Aquaculture 414–415 (2013) 229–234

Contents lists available at ScienceDirect

Aquaculture

j ourna l homepage: www.e lsev ie r .com/ locate /aqua-on l ine

Dietary arginine requirement of juvenile blunt snout bream,Megalobrama amblycephala

Mingchun Ren a, Yingjie Liao b, Jun Xie a,b, Bo Liu a,b, Qunlan Zhou a, Xianping Ge a,b,⁎, Honghong Cui b,Liangkun Pan a, Ruli Chen a

a Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences,Wuxi 214081, Chinab Wuxi Fisheries College, Nanjing Agricultural University, Wuxi 214081, China

⁎ Corresponding author at: Key Laboratory of FreshwResources Utilization, Ministry of Agriculture, FreshwaChinese Academy of Fishery Sciences, Wuxi 214081, Chin

E-mail address: gexp@ffrc.cn (X. Ge).

0044-8486/$ – see front matter © 2013 Elsevier B.V. All rihttp://dx.doi.org/10.1016/j.aquaculture.2013.08.021

a b s t r a c t

a r t i c l e i n f o

Article history:Received 20 May 2013Received in revised form 18 August 2013Accepted 19 August 2013Available online 27 August 2013

Keywords:Blunt snout breamArginine requirementGrowth performanceNOS

A 9-week feeding trialwas conducted to quantify the dietary arginine requirement of juvenile blunt snout bream.Six isonitrogenous and isoenergetic semi-purified diets (34.0% crude protein) were formulated to contain gradedarginine levels (0.83% to 3.36% of dry weight) at 0.5% increments replaced by equal proportions of glycine. At theend of the feeding trial, the results showed that survival rate was not significantly affected by dietary argininelevel. Final weight, specific growth rate (SGR), feed efficiency ratio (FER), protein efficiency ratio (PER) and pro-tein productive value (PPV) increased with increasing dietary arginine level from 0.83 to 1.81% (P b 0.05), there-after showed a declining trend but the differences were not significant. Whole body compositions wereindependent of dietary arginine levels (P N 0.05). Plasma arginine concentration increased with the increase ofdietary arginine from 0.83% to 1.81%, and thereafterwas relatively constant, while lower lysine content in plasmawas observed in fish fed the diet with 3.36% arginine level compared to those fed diets with 0.83–2.35% arginine(P b 0.05). Significantly higher plasma urea content was observed in fish fed diet with 3.36% arginine comparedwith those fed 0.83% arginine diet (0.83%). Significantly lower plasma total nitric oxide synthase (T-NOS) activ-ities were observed in fish fed 0.83% arginine diet than those fed diets with 2.35–3.36% arginine. Plasma super-oxide dismutase (SOD) activities and ammonia contents were not significantly affected by dietary argininelevels. Based on SGR, FER and PER, the optimal dietary arginine requirement of juvenile blunt snout bream wasestimated to be 2.46% of the diet (7.23% of dietary protein), 2.28% (6.71% of dietary protein) and 2.26% of thediet (6.65% of dietary protein), respectively.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Fish cannot synthesize all amino acids, and it is important to satisfythe essential amino acid requirements of fish by formulating balancednutrients in feed (NRC, 2011). Arginine has been shown to be an essen-tial amino acid for optimal growth of fish, which is the most limitingamino acid in some plant protein sources such as corn meal, sesamemeal and zein (Singh and Khan, 2007). Arginine is involved in manymetabolic pathways such as protein synthesis, urea production, metab-olism of glutamic acid and proline, and synthesis of creatine and poly-amines (Luo et al., 2004). In addition, as a precursor for nitric oxidesynthesis, arginine was identified to play a major role in the immunityresponse in some fish species (Buentello and Gatlin, 1999). The dietaryarginine requirement has been estimated for several fish species, and alarge range (from 3.0% of dietary protein for turbot, Pseua maxima to

ater Fisheries and Germplasmter Fisheries Research Center,a. Tel.: +86 51085557892.

ghts reserved.

8.1% of dietary protein for black sea bream, Sparus macrocephalus) hasbeen observed between species (NRC, 2011), and this wide rangemakes the extrapolation of dietary needs fromonefish species to anoth-er unfeasible (Luo et al., 2007).

Blunt snout bream, Megalobrama amblycephala, a freshwater fish,has a long history of cultivation in China because of its excellent fleshquality, rapid growth performance and high larval survival rate (Zhouet al., 2008). Its production has a fast increase and approximately0.63 million tons in 2010 (Ministry of Agriculture of the People'sRepublic of China, 2010). Nonetheless, the information of nutritionalrequirements in blunt snout bream is still quite limited and tradi-tional formulated feed production relies on formulas for grass carp(Ctenopharyngodon idella). Recently, dietary protein and lipid werequantified in blunt snout bream fingerlings based on growth perfor-mance (Li et al., 2010). To our knowledge, no information is availableconcerning the dietary essential amino acid requirement in blunt snoutbream up to now.

Therefore the present study was conducted to investigate the ef-fects of dietary arginine level on growth performance, feed utilization,whole body composition and immunity response in blunt snout bream,

Table 2Amino acid composition of ingredients (g·100 g−1 dry matter).

