi et al., 1996) may
be absent or the capacity of the enzymes converting 20:5n 3 to
22:6n 3 is insufcient to meet the requirement. Since the grouper
grows better with increasing dietary DHA:EPA ratio (Wu et al., 2002),
it is likely that the grouper, unlike freshwater shes, is limited or insufcient in the ability to convert 18:3n3 to 22:6n3, and thus both
18:3n3 and 22:6n3 are essential. In the present study, 18:2n6
and 18:3n6 were the only two n6 PUFAs that appeared in the tissues in signicant amounts. Their concentrations and dietary LA levels
are closely correlated. The tissue 20:4n6 levels of the different groups
were similar despite of the dietary disparity in LA levels, indicating
that the grouper were incapable of desaturating and elongating
18:2n 6 to 20:4n 6. In contrast to the insufcient capability of

116

F.-C. Wu, H.-Y. Chen / Aquaculture 324325 (2012) 111117

Table 7
The coefcients for correlations of dietary n3 fatty acids or n 6 fatty acid levels and
levels of important fatty acids in neutral and polar lipid fractions in the liver and muscle
of the juvenile groupers fed for 12 weeks on diets differing in compositions and ratios of
linoenic acid and linoleic acid.
Fatty
acids

Liver

Muscle
Polar lipid

Neutral lipid

Polar lipid

Dietary n 3 fatty acids

18:3n3
0.97**
18:4n 3
0.93**
20:3n 3
0.91**
20:4n 3
0.77**
20:5n3
0.81**
22:5n3
0.19NS

0.95**
0.92**
0.96**
0.93**
0.81**
0.86**

0.97**
0.97**
0.86**
0.95**
0.95**
0.83**

0.94**
0.90**
0.94**
0.93**
0.90**
0.80**

Dietary n 6 fatty acids

18:2n 6
0.93**
18:3n6
0.74**

0.96**
0.97**

0.98**
0.95**

0.96**
0.89**

**: P b 0.01,

Neutral lipid

NS

: P > 0.05.

the herbivorous milksh in bioconverting 18:2n 6 to 20:4n 6

(Kanazawa, 1985), the carnivorous grouper may lack totally the
capacity to synthesize 20:4n 6. Thus, and according to these
results, it seems that grouper needs 18:3n 3, DHA and n 6
HUFAs to maintain normal growth and vital metabolism.
The grouper had high tissue levels of 20:3n3 in both neutral and
polar fractions when the sh were fed on the LN diets, but not in
these fed on the LA diets. Bioconversion of 18:3n 3 to 20:3n 3
through elongation was possible, as reported in turbot (Tocher, 1993)
and grouper (Wu et al., 2002) because of the levels of 18:3n3 in the
diets. It is also possible that 18:3n3 levels in the diets were in excess
of the grouper requirements and, thus, resulted in unusually high
tissue levels of 20:3n3.
Wu et al. (2002) suggested that tissue levels of 20:1n 9 are a
good indicator of EFA deciency in E. malabaricus. In the present
study, after being fed with the basal diet containing no lipid mixture
for the 4 weeks of acclimation, the initial sh contained a signicant
amount of 18:1n9 in the polar fractions and 20:1n9 in both neutral
and polar fractions (data not shown). After the feeding trial, the amount
of 20:1n 9 in the liver of the experimental sh was apparently
reduced. Unlike 18:1n9 and 20:1n9, 20:3n9 was not detectable
before or after the dietary treatments. This reafrms that tissue concentration of 20:1n9 is a good indicator of EFA deciency in the grouper.
Accumulation of tissue 20:3n9 has been found to associate with EFA
deciency in rainbow trout (Castell et al., 1972) and bass (Webster et
al., 1994). EFA deciency in larval gilthead sea bream (Rodriguez et
al., 1994) is indicated by accumulation of 18:1n9.
From the limited available literatures, Williams (2009) suggested
that a dietary DHA content at least 0.75% is needed to optimize

Table 8
Phagocytic activity and respiratory burst activity of head-kidney leucocytes isolated
from the juvenile grouper fed for 12 weeks on diets differing in compositions and ratios
of linolenic acid and linoleic acid.
Diet1
N1
N2
NL3
NL1.4
NL0.7
NL0.4
L1
L2
Reference

Phagocytic activity2
b

0.484 0.005
0.432 0.009cd
0.521 0.013a
0.422 0.015d
0.449 0.018c
0.418 0.005d
0.429 0.001cd
0.413 0.018d
0.414 0.003d

Respiratory burst activity2

0.279 0.013b
0.183 0.001e
0.332 0.012a
0.190 0.014de
0.198 0.013de
0.208 0.008de
0.213 0.024d
0.204 0.009de
0.241 0.010c

