Aquaculture 317 (2011) 133137

Contents lists available at ScienceDirect

Aquaculture
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Dietary folic acid requirement of grouper, Epinephelus malabaricus, and its effects on
non-specic immune responses
Yu-Hung Lin a, Hui-You Lin a, Shi-Yen Shiau a,b,
a
b

Department of Food Science, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung 202, Taiwan, ROC
Department of Food and Nutrition, Providence University, 200 Chung-Chi Road, Shalu, Taichung 433, Taiwan, ROC

a r t i c l e

i n f o

Article history:
Received 16 February 2011
Received in revised form 2 April 2011
Accepted 5 April 2011
Available online 12 April 2011
Keywords:
Grouper
Folic acid
Nutrient requirement
Fish nutrition
Immune response

a b s t r a c t
To quantify the dietary folic acid (FA) requirement of grouper, Epinephelus malabaricus, and its effects on nonspecic immune responses, FA was added to a basal diet at 0, 0.4, 0.8, 1.2, 2, 4, 6 and 10 mg FA/kg, providing actual
dietary values of 0, 0.39, 0.72, 1.03, 1.89, 3.36, 5.85, and 9.52 mg FA/kg, respectively for a total of 8 experimental
diets. Each diet was fed to triplicate groups of sh (initial body weight: 7.28 0.03 g) in a recirculating seawater
rearing system for 8 weeks. Weight gain (WG) was highest (P b 0.05) in sh fed diets with 0.72, 1.03 and
3.36 mg FA/kg, followed by 0.39 mg FA/kg, and lowest in sh fed the FA-free control diet. Fish fed diets with
0.72 mg FA/kg had higher feed efciency (FE) but lower hepatosomatic index (HSI) than sh fed the control
diet. Fish fed diets with 0.72 mg FA/kg had highest hepatic FA concentration, followed by 0.39 mg FA/kg, and
lowest in sh fed the control diet. Hepatic thiobarbituric acid-reactive substance (TBARS) value was highest in
sh fed the control diet, followed by 0.39 mg FA/kg diet, and lowest in sh fed diets with 0.72 mg FA/kg.
Leukocyte superoxide anion (O
2 ) production ratio was higher in sh fed diets with 0.72 mg FA/kg than that in
sh fed diets with 0.39 mg FA/kg. Fish fed diets with 0.72 mg FA/kg had higher plasma lysozyme activity than
sh fed the control diet. Analyses of WG, HSI, hepatic FA concentration and hepatic TBARS value indicate that the
optimal dietary FA level for juvenile grouper is about 0.8 mg FA/kg diet, this dietary level is also adequate for nonspecic immune responses of the species.
2011 Elsevier B.V. All rights reserved.

1. Introduction
Folic acid (FA) is composed of a pteridine ring linked through a
methylene bridge to p-aminobenzoic acid to form pteroic acid, which is
in turn linked as an amide to glutamic acid. Folic acid, when converted to
active tetrahydrofolate coenzymes, functions as a one-carbon donor or
acceptor in a variety of reactions involved in amino acid and nucleotide
metabolism (NRC, 1993). It is necessary for normal cell division and
multiplication, and a deciency of folic acid is characterized in most
animals and in humans by impaired hematopoiesis.
A dietary requirement of FA has been reported for a few sh species
including 0.30.6 mg/kg diet for rainbow trout (Oncorhynchus mykiss)
(Cowey and Woodward, 1993), 1.2 mg/kg diet for channel catsh
(Ictalurus punctatus) (Duncan et al., 1993), and 0.82 mg/kg diet for
hybrid tilapia (Oreochromis aureus O. aureus) (Shiau and Huang, 2001a).
In sh nutrition research, nutrient requirements are often
quantied to maximize the animal's growth but largely ignored the

Corresponding author. Department of Food and Nutrition, Providence University,

200 Chung-Chi Road, Shalu, Taichung 433, Taiwan, ROC. Fax: + 886 2 2462 1684.
E-mail address: syshiau@pu.edu.tw (S.-Y. Shiau).
0044-8486/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2011.04.010

nutrient essentiality on its role in disease prevention. Vitamins have

been characterized to enhance resistance to infection by increasing
migration and proliferation of phagocytic cell. Four vitamins, i.e.,
vitamin A (Hernandez et al., 2007; Thompson et al., 1995; Yang et al.,
2008), vitamin C (Ai et al., 2004; Hardie et al., 1991; Li and Lovell,
1985; Lin and Shiau, 2005a; Roberts et al., 1995), vitamin E (Clerton et
al., 2001; Hardie et al., 1990; Lin and Shiau, 2005b; Ortuo et al., 2000)
and vitamin B6 (Albrektsen et al., 1995; Feng et al., 2010) have been
demonstrated to affect immune responses of sh. Vitamin C and E
have been shown quantitatively to have an immune stimulatory
effect, whereas deciency in vitamin A and B6has only been shown to
have a qualitatively immune depressive effect.
Groupers are popular and high value marine sh in many parts of
the world and they are also good candidates for intensive aquaculture
because of their desirable taste, hardiness and rapid growth (Chen and
Tsai, 1994; Millamena, 2002). The purpose of this study was to
quantify the dietary FA requirement of juvenile grouper, Epinephelus
malabaricus. Besides growth performance, other physiological and
immune parameters in sh including hepatic and plasma FA concentrations, hepatic superoxide dismutase (SOD) activity, thiobarbituric acid-reactive substances (TBARS) value, leukocyte superoxide
anion production ratio and plasma lysozyme activity were also
monitored.

