Aquaculture 437 (2015) 382–389

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Aquaculture

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Effect of dietary arginine levels on the growth performance, feed utilization, non-speciﬁc immune response and disease resistance of juvenile golden pompano Trachinotus ovatus

Heizhao Lin a,b,⁎, Xiaohong Tan a,b, Chuanpeng Zhou a,c, Jin Niu a,DongmeiXiaa,b, Zhong Huang a, Jun Wang a,YunWanga a Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guang- zhou 510300, PR China b School of Life Science and Technology, Shanghai Ocean University, Shanghai 201306, PR China c South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, PR China article info abstract

Article history: In order to assess dietary arginine requirement for juvenile golden pompano, an experiment of six different diets Received 12 September 2014 with six dietary arginine levels (2.05%, 2.38%, 2.65%, 2.98%, 3.25% and 3.58%) was conducted. The results showed Received in revised form 15 December 2014 that the content of dietary arginine had a significant effect on weight gain (WG), specific growth rate (SGR), feed Accepted 16 December 2014 conversion rate (FCR) and protein efficiency ratio (PER) of golden pompano (P b 0.05). Golden pompano fed diet Available online 20 December 2014 containing 2.65% arginine achieved the maximum WG, SGR and PER as well as the minimum FCR, however, the WG, SGR and PER decreased while FCR increased with a further increasing of dietary arginine (P b 0.05). Dietary Keywords: fi Trachinotus ovatus arginine level had no signi cant effect on viscerosomatic index, hepatosomatic index, condition factor, survival Arginine rate, whole body and muscle composition, serum glucose, triglyceride, cholesterol, alanine aminotransferase Growth performance and aspartate aminotransferase, and serum and hepatic total superoxide dismutase activities (P N 0.05). Dietary Serum biochemical parameters arginine content had a significant effect on serum and hepatic total nitric oxide synthase and lysozyme activities Non-specific immune responses as well as survival rate in a Vibrio harveyi challenge experiment (P b 0.05). Quadratic regression analysis on WG, SGR, FCR and PER indicated that the recommended optimum dietary arginine level for optimal growth of juvenile pompano was 2.73–2.74% of the dry diet, corresponding to 6.32–6.35% of dietary protein. © 2015 Elsevier B.V. All rights reserved.

1. Introduction animal bodies, arginine not only takes part in the synthesis of protein, carbamide and creatine as well as the metabolism of glutamic acid Golden pompano (Trachinotus ovatus) belongs to the family and proline, but also works as the synthetic precursor of NO and other Carangidae. It is a carnivorous fish that preys mainly on small crustaceans, substances. It also plays an important role in nutrition metabolism and shellfish, and fish. It is widely distributed in China, Japan, Australia, and control processes (Luo et al., 2004). Arginine can also improve the utili- other countries (Chen et al., 2007; Cheng and Zheng, 1987; Sun et al., zation efficiency of dietary protein and promote the development of 2014; Tang et al., 2013; Zheng et al., 2014). In recent years, interest in com- aquatic product cultivation in order to add a proper amount of arginine mercial culture of golden pompano in China and southeast Asian countries in an aquatic diet. At present, many scholars have conducted studies on such as Malaysia and Singapore has been increasing (Chen and Chen, the arginine requirements for many kinds of commercial fish, including 2011; Gu and Zhou, 2009; Liu et al., 2011a,b; Wang, 2009). To date, studies channel catfish (Ictalurus punctatus)(Buentello and Gatlin, 2000), of the nutritional requirements of golden pompano are limited. flounder (Paralichthys olivaceus)(Alam et al., 2002a), mrigal (Cirrhinus Arginine is one of the necessary amino acids for aquatic animals, as mrigala)(Ahmed and Khan, 2004), malabar grouper (Epinephelus well as the main limiting amino acid of plant-origin protein sources malabaricus)(Luo et al., 2007), hybrid catfish (Clarias such as corn meal, sesame meal and corn gluten meal (Singh and gariepinus × Clarias macrocephalus)(Singh and Khan, 2007), black sea Khan, 2007). Taking part in many different metabolic reactions in bream (Sparus macrocephalus)(Zhou et al., 2010), red drum (Sciaenops ocellatus)(Cheng et al., 2011), yellow grouper (Epinephelus awoara)(Zhou et al., 2012), cobia (Rachycentron canadum)(Ren et al., ⁎ Corresponding author at: Key Laboratory of South China Sea Fishery Resources 2014), tilapia (Oreochromis niloticus L.) (Yue et al., 2013) and blunt Exploitation & Utilization, Ministry of Agriculture, South China Sea Fisheries Research snout bream (Megalobrama amblycephala )(Ren et al., 2013). Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, PR China. Tel.: +86 755 84422048; fax: +86 510 84451442. Currently, the research on the nutritional requirements of golden E-mail address: [email protected] (H. Lin). pompano has involved quantities of protein and fat (Liu et al., 2011a;

