Dietary arginine affects growth performance, plasma amino acid contents and gene expressions of the TOR signaling pathway in juvenile blunt snout bream, Megalobrama amblycephala

Aquaculture 461 (2016) 1–8

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Dietary arginine affects growth performance, plasma amino acid contents and gene expressions of the TOR signaling pathway in juvenile blunt snout bream, Megalobrama amblycephala Hualiang Liang a,1, Mingchun Ren a,b,1, Habte-Michael Habte-Tsion a, Xianping Ge a,b,⁎, Jun Xie a,b, Haifeng Mi c, Bingwen Xi b, Linghong Miao a,b, Bo Liu a,b, Qunlan Zhou a,b, Wei Fang a a

Wuxi Fisheries College, Nanjing Agricultural University, Wuxi 214081, China Key Laboratory for Genetic Breeding of Aquatic Animals and Aquaculture Biology, Freshwater Fisheries Research Center (FFRC), Chinese Academy of Fishery Sciences (CAFS), Wuxi 214081, China c Tongwei Co. Ltd., Chengdu 610093, China b

a r t i c l e

i n f o

Article history: Received 6 February 2016 Received in revised form 12 April 2016 Accepted 13 April 2016 Available online 14 April 2016 Keywords: Blunt snout bream (Megalobrama amblycephala) Dietary arginine Growth performance Plasma amino acids TOR signaling pathway

a b s t r a c t An 8-week feeding trial was conducted to estimate the dietary arginine requirement and investigate the effects of dietary arginine levels on the growth performance, plasma essential amino acids contents and relative gene expressions of target of rapamycin (TOR) signaling pathway in juvenile blunt snout bream. Fish (initial weight: 20.0 ± 0.03 g) were randomly sorted into 18 cages (1 m × 1 m × 1 m) and each cage was stocked with 20 fish for farm pond culture. Six isonitrogenous and isoenergetic practical diets were formulated to contain graded arginine levels ranging from 0.87% to 2.70% of dry diet. At the end of the feeding trial, results showed that final weight (FW), weight gain (WG), specific growth rate (SGR) and protein efficiency ratio (PER) increased with increasing dietary arginine level up to 1.62%, and thereafter showed a decreasing trend, while feed conversion ratio (FCR) showed a converse trend. The hepatosomatic index (HSI) in the fish fed with 2.31 and 2.70% arginine diets were significantly higher than the other arginine diets. Viscerosomatic index (VSI) in the fish fed with 1.62 and 1.96% arginine diets were significantly lower than 0.87, 2.31 and 2.70% arginine diets. The highest whole body protein, lipid and lowest moisture contents were observed in fish fed with 1.96% dietary arginine compared to those fed with the other diets. Plasma arginine content increased with increasing dietary arginine level, whereas plasma lysine decreased with increasing dietary arginine level. Plasma histidine, isoleucine, valine, tryptophan and methionine contents were significantly affected by dietary arginine levels. Relative expression levels of TOR mRNA in the group fed with 1.96% dietary arginine was significantly higher compared to those fed with 0.87 and 1.22% arginine diets. Relative expression levels of insulin like growth factor-1 (IGF-1) mRNA in the groups fed with 1.62 and 1.96% dietary arginine were significantly higher than those fed with the other arginine diets. Significantly higher relative expression levels of ribosomal protein S6 kinase-polypeptide 1 (S6K1) mRNA were found in fish fed with 2.31 and 2.70% arginine diets. Based on FCR and SGR, the optimal dietary arginine level was determined to be 1.85% of diet (5.60% of dietary protein) and 1.84% of diet (5.58% of dietary protein), respectively, using quadratic regression analysis. Statement of relevance: The findings showed dietary arginine affected growth, plasma amino acid concentration, and gene expressions of TOR signaling pathway in juvenile blunt snout bream. The determination of dietary arginine requirement in practical diet would be useful in developing essential amino acids balanced commercial feed for blunt snout bream. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Arginine is an essential amino acid in all fish species studied so far (NRC, 2011) and the most versatile amino acid for fish. Arginine is ⁎ Corresponding author at: Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China. E-mail address: [email protected] (X. Ge). 1 These authors contributed equally to this study and share first authorship.

http://dx.doi.org/10.1016/j.aquaculture.2016.04.009 0044-8486/© 2016 Elsevier B.V. All rights reserved.

limited in some plant protein sources such as corn meal, sesame meal and zein or casein-based diets, and the demand for amino acids has to be met by exogenous supply (Mai et al., 1994; Wilson, 2002; Singh and Khan, 2007). Arginine supplementation has been shown to promote growth and feed utilization in several fish species, and the difference of species, the requirements of arginine vary from 1.0% to 3.1% diet, corresponding to 3.8% to 8.1% dietary protein (NRC, 2011). Arginine is not only involves in protein synthesis, but also participates in several metabolic pathways such as urea production, metabolism of glutamic acid and proline, and synthesis of creatine and polyamines (Luo et al.,

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H. Liang et al. / Aquaculture 461 (2016) 1–8

