Effect of proline supplementation on anti-oxidative capacity, immune response and stress tolerance of juvenile Pacific white shrimp, Litopenaeus vannamei

Aquaculture 448 (2015) 105–111

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Effect of proline supplementation on anti-oxidative capacity, immune response and stress tolerance of juvenile Pacific white shrimp, Litopenaeus vannamei Shi-Wei Xie, Li-Xia Tian, Yu-Ming Li, Weiwen Zhou, Shuai-Lin Zeng, Hui-Jun Yang, Yong-Jian Liu ⁎ Institute of Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China

a r t i c l e

i n f o

Article history: Received 1 April 2015 Received in revised form 22 May 2015 Accepted 23 May 2015 Available online 29 May 2015 Keywords: Litopenaeus vannamei Proline POX Immune response Environment stress

a b s t r a c t An 8-week feeding trial was conducted to evaluate the effect of proline on anti-oxidative capacity, immune response and stress tolerance of juvenile Pacific white shrimp, Litopenaeus vannamei. Six practical low fishmeal diets (15% fishmeal) were formulated to contain graded levels (2.02, 2.14, 2.27, 2.38, 2.49, 2.6%) of L-proline. Each diet was randomly assigned to triplicate groups of 30 shrimps (approximately 0.41 g) and the shrimps were fed 4 times a day to apparent satiation. Growth performance was not affected by proline levels (P N 0.05). But higher levels of proline increased hydroxyproline content in the whole body (P = 0.004). And survival after 60 h acute NH3 stress increased with the increasing dietary proline levels (P = 0.015). High levels of proline also increased the proline oxidase (POX), nitric oxide (NO), total antioxidant capacity (T-AOC) and phenoloxidase (PO) activities in hemolymph or hepatopancreas after the 60 h NH3 stress compared to basic group which had lowest proline level. Broken-line analysis showed that the optimal dietary proline requirement of L. vannamei was 2.29%, 2.32% and 2.34% based on PO, POX and 60 h survival after NH3 stress. These results clearly indicated that 2.29–2.34% of proline in the low fish meal diet could improve anti-oxidative capacity, immune response, NH3 stress tolerance of L. vannamei, and proline may be a conditionally essential AA for L. vannamei. Statement of relevance: This study indicates that proline may be a conditional amino acid for white shrimp under the environment stress, especially when fed with low fish meal diet. The results will contribute to the low fish meal diet study of the white shrimp. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Amino acids (AA) are not only building blocks for tissue proteins, but also essential substrates for the synthesis of many biologically active substances (e.g. polyamines, glutathione, creatine, carnitine, hormones, neurotransmitters) with crucial role in maintaining normal physiological and nutritional status of the body (Geraert and Mercier, 2010; Kaushik and Seiliez, 2010; Li et al., 2011; Rezaei et al., 2013b). Traditionally, AA were classified as nutritionally essential or nonessential based on nitrogen balance or growth. AA whose carbon skeletons are not synthesized de nove by animals or humans must be provided in diets, therefore, considered nutritionally essential. In contrast, AA that can be synthesized de novo in animals are thought to be nutritionally nonessential (Takeuchi and Gatlin, 2007; Wilson, 2002). But it was tactically assumed and without much evidence that animals or humans could synthesize sufficient amounts of all non-essential AA (NEAA) and did not need them in diets for optimal nutrition or health (Gaye-Siessegger ⁎ Corresponding author at: Nutrition laboratory, Institute of aquatic economic animals, School of life science, Sun Yat-Sen University, Guangzhou 510275, China. E-mail address: [email protected] (Y.-J. Liu).

http://dx.doi.org/10.1016/j.aquaculture.2015.05.040 0044-8486/© 2015 Elsevier B.V. All rights reserved.

et al., 2007; Mauriz et al., 2001). Recently, there have been growing evidences from cell culture and animal studies show that some of the traditionally classified NEAA (e.g. arginine, glycine, glutamine, taurine and proline) are important regulators of key metabolic pathways, which play enormous roles in multiple signaling pathways, thereby regulating gene expression, intracellular protein turnover, nutrient metabolism, and oxidative defense. These have led to the development of the concept of functional AA (FAA) (Kim et al., 2007; Li et al., 2009a; Wu, 2010, 2013). Proline is the only proteinogenic secondary amino acid, with its metabolite (hydroxyproline) constitute one-third of AA in collagen proteins (Phang et al., 2010; Wu et al., 2011). Proline is one of the Arginine Family AA, it plays important roles in protein synthesis (mammalian target of rapamycin activation) and structure, metabolism (synthesis of arginine, polyamines, NO, glutamate and ornithine via pyrroline-5-carboxylate), cellular energy sensing, cell differentiation, anti-oxidative reactions, and immune responses (Brunton et al., 2012; Tomlinson et al., 2011). Although most of mammals can synthesize proline from arginine, ornithine and glutamine/glutamate, but the rate of endogenous synthesis are inadequate for neonates, birds, and aquatic species (Hu et al., 2008; Wu et al., 2008, 2009, 2011). New development in the study of proline indicates that proline may be a functional AA or a conditionally essential AA, the