Wang, 2012), protein–energy ratios (Liu et al., 2011b) and demanded 2.2. Fish and experimental conditions quantities of diaminocaproic acid and methionine (Niuetal.,2013). However, the requirement of dietary arginine for golden pompano has Experimental fish were obtained from Shenzhen Trial Base of South not been studied. The purpose of this experiment was to examine the ef- China Sea Fisheries Research Institute (Shenzhen, China). Prior to the fect of dietary arginine on the growth performance, serum biochemical feeding trial, the fish were fed with a commercial diet for 2 weeks indices, immune responses and resistance to the Vibrio harveyi chal- toacclimate to the experimental diet and conditions. At the start of the lenge on golden pompano, in order to determine the optimum dietary experiment, the fish were fasted for 24 h and weighed after being arginine requirement for golden pompano. anesthetized with 10 mg L−1 eugenol (Shanghai Medical Instruments Co., Ltd., Shanghai, China). Juvenile gold pompano averages (18.81 ± 0.18 g, mean ± SD) were randomly sorted into eighteen floating cages 2. Materials and methods (1 m × 1 m × 1 m) with 20 fish each cage. Each diet was randomly assigned to cages in triplicate. Fish were fed two times daily at 8:00 2.1. Diet preparation and 16:00 until apparent satiation on the basis of visual observation. During the 8 week feeding trial, the number and weight of dead fish Six isonitrogenous and isolipidic diets (D1, D2, D3, D4, D5 and D6) and feed consumption were recorded every day. The water temperature −1 were formulated to contain graded levels of L-arginine at about 0.3% in- ranged from 21 to 29 °C, salinity about 13–17 g L ,pH7.6–7.8, ammo- crements (Table 1 and (2). Dietary arginine was quantitatively in- nia nitrogen lower than 0.1 mg L−1 and dissolved oxygen was more creased at the expense of glycine. Because the experimental diets than 5 mg L−1 throughout the feeding trial. contained about 43% protein, the amino acid (AA) contents of the experimental diets were made comparable to those of 43% whole body pro- 2.3. Sample collection tein AA contents (Niu et al., 2013). All the dietary AA contents were maintained nearly the same levels as the corresponding AA contents At the end of the feeding trial, fish were fasted for 24 h before sam- in 43% whole body protein except for arginine and glycine. A satisfying pling. Total numbers and mean body weight of fish in each cage were increase in arginine content was obtained in the diets, although it was determined. Three fish at the end of the experiment per cage were sam- not identical to the quantities added initially. The final levels of arginine pled and stored at −20 °C for the analysis of whole body composition. were 2.05%, 2.38%, 2.65%, 2.98%, 3.25% and 3.58% (dry weight), respec- Another four fish per cage were euthanized by 10 mg L−1 eugenol tively, by adding crystalline L-arginine, analyzed by reverse phase high (Shanghai Medical Instruments Co., Ltd., Shanghai, China) and then performance liquid chromatography (HPLC, HP1100, USA). All the in- blood samples were collected immediately from the caudal vein. gredients were ground into powder through 60-mesh and thoroughly Following centrifugation (3000 r/min, 10 min, 4 °C), the serum was mixed with oil and water, then forced through a pelletizer (F-26, separated and stored at −80 °C for analysis. Another three fish per South China University of Technology, Guangzhou, China) and air- cage were used to determine hepatosomatic index (HSI), dried to about 10% moisture. After drying, all diets were sealed in bags viscerosomatic index (VSI) and condition factor (CF). Viscera and liver and stored at −20 °C until used. were collected and weighed and the ratios were expressed as a percent- age of body weight, and the livers were rapidly removed and collected Table 1 for analysis of the activities of LYZ, T-NOS and T-SOD. Then the muscle Composition and nutrient levels of experimental diets (DM basis) %. (dorsal muscle on both sides of the fish) samples were removed and Ingredients D1 D2 D3 D4 D5 D6 stored frozen (−20 °C) until analysis for composition of the muscle.