2004; Wu et al., 2009). In recent years, arginine supplementation has been shown to affect energy metabolism, increasing lipid oxidation and enhancing deposition of both lipids and proteins in the muscle (Pirinen et al., 2007; Jobgen et al., 2009b; Tan et al., 2009; Clemmensen et al., 2012; McKnight et al., 2010) in mammal, and stimulating growth and protein deposition in fish (Cheng et al., 2012; Pohlenz et al., 2013; Ren et al., 2013). Protein synthesis is a key component of the processes involved in growth response, and is limited by translation initiation (Anthony et al., 2001). TOR (Target of Rapamycin) is a highly conserved protein kinase that is well known to initiate translation and stimulate protein synthesis by ribosomal protein S6 kinase-polypeptide 1 (S6K1) and the eukaryotic translation initiation factor 4E-binding proteins (4E-BPs) (Dennis et al., 2001). The TOR signaling pathway has become a hotspot in research in mammalian, and dietary arginine supplementation has been proven to increase TOR signaling activity (Hidetoshiban et al., 2004; Holz et al., 2005). This pathway has been described as an integration point of several inputs that modulate protein metabolism and growth, processes that involves signaling from nutrients such as amino acids, growth factors and energy status, and TOR protein was the central in TOR signaling pathway (Lansard et al., 2009; Belghit et al., 2013; Dai et al., 2014; Chen et al., 2012a). Arginine regulated TOR signaling pathway in Gibel carp (Carassis auratus gibelio) (Tu et al., 2015) and Jian carp (Cyprinus carpio var. Jian), (Chen et al., 2012a, 2012b). However, Yao et al. (2008) reported that dietary arginine supplementation did not affect TOR signaling activity in neonatal pigs. Thus, the mechanisms of the TOR signaling pathway are still not clear and need to be further investigated. Blunt snout bream, Megalobrama amblycephala, is a commercially important freshwater fish species in China with a long history of cultivation, because of its excellent flesh quality, rapid growth performance and high larval survival rate (Zhou et al., 2008; Ren et al., 2013). Furthermore, it is also distributed in North America (northern Canada to southern Mexico), Africa and Eurasia (Li et al., 2010). Commercial production of this fish species has been rapidly increased and reached approximately 0.70 million tons in 2012 (Ministry of Agriculture of the People's Republic of China's, 2013). In the last decade, there are reports available concerning the basic nutrient requirements of this species (Habte-Tsion et al., 2013; Li et al., 2010). In recent years, our research team has investigated dietary essential amino acid (EAAs) requirements of blunt snout bream including arginine (Ren et al., 2013), lysine (Liao et al., 2013), arginine (Liao et al., 2014), leucine (Ren et al., 2015a, 2015b, 2015c), valine (Ren et al., 2015a, 2015b, 2015c), isoleucine (Ren et al., 2015a, 2015b, 2015c), and threonine (Habte-Tsion et al., 2015a). In our previous study, arginine requirement of juvenile blunt snout bream in semi-single diet has been determined (2.26%, 2.28% and 2.46% of diet, Ren et al., 2013). However, dietary arginine requirement determination may be affected by dietary protein sources, because amino acid (AA) digestibility of the different feedstuffs was different. This raises a question whether previous recommended dietary arginine level is effective in maximizing performance of fish fed with commercial diet. Furthermore, there is no available knowledge concerning dietary arginine supplementation affects TOR signaling pathway in this fish species. Therefore, the aim of this study was to determine the dietary arginine requirement in practical diets, and to investigate the effects of dietary arginine levels on relative gene expression of TOR signaling pathway. 2. Materials and methods 2.1. Diet preparation Six isonitrogenous and isoenergetic practical diets (33.0% crude protein, 7.0% crude lipid) were formulated to contain graded arginine levels (0.87 (control), 1.22, 1.62, 1.96, 2.31 and 2.70% of dry diet). Dietary protein was supplied by fish meal, rapeseed meal and corn gluten, soybean

oil and lecithin as lipid sources (Table 1). A mixture of crystalline amino acids was supplemented to simulate the whole body amino acid pattern of blunt snout bream excepted for arginine (L-arginine 99%; Shanghai Feeer Technology Development Co. Ltd. China); dietary arginine was replaced by equal proportions of glycine (Table 2). Ingredients were ground into powder through 60 mesh sieve and mixed uniformly with soybean oil, lecithin and water to make sinking pellet feed. The pellet feed was forced though a pelletizer (F-26 (II), South China University of Technology, China), which were then dried at 45 °C overnight and then stored at −20 °C for further use. 2.2. Experimental procedure Juvenile blunt snout bream were obtained from the breeding farm of Freshwater Fisheries Research Centre (FFRC) of Chinese Academy of Fishery Sciences. Prior to the feeding trial, the juvenile blunt snout bream were selected for health and similar sizes, reared in cages (1 m × 1 m × 1 m), then fed with a commercial diet containing 33% protein and 7% lipid (Wuxi Tongwei feedstuffs Co. Ltd., Wuxi China) for two weeks to acclimatize with the experimental diet and conditions. During the acclimation period, fish were hand-fed three times daily at 8:00, 12:00 and 16:00 until apparent satiation on the basis of visual observation of fish feeding behavior. At the initiation of the experiment, fish were fasted for 24 h and weighed. The juvenile blunt snout bream (20.0 ± 0.03 g, 11.12 ± 0.05 cm) were randomly sorted into eighteen Table 1 Formulation and proximate composition of the experimental diets (% dry matter). Ingredients