134

Y.-H. Lin et al. / Aquaculture 317 (2011) 133137

2. Materials and methods

2.1. Diet preparation
Folic acid-free basal diet formulation and proximate composition
analysis (AOAC, 1995) are shown in Table 1. Vitamin-free casein
(Sigma Chemical Co., St. Louis, MO), sh oil (Semi-rened sh oil,
Oleaginosa Victoria S.A., Peru) and corn oil (Tai-Tang Industrial,
Taiwan), and corn starch (Sigma Chemical) were used as dietary
protein, lipid and carbohydrate sources, respectively. All diets were
kept isoenergetic at 360 kcal/100 g diet. An attractant that had a
similar chemical composition to squid mantle tissue (Mackie and
Mitchell, 1985) was added at 6% to all diets to increase palatability and
diet acceptance. Basal diet was supplemented with folic acid (Sigma
Chemical) at 0, 0.4, 0.8, 1.2, 2, 4, 6 and 10 mg FA/kg diet. Dietary FA
concentrations of the eight experimental diets were determined with
a microbiological assay using Lactobacillus casei (ATCC 7469) from the
Bioresource Collection and Research Center (Hsinchu, Taiwan) according to the procedure of Ryu et al. (1994). The determined values
are 0 (unsupplemented control), 0.39 (0.4 mg/kg), 0.72 (0.8 mg/kg),
1.03 (1.2 mg/kg), 1.89 (2 mg/kg), 3.36 (4 mg/kg), 5.85 (6 mg/kg), and
9.52 (10 mg/kg). The diets were prepared and stored as previously
described (Lin et al., 2010).
2.2. Experimental procedure
E. malabaricus juveniles obtained from a local hatchery (Pingtung,
Taiwan) were used in the study. Upon arrival, they were acclimated to
laboratory conditions for 4 weeks in a 1000 L plastic tank and fed a
commercial diet (Uni-President Enterprise Corp., Tainan, Taiwan). The
proximate composition (%) and FA concentration (mg FA/kg) of the
commercial diet were as follows: moisture, 11.7; crude protein, 43.3;
lipid, 8.8; ash, 9.3; and FA, 1.2. Commercial diet does not contain PABA.
At the beginning of the experiment, 12 sh (mean initial weight: 7.28
0.03 g) were stocked in each aquarium (0.305 0.610 0.555 m3). Each
Table 1
Formulation and proximate composition of the basal diet.
%
Ingredient
Casein
Fish oil
Corn oil
Corn starch
Vitamin mixturea
Mineral mixtureb
Attractantc
Carboxylmethylcellulose
Alpha-cellulose
Proximate composition
Moisture
Ash
Crude protein
Ether extract

51
4.5
4.5
16.7
2
4
6
3
8.3
10.46
3.17
47.61
8.79

a
Vitamin mixture (mg/g mixture): thiamin hydrochloride, 2.5; riboavin, 10; nicotinic
acid, 37.5; pyridoxine hydrochloride, 2.5; inositol, 18; L-ascorbyl-2-monophosphate Na,
1.66; calcium pantothenate, 0.75 mg, choline chloride, 250; menadione, 2; alphatocopheryl acetate, 5; retinyl acetate, 1; cholecalciferol, 0.0025; biotin, 0.25. All ingredients
were diluted with alpha-cellulose to 1 g.
b
Mineral mixture (mg/g mixture): calcium lactate, 327; K2PO4, 239.8; CaHPO4.2H2O,
135.8; MgSO4.7H2O, 132; Na2HPO4.2H2O, 87.2; NaCl, 43.5; ferric citrate, 29.7;
ZnSO4.7H2O, 3; MnSO4.H2O, 0.8; KI, 0.15; CuSO4, 0.15; AlCl3.6H2O, 0.15; CoCl2.6H2O,
1; selenomethionine, 0.02. All ingredients were diluted with alpha-cellulose to 1 g.
c
As mg/100 g diet: L-aspartic acid, 18; L-threonine, 44; L-serine, 33; L-glutamic
acid, 53; L-valine, 36; L-methionine, 36; L-isoleucine, 29; L-leucine, 55; L-tyrosine, 22; Lphenylalanine, 29; L-lysineHCl, 29; L-histidineHCl, 15; L-proline, 1456; L-alanine, 273;
L-arginine, 228; taurine, 337; glycine, 892; betainHCl, 910; trimethylamineHCl, 91;
trimethylamine n-oxide HCl, 1138; hypoxanthine, 47; inosine, 25; adenosine-5monophosphate, 40; L-(+)-lactic acid, 91; alpha-cellulose, 80 (Mackie and Mitchell,
1985).

experimental diet was fed to sh in three randomly chosen aquaria.

