Food Chemistry 167 (2015) 91–99

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Effects of dietary arginine supplementation on growth performance,

flesh quality, muscle antioxidant capacity and antioxidant-related
signalling molecule expression in young grass carp (Ctenopharyngodon
idella)
Biao Wang a, Yang Liu a,b,c,⇑, Lin Feng a,b,c, Wei-Dan Jiang a,b,c, Sheng-Yao Kuang d, Jun Jiang a,b,c,
Shu-Hong Li a, Ling Tang d, Xiao-Qiu Zhou a,b,c,⇑
a
Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan, China
b
Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, Sichuan, China
c
Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, Sichuan, China
d
Animal Nutrition Institute, Sichuan Academy of Animal Science, Chengdu 610066, Sichuan, China

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

Article history: Growth performance, flesh quality, antioxidant status and antioxidant-related signalling molecule
Received 28 February 2014 expression in the muscle of young grass carp, which were fed graded levels of arginine (6.9–24.5 g/kg
Received in revised form 19 May 2014 diet) for eight weeks, were investigated. Muscle protein, lipid and nitric oxide contents, shear force,
Accepted 23 June 2014
hydroxyproline concentration, and pH were significantly improved by appropriate arginine. Cooking loss,
Available online 28 June 2014
lactate content, cathepsins activities, malondialdehyde and protein carbonyl contents exhibited an oppo-
site tendency. Additionally, optimum arginine significantly enhanced glutathione content and the activ-
Keywords:
ities and gene expression of copper/zinc superoxide dismutase, catalase and glutathione peroxidase in
Grass carp (Ctenopharyngodon idella)
Arginine
muscle. Moreover, the expression levels of glutamate–cysteine ligase, target of rapamycin, ribosome pro-
Flesh quality tein S6 kinase 1, casein kinase 2 and NF-E2-related factor 2 in muscle were significantly elevated by
Antioxidant enzyme appropriate arginine. However, optimum arginine significantly decreased Kelch-like ECH-associated pro-
Gene expression tein 1 mRNA levels in muscle. In conclusion, arginine improved the flesh quality and muscle antioxidant
Signalling molecule capacity and regulated antioxidant-related signalling molecule expression.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction reported that optimum Vitamin E supplementation was shown to

Fish growth primarily depends on the growth of muscle which
is the major edible portion for consumers (Periago et al., 2005).
Hence, it is important to investigate flesh quality. Flesh quality
traits primarily involve the water-holding capacity (WHC), pH
and firmness (Brinker & Reiter, 2011). Nutrients play an important
role in the improvement of flesh quality (Khan, Qureshi, Nasir,
Rasool, & Iqbal, 2011). A previous study in our laboratory indicated
that myo-inositol deficiency resulted in Jian carp (Cyprinus carpio
var. Jian) muscle lesions, which decreased flesh quality (Jiang
et al., 2010). However, Buckley, Morrissey, and Gray (1995)
⇑ Corresponding authors at: Animal Nutrition Institute, Sichuan Agricultural
University, Chengdu 611130, Sichuan, China. Tel.: +86 835 2882422 (Y. Liu). Tel.:
+86 835 2886085; fax: +86 835 2885968 (X.-Q. Zhou).
E-mail addresses: kyckgk@hotmail.com (Y. Liu), xqzhouqq@tom.com, zhouxq@-
sicau.edu.cn (X.-Q. Zhou).
be effective in increasing pig muscle WHC, thereby improving
meat quality. A recent study from our laboratory indicated that
optimum zinc (Zn) supplementation improved grass carp (Cteno-
pharyngodon idella) muscle WHC and pH (Wu et al., 2014). Arginine
(Arg) has been demonstrated to be an important nutrient for fish
(Zhou, Zeng, Wang, Xie, & Zheng, 2012). However, few studies have
focused on the effects of dietary Arg on flesh quality in fish. In pig,
Arg improved muscle pH (Ma et al., 2010) and WHC (Tan et al.,
2009). This data suggested that Arg might also influence flesh qual-
ity in fish, which is valuable to investigate.
Metabolic processes, as well as other processes occurring in
muscle tissue, cause the formation of reactive oxygen species
(ROS) (Rowe, Maddock, Lonergan, & Huff-Lonergan, 2004). A high
level of ROS can interact with both lipids and protein and induce
oxidative stress (Tokur & Korkmaz, 2007). Importantly, fish muscle
tissue is more sensitive to oxidative stress due to excessively high
levels of polyunsaturated fatty acids (Martinez-Alvarez, Morales, &

