The optimal arginine requirement in diets for juvenile humpback grouper, Cromileptes altivelis

Journal Pre-proof The optimal arginine requirement in diets for juvenile humpback grouper, Cromileptes altivelis

Wei Mu, Xiao Wang, Xiaoyi Wu, Xiaojun Li, Yu Dong, Lina Geng, Lei Ma, Bo Ye PII:

S0044-8486(19)31500-5

DOI:

https://doi.org/10.1016/j.aquaculture.2019.734509

Article Number:

734509

Reference:

AQUA 734509

To appear in:

Aquaculture

Received Date:

12 June 2019

Accepted Date:

12 September 2019

Please cite this article as: Wei Mu, Xiao Wang, Xiaoyi Wu, Xiaojun Li, Yu Dong, Lina Geng, Lei Ma, Bo Ye, The optimal arginine requirement in diets for juvenile humpback grouper, Cromileptes altivelis, Aquaculture (0), https://doi.org/10.1016/j.aquaculture.2019.734509

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Journal Pre-proof The optimal arginine requirement in diets for juvenile humpback grouper, Cromileptes altivelis

Wei Mu, Xiao Wang, Xiaoyi Wu*, Xiaojun Li, Yu Dong, Lina Geng, Lei Ma, Bo Ye

State Key Laboratory of Marine Resource Utilization in South China Sea, Haikou 570228, China Hainan Provincial Key Laboratory for Tropical Hydrobiology and Biotechnology, Department of Aquaculture, Hainan University, Haikou, Hainan 570228, China

*Corresponding

author (Xiaoyi Wu): E-mail: [email protected]; Tel: +1186+898+66279184

1

Journal Pre-proof Abstract An 8-week feeding trial was conducted to evaluate the effects of dietary arginine (Arg) levels on growth performance, gut micromorphology, oxidation resistance and immune responses of humpback grouper, Cromileptes altivelis. Seven isoenergetic (340 kcal per 100g of dry matter), isoproteic (50% of dry matter) and isolipidic (10% of dry matter) experimental diets were formulated to contain 1.74%, 2.42%, 3.14%, 3.58%, 4.06%, 4.54% and 5.21% dietary Arg levels. Triplicate groups of 12 fish (average initial body weight: 6.55 ± 0.1g) were fed to apparent satiation by hand twice daily (08:00 h and 16:00 h) in an indoor recirculating seawater system which was consisted of 21 glass tanks (length 60 cm × width 45 cm × height 50 cm) and mechanical and biological water filters. After the feeding trial, all remaining fish were challenged by 2.5 mg Cu(II)·L-1, and survival rates were recorded for 12 h. Results showed that weight gain% (WG%), protein efficiency ratio (PER) as well as protein productive value (PPV) of fish were improved with the increments in dietary Arg from 1.74% to 3.58%, and thereafter, values of these parameters displayed a declining trend as dietary Arg level continued to rise from 3.58% to 5.21%. Fish fed 3.14% and 3.58% dietary Arg had lower FCR and daily feed intake (DFI) than fish fed other dietary Arg levels. Quadratic regression analysis of WG% against dietary Arg levels indicated that optimal dietary Arg level for maximum growth of humpback grouper was 3.39% of dry matter (6.78% of dietary protein). Hepatosomatic index (HSI) was reduced as dietary Arg level increased. Fish fed 3.58% dietary Arg had higher whole-body protein content than fish fed other dietary Arg levels. Expression of hepatic insulin-like growth factor-I (IGF-1), target of rapamycin (TOR), S6 kinase1 (S6K1) and hypothalamus growth hormone receptor (GHR) in fish fed 3.14% and 3.58% dietary Arg was higher than that in fish fed other dietary Arg levels. Gut micromorphology was significantly influenced by dietary Arg levels. After the exposure to 2.5mg Cu(II)·L-1 water for 12 h, fish fed 3.14% and 3.58% dietary Arg had higher survival ratios and expression of NF-E2-related factor 2 (Nrf2) and heat shock proteins genes 70 (HSP70) in head kiney. In serum, fish fed 3.58% dietary Arg had higher superoxide dismutase (SOD) and catalase (CAT) activities and immunoglobulinM (IgM) concentrations than fish fed other dietary Arg levels. In conclusion, the optimal dietary Arg requirement for maximal growth of humpback grouper was estimated to be 3.39% of dry matter (6.78% of dietary protein), and suitable dietary 2

Journal Pre-proof Arg supplementations improved growth performance, gut micromorphology, oxidation resistance as well as immunity of this species.

Keywords: Humpback grouper; Arginine; Growth; Gut micromorphology; Oxidation resistance; Immunity