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traditional view that the proline is a nonessential AA should be reevaluated. Ammonia (NH3) is a gas which dissolves in water where it ionizes to form the ammonium (NH+ 4 ). Most biological membranes are permeable to unionized ammonia (NH3) but relatively impermeable to ionized ammonium (ammonium ions, NH+ 4 ), so the toxicity is mostly expressed in terms of the concentration of unionized ammonia (NH3) (Eddy, 2005; Randall and Tsui, 2002). Acute NH3 toxicity has been well studied, and it was concluded that acute ammonia is very toxic to aquatic animals and has a deleterious effect on osmolality, ion concentrations, growth, survival and immune system in crustacean (Barbieri, 2010; Chen et al., 2012; Hsieh et al., 2008; Liu et al., 2008; Yue et al., 2010). There is almost no study about proline in the aquatic animals, the aim of this study is to evaluate the effect of dietary proline on growth performance, immune response and NH3 stress tolerance of juvenile Litopenaeus vannamei fed with low fishmeal diet. 2. Materials and methods 2.1. Diet preparation Six practical diets with graded levels of proline (2.02, 2.14, 2.27, 2.38, 2.49, 2.6%) were formulated and proximate analysis of the diets is given in Table 1. The diets were supplemented with lysine, methionine, threonine and taurine to satisfy the requirement for juvenile shrimps. Lysine, methionine, threonine, taurine and proline were mixed and precoated with 10 g kg−1 carboxymethyl cellulose in water at 60 °C to prevent leaching loss, and were then mingled with the other mixed dry ingredients. Lipids and water were then added and thoroughly mixed. The 1.2 mm diameter pellets were cold-extruded using a pelletizer (Institute of Chemical Engineering, South China University of Technology, Guangdong, China) and heated in an electric oven at 90 °C for 60 min, then air-dried to approximately 10% moisture, and stored at − 20 °C until used. 2.2. Shrimp and experimental conditions Juvenile L. vannamei were obtained from the Guangdong Evergreen Group Hatchery (Zhanjiang, China). Prior to start the experiment, the shrimps were acclimated to the culture environment for 3 weeks and fed with a commercial diet (44% crude protein, 8% crude lipid, Guangdong Evergreen Group, Zhanjiang, China). At the beginning of the experiment, 30 shrimps of the similar size were distributed randomly into 18 cylindrical fiberglass tanks (300 l), the initial body weight was approximately 0.41 g. Each diet was randomly assigned to triplicate tanks. All groups of shrimps were fed to apparent satiation four times daily at 7:00, 12:00, 17:00 and 21:00 with 5–8% body weight per day for 8 weeks. Any uneaten feeds were collected by siphoning, then dried, weighed and used to calculate feed intake. During the experimental period, water temperatures ranged from 28.63 ± 0.14 °C, the salinity was approximately 27.88 ± 0.4‰, pH 7.98 ± 0.05, the ammonia nitrogen was lower than 0.05 mg L−1, and dissolved oxygen was higher than 6.5 mg L−1. Natural light–dark cycle was used during the trial. 2.3. Sample collection and chemical analyses At the beginning of the feeding trial 100 shrimps were randomly sampled and stored frozen (−20 °C) until analysis of the initial proximate. At the end of the feeding trial, shrimps in each tank were weighed and sampled for analysis 24 h after the last feeding. Five shrimps from each tank were used to analyze the whole body composition. Another six shrimps per tank were dissected, hepatopancreas were immediately placed into liquid nitrogen and stored at −80 °C until analysis of the enzymatic activity, and muscles were taken to analyze proximate composition. Blood samples were centrifuged (8000 rpm, 10 min) at 4 °C.

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

Ingredients Fish meala Soybean mealb Peanut meal Wheat flour Beer yeast Shrimp meal Wheat gluten Soy oil Fish oil Choline chloride Ascorbic acid polyphosphate Soy lecithin Vitamin mixturec Mineral mixtured Monocalcium phosphate Carboxyl Methyl Cellulose DL-Mete e L-lysine monohydrochloride Taurinee Threoninee Prolinee

Diet 1

Diet 2

Diet 3

Diet 4

Diet 5

Diet 6

15 28 14 22.79 2 4 5 1.5 1.5 0.2 0.1 1 1 1 1 1 0.2 0.5

15 28 14 22.67 2 4 5 1.5 1.5 0.2 0.1 1 1 1 1 1 0.2 0.5

15 28 14 22.55 2 4 5 1.5 1.5 0.2 0.1 1 1 1 1 1 0.2 0.5

15 28 14 22.43 2 4 5 1.5 1.5 0.2 0.1 1 1 1 1 1 0.2 0.5

15 28 14 22.31 2 4 5 1.5 1.5 0.2 0.1 1 1 1 1 1 0.2 0.5

15 28 14 22.19 2 4 5 1.5 1.5 0.2 0.1 1 1 1 1 1 0.2 0.5

0.1 0.1 0

0.1 0.1 0.12

0.1 0.1 0.24

0.1 0.1 0.36

0.1 0.1 0.48

0.1 0.1 0.6

88.87 45.63 8.67 8.46 2.14

88.61 46.09 8.63 8.71 2.27

89.41 46.02 8.72 8.93 2.38

88.17 45.98 8.65 8.44 2.49

89.27 45.96 8.58 8.84 2.60

Proximate composition (% dry matter) Dry matter 88.66 Crude protein 45.70 Crude lipid 8.67 Ash 8.64 Proline 2.02

a Fish meal: 64.3% crude protein, 9.6% crude lipid, 2.83% proline, Haida Feed Corporation, Guangzhou, China. b Soybean meal: 42.9% crude protein, 1.2% crude lipid, 1.88% proline, Haida Feed Corporation, Guangzhou, China. c Vitamin mixture: (kg−1 of diet): vitamin A, 250,000 IU; riboflavin, 750 mg; pyridoxine HCL, 400 mg; cyanocobalamin, 1 mg; thiamin, 250 mg; menadione, 250 mg; folic acid, 125 mg; biotin, 10 mg; a-tocopherol, 2.5 g; myo-inositol, 8000 mg; calcium pantothenate, 1250 mg; nicotinic acid, 2000 mg; vitamin D3, 45,000 IU; vitamin C, 7000 mg. d Mineral mix (kg−1 of diet): ZnSO4 · 7H2O, 0.04 g; CaCO3, 37.9 g; KCL, 5.3 g; KI, 0.04 g; NaCl, 2.6 g; CuSO4 · 5H2O, 0.02 g; CoSO4 · 7H2O, 0.02 g; FeSO4 · 7H2O, 0.9 g; MnSO4 · H2O, 0.03 g; MgSO4 · 7H2O, 3.5 g; Ca(HPO4)2 · 2H2O, 9.8 g. e All the crystalline amino acids were supplied by Aladdin Industrial Corporation, Shanghai, China.