http://dx.doi.org/10.1016/j.foodchem.2014.06.091
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
92 B. Wang et al. / Food Chemistry 167 (2015) 91–99
Sanz, 2005; Periago et al., 2005). Oxidative stress is a major cause
of decreasing flesh quality (Buckley et al., 1995). Tokur and
Korkmaz (2007) reported that decreased flesh quality might be
related to the destruction of muscle structural integrity, which
resulted from oxidative damage in fish. Nutrients could improve
flesh quality by reducing oxidative damage. A previous study in
our laboratory demonstrated that dietary Zn improved grass carp
flesh quality through attenuating muscle oxidative damage (Wu
et al., 2014).However, there is no report concerning the effects of
Arg on lipid peroxidation and protein oxidation in fish, which
requires further investigation. To prevent oxidative damage, fish
have developed antioxidant systems (Martinez-Alvarez et al.,
2005). In general, fish antioxidant systems are composed of non-
enzymatic compounds (GSH) and antioxidant enzymes (including
superoxide dismutase (SOD), catalase (CAT) and glutathione perox-
idase (GPx)) (Martinez-Alvarez et al., 2005). These antioxidant
enzymes play an important role in eliminating ROS (Chen, Zhou,
Li, & Wu, 2013). To our knowledge, few studies have evaluated
the effects of Arg on antioxidant enzyme activity in fish. In rat mus-
cle tissue, it was shown that Arg significantly enhanced the activ-
ities of copper/zinc superoxide dismutase (SOD1), CAT and GPx
(Petrović et al., 2008). Lambertucci, Levada-Pires, Rossoni, Curi,
and Pithon-Curi (2007) reported that the changes in antioxidant
enzyme activities were partly dependent on antioxidant enzyme
gene transcription in rat muscle tissue. However, few studies have
examined the effects of Arg on antioxidant enzyme gene expres-
sion in fish. In rat brown adipose tissue, Arg elevated mRNA levels
of CAT and GPx (Otašević et al., 2011). The above data indicated
that Arg could affect antioxidant enzyme activity and gene expres-
sion in fish. This possibility requires further investigation.
Antioxidant enzyme gene expression is partly regulated by a
wide variety of transcription factors. Chen, Zou, Li, and Wu
(2013) reported that NF-E2-related nuclear factor 2 (Nrf2) is an
important transcription factor that can bind to the antioxidant-
responsive element (ARE) and induce transcriptional of antioxidant
enzyme genes, such as SOD, CAT and GPx, in mouse liver. However,
Kelch-like ECH-associated protein 1 (Keap1) was identified as a
Nrf2-binding protein, which depresses Nrf2 translocation to the
nucleus (Ma, 2013). To our knowledge, few studies have investi-
gated the effects of Arg on Nrf2 and Keap1 gene expression in fish.
A recent study from our laboratory cloned the cDNA of Nrf2 (Gen-
Bank accession number KF733814 and GenBank accession number
JX462955) and Keap1 (GenBank accession number KF811013 and
GenBank accession number JX470752) of grass carp and of Jian
carp for the first time and showed that some nutrients, such as
choline and myo-inositol, affected Nrf2 and Keap1 expression in
the intestine of Jian carp (unpublished data). In rats, Arg signifi-
cantly up-regulated the mRNA level of Nrf2 in left ventricular myo-
cardial tissue (Ramprasath et al., 2012). The effects of Arg on Nrf2
expression may be related to its metabolite. Nitric oxide (NO) is the
major metabolite of Arg and acts as an important signalling mole-
cule (Ramprasath et al., 2012). In vitro, NO up-regulated Nrf2 gene
expression in rat vascular smooth muscle cells (Liu et al., 2007).
Notably, these data indicated that Arg might regulate Nrf2 expres-
sion in fish through NO. Furthermore, several cellular signalling
molecules, such as target of rapamycin (TOR), could coordinately
regulate Nrf2 expression. In vitro, mTOR was reportedly involved
in the up-regulation of Nrf2 expression in human hepatic carci-
noma cells (Shay, Michels, Li, Kong, & Hagen, 2012). Recently, our
laboratory cloned the cDNA of TOR (GenBank accession number
FJ899680 and GenBank accession number JX854449) of Jian carp
and of grass carp for the first time and showed that the TOR gene
is expressed in fish muscle. Moreover, casein kinase 2 (CK2) is a
highly conserved serine/threonine kinase, and further studies have
shown that CK2 could regulate mTOR and its downstream target,
ribosome protein S6 kinase (S6K), in human glioblastoma cells
(Olsen, Svenstrup, & Guerra, 2012). Recently, the cDNA of CK2
(GenBank accession number KF914143) was cloned from grass
carp for the first time in our laboratory. A study in mice has shown
that polyamines, which are major metabolites of Arg, enhanced
CK2 activity in vitro and in vivo (Shore, Soler, & Gilmour, 1997).
These data indicated that a possible correlation exists between
Arg and the gene expression of antioxidant-related signalling mol-
ecules involved in the Nrf2 signalling pathway of the muscle in
fish. This possibility is worth investigating.
The present study was conducted to investigate the beneficial
effects of dietary Arg on fish growth and on flesh quality through
enhancing the muscle antioxidant capacity. In a further study, we
determine the relative expression of antioxidant enzyme genes
and antioxidant-related signalling molecules of fish, which could
provide partial theoretical evidence for the effects of Arg on fish
flesh quality and growth. Grass carp is an important economic
freshwater species that is widely cultured (Li, Tang et al., 2013).
The dietary Arg requirement was evaluated in juvenile grass carp
(Wang S., 2006). However, the requirement of Arg may be varied
at different growth stage in fish. In grass carp, lysine requirement
at the juvenile stage was higher (54.4 g/kg protein) (Wang, 2006)
than that at sub-adult stage (38.9 g/kg protein) (Li, Tang et al.,
2013). To date, no study has been conducted to evaluate the die-
tary Arg requirement in young grass carp. Hence, it is valuable to
investigate the dietary Arg requirement of young grass carp.
2. Materials and methods
2.1. Experimental diets and design
The isonitrogenous (300 g/kg protein) and isolipidic (41.7 g/kg
lipid) diets were formulated according to Ren et al. (2013). The
basal diet was constituted from the following ingredients (g/
100 g diet): fish meal (7.80), casein (3.00), gelatine (3.99), crystal
amino acid mix (18.86), arginine premix (5.00), fish oil (2.20), soy-
bean oil (1.89), a-starch (28.00), corn starch (13.34), vitamin pre-
mix (1.00), trace mineral premix (2.00), cellulose (10.00),
Ca(H2PO4)2 (2.27), choline chloride (0.60), ethoxyquin (0.05). The
dietary protein level was fixed at 30%, which supported the opti-
mal growth of grass carp (Khan, Jafri, & Chadha, 2004). Crystalline
amino acids were used to simulate the amino acid profile of whole
chicken egg protein, except for arginine, according to Li and Tang
(2013). A complete description of the ingredients and the compo-
sition of the basal diets were prepared according to Li et al.
(2014). Different concentrations of L-arginine were added to a basal
diet mixture to constitute the six levels of 6.0 (basal diet), 10.0,
14.0, 18.0, 22.0 and 26.0 g arginine/kg diet. Final arginine concen-
trations of the six experimental diets were measured using high-
performance liquid chromatography (Agilent Technologies, Palo
Alto, CA, USA) to be 6.9, 10.4, 14.1, 17.6, 21.4 and 24.5 g argi-
nine/kg diets, respectively. All ingredients were mixed, pelleted,
and stored at 20 °C until use, as described by Lee et al. (2011).
2.2. Feeding management
The procedures used in this study were approved by the Univer-
sity of Sichuan Agricultural Animal Care Advisory Committee.
Grass carp were obtained from Bai Long Lake (Sichuan, China).
Before beginning the experiment, grass carp were fed with the base
diet for 2 weeks to acclimate to the experimental diet and condi-
tions according to Zhang et al. (2009). After the acclimatisation
period, 540 grass carp with an average initial weight of
278.82 ± 0.68 g were randomly distributed into 18 experimental
cages (1.4  1.4  1.4 m3), each of which was equipped with a
100 cm diameter disc of 1-mm gauze in the bottom to collect the
 B. Wang et al. / Food Chemistry 167 (2015) 91–99
uneaten food, as described by Wu et al. (2014). Each experimental
diet was randomly assigned to cages in triplicate. The fish were fed
with their respective experimental diets to apparent satiation four
times a day for 8 weeks, and uneaten feed was collected 30 min
after the feeding. During the experimental period, dissolved oxy-
gen was not less than 6 mg/l, and water temperature and pH were
maintained at 26 ± 2 °C and 7.0 ± 0.5, respectively. The feeding trial
was completed under natural light.
2.3. Sample collection and analysis
Fish in each cage were weighed and counted at the initiation and
termination of the feeding trial to determine the percent weight
gain (PWG), specific growth rate (SGR), and feed efficiency (FE).
Six hours after the last feeding, blood samples of six fish from each
treatment were drawn from the caudal vein using heparinised
syringes for plasma ammonia determination according to
Fournier et al. (2003). Another 12 fish from each treatment were
anaesthetised in a benzocaine bath (50 mg/l) as described by
Berdikova Bohne, Hamre, and Arukwe (2007), and then fish were
sacrificed by a sharp blow to the head according to Hultmann,
Phu, Tobiassen, Aas-Hansen, and Rustad (2012). Immediately after
slaughtering, they were manually filleted and then muscle samples
were quickly obtained from the left side, immediately frozen in
liquid nitrogen, and then stored at 80 °C until analysis according
to Berdikova Bohne, Hamre, and Arukwe (2007). Meanwhile, from
the right side of the same fish, muscle samples from each treatment
were obtained for the immediate determination of flesh quality
parameters according to Wu et al. (2014). The muscle pH, shear
force and cooking loss were determined according to the method
described by Brinker and Reiter (2011). The determination of
hydroxyproline content was performed using the procedure
reported by Periago et al. (2005), with a slight modification. The
muscle moisture, crude protein and lipid contents were measured
using the method of Zhou et al. (2012).
Muscle samples were homogenised in 10 volumes (w/v) of ice-
cold physiological saline, and centrifuged at 6000g at 4 °C for
20 min. Then, the supernatant was collected for the analysis of
the following parameters. The contents of malondialdehyde
(MDA) and protein carbonyl (PC) were measured according to
Tokur and Korkmaz (2007). The activities of SOD and GPx were
determined by the method described by Petrović et al. (2008)
and Chen et al. (2013), respectively. CAT activity was measured
using the decomposition of hydrogen peroxide according to the
method of Petrović et al. (2008). The reduced GSH content was
measured according to the method described by Petrović et al.
(2009). The anti-superoxide anion (ASA) (O 2 scavenging ability)
and anti-hydroxyl radical (AHR) (OH scavenging ability) capacities
were determined by the method described by Jiang et al. (2010).
The muscle NO content was measured according to Fournier
et al. (2003). In addition, cathepsin B and L activities were mea-
sured using the fluorimetrically method according to Li, Zhou,
Zhang, Liu, and Ma (2008).The lactate content was measured
according to the method described by Hultmann et al. (2012).
The muscle amino acid composition and free ornithine content
were determined by using a modification of the HPLC method
described by Mostert and Hoffman (2007) and Berge, Sveier, and
Lied (1998), respectively. Arginase activity was measured as
described by Portugal and Aksnes (1983).
2.4. Real-time quantitative PCR analysis of gene expression in muscle
Total RNA was isolated from muscle using an RNAiso Plus Kit
(Takara, Dalian, China) according to the manufacturer’s instruc-
tions, followed by DNase I treatment. The quality and quantity of
the total RNA were assessed by agarose gel electrophoresis at 1%
93
and by spectrophotometric analysis at 260 and 280 nm according
to Chen et al. (2013). Subsequently, RNA was reverse transcribed
to cDNA using a PrimeScript™ RT reagent Kit (Takara, Dalian,
China) following the manufacturer’s instructions.
Real-time PCR were performed for GCL, SOD1, CAT, GPx, Nrf2,
Keap1, TOR, S6K1 and CK2 according to standard protocols with
the primer sequences and optimal annealing temperatures indi-
cated in Table 1. All real-time PCR reactions were performed on a
CFX96™ Real-Time PCR Detection System (Bio-Rad, Laboratories,
Inc.) using a SYBRÒ Prime Script RT-PCR Kit II (Takara, Dalian,
China). Amplification was performed in a final volume of 15 ll,
containing 2 ll cDNA template. The thermocycling conditions were
initiated with a denaturation step of 95 °C for 30 s and then sub-
jected to 40 cycles of PCR (denaturation at 95 °C for 5 s, annealing
at a different temperature for each gene for 30 s). After the ampli-
fication phase, a melt curve analysis was performed to confirm the
specificity of the amplification reaction. No template controls were
run for each PCR assay. The expression levels of these genes were
normalised to the expression levels of a grass carp housekeeping
gene (b-actin). The concentration of the target gene was based on
the threshold cycle number (CT), and the CT for each sample was
determined using CFX Manager™ software. In addition, the cDNA
concentration in the sample was determined according to the stan-
dard curves. A 10-fold serial dilution was used to generate stan-
dard curves for both targeted and endogenous control genes,
quantifying six concentrations (in triplicate). All primer amplifica-
tion efficiencies were approximately 100%. The expression results
were analysed using the 2DDCT method according to Shay et al.
(2012).
2.5. Calculations and statistical analysis
The data concerning the initial body weight, final body weight,
feed intake (FI) and proximate diet composition were used to cal-
culate the following parameters:
Percentage weight gain (PWG, %) = [(final body weight–initial
body weight)/initial body weight]  100%
Specific growth rate (SGR, %/d) = {[ln (mean final weight)–ln
(mean initial weight)]/days}  100%
Feed efficiency (FE) = (final body weight–initial body weight)/
feed intake
Protein efficiency ratio (PER) = (final body weight–initial body
weight)/protein intake
The results were presented as the means ± standard deviation
(SD). All data were subjected to a one-way analysis of variance
(ANOVA), which was followed by Duncan’s multiple-range test,
to determine significant differences among treatment groups at
the level of P < 0.05 using the software SPSS 18.0 (SPSS Inc., Chi-
cago, IL, USA). Dietary arginine requirements based on the PWG
were estimated using broken-line regression analysis.
3. Results
3.1. Growth performance
The effects of graded levels of dietary Arg on growth parameters
are provided in Table 2. The FBW, SGR, PWG and FI significantly
increased as dietary Arg levels increased from 6.9 to 14.1 g/kg diet
(P < 0.05), and plateaued thereafter (P > 0.05). The FE and PER were
significantly enhanced with increasing Arg levels up to 17.6 g/kg
diet and decreased thereafter (P < 0.05). The plasma ammonia con-
tent was the highest for fish fed on the basal diet, then significantly
decreased with increasing Arg levels up to 17.6 g/kg diet, and
94 B. Wang et al. / Food Chemistry 167 (2015) 91–99