Each aquarium was part of a closed recirculating system with a common
reservoir of seawater at 29 to 32 salinity. Water temperature of
the rearing system was held at 29 1 C. The water was circulated at
2 L/min through two separate biolters to remove impurities and
reduce ammonia concentrations. Half of the water in the system was
exchanged daily.
Fish were fed 3% of their body weight per day. This amount was
close to the maximal daily ration for grouper according to feed
consumption during the acclimation period of the study. The daily
ration was divided into two equal meals fed at 0830 and 1630 h. Fish
were weighed once every 2 weeks and the daily ration adjusted
accordingly. The remaining feed and feces were removed by a siphon
immediately after feeding. A photoperiod of 12 h light (0800 to
2000 h), 12 h dark was used. Any dead sh were removed and
not replaced during the experiment. Fish were fed the test diets for an
8-week period.
At the end of the feeding trial, the percentage of body weight
gain (WG) in each aquarium [100 (nal body weight initial body
weight) / initial body weight], feed efciency (FE) [(nal body weight
initial body weight) /feed intake], and survival [100 (nal sh number/
initial sh number] were calculated.
After the nal weight was noted, blood was collected from the
caudal vein by syringe with heparin as the anticoagulant from six
sh selected randomly per aquarium and pooled. The blood was
centrifuged at 3000 g for 15 min. Plasma was removed and stored
at 80 C. Plasma FA concentration was determined by the same
method of feed analysis (Ryu et al., 1994). The turbidimetric assay for
plasma lysozyme activity was carried out according to Obach et al.
(1993).
After blood collecting, the sh were sacriced. The livers were
removed from the same sh, and then weighed and pooled.
Hepatosomatic index (HSI) was calculated as [(liver weight / body
weight) 100]. Hepatic FA concentration and thiobarbituric acid-reactive
substances (TBARS) value were measured according to the method of
Ryu et al. (1994) and Uchiyama and Mihara (1978), respectively. The total
superoxide dismutase (SOD) activity was assessed using a commercial kit
(Merck Co., Germany).
Blood also was collected from other three sh. Leukocytes were
isolated from the blood of grouper by lymphoprep medium (density:
1.077; Gibco BRL, Life Technologies). The isolated leukocytes were
cultured in Aim V/Leibovitz's L15 medium (Gibco BRL) containing
5.5 mM glucose (Sigma Chemical). Intracellular superoxide anion
(O
2 ) production of leukocytes was quantied using the reduction of
nitroblue tetrazolium (NBT) to formazan as a measure of O
2 production
ratio as described by Secombes (1990). All analyses were conducted in
triplicate.
2.3. Statistical analysis
Each experimental diet was fed to three groups of sh according to
a completely randomized design. Results were analyzed by one-way
analysis of variance (ANOVA) using the SAS/PC statistical software
(SAS Inst. Inc., Cary, NC), and signicance was set at P b 0.05. Multiple
comparisons among means were made with Duncan's new multiple
range test. Dietary FA requirements for juvenile grouper were
estimated by the WG, HSI, hepatic FA concentration and TBARS
value with broken-line regression.
3. Results
Weight gain (WG) was highest (P b 0.05) in sh fed diets with 0.72,
1.03 and 3.36 mg FA/kg, followed by 0.39 mg FA/kg, and lowest in
sh fed the FA-free control diet (Table 2). Fish fed diets with
0.72 mg FA/kg had higher feed efciency (FE) but lower HSI than

Y.-H. Lin et al. / Aquaculture 317 (2011) 133137

151.9 31.2a
201.6 12.8b
255.3 8.7c
249.6 26.9c
238.5 27.0bc
258.4 3.8c
247.5 4.7c
247.0 15.2c

0.68 0.18a
0.81 0.05ab
0.90 0.04b
0.90 0.06b
0.86 0.12b
0.93 0.03b
0.91 0.05b
0.88 0.05b

HSI

86.1 17.4
86.1 17.4
80.6 17.4
91.7 8.3
91.7 14.4
97.2 4.8
88.9 12.7
94.4 4.8

2.05 0.03b
1.80 0.25ab
1.61 0.22a
1.64 0.14a
1.54 0.06a
1.81 0.17a
1.62 0.15a
1.53 0.13a

Different superscripts in the column indicate signicant (P b 0.05) difference between

different dietary treatments.
Values are means SD from three groups of sh (n = 3) with 12 sh per group.