Then, the supernatant was collected and stored at −80 °C until analysis of the enzymatic activity. Moisture, crude protein, crude lipid, and ash content in the diets and shrimp samples were determined using standard methods (AOAC, 1995). Moisture was determined by drying in an oven at 105 °C until constant weight. Crude protein (N × 6.25) was determined by the Kjeldahl method after acid digestion using an Auto Kjeldahl System (1030-Auto-analyzer; Tecator, Hoganos, Sweden). Crude lipid was determined by the ether-extraction method using a Soxtec System HT (Soxtec System HT6; Tecator, Sweden). Ash content was determined by muffle furnace at 550 °C for 12 h. Amino acid concentrations in diets, and free amino acid concentrations in whole body were determined using an automatic amino acid analyser (Hitachi Model 835–50; Japan) equipped with a column for physiological fluid analysis (2.6–150 mm, Hitachi custom ion exchange resin no.2619; Tokyo, Japan) by a professional laboratory, samples were hydrolyzed in the 6 N HCL at 110 °C for 22 h, then were separated by the ion exchange column and reaction with ninhydrin solution, and the amino acid concentrations were obtained through the spectrophotometer. Hematological parameters were obtained using an automatic blood analyzer (Hitachi 7170A; Tokyo, Japan). 2.4. NH3 exposure experiment After 24 h of feeding trial sampling, 8 shrimps from each tank were exposed to NH3 by adding NH4Cl into each tank to obtain NH+ 4 concentration of 12.64 mg L−1 which is equal to a concentration of 1.39 mg L−1

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Table 2 Growth performance and feed utilization of the juvenile L. vannamei fed with different dietary proline levels. Item

Proline level (% dry weight)

Initial weight (g) Final weight (g) Weight gain (%) SGR (% day−1) Survival (%) FE FI HSI PPV PER

2.02

2.14

2.27

2.38

2.49

2.6

0.41 6.03 ± 0.04 1370.28 ± 10.17 4.8 ± 0.01 93.33 ± 1.92 0.72 ± 0 8.41 ± 0.11 5.66 ± 0.13a 23.79 ± 0.28 1.57 ± 0.01

0.41 6.09 ± 0.07 1384.97 ± 16.96 4.82 ± 0.02 91.67 ± 5 0.7 ± 0 8.69 ± 0.31 5.85 ± 0.12ab 23.7 ± 0.28 1.48 ± 0.03

0.41 6.05 ± 0.07 1375.74 ± 18.12 4.81 ± 0.02 97.78 ± 1.11 0.71 ± 0.01 8.56 ± 0.05 5.9 ± 0.1ab 24.06 ± 0.22 1.53 ± 0.02

0.41 6.17 ± 0.06 1405.55 ± 14.1 4.84 ± 0.02 95 ± 5 0.7 ± 0 8.88 ± 0.14 6.1 ± 0.11b 23.4 ± 0.27 1.51 ± 0.01

0.41 6.29 ± 0.1 1434.09 ± 24.75 4.88 ± 0.03 92.22 ± 2.94 0.71 ± 0.01 8.81 ± 0.04 5.76 ± 0.06ab 24.32 ± 0.76 1.58 ± 0.02

0.41 6.25 ± 0.1 1424.7 ± 25.05 4.86 ± 0.03 96.67 ± 1.92 0.71 ± 0.03 8.76 ± 0.09 5.76 ± 0.03ab 24.35 ± 0.2 1.54 ± 0.01

Data represent mean ± SEM of three replicates. Values in the same line with different letters are significantly different (P b 0.05).

NH3 at a temperature of 30 °C, a pH of 8.2 and a salinity at 28‰ (Trussell, 1972; Zhang et al., 2012). During the 60 hours' NH3 exposure experiment, shrimps were carefully monitored and mortality was recorded every 12 h. Blood and hepatopancreas samples were collected at the termination of the experiment. 2.5. Enzyme activity assays Nitric oxide (NO) and nitric oxide synthase (NOS) were measured by following the introduction of the detection kit (Nanjing Jiancheng Bioengineering Institute, China). NO was measured by nitrate reductase method. NOS was measured by catalyze arginine to NO, one unit of enzyme activity was defined as 1 mL hemolymph or 1 mg hepatopancreas protein catalyze arginine to produce 1 μ mol NO in 1 min. Total antioxidant capacity (T-AOC) was measured by using the detection kits of another corporation (Beyotime Institute of Biotechnology, China). Phenoloxidase (PO) activity in plasma was measured by spectrophotometer according to the formation of dopachrome produced from L-dihydroxyphenylalanine (L-DOPA) (Zhang et al., 2012). Briefly, 10 μL hemolymph supernatant was evenly mixed with 200 μL phosphate buffer (0.1 mol L−1, pH 6.0) and 10 μL L-DOPA (0.1 mol L−1). Absorption at 490 nm was immediately recorded every 2 min for 30 min. One unit of enzyme activity was defined as a linear increase in absorbance of 0.001 per min per mL hemolymph. Proline oxidase (POX) activity was measured as previously described. Briefly, tissues (0.5 g) were homogenized at 4 °C in 6 mL of buffer [250 mmol L−1 sucrose, 1 mmol L−1 EDTA, 2.5 mmol L−1 dithiothreitol

in 50 mmol L−1 potassium phosphate mine.buffer (pH 7.2)] containing protease inhibitors (5 mg L−1 phenylmethyl sulfonyl fluoride, 5 mg L−1 aprotinin, 5 mg L−1 chymostatin and 5 mg L−1 pepstatin A). The homogenates were centrifuged at 600g for 10 min, and the supernatant fractions were centrifuged at 12,000 for 10 min at 4 °C. The pellets (mitochondria) were suspended buffer (pH 7.4, in 0.5 mL of 50 mmol L−1) potassium phosphate buffer (pH 7.5) and stored at −20 °C for 24 h before assay. The assay mixture (1.0 mL), which consisted of 15 mmol L−1 20 mmol L−1 ferricytochrome C, mitochondrial pellets and 50 mmol L−1 potassium phosphate buffer (pH 7.5), was incubated at 37 °C for 0 or 30 min. The reaction was terminated by addition of 0.5 mL of 10% trichloroacetic acid, followed by addition of 0.1 mL of 100 mmol L−1 o-aminobenzaldehyde. Then the mixture was centrifuged at 600g for 5 min, and the absorbance of the supernatant fraction was measured at 440 nm. One unit of enzyme activity was defined as 1 mg hepatopancreas protein catalyze proline to produce 1 nmol P5C in 1 min. L-proline,

2.6. Calculations and statistical analysis The parameters were calculated as follows: Percent weight gain (WG, %) = 100 × (Wt − Wi) / Wi Specific growth ratio (SGR, % day−1) = 100 × (LnWt − LnWi) / t Protein efficiency ratio (PER) = weight gain (g, wet weight)/protein intake (g, dry weight) Feed efficiency (FE) = weight gain (g, wet weight) / feed consumed (g, dry weight)