Table 1
Real-time PCR primer sequences.

Gene Sequences of primers Annealing temperature (°C) Amplification products (bp) Accession number
GCL
Forward 50 -CACGCTGCCAGAATACAA-30 56.9 144 KF998103
Reverse 50 -ATCACCACCTTTTCGCC-30
CK2
Forward 50 -CCCCAACCACAGTGACCT-30 57.9 118 KF914143
Reverse 50 -TCCCTGCTGATACTTCTCC-30
TOR
Forward 50 -TCCCACTTTCCACCAACT-30 61.4 177 JX854449
Reverse 50 -ACACCTCCACCTTCTCCA-30
S6K1
Forward 50 -TGGAGGAGGTAATGGACG-30 54.0 111 EF373673
Reverse 50 -ACATAAAGCAGCCTGACG-30
Nrf2
Forward 50 -CTGGACGAGGAGACTGGA-30 62.5 234 KF733814
Reverse 50 -ATCTGTGGTAGGTGGAAC-30
Keap1
Forward 50 -TTCCACGCCCTCCTCAA-30 63.0 205 KF811013
Reverse 50 -TGTACCCTCCCGCTATG-30
SOD1
Forward 50 -CGCACTTCAACCCTTACA-30 61.5 218 GU901214
Reverse 50 -ACTTTCCTCATTGCCTCC-30
GPx
Forward 50 -GGGCTGGTTATTCTGGGC-30 61.5 278 EU828796
Reverse 50 -AGGCGATGTCATTCCTGTTC-30
CAT
Forward 50 -GAAGTTCTACACCGATGAGG-30 58.7 158 FJ560431
Reverse 50 -CCAGAAATCCCAAACCAT-30
b-actin
Forward 50 -GGCTGTGCTGTCCCTGTA-30 61.4 101 M25013
Reverse 50 -GGGCATAACCCTCGTAGAT-30

GCL: glutamate-cysteine ligase, CK2: casein kinase 2, TOR: target of rapamycin, S6K1: ribosomal protein S6 kinase 1, Nrf2: NF-E2-related factor 2,
Keap1: Kelch-like-ECH-associated protein 1, SOD1: copper/zinc superoxide dismutase, GPx: glutathione peroxidase, CAT: catalase.

Table 2
Initial body weight (IBW, g/fish), final body weight (FBW, g/fish), feed intake (FI, g/fish), feed efficiency (FE), specific growth rate (SGR, %/d), percentage weight gain (PWG, %),
protein efficiency ratio (PER) and plasma ammonia content (PAC, lmol/L) of grass carp fed diets with graded levels of arginine (g/kg).

Dietary arginine levels (g/kg)

6.9 10.4 14.1 17.6 21.4 24.5
IBW 279.56 ± 1.39 278.44 ± 1.68 278.22 ± 0.38 278.22 ± 2.52 278.68 ± 2.67 279.78 ± 0.38
FBW 420.44 ± 7.95a 556.44 ± 9.71b 628.67 ± 6.57c 630.44 ± 14.15c 615.56 ± 14.49c 614.89 ± 5.35c
PWG 50.39 ± 2.24a 99.83 ± 2.42b 125.96 ± 2.66c 126.59 ± 3.91c 120.93 ± 6.68c 119.78 ± 2.17c
SGR 0.73 ± 0.03a 1.24 ± 0.02b 1.46 ± 0.02c 1.46 ± 0.03c 1.42 ± 0.05c 1.41 ± 0.02c
FI 318.17 ± 1.71a 479.10 ± 1.66b 562.96 ± 3.22c 559.61 ± 1.25c 549.08 ± 2.70c 561.54 ± 0.21c
FE 0.44 ± 0.01a 0.56 ± 0.01b 0.62 ± 0.01d 0.63 ± 0.02d 0.61 ± 0.01cd 0.60 ± 0.01c
PER 1.48 ± 0.07a 1.88 ± 0.01b 2.08 ± 0.03cd 2.10 ± 0.08d 2.05 ± 0.03cd 1.99 ± 0.37c
PAC 413.38 ± 12.34d 342.11 ± 21.38c 242.33 ± 12.34a 228.07 ± 12.34a 270.84 ± 12.34b 294.35 ± 16.38b

All data were expressed as mean ± SD. Mean values within the same row with different superscripts (a, b, c and d) are significantly different (P < 0.05).

increased thereafter (P < 0.05). The dietary Arg requirement esti-
mated by the broken-line model based on PWG was 13.45 g/kg
diet, corresponding to 43.64 g/kg dietary protein.
3.2. Muscle composition and flesh quality parameters
The muscle composition and flesh quality parameters of grass
carp fed diets containing graded levels of Arg are presented in
Tables 3 and 4. The moisture content was the maximum for fish
fed the Arg-unsupplemented diet and was the minimum for fish
fed the 24.5 g Arg/kg diet (P < 0.05). Cathepsin B activity in muscle
significantly decreased when dietary Arg levels increased to 14.1 g/
kg diet (P < 0.05) and plateaued thereafter (P > 0.05). The muscle
protein content, pH and shear force significantly increased with
increasing Arg levels from 6.9 to 10.4 g/kg diet (P < 0.05), and no
differences were found with a further increase in Arg levels
(P > 0.05). The lipid content was significantly enhanced with Arg
levels up to 10.4 g/kg diet and decreased thereafter (P < 0.05).
The hydroxyproline content was the highest for fish fed diets con-
taining Arg 14.1 g/kg diet and was the lowest for fish fed the Arg-
unsupplemented diet (P < 0.05). The cooking loss, lactate content
and cathepsin L activity in the muscles of fish fed diets with
14.1 g Arg/kg diet were significantly lower than that in fish fed
other diets (P < 0.05). Arginase activity in grass carp muscle was
significantly enhanced when dietary Arg levels increased to
14.1 g/kg diet (P < 0.05) and plateaued thereafter (P > 0.05). The
concentrations of ornithine in muscle was higher in fish fed diets
containing Arg21.4 g/kg diet and was the lowest for fish fed the
Arg-deficiency diet (P < 0.05). Meanwhile, histidine, arginine, tyro-
sine, glutamine and leucine content in muscle significantly
increased with Arg levels up to 17.6 g/kg diet (P < 0.05). Valine,
threonone and phenylalanine contents were higher for fish fed
diets containing Arg 10.4 g/kg diet and significantly decreased for
fish fed the Arg-excesses diet (24.5 g/kg diet) (P < 0.05). However,
 B. Wang et al. / Food Chemistry 167 (2015) 91–99 95

Table 3
Muscle composition (%), muscle shear force (N), cooking loss (%), pH (0 h), hydroxyproline concentration (mg/g tissue), lactate content (mmol/g protein) and cathepsin B and L
activities (U/g protein) and ornithine content (lg/g tissue) and arginase activity (lmol urea/min/g tissue) of grass carp fed diets with graded levels of arginine (g/kg).