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Journal Pre-proof 1. Introduction Arginine (Arg) is one of the essential amino acids in fish, and it takes part in multiple pathways of enormous nutritional and physiological importance, including the synthesis of protein, nitric oxide, creatine, proline, glutamate, polyamines, and agmatine (NRC, 2011; Yao et al., 2008; Wu et al., 1998). The role of Arg in protein synthesis is mainly through activation of the TOR signaling pathway and hence regulates fish protein synthesis and cell growth (Anthony et al., 2001; Wullschleger et al., 2005). Chen et al. (2011) and Wu et al. (2018) reported that expression of TOR and S6K1 in tissues and organs of fish was improved by the Arg supplementation to diets. Arg is also a potent secretagogue for insulin, growth hormone as well as IGF-1 (Newsholme et al., 2005), which can regulate the metabolism of glucose and amino acids in various tissues of animals, thus affecting animal growth (Wu et al., 2019). Furthermore, it was reported that the optimal amount of Arg in the diets can improve the fold height, enterocyte height and microvilli height of fish gut (Cheng et al., 2012; Rhoads et al., 2004; Wu et al., 2018). Besides the functions on fish growth performance and gut morphology, Arg also displays an important effect on the immunity and oxidation resistance. Arg supplementation can stimulate macrophage secretion of cytokines, increase cytotoxicity of macrophages and natural killer cells (Barbul, 1995; Choi et al., 2009) and enhance immune function in various immunological challenges (Field et al., 2000; Li et al., 2007). In addition, Arg serves as the sole substrate for synthesis of NO, which has many physiological functions including enhancement of macrophage cytotoxicity in channel catfish (Buentello et al., 1999; Buentello et al., 2001; Costas et al., 2011). Arg can also affect antioxidant enzyme (SOD, CAT) activities and IgM concentrations in serum of blunt snout bream (Liang et al., 2018a), yellow catfish (Zhou et al., 2015) and hybrid grouper (Wu et al., 2018). Wang et al. (2015) demonstrated that Arg supplementation blocked the apoptosis and antioxidant system and tight junction mRNA changes in the young grass carp tissues when fish was exposed to Cu stress. Dietary Arg requirement (of dietary protein) has been estimated for several fish species, such as hybrid grouper (6.64%-6.82%) (Wu et al., 2018), tilapia (4.84% and 6.24%) (Neu et al., 2016; Yue et al., 2013), black sea bream (7.74%) (Zhou et al., 2010), blunt snout bream (5.58%) (Liang et al., 2016), golden pompano (6.32%-6.35%) (Lin et al., 2015), yellow catfish (5.29%) (Zhou et 4

Journal Pre-proof al., 2015), channel catfish (3.3%-3.8%) (Buentello et al., 2000; Pohlenz et al., 2012a; Pohlenz et al., 2012b), hybrid striped bass (4.4%) (NRC, 2011) and red drum (4.1% and 4.2%) (Barziza et al., 2000; Cheng et al., 2011). These studies indicated that values of Arg requirement varied among different fish species, being from 3.0% to 8.1% of dietary protein. In recent years, the commercial farming scale of humpback grouper has gradually expanded, especially in south of China, Indonesia, Thailand and Malaysia (Williams et al., 2004). Although Arg has been demonstrated to be of importance for fish growth and health, information on Arg nutrition and immunity of this fish species is quite limited until now, so the aim of this study was to evaluate the effects of dietary Arg levels on growth performance, gut micromorphology, oxidation resistance and immunity of this fish species. 2. Materials and methods 2.1 Ingredients and experimental diets Seven isoenergetic (340 kcal per 100 g dry matter), isoproteic (50% of dry matter) and isolipidic (10% of dry matter) experimental diets were formulated to contain graded L-Arg levels, ranging from 1.74% to 5.21% (of dry matter) (Table 1). Since digestible energy coefficients for the ingredients used are not available for hybrid grouper, gross energy was calculated using physiological fuel values of 4.0, 4.0 and 9.0 kcal/g (16.7, 16.7 and 37.7 kJ / g) for carbohydrate, protein and lipid, respectively (Garling and Wilson, 1977; Lee and Putnam, 1973). The overall amino acid (AA) compositions except for Arg in diets were adjusted to the AA profile of whole-body protein of humpback grouper. A mixture of crystalline amino acids was supplemented to simulate the amino acid profile, leaving Arg out (Table 2), and equal proportions of L-alanine was used to substitute for Arg in the low Arg contained diets. Analyzed dietary Arg concentrations were 1.74%, 2.42%, 3.14%, 3.58%, 4.06%, 4.54% and 5.21% of dry matter, respectively (Table 3). All dry ingredients were carefully weighed and mixed in a Hobart mixer (A-200T MixerBench Model unit, Resell Food Equipment Ltd., Ottawa, Canada) for 30 minutes. CAAs were weighed individually for each of experimental diets. The CAA mixture (21.14 g) was pre-coated with 1.5 g carboxymethyl cellulose (CMC) and 15.51 g cellulose in water at 40°C to form a dough which subsequently was rubbed into powder by hands. The bound CAA was then 5

Journal Pre-proof mixed with other dry ingredients and recoated with 12.5 g corn starch and other 1.5 g CMC, and then lipid and water (30% - 50% dry ingredient mixture) were added gradually in sequence and mixed constantly. The diets were produced in a noodle-like shape of 3-mm in diameter using a twin-screw grinder (Institute of Chemical Engineering, South China University of Technology, Guangzhou, PR China). All diets were air-dried at about 21°C for 24 h, sieved, and then packaged and stored frozen (-20˚C). 2.2 Experimental conditions and procedure Humpback grouper juveniles were obtained from a commercial hatchery (Changjiang, Hainan, China). Before the growth trial, fish were acclimated to a commercial diet (50% crude protein, 10% crude lipid) for 3 weeks. Groups of 12 fish (average initial body weight of 6.55± 0.10 g) were stocked into glass tanks (length 60 cm × width 45 cm × height 50 cm) connected to mechanical and biological water filters as a recycling system. The water was oxygenated through air stones at the bottom of each tank, and all air stones were connected to an air-blower. Triplicate groups of fish were fed their prescribed diets to apparent satiation by hand twice daily (8:00 am, 16:30 pm). Feed intake was recorded every week and experimental aquaria were cleaned once a week. Water temperature (27-28°C), total ammonia (0-0.20 mg/L) and dissolved oxygen (5.8-6.2 mg/L) were monitored daily. Fish were exposed to a 12 h: 12 h light: dark cycle. The feeding trial lasted for 8 weeks. 2.3 Sampling and analysis At the beginning of the feeding trial, 10 fish were sampled and stored at -80 °C for analysis of initial whole-body proximate composition. At the end of the trial, experimental fish were starved for 14 hours prior to the sampling. After the fish were anaesthetized with MS-222 (0.1 g/L), two fish per tank were collected for whole-body composition analysis, and other three fish per tank were individually weighed, separately bled from the caudal vasculature using 1-mL heparinised (H6279; SIGMA) syringes and dissected to obtain viscera, liver and intraperitoneal fat (IPF) weights for computing body condition indices including hepatosomatic index (HSI) ((liver wt. / live wt.) × 100) and IPF ratio ((IPF wt. / live wt.) × 100), respectively. Intraperitoneal fat was obtained by removing and weighing the fat from the abdominal cavity as well as that adhering to the gut of the fish. Condition factor (CF) was also computed as (body weight × 100) / (body 6