Fish meala Rapeseed meala Corn starch Corn glutena Soybean oil Soybean lecithin Amino acid mixb Choline chloride Wheat meala Vitamin and mineral premixc Monocalcium phosphate Vitamin C Microcrystalline cellulose Ethoxy quinoline Glycine L-Arginine Bentonite Proximate analysis (% dry basis) L-Arginine Crude protein Crude lipid Ash

Diet number 1

2

3

4

5

6

5.00 5.00 12.10 22.00 3.00 2.00 9.24 0.10 22.00 1.50 3.00 0.05 10.00 0.01 2.00 0.00

5.00 5.00 12.10 22.00 3.00 2.00 9.24 0.10 22.00 1.50 3.00 0.05 10.00 0.01 1.60 0.40

5.00 5.00 12.10 22.00 3.00 2.00 9.24 0.10 22.00 1.50 3.00 0.05 10.00 0.01 1.20 0.80

5.00 5.00 12.10 22.00 3.00 2.00 9.24 0.10 22.00 1.50 3.00 0.05 10.00 0.01 0.80 1.20

5.00 5.00 12.10 22.00 3.00 2.00 9.24 0.10 22.00 1.50 3.00 0.05 10.00 0.01 0.40 1.60

5.00 5.00 12.10 22.00 3.00 2.00 9.24 0.10 22.00 1.50 3.00 0.05 10.00 0.01 0.00 2.00

3.00

3.00

3.00

3.00

3.00

3.00

0.87 33.64 7.73 5.01

1.22 33.68 7.62 5.02

1.62 33.84 7.61 5.05

1.96 33.34 7.27 5.01

2.31 34.76 7.28 5.1

2.70 34.59 7.23 5.08

a Rapeseed meal obtained from Wuxi Tongwei feedstuffs Co., Ltd, Wuxi, China, crude protein 37.5%, crude lipid 1.4%; corn gluten, obtained from Wuxi Tongwei feedstuffs Co., Ltd, Wuxi, China, crude protein 55.9%, crude lipid 3.3%; fish meal, obtained from Wuxi Tongwei feedstuffs Co., Ltd, Wuxi, China, crude protein 61.4%, crude lipid 9.3%; wheat meal obtained from Wuxi Tongwei feedstuffs Co., Ltd, Wuxi, China, crude protein 11.8%, crude lipid 1.2%. b Amino acid premix (g/100 g diet): L-histidine, 0.22; L-isoleucine, 0.50; L-lysine, 1.57; Lphenylalanine, 0.2; L-threonine, 0.53; L-valine, 0.44; L-aspartic acid, 1.18; serine, 0.31; glycine, 1.55; alanine; 0.39; L-tyrosine,0.07; tryptophan, 0.12; glumatic acid, 0.14; proline, 0.10. Amino acids obtained from Feeer Co., LTD (Shanghai, China). c Vitamin and mineral mix (IU or mg/kg of diet): Vitamin A, 900,000 IU; Vitamin D, 250,000 IU; Vitamin E, 4500 mg; Vitamin K 3, 220 mg; Vitamin B 1, 320 mg; Vitamin B 2, 1090 mg; Vitamin B 5, 2000 mg; Vitamin B 6, 5000 mg; Vitamin B 12, 116 mg; pantothenate, 1000 mg; folic acid, 165 mg; choline, 60,000 mg; biotin, 50 mg; niacin acid, 2500 mg; provided by Tongwei Feed Group Co. (Jiangsu, China). Supplied as L-form (99%, Shanghai Feeer Technology Development Co. Ltd., Shanghai, China). Mineral mix (g/kg of diet): calcium biphosphate, 20 g; sodium chloride, 2.6; potassium chloride, 5 g; magnesium sulfate, 2 g; ferrous sulfate, 0.9 g; zinc sulfate, 0.06 g; cupric sulfate, 0.02; manganese sulfate, 0.03 g; sodium selenate, 0.02 g; cobalt chloride, 0.05 g; potassium iodide, 0.004; and zeolite was used as a carrier.

H. Liang et al. / Aquaculture 461 (2016) 1–8 Table 2 Amino acid composition of ingredients (g 100 g−1 dry matter). Amino acid

EAA Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine Tryptophan NEAA Aspartic acid Serine Glycine Alanine Cystine Tyrosine Glutamic acid Proline