Amino acid Amount in Total 34%Whole body protein

15 g C 3.75 g G 5 g FW AAP

EAAArginine 0.43 0.24 0.18 0.00 0.85 2.01Histidine 0.33 0.01 0.11 0.31 0.76 0.76Isoleucine 0.63 0.05 0.14 0.68 1.49 1.49Leucine 1.21 0.09 0.23 0.87 2.40 2.40Lysine 1.01 0.11 0.23 1.09 2.43 2.43Methionine 0.37 0.02 0.09 0.43 0.90 0.90Phenylalanine 0.62 0.06 0.14 0.66 1.47 1.47Threonine 0.55 0.05 0.11 0.71 1.41 1.41Valine 0.78 0.08 0.16 0.56 1.57 1.57

NEAAAspartic acid 0.96 0.16 0.27 1.46 2.84 2.84Serine 0.70 0.09 0.12 0.55 1.45 1.45Glycine 0.24 0.69 0.19 1.37 2.50 2.50Alanine 0.43 0.27 0.20 1.25 2.14 2.14Cystine 0.03 0.00 0.02 0.14 0.19 0.19Tyrosine 0.68 0.02 0.10 0.27 1.07 1.07Gulmatic acid 2.79 0.32 0.42 1.11 4.64 4.64Proline 1.19 0.39 0.10 0.12 1.81 1.81

C, casein; G, gelatin; FM, fish meal; AAP, crystalline amino acid premix; EAA, essentialamino acid; NEAA, non-essential amino acid. Tryptophan could not be detected afteracid hydrolysis.

230 M. Ren et al. / Aquaculture 414–415 (2013) 229–234

and to quantify dietary arginine requirement in juvenile blunt snoutbream.

2. Materials and methods

2.1. Diet preparation

Six isonitrogenous and isoenergetic diets (34% crude protein), usingfish meal, casein and gelatin as protein sources and soybean oil as alipid source, were formulated to contain graded levels of arginine(0.83%, 1.30%, 1.81%, 2.35%, 2.82% and 3.36% of dry weight, respectively)whichwere replaced by equal proportions of glycine (Table 1). Amixtureof crystalline L-amino acids was supplemented to simulate the wholebody amino acid pattern of blunt snout bream except for arginine(Table 2). All the ingredients were ground into powder and thoroughlymixed with soybean oil and water, then forced through a pelletizer (4–2style, Xinchang Machinery LTD, China) and dried in a ventilated ovenat 30 °C. After drying, all diets were sealed in bags and stored at −15 °Cuntil used.

2.2. Experimental procedure

Experimental fish were obtained from a commercial farm (Jiangsu,China). Prior to the feeding trial, the fish were fed with diet 1 for2 weeks to acclimate to the experimental diet and conditions. Afterfasting for 24 h, juvenile blunt snout bream (initial weight 2.6 ±0.1 g) were randomly sorted into eighteen floating cages (1 m ×1 m × 1 m)with 30 fish in each cage. Each diet was randomly assigned

Table 1Formulation and proximate composition of the experimental diets (% dry matter).

Ingredients Diet number

1 2 3 4 5 6

White fish meala 5.00 5.00 5.00 5.00 5.00 5.00Caseina 15.00 15.00 15.00 15.00 15.00 15.00Gelatina 3.75 3.75 3.75 3.75 3.75 3.75Soybean oil 6.00 6.00 6.00 6.00 6.00 6.00Soybean lecithin 1.00 1.00 1.00 1.00 1.00 1.00Amino acid premixb 11.56 11.56 11.56 11.56 11.56 11.56Vitamin premixc 2.00 2.00 2.00 2.00 2.00 2.00Mineral premixd 5.00 5.00 5.00 5.00 5.00 5.00Corn starch 35.00 35.00 35.00 35.00 35.00 35.00Cellulose 7.69 7.69 7.69 7.69 7.69 7.69Carboxymethyl cellulose 5.00 5.00 5.00 5.00 5.00 5.00Ethoxyquin 0.50 0.50 0.50 0.50 0.50 0.50Glycine 2.50 2.00 1.50 1.00 0.50 0.00L-Arginine 0.00 0.50 1.00 1.50 2.00 2.50

Proximate analysis (% of dry diet)Arginine 0.83 1.30 1.81 2.35 2.82 3.36Crude protein 33.8 34.0 33.9 33.6 33.8 34.3Crude lipid 8.31 8.40 8.54 8.26 8.35 8.42Gross energy (KJ g−1) 18.8 18.8 18.9 18.8 18.9 18.8

a Casein, obtained from Hua'an Biological Products Lit. (Gansu, China), crude protein90.2%; gelatin, obtained from Zhanyun Chemical Lit. (Shanghai, China), crude protein91.3%; white fish meal, obtained from Copeinca (Lima, Peru), crude protein 67.4%, andcrude lipid 9.3%.

b Amino acid premix (g/100 g diet): L-histidine, 0.31; L-isoleucine, 0.68; leucine, 0.87;L-lysine, 1.09; L-methionine, 0.43; L-phenylalanine, 0.66; L-threonine, 0.71; L-valine, 0.56;L-aspartic acid, 1.46; serine, 0.55; glycine, 1.37; alanine; 1.25; L-cystine 0.14; L-tyrosine,0.27; tryptophan, 0.12; glumatic acid, 1.11; proline 0.12. Amino acids obtained fromFeeer Co., LTD (Shanghai, China).

c Vitamin premix (IU or mg/kg of diet): vitamin A, 25,000 IU; vitamin D3, 20,000 IU;vitamin E, 200 mg; vitamin K3, 20 mg; thiamin, 40 mg; riboflavin, 50 mg; calcium panto-thenate, 100 mg; pyridoxine HCl, 40 mg; cyanocobalamin, 0.2 mg; biotin, 6 mg; folic acid,20 mg; niacin, 200 mg; inositol, 1000 mg; vitamin C, 2000 mg; choline, 2000 mg, andcellulose was used as a carrier.