Journal Pre-proof length)3. At this dissection, the hypothalamus, liver and head kidney samples for gene expression assay were also obtained and immediately frozen in liquid nitrogen and then stored at -80 °C. For morphological analysis, one fish from each tank was randomly collected and gut was removed and then immersed in Davidson's fixative solution (water/formalin/ethanol/acetic acid, 3/2/3/1, v/v). After 24 h, the samples for histological analysis were dehydrated and transferred to an ethanol solution (70%). The foregut and midgut were embedded in paraffin and sectioned into 4-μm transverse cuts following the axis of the gut lumen. The samples were then mounted on glass slides, and stained with hematoxylin and eosin. Slides were examined on a light microscope (Olympus IX71) equipped with the Image-Pro Plus 7.0 software. Digitalized images were analyzed to measure the micrometer length of various enteric structures. Fold height (hF), fold width (wF), enterocyte height (hE) and microvillus height (hMV) were measured (10 fields per individual sample) according to the procedures described by Escaffre et al. (2007). Crude protein (N × 6.25) was determined by Dumas combustion method using a rapid MAX N exceed system (Elementar,Germany). Crude lipid was determined by ether extraction using a Soxtec System HT (Soxtec System HT6, Haineng SOX406, Shandong, China). Dry matter was determined by heating ~2 g samples at 125 °C for 3 h (AOAC, 1990). For amino acid (AA) analysis, the diets were hydrolyzed at 110 °C with 6 N of hydrochloric acid, being under an atmosphere of N2 for 24 h. Serum samples were deproteinized with absolute ethyl alcohol. Amino acid analysis was carried out utilizing a pre-column derivatization procedure with amino quinolyl-N-hydrosysuccinimidyl carbamate (AQC) by HPLC (Waters 2690 system, Massachusetts, America). 2.4 Oxidative stress challenge test After sampling, all remaining fish were fed their prescribed diets for 2 d and then exposed to 2.5 mg Cu(II)·L-1 water for 12 h by the addition of CuSO4·5H2O to water. The dose of Cu(II) exposure used in this study was found to induce oxidative stress in a preliminary experiment. During the challenge test, the survival rate per aquarium was recorded, and then remaining fish per aquarium were randomly selected and individually sampled as above to obtain serum and head kidney. 2.5 Total RNA extraction and reverse transcription 7

Journal Pre-proof Total RNA was extracted from humpback grouper hypothalamus, head kidney and liver using Trizol Reagent (Invitrogen, America) followed by quality measurement on a 1.0% denaturing agarose gel and yield determination on NanoDrop® ND-1000 (Wilmington, DE).The RNA was treated with RNA-FreeDNase (Takara, Japan) to remove DNA contamination and reversely transcribed to cDNA by Prime Script™ RT reagen Kit with gDNA Eraser (Takara, Japan) following the instructions provided by the manufacturer. 2.6 Real-time quantitative PCR analysis of IGF-1, TOR, S6K1 in liver, GHR in hypothalamus as well as Nrf2, Keap1, HSP70 in head kidney Real-time RT-PCR was carried out in a quantitative thermal cycler (QuantStudio 6 Flex, Applied Biosystems, Singapore). The amplification was performed in a total volume of 10 μL containing 5 μL TB Green™Premix Ex Taq™ II (Takara, Japan), 0.2 μL of each primer (10 μmol/L), 4.1 μL of nuclease-free water and 0.5 μL of cDNA mix. The real-time RTPCR program was as follows: 95 °C for 30s, followed by 40 cycles of 95 °C for 5 s, 56 °C for 30 s, and 72 °C for 30 s, then 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s. The real-time RT-PCR primer pairs for GHR, IGF-1, TOR, S6K1, Nrf2, Kelch-like-ECH associated protein 1 (Keap1), HSP70 and β-actin were designed by Primer Premier5.0 based on the published nucleotide sequences and listed in Table 4. At the end of each PCR reaction, melting curve analysis of amplification products was carried out to confirm that a single PCR product was present in these reactions. Standard curves were made with five different dilutions (in triplicate) of the cDNA samples and amplification efficiency was analyzed according to the following equation E=10(-1/slope)-1. The expression levels of the target genes were calculated followed the 2-ΔΔct method described by Yao et al. (2009). 2.7 Analysis of activities of SOD, CAT and IgM concentrations in serum Activities of SOD (Kitno. E25019758), CAT (Kit no. F01019757) and IgM concentrations (Kit no. C0197170176) in serum were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA) according to the kit instructions of the manufacturer (CUSABIO, Hubei, China). 2.8 Statistic analysis Normality and homoscedasticity assumptions were confirmed before any statistical analysis. All evaluated variables were subjected to an ANOVA to determine whether dietary Arg levels 8