Amount in 5g FM

5g RM

22 g CG

22 g WM

APP

Total 33% whole body protein

0.16 0.07 0.13 0.21 0.22 0.08 0.12 0.12 0.15 0.03

0.11 0.05 0.08 0.13 0.1 0.04 0.07 0.08 0.1 0.03

0.41 0.27 0.54 2.21 0.2 0.35 0.83 0.45 0.6 0.07

0.19 0.09 0.11 0.2 0.13 0.04 0.12 0.1 0.15 0.06

⁎ 0.26 0.60 0.00 1.71 0.37 0.29 0.62 0.53 0.14

0.87 0.48 0.85 2.75 0.65 0.51 1.14 0.75 1.00 0.18

0.27 0.11 0.18 0.18 0.02 0.1 0.39 0.11

0.13 0.08 0.09 0.08 0.04 0.04 0.33 0.11

0.8 0.69 0.35 1.19 0.23 0.68 2.95 1.21

0.2 0.12 0.11 0.11 0.07 0.09 0.42 0.22

1.35 0.39 1.69 0.52 0.00 0.13 0.41 0.21

1.41 1.01 0.73 1.56 0.37 0.91 4.09 1.65

1.95 0.74 1.45 2.33 2.36 0.88 1.43 1.37 1.53 0.32 0.00 2.76 1.40 2.42 2.08 0.19 1.04 4.50 1.86

FM, fish meal; RM, rapeseed meal; CG, corn gluten; WM, wheat meal; AAP, crystalline amino acid premix; EAA, essential amino acid; NEAA, non-essential amino acid. Tryptophan could not be detected after acid hydrolysis.

cages with 20 fish in each cage for farm pond culture. Each diet was randomly assigned to triplicate cages for 8 weeks. Fish were hand-fed three times daily at 8:00, 12:00 and 16:00 until apparent satiation on the basis of visual observation of fish feeding behavior. During the experimental period, water temperature ranged from 26 to 28 °C, pH from 7.3 to 7.8, dissolved oxygen from 6.0 to 7.5 mg/L, ammonia nitrogen from 0.005 to 0.009 mg/L and hydrogen sulfide from 0.005 to 0.008 mg/L. 2.3. Sample collection and chemical analysis At the end of the feeding trial, six experimental blunt snout bream from each cage were collected, anesthetized with 100 mg/L MS-222, weighed, and then blood samples were collected immediately from the caudal vein with disposable medical syringes. At the same time, liver samples were collected from the sampled fish. Plasma was separated by centrifugation (3500 ×g, 10 min, 4 °C). Plasma and liver samples were stored at − 80 °C until analysis. Another five fish per cage were sampled and stored at −20 °C for body composition analysis. Individual body weight, body length, liver weight and visceral weight were recorded to calculate condition factor, hepatosomatic index and viscerosomatic index. Moisture, crude protein, crude lipid and ash contents were analyzed for diet and fish whole body according to the established methods of AOAC (2003): dry matter after drying in an oven at 105 °C until constant weight; crude protein (N × 6.25) by Kjeldahl method after acid digestion; lipid by ether extraction using Soxhlet; ash by combustion at 550 °C for 5 h. Duplicate analyses were conducted for each sample. According to the method described by Ren et al. (2013), amino acid concentrations were determined by a professional laboratory (Evonik Degussa Co., Ltd., China). The diet and ingredients were freeze-dried overnight, and then hydrolyzed for 24 h in 6 N HCl at 110 °C for total amino acid contents analysis. The plasma samples were deproteinized by trichloroacetic acid (5%). After pretreatment, all the samples were carried out on Agilent-1100 amino acid analyzer (Agilent Technologies Co., Ltd., Santa Clara, USA). Tryptophan could not be detected after acid hydrolysis. Relative gene expressions of TOR pathway was determined using Real-time PCR analysis as described in our previous study (HabteTsion et al., 2015b). Briefly, total RNA was extracted from the liver of juvenile blunt snout bream using an RNAiso plus kit (Takara, Dalian,

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China). After quality and quantity of RNA were checked, complementary DNA (cDNA) was synthesized using a PrimeScript TM RT reagent kit (Takara, Dalian, China). Specific primers for TOR, β-actin, 4E-BP2, IGF-1 genes (primer sequences of TOR: 5′-TTTACACGAGCAAGTCTACG GA-3′ and 5′-CTTCATCTTGGCTCAGCTCTCT-3′; primer sequences of βactin: 5′-TCGTCCACCGCAAATGCTTCTA-3′ and 5′-CCGTCACCTTCACCGT TCCAGT-3′; primer sequences of 4E-BP2: 5′-ATGTCGTCCAGTCGTCAG TTT-3′ and 5′-AGGAGTGGTGCAATAGTCGTG-3′; primer sequences of IGF-1: 5′-CTCACTGGTGCTGTTCGTCCTC-3′ and 5′-TGAAAGCAGCATTC GTCCACA-3′; primer sequences of S6K1: 5′-GGTGCATGTCACCTTATG GG-3′ and 5′-AGCTGGCAGCACTTCTAGTC-3′) were designed according to the partial cDNA sequences of the TOR genes using the M. amblycephala transcriptome analysis (Gao et al., 2012; Habte-Tsion et al., 2015b). β-Actin was employed as a nonregulated reference gene, as previously used in blunt snout bream studies (Habte-Tsion et al., 2015b; Ren et al., 2015a, 2015b, 2015c). No changes in β-actin gene expression were observed in our investigations. Relative quantification of target gene expression was performed using the Pfaffl's mathematical model (Pfaffl, 2001).