Dietary arginine levels (g/kg)

6.9 10.4 14.1 17.6 21.4 24.5
Moisture (%) 77.18 ± 1.08b 76.29 ± 0.60ab 76.32 ± 0.63ab 76.10 ± 0.92ab 75.66 ± 2.18a 75.62 ± 0.75a
Protein (%) 17.34 ± 0.76a 18.46 ± 0.50b 18.46 ± 0.52b 18.71 ± 0.66b 18.90 ± 1.61b 18.46 ± 0.55b
Lipid (%) 2.28 ± 0.13a 2.71 ± 0.11c 2.49 ± 0.19b 2.52 ± 0.16b 2.51 ± 0.21b 2.39 ± 0.13ab
Shear force 2.06 ± 0.06a 2.20 ± 0.09b 2.30 ± 0.08b 2.31 ± 0.10b 2.30 ± 0.07b 2.28 ± 0.09b
Cooking loss (%) 21.08 ± 0.85c 18.39 ± 0.33b 15.56 ± 0.32a 17.92 ± 0.38b 20.76 ± 0.72c 20.73 ± 0.49c
pH 6.00 ± 0.06a 6.20 ± 0.02b 6.18 ± 0.03b 6.20 ± 0.05b 6.19 ± 0.05b 6.18 ± 0.05b
Hydroxyproline 0.08 ± 0.01a 0.12 ± 0.01b 0.21 ± 0.02e 0.19 ± 0.01d 0.14 ± 0.01c 0.15 ± 0.01c
Cathepsin B 5.15 ± 0.37b 5.34 ± 0.53b 4.19 ± 0.20a 4.51 ± 0.47a 4.40 ± 0.10a 4.44 ± 0.07a
Cathepsin L 2.11 ± 0.13b 1.85 ± 0.18a 1.70 ± 0.08a 2.01 ± 0.19b 2.01 ± 0.04b 2.08 ± 0.06b
Lactate content 3.48 ± 0.06c 1.52 ± 0.12ab 1.32 ± 0.05a 1.46 ± 0.22ab 1.67 ± 0.23b 1.64 ± 0.08ab
Arginase 0.10 ± 0.01a 0.14 ± 0.01b 0.26 ± 0.03c 0.26 ± 0.01c 0.24 ± 0.01c 0.25 ± 0.01c
Ornithine 7.79 ± 0.63a 8.55 ± 0.54a 10.90 ± 0.57b 11.96 ± 0.60bc 12.96 ± 0.39c 12.25± 0.65bc

Values are mean ± SD (n = 6). Values within the same row having different superscripts (a, b, c and d) are significantly different (P < 0.05).

Table 4
Amino acid composition of grass carp muscle (g/100 g dry).

Dietary arginine levels (g/kg)

6.9 10.4 14.1 17.6 21.4 24.5
a a b a a
Aspartic acid 8.11 ± 0.50 8.39 ± 0.43 9.78 ± 0.62 8.42 ± 0.20 8.85 ± 0.36 8.13 ± 0.13a
Threonine 3.30 ± 0.29ab 3.79 ± 0.01b 3.48 ± 0.02ab 3.67± 0.03ab 3.69 ± 0.39ab 3.22 ± 0.14a
Serine 3.26 ± 0.20a 3.60 ± 0.15a 3.75 ± 0.43a 3.67 ± 0.03a 3.71 ± 0.15a 3.62 ± 0.24a
Glutamine 12.29 ± 0.02a 14.43 ± 0.01b 14.35 ± 1.29b 14.78 ± 0.38b 15.34 ± 0.59b 14.66 ± 0.09b
Glycine 3.78 ± 0.14a 3.97 ± 0.21a 3.97 ± 0.15a 3.94 ± 0.05a 4.11 ± 0.19a 4.02 ± 0.10a
Alanine 4.24 ± 0.15a 4.54 ± 0.32a 4.36 ± 0.08a 4.68± 0.10a 4.58 ± 0.54a 4.56 ± 0.14a
Valine 3.91 ± 0.02ab 4.42 ± 0.37b 4.10 ± 0.31ab 3.99 ± 0.07ab 3.78 ± 0.11ab 3.60 ± 0.38a
Cystine 0.54 ± 0.06a 0.55 ± 0.03a 0.57 ± 0.05a 0.60 ± 0.03a 0.51 ± 0.01a 0.57 ± 0.06a
Methionine 2.00 ± 0.15a 2.17 ± 0.16a 2.24 ± 0.01a 2.40 ± 0.05a 2.44 ± 0.09a 2.13 ± 0.01a
Isoleucine 3.46 ± 0.24a 3.54 ± 0.09a 3.43 ± 0.07a 3.67 ± 0.17a 3.80 ± 0.18a 3.58 ± 0.18a
Leucine 5.22 ± 0.19a 5.72 ± 0.01b 6.10 ± 0.02c 6.49 ± 0.13d 6.39 ± 0.10d 5.95 ± 0.03bc
Tyrosine 2.55 ± 0.07a 2.90 ± 0.32ab 2.74 ± 0.02ab 2.97 ± 0.03b 3.07 ± 0.16b 3.07 ± 0.03b
Phenylalanine 3.30 ± 0.36ab 3.75 ± 0.17b 3.56 ± 0.03ab 3.33 ± 0.04ab 3.46 ± 0.16ab 3.15 ± 0.14a
Lysine 7.00 ± 0.31a 7.61 ± 0.80a 7.95 ± 0.93a 7.74 ± 0.02a 6.83 ± 0.27a 6.57 ± 0.19a
Histidine 1.58 ± 0.09a 1.67 ± 0.14a 1.74 ± 0.01a 1.97 ± 0.04b 1.64 ± 0.04a 1.59 ± 0.01a
Arginine 4.47 ± 0.12a 5.02 ± 0.47ab 5.52 ± 0.46ab 5.74 ± 0.59b 5.82 ± 0.10b 5.62 ± 0.15b
P
EAA 34.24 ± 1.58a 37.68 ± 1.47bc 38.13 ± 1.63bc 39.00 ± 1.08c 37.84 ± 1.44bc 35.42 ± 0.24ab

Values are mean ± SD (n = 6). Values within the same row having different superscripts (a, b, c and d) are significantly different (P < 0.05).

Table 5
Effects of graded levels of arginine (g/kg) on malondialdehyde (MDA, nmol/mg protein) and protein carbonyl (PC, nmol/mg protein) contents, copper/zinc superoxide dismutase
(SOD1, U/mg protein), catalase (CAT, U/mg protein) and glutathione peroxidase (GPx, U/mg protein) activities, glutathione (GSH, mg/g protein) content, anti-superoxide anion
(ASA, U/g protein) and anti-hydroxyl radical (AHR, U/mg protein) capacities and nitric oxide (NO, nmol/g tissue) concentration in grass carp muscle.

Dietary arginine levels (g/kg diet)

6.9 10.4 14.1 17.6 21.4 24.5
MDA 2.42 ± 0.19e 2.05 ± 0.17d 0.88 ± 0.07a 0.98 ± 0.09a 1.30 ± 0.05b 1.51 ± 0.08c
PC 5.44 ± 0.55e 4.12 ± 0.22d 2.17 ± 0.16b 1.40 ± 0.20a 3.11 ± 0.22c 3.11 ± 0.28c
CAT 0.97 ± 0.05a 1.21 ± 0.06b 1.70 ± 0.13e 1.51 ± 0.03bd 1.44 ± 0.14cd 1.31 ± 0.08bc
SOD1 7.73 ± 0.32a 9.00 ± 0.83b 10.42 ± 0.38c 9.18 ± 0.15b 8.69 ± 0.31b 8.00 ± 0.67a
GPx 96.90 ± 8.58a 124.62 ± 12.04b 146.70 ± 4.95c 145.96 ± 11.72c 144.49 ± 6.01c 137.28 ± 4.72c
GSH 3.30 ± 0.30a 4.76 ± 0.39b 7.12 ± 0.35c 7.03 ± 0.41c 4.59 ± 0.24b 3.60 ± 0.20a
ASA 33.86 ± 2.85a 38.44 ± 0.90b 41.31 ± 1.13c 34.66 ± 2.56a 34.50 ± 1.93a 33.82 ± 3.00a
AHR 59.17 ± 3.80a 70.49 ± 2.90c 79.26 ± 2.24d 77.47 ± 3.49d 62.05 ± 1.48ab 63.32 ± 2.94b
NO 19.08 ± 1.40a 27.36 ± 2.44b 35.97 ± 1.64c 36.63 ± 0.93c 35.27 ± 1.65c 35.11 ± 0.96c

Values are mean ± SD (n = 6). Values within the same row having different superscripts (a, b, c and d) are significantly different (P < 0.05).