Journal Pre-proof significantly affected the observed responses (P<0.05). In addition, to determine whether the effect was quadratic, a follow-up trend analysis using orthogonal polynomial contrasts was performed (Davis, 2010) using the SPSS 18.0. The adjusted R2 was calculated as previously described by Kvalseth(1985) (Kvalseth, 1985). The optimum dietary Arg requirement based on maximal WG%, FCR, PER and PPV was established through the quadratic regression model. 3. Results 3.1 Growth performance and feed utilization WG% of experimental fish was increased with the increasing dietary Arg levels, up to a peak value at 3.58% dietary Arg level (Table 5), above which WG% was reduced. Survival rate of fish in each experimental treatment was 100%. Fish fed 3.58% dietary Arg had lower DFI and FCR than fish fed other dietary Arg levels. Fish fed 3.14% and 3.58% dietary Arg levels had higher PER or PPV than fish fed other dietary Arg levels. Quadratic regression analysis of WG% and FCR against dietary Arg levels indicated that optimal dietary Arg level was 3.33%-3.39% of dry matter (6.66%-6.78% of dietary protein) for humpback grouper (Fig. 1). 3.2 Body condition indices and whole-body proximate compositions HSI of fish fed 1.74% dietary Arg was significantly higher than that of fish fed any of other dietary Arg level (Table 6). The protein and lipid contents in whole body were also significantly influenced by different experimental treatments, and fish fed 3.58% dietary Arg had the highest values of these compositions. There were no significant differences in IPF ratio, CF and whole-body moisture among all experimental treatments. 3.3 Gut micromorphology Gut micromorphology of experimental fish were significantly affected by different dietary Arg levels (Table 7). Values of hF, wF, hE and hMV in foregut of fish were improved as dietary Arg level increased from 1.74% to 3.58%, and thereafter, they were reduced with the further increments in dietary Arg levels. For the micromorphology of midgut, fish fed 3.58% dietary Arg had higher hF, wF and hE than fish fed other dietary Arg levels, although the differences in wF and hE were not significant. Values of hMV in midgut of fish fed 3.14% and 3.58% dietary Arg were higher than those of fish fed 1.74%, 2.42%, 4.06%, 4.54% and 5.21% dietary Arg. 3.4 Expression of IGF-1, TOR, S6K1 in liver and GHR in hypothalamus 9

Journal Pre-proof Expression of hepatic IGF-1 and hypothalamus GHR in fish fed 3.14% and 3.58% dietary Arg were higher than those in fish fed other dietary Arg levels (Fig. 2). For TOR-related signaling molecules, fish fed 1.74%, 4.54% and 5.21% dietary Arg levels had lower mRNA levels of hepatic TOR and S6K1 compared to fish fed 2.42%, 3.14%, 3.58%, 4.06% dietary Arg levels. 3.5 Survival ratios and antioxidant parameters 3.5.1 Before challenge (exposure to Cu(II)·L-1 water for 12 h) Survival ratios of fish kept unvaried among all experimental treatments after they were fed experimental diets for 8 weeks (Table 5). Expression of Nrf2 in head kidney of fish fed 3.14%, 3.58% and 4.06% dietary Arg were higher than those of fish fed 1.74%, 2.42%, 4.54% and 5.21% dietary Arg (Fig. 3). Keap1 expression in head kidney of fish fed 3.14%, 3.58% and 4.06% dietary Arg were lower than that of fish fed 1.74%, 2.42%, 4.54% and 5.21% dietary Arg. Fish fed 1.74%, 4.54% and 5.21% dietary Arg had lower SOD activity in serum than fish fed 2.42%, 3.14%, 3.58% and 4.06% dietary Arg. Serum CAT activity showed no remarkable variations among all experimental treatments. 3.5.2 After challenge (exposure to Cu(II)·L-1 water for 12 h) After exposure to 2.5mg Cu(II)·L-1 water for 12 h, survival ratios of fish fed 1.74%, 2.42%, 3.14%, 3.58%, 4.06%, 4.54%, 5.21% dietary Arg were 50%, 56%, 61%, 72%, 67%, 67% and 67%, and they were significantly affected by different dietary Arg levels. Nrf2 expression in head kidney of fish fed 1.74% and 2.42% dietary Arg was lower than those in fish fed other dietary Arg levels. Fish fed 1.74% dietary Arg had higher expression of Keap1 in head kidney than fish fed other dietary Arg levels. Activities of CAT and SOD in serum of fish fed 3.58% dietary Arg were higher than those in fish fed other dietary Arg levels. 3.6 Expression of Hsp70 in head kidney and IgM concentration in serum 3.5.1 Before challenge (exposure to Cu(II)·L-1 water for 12 h) Fish fed 3.58% dietary Arg displayed lower expression of Hsp70 in head kidney than fish fed other dietary Arg levels (Fig. 4). IgM concentration in serum was significantly influenced by dietary Arg levels, and it was the highest in fish fed 3.58% dietary Arg. Fish fed 1.74% dietary Arg had the lowest IgM concentrations among all experimental treatments. 3.5.2 After challenge (exposure to Cu(II)·L-1 water for 12 h) 10