4. Discussion
In the present study, growth performance was poor in FA-decient
grouper and improved by dietary FA supplementation, which clearly
demonstrated that dietary folic acid is essential for grouper.
It is considered that the typical FA deciency sign is megaloblastic
anemia (NRC, 1993). In the present study, red blood cell (RBC) count,
hematocrit (Hct) and hemoglobin (Hb) concentration were similar in

Plasma

Hepatic

Folic acid

Folic acid

TBARS value

mg/kg diet

ng/L

g/g tissue

nmol MDA1/g tissue

0
0.39
0.72
1.03
1.89
3.36
5.85
9.52

27.88 8.96a
33.28 2.96ab
48.15 6.57bc
56.07 7.88c
52.77 5.71c
56.39 5.08c
57.07 13.40c
60.60 11.26c

1.16 0.11a
1.67 0.26b
2.20 0.16c
2.38 0.20c
2.46 0.03c
2.39 0.21c
2.51 0.29c
2.44 0.26c

55.56 3.56c
42.83 5.06b
30.58 3.62a
31.89 6.62a
32.50 5.41a
28.31 3.90a
26.50 4.07a
32.01 2.85a

Values are means SD from three groups of sh (n = 3) with 6 sh per group.

1
MDA: malonaldehyde.
Different superscripts in the column indicate signicant (P b 0.05) difference between
different dietary treatments.

units/g tissue

0
0.39
0.72
1.03
1.89
3.36
5.85
9.52

439.38 11.22a
486.62 32.63b
507.42 24.58b
610.68 21.80c
561.26 20.54c
594.45 31.90c
550.50 20.05c
566.21 18.89c

Lysozyme

1.05 0.03a
1.12 0.09a
1.45 0.14b
1.51 0.11b
1.35 0.13b
1.33 0.09b
1.42 0.05b
1.33 0.10b

128.86 3.50a
150.78 16.40ab
172.78 11.55b
170.38 1.13b
181.42 23.84b
189.32 20.16b
189.32 18.29b
188.17 17.39b

units/mL

Values are means SD from three groups of sh (n = 3) with 6 sh per group for SOD
and lysozyme; 3 sh per group for O
2 production ratio.
Different superscripts in the column indicate signicant (P b 0.05) difference between
different dietary treatments.

300
250
Ymax=249.4

200
150

2.0
1.8

0.70

0.69

1.6

Ymin=1.63

1.4

2.5
Ymax=2.40

2.0
1.5

50

.80

Analyzed folic acid

concentration

mg/kg diet

O
2 production
ratio

0.88

+ 55
4.63X
Y=2--3 9)
.9
(R =0

Table 3
Plasma and hepatic total folic acid concentrations and hepatic thiobarbituric acidreactive substance (TABRS) value of grouper fed different diets for 8 weeks.

SOD

.05
X+ 2
0.61
Y=-2 0.99)
(R =

sh fed the control diet. Survival was not different among all dietary
treatments.
Plasma FA concentration was higher in sh fed diets with
0.72 mg FA/kg than sh fed the control diet (Table 3). Fish fed
diets with 0.72 mg FA/kg had highest hepatic FA concentration,
followed by 0.39 mg FA/kg, and lowest in sh fed the control diet.
Hepatic TBARS value was highest in sh fed the control diet, followed by
0.39 mg FA/kg diet, and lowest in sh fed diets with 0.72 mg FA/kg.
Hepatic total superoxide dismutase (SOD) activity was highest in sh
fed diets with 1.03 mg FA/kg, followed by 0.39 and 0.72 mg FA/kg
diet, and lowest in sh fed the control diet (Table 4). Leukocyte superoxide anion (O
2 ) production ratio was higher in sh fed diets with
0.72 mg FA/kg than that in sh fed diets with 0.39 mg FA/kg. Fish fed
diets with 0.72 mg FA/kg had higher plasma lysozyme activity than
sh fed the control diet.
Analysis by broken-line regression of WG, HSI, hepatic FA concentration and TBARS value indicated that the adequate requirements
of dietary FA for of juvenile grouper are 0.70, 0.69, 0.88 and 0.74 mg/kg
diet, respectively (Fig. 1).

Analyzed folic acid

concentration

Y=1
4
(R 2= 3.1X+1
50.0
0.99
)

0
0.39
0.72
1.03
1.89
3.36
5.85
9.52

Survival

Y=1
(R 2= .44X+1
.1 4
0.99
)

mg/kg diet

FE

Weight gain (%)

WG

HSI (%)

Analyzed folic acid

concentration

Table 4
Hepatic total superoxide dismutase (SOD) activity, leukocyte superoxide anion (O-2)
production ratio and plasma lysozyme activity of grouper fed different diets for
8 weeks.