increasing the levels of Arg in diets did not significantly affect the
concentration of serine, glycine, alanine, cystine, methionine, iso-
leucine and lysine in grass carp muscle.
3.3. Muscle lipid peroxidation, protein oxidant and antioxidant status
As shown in Table 5, MDA and PC contents in muscle signifi-
cantly decreased with dietary Arg levels up to 14.1 and 17.6 g/kg
diet (P < 0.05), respectively, and increased with a further increase
in Arg (P < 0.05). The CAT and SOD1 activities, GSH content and
ASA and AHR capacities in muscle significantly increased with
Arg levels up to 14.1 g/kg diet (P < 0.05) and decreased thereafter
(P < 0.05). The GPx activity and NO content significantly increased
with increasing Arg levels up to 14.1 g/kg diet (P < 0.05), then
remained plateaued with incremental Arg levels from 14.1 to
24.5 g/kg diet.
96 B. Wang et al. / Food Chemistry 167 (2015) 91–99

3.4. Gene expression in muscle
The SOD1, CAT, GPx, GCL, Nrf2, Keap1, TOR, S6K1 and CK2 gene
expression levels in the muscles of grass carp fed diets containing
different levels of Arg are presented in Fig. 1. The expression levels
(a)
Relative expression level (fold)
of SOD1, GPx, CAT, Nrf2, S6K1 and GCL significantly increased with
increasing Arg levels up to 14.1 g/kg diet and decreased thereafter
(P < 0.05). Fish fed diets containing 10.4, 14.1 and 17.6 g Arg/kg
diet had significantly higher levels of TOR mRNA in muscle than
those fish fed other dietary levels (P < 0.05). The Keap1 mRNA level
significantly decreased with increasing Arg levels up to 10.4 g/kg
diet and increased thereafter (P < 0.05).
SOD1 CAT GPx GCL

3.5
3.0 d 4. Discussion
2.5
c 4.1. Arg improved fish growth and its requirement in young grass carp
c d
2.0 c bc
bc c
ab c bc
1.5 a ab b b The results of the current study have revealed that the growth
ab ab a ab a a a
1.0 a a performance of grass carp was influenced by Arg. The present
study showed that the PWG, FI and FE of grass carp significantly
0.5
improved with optimal Arg supplementation. The correlation anal-
0.0 ysis showed that the grass carp PWG positively correlated with the
6.9 10.4 14.1 17.6 21.4 24.5 FI (r = +0.997, P < 0.05) and FE (r = +0.989, P < 0.05), suggesting that
Dietary arginine levels (g/kg diet)
the enhancement of fish growth may be attributed to the fact that
the FI and FE were improved with appropriate dietary Arg. Based
2.5 (b) on the PWG, the dietary Arg requirement for young grass carp
Relative expression level (fold)

Nrf2 Keap1
c was determined to be the 13.45 g/kg diet (corresponding to
2.0 d c c 43.64 g/kg dietary protein) using the broken-line analysis, which
1.5 c c was slight lower than the requirement reported by Wang (2006)
b bc
a ab forjuvenile grass carp at the18.9 g/kg diet (corresponding to
1.0 ab 49.7 g/kg dietary protein). Fish weight gain is primarily attributed
a to the accretion of lipids and proteins in muscle (Bureau, Azevedo,
0.5 Tapia-Salazar, & Cuzon, 2000). The present study showed that the
optimum concentration of Arg significantly enhanced grass carp
0.0 muscle lipid and protein contents, which were in accordance with
6.9 10.4 14.1 17.6 21.4 24.5 the results for juvenile yellow grouper (Zhou et al., 2012). Fish pro-
Dietary arginine levels (g/kg diet) tein synthesis is partly related to the balance of amino acids in the
diet. In fish, plasma ammonia is the main end product of amino
TOR S6K1 acid catabolism, which reflects the amino acid balance (Fournier
2.0
(c) b et al., 2003). Fournier et al. (2003) reported that the lower plasma
Relative expression level (fold)

b
1.8 b b b ammonia content in rainbow trout resulted from a greater balance
1.6 c of amino acid profiles in diets. Our study showed that the plasma
1.4 a c
a a
ammonia content was the lowest for grass carp fed with optimum
1.2
a levels of Arg, suggesting that amino acids were available in an
1.0 a
appropriate balance for fish protein synthesis with the optimal
0.8
0.6 Arg supplementation. Studies showed that fish muscle EAA (essen-
0.4 tial amino acid) levels have been used as an index of dietary amino
0.2 acid status (Cara, Moyano, Zambonino, & Alarcón, 2007). In the
0.0 present study, total EAA in grass carp muscle was significantly
6.9 10.4 14.1 17.6 21.4 24.5 increased with 10.4 g/ kg Arg supplementation, suggesting that
Dietary arginine levels (g/kg diet) optimum Arg could balance AA and promote protein synthesis. A
similar result has been reported in juvenile yellow grouper (Zhou
CK2 et al., 2012). Fish muscle is the main edible portion (Periago
(d)
Relative expression level (fold)