Fish meal 15.00 15.00 15.00 15.00 15.00 15.00 Corn gluten meal 25.00 25.00 25.00 25.00 25.00 25.00 2.4. Measurement and analysis Rapeseed meal 5.00 5.00 5.00 5.00 5.00 5.00 Peanut meal 9.00 9.00 9.00 9.00 9.00 9.00 Corn starch 19.00 19.00 19.00 19.00 19.00 19.00 Dry matter, crude protein, lipid and ash were determined according Beer yeast powder 2.00 2.00 2.00 2.00 2.00 2.00 to the established methods of AOAC (2005). Dry matter after drying in a Amino acid mixture 8.56 8.56 8.56 8.56 8.56 8.56 an oven at 105 °C until constant weight; crude protein (N × 6.25) was Fish oil 3.72 3.85 3.85 3.85 3.85 3.85 analyzed by Kjeldahl method using Kjeltec (FOSS 2300, Hoganas, Bean oil 3.72 3.84 3.84 3.84 3.84 3.84 Lecithin 1.00 1.00 1.00 1.00 1.00 1.00 Sweden) after acid digestion; and lipid was determined by petroleum Vitamin premixb 2.00 2.00 2.00 2.00 2.00 2.00 ether extraction using Soxtec™ 205 (FOSS, Hoganas, Sweden). For ash Mineral premixc 4.00 4.00 4.00 4.00 4.00 4.00 content analysis, samples were placed in a mufﬂe furnace (FO610C, Choline chloride (50%) 0.50 0.50 0.50 0.50 0.50 0.50 Yamato Scientiﬁc Co., Ltd., Tokyo, Japan) at 550 °C for 8 h. Amino acids Crystalline arginine 0.00 0.30 0.60 0.90 1.20 1.50 Crystalline glycine 1.50 1.20 0.90 0.60 0.30 0.00 in diets and muscle were determined after acid hydrolysis. For total Total 100.00 100.00 100.00 100.00 100.00 100.00 amino acid content analysis, the diet was freeze-dried overnight, and then hydrolyzed for 24 h in 6 N HCl at 110 °C. For free amino acid con- Nutrient levelsd Moisture 4.15 4.37 4.82 4.71 4.85 4.50 tent analysis, after pretreatment, all the samples were analyzed with an Crude protein 42.74 43.26 42.89 42.61 43.72 43.82 L-8900 amino acid analyzer (Hitachi, Tokyo, Japan). Tryptophan could Crude fat 12.19 12.32 12.82 12.48 12.73 12.42 not be detected after acid hydrolysis. Serum cholesterol, triglyceride, Ash 11.31 11.41 11.60 11.53 11.44 11.48 and glucose contents were measured using the enzymatic (cholesterol a Amino acid mixture provides the following per kg of diet: Thr 0.54 g, Val 7.52 g, Met oxidase) and colorimetric methods, the enzymatic (glycerol phosphate 4.77 g, Ile 9.24 g, Leu 7.36 g, Phe 4.94 g, Lys 8.70 g, Asp 5.62 g, Glu 20.27 g, Pro 4.56 g, Ala oxidase) and colorimetric (PAP) methods, and the glucose oxidase 10.26 g, and Tyr 1.82 g. method, respectively, using test kits purchased from Junshi Biotechnol- b Vitamin premix provides the following per kg of diet: VB 25 mg, VB 45 mg, VB 1 2 6 ogy Co., Ltd. (Shanghai, China). Serum glutamic–pyruvic transaminase 20 mg, VB12 0.1 mg, VK3 10 mg, inositol 800 mg, pantothenic acid 60 mg, nicotinic acid 200 mg, folic acid 1.2 mg, biotin 32 mg, VD3 5mg,VE120mg,VC2.0g,choline (ALT) and glutamic-oxaloacetic transaminase (AST) activities were chloride 2.0 g, ethoxyquin 150 mg, and manna-croup 14.52 g. both tested by a ROCHE-P800 automatic biochemical analyzer (Roche, c Mineral premix provides the following per kg of diet: NaF 4 mg, KI 1.6 mg, Basel, Switzerland). Serum total protein content was tested by a CoCl ·6H O(1%) 100 mg, CuSO ·5H O 20 mg, FeSO ·H O 160 mg, ZnSO ·H O 100 mg, 2 2 4 2 4 2 4 2 ROCHE-P800 automatic biochemical analyzer (Roche, Basel, MnSO4·H2O 120 mg, MgSO4·7H2O 2.4 g, ZnSO4·H2O 100 mg, Ca(H2PO4)2·H2O 6.0 g, NaCl 200 mg, and zeolite power 30.90 g. Switzerland). T-SOD, T-NOS and LYZ activities were measured with d Measured values. commercial assay kits (Nanjing Jiancheng Bioengineering Institute, 384 H. Lin et al. / Aquaculture 437 (2015) 382–389

Table 2 protein in whole body) / protein intake; Amino acid composition of experimental diets (DM basis) %. • Condition factor (CF, g/cm3) = 100 × (body weight, g) / (body length, 3 Amino acids Diets cm ); • Viscerosomatic index (VSI, %) = 100 × (viscera weight, g) / (whole D1 D2 D3 D4 D5 D6 body weight, g); Thr 1.14 1.13 1.13 1.14 1.12 1.12 • Hepatosomatic index (HSI, %) = 100 × (liver weight, g) / (whole body Val 1.94 1.91 1.94 1.97 1.95 1.92 Met 0.80 0.80 0.84 0.78 0.78 0.78 weight, g); Ile 2.04 2.01 2.05 2.08 2.10 2.02 • Survival rate (%) = 100 × (final number of fish) / (initial number of Leu 2.98 2.90 2.95 2.91 2.95 2.93 fish). Phe 1.65 1.63 1.66 1.63 1.64 1.62 Arg 2.05 2.38 2.65 2.98 3.25 3.58 His 0.61 0.61 0.61 0.61 0.60 0.60 Lys 1.62 1.58 1.61 1.60 1.60 1.57 Total EAA 14.83 14.95 15.44 15.70 15.99 16.14 2.7. Statistical analysis Asp 3.09 3.07 3.10 3.11 3.04 3.02 Ser 1.35 1.34 1.35 1.34 1.31 1.32 Glu 6.27 6.24 6.31 6.29 6.19 6.13 All data were presented as means ± SD and subjected to one-way Pro 1.97 1.94 1.95 1.94 1.92 1.89 analysis of variance (ANOVA) to test the effects of experimental diets Gly 3.28 2.96 2.66 2.38 2.06 1.76 using the software of the SPSS for windows (Ver 13.0; SPSS, Inc., Ala 2.63 2.61 2.62 2.61 2.58 2.57 Chicago, IL, USA). Duncan's new multiple range test was used to resolve Tyr 0.93 0.91 0.93 0.90 0.91 0.89 Total NEAA 19.52 19.07 18.92 18.57 18.01 17.58 the differences among treatment means (Duncan, 1955). Statistical Total 34.35 34.02 34.36 34.27 34.00 33.72 significance was examined at P b 0.05 unless otherwise noted. A quadratic regression analysis method (Snedecor and Cochran, 1978) was employed to estimate the optimum arginine requirement.