d Mineral premix (g/kg of diet): calciumbiphosphate, 20 g; sodium chloride, 2.6; potas-sium chloride, 5 g;magnesium sulphate, 2 g; ferrous sulphate, 0.9 g; zinc sulphate, 0.06 g;cupric sulphate, 0.02; manganese sulphate, 0.03 g; sodium selenate, 0.02 g; cobalt chlo-ride, 0.05 g; potassium iodide, 0.004; and zeolite was used as a carrier.

to triplicate cages. Fish were hand-fed three times daily at 8:00, 12:00and 16:00 until apparent satiation on the basis of visual observation.During the 9 week feeding trial, the number and weight of dead fishand feed consumptionwere recorded every day. Thewater temperaturefluctuated from 21 to 24 °C and dissolved oxygen was approximately6 mg L−1 throughout the feeding trial.

2.3. Sample collection and analysis

2.3.1. Sample collectionAt the end of the feeding trial, fish were fasted for 24 h before

sampling. Total numbers and mean body weight of fish in each cagewere determined. Five fish per cage were euthanized by MS-222(100 mg·L−1), and then blood samples were collected immediatelyfrom the caudal vein using heparinized syringes. Following centrifuga-tion (3500 ×g, 10 min, 4 °C), the plasmawas separated. All the sampleswere stored at−80 °C until analysis. Ten fish at the beginning and fivefish at the end of the experiment per cage were sampled and stored at−20 °C for the analysis of whole body composition.

2.3.2. Laboratory analysisDry matter, crude protein and lipid were determined according to

the established methods of AOAC (2003): dry matter after drying inan oven at 105 °C until constant weight; crude protein (N × 6.25) byKjeldahl method after acid digestion; lipid by ether extraction usingSoxhlet. Gross energy of the experimental diets was analyzed by anadiabatic bomb calorimeter (PARR1281, USA).

Amino acid concentrations were determined by a professional labo-ratory (at the Institute of Feed Science, Jiangnan University, China). Fortotal amino acid content analysis, the diet and ingredients were freeze-dried overnight, and then hydrolyzed for 24 h in 6 N HCl at 110 °C. Forfree amino acid content analysis, the plasma were deproteinized bytrichloroacetic acid (5%). After pretreatment, all the samples were ana-lyzed with an Agilent-1100 amino acid determination system (AgilentTechnologies Co., Ltd., Santa Clara, USA). Tryptophan could not bedetected after acid hydrolysis.

The plasma urea content was determined by the diacetyl monoximemethod using a medical detection kit (Nanjing Jiancheng Bioengineer-ing Institute, China). The determination of plasma ammonia contentwas performed using a diagnostic kit (Nanjing Jiancheng Bioengineer-ing Institute, China) by Berthelot reaction. Total nitric oxide synthase

Table 3Effects of dietary arginine level on growth performance of juvenile blunt snout bream fed experimental diets for 9 weeks (means ± S.E.M.)1.

Item Arginine level (% of dry diet)

0.83 1.30 1.81 2.35 2.82 3.36

Initial weight 2.68 ± 0.01 2.68 ± 0.01 2.68 ± 0.01 2.68 ± 0.01 2.68 ± 0.01 2.68 ± 0.01Survival (%)2 95.0 ± 1.67 94.4 ± 5.56 91.1 ± 5.56 95.6 ± 2.22 90.0 ± 5.77 91.7 ± 1.67Final weight (g) 8.23 ± 0.29a 9.94 ± 0.28b 12.0 ± 0.50c 11.9 ± 0.33c 11.7 ± 0.41c 11.0 ± 0.26bc

SGR (% day−1)3 1.78 ± 0.05a 2.08 ± 0.06b 2.38 ± 0.07c 2.36 ± 0.04bc 2.34 ± 0.06c 2.24 ± 0.03bc

FER4 0.45 ± 0.02a 0.54 ± 0.02ab 0.60 ± 0.03b 0.60 ± 0.01b 0.59 ± 0.03b 0.52 ± 0.03ab

PER5 1.21 ± 0.04a 1.46 ± 0.09ab 1.63 ± 0.12b 1.61 ± 0.03ab 1.57 ± 0.08ab 1.34 ± 0.11ab

PPV (%)6 18.9 ± 0.49a 24.1 ± 1.90ab 27.6 ± 2.43b 26.9 ± 0.74ab 27.1 ± 2.47ab 22.9 ± 1.58ab

1Data are means of triplicate. Means in the same column sharing a same superscript letter are not significantly different determined by Tukey's test (P N 0.05).2Survival rate (%) = 100 × (finial number of fish) / (initial number of fish).3SGR: specific growth rate = 100 × (Ln finial individual weight − Ln initial individual weight) / number of days.4FER: feed efficiency ratio = wet weight gain/dry diet fed.5PER: protein efficiency ratio = wet weight gain/protein intake.6PPV: protein productive value = protein gain/protein intake.

231M. Ren et al. / Aquaculture 414–415 (2013) 229–234

(T-NOS) activities were determined using the enzymatic double cyclemethod as described by Wang et al. (2000). Superoxide dismutase(SOD) activity was measured by its ability to inhibit superoxide aniongenerated by a xanthine and xanthine oxidase reaction system using aSOD detection kit (Nanjing Jiancheng Bioengineering Institute, China)as described by Zhou et al. (2012).

