Aquaculture 422–423 (2014) 84–90

Contents lists available at ScienceDirect

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

Interactive effects of vitamin C and E supplementation on growth

performance, fatty acid composition and reduction of oxidative stress in
juvenile Japanese flounder Paralichthys olivaceus fed dietary oxidized
fish oil
Jian Gao a,b,⁎, Shunsuke Koshio c, Manabu Ishikawa c, Saichiro Yokoyama c, Roger Edward P. Mamauag d
a
Laboratory of Aquatic Animal Nutrition, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, Hubei,
People's Republic of China
b
Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, People's Republic of China
c
Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University, Kagoshima 890-0056, Japan
d
Southeast Asian Fisheries Development Center, Aquaculture Department, Tigbauan 5021, Iloilo, Philippines

a r t i c l e i n f o a b s t r a c t

Article history: A study was conducted to determine the interactive effects of vitamin C (VC) and E (VE) supplementation on
Received 3 July 2013 growth, fatty acid composition and oxidative status of Japanese flounder juveniles. Fish (initial average body
Received in revised form 25 November 2013 weight of 1.1 ± 0.1 g) in triplicate were fed five test diets for 60 days. Control diet contained fresh fish oil
Accepted 25 November 2013
(FFO, 8.9 meq/kg) with 100 mg α-tocopherol (α-Toc) equivalents/kg of VE and 500 mg ascorbic acid (AsA)
Available online 12 December 2013
equivalents/kg of VC (FFO100E/500C). The other four diets contained oxidized fish oil (OFO, 167.8 meq/kg)
Keywords:
with varying levels of VE (mg/kg) and VC (mg/kg) (OFO100E/500C, OFO200E/500C, OFO100E/1000C and
Paralichthys olivaceus OFO200E/1000C). Fish fed FFO100E/500C and OFO100E/500C had no differences in body weight gain (BWG).
Vitamin C However, fish fed OFO200E/1000C diet had a significantly lower BWG than FFO100E/500C. Fish fed OFO200E/
Vitamin E 500C and OFO100E/1000C showed no differences in thiobarbituric acid-reactive substance values compared
Lipid peroxidation with FFO100E/500C. Increasing the levels of VC and VE supplementation increased liver AsA and α-Toc contents,
Oxidative stress respectively. Liver α-Toc content was significantly increased with incremental dietary VC levels, indicating a
Fatty acid composition sparing effect of VC on liver α-Toc content of fish. Increasing the levels of dietary VC and VE supplementations
decreased concentrations of 20:5n-3, 22:5n-3 and 22:6n-3 in fish liver. Fish fed OFO100E/500C and OFO200E/
1000C diets showed higher oxidative stress condition than those fed FFO100E/500C. In conclusion, dietary VC
and VE supplementation could maintain normal growth and health condition of juvenile Japanese flounder fed
OFO. However, high doses of both vitamin supplements induced fish lipid peroxidation under oxidative stress
condition.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction
Oxidative stress is an increase in free radical production by cells,
resulting in cell and tissue damage (Kohen and Nyska, 2002). The ma-
jority of these radicals are reactive oxygen species, such as the hydroxyl
radical, hydrogen peroxide, and superoxide anion, all of which have the
propensity to damage crucial cellular components like lipids, carbohy-
drates, proteins, and DNA (Finkel and Holbrook, 2000). Fish oil contains
high amounts of n-3 highly unsaturated fatty acids (n-3 HUFA) such
as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid
(DHA, 22:6n-3), which are required in sufficient amounts to supply
essential fatty acids (EFA) in order to support the normal growth
and survival of marine fish species. However, EFA are susceptible to
⁎ Corresponding author at: Key Lab of Freshwater Animal Breeding, Ministry of
Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, People's Republic
of China.
lipid peroxidation. Oxidized fish oil (OFO) is harmful to humans and
animals (Frankel, 1998).
Vitamins C (ascorbic acid, AsA) and E (α-tocopherol, α-Toc) are
major antioxidant additives used in the food industry and have been
shown to reduce the oxidative stress in animals (Baker and Davies,
1996, 1997; Chien et al., 1999; Gao et al., 2012a, 2013a; Hamre et al.,
2001; Tocher et al., 2003). Interactive effects between vitamins C and
E in preventing lipid peroxidation have been investigated (Niki et al.,
1982). Vitamin C has the ability to regenerate and/or spare vitamin E
in fish, such as in Atlantic salmon Salmo salar (Hamre et al., 1997),
gilthead sea bream Sparus aurata (Montero et al., 1999), juvenile lake
sturgeon Acipenser fulvescens (Moreau et al., 1999), channel catfish
Ictalurus punctatus (Lim et al., 2000), yellow perch Perca flavescens (Lee
and Dabrowski, 2003) and red sea bream Pagrus major (Gao et al., 2012b).
In addition to the protective effects of both vitamins against lipid
peroxidation, high doses of vitamin E may even exert pro-oxidative ef-
fects in human low density lipoproteins (Bowry and Stocker, 1993).