Table 3 Free amino acid ratios of the juvenile L. vannamei fed with different dietary proline levels in whole body. Item

Asp Thr Ser Glu Pro Hyp Gly Ala Cys Val Met Ile Leu Tyr Phe Lys His Arg

Proline level (% dry weight) 2.02

2.14

2.27

2.38

2.49

2.6

10.3 ± 0.02 3.84 ± 0.08 3.15 ± 0.13 15.7 ± 0.04 6.46 ± 0.15 1.44 ± 0.02a 7.63 ± 0.12 7.04 ± 0.06 0.59 ± 0.01 4.93 ± 0.01 2.51 ± 0.04 4.22 ± 0.01 7.18 ± 0.03 3.31 ± 0.01 4.51 ± 0.01 7.72 ± 0.05 2.02 ± 0 7.45 ± 0.06

10.19 ± 0.06 3.87 ± 0.06 3.29 ± 0.15 15.67 ± 0.11 6.35 ± 0.14 1.48 ± 0.02ab 7.91 ± 0.24 6.77 ± 0.15 0.61 ± 0.02 4.89 ± 0.03 2.49 ± 0.01 4.24 ± 0.02 7.14 ± 0.05 3.27 ± 0.01 4.48 ± 0.01 7.71 ± 0.1 2.01 ± 0.03 7.63 ± 0.14

10.12 ± 0.02 3.89 ± 0.05 3.23 ± 0.08 15.62 ± 0.09 6.64 ± 0.18 1.51 ± 0.02b 7.83 ± 0.07 6.83 ± 0.07 0.59 ± 0.03 4.94 ± 0.03 2.45 ± 0.02 4.2 ± 0.02 7.09 ± 0.02 3.32 ± 0.05 4.52 ± 0.04 7.65 ± 0.02 2.02 ± 0.01 7.55 ± 0.02

10.19 ± 0.02 3.92 ± 0.04 3.32 ± 0.08 15.7 ± 0.02 6.12 ± 0.17 1.55 ± 0.01b 8.08 ± 0.19 6.72 ± 0.1 0.59 ± 0.01 4.92 ± 0.05 2.45 ± 0.07 4.2 ± 0.04 7.14 ± 0.05 3.3 ± 0.04 4.49 ± 0.04 7.65 ± 0.04 2 ± 0.02 7.64 ± 0.06

10.17 ± 0.1 3.88 ± 0.03 3.24 ± 0.09 15.66 ± 0.08 6.5 ± 0.19 1.52 ± 0.02b 7.9 ± 0.06 6.7 ± 0.1 0.6 ± 0 4.94 ± 0.05 2.48 ± 0.02 4.21 ± 0.02 7.12 ± 0.01 3.27 ± 0.04 4.49 ± 0.03 7.73 ± 0.02 2.04 ± 0.02 7.55 ± 0.08

10.16 ± 0.01 3.88 ± 0.05 3.31 ± 0.13 15.73 ± 0.05 6.15 ± 0.21 1.55 ± 0.03b 8.08 ± 0.1 6.75 ± 0.08 0.61 ± 0.01 4.97 ± 0.05 2.47 ± 0.02 4.22 ± 0.01 7.12 ± 0.01 3.3 ± 0.02 4.52 ± 0.02 7.61 ± 0.02 1.99 ± 0.02 7.57 ± 0.05

Data represent mean ± SEM of three replicates. Values in the same line with different letters are significantly different (P b 0.05).

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Table 4 Hematological parameters of the juvenile L. vannamei fed with different dietary proline levels. Item

Proline level (% dry weight)

AST(U L−1) ALT(U L−1) Urea (mmol L−1)

2.02

2.14

2.27

2.38

2.49

2.6

525.7 ± 16.18 460.33 ± 11.03 1.93 ± 0.2

514.55 ± 26.74 444.85 ± 22.55 1.8 ± 0.25

521.93 ± 35.87 439 ± 22.99 1.77 ± 0.22

515.23 ± 23.22 429.2 ± 7.35 1.4 ± 0.15

493.33 ± 35.93 411.45 ± 4.05 1.5 ± 0.1

490.93 ± 18.28 412 ± 17.36 1.73 ± 0.07

Data represent mean ± SEM of three replicates.

Protein productive value (PPV, %) = 100 × protein gain (g) / protein intake (g) Hepatosomatic index (HSI) = 100 × wet hepatopancreas weight (g) / wet body weight (g) Survival (%) = 100 × (final amount of shrimps) / (initial amount of shrimps)

3.3. Hematological parameters Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and urea levels in hemolymph decreased as the dietary proline levels increased (Table 4), although there is no significant difference. The lowest AST and AST activity observed in 2.49% proline group. And the lowest urea content observed in 2.38% proline group.

Where Wt is the final body weight (g), Wi is the initial body weight (g), and t is the experimental duration in days. The results are presented as the means ± SEM. All the data were statistically analyzed by SPSS 19.0 (SPSS, Chicago, IL, USA). The data were first tested for homogeneity, if the data had similar variances, then one-way ANOVA was used to test the main effect of dietary manipulation. When there were significant differences (P b 0.05), the group means were further compared using Duncan's multiple range test. But if the data did not have similar variances, the non-parameter Kruskal– Wallis test was applied, and followed by all pairwise multiple comparisons if the results of Kruskal–Wallis test shown significant difference (P b 0.05). Broken-line analysis (Robbins et al., 1979) was used to estimate the optimum dietary level of proline.

3. Results 3.1. Growth performance and feed utilization The growth performance and feed utilization of the shrimp fed with different proline levels are shown in Table 2. After the 8-week feeding trial, HSI significantly increased as the dietary proline levels increased from 2.02% to 2.38%. However, it decreased with a further increase in proline level. Weight gain (WG), specific growth ratio (SGR), survival, feed efficiency (FE), feed intake (FI), protein productive value (PPV) and protein efficiency ratio (PER) were not significantly affected by dietary proline levels.

3.2. Whole body free amino acid ratio Effect of dietary proline levels on free amino acid ratio (% total AA) of whole body is presented in Table 3. Hydroxyproline ratios linearly increased with the increasing dietary proline levels (P = 0.006), arginine and glycine ratios also show an increase trend with the increasing proline levels, although there were no significant differences (P = 0.15, 0.07).