Hepatic TBARS value Hepatic FA concentration

(g/g tissue)
(nmol MDA/g tissue)

Table 2
Weight gain (WG), feed efciency (FE), survival and hepatosomatic index (HSI) of
grouper fed different diets for 8 weeks.

135

40

0.74
Ymin=30.30

30
20
0

10

Analyzed dietary FA concentration (mg/kg diet)

Fig. 1. Analysis by broken-line regression of weight gain (WG), hepatosomatic index
(HSI), hepatic folic acid (FA) concentration and thiobarbituric acid-reactive substance
(TBARS) value indicated that the adequate requirements of dietary FA of juvenile
grouper are 0.70, 0.69, 0.88 and 0.74 mg/kg diet, respectively. Each point represents
the mean SD of three aquaria within a treatment with 12 sh per aquarium for WG
and with 6 sh per aquarium for HSI, hepatic FA concentration and TBARS value,
respectively.

136

Y.-H. Lin et al. / Aquaculture 317 (2011) 133137

all dietary treatments (data not shown). How long would need for sh
to develop FA deciency sign is an open issue. Nevertheless, after
8 weeks of growth trial WG and all measured physiological parameters including tissue FA concentration, TBARS value, SOD activity and
immune responses were well respond to the dietary FA supplementation. No FA deciency signs were also reported in common carp
(Aoe et al., 1967) and hybrid tilapia (Shiau and Huang, 2001a). Deciency
signs were not observed in sh fed a FA-free diet, presumably due to
bacterial synthesis of FA in the intestine (Duncan et al., 1993; Kashiwada
et al., 1971). It has been generally believed that intestinal microorganisms may contribute a considerable quantity of folic acid to the host.
Duncan et al. (1993) demonstrated that intestinal microorganisms are a
signicant source of folic acid for channel catsh. Kashiwada et al. (1971)
isolated folic acid-synthesizing bacteria from the intestine of common
carp and concluded that this explained why common carp do not require
a dietary source of FA. Folic acid content of erythrocyte was not measured
in our study, this value may conrm that FA was synthesized in zero level
fed groups of groupers. Future study in this regard is needed.
The PABA is recognized as the component of folic acid used as a
source for microorganisms synthesizing FA (Camilo et al., 1996; Rong
et al., 1991; Russell et al., 1986). In the present study, PABA was not
added to the vitamin mixture, nevertheless approximately 0.1 mg/kg
diet of PABA was detected in the basal diet. This PABA background
level may partially attribute to no deciency sign of the FA-decient
grouper.
Both folic acid and vitamin B12 involve in blood cell formation. In cases
where anemia has been produced by an inadequacy of vitamin B12,
treatment with large amounts of FA will normalize the red blood cell
(Christian and Greger, 1994). It has been reported that the combined
deciency of FA and vitamin B12 resulted in a more pronounced anemia
in rohu (Labeo rohita Ham) suggesting that FA and vitamin B12 have a
supplementary and complementary role in sh metabolism (John and
Mahajan, 1979). Cobalt (Co) was recognized as the mineral in the large
vitamin B12 molecule. Our previous study (Lin et al., 2010) demonstrated
that in the presence of 10 mg Co/kg diet, intestinal microorganism of
grouper can synthesize enough vitamin B12 for sh growth. In the present
study, basal diet contained 10 mg Co/kg, thus a deciency of dietary
vitamin B12 should not interfere with the status of FA requirement for
grouper.
Higher HSI in grouper fed the FA-free diet was also reported in
rainbow trout (Kitamura et al., 1967) and tiger shrimp (Shiau and
Huang, 2001b). The reason of higher HSI in sh fed FA-decient diet
than other FA-supplemented groups was not known. Nevertheless
HSI value decreased as dietary FA supplementation level increased up
to 0.72 mg FA/kg, then leveled off thereafter (Table 2). This suggests
that HSI can be used to estimate the dietary folic acid requirement of
grouper. The value (0.69 mg FA/kg) obtained by broken-line regression analysis from HSI (Fig. 1) was in agreement with the value
(0.70 mg FA/kg) obtained from growth performance.
Thiobarbituric acid-reactive substance (TBARS) analysis is one of
the most popular and commonly used indicators of oxidative stress,
which works by detecting lipid oxidation (Rosmini et al., 1996). It has
been found that FA-decient animals increased the homocysteine
from methylation cycle and induced lipid peroxidation (Bays et al.,
2001; Dez et al., 2005; Huang et al., 2001; Moat et al., 2003).
Homocysteine, a sulphydryl-containing amino acid derived from the
demethylation of methionine, could generate reactive oxygen species,
such as hydrogen peroxide, superoxide anion and hydroxyl radical
during the auto-oxidation of homocysteine to homocystine or other
mixed disulphides (Heydrick et al., 2004; Welch et al., 1997). This may
explain the high TBARS value in FA-decient grouper found in the
present study.
One family of antioxidant enzymes, the superoxide dismutase
(SOD), function to remove damaging reactive oxygen species (ROS)
from the cellular environment by catalyzing the dismutation of two
superoxide radicals to hydrogen peroxide and oxygen (Fattman et al.,