0044-8486/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.aquaculture.2013.11.031
 J. Gao et al. / Aquaculture 422–423 (2014) 84–90
Vitamin C may also act as both pro- and anti-oxidant in biological sys-
tems depending on the physiological conditions (Lee et al., 2001;
Roginsky and Stegmann, 1994). Japanese flounder Paralichthys olivaceus
is one of the most valuable fish species in eastern Asia, particularly in
Japan as an aquaculture species.
A few studies about vitamin C and E requirements of this species
have been reported. Wang et al. (2006a) found that Japanese flounder
fed diets containing about 96.0 and 213 mg/kg vitamin E supplementa-
tion increased respiratory burst activity than those fed diet contained
about 20 mg/kg vitamin E at 1.0% n-3 HUFA level. Panganiban et al.
(2004) observed that supplementation of 500 mg/kg vitamin C could
increase red blood cell counts of juvenile Japanese flounder. Dietary
lipid oxidation reducing the contents of fish antioxidant nutrients like
vitamins C and E has been reported in a number of studies (Baker and
Davies, 1997; Lewis-McCrea and Lall, 2007; Mourente et al., 2002). A
higher dose of antioxidants needs to be provided in diets when oxidized
lipid is used (Gao et al., 2012a). Therefore, the purpose of the present
study was to determine the effects of various levels of vitamins C and
E, and their interactive effects on growth performance, liver fatty acid
composition and oxidative stress condition of juvenile Japanese floun-
der fed dietary oxidized fish oil.
2. Materials and methods
2.1. Experimental diets
The oxidized fish oil was prepared by the following procedure. Cod
liver oil without any antioxidant was obtained from Nippon Suisan
Kaisha Ltd. (Tokyo, Japan) and was oxidized by heating at 70 °C with
vigorous aeration. Peroxide value (POV) was monitored every 8 h
until the values of 167.8 meq/kg was achieved. Five diets were formu-
lated to contain about 55.0% protein, 18.7% lipid and 11.3% ash
(Table 1). According to previous studies (Panganiban et al., 2004;
Wang et al., 2006a, 2006b), 500 mg/kg of vitamin C and 100 mg/kg of
vitamin E supplementation in diet are essential to maintain the normal
physiological function for juvenile Japanese flounder. Therefore, we de-
signed that the control diet contained fresh fish oil (FFO) with 100 mg
α-Toc equivalents/kg of vitamin E and 500 mg AsA equivalents/kg of
Table 1
Ingredient and proximate composition of basal diets.
Ingredients (g/kg)
Brown fish meala
Activated gluten
Starch
Fresh fish oilb or oxidized fish oilc
Soybean lectin
Mineral mixtured
Vitamin mixturee
α-Tocopherol + AMP-Na/Caf + α-cellulose
Proximate composition
Moisture (%)
Lipid (% D.M.)
Ash (% D.M.)
Protein (% D.M.)
a
Nippon Suisan Co. Ltd., Japan.
b
Cod liver oil without antioxidants, Nippon Suisan, Miyazaki, Japan.
c
Peroxide value of oxidized fish oil was 167.8 meq/kg.
d glutamyl oxaloacetic transaminase (GOT) and glutamic-pyruvate trans-
Mineral mixture (mg/kg diet): MgSO4 5070; Na2HPO4 3230; K2HPO4 8870; Fe Cit-
rate 1100; Ca Lactate12090; Al (OH)3 10; ZnSO4 130; CuSO4 4; MnSO4 30; Ca (IO3)2 10;
CoSO4 40.
e
Vitamin mixture (mg/kg diet): β-carotene 96.26; Vitamin D3 9.68; Menadione
NaHSO3·3H2O (K3) 45.83; Thiamine-Nitrate (B1) 57.75; Riboflavin (B2) 192.37;
Pyridoxine-HCl (B6) 45.83; Cyanocobalamine (B12) 0.07; D-Biotin 5.78; Inositol
3212.83; Niacine (Nicotic acid) 769.73; Ca Panthothenate 269.49; Folic acid 14.40;
Choline chloride 7869.30; ρ-Aminobenzoic acid 383.25.
f
L-Ascorbyl-2-Monophosphate (AMP-Na/Ca, DSM Nutrition Japan K.K.).
85
vitamin C (FFO100E/500C). The other four diets contained oxidized
fish oil (OFO) with varying levels of vitamin E (mg α-Toc equivalents/
kg) and vitamin C (mg AsA equivalents/kg) at the following dietary
levels: OFO100E/500C, OFO200E/500C, OFO100E/1000C and OFO200E/
1000C. POV, AsA and α-Toc concentrations, thiobarbituric acid-
reactive substances (TBARS) and fatty acid composition of test diets
are presented in Table 2. All test diets were stored at −80 °C prior to
the start of the growth experiment, and were stored at −20 °C in a re-
frigerator during the feeding trial.
2.2. Fish and sampling procedures
Juvenile Japanese flounder were obtained from a local hatchery, in
Miyazaki prefecture, Japan, and transported to the Kamoike Marine Pro-
duction Laboratory, Faculty of Fisheries, Kagoshima University, Kagoshi-
ma, Japan. Prior to the feeding trial, all fish were acclimated to the
indoor rearing conditions for 2 weeks and fed a commercial pelleted
fish feed (Higashimaru Co., Ltd., Kagoshima, Japan). A total of 180 fish
(initial body weight 1.1 ± 0.1 g, mean ± SD) were divided into 15
tanks (100-L) with a flow-through system using filtered seawater as
the water source at a stocking density of 12 fish/tank. The experiment
condition was maintained at optimum to ensure the survival of the
fish. The water flow to the tanks was at 1.9 l/min, artificial aeration
and natural light/dark regime was maintained throughout the growth
trial. The water temperature was 16.6 ± 0.5 °C during the feeding peri-
od. Fish were fed test diets at 08:00 and 16:00 h to apparent satiation
level for 60 days. Uneaten diets were collected every day, dried and
weighted to determine the daily feed intake.
At the end of the feeding trial, all fish were measured for weight and
length gain. Three fish from each tank were randomly sampled and
stored at −20 °C for proximate analysis. Blood was collected using hep-
arinized (1800 IU/ml) needle (25 G × 2.5 cm) and syringes (1 ml) from
three fish per tank and pooled into one tube as a blood sample. Liver was
dissected out from six fish in each replicate tank, weighed individually
to calculate hepatosomatic index (HSI) and finally pooled together
and kept at − 80 °C for measurement of AsA and α-Toc concentra-
tions, TBARS value and fatty acid composition analysis. Muscle (with-
out skin) and intestine samples were collected, pooled together and
stored at −80 °C until analysis of TBARS.
2.3. Analysis
Proximate compositions of diets and whole body were analyzed in
% triplicates for protein, ash and moisture (A.O.A.C., 1990). Total lipid
74.0
was measured following the method of Bligh and Dyer (1959). Peroxide
5.0 value (POV) of fish oil and diets (extracted with acetone) were deter-
4.0 mined by the I.U.P.A.C. (1987). The measurement of TBARS was carried
6.0 out using a method adapted from Yagi (1987). Values of AsA contents of
4.0
diets and whole-body were quantified by the method of Gao et al.
3.0
3.0 (2013a). Methods of high-performance liquid chromatography (HPLC)
1.0 analysis for AsA concentrations in fish tissues or diets were those previ-
ously described by Sakakura et al. (1998). The α-Toc concentrations of
9.8 diets and liver were determined by HPLC with a fluorescence detector
18.7 (FLD) according to Gao et al. (2012a). The fatty acid compositions of
11.3 the total lipid fractions of the diet and liver were determined using
55.0 gas chromatography (GC-17A; Shimadzu Corp, Kyoto, Japan; column:
OmegawaxTM320) according to the method of Teshima et al. (1986).
Plasma chemical parameters including glucose, total cholesterol,
aminase (GPT) were measured spectro-photometrically with an auto-
mated analyzer (SPOTCHEM™ EZ model SP-4430, Arkray, Inc. Kyoto,
Japan). Biological antioxidant potential (BAP) and reactive oxygen me-
tabolites (d-ROMs) were also measured spectrophotometrically from
blood plasma with an automated analyzer (FRAS, Diacron International
s.r.l., Grosseto, Italy) by following the methods of our previous study
(Gao et al., 2012a).
86 J. Gao et al. / Aquaculture 422–423 (2014) 84–90

Table 2
Peroxide value (POV, meq/kg), analyzed ascorbic acid concentration (mg/kg), analyzed α-tocopherol (mg/kg), thiobarbituric acid reactive substances (TBARS, μmol MDAa/mg dry mass)
and fatty acid composition (% of total fatty acids) of test diets.

Experimental dietsb

FFO100E/500C OFO100E/500C OFO200E/500C OFO100E/1000C OFO200E/1000C

Analyzed ascorbic acid 470.4 431.9 443.7 731.7 719.1

Analyzed α-tocopherol 85.6 73.2 140.3 67.7 131.2
POV 5.8 28.6 29.1 28.5 28.1
TBARS 21.1 45.5 46.7 48.8 48.4
Fatty acid composition
(% of total fatty acids)
ΣSaturates 26.3 28.6 29.5 28.8 28.9
ΣMonoenes 31.6 32.0 31.8 31.6 33.2
Σn-6 14.2 15.1 14.8 15.1 15.4
Σn-3 24.6 20.5 20.0 20.8 19.6

ΣSaturates includes 14:0, 16:0 and 18:0.