2.3.3. Statistical analysisData were transformed if necessary after evaluating assumptions

of normality, equality of variances and outliers, and subjected to one-way analysis of variance (ANOVA) using the software SPSS 13.0 forWindows. Significant differences in the means between dietary treat-ments were evaluated by Tukey's multiple range test. Mean differenceswere considered significant at a P value equal or less than 0.05. Thesecond-order polynomial regression model (Zeitoun et al., 1976) wasused to estimate the optimumdietary arginine requirement for juvenileblunt snout bream on the basis of SGR, FER and PER after comparing theestimation coefficient (R2) between broken-line regression model andsecond-order polynomial regression model.

3. Results

3.1. Growth performance and feed utilization

During the 9 week feeding trial, the experimental diets were wellaccepted by juvenile blunt snout bream. Survival rates for all treatmentswere over 90% and independent of dietary arginine level (P N 0.05).Growth performance and feed utilization were significantly affectedby dietary arginine level. Final weight, SGR, FER, PER and PPV were sig-nificantly increasedwith the increase of dietary arginine level from0.83to 1.81%. The highest final weight (12.0 g), SGR (2.38%·day−1), FER

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0.5 1 1.5 2 2.5 3 3.5

Dietary arginine level (%)

Spec

ific

gro

wth

rat

e (%

day

-1)

X=2.46

Fig. 1. Effect of dietary arginine level on specific growth rate of juvenile blunt snout bream.

(0.60), PER (1.63) and PPV (27.6) were observed in the fish fed dietwith 1.81% arginine. Thereafter, performance values tended to declinebut not in a significant manner (Table 3). Based on SGR, the optimumarginine requirement for juvenile blunt snout bream was estimated tobe 2.46% of the diet (7.23% of dietary protein), 2.28% (6.71% of dietaryprotein), and 2.26% of the diet (6.65% of dietary protein), respectively(Figs. 1, 2 and 3).

3.2. Whole body compositions

There were no significant differences in whole body moisture, pro-tein, lipid and ash contents of juvenile blunt snout breamamong dietarytreatments (Table 4).

3.3. Plasma free amino acid profile

Plasma arginine concentration significantly increased with increas-ing dietary arginine from 0.83% to 1.81%. While significantly lowerlysine content in plasma was observed in fish fed the diet containinghighest arginine level compared with those fed diets with argininefrom 0.83 to 2.35%. However dietary arginine level did not affect plasmahistidine, isoleucine, leucine, methionine, phenylalanine, threonine orvaline concentration (Table 5).

3.4. Plasma urea–N and ammonia content

Significantly higher plasma urea–N content was observed in fish feddiet with 3.36% arginine than those fed diet with 0.83% arginine. Plasmaammonia content showed an increasing trend with the increase ofdietary arginine level but not in a significant manner (Table 6).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.5 1 1.5 2 2.5 3 3.5

Dietary arginine level (%)

Fee

d ef

fici

ency

rat

io

X=2.28

Fig. 2. Effect of dietary arginine level on feed efficiency ratio of juvenile blunt snout bream.

0

0.4

0.8

1.2

1.6

2

0.5 1 1.5 2 2.5 3 3.5

Dietary arginine level (%)

Pro

tein

eff

icie

ncy

rati

o

X=2.26

Fig. 3. Effect of dietary arginine level on protein efficiency ratio of juvenile blunt snoutbream.

232 M. Ren et al. / Aquaculture 414–415 (2013) 229–234

3.5. Plasma T-NOS and SOD activities

Significantly lower total nitric oxide synthase (T-NOS) activitieswere observed in fish fed arginine deficient diet (0.83% arginine) com-pared with those fed diets with 2.35–3.36% arginine. No significantdifferences were observed in SOD activities among the treatments(Table 6).

4. Discussion

In the present study, juvenile blunt snout bream fed the argininedeficient diet showed the lower growth performance and feed utiliza-tion, and these values improved with the supplementation of dietarycrystalline arginine, which indicated that arginine is an essentialamino acid for blunt snout bream and the juvenile fish are able to utilizethe crystalline arginine.

In this study, the highest growth and feed utilization were observedin fish fed diet with 1.81% arginine level, and the dietary argininerequirement of juvenile blunt snout bream was determined to be 2.46%of the diet (7.23% of dietary protein), 2.28% (6.71% of dietary protein),and 2.26% of the diet (6.65% of dietary protein), respectively, usingsecond-order polynomial regression model, which was similar to the re-quirements reported for silver perch, Bidyanus bidyanus (6.8% of dietaryprotein, Ngamsnae et al., 1999) and yellow grouper, Epinephelus awoara(6.5% of dietary protein, Zhou et al., 2012), and higher than the valuesfor common carp, Cyprinus carpio (4.3% of dietary protein, Nose, 1979),yellow perch, Perca flavescens (4.9% CP, Twibell and Brown, 1997),Asian sea bass, Lates calcarifer (3.8% of dietary protein, Murillo-Gurreaet al., 2001), Mrigal carp, Cirrhinus mrigala (4.6% of dietary protein,Ahmed and Khan, 2004) and Nile tilapia, Oreochromis niloticus (3.4% ofdietary protein, Santiago and Lovell, 1988), while lower than the valuesreported for black sea bream, Acanthopagrus schlegelii (7.7–8.1% of CP,Zhou et al., 2010). The dietary arginine requirements of cultured fishhave a large spread (from 3.0% to 8.1%), and even in the same species

Table 4Effect of dietary arginine on body composition in juvenile blunt snout bream fed different leve

Whole body composition Arginine level (% of dry diet)

0.83 1.30 1.