2.4. Statistics analysis Parameters were calculated as follows: Specific growth rate (SGR) (%/d) = 100 × [(ln(final body weight (g)) − ln(initial body weight (g))) / days] Feed conversion ratio (FCR) = dry feed fed (g) / wet weight gain (g) Weight gain (WG) (%) = 100 × (final weight (g) − initial weight (g)) / initial weight (g) Condition factor (CF) (%) = 100 × fish weight (g) / [body length (cm)]3 Hepatosomatic index (HSI) (%) = 100 × (liver weight (g) / body weight (g)) Viscerosomatic index (VSI) (%) = 100 × (visceral weight (g) / body weight (g)) Protein efficiency ratio (PER) = wet weight gain / protein intake. All data were subjected to one-way analysis of variance (ANOVA) using the software of the SPSS 16.0 for Windows. Significant differences between means were evaluated by Turkey's Multiple Range Test. Probabilities of P b 0.05 were considered significant. Data are expressed as means with SEM. The quadratic regression model was used to estimate the optimum dietary arginine requirement on the basis of SGR and FCR.

3. Results 3.1. Growth performance Results of growth performance of the juvenile blunt snout bream are showed in Table 3. Final weight (FW), weight gain (WG), specific growth rate (SGR) and protein efficiency ratio (PER) increased with increasing dietary arginine level up to 1.62% dietary arginine, and thereafter showed a decreasing trend. The HSI in the fish fed with 2.31 and 2.70% arginine diets were significantly higher than those fed with the other arginine diets (P b 0.05). Dietary arginine levels significantly affected the VSI in the groups fed with 1.62 and 1.96% arginine diets compared with 0.87, 2.31 and 2.70% arginine diets (P b 0.05), but CF was not significantly affected by the graded dietary arginine levels (P N 0.05). Based on FCR and SGR, the optimal dietary arginine level for juvenile blunt snout bream was determined to be 1.85% of diet (5.60% of dietary protein) (Fig. 1) and 1.84% of diet (5.58% of dietary protein) (Fig. 2), respectively, using quadratic regression analysis.

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H. Liang et al. / Aquaculture 461 (2016) 1–8

Table 3 Growth performance and morphological index of juvenile blunt snout bream fed experimental diets for 8 weeks (means ± S.E.M.)1. Arginine % diet

Initial weight (g)

Final weight (g)

WG (%)2

FCR3

SGR (% day−1)4

HSI (%)5

VSI (%)6

CF (%)7

PER8

0.87 1.22 1.62 1.96 2.31 2.70

20.00 ± 0.05 20.08 ± 0.03 19.97 ± 0.02 20.02 ± 0.06 19.96 ± 0.05 20.02 ± 0.02

64.33 ± 1.33a 82.92 ± 4.11bc 88.62 ± 4.52c 85.55 ± 2.49c 77.82 ± 1.17bc 70.57 ± 0.02ab

231.6 ± 8.25a 321.5 ± 22.84bc 358.1 ± 14.48c 342.2 ± 4.01c 306.3 ± 7.56bc 272.6 ± 7.97ab

1.41 ± 0.04c 1.21 ± 0.09abc 1.03 ± 0.04a 1.11 ± 0.01ab 1.21 ± 0.03abc 1.31 ± 0.02bc

2.14 ± 0.04a 2.56 ± 0.10bc 2.72 ± 0.05c 2.65 ± 0.02c 2.50 ± 0.03bc 2.35 ± 0.04ab

1.13 ± 0.11a 1.01 ± 0.06a 1.05 ± 0.22a 1.08 ± 0.05a 1.70 ± 0.15b 1.74 ± 0.09b

11.48 ± 1.25b 9.15 ± 0.71ab 6.80 ± 0.58a 6.58 ± 0.54a 12.55 ± 1.20b 11.78 ± 1.33b

2.10 ± 0.04 2.16 ± 0.04 2.18 ± 0.04 2.19 ± 0.05 2.18 ± 0.04 2.20 ± 0.03

2.12 ± 0.07a 2.49 ± 0.19abc 2.89 ± 0.10c 2.71 ± 0.03bc 2.38 ± 0.06ab 2.20 ± 0.03a

1 2 3 4 5 6 7 8

Data are means of triplicate, value in the same column with different superscripts are significantly different (P b 0.05). Weight gain (WG) (%) = 100 × (final weight (g) − initial weight (g)) / initial weight (g). Feed conversion ratio (FCR) = dry feed fed (g) / wet weight gain (g). Specific growth rate (SGR) (%/d) = 100 × [(ln(final body weight (g)) − ln(initial body weight (g))) / days]. Hepatosomatic index (HSI) (%) = 100 × (liver weight (g) / body weight (g)). Viscerosomatic index (VSI) (%) = 100 × (visceral weight (g) / body weight (g)). Condition factor (CF) (%) = 100 × fish weight (g) / [body length (cm)]3. PER: protein efficiency ratio = wet weight gain / protein intake.