Nanjing, China) in accordance with the instructions of the manufacturer. 3. Results

2.5. Challenge test 3.1. Effect of dietary arginine level on growth performance and feed utilization of T. ovatus After 1 week of initial sampling, all fish (10 per cage) were injected intraperitoneally with 0.1 mL V. harveyi (concentration of 3.0 × 108 CFU/ The results showed that dietary arginine levels had a significant mL), and recorded the cumulative mortality of 10 days after tapping the effect on final body weight, WG, SGR, PER and PR of golden pompano bacteria. V. harveyi strains were provided by the South China Sea Fisher- (P b 0.05) (Table 3). WG, SGR, PER and PR increased with increasing ies Research Institute, expanded the strains in 28 °C after training, with of dietary arginine level up to 2.65%, and thereafter declined. The sterilized saline elution, count, then diluted to the concentration needed highest values of WG and SGR occurred at the 2.65% dietary arginine for the experiment. level (P b 0.05). FCR decreased with increasing of dietary arginine level up to 2.65%, and then increased. The FCR in 2.38%, 2.65% and 2.6. Calculations 2.98% arginine-containing groups was significantly lower than that in 2.05%, 3.25% and 3.58% arginine-containing groups (P b 0.05). The The parameters were calculated as follows: PER in 2.65% and 2.98% arginine-containing groups was significantly higher than that in 2.05%, 3.25% and 3.58% arginine-containing • Weight gain rate (WG, %) = 100 × (final body weight − initial body groups (P b 0.05). The PR in 2.65% arginine-containing group was sig- weight) / initial body weight; nificantly higher than that in 2.05%, 2.38%, 3.25% and 3.58% arginine- • Specific growth rate (SGR, % day−1)=100×(Ln final individual containing groups (P b 0.05). There were no differences in CF, VSI, HSI weight − Ln initial individual weight) / number of days; and SR among all treatments (P N 0.05). Quadratic regression analysis • Feed conversion ratio (FCR) = dry diet fed / wet weight gain; on WG (Fig. 1), SGR (Fig. 2), FCR (Fig. 3) and PER (Fig. 4)indicated • Protein efficiency ratio (PER) = 100 × wet weight gain / protein in- that the recommended optimum dietary arginine level for optimal take; growth of juvenile pompano was 2.73–2.74% of the dry diet, corre- • Protein retention (PR) = 100 × (final protein in whole body − initial sponding to 6.32–6.35% of dietary protein.

Table 3 Effects of dietary arginine level on growth performance and feed utilization in pompano (Trachinotus ovatus).

Diets (Arg %) Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6 P value

2.05 2.38 2.65 2.98 3.25 3.58

Initial weight/g 18.71 ± 0.28 18.98 ± 0.05 18.75 ± 0.19 18.73 ± 0.07 18.97 ± 0.15 18.81 ± 0.18 0.18 Final weight/g 67.0 ± 1.06ab 75.74 ± 2.24c 84.21 ± 3.55d 77.78 ± 1.80c 69.23 ± 1.18b 63.14 ± 2.59a 0.00 WG/% 258.2 ± 9.97b 299. ± 10.79c 349. ± 16.34d 315. ± 10.16c 264.9 ± 7.27b 237. ± 13.35a 0.01 SGR/(% /d) 2.28 ± 0.05b 2.47 ± 0.05c 2.68 ± 0.06d 2.54 ± 0.04c 2.31 ± 0.03b 2.17 ± 0.07a 0.03 FCR 2.00 ± 0.09b 1.63 ± 0.17a 1.47 ± 0.13a 1.55 ± 0.21a 1.88 ± 0.04b 2.13 ± 0.15b 0.04 PER/(%) 1.64 ± 0.07ab 1.91 ± 0.19bc 2.06 ± 0.18c 2.02 ± 0.23c 1.67 ± 0.05ab 1.53 ± 0.08a 0.00 PR/(%) 17.48 ± 1.94a 21.85 ± 2.89ab 26.90 ± 4.10c 23.79 ± 2.75bc 19.39 ± 0.40ab 17.62 ± 1.55a 0.02 CF/(g/cm3) 3.41 ± 0.15 3.37 ± 0.19 3.45 ± 0.16 3.41 ± 0.22 3.60 ± 0.35 3.52 ± 0.28 0.78 VSI/% 1.71 ± 0.43 1.52 ± 0.26 1.50 ± 0.26 1.73 ± 0.28 1.88 ± 0.51 1.58 ± 0.45 0.85 HSI/% 6.72 ± 0.60 6.74 ± 0.81 6.78 ± 0.41 6.78 ± 0.41 6.97 ± 0.68 6.2 ± 0.62 0.47 SR/% 98.33 ± 2.89 100.00 ± 0.00 98.33 ± 2.89 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 0.57

Values are means ± SD of three replications. Means in the same raw with different superscripts are significantly different (P b 0.05). WG: weight gain; SGR: specificgrowthrate;FCR:feed conversion ratio; PER: protein efficiency ratio; PR: protein retention; CF: condition factor; VSI: viscerosomatic index; HSI: hepatosomatic index; SR: survival rate. H. Lin et al. / Aquaculture 437 (2015) 382–389 385

Fig. 2. Effects of dietary arginine level on speciﬁc growth rate of pompano.

Fig. 1. Effects of dietary arginine level on weight gain of pompano.