Moisture (%) 70.4 ± 0.22 70.3 ± 0.26 69Crude protein (% w.w.) 16.6 ± 0.54 16.9 ± 0.14 17Crude lipid (% w.w.) 8.66 ± 0.30 9.22 ± 0.37 9.Ash (% w.w.) 3.54 ± 0.08 3.39 ± 0.05 3.

w.w., wet weight.a Data are means of triplicate. Means in the same column sharing a same superscript letter a

different values have been reported (NRC, 2011). The large variationwithin or among fish species is possibly affected by fish species, fishsize, dietary protein sources and levels, and experimental conditions(Kim et al., 1992; Luo et al., 2004). However, the mathematical modelused to estimate the requirement might affect the estimation values(Zhou et al., 2010).Moreover, glutamate has been proved to be an impor-tant precursor for endogenous arginine, and the dietary glutamate levelmay affect the dietary arginine requirement estimation in fish, such asthe arginine requirement estimate was 33% higher when glycine re-placed glutamate in the diet in catfish (Buentello and Gatlin, 2000). Inthis study, equal proportions of glycine were used to replace the dietaryarginine to avoid the potential influence of arginine requirement estima-tion from glutamic acid.

Although there were no statistical differences, lower SGR, FER, PERand PPVwere observed in fish fed diet with the highest dietary argininelevel (3.36% of dry diet) compared to those fed diet with optimumarginine level (1.81%). Similarly, some studies indicated excess dietaryarginine level resulting in adverse growth performance and feed utiliza-tion in fish, such as in hybrid Clarias (Singh and Khan, 2007), Indianmajor carp, Labeo rohita (Abidi and Khan, 2009), rainbow trout(Fournier et al., 2003) and yellow grouper (Zhou et al., 2012). Waltonet al. (1986) indicated that the excess dietary arginine may lead toextra energy expenditure toward deamination, probably resulting intoxic effects and stress in the fish that have adverse effects on growth.

Furthermore, the reduced growth in fish fed diets with excessivearginine may be due to the lysine–arginine antagonism resulting inthe imbalance amino acid profiles as well. The antagonism betweenlysine and arginine is well known in poultry and rats embodying inreduced growth, competing for absorptive or re-absorptive site and byincreasing amino acid degradation through interference with their nor-mal intermediate metabolism (Luo et al., 2004). However, the lysine–arginine antagonism in fish is still far from clear. In the present study,besides the reduced growth and feed utilization, significantly lowerlysine content in the plasma was observed in fish fed diet with highestdietary arginine (3.36%) compared with those fed diets with argininefrom 0.83 to 2.35%, which indicated that to some extent antagonism be-tween lysine and arginine existed in juvenile blunt snout bream fed theimbalance arginine/lysine ratio diet. Since arginine and lysine share thesame brush border membrane carrier, competitive inhibition betweentwo amino acids can affect their absorption, transport and metabolism(Kaushik and Fauconneau, 1984). Similar to this study, increased levelsof dietary arginine resulted in reduced uptake of lysine in the intestinein rainbow trout and Atlantic salmon, indicating competition betweenarginine and lysine at the intestinal level (Berge et al., 1999; Kaushiket al., 1988). However, Davies et al. (1997) considered that the lysine–arginine interaction existed during post-absorptive assimilation andmetabolism, but not at the absorptive phase because of high arginineand lysine digestibility in rainbow trout. In contrast, some authors indi-cated that no lysine–arginine antagonism existed in European sea bassand Japanese flounder due to no negative effect of growth or plasmaamino acid levels (Alam et al., 2002b; Tibaldi et al., 1994).

In the present study, whole body composition of juvenile bluntsnout bream was not significantly affected by dietary arginine level,which was agreed with the reports in juvenile grouper (Luo et al.,

ls of dietary arginine for 9 weeks (means ± S.E.M.).a

81 2.35 2.82 3.36

.9 ± 0.38 70.3 ± 0.18 70.2 ± 0.16 70.1 ± 0.16

.1 ± 0.20 17.4 ± 0.27 17.2 ± 0.11 17.0 ± 0.4135 ± 0.47 9.19 ± 0.32 9.30 ± 0.29 9.19 ± 0.1830 ± 0.11 3.23 ± 0.12 3.26 ± 0.06 3.18 ± 0.07

re not significantly different determined by Tukey's test (P N 0.05).

Table 5Free amino acid content in the plasma of blunt snout bream fed different levels of dietary arginine for 9 weeks (means ± S.E.M.)1.

Essential amino acid concentration (mg L−1) Arginine level (% of dry diet)

0.83 1.30 1.81 2.35 2.82 3.36

Arg 5.87 ± 0.58a 8.13 ± 0.88a 12.6 ± 0.70b 11.2 ± 1.14ab 13.2 ± 2.31b 12.9 ± 1.55b

His 23.0 ± 3.56 24.1 ± 5.46 25.4 ± 2.31 24.5 ± 1.82 26.1 ± 5.34 25.7 ± 4.64Ile 8.60 ± 2.25 10.4 ± 2.99 12.7 ± 3.64 10.5 ± 0.81 8.53 ± 2.78 9.37 ± 1.47Leu 12.8 ± 1.36 10.3 ± 0.23 11.5 ± 1.32 10.2 ± 2.57 8.13 ± 2.67 9.57 ± 2.75Lys 19.4 ± 3.13b 19.9 ± 1.65b 20.2 ± 0.68b 17.8 ± 1.02b 12.9 ± 2.19ab 9.10 ± 0.98a

Met 3.53 ± 0.26 3.17 ± 0.75 3.37 ± 0.27 3.00 ± 0.45 3.30 ± 0.95 2.13 ± 0.22Phe 4.60 ± 0.17 4.20 ± 0.93 4.53 ± 0.23 4.07 ± 0.61 3.97 ± 1.52 4.37 ± 0.68Thr 24.8 ± 6.61 21.9 ± 5.61 23.2 ± 3.31 17.7 ± 2.19 14.7 ± 2.28 16.3 ± 4.01Val 11.4 ± 3.34 12.3 ± 0.09 13.8 ± 1.64 8.40 ± 2.80 10.0 ± 3.42 10.7 ± 2.23

1Data are means of triplicate. Means in the same column sharing a same superscript letter are not significantly different determined by Tukey's test (P N 0.05).