1
Diet nomenclature: N (-linolenic acid); L (linoleic acid); NL (N:L); Subscript
numbers indicate amount or ratio of fatty acids.
2
Values are optical density (OD620 nm); Means ( SEM, n = 9) in a same column
without a common superscript letter differ signicantly (P b 0.05).

immunocompetence of groupers if 11.5% n3 HUFA is present in

the diet. Dietary DHA has been found to enhance the phagocytic functions of leucocytes and T cell proliferation of juvenile E. malabaricus
(Wu et al., 2003). Our results showed that dietary inclusion of LN
and a higher LN:LA ratio could enhance non-specic cellular immune
responses of the juvenile grouper. Both phagocytic and respiratory
burst activities were higher in sh fed diets containing 1% than 2%
LN. The highest responses, however, were found in the sh fed the
diet that contained 3.35% diet of the LN:LA mixture at a ratio of 3:1.
These results indicate that optimal levels of dietary LN or optimal
ratios of dietary LN:LA ratio are benecial in enhancing non-specic
cellular defense response in the grouper. Intake of high levels of
dietary n 3 PUFAs has been known to suppress immune responses
in human (Payan et al., 1986; Soyland et al., 1994; Virella et al., 1989)
and several shes including rainbow trout (Kiron et al., 1995) and
Atlantic salmon (Thompson et al., 1996). Channel catsh, in contrast,
have been shown to exhibit increased intracellular killing by macrophages when their diet contained increased levels of dietary n3
PUFA (Sheldon and Blazer, 1991). The killing ability of salmon
macrophages however is not greatly inuenced by ratios of n3 to
n 6 PUFA (Thompson et al., 1996). Dietary PUFAs can affect the
immune function (Hwang, 1989), however, the mechanisms involved
are still unclear. In grouper, it is probable that 1% diet of LN is sufcient
to meet the metabolic need, and excessive levels reduced defense
response. The ratios of LN:LA may affect the requirement of grouper
for dietary PUFA and, in turn, modulate their non-specic cellular
immune responses (Tan et al., 2009).
It is thus proper to conclude that the grouper have a dietary need
for LN. The grouper may be capable of transforming LN into 20:5(n3),
but not further to 22:6(n3). Presence of dietary LN and a high dietary
LN to LA ratio of 3:1 are benecial in improving weight gain and
enhancing non-specic cellular immune responses of the juvenile
grouper.
Acknowledgments
The authors thank Miss Lily Chen and Mr. Min-Lung Chen for their
technical assistance and the two anonymous reviewers for their
constructive comments. This research was in part supported by research
grants NSC 93-2313-B-110-001 from the National Science Council and
099C0020085-AA from the Council of Agriculture.
References
Benitez, L.V., Gorriceta, I.R., 1983. Lipid composition of milksh grown in ponds by traditional aquaculture. In: Cho, C.Y., Cowey, C.B., Watanabe, T. (Eds.), Finsh Nutrition in AsiaMethodological Approaches to Research and Development.
International Development Research Center of Canada, Ottawa, Canada,
pp. 145152.
Blazer, V.S., 1992. Nutrition and disease resistance in sh. Annual Review of Fish Diseases 2, 309323.
Borlongan, I.G., 1992. The essential fatty acid requirement of milksh (Chanos chanos).
Fish Physiology and Biochemistry 9, 401407.
Braun-Nesje, R., Bertheuseen, K., Kaplan, G., Seljelid, R., 1981. Salmonid macrophages:
separation, in vitro culture and characterization. Journal of Fish Diseases 4,
141151.
Brenner, R.R., Peluffo, R.O., 1966. Effects of saturated and unsaturated fatty acids on the
desaturation in vitro of palmitic, stearic, oleic, linoleic and linolenic acid. Journal of
Biological Chemistry 241, 52135219.
Buzzi, M., Henderson, R.J., Sargent, J.R., 1996. The desaturation and elongation of linolenic acid and eicosapentaenoic acid by hepatocytes and liver microsomes from
rainbow trout fed diets containing sh oil or olive oil. Biochemical and Biophysical
Acta 1299, 235244.
Castell, J.D., Lee, D.J., Sinnhuber, R.O., Wales, J.H., Lee, D.J., 1972. Essential fatty acids in
the diet of rainbow trout (Salmo gairdneri): growth, feed conversion and some
gross deciency symptoms. Journal of Nutrition 102, 7786.
Cowey, C.B., Adron, J.W., Robert, R.J., 1976. The effect of different dietary oils on tissue
fatty acid and tissue pathology in the turbot Scopthalmus maximus. Comparative
Biochemistry and Physiology 53B, 399403.
Dhert, P., Lavens, P., Duray, M., Soregeloos, P., 1990. Improved larval survival at metamorphosis of Asian seabass (Lates calcarifer) using omega 3-HUFA-enriched live
food. Aquaculture 90, 6374.