3.2. Effect of dietary arginine level on whole body and muscle composition 3.5. Effect of dietary arginine level on survival rate of T. ovatus of T. ovatus The post-challenge survival was presented in Fig. 5. The challenge Table 4 showed that whole body and muscle crude protein contents test showed that the post-challenge survival increased with increasing were not significantly affected by dietary arginine levels (P N 0.05). The of dietary arginine up to 2.65%, and thereafter declined after infection 2.65% arginine-containing group showed a slightly increase in whole with V. harveyi. Compared with 2.05% arginine-containing group, the body and muscle crude protein contents though no significant differ- 2.65%, 2.98% and 3.25% arginine-containing groups significantly in- ences were found among diet groups (P N 0.05). There were no differ- creased the post-challenge survival (P b 0.05). Compared with the con- ences among treatments in whole body and muscle moisture, crude trol, the 2.38% and 3.58% arginine-containing groups had a tendency fat and ash contents, respectively (P N 0.05). toward increase in the post-challenge survival. The highest post- challenge survival was recorded in the 2.65% arginine-containing group. 3.3. Effect of dietary arginine level on serum biochemical indices of T. ovatus

4. Discussion No significant differences were observed in serum total cholesterol (CHO), triglyceride (TG), alanine aminotransferase (ALT), aspartate Previous studies on amino acid requirements of aquatic animals aminotransferase (AST) and glucose (GLU) contents among all dietary have usually been relied on broken-line models and binomial curve treatments (P N 0.05) (Table 5). However, dietary arginine levels had a models to assess the amino acid requirements. The broken-line model significant effect on serum total protein (TP) content (P b 0.05). Serum was effectively utilized to study the amino acid requirement in TP content increased with increasing of dietary arginine levels up to largemouth bass (Micropterus salmoides)(Zhou, 2011a), black sea 2.98% and then declined. The serum TP contents in 2.65%, 2.98% and bream (Acanthopagrus schlegelii)(Zhou, 2011b), darkbarbel catfish 3.25% groups were significantly higher than those in 2.05%, 2.38% and (Pelteobagrus vachelli)(Feng, 2011), and coho salmon (Oncorhynchus 3.58% groups (P b 0.05). kisutch)(Luzzana et al., 1998), while the binomial curve model was utilized in studies on turbot (Scophthalmus maximus)(Wei, 2010), cobia 3.4. Effect of dietary arginine level on non-specific immune responses of (R. canadum)(Zhao et al., 2007), blunt snout bream (M. amblycephala) T. ovatus (Ren et al., 2013), tilapia (O. niloticus)(Yue et al., 2013), and yellow grouper (E. awoara)(Zhou et al., 2012). According to the results in Dietary arginine levels had no significant effect on the total superox- this experiment, WG, SGR, PER and PR increased with increasing of die- ide dismutase (SOD) activities of serum and liver (P N 0.05) in Table 6. tary arginine level up to 2.65%, and thereafter declined. The highest WG Dietary arginine levels had significant effects on the activities of serum and SGR occurred at the 2.65% dietary arginine level (P b 0.05). There- and liver total nitric oxide synthase (T-NOS) and lysozyme (LYZ) (P b fore, the binomial model was suitable for determining the requirement 0.05). The activity of serum T-NOS increased with increasing of dietary of amino acid for golden pompano. A quadratic regression analysis on arginine level up to 3.25%, and then leveled off. The activity of serum WG and SGR against the dietary arginine levels indicated that the opti- T-NOS in 3.25% arginine-containing group was significantly higher mum dietary arginine level for maximum growth was estimated to be than that in 2.05%, 2.38%, 2.65% and 2.98% arginine-containing groups 2.73% of the dry diet, corresponding to 6.32% of dietary protein. The re- (P b 0.05). The serum LYZ activity increased with increasing dietary ar- sults in our experiment were similar to those in previous studies on tur- ginine level up to 2.65%, and thereafter remained the same. Serum LYZ bot (S. maximus, 6.39%) (Wei, 2010), tilapia (O. niloticus, 6.24%) (Yue activities in 2.05% and 2.38% arginine-containing groups were signifi- et al., 2013), cobia (R. canadum, 6.20%) (Ren et al., 2014) and yellow cantly lower than those in 2.65% arginine-containing group (P b 0.05). grouper (E. awoara, 6.5%) (Zhou et al., 2012), but higher than those in There were no differences in serum LYZ activities among all groups ex- largemouth bass (M. salmoides, 4.31%–4.79%) (Zhou, 2011a), darkbarbel cept 2.65% arginine-containing group. On the contrary, the liver T-NOS catfish (P. vachelli, 5.17%) (Feng, 2011), catla (Catla catla,5.57%)(Zehra activity decreased with increasing of dietary arginine level up to and Khan, 2013), rohu (Labeo rohita,3.05%–3.47%) (Abidi and Khan, 2.38%, and thereafter increased. The liver T-NOS activities in 2.38% and 2009), hybrid catfish (C. gariepinus × C. macrocephalus,4.45%–5.0%) 2.65% arginine-containing groups were significantly lower than those (Singh and Khan, 2007), flounder (P. olivaceus,4.08%)(Alam et al., of the other arginine-containing groups (P b 0.05). The liver LYZ activity 2002a), channel catfish (I. punctatus,3.3%–3.8%) (Buentello and Gatlin, increased with increasing dietary arginine level up to 2.98%, and then 2000) and coho salmon (O. kisutch, 4.9%) (Luzzana et al., 1998), lower leveled off. The liver LYZ activities in the 2.05% and 2.38% arginine- than those in silver perch (Bidyanus bidyanus, 6.8%) (Ngamsnae et al., containing groups were signifi cantly lower than those in 2.65%, 2.98% 1999), black sea bream (S. macrocephalus, 7.74%) (Zhou, 2011b) and and 3.25% arginine-containing groups (P b 0.05). blunt snout bream (M. amblycephala,7.23%)(Ren et al., 2013). The 386 H. Lin et al. / Aquaculture 437 (2015) 382–389