ΣMonoences includes 16:1n-7, 18:1n-9, 18:1n-7, 20:1n-9, 22:1n-11 and 22:1n-9.
Σn-6 includes 18:2n-6, 20:2n-6 and 20:4n-6.
Σn-3 includes 18:3n-3, 18:4n-3, 20:4n-3, 20:5n-3, 22:5n-3 and 22:6n-3.
a
MDA, malondialdehyde.
b
FFO, fresh fish oil; OFO, oxidized fish oil.

2.4. Statistical analysis
Statistical analysis was performed with analysis of variance
(ANOVA) using a program (package super-ANOVA, Abacus 19 Concepts,
Berkeley, California, USA). The data from each treatment group were
compared to the control group (FFO100E/500C) using the Tukey–
Kramer test (one-way ANOVA). Differences among treatments were
considered significant when P b 0.05. Two-way ANOVA was also used
to test the effects of dietary vitamin C and E levels, and their interactions
excluding FFO100E/500C.
3. Result
There were no observed significant differences in survival rate and
feed intake among treatments. The final weight of fish fed test diets
ranged from 8.2 to 10.5 g after 60 days (Table 3). Body weight gain of
fish fed OFO100E/500C, OFO200E/500C and OFO100E/1000C had no
significant differences compared with those of fish fed FFO100E/500C.
However, fish fed OFO200E/1000C had a significantly lower growth
rate compared to fish from the control group. Within fish fed OFO
diets, the supplementation of vitamin C and the interaction of both vita-
mins were a significant factor to affect growth performance. No signifi-
cant difference on condition factor among treatment groups was found.
Proximate composition analysis (crude fat, moisture and crude
protein) of whole body had no significant difference among treatment
groups (Table 4). Whole body ash contents of fish fed OFO100E/500C,
OFO100E/1000C and OFO200E/1000C were significantly higher than
those fed FFO100E/500C. Supplementation of vitamin C or E was a sig-
nificant factor on whole body ash content. The HSI (hepatosomatic
index) was also increased by dietary OFO, but fish fed OFO200E/500C
showed significantly lower value than fish from the control group,
whereas the highest value was found in fish fed OFO100E/500C and
OFO200E/1000C.
Liver AsA and α-Toc concentrations are shown in Table 5. Fish fed
OFO100E/500C had lower liver AsA and α-Toc concentrations than
those fed FFO100E/500C in equivalent vitamin E and C supplementa-
tion. Liver AsA and α-Toc concentrations significantly increased with in-
creasing dietary vitamin C and E levels, respectively. Increasing the
dietary vitamin C concentration from 500 to 1000 mg/kg significantly
increased liver α-Toc concentration. However, fish fed OFO200E/
1000C had significantly lower liver α-Toc concentration than fish from
the control group. The TBARS values in intestine, liver and muscle are
also presented in Table 5. There were no significant differences in intes-
tine and muscle TBARS values among treatment groups. Fish fed OFO
diets had higher TBARS value than those from the control group. Fish
fed OFO200E/1000C had higher TBARS value than other groups.
Fatty acid compositions of liver are shown in Table 6. In the compar-
ison between FFO100E/500C and OFO100E/500C, dietary OFO signifi-
cantly increased liver concentrations of 18:2n-6, 20:4n-6 and total n-6
fatty acids and significantly decreased 18:0 18:4n-3, 20:4n-3 and

Table 3
Growth performances of Japanese flounder fed with different diets for 60 days.⁎

Experimental dietsa Two way-ANOVA

FFO100E/500C OFO100E/500C OFO200E/500C OFO100E/1000C OFO200E/1000C C E C×E

Final weight (g) 10.5 ± 0.6 9.6 ± 0.4 10.1 ± 0.8 9.8 ± 0.5 8.2 ± 0.7⁎ 0.037 – 0.013
BWGb 799.8 ± 60.7 719.8 ± 39.7 766.4 ± 66.8 737.5 ± 43.2 597.9 ± 60.3⁎ 0.041 – 0.017
SGRc 3.7 ± 0.1 3.5 ± 0.1 3.6 ± 0.1 3.5 ± 0.1 3.2 ± 0.1⁎ 0.039 – 0.017
FCRd 1.1 ± 0.1 1.3 ± 0.1 1.3 ± 0.1 1.4 ± 0.1 1.6 ± 0.1⁎ 0.013 0.044 0.043
SRe 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 97.2 ± 4.8 – – –
FIf (g/fish) 10.7 ± 0.2 11.1 ± 0.8 11.8 ± 0.7 11.8 ± 0.8 11.3 ± 0.8 – – –
CFg 1.1 ± 0.1 1.0 ± 0.2 1.0 ± 0.1 1.1 ± 0.2 1.0 ± 0.1 – – –
⁎ Values are expressed as mean ± SE from triplicate groups (twelve fish per tank and three thanks per treatment). Data with an asterisk in a row are significantly different from those of
FFO100E/500C (P b 0.05). FFO100E/500C is not included in two-way ANOVA.
a
FFO, fresh fish oil; OFO, oxidized fish oil.
b
BWG, body weight gain (%) = 100 × (final weight − initial weight) / (initial weight).
c
SGR, specific growth rate = 100 × (ln final weight − ln initial weight) / (duration).
d
FCR, feed conversion ratio = dry feed intake (g) / weight gain (g).
e
SR, survival rate (%) = 100 × (initial fish number − dead fish number) / (initial fish number).
f
FI, Feed intake (g/fish) = (total feed intake (g) / number of fishes) in the 60 day feeding period.
g
CF: condition factor (%) = Final weight / length gain.
 J. Gao et al. / Aquaculture 422–423 (2014) 84–90 87

Table 4
Whole body proximate analysis (%) and somatic parameters in Japanese flounder fed with different diets for 60 days.⁎

Experimental dietsa Two way-ANOVA

FFO100E/500C OFO100E/500C OFO200E/500C OFO100E/1000C OFO200E/1000C C E C×E

Crude Fat (%) 14.3 ± 0.5 14.3 ± 0.6 13.7 ± 0.6 14.4 ± 0.5 13.7 ± 0.8 – – –
Moisture (%) 75.1 ± 0.9 74.8 ± 0.6 75.5 ± 0.8 74.1 ± 0.7 75.0 ± 0.7 – – –
Crude ash (%) 12.6 ± 0.2 13.2 ± 0.3⁎ 13.0 ± 0.1 13.2 ± 0.2⁎ 13.4 ± 0.2⁎ 0.001 0.013 –
Crude protein (%) 66.2 ± 0.7 64.9 ± 1.3 67.0 ± 0.5 65.9 ± 1.4 66.6 ± 1.5 – – –
HSIb 2.2 ± 0.4 2.5 ± 0.4 1.7 ± 0.1⁎ 2.3 ± 0.3 2.5 ± 0.2 – – 0.012
⁎ Values are expressed as mean ± SE from triplicate groups (three fish per tank and three thanks per treatment). Data with an asterisk in a row are significantly different from those of
FFO100E/500C (P b 0.05). FFO100E/500C is not included in two-way ANOVA. Crude protein, crude lipid and ash are expressed on a dry weight basis.
a
FFO, fresh fish oil; OFO, oxidized fish oil.
b
HSI: hepatosomatic index (%).