3.2. Whole body composition Whole body compositions are presented in Table 4. Significantly higher whole body protein contents and significantly lower moisture contents of fish fed with 1.96% dietary arginine compared to those fed with the other diets (P b 0.05). Fish fed with 1.62% and 1.96% arginine diets showed significantly higher body lipid compared to those fed with 0.87, 2.31 and 2.70% arginine diets (P b 0.05). Whole body ash of juvenile blunt snout bream were not significantly affected by the graded dietary arginine levels (P N 0.05). 3.3. Plasma amino acid Plasma essential amino acids (EAAs) concentration of blunt snout bream are shown in Table 5. Plasma arginine concentration increased with increasing dietary arginine level. Plasma lysine content decreased with increasing dietary arginine level. Plasma threonine and leucine content showed relatively stable level. Plasma histidine, isoleucine, valine, tryptophan and methionine contents were significantly affected by dietary arginine levels (P b 0.05) (Table 5). 3.4. Relative expressions of TOR in the liver As presented in Fig. 3, the relative expression levels of TOR mRNA in the liver of fish fed with 1.96% arginine diet was significantly higher than those fed with 0.87 and 1.22% arginine diets. Relative expression levels of IGF-1 mRNA in the liver of fish fed with 1.62 and 1.96% arginine diets were significantly higher compared to other arginine diets

Fig. 1. Quadratic regression analysis of feed conversion ratio (FCR) against varying levels of dietary arginine.

(P b 0.05). Significantly higher relative expression levels of S6K1 mRNA in liver was observed in fish fed with 2.31 and 2.70% arginine diets (P b 0.05). Dietary arginine level didn't affect significantly to the relative expression levels of 4E-BP2 mRNA in liver (P N 0.05). 4. Discussion In the present study, fish fed with the arginine deficient diet (0.87%) showed poor growth performance and feed utilization, while these values were improved with dietary L-arginine supplementation, which indicated that arginine is an essential amino acid for blunt snout bream, and fish were able to utilize the L-arginine. Based on FCR and SGR, dietary arginine requirement for juvenile blunt snout bream were determined to be 1.85% and 1.84% of the diet (5.60% and 5.58% of dietary protein), which was similar to the requirements reported for Chinook salmon (Oncorhynchus tshawytscha) (6.0% of dietary protein, Klein and Halver, 1970) and coho salmon (Oncorhynchus kisutch) (5.8% of dietary protein, Klein and Halver, 1970), but lower than the reports in Asian sea bass (Lates calcarifer) (3.8% of dietary protein, MurilloGurrea et al., 2001) and Nile tilapia (Oreochromis niloticus) (3.4% of dietary protein, Santiago and Lovell, 1988), higher than the reports in silver perch (Bidyanus bidyanus) (6.8% of dietary protein, Ngamsnae et al., 1999) and yellow grouper (Epinephelus awoara) (6.5% of dietary protein, Zhou et al., 2012b). In the present study, dietary arginine requirement for juvenile blunt snout bream was also lower than our previous results (7.23% of dietary protein; 6.71% of dietary protein or 6.65% of

Fig. 2. Quadratic regression analysis of specific growth rate (SGR, % day−1) against varying levels of dietary arginine.

H. Liang et al. / Aquaculture 461 (2016) 1–8 Table 4 Effect of dietary arginine levels on the body composition of juvenile blunt snout bream fed experimental diets for 8 weeks (means ± S.E.M.)1. Arginine % diet

Moisture (% )

Crude protein (% w.w.)

Crude lipid (% w.w.)

Ash (% w.w.)

0.87 1.22 1.62 1.96 2.31 2.70

70.66 ± 0.81b 70.16 ± 0.18b 70.14 ± 0.54b 67.73 ± 0.59a 71.37 ± 0.40b 71.99 ± 0.44b

17.43 ± 0.54a 17.17 ± 0.34a 17.11 ± 0.30a 18.82 ± 0.19b 17.21 ± 0.37a 17.44 ± 0.39a

8.98 ± 0.63a 9.90 ± 0.13ab 10.26 ± 0.27b 10.45 ± 0.17b 9.00 ± 0.15a 8.56 ± 0.29a

3.51 ± 0.19 3.20 ± 0.14 3.28 ± 0.09 3.14 ± 0.18 3.36 ± 0.12 3.48 ± 0.32

w.w., wet weight. 1 Data are means of triplicate, value in the same column with different superscripts are significantly different (P b 0.05).