2003). Outinen et al. (1999) demonstrated that homocysteine

inhibited gene expression of SOD in human cell line. Depressed
hepatic SOD activity (Table 4), along with higher hepatic TBARS value
(Table 3) in grouper fed diet deplete of FA may suggest that FA is
involved in the antioxidant system of the sh.
Phagocytes possess a unique membrane enzyme, NADPH oxidase,
capable of oxidation of molecular oxygen into superoxide anion (O
2 )
during a process termed the respiratory burst. Superoxide anion
production is considered to be one of the most important microbicidal
components in the armoury of phagocytes (Secombes, 1990). Lysozyme is known to act as a non-specic immune mediator against
parasitic, bacterial and viral infections, sh blood is considered as a
good indicator of its immune function (Puangkaew et al., 2004). These
two immune parameters were measured in the present study. Folic
acid functions as an intermediate carrier of one-carbon groups in a
number of complex enzymatic reactions. In these reactions, methyl,
methylene, and other one-carbon groups are transferred from one
molecule to another. These reactions are found in the metabolism of
certain amino acids and the biosynthesis of purines and pyrimidines
along with the nucleotides found in DNA and RNA (NRC, 1993). In
other words, production of superoxide anion and lysozyme should be
carried out in the presence of FA. This has been evidenced by depression of both leukocyte O
2 production ratio and plasma lysozyme
activity in grouper fed FA-decient diet (Table 4). When leukocyte
O
2 production ratio and plasma lysozyme activity were each plotted
against hepatic TBARS value, negative regression (Y = 0.014X +
2
1.82, R2 = 0.76, O
2 production ratio; Y = 2.15X +246.61, R = 0.91,
lysozyme activity) indicate that oxidative stress depress immune
response in grouper. This may also explain depression of leukocyte O
2
production ratio and plasma lysozyme activity in grouper fed FA-decient
diet.
It has been reported that the level of dietary vitamin C and vitamin E
required to enhance immune response in sh are much higher than that
required for growth. For instance, 6 (grouper) (Lin and Shiau, 2005a),
10 (Japanese seabass) (Ai et al., 2004), 50 (turbot) (Roberts et al.,
1995), 138 (Atlantic salmon) (Hardie et al., 1991), and 200 (channel
catsh) (Li and Lovell, 1985) of vitamin C; 4 (grouper) (Lin and Shiau,
2005b), 5 (Atlantic salmon) (Hardie et al., 1990), 6 (rainbow trout)
(Clerton et al., 2001), and 12 (gilthead seabream) (Ortuo et al., 2000)
of vitamin E are required in diet for enhancing immune response. As for
folic acid, our results suggest that the amount of dietary FA needs to
maximize grouper growth also adequate to stimulate immune responses
of the sh.
Acknowledgement
This work was supported by a grant from the National Science
Council of the Republic of China, grant number NSC 98-2321-B-126001-MY3.
References
Ai, Q., Mai, K., Zhang, C., Xu, W., Duan, Q., Tan, B., Liufu, Z., 2004. Effects of dietary
vitamin C on growth and immune response of Japanese seabass, Lateolabrax
japonicus. Aquaculture 242, 489500.
Albrektsen, S., Sandnes, K., Glette, J., Waagb, R., 1995. Inuence of dietary vitamin B6
on tissue vitamin B6 contents and immunity in Atlantic salmon, Salmo salar L.
Aquac. Res. 26, 331339.
Aoe, H., Masuda, I., Saito, T., Takada, T., 1967. Water-soluble vitamin requirements of
carp. 5. Requirement for folic acid. Bull. Jpn. Soc. Sci. Fish. (Currently Fisheries Sci.)
33, 10681071.
Association of Ofcial Analytical Chemists (AOAC), 1995. Ofcial Methods of Analysis,
16th ed. AOAC, Arlington, VA.
Bays, B., Cruz Pastor, M., Bonal, J., Junc, J., Romero, R., 2001. Homocysteine and lipid
peroxidation in haemodialysis: role of folinic acid and vitamin E. Nephrol. Dial.
Transplant. 16, 21722175.
Camilo, E., Zimmerman, J., Mason, J.B., Golner, B., Russell, R., Selhub, J., Rosenberg, I.,
1996. Folate synthesized by bacteria in human upper small intestine is assimilated
by the host. Gastroenterology 110, 991998.