20:5n-3. Incremental dietary vitamin C from 500 to 1000 mg/kg signif-
icantly decreased in the percentages of 20:4n-6, 20:4n-3, EPA, DHA,
22:5n-3 and total n-3 fatty acids and significantly increased the percent-
ages of 17:0, 18:0, total saturated fatty acids, 18:1n-9, 20:1n-9 and total
monoenes in liver of fish fed OFO diets. Meanwhile, incremental dietary
vitamin E from 100 to 200 mg/kg significantly decreased the percent-
ages of 20:4n-3 and 22:5n-3 and significantly increased 14:0, 16:0,
total saturated fatty acids and 18:1n-9 in liver of fish. Some fatty acid
like 16:0, 18:2n-6, 20:2n-6 and total n-6 fatty acid were influenced by
the interaction of both vitamins.
Plasma glucose, T-Cho, GOT, GPT and oxidative status (d-ROMs and
BAP) of fish fed with test diets for 60 days are shown in Table 7. There
were no significant differences in glucose and T-Cho among treatment
groups. Fish fed OFO100E/500C had higher GOT and GPT values than
those fed FFO100E/500C in equivalent vitamin E and C supplementa-
tion. GOT was significantly reduced by vitamin C supplementation.
However, fish fed OFO200E/1000C diet had significantly higher GOT
and GPT values than fish from the control group. Fish fed OFO100E/
500C had higher d-ROMs and lower BAP values than those fed
FFO100E/500C. Fish fed OFO200E/500C and OFO100E/1000C diets
showed lower d-ROM value and higher BAP values. However, fish fed
OFO200E/1000C diet had significantly higher d-ROM value than fish
from the control group. Fig. 1 shows the pattern of combined effects of
d-ROMs and BAP. Diets with FFO100E/500C, OFO200E/500C and
OFO100E/1000C were located in zone A, while OFO100E/500C and
OFO200E/1000C were located in zone D.
4. Discussion
Dietary oxidized lipid results in the accumulation of lipid peroxides
and intermediates, lower unsaturation index of tissues, tissue oxidative
stress and a decrease of antioxidants. A number of studies have shown
that the growth of fish was decreased by dietary oxidized lipid (Koshio
et al., 1994; Lewis-McCrea and Lall, 2007; Mourente et al., 2002). In the
present study, the comparison of fish fed FFO and OFO diets with equiva-
lent vitamin E and vitamin C contents (FFO100E/500C and OFO100E/
500C) showed no significant difference on the growth performance
of fish. However, increasing the dietary vitamin E level from 100 to
200 mg/kg or dietary vitamin C level from 500 to 1000 mg/kg, did not sig-
nificantly affect growth, suggesting that supplementation of 500 mg/kg of
vitamin C and 100 mg/kg of vitamin E was high enough for prevention of
lipid peroxidation in juvenile Japanese flounder cultured in 60 days. Sim-
ilar results were also found by Tocher et al. (2003), who reported that
supplementation of vitamin E in the diet could improve growth in
gilthead sea bream Sparus aurata fed with dietary oxidized oil. Moreover,
our previous study (Gao et al., 2013a) indicated that supplementation of
more than 500 mg/kg vitamin C could improve growth performance
and reduce oxidative stress of red sea bream P. major fed dietary oxidized
lipid. These results can be assumed that vitamins C and E play an impor-
tant role in protecting biological membranes and lipid components
against attack from oxygen free radicals. However, our results showed
that the increasing dietary vitamin E level from 100 to 200 mg/kg and vi-
tamin C level from 500 to 1000 mg/kg decreased growth performance. A
similar result was found by Ji et al. (2003), who found a lower growth of
red sea bream fed high concentrations of vitamin C and E supplemented
diets. Welker and Congleton (2009) reported that the erythrocyte mem-
branes of smolts were more susceptible to peroxidation, when dietary
vitamin C and/or E supplementation was higher than 615 mg/kg. In crus-
tacean species, Celada et al. (2013) also found that the levels of vitamin C
at 400 mg/kg diet or higher were unnecessary and could involve harmful
effects on the growth rate of juvenile crayfish. Moreover, the pro-
oxidative effect of an excess dose (1000 mg/kg) of dietary α-tocopherol
promoted lipid peroxidation, leading to an increase in the accumulation
of hydroperoxides in sweet smelt blood (Kaewsrithong et al., 2001).
These reports indicated that a high dose of vitamin C and/or E supplemen-
tations in diets had a deleterious effect on fish and crustaceans.
The comparison between fish fed FFO100E/500C and OFO100E/500C
diets showed that liver AsA and α-Toc contents were significantly re-
duced by dietary OFO. Similar results have been observed in previous
studies (Baker and Davies, 1997; Mourente et al., 2002; Tocher et al.,
2003). These results indicated that vitamin C and E can be an effective
antioxidant to prevent lipid peroxidation in fish. The interactive effects
of vitamins C and E on preventing lipid peroxidation have been reported
by several studies (Chen et al., 2004; Gao et al., 2013b; Lewis-McCrea
and Lall, 2010; Lim et al., 2000; Montero et al., 1999; Shiau and Hsu,
2002). The present results showed that the liver α-Toc concentration

Table 5
Liver ascorbic acid and α-tocopherol concentrations (mg/kg wet) and tissue TBARS (μmol MDA/mg wet) in Japanese flounder fed test diets for 60 days.⁎

Experimental dietsa Two way-ANOVA

FFO100E/500C OFO100E/500C OFO200E/500C OFO100E/1000C OFO200E/1000C C E C×E

Liver AsA 102.1 ± 9.8 66.7 ± 5.4⁎ 63.5 ± 3.6⁎ 127.8 ± 8.2⁎ 119.0 ± 12.9 b0.001 – –
Liver α-Toc 160.8 ± 9.1 82.0 ± 16.6⁎ 90.4 ± 10.2⁎ 113.5 ± 5.7⁎ 81.0 ± 7.3⁎ 0.010 0.008
TBARS
Intestine 7.1 ± 0.2 10.0 ± 0.4 6.5 ± 2.2 7.1 ± 1.9 9.1 ± 1.0 – – 0.018
Liver 14.4 ± 3.1 27.0 ± 5.5⁎ 22.6 ± 4.4 23.6 ± 1.2 36.0 ± 4.9⁎ – – 0.010
Muscle 1.6 ± 0.6 2.6 ± 0.8 1.9 ± 0.1 1.3 ± 0.2 2.3 ± 0.6 – – –
⁎ Values are expressed as mean ± SE from triplicate groups (pooled tissues of six fish per tank and three thanks per treatment). Data with an asterisk in a row are significantly different
from those of FFO100E/500C (P b 0.05). FFO100E/500C is not included in two-way ANOVA.
a
FFO, fresh fish oil; OFO, oxidized fish oil.
88 J. Gao et al. / Aquaculture 422–423 (2014) 84–90