3.4. NH3 exposure stress The survivals of the shrimps after NH3 exposure stress is presented in Table 5. Survival of shrimps after 60 h NH3 exposure stress were significantly increased with the dietary proline levels up to 2.38% (P = 0.015). Survival of shrimps after 12, 24 and 36 h did not show any statistical differences among the dietary treatments. Broken-line analysis based on survival of shrimps after 60 h NH3 stress shown that the optimal dietary proline level should be 2.34% of diet (Fig. 1).

3.5. Anti-oxidant and immune related parameters of hemolymph before and after NH3 exposure stress As shown in Table 6, before the NH3 exposure stress, NO, NOS, TAOC and PO show no significant differences among all the treatments (P = 0.103, 0.251, 0.861 and 0.161, respectively). While after the NH3 exposure stress, PO significantly increased with increasing dietary proline levels (P = 0.016), the highest values were observed in 2.49% proline group. Broken-line analysis based on PO after NH3 stress shown that the optimal dietary proline level should be 2.29% of diet (Fig. 2).

3.6. Anti-oxidant and immune related parameters of hepatopancreas before and after NH3 exposure stress As shown in Table 7, after the NH3 exposure, NO and POX activity significantly increased with increasing dietary proline levels (P = 0.026, 0.016), the highest values were observed in 2.49% proline group. And the shrimps fed with control diet had the lowest T-AOC and NOS than other groups no matter before or after the NH3 exposure. Broken-line analysis based on POX after NH3 stress shown that the optimal dietary proline level should be 2.32% of diet (Fig. 3).

Table 5 Survival of the juvenile L. vannamei fed with different dietary proline levels after the NH3 stress. Time

12 h 24 h 36 h 48 h 60 h

Proline level (% dry weight) 2.02

2.14

2.27

2.38

2.49

2.6

90.48 ± 9.52 90.48 ± 9.52 85.71 ± 14.29 76.19 ± 9.52 61.9 ± 9.52a

95.24 ± 4.76 82.14 ± 8.99 77.38 ± 4.29 72.62 ± 1.19 73.21 ± 1.79abc

100 ± 0 85.71 ± 8.25 76.19 ± 4.76 66.67 ± 9.52 64.29 ± 12.6ab

100 ± 0 100 ± 0 95.83 ± 4.17 91.07 ± 4.49 86.31 ± 8.27bc

95.24 ± 4.76 95.24 ± 4.76 95.24 ± 4.76 83.81 ± 1.9 83.81 ± 1.9abc

93.75 ± 6.25 93.75 ± 6.25 93.75 ± 6.25 89.17 ± 5.83 89.17 ± 6.25c

Data represent mean ± SEM of three replicates. Values in the same line with different letters are significantly different (P b 0.05).

Hematological PO activity after NH3 stress

Survival rate (%) after 60h NH3stress

S.-W. Xie et al. / Aquaculture 448 (2015) 105–111

100 90 80 70

y = 65.917x - 69.892 Y=84.51 R² = 0.9783

60

50

Xopt=2.34

40 2

2.2

2.4

2.6

109

2.1 1.9 1.7 1.5 y = 2.387x - 3.7195 Y= R² = 0.8066

1.3 1.1 0.9 0.7

Xopt=2.29

0.5

2.8

Dietary proline level (% dry diet)

Dietary proline level (% dry diet) Fig. 1. Survival (%) of L. vannamei fed graded levels of proline after the 60 h NH3 stress.

Fig. 2. Hematological PO activity of L. vannamei fed graded levels of proline after NH3 stress.

4. Discussion Many previous studies have shown that proline is a nutritionally essential AA for poultry and young mammals (Baker, 2009; Ball et al., 1986; Graber et al., 1970; Wu et al., 2011) due to inadequate endogenous synthesis. But whether proline can be sufficiently synthesized in fish to meet requirements for maintenance, maximal growth or optimal health remains largely unknown (Li et al., 2009a). Zhang et al. (2006) reported proline concentration in muscle of rainbow trout alevin was dependent on dietary proline and that endogenous synthesis of proline from glutamate could not meet the requirement for proline. Aksnes et al. (2008) found that dietary supplementation of 0.28% hydroxyproline, but not 0.28% proline, increased growth rate and modified bone composition of salmon (about 110 g) fed high plant protein diet. These studies indicate that effect of dietary proline on growth may depend on many factors such as diet composition, fish species, fish size and the supplemented concentration. Since proline is abundant in fish meal and scarce in plant protein sources (Rezaei et al., 2013a; Wu et al., 2011), proline is insufficient in the low fish meal diet used in this study. The present study demonstrated that dietary proline has no significantly effect on the growth performance of L. vannamei. But the highest value of WG observed in the 2.49% proline group was 4.66% higher than the value in the basic group, the improvement to growth mainly due to proline could enhance the FI (5.6% higher than the basic group), which is similar with traditional thought that proline is a dispensable AA for fish and promotes feed intake (Li et al., 2009a). These indicated that effect of dietary proline on growth may depend on many factors such as diet composition, fish species, fish size and the supplemented concentration. The present result indicated that, the rate of proline synthesis in L. vannamei could meet the requirement for maximal growth even fed with low proline diet. Hydroxyproline, arginine and glycine are three functional AA, which play important role in nutrient utilization and immune response (Aksnes et al., 2008; Wang et al., 2013; Wu et al., 2009). In the present study, hydroxyproline ratios (% total AA) in the whole body were