Journal Pre-proof Expression of HSP70 of fish fed 3.14%, 3.58%, 4.06% and 4.58% dietary Arg was higher than that of fish fed 1.74%, 2.42% and 5.12% dietary Arg. Fish fed 1.74% dietary Arg had lower HSP70 expression than fish fed other dietary Arg levels. Fish fed 3.58% dietary Arg levels had higher IgM concentrations in serum compared to fish fed other dietary Arg levels. 4. Discussion This study indicated that optimal dietary Arg requirement for maximal WG% of humpback grouper was estimated to be 3.39% of dry matter, corresponding to 6.78% of dietary protein. This value was similar to those (of dietary protein) observed in hybrid grouper (6.64%-6.82%) (Wu et al., 2018), golden pompano (6.32%-6.35%) (Lin et al., 2015), yellow grouper (6.5%) (Zhou et al., 2012), tilapia (6.24%) (Yue et al., 2013), silver perch (6.8%) (Ngamsnae et al., 1999), and higher than the values observed in rainbow trout (4%) (Fournier et al., 2003), red sea bream (4.74%) (Rahimnejad et al., 2014), Asian sea bass (3.8%) (Murillo-Gurrea et al., 2001), Atlantic salmon (4.1%-4.8%) (Berge et al., 1999), channel catfish (3.3%-3.8%) (Buentello et al., 2000), while lower than the values obtained in black sea bream (7.2%) (Zhou et al., 2010) and blunt snout bream (7.23%) (Ren et al., 2013). Reasons for the difference may include species and growth stage of fish (NRC, 2011). The higher WG% obtained in fish fed 3.58% dietary Arg compared to those of fish fed other dietary Arg levels was attributed to the lower FCR, higher protein utilization efficiency, higher expression of IGF-1 and GHR and better gut micromorphology as observed in 3.58% dietary Arg fed fish. The reduction in growth performance of fish fed excessive Arg in this study could be due to toxic effects and stress (Walton et al., 1986) and lysine-arginine antagonism (Luo et al., 2004) resulted from excessive Arg. It is well known that the supplementation of individual essential amino acid in the diet below fish requirement will lead to an imbalanced proportion of necessary amino acids, thus affecting the absorption and utilization of other amino acids. This may explain the poor growth of fish fed low Arg in the present study. The growth axis of GH-GHR-IGF plays an important role in regulating growth and development in vivo (Fuentes et al., 2013). Arg is a stimulant for GH, IGF-I and glucagon, and a precursor for polyamine synthesis, and hence it is involved in fish growth related processes (Hird et al., 1986; Baoñs et al., 1999). Results from this study further demonstrated that suitable dietary 11

Journal Pre-proof Arg levels significantly promote the expression of hypothalamus GHR and hepatic IGF-I. However, our results also showed that excess dietary Arg had a negative effect on the expression of hepatic IGF-1. The possible explanation was that excess dietary Arg led to insulin resistance through negative feedback system, caused serine/threonine phosphorylation as in higher animals, which might affect the expression of IGF-1 level (Liang et al., 2016; Harrington et al., 2004). It was found by Yao et al. (2008) that in case of deficient or excessive Arg, expression of TOR and S6K1 in animals is decreased, resulting in reduced protein biosynthesis and increased proteolysis of cellular proteins, and Tomé (2018) reported that the TOR pathway plays a crucial role in upregulating cellular anabolic pathways including protein, lipid, and pyrimidine biosynthesis, so in the present study, the high whole-body protein and lipid contents observed in fish fed 3.58% dietary Arg may be due to their high expression of hepatic TOR and S6K1. . In the present study, fish fed 1.74% dietary Arg had higher HSI than fish other dietary Arg levels. This possibly was due to the fact that more amino acids were converted to energy and stored as hepatic glycogen when imbalanced amino acids existed in the diets. Gut morphology is an important parameter to evaluate the digestibility, absorption efficiency of nutrients and fish growth performance (Moriyama et al., 2000; Sørensen et al., 2011). Results from this study indicated that appropriate Arg supplementation can promote gut development of humpback grouper. Similar results were also observed in red drum (Cheng et al., 2011), hybrid striped bass (Cheng et al., 1995) and hybrid grouper (Wu et al., 2018). Besides the influence on growth performance and gut micromorphology observed in this study, dietary Arg levels were also found to significantly affect the antioxidant system and immune responses of humpback grouper. In the current study, the supplementation of dietary Arg significantly improved the survival rate of humpback grouper after a Cu(II) challenge, suggesting that dietary Arg improved their resistance to Cu stress. Antioxidant genes and enzymes are the principal cellular protective mechanisms against oxidative stress in fish and shellfish (Giuliani et al., 2017; Yuan et al., 2019). Nrf2 and Keap1 are cellular sensors of chemical and radiation induced oxidative and electrophilic stress, and play an important role in the transcriptional activation of an array of antioxidant and detoxification genes (Wang et al., 2018). Nrf2 has been demonstrated to be a critical transcription factor that controls the expression and coordinated 12