Table 6
Liver fatty acid composition (% of total fatty acid) of Japanese flounder fed test diets for 60 days.⁎

Experimental dietsa Two way-ANOVA

FFO100E/500C OFO100E/500C OFO200E/500C OFO100E/1000C OFO200E/1000C VC VE VC × VE

14:0 4.5 ± 0.2 4.7 ± 0.1 5.2 ± 0.1 4.8 ± 0.1⁎ 5.5 ± 0.4⁎ 0.002
16:0 14.2 ± 0.3 14.3 ± 0.1 15.2 ± 0.2⁎ 14.7 ± 0.4 14.9 ± 0.3 0.007 0.036
17:0 1.7 ± 0.2 1.8 ± 0.1 1.7 ± 0.0 2.1 ± 0.2 2.3 ± 0.2⁎ 0.001
18:0 2.2 ± 0.0 2.0 ± 0.0⁎ 2.0 ± 0.1 1.9 ± 0.1⁎ 2.1 ± 0.2 0.037
Σsaturates 22.6 ± 0.4 22.8 ± 0.2 24.1 ± 0.2⁎ 23.5 ± 0.2⁎ 24.8 ± 0.3⁎ b0.001 b0.001
16:1n-7 7.7 ± 0.2 7.8 ± 0.2 8.0 ± 0.4 8.1 ± 0.0 8.2 ± 0.5 –
18:1n-9 16.6 ± 0.5 16.5 ± 0.1 16.2 ± 0.3 17.7 ± 0.3⁎ 16.8 ± 0.2 0.003 b0.001
18:1n-7 3.9 ± 0.1 4.0 ± 0.3 4.1 ± 0.1 3.7 ± 0.1 3.8 ± 0.3 –
20:1n-9 4.0 ± 0.1 3.9 ± 0.1 3.6 ± 0.3 4.2 ± 0.2 4.2 ± 0.3 0.019
22:1n-11 2.5 ± 0.0 2.6 ± 0.2 2.5 ± 0.1 2.6 ± 0.3 2.8 ± 0.1 –
Σmonoenes 35.4 ± 0.7 35.4 ± 0.4 34.8 ± 0.8 37.0 ± 0.8 36.4 ± 0.7 0.005
18:2n-6 17.7 ± 0.2 19.5 ± 0.4⁎ 19.2 ± 0.1⁎ 19.4 ± 0.1⁎ 19.8 ± 0.1⁎ 0.033
20:2n-6 0.6 ± 0.0 0.6 ± 0.1 0.5 ± 0.0 0.5 ± 0.0 0.6 ± 0.1 0.005
20:4n-6 0.9 ± 0.1 1.2 ± 0.1⁎ 1.1 ± 0.1 1.0 ± 0.0 1.0 ± 0.1⁎ 0.007
Σn-6 FA 19.2 ± 0.2 21.2 ± 0.3⁎ 20.8 ± 0.1⁎ 20.9 ± 0.1⁎ 21.3 ± 0.2⁎ 0.004
18:3n-3 1.9 ± 0.1 1.8 ± 0.0 1.9 ± 0.0 1.8 ± 0.0 1.8 ± 0.1 –
18:4n-3 0.9 ± 0.1 0.6 ± 0.0⁎ 0.7 ± 0.0⁎ 0.7 ± 0.0⁎ 0.6 ± 0.1⁎ –
20:4n-3 0.5 ± 0.0 0.4 ± 0.0⁎ 0.4 ± 0.0⁎ 0.4 ± 0.0⁎ 0.3 ± 0.0⁎ 0.028 0.004
20:5n-3 (EPA) 8.1 ± 0.1 7.0 ± 0.1⁎ 7.0 ± 0.3⁎ 6.3 ± 0.3⁎ 5.8 ± 0.3⁎ b0.001
22:5n-3 1.7 ± 0.3 1.4 ± 0.1 1.3 ± 0.1 1.2 ± 0.0⁎ 1.0 ± 0.0⁎ b0.001 b0.001
22:6n-3 (DHA) 6.5 ± 0.8 6.1 ± 0.3 5.9 ± 0.6 5.1 ± 0.5 4.2 ± 0.8⁎ 0.003
Σn-3 FA 19.5 ± 1.1 17.5 ± 0.3 17.1 ± 0.9⁎ 15.4 ± 0.8⁎ 13.7 ± 1.0⁎ b0.001

VC; vitamin C, VE; vitamin E, VC × VE; interaction.

⁎ Values are expressed as mean ± SE from triplicate groups (pooled livers of six fish per tank and three tanks per treatment). Data with an asterisk in a row are significantly different
from those of FFO100E/500C (P b 0.05). FFO100E/500C is not included in two-way ANOVA.
a
FFO, fresh fish oil; OFO, oxidized fish oil.

was increased by dietary vitamin C supplementation. This was also ob-
served by Lim et al. (2010), who reported that increasing dietary vita-
min C level from 100 mg/kg to 2000 mg/kg significantly increased
liver α-Toc concentration of Nile tilapia Oreochromis niloticus compared
to that of fish fed vitamin C un-supplemented diets. A synergistic effect
of simultaneous protection in the water and lipid phases against oxida-
tion, and a regenerating effect of vitamin C on tocopheroxyl radicals
have been described by Hamre et al. (1997). In contrast, no effects on
tissue α-Toc have been observed in a number of studies (Ruff et al.,
2003). The sparing effect of AsA on tissue α-Toc was found only when
vitamin E was deficient or near deficient (Gao et al., 2012a; Hamre
et al., 1997).
Fish fed OFO200E/1000C led to a 2-fold decrease in liver α-Toc con-
centration compared to fish fed the FFO100E/500C diet. A decrease in
antioxidant vitamin in animal tissues is caused by its utilization in halt-
ing the free radical cascade initiated by lipid peroxyl radicals and other
assorted products of lipid peroxidation (Sheehy et al., 1994). Lee et al.
(2001) determined that the efficiency of vitamin C in inducing the de-
composition of lipid hydroperoxides suggested that this process could
give rise to substantial amounts of DNA damage in vivo. On the other
hand, some studies have suggested that excess doses of α-Toc might in-
duce lipid peroxidation in tissues of fish (Kaewsrithong et al., 2001;
Tokuda and Takeuchi, 1995, 1999). The pro-oxidant mechanisms of ox-
idized α-Toc may be due to α-Toc peroxyl radical, α-Toc oxy radical, hy-
droxyl radical, and singlet oxygen formed from the α-Toc (Kim et al.,
2007). Therefore, the finding in this study suggested that high doses of
vitamin C and E supplementation in diet may produce a certain amount
of AsA and α-Toc radicals, which may act as pro-oxidants under oxida-
tive stress conditions in fish. TBARS is an important indicator of lipid
peroxidation. Our results indicated that a negative correlation was ob-
served between liver α-Toc concentration and value of TBARS in liver.
However, the highest value of TBARS was observed in fish fed
OFO200E/1000C. These results clearly indicated that high doses of vita-
min E and C supplementation induced lipid peroxidation in tissues
of fish.
It is generally accepted that maintaining high levels of PUFA is asso-
ciated with the presence of vitamin E, which is added to diets not only to
improve nutritional properties, but also to protect against free radicals
before they oxidize these fats in cell membranes. Our previous study
showed that the percentages of EPA, DHA and 22:5n-3 in liver of