Y.-H. Lin et al. / Aquaculture 317 (2011) 133137

Chen, H.Y., Tsai, J.C., 1994. Optimum dietary protein level for the growth of juvenile
grouper, Epinephelus malabaricus, fed semipuried diets. Aquaculture 119,
265271.
Christian, J.L., Greger, J.L., 1994. Nutrition for living. The Benjamin/Cummings
Publishing Company, Redwood City, California, USA. 621 pp.
Clerton, P., Troutaud, D., Verlhac, V., Gabaudan, J., Deschaux, P., 2001. Dietary vitamin E
and rainbow trout (Oncorhynchus mykiss) phagocyte functions: effect on gut and
on head kidney leukocyte. Fish Shellsh Immunol. 11, 113.
Cowey, C.B., Woodward, B., 1993. The dietary requirement of young rainbow trout
(Oncorhynchus mykiss) for folic acid. J. Nutr. 123, 15941600.
Dez, N., Prez, R., Hurtado, V., Santidrin, S., 2005. Hyperhomocysteinaemia induced by
dietary folate restriction causes kidney oxidative stress in rats. Br. J. Nutr. 94,
204210.
Duncan, P.L., Lovell, R.T., Butterworth, C.E., Freeberg, L.F., Tamura, T., 1993. Dietary
folate requirement determined for channel catsh, Ictalurus punctatus. J. Nutr. 123,
18881897.
Fattman, C.L., Schaefer, L.M., Oury, T.D., 2003. Extracellular superoxide dismutase in
biology and medicine. Free Radic. Biol. Med. 35, 236256.
Feng, L., He, W., Jiang, J., Liu, Y., Zhou, X.Q., 2010. Effects of dietary pyridoxine on disease
resistance, immune responses and intestinal microora in juvenile Jian carp
(Cyprinus carpio var. Jian). Aquac. Nutr. 16, 254261.
Hardie, L.J., Feletcher, T.C., Secombes, C.J., 1990. The effect of vitamin E on the immune
response of the Atlantic salmon (Salmo salar L.). Aquaculture 87, 113.
Hardie, L.J., Feletcher, T.C., Secombes, C.J., 1991. The effect of vitamin C on the immune
response of the Atlantic salmon Salmo salar L. Aquaculture 95, 201214.
Hernandez, L.H.H., Teshima, S.I., Koshio, S., Ishikawa, M., Tanaka, Y., Alam, S., 2007.
Effects of vitamin A on growth, serum anti-bacterial activity and transaminase
activities in the juvenile Japanese ounder, Paralichthys olivaceus. Aquaculture 262,
444450.
Heydrick, S.J., Weiss, N., Thomas, S.R., Cap, A.P., Pimentel, D.R., Loscalzo, J., Keaney Jr., J.F.,
2004. L-Homocysteine and L-homocystine stereospecically induce endothelial nitric
oxide synthase-dependent lipid peroxidation in endothelial cell. Free Radic. Biol. Med.
36, 632640.
Huang, R.F.S., Hsu, Y.C., Lin, H.L., Yang, F.L., 2001. Folate depletion and elevated plasma
homocysteine promote oxidative stress in rat livers. J. Nutr. 131, 3338.
John, M.J., Mahajan, C.L., 1979. The physiological response of shes to a deciency of
cyanocobalamin and folic acid. J. Fish Biol. 14, 127133.
Kashiwada, K., Kanazawa, A., Techima, S., 1971. Studies on the production of B vitamins
by intestinal bacteria of carp. Mem. Fac. Fish. Kagoshima Univ. 20, 185189.
Kitamura, S., Suwa, T., Ohara, S., Nakagawa, K., 1967. Studies on vitamin requirements of
rainbow trout. 2. The deciency symptoms of fourteen kinds of vitamin. Bull. Jpn.
Soc. Sci. Fish. (Currently Fisheries Sci.) 33, 11201125.
Li, Y., Lovell, R.T., 1985. Elevated levels of dietary ascorbic acid increase immune
responses in channel catsh. J. Nutr. 115, 123131.
Lin, M.F., Shiau, S.Y., 2005a. Dietary L-ascorbic acid affects growth, non-specic immune
responses and disease resistance in juvenile grouper, Epinephelus malabaricus.
Aquaculture 244, 215221.
Lin, Y.H., Shiau, S.Y., 2005b. Dietary vitamin E requirements of grouper, Epinephelus
malabaricus, under two lipid levels, and their effects on immune responses.
Aquaculture 248, 235244.
Lin, Y.H., Wu, J.Y., Shiau, S.Y., 2010. Dietary cobalt can promote gastrointestinal bacterial
production of vitamin B12 in sufcient amounts to supply growth requirements of
grouper, Epinephelus malabaricus. Aquaculture 302, 8993.
Mackie, A.M., Mitchell, A.I., 1985. Identication of gustatory feeding stimulants for sh
applications in aquaculture. In: Cowey, C.B., Mackie, A.M., Bell, J.B. (Eds.), Nutrition and
Feeding in Fish. Academic Press, London, pp. 177189.