Fig. 3. Effects of dietary arginine level on feed conversion ratio of pompano. Fig. 4. Effects of dietary arginine level on protein efficiency rate of pompano. requirement of dietary arginine for fish varied between 3.30% and 8.64% that there was no antagonism between arginine and lysine in fish such (of dietary protein). The reasons for this significant variation included as channel catfish (I. punctatus)(Robinson et al., 1981), flounder species and growth stage of fish, dietary protein source and content, (P. olivaceus)(Alam et al., 2002b) and European sea bass (Dicentrarchus composition of dietary protein amino acid, water temperature, salinity, labrax)(Tibaldi et al., 1994). Walton et al. (1986) held that excessive ar- and experimental assessment indices. In addition, glutamic acid was the ginine resulting in slow growth of fish could be due to fish requiring extra precursor substance of arginine, therefore dietary glutamic acid content energy for deamination and ammonia excretion, so leading to toxic ac- has an effect on the requirement of arginine for fish. Buentello and tion and increased survival pressure. Gatlin (2000) found that arginine requirements increased by 33% In this experiment, CF, HSI and VSI in different groups had no signif- when using glycine to replace dietary glutamic acid. In our experiment, icant difference, similar to the result in largemouth bass (M. salmoides) dietary arginine was quantitatively increased at the expense of glycine (Zhou, 2011a). Zhou (2011b) reported that dietary arginine content had but not glutamic acid in order to avoid the potential effect of glutamic significant effects on CF of black sea bream (S. macrocephalus) but with- acid. Two kinds of common indices were often used in the experiment out significant effects on its HSI. Zhou et al. (2012) showed that dietary of amino acid requirement for fish, such as growth performance indices arginine content had significant effect on CF and HSI of yellow grouper (WG and SGR), and other indices reflecting the utilization efficiency of (E. awoara) but without significant effects on VSI. Different kinds of diet (FCR, PER). This experiment used WG, SGR and PER to assess the di- studies showed that CF, VSI and HSI not only relate to dietary arginine etary arginine requirement for golden pompano. The results from differ- levels, but also relate to fish species, size and other aspects in the ent methods were similar, excluding the effect of different experimental experiment. assessment indices on dietary arginine requirement for golden In the present experiment, dietary arginine levels had no significant pompano. effect on whole body and muscle compositions (moisture, crude pro- Interestingly, the survival rates in all groups were not signifi- tein, crude fat and ash) of golden pompano. Until now, no definite con- cantly affected by dietary arginine levels in this experiment and clusion whether dietary arginine levels had an effect on fish nutrient golden pompano in all groups had no pathological state of defi- composition exists and the conclusions varied among different studies. ciency, which possibly due to 2.05% dietary arginine could basi- Zhou (2011a) observed that dietary arginine level had a signifi cant cally satisfy the demand of golden pompano. WG, SGR and PER effect on whole body crude protein, but no significant effect on whole of fish in the high arginine level groups of 3.25% and 3.58% were body moisture, crude fat and ash, nor did it have an effect on muscle significantly lower than those of fish in the 2.65% arginine- moisture, crude protein, crude fat and ash in largemouth bass containing group, while the FCR showed the opposite trend. Ex- (M. salmoides). Zhou (2011b) found that crude protein content in cessive dietary arginine content caused golden pompano to whole body and muscle increased with increasing of dietary arginine grow at lower rates, similar to the results of silver perch levels, but crude fat showed an opposite trend in black sea bream (B. bidyanus)(Ngamsnae et al., 1999), rohu (L. rohita)(Abidi (S. macrocephalus). Dietary arginine had no significant effect on body and Khan, 2009), cobia (R. canadum)(Zhao et al., 2007), black nutrition in darkbarbel catfish (P. vachelli)(Feng, 2011). Previous stud- sea bream (S. macrocephalus)(Zhou, 2011b), yellow grouper ies showed that dietary arginine level had no significant effect on whole (E. awoara)(Zhou et al., 2012) and blunt snout bream body moisture, crude protein, crude fat and ash in tilapia (O. niloticus) (M. amblycephala)(Ren et al., 2013). Ji (2000) observed that the and blunt snout bream (M. amblycephala), respectively (Ren et al., balance of dietary amino acid could improve PER and lower FCR 2013; Yue et al., 2013). Dietary arginine levels could significantly affect in black sea bream (S. macrocephalus). Excessive arginine in the whole body dry matter, muscle crude protein and crude fat, while diet could result in an imbalanced proportion of necessary having no significant effect on whole body crude protein, crude fat amino acids affecting the absorption and utilization of other and ash, muscle dry matter and ash in yellow grouper (E. awoara) amino acids by golden pompano. This might have the effect of (Zhou et al., 2012). However, dietary arginine levels had no significant hindering the growth of fish and lowering FCR. Excessive arginine effect on whole body moisture, crude protein, crude fat and ash, but could generate antagonism with lysine and hinder the absorption had a significant effect on muscle moisture, crude protein and crude and utilization of lysine. Many studies showed that an antago- fat in malabar grouper (E. malabaricus)(Luo et al., 2007). The effect of nism between arginine and lysine generally exists in mammals, dietary arginine level on body composition in fish might also relate to but there was no definite conclusion in fish (Sun et al., 2014). An- fish species, due to the fact that some fish are capable of arginine tagonism between arginine and lysine can appear at the level of synthesis. digestion and absorption, as well as at the metabolic level after In this experiment, the dietary arginine levels had a significant effect absorption. Some researchers found an antagonism between argi- on serum TP content of golden pompano, but have no significant effect nine and lysine in fish, such as rainbow trout (Oncorhynchus mykiss) on CHO, TG, ALT, AST and GLU contents. The serum biochemical indices (Kim et al., 1992), Atlantic salmon (Salmo salar)(Berge et al., 2002)and of fish reflected metabolism, nutrition, and health status of fish black sea bream (S. macrocephalus)(Zhou, 2011b), while others held (Ruchimat et al., 1997a). Excessively low or high dietary arginine H. Lin et al. / Aquaculture 437 (2015) 382–389 387