1.8 b b et al., 2005). A previous study has shown that nutrients play an
1.6 b
b important role in the improvement of flesh quality (Khan et al.,
1.4 2011). However, few studies have focused on the effects of dietary
1.2 a
a
Arg on flesh quality in fish. Thus, we next assayed the effects of Arg
1.0
on the flesh quality of grass carp.
0.8
0.6
0.4 4.2. Arg improved the flesh quality of fish
0.2
0.0
Muscle firmness is considered an important flesh quality trait in
6.9 10.4 14.1 17.6 21.4 24.5
fish (Brinker & Reiter, 2011). In general, the enhancement of fish
Dietary arginine levels (g/kg diet)
fillet firmness resulted in improving the flesh quality (Johnston
Fig. 1. Relative gene expression levels of copper/zinc superoxide dismutase, et al., 2006). Shear force is a biomarker that represents the flesh
catalase, glutathione peroxidase and glutamate-cysteine ligase (a); NF-E2-related firmness of fish (Johnston et al., 2006). Our study showed that grass
nuclear factor 2, Kelch-like ECH-associated protein 1 (b); target of rapamycin and carp muscle shear force was elevated with optimum Arg supple-
ribosome protein S6 kinase 1 (c); and casein kinase 2 (d) in the muscle of grass carp
fed diets containing graded levels of arginine (g/kg). The values are the means ± SD
mentation, suggesting that Arg enhanced fish muscle firmness,
(n = 6), and different letters above bars indicate the significant difference among thereby improving the flesh quality. The increment of flesh firm-
treatments (P < 0.05). ness partly related to Arg metabolism. Wu and Morris (1998)
 B. Wang et al. / Food Chemistry 167 (2015) 91–99
reported that glutamate is an important metabolite of Arg. One
study showed that glutamate enhanced muscle firmness in Atlan-
tic salmon (Larsson et al., 2014). In the current study, the appropri-
ate Arg supplementation increased grass carp muscle glutamine
content, which showed similar patterns compared with shear
force, suggesting that Arg increased muscle firmness through
increment glutamine content in fish. On the other hand, the
enhancement of fish firmness by Arg may be linked to an increase
in the collagen content. A study of Atlantic salmon indicated that
increased flesh firmness could be partially attributed to a higher
collagen content, which could be quantified by the hydroxyproline
concentration (Johnston et al., 2006). The result of the present
study showed that the hydroxyproline concentration in the muscle
of grass carp significantly improved with increasing Arg up to
14.1 g/kg diet. The correlation analysis indicated that grass carp
muscle shear force positively correlated with the hydroxyproline
concentration (r = +0.813, P < 0.05), suggesting that the increased
firmness may attributed to the fact that Arg increased the content
of collagen in fish fillets. The increment of muscle collagen content
by Arg may be ascribed to the metabolism of Arg. One study
showed that arginase catalysed the transformation of Arg into
ornithine which served as a precursor for synthesis of collagen syn-
thesis (Flynn, Meininger, Haynes, & Wu, 2002). In the present
study, ornithine content and arginase activity in grass carp muscle
were significantly increased by 14.1 g/kg Arg, which showed simi-
lar patterns compared with hydroxyproline content, suggesting
that Arg increased muscle ornithine to promote collagen content
by increment arginase activity in fish. Furthermore, the Arg-associ-
ated increased collagen content may be related to the decrease in
cathepsin B and L activities. Maciewicz, Wotton, Etherington, and
Duance (1990) reported that cathepsin B and L could degrade rat
collagen content in vitro. Our study indicated that the activities
of cathepsin B and L in grass carp muscle significantly decreased
with optimum Arg supplementation. The correlation analysis
showed that the grass carp flesh hydroxyproline concentration
negatively correlated with the activities of cathepsin B
(r = 0.779, P = 0.068) and L (r = 0.609, P = 0.200), suggesting that
the Arg-enhanced flesh collagen content may be partly through
decreasing cathepsin B and L activities to inhibit the degradation
of collagen in fish. In contrast, post-mortem muscle pH is another
important flesh quality parameter (Periago et al., 2005). In general,
the grass carp post-mortem muscle pH value can vary from 6.2 to
6.7 (Li, Zhou et al., 2013). Our study showed that the grass carp
post-mortem muscle pH in the Arg-unsupplemental group was
6.0 and then increased to 6.18–6.20 with the Arg supplementation
with the 10.4–24.5 g/kg diet, which indicated that Arg could
increase the fish muscle pH. The positive influence of Arg on the
muscle pH may be partly related to the decrease in the lactate con-
tent. Hultmann et al. (2012) reported that the decreased lactate
content led to an improvement in the post-mortem pH in Atlantic
cod. The result of the present study showed that the lactate content
in the muscle of grass carp significantly decreased with increasing
dietary Arg up to the 14.1 g/kg diet. Correlation analysis showed
that the grass carp muscle pH negatively correlated with the lac-
tate content (r = 0.981, P < 0.01), suggesting that dietary Arg
may have improved the muscle pH value through decreasing the
lactate content in fish. This possibility is consistent with the
reports using pig muscle (Tan et al., 2009). In addition to firmness
and pH, the muscle WHC is also an important flesh quality param-
eter, which can be evaluated by the cooking loss in fish (Skipnes,
Østby, & Hendrickx, 2007). The decreased cooking loss resulted
from elevating the WHC of cod muscle (Skipnes et al., 2007). Our
study showed that Arg deficiency significantly increased the cook-
ing loss of muscle in grass carp, and with the increment of the
graded Arg to the 14.1 g/kg diet, the cooking loss gradually
decreased, suggesting that Arg could enhance the WHC of fish
97
muscle to improve the flesh quality. A similar result has been
reported in pig (Ma et al., 2010). Arg-increased grass carp muscle
WHC may be ascribed to a decrease in cathepsin activity. Hagen,
Solberg, and Johnston (2008) reported that cathepsin could hydro-
lyse major muscle structural proteins, which could cause decreas-
ing muscle WHC in Atlantic Halibut. In our study, the correlation
analysis showed that the grass carp muscle cooking loss positively
correlated with cathepsin L activity (r = +0.900, P < 0.05), suggest-
ing that Arg may have partially increased flesh WHC via decreasing
cathepsin L activity in fish. These results indicated that Arg
improved the fish flesh quality. Tokur and Korkmaz (2007)
reported that the decreased flesh quality was partly related to
the destruction of muscle structural integrity, which resulted from
oxidative damage in fish. A previous study in our laboratory indi-
cated that the enhancement of flesh quality might be through ele-
vating the muscle antioxidative capacity of grass carp (Wu et al.,
2014). According to this possibility, we next investigated the
effects of Arg on the muscle antioxidative capacity of grass carp.
4.3. Arg elevated fish muscle antioxidative capacity
In fish, PC and MDA contents are widely used as indices for pro-
tein oxidation and lipid peroxidation, respectively (Tokur &
Korkmaz, 2007). The data presented have shown that the highest
muscle PC and MDA contents occurred in grass carp fed the Arg-
deficient diet and significantly reduced with the increase of the
graded Arg levels to optimum levels, suggesting that Arg may
maintain muscle structural integrity through suppressing oxida-
tive damage in fish. Similar results in muscle were obtained for Jian
carp fed with appropriate dietary myo-inositolin our laboratory
(Jiang et al., 2010). However, no report was conducted to investi-
gate the influence of Arg on protein oxidation and lipid peroxida-
tion in fish. In general, the protection effects against oxidative
damage may be related to an increase in the free radical scaveng-
ing capacity (Chen et al., 2013). The superoxide radical and hydro-
xyl radical are the primary free radicals strongly involved in
oxidative damage (Martinez-Alvarez et al., 2005). Thus, we deter-
mined the scavenging abilities of the superoxide radical and hydro-
xyl radical. The data from the present study demonstrated that
optimum Arg significantly improved grass carp muscle ASA and
AHR capacities, which showed an opposite pattern with MDA
and PC, further supporting the argument that Arg decreases oxida-
tive damage through elevating the radical scavenging ability in
fish. The beneficial effects of Arg on the free radical scavenging
ability were partly attributed to non-enzymatic antioxidants such
as GSH and antioxidant enzymes such as SOD1, CAT and GPx in
fish. The current study demonstrated that optimum Arg signifi-
cantly elevated the GSH content in grass carp muscle, suggesting