Table 7
Blood parameter of Japanese flounder fed test diets for 60 days.⁎

Experimental diets Two way-ANOVA

FFO100E/500C OFO100E/500C OFO200E/500C OFO100E/1000C OFO200E/1000C C E C×E

Glucose (mg/dl) 30.7 ± 0.6 31.3 ± 2.1 29.7 ± 1.2 31.3 ± 0.6 29.5 ± 0.7 – – –
Total cholesterol (mg/dl) 299.7 ± 29.6 377.7 ± 29.4 298.7 ± 32.6 283.0 ± 62.4 328.0 ± 50.9 – – 0.049
GOTa (IU/l) 94.3 ± 8.3 118.7 ± 15.5 117.3 ± 5.5 85.3 ± 10.0 151.0 ± 10.6⁎ b0.001 – b0.001
GPTb (IU/l) 19.3 ± 1.5 21.7 ± 4.9 17.3 ± 1.5 19.3 ± 2.5 40.0 ± 12.7⁎ – – 0.004
Oxidative status
d-ROMsc (U.Carr) 280.3 ± 43.7 334.0 ± 56.0 196.0 ± 63.2 204.7 ± 26.8 438.3 ± 32.9⁎ – – b0.001
BAPd (μmol/l) 1770.7 ± 176.1 1113.7 ± 251.2 2094.3 ± 187.0 2029.7 ± 455.1 1744.7 ± 339.5 – – 0.010
⁎ Values are expressed as mean ± SE from triplicate groups (pooled blood of three fish per tank and three tanks per treatment). Data with an asterisk in a row are significantly different
from those of FFO100E/500C (P b 0.05). FFO100E/500C is not included in two-way ANOVA.
a
GOT: glutamyl oxaloacetic transaminase.
b
GPT: glutamic-pyruvate transaminase.
c
d-ROMs: reactive oxygen metabolites.
d
BAP: biological antioxidant potential.
 J. Gao et al. / Aquaculture 422–423 (2014) 84–90
24.0 A B
22.0
BAP (µmoli/L) ,*100
OFO200E/500C
20.0 OFO100E/1000C
18.0
FFO100E/500C OFO200E/1000C
16.0
14.0
12.0
C OFO100E/500C D
10.0
0 100 200 300 400 500 600
d-ROMs (U CARR)
Fig. 1. Oxidative stress parameter in blood of Japanese flounder fed test diets for 60 days.
Combined values of d-ROMs and BAP from each treatment are expressed as means. Zone
A: High antioxidant potential and low reactive oxygen metabolites (highly preferred).
Zone B: High antioxidant potential and high reactive oxygen metabolites (acceptable).
Zone C: Low antioxidant potential and low reactive oxygen metabolites (acceptable).
Zone D: Low antioxidant potential and high reactive oxygen metabolites (not acceptable).
juvenile red sea bream were increased with incremental levels of die-
tary vitamin E (Gao et al., 2012a) and C (Gao et al., 2013a), respectively.
Similarly, Navarro et al. (2012) also reported that supplementation of
100 or 150 mg vitamin E/kg diet in Nile tilapia had the highest levels
of omega-3 and omega-6 PUFA. However, the present results showed
that increasing the levels of dietary vitamin C and E supplementation
decreased EPA, 22:5n-3, DHA and n-3 HUFA contents in liver of fish, re-
spectively. A similar result was found by Zhong et al. (2007) who report-
ed that PUFA content in muscle phospholipids of fish fed oxidized oil
was decreased by vitamin E supplementation. Chien and Hwang
(2001) observed that vitamin C significantly reduced the proportions
of DHA and other PUFA in liver lipid of thornfish. Nevertheless, Tocher
et al. (2002) found that the proportions of EPA, DHA, total n-3 PUFA
and total PUFA were lower in sea bream fed with 100 mg/kg vitamin
E supplementation compared with fish fed with the other diets with 0
and 1000 mg/kg vitamin E supplementation. The mechanism behind
vitamin E supplementation effect on tissues fatty acid compositions of
fish needs further research.
The blood parameters of a fish species are true indicators of the state
of health of the organism. In the present study, fish fed dietary OFO sig-
nificantly increased plasma T-Cho level. Our previous study also found
that blood T-Cho could be increased by dietary oxidized lipid in red
sea bream (Gao et al., 2012a). However, the level of T-Cho was reduced
with incremental levels of vitamin E or vitamin C supplementation in
diets. Plasma GOT and GPT are indicators of liver damage. In this
study, fish fed OFO, increasing the dietary vitamin C supplementation
from 500 to 1000 mg AsA equivalents/kg with 100 mg α-Toc equiva-
lents/kg showed lower GOT value. This is in agreement with Ren et al.
(2007), who found that feeding higher dietary vitamin C supplementa-
tion reduced GOT level in Japanese eel. Increased value of GOT in fish fed
the OFO200E/1000C diet can be due to high doses of vitamin E and C
supplementation which induced lipid peroxidation, resulting in a poor
liver health condition. Oxidative stress was measured using the free rad-
ical analytical system assessing the derivatives of oxidative stress by
measuring reactive oxygen metabolites (using the d-ROM test) and bi-
ological antioxidant potential (using the BAP test) in whole plasma
samples. Measurement of d-ROM and BAP has been used for evaluation
of the oxidative stress condition of fish (Gao et al., 2012a, 2013a). As in-
dicated in Fig. 1, it is interesting to note that OFO200E/500C and
OFO100E/1000C groups with the control group (FFO100E/500C) located
in zone A. Dietary treatments OFO100E/500C and OFO200E/1000C
groups were located in zone D. Therefore, the high oxidative stress
caused by fish fed a high dose of vitamin C and E supplemented OFO
diet may explain why the growth of Japanese flounder in the OFO200E/
1000C group was low.
89
5. Conclusions
In summary, our findings showed that fish fed FFO and OFO diets
with equivalent vitamin E and vitamin C concentrations did not signifi-
cantly affect growth performance. Fish fed OFO100E/500C diet had
higher TBARS values in intestine, liver and muscle than those fed
FFO100E/500C diet. Increasing the levels of dietary vitamins C and E ex-
cept OFO200E/1000C had similar tissues TBARS values. Liver α-Toc con-
tent was significantly increased with incremental dietary vitamin C
levels, indicating a sparing effect of vitamin C on liver α-Toc content
of fish. Results also showed that increasing the levels of dietary vitamin
C and E supplementation decreased EPA, 22:5n-3, DHA and n-3 HUFA
contents in fish liver. Fish fed OFO200E/1000C diet had significantly de-
creased growth performance, liver α-Toc concentration and increased
liver TBARS value and oxidative status of blood parameters. In conclu-