Journal Pre-proof induction of a battery of defensive genes encoding detoxifying enzymes and antioxidant proteins (Kaspar et al., 2009). Keap1 was identified as an Nrf2-binding protein, which depresses Nrf2 translocation to the nucleus (Ma, 2013). In this study, it was observed that Nrf2 expression in head kidney robustly responded to different dietary Arg levels before/after Cu challenge, and expression of Keap1 showed an opposite variation trend to Nrf2. The possible cause is that the Arg supplementation activated the Nrf2 pathway in head kidney of humpback grouper, resulted in the up-regulation of ARE-driven antioxidant expressions via Nrf2-Keap1 pathway, thereby, suppressed oxidative stress and induced an endogenous antioxidant response (Liang et al., 2018b). Additionally, the high activities of SOD and CAT in serum of fish fed 3.58% dietary Arg also contributed to fish ability of antioxidation, because these two antioxidant enzymes play an important role in the self-defense system, possessing the vital function in the immune system (Zhou et al., 2015), and Jiang et al. (2016) reported that the protective effect of antioxidant enzymes against oxidative damage may be related to their ability to scavenge reactive oxygen species. Immune system is fundamental for survival of an organism against invading pathogens and other harmful agents (Banergee et al., 2015). Heat shock protein (HSP) is a highly conserved special protein synthesized by cells, and it plays an important role in immunomodulation, apoptosis and influences the immune responses (Chow et al., 1998). In aquatic animals, HSP70 has been shown to play an important role in health, in relation to the host response to environmental pollutants (Kakkar et al., 2014). Our results indicated that proper Arg supplementations can increase the expression of HSP70 in head kidney of humpback grouper after Cu(II) challenge. IgM was the major component of specific humoral immune system (Watts et al., 2001; Uribe et al., 2011). In this study, fish fed 3.58% dietary Arg displayed the highest IgM concentration in serum among all treatments before/after the Cu(II) challenge, indicating an improvement in humoral immunity. In hybrid grouper, it was also observed that suitable Arg supplementations could improve the IgM concentration in serum (Wu et al., 2018). Pohlenz et al. (2012c) reported that in head kidney and spleen of vaccinated channel catfish, Arg can improve B cell populations which play an important role in antibody production in fish. 13

Journal Pre-proof In conclusion, results of this study indicated that the optimal dietary Arg requirement for best growth of juvenile humpback grouper was estimated to be 3.39% of dry matter, corresponding to 6.78% of dietary protein. The oxidation resistance, immunity as well as gut micromorphology of humpback grouper could be improved by the suitable Arg supplementations to diets.

Acknowledgements This study was supported by a grant (no.: ZDYF2018055) from Hainan key research and development Projects and a grant (no.: 31760760) from The National Natural Science Fund of China. Authors wish to appreciate the Editor and anonymous reviewers for their valuable suggestions on our manuscript.

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21

Fig. 1. Relationship of weight gain %, and feed conversion ratio with dietary Arg levels based on quadratic regression analysis, where Xopt represents the dietary arginine level for the maximum weight gain %, feed conversion ratio, protein efficiency ratio and protein productive of humpback grouper.

Fig. 2. Relative expression of GHR in hypothalamus and IGF-I, TOR and S6K1 in liver of humpback grouper juveniles fed different dietary Arg levels for 8 weeks (n = 9). Relative mRNA expression was evaluated by real-time quantitative PCR. GHR: growth hormone receptor; IGF-I: insulin like growth factor-I; TOR: target of rapamycin; S6K1: S6 kinase 1. SOP= second order

polynomial trend; Adj. R2= adjusted R square.

Fig. 3. Survival, relative expression of Nrf2, Keap1 in head kidney and activities of SOD, CAT in serum of humpback grouper juveniles fed different dietary Arg levels for 8 weeks before/after exposure to 2.5 mg Cu(II)·L-1 water for 12h (n = 9). Nrf2: NF-E2-related factor 2; Keap1: Kelch-like-ECH associated protein 1; SOD: superoxide dismutase; CAT: catalase.

Fig. 4. Relative expression of HSP70 in head kidney and IgM concentrations in serum of humpback grouper juveniles fed different dietary Arg levels for 8 weeks before/after exposure to 2.5 mg Cu(II)·L-1 water for 12h (n = 9). HSP70: heat shock proteins genes 70; IgM: Immunoglobulin M.

Journal Pre-proof The optimal dietary Arg requirement of humpback grouper was 3.39% of dry matter. Suitable dietary Arg supplementations improved growth and gut micromorphology of fish. Suitable dietary Arg supplementations improved oxidation resistance and immunity of fish.

Table 1. Formulations and analyzed composition of experimental diets (dry-matter basis) Ingredients

Dietary Arg levels % 1.74

2.42

3.14

3.58

4.06

4.54

5.21

Peruvian fishmeal (Anchovy)1

30

30

30

30

30

30

30

Casein

10

10

10

10

10

10

10

17.54

17.54

17.54

17.54

17.54

17.54

17.54

0

0.6

1.2

1.8

2.4

3.0

3.6

Ala

3.6

3.0

2.4

1.8

1.2

0.6

0

Chile fish oil (Salmon)

6.35

6.35

6.35

6.35

6.35

6.35

6.35

Corn starch

12.5

12.5

12.5

12.5

12.5

12.5

12.5

Vitamin mixture3

1

1

1

1

1

1

1

Mineral mixture4

0.5

0.5

0.5

0.5

0.5

0.5

0.5

3

3

3

3

3

3

3

15.51

15.51

15.51

15.51

15.51

15.51

15.51

85

84.5

84.9

84.7

84.3

87.9

85.4

48.4

49

48.6

49.9

49.3

48.5

49.1

Amino acid mixture2 L-arginine

Carboxymethy cellulose Cellulose Analyzed composition of diets5 Dry matter % Crude protein %

Crude lipid %

9.6

9.6

9.8

9.9

9.8

9.8

9.4

Ash %

9.2

9.2

9.7

9.9

9.3

9.5

9.3

(Crude fiber + NFE)6

32.8

32.2

31.9

30.3

31.6

32.2

32.2

Gross energy (kcal/100g)7

330

332.4

332.6

338.7

335.4

332.2

331

Arg %

1.74

2.42

3.14

3.58

4.06

4.54

5.21

1Tecnologica 2Amino

de Alimentos S. A., Peru; proximate composition (100% dry matter): moisture, 7.3; crude protein, 71.29; amino acid, 70.44; crude lipid, 12.1.