F.-C. Wu, H.-Y. Chen / Aquaculture 324325 (2012) 111117

ElHusseiny, O., Abdul-Aziz, G., Goda, A., Suloma, A., 2010. Effect of altering linoleic
acid and linolenic acid dietary levels and ratios on the performance and tissue
fatty acid proles of Nile tilapia Oreochromis niloticus fry. Aquaculture International
18, 11051119.
Erdal, J.I., Evensen, O., Kaurstad, O.K., Lillehaug, A., Solbakken, R., Thorud, K., 1991. Relationship between diet and immune response in Atlantic salmon (Salmo salar L.)
after feeding various levels of ascorbic acid and omega-3 fatty acids. Aquaculture
98, 363379.
Gebran, S.J., Romano, E.L., Pons, H.A., Cariani, L., Sonayo, A.N., 1992. A modied colorimetric method for the measurement of phagocytosis and antibody-dependent cell
cytotoxicity using 2,7-diaminouorene. Journal of Immunological Methods 15,
255260.
Greene, D.H.S., Selivonchick, D.P., 1987. Lipid metabolism in sh. Progress in Lipid Research 26, 5385.
Henderson, R.J., Sargent, J.R., 1985. Fatty acid metabolism in sh. In: nutrition and feeding in sh. In: Cowey, C.B., Mackie, A.M., Bell, J.G. (Eds.), Academic Press, London,
pp. 349364.
Hwang, D.H., 1989. Essential fatty acids and immune response. The FASEB Journal 3,
20522061.
Izquierdo, M.S., Watanabe, T., Takeuchi, T., Arakawa, T., Kitajima, C., 1989. Requirement
of larval red seabream Pagrus major for essential fatty acids. Nippon Suisan Gakkaishi 55, 859867.
Kanazawa, A., 1985. Nutritional factors in sh reproduction. In: Lee, C.S., Liao, I.C.
(Eds.), Reproduction and Culture of Milksh. The Oceanic Institute, Waimanalo,
Hawaii, USA, pp. 115125.
Kelley, D.S., 2001. Modulation of human immune and inammatory responses by dietary fatty acids. Nutrition 17, 669673.
Kiron, V., Fukuda, H., Takeushi, T., Watanabe, T., 1995. Essential fatty acids nutrition
and defense mechanisms in rainbow trout Oncorhynchus mykiss. Comparative Biochemistry and Physiology 111A, 361367.
Lemaire-Gony, S., Lemaire, P., Pulsford, A.L., 1995. Effects of cadmium and benzo(a)pyrene on the immune system, gill ATPase and EROS activity of European sea bass
Dicentrarchus labrax. Aquatic Toxicology 31, 297313.
Lochmann, R.T., Gatlin III, D.M., 1993. Essential fatty acid requirement of juvenile red
drum (Sciaenops ocellatus). Fish Physiology and Biochemistry 12, 221235.
Dietary requirements, In: Lovell, T. (Ed.), Nutrition and Feeding of Fish, 2nd ed. Kluwer
Academic, London, UK, pp. 1370.
Metacalfe, L.D., Schmitz, A.A., 1961. The rapid preparation of fatty acid esters for gas
chromatographic analysis. Analytical Chemistry 33, 363365.
Owen, J.M., Adron, J.W., Middleton, C., Cowey, C.B., 1975. Elongation and desaturation
of dietary fatty acids in turbot, Scophthalmus maximus L., and rainbow trout,
Salmo gairdneri Rich. Lipids 10, 528531.
Payan, D.G., Wong, M.Y.S., Chernov-Rogan, T., Valone, F.H., Pickett, W.C., Blake, V., Gold,
W.M., 1986. Alteration in human leukocyte function induced by induced by ingestion of eicosapentaenoic acid. Journal of Clinical Immunology 6, 402410.
Pick, E., Mizel, D., 1981. Rapid microassay for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader. Journal of Immunological Methods 46, 211216.
Rodriguez, C., Perez, J.A., Izquierdo, M.S., Mora, J., Lorenzo, A., Fernandez-Palacious, H.,
1994. Essential fatty acid requirements of larval gilthead sea bream, Sparus aurata
(L.). Aquaculture and Fisheries Management 25, 295304.
Sargent, J., Henderson, R.J., Tocher, D.R., 1989. The lipids. In: Halver, J.E. (Ed.), Fish nutrition. 2nd ed. Academic Press, London.
Secombes, C.J., 1990. Isolation of salmonid macrophages and analysis of their killing activity. In: Stolen, J.S., Fletcher, T.C., Anderson, D.P., Robertson, B.S., van Muiswinkel,