233M. Ren et al. / Aquaculture 414–415 (2013) 229–234

2007; Zhou et al., 2012). However, the whole body protein contentexhibited a similar trend with PER and PPV, which indicated that theoptimal dietary arginine supplementation might improve the N reten-tion and protein deposition, and reduce the nitrogen waste in juvenileblunt snout bream.

In the current study, plasma arginine concentrationwas significantlyincreasedwith increasing dietary arginine level from0.83 to 1.81%. Sim-ilar results have been reported in the plasma, muscle or whole body ofseveral fish (Berge et al., 1997, 2002; Schwarz et al., 1998; Yamamotoet al., 2000). However, no further increases were found in fish feddiets containing arginine beyond 1.81%, and the possible explanationis that the deamination of arginine was up-regulated in these groupsand excess arginine was excreted through soluble ammonia, such asthe previous reports in Japanese flounder (Alam et al., 2002a). In thepresent study, the plasma ammonia contents showed an increasedtrend, but there were no significant differences among the treatments,which is possibly due to the fast ammonia clearance from plasma bythe gills (Berge et al., 2002). In fish, it is mostly used as an osmolyte orexcreted as a waste product of ammonia detoxification, thus most fishdo not excrete high levels of urea and thus have nephrons involved inurine concentration (Mathai, 2005). In this study, plasma urea contentskept relatively constant in fish fed diets with arginine from 0.83 to2.26%. However, significantly higher plasma urea content was observedin fish fed diet with 3.36% arginine compared with those fed 0.83% argi-nine diet,which agreedwith the reports in Atlantic salmon and rainbowtrout that the excess dietary arginine supplementation did not improvenitrogen utilization, but affected the urea production by arginase-mediated degradation of arginine (Berge et al., 2002; Fournier et al.,2003).

Arginine is well established to be an immuno-nutrient in higheranimals, such as humans (Evoy et al., 1998). In recent years, the immunefunction of arginine has received a body of attention in fish becausearginine is a precursor for nitric oxide synthesis and nitric oxide is im-portant in physiological processes controlling blood vessel tone, neuro-transmission, platelet aggregation and adhesion, cell proliferation andmacrophage activity (Eddy and Tibbs, 2003; NRC, 2011). In the presentstudy, there were significantly lower plasma T-NOS activities in fish fedarginine deficient diets (0.83%) compared with those fed diets witharginine from 2.35 to 3.36%. Similar results have been observed in

Table 6Plasma ammonia, urea–N, total nitric oxide synthase and superoxide dismutase activities of bl

Item Arginine level (% of dry diet)

0.83 1.30 1.81

Ammonia (μmol/L) 178.1 ± 6.25 171.6 ± 5.46 175.5 ±Urea–N (mmol/L) 0.42 ± 0.05a 0.45 ± 0.07ab 0.45 ±T-NOS (U/mL) 1.43 ± 0.30a 1.47 ± 0.39ab 2.41 ±SOD (U/L) 45.8 ± 2.17 48.8 ± 2.50 46.3 ±

1Data are means of triplicate. Means in the same column sharing a same superscript letter are

darkbarbel catfish and yellow grouper (Feng et al., 2011; Zhou et al.,2012). In response to bacterial lipopolysaccharide, the arginine appearsto play an important role in providing the needed substrate for NObiosynthesis and cultured macrophage NO production was increasedwhen arginine, citrulline and arginine plus glutamine were used assubstrate in channel fish (Buentello and Gatlin, 1999). L-Argininesupplementation could enhance eNOS expression in experimentalmodel of hypercholesterolemic rabbit aorta (Javanmard et al., 2009).However, the mechanisms of the regulatory effects of dietary arginineon immune response in fish are still unclear and need to be furtherinvestigated.

Superoxide dismutase (SOD) plays an important role in the self-defense system and the immune system (Calvin and Muscatine,1997). In this study, dietary arginine level did not regulate the plasmaSOD activities in juvenile blunt snout bream, which indicated that thedietary arginine supplementation did not influence the defense againstoxidative stress in juvenile blunt snout bream. The results in this studywere consistent with the reports in yellow grouper and turbot (Li et al.,2008; Zhou et al., 2012). However, in catfish SOD activities were en-hanced by increasing dietary arginine level (Buentello et al., 2007).

In conclusion, the dietary arginine requirement of juvenile bluntsnout bream was estimated to be 2.46% of the diet (7.23% of dietaryprotein), 2.28% (6.71% of dietary protein), and 2.26% of the diet (6.65%of dietary protein) on the basis of SGR, FER and PER, respectively. Thelysine–arginine antagonism appears to exist in juvenile fish.