acid mixture (g/100g): L-lysine, 3.62; L -methionine, 0.08; L -threonine, 0.86; L -isoleucine, 0.65; L -leucine, 1.22; L -phenylalanine, 0.69; L -valine, 0.70; L -histidine, 0.12; L -aspartic

acid, 2.56; L -serine, 0.66; L -glumatic acid, 2.67; Glycine, 2.52; L -cystine, 0.05; L -tyrosine, 0.19; L -proline, 0.64. 3Vitamin

mixture (mg/g mixture): thiamin hydrochloride, 2.5; riboflavin, 10; calcium pantothenate, 25; nicotinic acid, 37.5; pyridoxine hydrochloride, 2.5; folic acid, 0.75; inositol, 100;

ascorbic acid, 50; choline chloride, 250; menadione, 2; alpha-tocopheryl acetate, 20; retinol acetate, 1; cholecalciferol, 0.0025; biotin, 0.25; vitamin B12, 0.05. All ingredients were diluted with alpha-cellulose to 1 g (Lin et al., 2003). 4Mineral

mixture (mg/g mixture): calcium lactate, 327; K2PO4, 239.8; CaHPO4·2H2O, 135.8; MgSO4·7H2O, 132; Na2HPO4·2H2O, 87.2; NaCl, 43.5; ferric citrate, 29.7; ZnSO4·7H2O, 3;

CoCl2·6H2O, 1; MnSO4·H2O, 0.8; KI, 0.15; AlCl3·6H2O, 0.15; CuCl2, 0.1 (Lin et al., 2003). 5Values

represent means of duplicate samples.

6 [Crude

fiber + Nitrogen free extract (NFE)] = 100 − (% ash + % protein + % lipid). 7By calculation: Fuel values for carbohydrate, protein and lipid were 4.0, 4.0 and 9.0 kcal/g (16.7, 16.7 and

37.7 kJ /g), respectively.

Table 2. Amino acid composition of the ingredients, required supplemental crystalline amino acids in the diets and the amino acid profile of whole-body protein of humpback grouper (dry-matter basis)1. Amount in

AA profile of whole-body protein of humpback grouper

30g FM2

10g CS

CAA

total

Lysine

1.74

0.74

1.99

4.47

4.47

Arginine

1.43

0.39

2.19

4.01

4.01

Methionine

0.72

0.23

0.08

1.03

1.03

Threonine

1.04

0.40

0.86

2.30

2.30

Isoleucine

1.06

0.49

0.65

2.20

2.20

Leucine

1.70

0.86

1.22

3.78

3.78

Phenylalanine

1.00

0.49

0.69

2.18

2.18

Valine

1.23

0.62

0.70

2.55

2.55

Histidine

0.75

0.27

0.12

1.14

1.14

1.98

0.65

2.56

5.19

5.19

Amino acids EAA

NEAA Aspartic acid

Serine

0.94

0.52

0.66

2.12

2.12

Glutamic acid

2.77

0.53

2.67

7.24

7.24

Glycine

1.42

0.19

2.52

4.13

4.13

Alanine

1.39

0.28

1.72

3.39

3.39

Tyrosine

0.83

0.53

0.19

1.55

1.55

Proline

1.06

0.89

0.64

2.59

2.59

1Values 2FM:

represent means of duplicate samples.

Fishmeal; CS: Casein; CAA: Crystalline amino acid.

Table 3. Amino acid compositions (%) of experimental diets (dry-matter basis)1. Dietary Arg levels % 1.74

2.42

3.14

3.58

4.06

4.54

Lysine

3.86

3.89

4.03

4.00

3.91

3.97

3.95

Arginine

1.74

2.42

3.14

3.58

4.06

4.54

5.21

Methionine

1.00

0.94

1.02

0.61

0.93

0.93

0.73

Threonine

2.15

2.14

2.37

2.34

2.19

2.06

2.26

Isoleucine

2.70

2.03

2.22

2.28

2.08

2.02

2.08

Leucine

3.65

3.63

3.86

3.84

3.59

3.49

3.67

Phenylalanine

2.15

2.01

2.35

2.30

1.94

2.16

2.18

Valine

2.42

2.43

2.55

2.60

2.41

2.34

2.47

Histidine

1.10

1.08

1.19

1.20

1.08

1.03

1.13

∑EAA

20.77

20.57

22.73

22.75

22.19

22.54

23.68

Aspartic acid

4.55

4.65

4.79

4.73

4.64

4.77

4.71

Serine

1.94

1.90

2.11

2.08

1.93

1.87

1.96

AA / ∑ AA

5.21

EAA

NEAA

Glutamic acid

6.63

6.60

6.98

6.92

6.66

6.68

6.72

Glycine

3.91

3.87

4.20

4.06

3.83

3.74

3.91

Alanine

4.75

4.32

4.11

3.59

3.07

2.69

2.25

Tyrosine

1.43

1.41

1.61

1.45

1.44

1.34

1.43

Proline

2.88

2.65

2.83

2.90

2.66

2.55

2.73

∑NEAA

26.09

25.4

26.63

25.73

24.23

23.64

23.71

∑AA

46.87

45.97

49.36

48.48

46.42

46.18

47.39

1Values

represent means of duplicate samples.