that Arg improved the non-enzymatic antioxidative capacity in fish
muscle. Similar results in muscle were obtained for Jian carp fed
with appropriate myo-inositolin our laboratory (Jiang et al.,
2010). The effects of maintaining the fish muscle GSH content by
Arg may be partly associated with GSH synthesis. Petrović et al.
(2009) reported that glutamate-cysteine ligase (GCL) is the rate-
limiting enzyme for the biosynthesis of GSH in mice. In human
brain endothelial cells, the enhancement of GCL expression pro-
moted GSH synthesis (Okouchi, Okayama, Steven Alexander, &
Yee Aw, 2006). A recent study from our laboratory cloned the cDNA
of GCL (GenBank accession number KF998103) for the first time,
and our study showed that optimum Arg significantly elevated
GCL expression in the muscle of grass carp. The correlation analysis
showed a strong positive correlation between the grass carp mus-
cle GSH content and GCL gene expression (r = +0.769, P = 0.074),
suggesting that Arg partly elevated the GSH content through
enhancing GCL expression to promote GSH synthesis in fish. A sim-
ilar result has been reported in rat brown adipose tissue (Petrović
98 B. Wang et al. / Food Chemistry 167 (2015) 91–99
et al., 2009). Moreover, the data in this study also showed that
optimum Arg significantly enhanced grass carp muscle SOD1,
CAT and GPx activities, which had similar patterns compared with
ASA and AHR capacities, suggesting that Arg-enhanced fish muscle
superoxide radical and hydroxyl radical scavenging abilities may
be ascribed to the improvement of SOD1, CAT and GPx activities.
Although no report has been conducted in fish, similar results con-
cerning increased antioxidant enzyme activities by Arg were
observed in rat muscle (Petrović et al., 2008). Arg-enhanced antiox-
idant enzyme activities in fish muscle may be a consequence of the
improvement of the gene transcription of antioxidant enzymes and
of antioxidant-related signalling molecules. Lambertucci et al.
(2007) reported that the enhancement of antioxidant enzyme
activities occurred due to an increase in mRNA levels in rats. Thus,
we next detected the influence of Arg on antioxidant enzyme and
signalling molecule gene expression in the muscle.
4.4. Arg regulated antioxidant enzyme gene and signalling molecule
expression
The results of our current study first demonstrated that optimum
Arg supplementation significantly elevated the mRNA levels of
SOD1, CAT and GPx in grass carp muscle, which showed a similar
pattern with antioxidant enzyme activities, suggesting that the
enhancement of antioxidant enzyme activities is partly attributed
to the fact that Arg up-regulated the gene transcription of antioxi-
dant enzymes in fish muscle. This result is in agreement with the
report in rat adipose tissue (Otašević et al., 2011). The up-regulation
of the antioxidant enzyme gene expression by dietary Arg may result
from activating signalling molecules of regulating antioxidant
enzyme genes in fish. Thus far, the molecular mechanism for the
effects of Arg on antioxidant enzyme gene expression in fish has
not been reported. Therefore, we next investigated the effects of
Arg on the expression of these antioxidant-related signalling mole-
cules. Nrf2 has been demonstrated to be a critical transcription fac-
tor that promotes the transcription of antioxidant enzyme genes,
including SOD, CAT and GPx, through binding to the antioxidant
response element in the promoter region of these antioxidant
enzyme genes in fish (Ma, 2013). Chen et al. (2013) reported that
the up-regulation of Nrf2 expression could elevate SOD, CAT and
GPx gene expression in mouse liver. Our results first showed that
mRNA levels of Nrf2 in grass carp muscle were up-regulated with
increasing dietary Arg up to the optimum level. The correlation anal-
ysis indicated that the mRNA levels of the grass carp muscle antiox-
idant enzyme genes positively correlated with the Nrf2 mRNA level
(rSOD1 = + 0.980, P < 0.05; rCAT = +0.922, P < 0.05; rGPx = +0.851,
P < 0.05), suggesting that Arg-elevated fish muscle antioxidant
enzyme gene expression may be through up-regulating Nrf2 gene
transcription. A similar result has been reported in rat myocardial
tissue (Ramprasath et al., 2012). The positive influence of Arg on
the regulation of grass carp muscle Nrf2 expression may be related
to NO. NO is the major metabolite of Arg and acts as an important sig-
nalling molecule (Ramprasath et al., 2012). In vitro, the increased NO
content up-regulated Nrf2 gene expression in rat vascular smooth
muscle cells (Liu et al., 2007). The result of our current study demon-
strated that optimum Arg was effective in increasing the NO content
in grass carp muscle. The correlation analysis indicated that grass
carp muscle Nrf2 mRNA levels positively correlated with NO
(r = +0.627, P = 0.182), suggesting that up-regulation of Nrf2 expres-
sion by Arg may partially act via the enhancement of the NO content
in fish. In contrast, the promotion of Nrf2 translocation to the
nucleus also plays an important role in the up-regulation of fish anti-
oxidant enzyme gene expression. Keap1 has been identified as an
Nrf2-binding protein, which prevents Nrf2 translocation to the
nucleus and facilitates Nrf2 degradation via the proteasome (Ma,
2013). In HaCaT cells, the down-regulation of Keap1 expression
resulted in the promotion of the nuclear translocation of Nrf2,
thereby up-regulating downstream antioxidant gene expression
(Devling, Lindsay, McLellan, McMahon, & Hayes, 2005). The current
study first showed that appropriate Arg supplementation signifi-
cantly decreased the Keap1 gene expression in grass carp muscle,
suggesting that Arg may promote Nrf2 translocation to the nucleus
to enhance antioxidant enzyme gene expression by the down-regu-
lation of Keap1 expression in fish.
Moreover, Nrf2 expression is regulated by its upstream signal-
ling molecules, such as TOR (Shay et al., 2012). A recent in vitro
study has shown that the elevated mTOR and S6K expression could
up-regulate Nrf2 expression in human brain endothelial cells
(Okouchi et al., 2006). The present study indicated that optimum
Arg supplementation significantly up-regulated grass carp muscle
mRNA levels of TOR and S6K1. The correlation analysis showed
that the grass carp muscle Nrf2 mRNA level positively correlated
with TOR (r = +0.772, P = 0.072) and with S6K1 (r = +0.732,
P = 0.098), suggesting that the up-regulation of fish muscle Nrf2
expression by appropriate Arg may be partly related to the up-reg-
ulation of TOR and S6K1 expression. Furthermore, Arg-up-regu-
lated TOR may be partly due to the up-regulation of CK2. In vitro,
the up-regulation of CK2 caused the up-regulation of the expres-
sion of mTOR and S6K in human glioblastoma cells (Olsen et al.,
2012). The present study first showed that the CK2 mRNA level
was significantly enhanced in grass carp muscle with increasing
Arg levels up to the 10.4 g/kg diet. The correlation analysis indi-
cated that grass carp muscle TOR gene expression positively corre-
lated with CK2 gene expression (r = +0.651, P = 0.162), suggesting
that up-regulation of TOR expression in muscle by Arg may be
related to the up-regulation of CK2 expression in fish. However,
more studies are required to thoroughly explore the mechanism
by which Arg regulates the expression of these antioxidant-related
signalling molecules.
5. Conclusions
The results of the present study demonstrated that dietary Arg
supplementation was effective in promoting grass carp growth and
in enhancing flesh quality through improving non-enzymatic and
enzymatic antioxidative capacities. The enhancement of antioxi-
dant enzyme activities by Arg was partly attributed to the up-reg-
ulation of antioxidant enzyme gene expression, which could be
regulated by several signalling molecules, such as Nrf2, Keap1,
CK2, TOR and S6K1. These results could provide a partial molecular
mechanism for the improvement of flesh quality by Arg in fish.
However, further studies should be performed to reveal the under-
lying molecular mechanism of dietary Arg on flesh quality. In this
study, the dietary Arg requirement for the optimal growth of young
grass carp was determined to be 13.45 g/kg diet (corresponding to
43.64 g/kg dietary protein) using the broken-line analysis.
6. Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This research was financially supported by the National 973
Project of China (2014CB138600), National Department Public
Benefit Research Foundation (Agriculture) of China (201003020),
Science and Technology Support Programmer of Sichuan Province
(2014NZ0003) and Major Scientific and Technological Achieve-
ment Transformation Project of Sichuan Province of China
(2012NC0007). The authors would like to thank the personnel of
these teams for their kind assistance.
 B. Wang et al. / Food Chemistry 167 (2015) 91–99 99