Acknowledgment

The authors gratefully thank the post graduate students and thosefrom the Fish Disease and Nutrition Department, Freshwater FisheriesResearch Center (FFRC) for their help throughout the research periodand the Key Laboratory Freshwater Fisheries and Germplasm ResourcesUtilization, FFRC, Chinese Academy of Fishery Sciences for their assis-tance in preparation of the test animal and experimental facility. Thefunding of this study was financially supported by the Modern Agricul-ture Industrial Technology System special project—the National StapleFreshwater Fish Industrial Technology System (Nycytx-46), by a SpecialFund for Agro-scientific Research in the Public Interest (201003020).

unt snout bream fed the experimental diets for 9 weeks (means ± S.E.M.)1.

2.35 2.82 3.36

10.5 197.2 ± 17.7 194.9 ± 12.8 207.8 ± 8.130.07ab 0.46 ± 0.09ab 0.46 ± 0.07ab 0.54 ± 0.05b

0.46ab 3.62 ± 0.89b 4.68 ± 0.73b 3.88 ± 0.53b

3.01 47.1 ± 4.54 46.2 ± 3.65 48.5 ± 1.53

not significantly different determined by Tukey's test (P N 0.05).

234 M. Ren et al. / Aquaculture 414–415 (2013) 229–234

References

Abidi, S.F., Khan, M.A., 2009. Dietary arginine requirement of fingerling Indianmajor carp,Labeo rohita (Hamilton) based on growth, nutrient retention efficiencies, RNA/DNAratio and body composition. J. Appl. Ichthyol. 25, 707–714.

Ahmed, I., Khan, M.A., 2004. Dietary arginine requirement of fingering India major carp,Cirrhinus mrigala (Hamilton). Aquaculture 235, 499–511.

Alam, M.S., Teshima, S., Koshio, S., Ishikawa, M., 2002a. Arginine requirement of juvenileJapanese flounder Paralichthys olivaceus estimated by growth and biochemicalparameters. Aquaculture 205, 127–140.

Alam, M.S., Teshima, S., Ishikawa, M., Hasengawa, D., Koshio, S., 2002b. Effects of dietaryarginine and lysine levels on growth performance and biochemical parameters ofjuvenile Japanese flounder Paralichthys olivaceus. Fish. Sci. 68, 509–516.

AOAC, 2003. Official Methods of Analysis of the Association of Official Analytical Chemists,15th ed. Association of Official Analytical Chemists, Inc., Arlington, VA.

Berge, G.E., Lied, E., Sveier, H., 1997. Nutrition of Atlantic salmon (Salmo salar): the re-quirement and metabolism of arginine. Comp. Biochem. Physiol. 117A, 501–509.

Berge, G.E., Bakke-McKellep, A.M., Lied, E., 1999. In vitro uptake and interaction betweenarginine and lysine in the intestine of Atlantic salmon (Salmo salar). Aquaculture179, 181–193.

Berge, G.E., Sveier, H., Lied, E., 2002. Effects of feeding Atlantic salmon (Salmo salar L.)imbalanced levels of lysine and arginine. Aquac. Nutr. 8, 239–248.

Buentello, J.A., Gatlin III, D.M., 1999. Nitric oxide production in activated macrophagesfrom channel catfish (Ictalurus punctatus): influence of dietary arginine and culturemedia. Aquaculture 179, 513–521.

Buentello, J.A., Gatlin III, D.M., 2000. The dietary arginine requirement of channel catfish(Ictalurus punctatus) is influenced by endogenous synthesis of arginine from glutamicacid. Aquaculture 188, 311–321.

Buentello, J.A., Reyes-Becerril, M., Romero-Geraldo, M.J., Ascencio-Valle, F.J., 2007. Effectsof dietary arginine on hematological parameters and innate immune function ofchannel catfish. J. Aquat. Anim. Heal. 19, 195–203.

Calvin, M.N.I., Muscatine, Y.L., 1997. Oxidative stress in the symbiotic sea anemoneAiptasia pulchella (Carlgren, 1943): contribution of the animal to superoxide ion pro-duction at elevated temperature. Biol. Bull. 192, 444–456.

Davies, S.J., Morris, P.C., Baker, R.T.M., 1997. Partial substitution of fish meal and full-fatsoya bean meal with wheat gluten and influence of lysine supplementation in dietsfor rainbowtrout, Oncorhynchus mykiss (Walbaum). Aquac. Res. 28, 317–328.

Eddy, F.B., Tibbs, P., 2003. Effects of nitric oxide synthase inhibitors and a substrate,L-arginine, on the cardiac function of juvenile salmonid fish. Comp. Biochem. Physiol.135C, 137–144.

Evoy, D., Lieberman, M.D., Fahey III, T.J., Daly, J.M., 1998. Immunonutrition: the role ofarginine. Nutrition 14, 611–617.

Feng, F.X., Ai, Q.H., Xu, Wei, Mai, K.S., Zhang, W.B., 2011. Effects of dietary arginine andlysine on growth and non-specific immune responses of juvenile darkbarbel catfish(Pelteobagrus vachelli). J. Fish. China 7, 120–128.

Fournier, V., Gouillou-Coustans, M.F., Métailler, R., Vachot, C., Moriceau, J., Le Delliou, H.,Huelvan, C., Desbruyeres, E., Kaushik, S.J., 2003. Excess dietary arginine affects ureaexcretion but does not improve N utilisation in rainbow trout Oncorhynchus mykissand turbot Psetta maxima. Aquaculture 217, 559–576.

Javanmard, S.H., Nematbakhsh, M., Mahmoodi, F., Mohajeri, F.R., 2009. L-Arginine supple-mentation enhances eNOS expression in experimental model of hypercholesterol-emic rabbits aorta. Pathophysiology 16, 9–13.