Table 4 Effects of dietary arginine level on whole body and muscle proximate composition of pompano (T. ovatus).

Diets (Arg %) Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6 P value

2.05 2.38 2.65 2.98 3.25 3.58

Whole body Moisture 65.48 ± 1.55 65.0 ± 1.90 65.8 ± 0.57 65.0 ± 0.72 64.6 ± 0.70 65.2 ± 0.94 0.85 Crude protein 48.00 ± 4.07 48.4 ± 2.55 52.1 ± 2.54 48.1 ± 1.41 50.04 ± 1.31 51.77 ± 1.84 0.21 Crude fat 39.01 ± 0.54 39.07 ± 0.93 38.66 ± 1.27 38.94 ± 0.93 39.58 ± 0.44 38.89 ± 1.12 0.30 Ash 11.28 ± 1.12 11.11 ± 0.70 11.39 ± 0.67 10.94 ± 0.42 11.06 ± 0.32 11.52 ± 0.50 0.89

Muscle Moisture 71.86 ± 0.27 71.74 ± 0.48 72.29 ± 0.77 72.02 ± 0.06 71.63 ± 0.33 72.19 ± 0.52 0.16 Crude protein 73.23 ± 2.08 72.03 ± 2.19 73.28 ± 1.84 72.54 ± 1.82 72.53 ± 1.36 71.64 ± 2.25 0.35 Crude fat 19.81 ± 1.04 20.31 ± 0.81 18.70 ± 1.37 19.59 ± 2.52 21.32 ± 1.09 20.5 ± 0.54 0.12 Ash 6.22 ± 0.17 6.25 ± 0.07 6.14 ± 0.24 6.22 ± 0.18 6.14 ± 0.04 6.44 ± 0.28 0.08

Values are means ± SD of three replications. Means in the same raw with different superscripts are significantly different (P b 0.05). content would result in a decrease of serum TP content of golden pom- or leading to illness. Superoxide dismutase was an important enzyme pano, a tendency of change consistent with PER. An increase of serum TP to eliminate reactive oxygen in animal body that can eliminate the content was in favor of animal's metabolic level and immunity, promot- superoxide anion free radical generated in the animal's oxidation ing synthesis of protein and increasing nitrogen deposition (Zhou et al., processes, an important part of animal body's antioxidant system (Hu 2009). In the experiment, serum TP content of golden pompano firstly and Niu, 2005), playing an important role in animal body protection. increased along with increase of dietary arginine levels, and then In this experiment, dietary arginine had no significant effect on the decreased past a certain point with increasing dietary arginine levels. antioxidant abilities of golden pompano. The results of this experiment This finding was consistent with the results of black sea bream were consistent with the results in turbot (S. maximus)(Li et al., 2008), (S. macrocephalus)(Zhou, 2011a,b). Cholesterol takes part in synthe- yellow grouper (E. awoara)(Zhou et al., 2012) and blunt snout bream sis of cell membranes, bile, vitamin D and hormone within animal (M. amblycephala)(Ren et al., 2013). However, other study on channel bodies, and can reflecttheinternalfatmetabolicstatustoacertain catfish (I. punctatus) indicated that dietary arginine could improve the extent. Similar to other vertebrates, fish can synthesize cholesterol, activity of hepatic SOD (Buentello et al., 2007). The reason of this and most of cholesterol in the blood is from the liver and the other difference required further studies. from the digestive tract, the concentration of cholesterol in the Arginine, the precursor for NO synthesis in animal bodies, was an blood would increase rapidly in poor liver cell function (Luo, important immunoregulation factor discovered in recent years. This 2005). The concentration of golden pompano serum cholesterol immunoregulation factor played an important part in regulation of was not affected by dietary arginine content. This is consistent animal body's immune system. NO regulates T lymphocyte proliferation with the results of black sea bream (S. macrocephalus)(Wang, and antibody immune response, enhanced the activity of natural killer 2012). Alanine aminotransferase and aspartate aminotransferase cells, and takes part in apoptotic response of macrophage (Ye and were two kinds of enzymes playing an important part in the metab- Tian, 2010). Buentello et al. (2007) found that after injecting LPS in olism of amino acid, and are important indices to evaluate the health enterocoelia, the production of peritoneal macrophage NO was of the liver (Regost et al., 1999). Alanine aminotransferase and as- increased along with an increase in dietary arginine levels in channel partate aminotransferase were found in small amounts in the catfish (I. punctatus). In this experiment, dietary arginine content had serum of healthy animals, but when animals' liver was damaged asignificant effect on the activities of serum and liver T-NOS of golden and hepatic cells are broken, alanine aminotransferase and aspartate pompano, similar to the result of yellow grouper (E. awoara)(Zhou aminotransferase in hepatic cells would be elevated in the blood et al., 2012) and blunt snout bream (M. amblycephala)(Ren et al., 2013). (Hemre et al., 1996; Sheikhzadeh et al., 2012). In this experiment, Lysozyme was also called muramidase, an effective antibacterial dietary arginine level had no significant effect on the activity of agent, which could destroy peptidoglycan support and cause bacterial ALTandASTofgoldenpompano,aninconsistentwiththeresultsof splitting under osmotic pressure within bacteria (Saurabh and Sahoo, black sea bream (S. macrocephalus)(Wang, 2012). So far, little stud- 2008). In the experiment, dietary arginine content had a significant ies on the effect of dietary arginine on serum biochemical indices effect on the activity of golden pompano serum and liver lysozyme had been conducted in fish. Some researchers held that the change activities, similar to the result in largemouth bass (M. salmoides) of fish serum biochemical indices might be caused by stress, or by (Zhou, 2011a), which indicated that adding a proper amount of arginine some change of amino acids in diet. in the diet could improve non-specificimmunityoffish. During normal metabolic process, animal bodies would generate Bacterial challenge tests have been used as a final indicator of fish reactive oxygen and the accumulation of large amounts of reactive disease resistance after nutrition experiments (Lim et al., 2009; oxygen resulted in oxidative damage, accelerating animal body's aging Waagbø et al., 1994; Yildirim-Aksoy et al., 2009). In an Edwardsiella