dietary protein, Ren et al., 2013). Compared with our previous study in semi-single diet, different intact protein sources used in practical diets in this experiment, which might affect the dietary arginine requirement determination. Furthermore, compared with previous work, present work was different in fish size (20.0 g vs 2.68 g), which might affect the dietary arginine requirement determination, in addition, the variation is also possibly affected by experimental conditions (Kim et al., 1992; Luo et al., 2004). In the present study, excess dietary arginine showed a negative effect on growth and feed utilization. Similarly, Zhou et al. (2012b) reported the same phenomenon in yellow grouper, and Chen et al. (2012a, 2012b) reported that excess dietary arginine has a negative effect on growth and feed utilization in Jian carp (Cyprinus carpio var. Jian). This could be due to unbalanced dietary amino acids profile and extra energy expenditure towards deamination or antagonism between arginine and lysine (Kaushik and Fauconneau, 1984; Kaushik et al., 1988). In the present study, plasma arginine concentration increased with increasing dietary arginine level, which was similar to our previous research (Ren et al., 2013). Berge et al. (1997) and Yamamoto et al. (2000) reported similar results in Atlantic salmon (Salmo salar) and fingerling rainbow trout. In this study, plasma lysine decreased with increasing dietary arginine level by increasing amino acid degradation through interference with their normal intermediate metabolism (Luo et al., 2004), which was the same with our previous study (Ren et al., 2013), which confirmed that excess dietary arginine caused antagonism between lysine and arginine in juvenile blunt bream snout. Similar to this study, with increased levels of dietary arginine, uptake of lysine was reduced in rainbow trout and Atlantic salmon (Berge et al., 1999; Kaushik et al., 1988). In contrast, Tibaldi et al. (1994); Alam et al. (2002) and Tu et al. (2015) reported that lysine-arginine didn't have antagonism in European sea bass, Japanese flounder (Paralichthys olivaceus) and Gibel carp (Carassius auratus gibelio). In this study, in 1.96% dietary arginine diet, whole body protein of juvenile blunt snout bream was significantly higher than other dietary arginine groups, which is in agreement with the reports in several other fish species (Cheng et al., 2012; Pohlenz et al., 2013). The whole body protein content of juvenile blunt snout bream showed a similar trend with SGR and an opposite trend with FCR, which indicated that the

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optimal dietary arginine supplementation improved the protein synthesis of juvenile blunt snout bream. Insulin-like growth factor 1 (IGF-1), also called somatomedin C, and is a hormone similar in molecular structure to insulin. IGF-1 plays an important role in growth and anabolic effects (Keating, 2008). Wullschleger et al. (2006) reported that growth factors like IGFS via the PI3K pathway could regulate cell growth. Dyer et al. (2004) and Li et al. (2006) found that IGF-1 level was very effective in the evaluation of fish growth rate and the response about alterative feed nutrition composition. Some researchers have reported that IGF-1 could promote the growth in coho salmon (McCormick et al., 1992; Duan et al., 1995), tilapia (Oreochromis mossambicus) (Chen et al., 2000). In the present study, optimal dietary arginine levels significantly affected the gene expression of IGF-1, which was similar trend with growth performance. The result revealed that optional dietary arginine levels could activate IGF-1 to promote the growth of juvenile blunt snout bream. Furthermore, growth factors such as insulin and IGFs via the PI3K pathway regulate cell growth (Wullschleger et al., 2006). Tu et al. (2015) has reported that IGF-1 perhaps activated TOR signaling pathway in dietary arginine supplementation. In previous studies in Atlantic salmon myocytes and rainbow trout hepatocytes, IGF-1 levels slightly increased TOR signaling but when combined with amino acids caused a greater stimulation (García de la serrana and Johnston, 2013; Lansard et al., 2010). In this study, relative expression levels of IGF-1 mRNA showed a similar trend with relative expression levels of TOR. The result indicated that IGF-1 could activate TOR signaling pathway with optional dietary arginine levels. But, the mechanism of improved TOR signaling pathway by IGF-1 is complex and the literature is limited. In the present study, excess dietary arginine showed a negative effect on the expression of IGF-1 level. The possible explanation is that excess dietary arginine led to insulin resistance through negative feedback system caused serine/threonine phosphorylation as in higher animals (Harrington et al., 2004; Um et al., 2006; Klebs, 2005; Krebs et al., 2003), which might affect the expression of IGF-1 level. Furthermore, in this study, we confirmed that excess dietary arginine caused antagonism between lysine and arginine in juvenile blunt bream snout. The deficiency of dietary lysine might also affect the expression of IGF-1 level. However, a mechanism of that arginine affect IGF-1 is unclear, and need to be further investigated. Protein synthesis is a key component of the processes involved in growth response (Anthony et al., 2001). However, protein synthesis is limited by translation initiation. Recent studies indicated that the TOR pathway, as an amino acid-sensing mechanism, play a crucial role in translation and protein synthesis process (Wullschleger et al., 2006). Meanwhile, amino acids are important precursors that stimulate protein synthesis mainly activating the target of rapamycin (TOR) nutrient-sensitive signaling pathway (Kim, 2009; Meijer, 2003). In mammalian, some reports showed that dietary arginine supplementation can increase TOR signaling activity (Hidetoshiban et al., 2004; Holz et al., 2005). However, Yao et al. (2008) has reported that dietary arginine supplementation didn't affect TOR signaling activity in liver. In rainbow trout, it has been discovered that protein accumulation

Table 5 Effect of dietary arginine levels on the plasma essential amino acids concentration of juvenile blunt snout bream fed experimental diets for 8 weeks (means ± S.E.M.)1. Arginine % diet

0.87 1.22 1.62 1.96 2.31 2.70 1

Essential amino acids (EAAs) (mg/L) Methionine

Lysine

Threonine

Arginine

Isoleucine

Leucine

Valine

Histidine

Tryptophan

8.72 ± 1.12a 11.73 ± 1.13ab 13.21 ± 2.10ab 14.95 ± 1.11ab 16.27 ± 2.60ab 17.63 ± 1.71b

56.07 ± 1.12b 53.81 ± 0.47b 49.14 ± 0.56ab 46.47 ± 2.96ab 42.91 ± 4.57a 41.63 ± 1.13a