sion, supplementation of vitamins C and E in diet could maintain normal
growth and health condition of juvenile Japanese flounder fed dietary
OFO compared with those fed dietary FFO. However, a high dose of
both vitamin supplementations may induce lipid peroxidation in fish
tissues under oxidative stress condition.
Acknowledgments
The first author wish to acknowledgment the Ministry of Education,
Culture, Sports, Science and Technology of Japan (MONBUKAGAKUSHO)
for supporting this research work.
References
A.O.A.C., 1990. Official Methods of Analysis of the Association of Official Analytical Chem-
ists, 15th ed. Association of Official Analytical Chemists, Arlington, VA, USA.
Baker, R.T.M., Davies, S.J., 1996. Changes in tissue α-tocopherol status and degree of lipid
peroxidation with varying α-tocopheryl acetate inclusion in diets for the African cat-
fish. Aquac. Nutr. 2, 71–79.
Baker, R.T.M., Davies, S.J., 1997. Modulation of tissue α-tocopherol in African catfish,
Clarias gariepinus (Burchell), fed oxidized oils, and the compensatory effect of supple-
mental dietary vitamin E. Aquaculture 3, 91–97.
Bligh, E.G., Dyer, W.J., 1959. A rapid method for total lipid extraction and purification. Can.
J. Biochem. Physiol. 37, 911–917.
Bowry, V.W., Stocker, R., 1993. Tocopherol-mediated meroxidation —the prooxidant effect
of vitamin-E on the radical-initiated oxidation of human low-density-lipoprotein.
J. Am. Chem. Soc. 115, 6029–6044.
Celada, J.D., Fuertes, J.B., Carral, J.M., Sáez-Royuela, M., González, Á., González-Rodríguez,
Á., 2013. Effects of vitamin C inclusion in practical diets on survival and growth of ju-
venile crayfish (Pacifastacus leniusculus Dana, Astacidae) from the onset of exogenous
feeding. Aquac. Nutr. 19, 110–116.
Chen, R.G., Lochmann, R., Goodwin, A., Praveen, K., Dabrowski, K., Lee, K.J., 2004. Effects of
dietary vitamins C and E on alternative complement activity, hematology, tissue com-
position, vitamin concentrations and response to heat stress in juvenile golden shiner
(Notemigonus crysoleucas). Aquaculture 242, 553–569.
Chien, L.T., Hwang, D.F., 2001. Effects of thermal stress and vitamin C on lipid peroxidation
and fatty acid composition in the liver of thornfish Terapon jarbua. Comp. Biochem.
Physiol. B 128, 91–97.
Chien, L.T., Hwang, D.F., Jeng, S.S., 1999. Effect of thermal stress on dietary requirement of
vitamin C in thornfish Terapon jarbua. Fish. Sci. 65, 731–735.
Finkel, T., Holbrook, N.J., 2000. Oxidants, oxidative stress and the biology of ageing. Nature
408, 239–247.
Frankel, E.N., 1998. Lipid Oxidation. The Oily Press Ltd., Dundee, UK.
Gao, J., Koshio, S., Ishikawa, M., Yokoyama, S., Mamauag, R.E.P., Han, Y., 2012a. Effects of
dietary oxidized fish oil with vitamin E supplementation on growth performance
and reduction of lipid peroxidation in tissues and blood of red sea bream Pagrus
major. Aquaculture 356–357, 73–79.
Gao, J., Koshio, S., Ishikawa, M., Yokoyama, S., Kader, M.A., Laining, A., 2012b. Interaction
between vitamin C and E on oxidative stress in tissues and blood of red sea bream
(Pagrus major). Aquac. Sci. 60, 119–126.
Gao, J., Koshio, S., Ishikawa, M., Yokoyama, S., Mamauag, R.E.P., Nguyen, B.T., 2013a. Effect
of dietary oxidized fish oil and vitamin C supplementation on growth performance
and reduction of oxidative stress in Red Sea Bream Pagrus major. Aquac. Nutr. 19,
35–44.
Gao, J., Koshio, S., Ishikawa, M., Yokoyama, S., Daisuke, N., Ren, T.J., 2013b. Interactive ef-
fects of vitamin C and E supplementation on growth, fatty acid composition, and lipid
peroxidation of sea cucumber, Apostichopus japonicus, fed with dietary oxidized fish
oil. J. World Aquac. Soc. 44, 536–546.
Hamre, K., Waagbo, R., Berge, R.K., Lie, O., 1997. Vitamins C and E interact in juvenile
Atlantic salmon (Salmo salar, L). Free Radic. Biol. Med. 22, 137–149.
90 J. Gao et al. / Aquaculture 422–423 (2014) 84–90
Hamre, K., Kolås, K., Sandnes, K., Julshamn, K., Kiessling, A., 2001. Feed intake and absorp-
tion of lipid oxidation products in Atlantic salmon (Salmo salar) fed diets coated with
oxidized fish oil. Fish Physiol. Biochem. 25, 209–219.
I.U.P.A.C. (International Union of Pure and Applied Chemistry), 1987. Standard Methods
for the Analysis of Oils, Fats and Derivatives.
Ji, H., Om, A.D., Yoshimatsu, T., Hayashi, M., Umino, T., Nakagawa, H., Asano, M.,
Nakagawa, A., 2003. Effect of dietary vitamins C and E fortification on lipid metabo-
lism in red sea bream Pagrus major and black sea bream Acanthopagrus schlegeli.
Fish. Sci. 69, 1001–1009.
Kaewsrithong, J., Ohshima, T., Ushio, H., Nagasaka, R., Maita, M., Sawada, M., 2001. Effects
of an excess dose of dietary a-tocopherol on hydroperoxide accumulation and eryth-
rocyte osmotic fragility of sweet smelt Plecoglossus altivelis. Aquac. Res. 32, 191–198.
Kim, H.J., Lee, H.O., Min, D.B., 2007. Effects and prooxidant mechanisms of oxidized α-
tocopherol on the oxidative stability of soybean oil. J. Food Sci. 72, 223–230.
Kohen, R., Nyska, A., 2002. Oxidation of biological systems: oxidative stress phenomena,
antioxidants, redox reactions, and methods for their quantification. Toxicol. Pathol.
30, 1164–1169.
Koshio, S., Ackman, R.G., Lall, S.P., 1994. Effects of oxidized herring and canola oils in diets
on growth, survival, and flavor of Atlantic salmon, Salmo salar. J. Agric. Food Chem. 42,
1164–1169.
Lee, K.J., Dabrowski, K., 2003. Interaction between vitamins C and E affects their tissue
concentrations, growth, lipid oxidation, and deficiency symptoms in yellow perch
(Perca flavescens). Br. J. Nutr. 89, 589–596.
Lee, S.H., Tomoyuki, O., Blair, I.A., 2001. Vitamin C-induced decomposition of lipid hydro-
peroxides to endogenous genotoxins. Science 292, 2083–2086.
Lewis-McCrea, L.M., Lall, S.P., 2007. Effects of moderately oxidized dietary lipid and the