significantly increased with the proline supplementation, arginine and glycine ratios also show a tendency of increase. Hydroxyproline can be synthesized from proline via hydroxylation (Wu et al., 2011), arginine can be synthesized from proline through ornithine aminotransferases (Tomlinson et al., 2011), and glycine also could be synthesized from proline and hydroxyproline through several steps (Wu et al., 2011; Xie et al., 2014). So that the increase of these AA is mainly due to the proline supplementation. The AST and ALT are normally used as helpful indicators of general health status of animals, high levels of them in plasma indicate the liver damage (Hemre et al., 1996; Jin et al., 2013). In the present study, both AST and ALT decreased with the dietary proline levels increasing to 2.49%, together with the result that HSI significantly increased with the dietary proline levels increasing to 2.38%, may indicate high levels of proline improve the liver health status of L. vannamei. In the present study, the survivals of shrimps after 60 h NH3 exposure stress were significantly increased with the dietary proline levels increased to 2.38% (P = 0.015). It was the first time we evaluate the attenuate effect of dietary proline on environment stress in aquatic animals, similar studies have done in bacteria, plants, chicks, piglets and humans before (Hamasu et al., 2009; Lopez-Carrion et al., 2008; Madan et al., 1995; Phang et al., 2008a). The present and previous studies suggested that L-proline may have a critical role to attenuate stress response in a variety of species. The present data of enzyme activity in hemolymph and hepatopancreas indicated that the function of proline to attenuate stress mainly due to four pathways: 1) Produce NO via arginine, arginine is one of the most important precursor for synthesis of NO (Kleinert et al., 2004; Li et al., 2009b; Wu, 2010), which plays crucial roles in many diverse processes, including vasodilation, immune responses, neurotransmission and adhesion of leucocytes (Bogdan et al., 2000; Li et al., 2007). In response to inflammatory conditions, hepatocytes can be induced to

Table 6 Hematological enzyme activity of the juvenile L. vannamei fed with different dietary proline levels before and after the NH3 stress. Proline level (% dry weight) 2.02 Before the NH3 stress NO (mmol mL−1) NOS (U mL−1) T-AOC (U mL−1) PO (U mL−1) After the NH3 stress NO (mmol mL−1) NOS (U mL−1) T-AOC (U mL−1) PO (U mL−1)

78.47 ± 5.66 25.72 ± 0.65 0.68 ± 0.18 1.08 ± 0.09

359.67 ± 15.57 13.82 ± 1.84 1.95 ± 0.4 1.19 ± 0.19a

2.14 85.6 ± 12.78 24.73 ± 1.9 0.59 ± 0.05 1.3 ± 0.3

364.02 ± 31.2 14.66 ± 2.43 2.38 ± 0.15 1.22 ± 0.21a

2.27

2.38

2.49

2.6

106.67 ± 11.09 25.27 ± 2.1 0.59 ± 0.08 1.19 ± 0.15

92.46 ± 2.43 25.89 ± 2.2 0.59 ± 0.05 0.88 ± 0.18

94.08 ± 13.97 26.21 ± 1.49 0.73 ± 0.1 1.03 ± 0.24

79.17 ± 3.2 23.48 ± 0.25 0.6 ± 0.19 0.91 ± 0.12

396.08 ± 3.12 14.86 ± 2.15 2.43 ± 0.18 1.78 ± 0.17ab

375.28 ± 18.31 13.64 ± 0.62 2.4 ± 0.06 1.6 ± 0.21ab

412.24 ± 18.07 11.93 ± 0.21 2.66 ± 0.1 1.93 ± 0.14b

391.57 ± 4.94 12.55 ± 0.2 2.46 ± 0.07 1.6 ± 0.1ab

Data represent mean ± SEM of three replicates. Values in the same line with different letters are significantly different (P b 0.05).

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Table 7 Hepatopancreas enzyme activity of the juvenile L. vannamei fed with different dietary proline levels before and after the NH3 stress. Proline level (% dry weight) 2.02

2.14

2.27

Before the NH3 stress NO (mmol mg protein−1) NOS (U mg protein−1) POX (U mg protein−1) T-AOC (U mg protein−1)

5.19 ± 0.39 1.86 ± 0.07 5.27 ± 0.16 0.24 ± 0.03

5.5 ± 0.26 2.11 ± 0.45 5.32 ± 0.25 0.27 ± 0.01

After the NH3 stress NO (mmol mg protein−1) NOS (U mg protein−1) POX (U mg protein−1) T-AOC (U mg protein−1)

10.72 ± 2.24ab 1.99 ± 0.1 10.1 ± 0.78a 0.21 ± 0.02

9.52 ± 1.42a 2.02 ± 0.17 10.59 ± 0.57ab 0.21 ± 0.04

2.38

5.61 ± 0.39 2.22 ± 0.16 5.55 ± 0.33 0.25 ± 0.03

15.81 ± 2.35b 2.1 ± 0.06 13.7 ± 1.37abc 0.19 ± 0.03

2.49

2.6

5.8 ± 0.25 2.24 ± 0.11 5.65 ± 0.19 0.25 ± 0.05

6.02 ± 0.52 2.11 ± 0.1 5.38 ± 0.21 0.23 ± 0.02

5.64 ± 0.29 2.04 ± 0.09 5.64 ± 0.29 0.23 ± 0

14.27 ± 1.8ab 2.2 ± 0.24 14.27 ± 1.8bc 0.23 ± 0.05

16.44 ± 1.05b 2.12 ± 0.13 14.6 ± 1.2c 0.22 ± 0.04

12.64 ± 0.7ab 2.09 ± 0 13.31 ± 0.5abc 0.2 ± 0.02

Data represent mean ± SEM of three replicates. Values in the same line with different letters are significantly different (P b 0.05).

Hematological POX activity after NH3 stress

produce NO through NOS (Wu and Morris, 1998). In the present study, NO contents in hemolymph and hepatopancreas after the NH3 stress increased with the dietary proline up to 2.49%, the NOS activity in hepatopancreas before and after the NH3 stress also show a tendency of increase with the increasing dietary proline, which indicated that proline supplementation promoted the NO synthesis through NOS in shrimp. 2) Enhance POX activity, one of the central enzymes in the proline cycle is POX, which oxidate proline to P5C (Phang et al., 2008b). In the present study, the POX activity in hepatopancreas after NH3 stress significantly increased with the increasing dietary proline levels, similar with the recent advances in proline metabolism that proline metabolic pathway was mobilized primarily and POX is upregulated directly by both genotoxic, nutrition and inflammatory stress (Hamasu et al., 2009; Phang et al., 2008a, 2010). 3) Scavenge free radicals, a common response to acute NH3 stress is the stimulated generation of free radicals particularly the reactive oxygen species (Hegazi et al., 2010), they are capable of causing considerable cellular damage through peroxidation of membrane lipid components, both low molecular weight antioxidant metabolites and antioxidative enzymes contribute to scavenging of free radicals (Kaul et al., 2008). The T-AOC activity in the hemolymph after stress in the present study increased with the increasing dietary proline levels, indicated that high levels of proline enhanced the capacity of juvenile L. vannamei to scavenge the free radicals. Similar reports have also shown that proline could accumulate in many organisms when they are exposed to environment stresses, and proline mainly to react with OH• and singlet oxygen (1O2) (Mohanty and Matysik, 2001; Smirnoff and Cumbes, 1989), the low ionization potential and the secondary amine of the pyrrolidine ring are key factors in the chemical and physical quenching activity of proline with OH• and 1O2 (Krishnan et al., 2008), so increased levels of proline may activate glutathione synthesis or stabilize antioxidant enzymes during oxidative stress and invoke signaling pathways that upregulate cellular antioxidant defenses. 4) Enhance PO activity, PO plays a crucial role in immune defense of 16 15 14 13

y = 14.532x - 19.684 R² = 0.8661

12 11 10

Xopt=2.32

9 8

2

2.1

2.2

2.3

2.4

2.5

2.6

2.7

Dietary proline level (% dry diet) Fig. 3. Hematological POX activity of L. vannamei fed graded levels of proline after NH3 stress.