49.33 ± 5.12 47.23 ± 9.83 50.64 ± 5.94 40.62 ± 3.57 44.84 ± 1.05 45.87 ± 4.26

2.90 ± 0.74a 5.43 ± 0.73a 17.11 ± 2.05ab 34.31 ± 5.71bc 38.53 ± 3.07c 40.72 ± 3.10c

6.30 ± 1.20a 7.01 ± 0.62a 5.23 ± 1.31a 6.35 ± 1.23a 10.93 ± 3.11ab 16.13 ± 2.63b

23.11 ± 6.53 25.33 ± 4.92 23.41 ± 2.53 27.53 ± 1.62 27.81 ± 5.00 37.53 ± 2.27

14.91 ± 1.82ab 15.02 ± 1.13ab 12.23 ± 1.34a 13.94 ± 2.04a 20.05 ± 4.53ab 27.36 ± 3.77b

36.91 ± 9.01a 43.31 ± 5.63ab 58.56 ± 3.06ab 58.63 ± 2.40ab 56.32 ± 3.76ab 63.50 ± 6.20b

12.51 ± 1.90a 14.22 ± 1.43ab 16.31 ± 1.24ab 20.2 ± 1.56ab 23.13 ± 3.21b 23.14 ± 1.85b

Data are means of triplicate, value in the same column with different superscripts are significantly different (P b 0.05).

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Fig. 3. Relative expression of TOR (A), 4E-BP2 (B), S6K1 (C) and IGF-I (D) genes of blunt snout bream fed diets with different arginine levels. Data are expressed as means with S.E.M., value with different superscripts are significantly different (P b 0.05).

through stimulation of amino acid sensitive TOR signaling pathway (Seiliez et al., 2008). It has been discovered that fish accumulate protein through a stimulation of amino acid sensitive TOR signaling pathway (Seiliez et al., 2008; Lansard et al., 2009, 2010, 2011). Furthermore, Chen et al. (2012a, 2012b) and Tu et al. (2015) reported that arginine might decrease the inhibition of translation and increase TOR activity, thus improving the synthesis of proteins in Jian carp and Gibel carp. Nevertheless, a different pattern of TOR regulation was reported by Wullschleger et al. (2006), when insulin and amino acids were added together. TOR signaling pathway was also activated by combination of insulin and amino acids and not by amino acids alone in rainbow trout (Lansard et al., 2010). In the present study, relative expression of TOR mRNA was significantly affected by dietary arginine levels, which showed a similar trend with whole body protein content in juvenile blunt snout bream. The relationship indicated that the balanced dietary arginine could activate TOR signaling pathway, which could promote protein synthesis in this fish. This study indicated that, IGF-1 could activate TOR signaling pathway with optional dietary arginine levels. Nevertheless, the underlying mechanisms by which arginine activated TOR signaling pathway are still not clear in fish and need further investigation. TOR promotes cap dependent translation initiation through the phosphorylation and inactivation of its downstream effector 4E-BPs or increases translation of 5′ TOP mRNAs via phosphorylation of S6K1 (Wullschleger et al., 2006). In the present study, dietary arginine level affected significantly the expressions of S6K1. Relative S6K1 mRNA level in the liver of juvenile blunt snout bream increased with increasing dietary arginine levels, which is similar to the result reported for juvenile Gibel carp (Tu et al., 2015). Increasing evidence has emerged in recent years to show that amino acids may interfere with insulin function (Tremblay et al., 2007b). In this study, we also found that S6K1 could over express when high dietary arginine levels, which might affect

glucose metabolism in juvenile blunt snout bream. It is well-known that morbidly obese patients are often affected by increased insulin resistance, resulting in the development of type 2 diabetes, because excess levels of nutrients and high concentrations of amino acids in body, S6K1 can be activated for a long time, through negative feedback system caused serine/threonine phosphorylation and then led to insulin resistance (Harrington et al., 2004; Um et al., 2006; Klebs, 2005; Krebs et al., 2003). To our knowledge, there are few reports to determine the effect of dietary arginine on mRNA expression of S6K1 and insulin resistance in fish. Further study is required to make clear the relationship between glucose metabolism and S6K1 in fish. Arginine supplementation didn't affect significantly the expressions of 4E-BP2. Similarly, Tu et al. (2015) reported that arginine supplementation had no effect on 4EBP2 expression in juvenile Gibel carp. In conclusion, results of the present investigation indicated that dietary arginine levels significantly influenced the growth performance, whole body composition (protein and lipid contents). Meanwhile, dietary arginine levels significantly influenced relative gene expressions of TOR, IGF-1 and S6K1, which could activate TOR pathway and promote TOR-related protein synthesis. The dietary arginine requirement of juvenile blunt snout bream was estimated to be 1.85% and 1.84% of diet (5.60% and 5.58% of dietary protein) based on FCR and SGR, which would be useful in developing essential amino acids balanced diet and promoting the development of commercial feed formula.

Acknowledgment This study was financially supported by the National Natural Science Foundation of China (31302199), the Modern Agriculture Industrial Technology System special project-the National Technology System for Conventional Freshwater Fish Industries (CARS-46), and the Special Fund for Agro-scientific Research in the Public Interest (20100302).

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