References Liu, X. M., Peyton, K. J., Ensenat, D., Wang, H., Hannink, M., Alam, J., et al. (2007).
Nitric oxide stimulates heme oxygenase-1 gene transcription via the Nrf2/ARE
complex to promote vascular smooth muscle cell survival. Cardiovascular
BerdikovaBohne, V. J., Hamre, K., & Arukwe, A. (2007). Hepatic metabolism, phase I
Research, 75, 381–389.
and II biotransformation enzymes in Atlantic salmon (Salmo Salar, L) during a
Ma, X., Lin, Y., Jiang, Z., Zheng, C., Zhou, G., Yu, D., et al. (2010). Dietary arginine
12 week feeding period with graded levels of the synthetic antioxidant,
supplementation enhances antioxidative capacity and improves meat quality of
ethoxyquin. Food and chemical toxicology, 45, 733–746.
finishing pigs. Amino Acids, 38, 95–102.
Berge, G. E., Sveier, H., & Lied, E. (1998). Nutrition of Atlantic salmon (Salmo salar);
Ma, Q. (2013). Role of Nrf2 in oxidative stress and toxicity. Annual review of
the requirement and metabolic effect of lysine. Comparative Biochemistry and
pharmacology and toxicology, 53, 401–426.
Physiology Part A: Molecular & Integrative Physiology, 120, 477–485.
Maciewicz, R. A., Wotton, S. F., Etherington, D. J., & Duance, V. C. (1990).
Brinker, A., & Reiter, R. (2011). Fish meal replacement by plant protein substitution
Susceptibility of the cartilage collagens types II, IX and XI to degradation by
and guar gum addition in trout feed, Part I: effects on feed utilization and fish
the cysteine proteinases, cathepsins B and L. FEBS Letters, 269, 189–193.
quality. Aquaculture, 310, 350–360.
Martinez-Alvarez, R. M., Morales, A. E., & Sanz, A. (2005). Antioxidant defenses in
Buckley, D., Morrissey, P., & Gray, J. (1995). Influence of dietary vitamin E on the
fish: biotic and abiotic factors. Reviews in Fish Biology and Fisheries, 15, 75–88.
oxidative stability and quality of pig meat. Journal of Animal Science, 73,
Mostert, R., & Hoffman, L. (2007). Effect of gender on the meat quality
3122–3130.
characteristics and chemical composition of kudu (Tragelaphus strepsiceros),
Bureau, D. P., Azevedo, P. A., Tapia-Salazar, M., & Cuzon, G. (2000). Pattern and cost
an African antelope species. Food Chemistry, 104, 565–570.
of growth and nutrient deposition in fish and shrimp: potential implications
Okouchi, M., Okayama, N., Steven Alexander, J., & Yee Aw, T. (2006). NRF2-
and applications. Avances en Nutrición Acuícola V. Memorias del V Simposium
dependent glutamate-L-cysteine ligase catalytic subunit expression mediates
Internacional de Nutrición Acuícola, 19, 111–140.
insulin protection against hyperglycemia-induced brain endothelial cell
Cara, J., Moyano, F., Zambonino, J. L., & Alarcón, F. (2007). The whole amino acid
apoptosis. Current Neurovascular Research, 3, 249–261.
profile as indicator of the nutritional condition in cultured marine fish larvae.
Olsen, B. B., Svenstrup, T. H., & Guerra, B. (2012). Downregulation of protein kinase
Aquaculture Nutrition, 13, 94–103.
CK2 induces autophagic cell death through modulation of the mTOR and MAPK
Chen, S., Zou, L., Li, L., & Wu, T. (2013). The protective effect of glycyrrhetinic acid on
signaling pathways in human glioblastoma cells. International Journal of
carbon tetrachloride-induced chronic liver fibrosis in mice via upregulation of
Oncology, 41, 1967–1976.
Nrf2. PLoS One, 8, e53662.
Otašević, V., Buzadžić, B., Korać, A., Stančić, A., Janković, A., Vučetić, M., et al. (2011).
Devling, T. W., Lindsay, C. D., McLellan, L. I., McMahon, M., & Hayes, J. D. (2005).
Effects of L-arginine and NAME supplementation on mRNA, protein expression
Utility of siRNA against Keap1 as a strategy to stimulate a cancer
and activity of catalase and glutathione peroxidase in brown adipose tissue of
chemopreventive phenotype. Proceedings of the National Academy of Sciences
rats acclimated to different temperatures. Journal of Thermal Biology, 36,
of the United States of America, 102, 7280–7285.
269–276.
Flynn, N., Meininger, C., Haynes, T., & Wu, G. (2002). The metabolic basis of arginine
Periago, M. J., Ayala, M. D., López-Albors, O., Abdel, I., Martínez, C., García-Alcázar,
nutrition and pharmacotherapy. Biomedicine and Pharmacotherapy, 56,
A., et al. (2005). Muscle cellularity and flesh quality of wild and farmed sea bass,
427–438.
Dicentrarchus labrax L. Aquaculture, 249, 175–188.
Fournier, V., Gouillou-Coustans, M., Metailler, R., Vachot, C., Moriceau, J., Le Delliou,
Petrović, V., Buzadžić, B., Korać, A., Vasilijević, A., Janković, A., & Korać, B. (2009). L-
H., et al. (2003). Excess dietary arginine affects urea excretion but does not
Arginine supplementation induces glutathione synthesis in interscapular
improve N utilisation in rainbow trout (Oncorhynchus mykiss) and turbot (Psetta
brown adipose tissue through activation of glutamate-cysteine ligase
maxima). Aquaculture, 217, 559–576.
expression: the role of nitric oxide. Chemico-Biological Interactions, 182,
Hagen, Ø., Solberg, C., & Johnston, I. A. (2008). Activity of aspargate (cathepsin D),
204–212.
cysteine proteases (cathepsins B, B+ L, and H), and matrix metallopeptidase
Petrović, V., Buzadžić, B., Korać, A., Vasilijević, A., Janković, A., Mićunović, K., et al.
(collagenase) and their influence on protein and water-holding capacity of
(2008). Antioxidative defence alterations in skeletal muscle during prolonged
muscle in commercially farmed Atlantic halibut (Hippoglossus hippoglossus L).
acclimation to cold: role of L-Arg/NO-producing pathway. Journal of
Journal of agricultural and food chemistry, 56, 5953–5959.
Experimental Biology, 211, 114–120.
Hultmann, L., Phu, T. M., Tobiassen, T., Aas-Hansen, Ø., & Rustad, T. (2012). Effects of
Portugal, T. R., & Aksnes, A. (1983). Arginase activity in different fish species and
pre-slaughter stress on proteolytic enzyme activities and muscle quality of
tissues. Comparative Biochemistry and Physiology Part B: Comparative
farmed Atlantic cod (Gadusmorhua). Food Chemistry, 134, 1399–1408.
Biochemistry, 76, 15–16.
Jiang, W. D., Feng, L., Liu, Y., Jiang, J., Hu, K., Li, S. H., et al. (2010). Lipid peroxidation,
Ramprasath, T., Kumar, P. H., Puhari, S. S. M., Murugan, P. S., Vasudenvan, V., &
protein oxidant and antioxidant status of muscle, intestine and hepatopancreas
Selvam, G. S. (2012). L-Arg ameliorates cardiac left ventricular oxidative stress
for juvenile Jian carp (Cyprinus carpio var. Jian) fed graded levels of myo-
by upregulating eNOS and Nrf2 target genes in alloxan-induced hyperglycemic
inositol. Food Chemistry, 120, 692–697.
rats. Biochemical and Biophysical Research Communications, 428, 389–394.
Johnston, I. A., Li, X., Vieira, V. L., Nickell, D., Dingwall, A., Alderson, R., et al. (2006).
Ren, M., Liao, Y., Xie, J., Liu, B., Zhou, Q., Ge, X., et al. (2013). Dietary arginine
Muscle and flesh quality traits in wild and farmed Atlantic salmon. Aquaculture,
requirement of juvenile blunt snout bream, Megalobrama amblycephala.
256, 323–336.
Aquaculture, 414, 229–234.
Khan, M. A., Jafri, A. K., & Chadha, N. K. (2004). Growth, reproductive performance,
Rowe, L., Maddock, K., Lonergan, S., & Huff-Lonergan, E. (2004). Influence of early
muscle and egg composition in grass carp, Ctenopharyngodon idella
postmortem protein oxidation on beef quality. Journal of Animal Science, 82,
(Valenciennes), fed hydrilla or formulated diets with varying protein levels.
785–793.
Aquaculture Research, 35, 1277–1285.
Shay, K. P., Michels, A. J., Li, W., Kong, A. N. T., & Hagen, T. M. (2012). Cap-
Khan, N., Qureshi, N. A., Nasir, M., Rasool, F., & Iqbal, K. J. (2011). Effect of artificial
independent Nrf2 translation is part of a lipoic acid-stimulated detoxification
diet and culture systems on sensory quality of fried fish flesh of Indian major
stress response. Biochimicaet Biophysica Acta (BBA)-Molecular Cell Research, 1823,
carps.. Pakistan Journal of Zoology, 43, 1177–1182.
1102–1109.
Lambertucci, R. H., Levada-Pires, A. C., Rossoni, L. V., Curi, R., & Pithon-Curi, T. C.
Shore, L. J., Soler, A. P., & Gilmour, S. K. (1997). Ornithine decarboxylase expression
(2007). Effects of aerobic exercise training on antioxidant enzyme activities and
leads to translocation and activation of protein kinase CK2 in vivo. Journal of
mRNA levels in soleus muscle from young and aged rats. Mechanisms of ageing
Biological Chemistry, 272, 12536–12543.
and development, 128, 267–275.
Skipnes, D., Østby, M. L., & Hendrickx, M. E. (2007). A method for characterising
Larsson, T., Koppang, E. O., Espe, M., Terjesen, B. F., Krasnov, A., Moreno, H. M., et al.
cook loss and water holding capacity in heat treated cod (Gadusmorhua) muscle.
(2014). Fillet quality and health of Atlantic salmon (Salmo salar L.) fed a diet
Journal of Food Engineering, 80, 1078–1085.
supplemented with glutamate. Aquaculture, 426, 288–295.
Tan, B., Yin, Y., Liu, Z., Li, X., Xu, H., Kong, X., et al. (2009). Dietary L-arginine
Lee, J. W., De Riu, N., Lee, S., Bai, S. C., Moniello, G., & Hung, S. S. (2011). Effects of
supplementation increases muscle gain and reduces body fat mass in growing-
dietary methyl mercury on growth performance and tissue burden in juvenile
finishing pigs. Amino Acids, 37, 169–175.
green (Acipenser medirostris) and white sturgeon (A. transmontanus). Aquatic
Tokur, B., & Korkmaz, K. (2007). The effects of an iron-catalyzed oxidation system on
Toxicology, 105, 227–234.
lipids and proteins of dark muscle fish. Food Chemistry, 104, 754–760.
Li, S. H., Zhou, X. Q., Zhang, N., Liu, H., & Ma, C. W. (2008). Purification and
Wang S. (2006). Studies on protein and essential amino acid requirements of grass
characterisation of cathepsin L2 from dorsal muscle of silver carp
carp (Ctenopharyngodonidella). Doctor thesis, Sun Yat-sen University,
(Hypophthalmichthys molitrix). Food Chemistry, 111, 879–886.
Guangzhou, China.
Li, L., Zhou, J. S., He, Y. L., Yang, Y. H., Wang, L. Z., Yang, J. N., et al. (2013a).
Wu, G., & Morris, J. S. (1998). Arginine metabolism: nitric oxide and beyond.
Comparative study of muscle physicochemical characteristics in common
Biochemistry Journal, 336, 1–17.
cyprinus carpio, silurus asotus and ctenopharyngodon idellus. Journal of
Wu, Y. P., Feng, L., Jiang, W. D., Liu, Y., Jiang, J., Li, S. H., et al. (2014). Influence of
Hydroecology, 34, 82–86.
dietary zinc on muscle composition, flesh quality and muscle antioxidant status
Li, X.-Y., Tang, L., Hu, K., Liu, Y., Jiang, W.-D., Jiang, J., et al. (2013b). Effect of dietary
of young grass carp (Ctenopharyngodon idella). Aquaculture Research. http://
lysine on growth, intestinal enzymes activities and antioxidant status of sub-
dx.doi.org/10.1111/are.12392.
adult grass carp (Ctenopharyngodon idella). Fish physiology and biochemistry, 7,
Zhang, L., Mai, K., Tan, B., Ai, Q., Qi, C., Xu, W., et al. (2009). Effects of dietary
1–13.
administration of probiotic Halomonas sp. B12 on the intestinal microflora,
Li, W., Feng, L., Liu, Y., Jiang, W. D., Kuang, S. Y., Jiang, J., Li, S. H., Tang, L., & Zhou, X.
immunological parameters, and midgut histological structure of shrimp,
Q. (2014). Effects of dietary phenylalanine on growth, digestive and brush
Fenneropenaeus chinensis. Journal of the World Aquaculture Society, 40, 58–66.
border enzyme activities and antioxidant capacity in the hepatopancreas and
Zhou, Q. C., Zeng, W.-P., Wang, H. L., Xie, F. J., & Zheng, C. Q. (2012). Dietary arginine
intestine of young grass carp (Ctenopharyngodon idella). Aquaculture Nutrition, in
requirement of juvenile yellow grouper (Epinephelusawoara). Aquaculture, 350,
press.
175–182.