Kaushik, S.J., Fauconneau, B., 1984. Effects of lysine administration on plasma arginine and onsome nitrogenous catabolites in rainbow trout. Comp. Biochem. Physiol. 79A, 459–462.

Kaushik, S.J., Fauconneau, B., Terrier, L., Gras, J., 1988. Arginine requirement and statusassessed by different biochemical indices in rainbow trout (Salmo gairdneri R.). Aqua-culture 70, 75–95.

Kim, K.I., Kayes, T.B., Amundson, C.H., 1992. Requirements for lysine and arginine by rain-bow trout (Oncorhynchus mykiss). Aquaculture 106, 333–344.

Li, Y., Wang, Y.J., Jiang, K.Y., 2008. Influence of several non-nutrient additives onnonspecific immunity and growth of juvenile turbot, Scophthalmus maximus L.Aquac. Nutr. 14, 387–395.

Li, X.F., Liu, W.B., Jiang, Y.Y., Zhu, H., Ge, X.P., 2010. Effects of dietary protein andlipid levels in practical diets on growth performance and body composition of bluntsnout bream (Megalobrama amblycephala) fingerlings. Aquaculture 303, 65–70.

Luo, Z., Liu, Y.J., Mai, K.S., Tian, L.X., 2004. Advance in researches on arginine requirementfor fish: a review. J. Fish. China 28, 450–459.

Luo, Z., Liu, Y.J., Mai, K.S., Tian, L.X., Tan, X.Y., Yang, H.J., 2007. Effects of dietary argininelevels on growth performance and body composition of juvenile grouper Epinepheluscoioides. J. Appl. Ichthyol. 23, 252–257.

Mathai, J.C., 2005. Ammonotelic teleosts andurea transporters. Am. J. Physiol. Renal Physiol.288, F453–F454.

Ministry of Agriculture of the People's Republic of China, 2010. Chinese FisheriesYearbook. Chinese Agricultural Press, Beijing, China.

Murillo-Gurrea, D.P.R., Coloso, R.M., Borlongan Jr., I.G., 2001. Lysine and arginine require-ment s of juvenile Asian sea bass (Lates calcarifer). J. Appl. Ichthyol. 17, 49–53.

National Research Council (NRC), 2011. Nutrient Requirements of Fish and Shrimp.National Academy Press, Washington, DC 57–101.

Ngamsnae, P., De Silva, S.S., Gunasekera, R.M., 1999. Arginine and phenylalanine require-ment of juvenile silver perch Bidyanus bidyanus and validation of the use of bodyamino acid composition for estimating individual amino acid requirements. Aquac.Nutr. 5, 173–180.

Nose, T., 1979. Summary report on the requirements of essential amino acids for carp. In:Halver, J.E., Tiews, K. (Eds.), Finfish Nutrition and Fishfeed Technology. HeenemannGmbH, Berlin, Germany, pp. 145–156.

Santiago, C.B., Lovell, R.T., 1988. Amino acid requirements for growth of Nile tilapia.J. Nutr. 118, 1540–1546.

Schwarz, F.J., Kirchgessner, M., Deuringer, U., 1998. Studies on the methionine require-ment of carp (Cyprinus carpio L.). Aquaculture 161, 121–129.

Singh, S., Khan, M.A., 2007. Dietary arginine requirement of fingerling hybrid Clarias(Clarias gariepinus × Clarias macrocephalus). Aquac. Res. 38, 17–25.

Tibaldi, E., Tulli, F., Lanari, D., 1994. Arginine requirement and effect of different dietaryarginine and lysine levels for fingerling sea bass (Dientrarchus labrus). Aquaculture127, 207–218.

Twibell, R.G., Brown, P.B., 1997. Dietary arginine requirement of juvenile yellow perch.J. Nutr. 127, 1838–1841.

Walton, M.J., Cowey, C.B., Coloso, R.M., Adron, J.W., 1986. Dietary requirements of rain-bow trout for tryptophan, lysine and arginine determined by growth and biochemicalmeasurements. Fish Physiol. Biochem. 2, 161–169.

Wang, W., Inoue, N., Nakayama, T., 2000. An assay method for nitric oxide synthase incrude samples by determining product NAPH. Anal. Biochem. 227, 274–280.

Yamamoto, T., Unuma, T., Akiyama, T., 2000. The influence of dietary protein and fat levelson tissue free amino acid levels of fingerling rainbow trout (Oncorhynchus mykiss).Aquaculture 182, 353–372.

Zeitoun, I.H., Ullrey, D.E., Magee, W.T., Gill, J.L., Bergen, W.G., 1976. Quantifying nutrientrequirement of fish. J. Fish. Res. Board Can. 33, 167–172.

Zhou, Z., Ren, Z., Zeng, H., Yao, B., 2008. Apparent digestibility of various feedstuffs forblunt snout bream, Megalobrama amblycephala. Aquac. Nutr. 4, 153–165.

Zhou, F., Xiong, W., Xiao, J.X., Shao, Q.J., Bergo, O.N., Hua, Y., Chai, X., 2010. Optimum argi-nine requirement of juvenile black sea bream, Sparus macrocephalus. Aquac. Res. 41,e418–e430.

Zhou, Q.C., Zeng, W.P., Wang, H.L., Xie, F.J., Wang, T., Zheng, C.Q., 2012. Dietary argininerequirement of juvenile yellow grouper Epinephelus awoara. Aquaculture 350–353,175–182.