Journal Pre-proof Table 4. Primers used for quantitative RT-PCR (qPCR). Used for

Gene name

qPCR

GHR1

Genebank accession no. KR269817.1

Primer sequence(5’-3’) F8: CACAGACTTCTATGCCCAGGT R9: GTGTAGCCGCTTCCTTCAG

IGF-I2

AY776159.1

F: TATTTCAGTAAACCAACAGGCTATG R: TGAATGACTATGTCCAGGTAAAGG

TOR3

JN850959.1

F: TCTCCCTGTCCAGAGGCAATAA R: CAGTCAGCGGGTAGATCAAAGC

S6K14

XM_020085100.1

F: TCCTTCTCCGTCTGTAAACGA R: CATGAACACCTGCTTACCAT

Nrf25

KU892416.1

F: TATGGAGATGGGTCCTTTGGTG R: GCTTCTTTTCCTGCGTCTGTTG

Keap16

XM_023419210.1

F: TCCACAAACCCACCAAAGTAA R: TCCACCAACAGCGTAGAAAAG

HSP707

MF176884.1

F: GTCCTGATCAAACGAAACACCA R: CACGCTCACCCTCATAAACCT

β-actin

AY510710.2

F: CTCTGGGCAACGGAACCTCT R: GTGCGTGACATCAAGGAGAAGC

1GHR:

growth hormone receptor;

2IGF-I:

insulin like growth factor-I;

3TOR:

target of rapamycin;

4S6K1: 5Nrf2:

ribosomal protein S6 kinase 1;

NF-E2-related factor 2;

6Keap1: 7HSP70:

Kelch-like-ECH and associated protein 1; heat shock proteins genes 70

8F:

Forward sequence;

9R:

Reverse sequence;

Table 5. Growth performance and feed utilization of humpback grouper juveniles fed different dietary Arg levels for 8 weeks1.

Dietary Arg levels %

WG%2

DFI3

FCR4

PER5

PPV6

Survival %

1.74 2.42 3.14 3.58 4.06 4.54 5.21 PSE1

196 206 221 244 213 203 196 3.9

2.36 2.07 2.07 1.89 2.22 2.27 2.38 0.041

1.32 1.16 1.16 1.06 1.24 1.27 1.34 0.023

1.57 1.76 1.77 1.90 1.64 1.63 1.53 0.298

29.1 34.3 33.3 38.3 31.6 31.6 29.0 0.71

100 100 100 100 100 100 100 0.00

ANOVA (P-Value)

<0.001

<0.001

<0.001

<0.001

<0.001

Regression analysis of SOP (N = 3) Adj. R2 P-Value

0.536 0.001

0.612 <0.001

0.611 <0.001

0.600 <0.001

0.567 0.001

1PSE=

pooled standard error of treatment means (n = 3); Adj. R2= adjusted R square; SOP= second order polynomial trend.

2Weight 3Daily 4Feed

Gain%: 100 × (final mean body weight - initial mean body weight) / initial mean body weight.

Feed Intake = 100 × feed offered / (final mean body weight - initial mean body weight) /days.

Conversion Ratio: g dry feed / g weight gain.

5Protein

Efficiency Ratio: g weight gain / g protein fed.

6Protein

Productive Value: g protein gain / g protein fed.

Table 6. Body condition indices and whole-body compositions (fresh-wt. basis) of humpback grouper juveniles fed different dietary Arg levels for 8 weeks. Dietary Arg levels %

Whole-body composition4 (n = 12)

Body condition indices (n = 12) HSI1

IPF2

CF3

Moisture

Protein

Lipid

1.74

4.26

0.98

2.17

72

17.47

5.00

2.42

2.93

1.09

2.00

72

18.02

5.15

3.14

2.84

1.11

2.11

73

17.74

5.01

3.58

2.85

1.12

2.15

71

18.75

5.91

4.06

2.64

1.04

2.10

72

18.03

5.63

4.54

2.36

1.04

2.17

72

18.05

5.54

5.21

1.90

1.04

2.09

73

17.74

4.34

PSE

0.16

0.02

0.02

0.01

0.095

0.383

ANOVA (P-Value)

<0.001

0.675

0.235

0.294

0.001

0.01

Adj. R2

0.802

0.043

0.008

0.070

0.315

0.317

P-Value

<0.001

0.262

0.929

0.227

0.013

0.013

Regression analysis of SOP

1Hepatosomatic

index (HSI) =100×liver weight (g)/whole-body weight (g).

2Intraperitoneal

fat ratio (IPF) =100×intraperitoneal fat weight (g)/body weight (g).

3Condition 4Initial

factor (CF) =100×body weight (g)/body length (cm)3.

whole-body composition (%): Moisture=78.20; Protein=15.25.

Table 7. Gut micromorphology of humpback grouper juveniles fed different dietary Arg levels for 8weeks. Dietary Arg levels %

Foregut (μm)

Midgut (μm)

hF1

wF

hE

hMV

hF

wF

hE

hMV

1.74

372.55

60.37

29.79

1.70

363.78

65.66

31.93

2.82

2.42

451.20

63.84

30.40

1.79

346.52

63.49

27.44

2.83

3.14

483.14

67.30

35.14

2.01

428.78

66.13

27.93

3.22

3.58

636.89

73.39

37.10

2.22

466.67

76.53

34.73

3.14

4.06

546.96

66.95

34.15

2.07

388.23

60.17

27.25

2.90

4.54

516.43

60.11

31.41

1.78

378.89

58.16

28.48

2.78

5.21

373.32

53.49

26.15

1.36

302.94

61.15

28.37

2.65

PSE

20.46

1.40

0.80

0.06

12.28

1.714

0.807

0.053

ANOVA (P-Value)

<0.001

<0.001

<0.001

<0.001

<0.001

0.06

0.072

0.014

Adj. R2

0.690

0.74

0.762

0.703

0.51

0.034

0.035

0.395

P-Value

<0.001

<0.001

<0.001

<0.001

0.001

0.283

0.723

0.004

Regression analysis of SOP

1hF=fold

height; wF=fold width; hE=enterocyte height; hMV=microvillus height.