invertebrate, and higher PO activity means less susceptible to infection, NH3 stress has shown to significantly decrease PO activity of Macrobrachium rosenbergii and L. vannamei (Cheng and Chen, 2002; Liu and Chen, 2004; Zhang et al., 2012). In the present study, the higher PO activity in the hemolymph after NH3 stress indicated that proline could enhance the immune defense of juvenile L. vannamei. In conclusion, the results from the present study indicated that proline supplementation improved amino acid constituent, anti-oxidative capacity, immune response, NH3 stress tolerance of juvenile L. vannamei, but have no effect on growth. Broken-line analysis based on PO, POX and 60 h survival after NH3 stress showed that the shrimps fed with 2.29– 2.34% proline diet show a better immune performance after NH3 stress. These result indicated that based on health status especially when under stress, proline may be a conditionally essential AA for L. vannamei. Acknowledgments This work was supported by the fund of China Agriculture Research Systerm-47 (CARS-47). References Aksnes, A., Mundheim, H., Toppe, J., Albrektsen, S., 2008. The effect of dietary hydroxyproline supplementation on salmon (Salmo salar L.) fed high plant protein diets. Aquaculture 275, 242–249. AOAC, A.O.O.A., 1995. Official Methods of Analysis of Official Analytical Chemists International. 16th ed. Association of Official Analytical Chemists, Arlington, VA. Baker, D.H., 2009. Advances in protein–amino acid nutrition of poultry. Amino Acids 37, 29–41. Ball, R.O., Atkinson, J.L., Bayley, H.S., 1986. Proline as an essential amino acid for the young pig. Br. J. Nutr. 55, 659–668. Barbieri, E., 2010. Acute toxicity of ammonia in white shrimp (Litopenaeus schmitti) (Burkenroad, 1936, Crustacea) at different salinity levels. Aquaculture 306, 329–333. Bogdan, C., Röllinghoff, M., Diefenbach, A., 2000. The role of nitric oxide in innate immunity. Immunol. Rev. 173, 17–26. Brunton, J.A., Baldwin, M.P., Hanna, R.A., Bertolo, R.F., 2012. Proline supplementation to parenteral nutrition results in greater rates of protein synthesis in the muscle, skin, and small intestine in neonatal Yucatan miniature piglets. J. Nutr. 142, 1004–1008. Chen, Y., Sim, S.S., Chiew, S.L., Yeh, S., Liou, C., Chen, J., 2012. Dietary administration of a Gracilaria tenuistipitata extract produces protective immunity of white shrimp Litopenaeus vannamei in response to ammonia stress. Aquaculture 370, 26–31. Cheng, W., Chen, J., 2002. The virulence of Enterococcus to freshwater prawn Macrobrachium rosenbergii and its immune resistance under ammonia stress. Fish Shellfish Immunol. 12, 97–109. Eddy, F.B., 2005. Ammonia in estuaries and effects on fish. J. Fish Biol. 67, 1495–1513. Gaye-Siessegger, J., Focken, U., Abel, H., Becker, K., 2007. Influence of dietary non-essential amino acid profile on growth performance and amino acid metabolism of Nile tilapia, Oreochromis niloticus (L.). Comp. Biochem. Physiol. A Mol. Integr. Physiol. 146, 71–77. Geraert, P., Mercier, Y., 2010. Amino Acids: Beyond the Building Blocks. ADISSEO France SAS, Antony. Graber, G., Allen, N.K., Scott, H.M., 1970. Proline essentiality and weight gain. Poult. Sci. 49, 692–697. Hamasu, K., Haraguchi, T., Kabuki, Y., Adachi, N., Tomonaga, S., Sato, H., Denbow, D.M., Furuse, M., 2009. L-proline is a sedative regulator of acute stress in the brain of neonatal chicks. Amino Acids 37, 377–382. Hegazi, M.M., Attia, Z.I., Ashour, O.A., 2010. Oxidative stress and antioxidant enzymes in liver and white muscle of Nile tilapia juveniles in chronic ammonia exposure. Aquat. Toxicol. 99, 118–125.