Table 5 Effects of dietary arginine level on serum biochemical indices of pompano (T. ovatus).

Diets (Arg %) Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6 P value

2.05 2.38 2.65 2.98 3.25 3.58

CHO/(m mol/L) 3.73 ± 0.30 3.09 ± 0.52 3.93 ± 0.03 3.57 ± 0.80 3.81 ± 0.63 3.96 ± 0.66 0.44 TG/(m mol/L) 0.83 ± 0.09 0.84 ± 0.12 1.10 ± 0.39 1.13 ± 0.38 0.94 ± 0.21 1.06 ± 0.29 0.65 ALT/(U/L) 5.00 ± 1.00 5.00 ± 1.00 5.33 ± 1.53 5.67 ± 1.53 5.00 ± 2.00 6.33 ± 1.53 0.85 AST/(U/L) 33.67 ± 4.04 37.67 ± 0.58 31.67 ± 4.16 35.33 ± 1.53 34.00 ± 4.00 36.33 ± 3.79 0.92 GLU/(m mol/L) 6.66 ± 1.74 7.16 ± 1.12 5.54 ± 0.68 7.69 ± 1.77 6.09 ± 1.36 7.47 ± 1.45 0.27 TP/(g/L) 21.20 ± 2.69ab 22.30 ± 1.06b 27.10 ± 0.28c 27.35 ± 1.06c 26.75 ± 0.07c 18.90 ± 0.00a 0.02

Values are means ± SD of three replications. Means in the same raw with different superscripts are signiﬁcantly different (P b 0.05). CHO: cholesterol; TG: triglyceride; ALT: alanine aminotransferase, AST: aspartate aminotransferase; GLU: glucose; TP: total protein. 388 H. Lin et al. / Aquaculture 437 (2015) 382–389

Table 6 Effect of dietary arginine level on serum and liver enzyme activities in pompano (T. ovatus).

Diets (Arg %) Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6 P value

2.05 2.38 2.65 2.98 3.25 3.58

Serum T-SOD (U/mL) 66.02 ± 9.45 70.30 ± 4.21 80.18 ± 4.35 72.66 ± 4.49 71.55 ± 12.61 67.81 ± 2.90 0.29 T-NOS (U/mL) 10.10 ± 2.03a 13.30 ± 1.15ab 12.40 ± 2.40ab 10.52 ± 3.27ab 17.83 ± 2.53c 16.77 ± 2.52bc 0.02 LYZ (U/mL) 91.10 ± 0.61a 91.69 ± 0.39a 98.62 ± 3.19b 96.50 ± 5.30ab 95.79 ± 3.36ab 96.72 ± 2.68ab 0.04

Liver T-SOD (U/mg protein) 124.41 ± 14.86 121.56 ± 18.92 131.07 ± 16.49 118.35 ± 1.56 115.45 ± 14.97 105.63 ± 0.88 0.35 T-NOS (U/mg protein) 10.17 ± 1.82c 4.37 ± 0.33a 4.51 ± 0.80a 6.70 ± 0.57b 8.29 ± 1.37bc 8.54 ± 0.95bc 0.03 LYZ (U/mg protein) 28.02 ± 0.71a 30.63 ± 2.21ab 34.55 ± 0.94cd 37.72 ± 2.06d 36.17 ± 1.61d 31.90 ± 2.30bc 0.01

Values are means ± SD of three replications. Means in the same raw with different superscripts are signiﬁcantly different (P b 0.05). T-SOD: total superoxide dismutase; T-NOS: total nitric oxide synthase; LYZ: lysozyme.

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