137

Millamena, O.M., 2002. Replacement of sh meal by animal by-product meals in a practical

diet for grow-out culture of grouper, Epinephelus coioides. Aquaculture 204, 7584.
Moat, S.J., Hill, M.H., McDowell, I.F.W., Pullin, C.H., Asheld-Watt, P.A.L., Clark, Z.E.,
Whiting, J.M., Newcombe, R.G., Lewis, M.J., Powers, H.J., 2003. Reduction in plasma
total homocysteine through increasing folate intake in healthy individuals is not
associated with changes in measures of antioxidant activity or oxidant damage.
Eur. J. Clin. Nutr. 57, 483489.
NRC (National Research Council), 1993. Nutrient Requirements of Fish. National
Academy Press, Washington, DC, USA. 114 pp.
Obach, A., Quentel, C., Baudin Laurencin, F., 1993. Effects of alpha-tocopherol and
dietary oxidized sh oil on the immune response of sea bass Dicentrarchus labrax.
Dis. Aquat. Organ. 15, 175185.
Ortuo, J., Esteban, M.A., Meseguer, J., 2000. High dietary intake of -tocopherol acetate
enhances the non-specic immune response of gilthead seabream (Sparus aurata
L.). Fish Shellsh Immunol. 10, 293307.
Outinen, P.A., Sood, S.K., Pfeifer, S.I., Pamidi, S., Podor, T.J., Li, J., Weitz, J.I., Austin, R.C.,
1999. Homocysteine-induced endoplasmic reticulum stress and growth arrest
leads to specic changes in gene expression in human vascular endothelial cells.
Blood 94, 959967.
Puangkaew, J., Kiron, V., Somamoto, T., Okamoto, N., Satoh, S., Takeuchi, T., Watanabe,
T., 2004. Nonspecic immune response of rainbow trout (Oncorhynchus mykiss
Walbaum) in relation to different status of vitamin E and highly unsaturated fatty
acids. Fish Shellsh Immunol. 16, 2539.
Roberts, M.L., Davies, S.J., Pulsford, A.L., 1995. The inuence of ascorbic acid (vitamin C)
on non-specic immunity in the turbot (Scophthalmus maximus L.). Fish Shellsh
Immunol. 5, 2738.
Rong, N., Selhub, J., Goldin, B.R., Rosenberg, I.H., 1991. Bacterially synthesized folate in
rat large intestine is incorporated into host tissue folyl polyglutamates. J. Nutr. 121,
19551959.
Rosmini, M.R., Perlo, F., Prez-Alvarez, J.A., Pagn-Moreno, M.J., Gago-Gago, A., LpezSantovea, F., Aranda-Catal, V., 1996. TBA test by an extractive method applied to
Pat. Meat Sci. 42, 103110.
Russell, R.M., Krasinski, S.D., Samloff, I.M., Jacob, R.A., Dallal, G.E., McGandy, R.B., Hartz,
S.C., 1986. Fundic atrophic gastritis in an elderly population. J. Am. Geriatr. Soc. 34,
800806.
Ryu, K.S., Eitenmiller, R.R., Pesti, G.M., 1994. A comparison of enzyme preparations to
liberate folic acid for the microbiological assay of feed ingredients. J. Sci. Food Agric.
64, 389392.
Secombes, C.J., 1990. Isolation of salmonid macrophages and analysis of their killing
activity. In: Stolen, J.S., Fletcher, T.C., Anderson, D., Robertson, B.S., van Muiswinkel,
W.B. (Eds.), Techniques in Fish Immunology, Vol. I. SOS Publications, Fair Haven, N.J,
pp. 137154.
Shiau, S.Y., Huang, S.Y., 2001a. Dietary folic acid requirement for maximal growth of
juvenile tilapia, Oreochromis niloticus O. aureus. Fisheries Sci. 67, 655659.
Shiau, S.Y., Huang, S.Y., 2001b. Dietary folic acid requirement determined for grass
shrimp, Penaeus monodon. Aquaculture 200, 339347.
Thompson, I., Ghoubert, G., Houlihan, D.F., Secombes, C.J., 1995. The effect of dietary
vitamin A and astaxanthin on the immunocompetence of rainbow trout.
Aquaculture 133, 91102.
Uchiyama, M., Mihara, M., 1978. Determination of malonaldehyde precursor in tissue
by thiobarbituric acid test. Anal. Biochem. 86, 271278.
Welch, G.N., Upchurch Jr., G.R., Loscalzo, J., 1997. Homocysteine oxidative stress, and
vascular disease. Hosp. Pract. 32, 8192.
Yang, Q., Zhou, X., Jiang, J., Liu, Y., 2008. Effect of dietary vitamin A deciency on growth
performance, feed utilization and immune responses of juvenile Jian carp (Cyprinus
carpio var. Jian). Aquac. Res. 39, 902906.