role of vitamin E on the development of skeletal abnormalities in juvenile Atlantic
halibut (Hippoglossus hippoglossus). Aquaculture 262, 142–155.
Lewis-McCrea, L.M., Lall, S.P., 2010. Effects of phosphorus and vitamin C deficiency, vita-
min A toxicity, and lipid peroxidation on skeletal abnormalities in Atlantic halibut
(Hippoglossus hippoglossus). J. Appl. Ichthyol. 26, 334–343.
Lim, C., Klesius, P.H., Li, M.H., Robinson, E.H., 2000. Interaction between dietary levels of iron
and vitamin C on growth, hematology, immune response and resistance of channel cat-
fish (Ictalurus punctatus) to Edwardsiella ictaluri challenge. Aquaculture 185, 313–327.
Lim, C., Yildirim-Aksoy, M., Welker, T., Klesius, P.H., Li, M.H., 2010. Growth performance, im-
mune response, and resistance to Streptococcus iniae of Nile Tilapia, Oreochromis niloticus,
fed diets containing various levels of vitamins C and E. J. World Aquacult. Soc. 41, 35–48.
Montero, D., Marrero, M., Izquierdo, M.S., Robaina, L., Vergara, J.M., Tort, L., 1999. Effect of vi-
tamin E and C dietary supplementation on some immune parameters of gilthead
seabream (Sparus aurata) juveniles subjected to crowding stress. Aquaculture 171,
269–278.
Moreau, R., Dabrowski, K., Czesny, S., Cihla, F., 1999. Vitamin C–vitamin E interaction in
juvenile lake sturgeon (Acipenser fulvescens R.), a fish able to synthesize ascorbic
acid. J. Appl. Ichthyol. 15, 250–257.
Mourente, G., Diaz-Salvago, E., Bell, J.G., Tocher, D.R., 2002. Increased activities of hepatic
antioxidant defence enzymes in juvenile gilthead sea bream (Sparus aurata L.) fed
dietary oxidised oil: attenuation by dietary vitamin E. Aquaculture 214, 343–361.
Navarro, R.D., Navarro, F.K.S.P., Filho, O.P.R., Ferreira, W.M., Pereira, M.M., Filho, J.T.S.,
2012. Quality of polyunsaturated fatty acids in Nile tilapias (Oreochromis niloticus)
fed with vitamin E supplementation. Food Chem. 134, 215–218.
Niki, E., Tsuchiya, J., Tanimura, R., Kamiya, Y., 1982. Regeneration of vitamin E from alpha-
chromanoxyl radical by glutathione and vitamin C. Chem. Lett. 11, 789–792.
Panganiban, A., Koshio, S., Teshima, S., Ishikawa, M., Moe, Y.Y., Ren, T., 2004. Supplemen-
tation of ascorbyl monophosphate in diets increases stress tolerance in Japanese
flounder, Paralichthys olivaceus juveniles. Abstracts of the Annual Meeting of the
Japanese Society of Fisheries Science. Japanese Society of Fisheries Science, Tokyo,
Japan, p. 133.
Ren, T., Koshio, S., Ishikawa, M., Yokoyama, S., Micheal, F.R., Uyan, O., Tung, H.T., 2007. In-
fluence of dietary vitamin C and bovine lactoferrin on blood chemistry and non-
specific immune responses of Japanese eel, Anguilla japonica. Aquaculture 267, 31–37.
Roginsky, V.A., Stegmann, H.B., 1994. Ascorbyl radical as natural indicator of oxidative
stress —quantitative regularities. Free Radic. Biol. Med. 17, 93–103.
Ruff, N., Fitzgerald, R.D., Cross, T.F., Hamre, K., Kerry, J.P., 2003. The effect of dietary vita-
min E and C level on market-size turbot (Scophthalmus maximus) fillet quality.
Aquac. Nutr. 9, 91–103.
Sakakura, Y., Koshio, S., Iida, Y., Tsukamoto, K., Kida, T., Blom, J.H., 1998. Dietary vitamin C
improves the quality of yellowtail (Seriola quinqueradiata) seedlings. Aquaculture
161, 427–436.
Sheehy, P.J.A., Morrissey, P.A., Flynn, A., 1994. Consumption of thermally-oxidized sun-
flower oil by chicks reduces alpha-tocopherol status and increases susceptibility of
tissues to lipid oxidation. Br. J. Nutr. 71, 53–65.
Shiau, S.Y., Hsu, C.Y., 2002. Vitamin E sparing effect by dietary vitamin C in juvenile hybrid
tilapia, Oreochromis niloticus xO. aureus. Aquaculture 210, 335–342.
Teshima, S., Kanazawa, A., Yamashita, M., 1986. Effects of dietary phospholipids on lipid
transport in the juvenile prawn. Nippon Suisan Gakkaishi 52, 159–163.
Tocher, D.R., Mourente, G., Van der Eecken, A., Evjemo, J.O., Diaz, E., Bell, J.G., Geurden, I.,
Lavens, P., Olsen, Y., 2002. Effects of dietary vitamin E on antioxidant defence mech-
anisms of juvenile turbot (Scophthalmus maximus L.), halibut (Hippoglossus
hippoglossus L.) and sea bream (Sparus aurata L.). Aquac. Nutr. 8, 195–207.
Tocher, D.R., Mourente, G., Van der Eecken, A., Evjemo, J.O., Diaz, E., Wille, M., Bell, J.G.,
Olsen, Y., 2003. Comparative study of antioxidant defence mechanisms in marine
fish fed variable levels of oxidised oil and vitamin E. Aquac. Int. 11, 195–216.
Tokuda, M., Takeuchi, M., 1995. Effects of excess doses of a-tocopherol on the lipids and
function of rainbow trout liver. J. Nutr. Sci. Vitaminol. 41, 25–32.
Tokuda, M., Takeuchi, M., 1999. Effects of excess doses of a-tocopherol on the lipid in
serum and muscle of rainbow trout. Fish. Sci. 65, 496–497.
Wang, Z.L., Mai, K.S., Liufu, Z.G., Ma, H.M., Xu, W., Ai, Q.H., Zhang, W.B., Tan, B.P., Wang,
X.J., 2006a. Effect of high dietary intakes of vitamin E and n-3 HUFA on immune re-
sponses and resistance to Edwardsiella tarda challenge in Japanese flounder
(Paralichthys olivaceus, Temminck and Schlegel). Aquac. Res. 37, 681–692.
Wang, Z.L., Mai, K.S., Liufu, Z.G., Ai, Q.H., Xu, W., Ma, H.M., Zhang, W.B., Tan, B.P., 2006b.
Effect of dietary vitamin C and beta-glucan on immunity and disease resistance of
Japanese flounder (Paralichthys olivaceus). High Technol. Lett. 16, 757–762.
Welker, T.L., Congleton, J.L., 2009. Effect of dietary alpha-tocopherol plus ascorbic acid, se-
lenium, and iron on oxidative stress in sub-yearling Chinook salmon (Oncorhynchus
tshawytscha Walbaum). J. Anim. Physiol. Anim. Nutr. 93, 15–25.
Yagi, K., 1987. Lipid peroxides and human disease. Chem. Phys. Lipids 45, 337–351.
Zhong, Y., Lall, S.P., Shahidi, F., 2007. Effects of oxidized dietary oil and vitamin e supple-
mentation on lipid profile and oxidation of muscle and liver of juvenile Atlantic cod
(Gadus morhua). J. Agric. Food Chem. 55, 6379–6386.