117

W.B. (Eds.), Techniques in Fish Immunology, vol. 1. SOS Publications, Fair Haven,
NJ, USA, pp. 137154.
Senadheera, S.P.S.D., Turchini, G.M., Thanuthong, T., Francis, D.S., 2010. Effects of dietary R-linolenic acid (18:3n 3)/linoleic acid (18:2n 6) ratio on growth performance, llet fatty acid prole and nishing efciency in Murray cod. Aquaculture
309, 222230.
Sheldon, W.M., Blazer, V.S., 1991. Inuence of dietary lipid and temperature on bacterial activity of channel catsh macrophages. Journal of Aquatic Animal Health 3,
8793.
Solem, S.T., Jorgensen, J.B., Robertsen, B., 1995. Stimulation of respiration of respiratory
burst and phagocytic activity in Atlantic salmon (Salmo salar L.) macrophages by
lipopolysaccharide. Fish & Shellsh Immunology 5, 475491.
Soyland, E., Lea, T., Sandstad, B., Drevon, A., 1994. Dietary supplementation with very
long-chain n 3 fatty acids in man decreases expression of the interleukin-2 receptor (CD25) on mitogens-stimulated lymphocytes from patients with inammatory skin diseases. European Journal of Clinical Investigation 24, 236242.
Stowell, S.L., Gatlin III, D.M., 1992. Effects of dietary pantethine and lipid levels on
growth and body composition of channel catsh, Ictalurus punctatus. Aquaculture
108, 177188.
Tan, X.Y., Luo, Z., Xie, P., Liu, X.J., 2009. Effect of dietary linolenic acid/linoleic acid ratio
on growth performance, hepatic fatty acid proles and intermediary metabolism of
juvenile yellow catsh Pelteobagrus fulvidraco. Aquaculture 296, 96101.
Thompson, K.D., Tatner, M.F., Henderson, R.J., 1996. Effects of dietary n 3 and n 6
polyunsaturated fatty acid ratio on the immune response of Atlantic salmon,
Salmo salar L. Aquaculture Nutrition 2, 2131.
Tocher, D.R., 1993. Elongation predominates over desaturation in the metabolism of
18:3n 3 and 20:5n 3 in turbot (Scophthalmus maximus) brain astroglial cells
in primary culture. Lipids 28, 267272.
Virella, G., Kilpatrick, J.M., Rugeles, M.T., Hyman, B., Russell, R., 1989. Depression of humoral responses and phagocytic functions in vivo and in vitro by sh oil and eicosapentaenoic acid. Clinical Immunology and Immunopathology 52, 257270.
Ways, P., Hanahan, D.J., 1964. Characterization and quantication of red cells lipids in
normal man. Journal of Lipid Research 5, 318328.
Webster, C.D., Lovell, R.T., Clawson, J.A., 1994. Ratio of 20:3(n 9) to 20:5n 3 in phospholipids as an indicator of dietary essential fatty acid sufciency in striped bass,
Morone saxatilis, and palmetto bass, M. Saxatilis M. chrysops. Journal of Applied
Aquaculture 4, 7590.
Williams, K.C., 2009. A review of feeding practices and nutritional requirements of
postlarval groupers. Aquaculture 292, 141152.
Wu, F.-C., Ting, Y.-Y., Chen, H.-Y., 2002. Docosahexaenoic acid is superior to eicosapentaenoic acid as the essential fatty acid for growth of grouper, Epinephelus malabaricus. Journal of Nutrition 132, 7279.
Wu, F.-C., Ting, Y.-Y., Chen, H.-Y., 2003. Dietary docosahexaenoic acid is more optimal
than eicosapentaenoic acid affecting the level of cellular defense responses of the
juvenile grouper Epinephelus malabaricus. Fish & Shellsh Immunology 14,
223238.
Xu, R., Hung, S.O.O., German, J.B., 1993. White sturgeon tissue fatty acid compositions
are affected by dietary lipids. Journal of Nutrition 123, 16851692.
Yu, T.C., Sinnhuber, R.O., 1976. Growth response of rainbow trout (Salmo gairdneri) to
dietary 3 and 6 fatty acids. Aquaculture 8, 309317.
Yu, T.C., Sinnhuber, R.O., 1979. Effects of dietary 3 and 6 fatty acids on growth and
feed conversion efciency of coho salmon (Oncorhynchus kisutch). Aquaculture 16,
3138.