S.-W. Xie et al. / Aquaculture 448 (2015) 105–111 HEMRE, G.I., Waagbø, R., Hjeltnes, B., Aksnes, A., 1996. Effect of gelatinized wheat and maize in diets for large Atlantic salmon (Salmo salar L.) on glycogen retention, plasma glucose and fish health. Aquac. Nutr. 2, 33–39. Hsieh, S., Ruan, Y., Li, Y., Hsieh, P., Hu, C., Kuo, C., 2008. Immune and physiological responses in Pacific white shrimp (Penaeus vannamei) to Vibrio alginolyticus. Aquaculture 275, 335–341. Hu, C.A., Phang, J.M., Valle, D., 2008. Proline metabolism in health and disease. Amino Acids 35, 651–652. Jin, Y., Tian, L., Zeng, S., Xie, S., Yang, H., Liang, G., Liu, Y., 2013. Dietary lipid requirement on non-specific immune responses in juvenile grass carp (Ctenopharyngodon idella). Fish Shellfish Immunol. 34, 1202–1208. Kaul, S., Sharma, S.S., Mehta, I.K., 2008. Free radical scavenging potential of L-proline: evidence from in vitro assays. Amino Acids 34, 315–320. Kaushik, S.J., Seiliez, I., 2010. Protein and amino acid nutrition and metabolism in fish: current knowledge and future needs. Aquac. Res. 41, 322–332. Kim, S.W., Mateo, R.D., Yin, Y., Wu, G., 2007. Functional amino acids and fatty acids for enhancing production performance of sows and piglets. Asian Aust. J. Anim. Sci. 20, 295. Kleinert, H., Pautz, A., Linker, K., Schwarz, P.M., 2004. Regulation of the expression of inducible nitric oxide synthase. Eur. J. Pharmacol. 500, 255–266. Krishnan, N., Dickman, M.B., Becker, D.F., 2008. Proline modulates the intracellular redox environment and protects mammalian cells against oxidative stress. Free Radic. Biol. Med. 44, 671–681. Li, P., Yin, Y., Li, D., Woo Kim, S., Wu, G., 2007. Amino acids and immune function. Br. J. Nutr. 98, 237–252. Li, P., Mai, K., Trushenski, J., Wu, G., 2009a. New developments in fish amino acid nutrition: towards functional and environmentally oriented aquafeeds. Amino Acids 37, 43–53. Li, X., Bazer, F.W., Gao, H., Jobgen, W., Johnson, G.A., Li, P., McKnight, J.R., Satterfield, M.C., Spencer, T.E., Wu, G., 2009b. Amino Acids and Gaseous Signaling. pp. 65–78. Li, X., Rezaei, R., Li, P., Wu, G., 2011. Composition of amino acids in feed ingredients for animal diets. Amino Acids 40, 1159–1168. Liu, C., Chen, J., 2004. Effect of ammonia on the immune response of white shrimpLitopenaeus vannamei and its susceptibility to Vibrio alginolyticus. Fish Shellfish Immunol. 16, 321–334. Liu, H., Xie, S., Zhu, X., Lei, W., Han, D., Yang, Y., 2008. Effects of dietary ascorbic acid supplementation on the growth performance, immune and stress response in juvenile Leiocassis longirostris Günther exposed to ammonia. Aquac. Res. 39, 1628–1638. Lopez-Carrion, A.I., Castellano, R., Rosales, M.A., Ruiz, J.M., Romero, L., 2008. Role of nitric oxide under saline stress: implications on proline metabolism. Biol. Plant. 52, 587–591. Madan, S., Nainawatee, H.S., Jain, R.K., Chowdhury, J.B., 1995. Proline and proline metabolising enzymes in in-vitro selected NaCl-tolerant Brassica juncea L. under salt stress. Ann. Bot. 76, 51–57. Mauriz, J.L., Matilla, B., Culebras, J.M., Gonzalez, P., Gonzalez-Gallego, J., 2001. Dietary glycine inhibits activation of nuclear factor kappa B and prevents liver injury in hemorrhagic shock in the rat. Free Radic. Biol. Med. 31, 1236–1244. Mohanty, P., Matysik, J., 2001. Effect of proline on the production of singlet oxygen. Amino Acids 21, 195–200. Phang, J.M., Pandhare, J., Liu, Y., 2008a. The metabolism of proline as microenvironmental stress substrate. J. Nutr. 138. Phang, J.M., Donald, S.P., Pandhare, J., Liu, Y., 2008b. The metabolism of proline, a stress substrate, modulates carcinogenic pathways. Amino Acids 35, 681–690.

111

Phang, J.M., Liu, W., Zabirnyk, O., 2010. Proline metabolism and microenvironmental stress. Annu. Rev. Nutr. 30, 441–463. Randall, D.J., Tsui, T., 2002. Ammonia toxicity in fish. Mar. Pollut. Bull. 45, 17–23. Rezaei, R., Wang, W.W., Wu, Z.L., Dai, Z., Wang, J., Wu, G., 2013a. Biochemical and physiological bases for utilization of dietary amino acids by young pigs. J. Anim. Sci. Biotechnol. 4. Rezaei, R., Wang, W., Wu, Z., Dai, Z., Wang, J., Wu, G., 2013b. Biochemical and physiological bases for utilization of dietary amino acids by young pigs. J. Anim. Sci. Biotechnol. 4, 7. Robbins, K.R., Norton, H.W., Baker, D.H., 1979. Estimation of nutrient requirements from growth data. J. Nutr. 109, 1710–1714. Smirnoff, N., Cumbes, Q.J., 1989. Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry 28, 1057–1060. Takeuchi, T., Gatlin III, D.M., 2007. Amino Acids, Peptides. Dietary Supplements for the Health and Quality of Cultured Fish. CAB International, Oxon, UK, pp. 47–63. Tomlinson, C., Rafii, M., Ball, R.O., Pencharz, P.B., 2011. Arginine can be synthesized from enteral proline in healthy adult humans. J. Nutr. 141, 1432–1436. Trussell, R.P., 1972. The percent un-ionized ammonia in aqueous ammonia solutions at different pH levels and temperatures. J. Fish. Board Can. 29, 1505–1507. Wang, W., Wu, Z., Dai, Z., Yang, Y., Wang, J., Wu, G., 2013. Glycine metabolism in animals and humans: implications for nutrition and health. Amino Acids 1–15. Wilson, R.P., 2002. Amino acids and proteins. Fish Nutr. 143. Wu, G., 2010. Functional amino acids in growth, reproduction, and health. Adv. Nutr. 1, 31–37. Wu, G., 2013. Functional amino acids in nutrition and health. Amino Acids 45, 407–411. Wu, G., MORRIS, J.S., 1998. Arginine metabolism: nitric oxide and beyond. Biochem. J. 336, 1–17. Wu, G., Bazer, F.W., Datta, S., Johnson, G.A., Li, P., Satterfield, M.C., Spencer, T.E., 2008. Proline metabolism in the conceptus: implications for fetal growth and development. Amino Acids 35, 691–702. Wu, G., Bazer, F.W., Davis, T.A., Kim, S.W., Li, P., Rhoads, J.M., Satterfield, M.C., Smith, S.B., Spencer, T.E., Yin, Y., 2009. Arginine metabolism and nutrition in growth, health and disease. Amino Acids 37, 153–168. Wu, G., Bazer, F.W., Burghardt, R.C., Johnson, G.A., Kim, S.W., Knabe, D.A., Li, P., Li, X., McKnight, J.R., Satterfield, M.C., 2011. Proline and hydroxyproline metabolism: implications for animal and human nutrition. Amino Acids 40, 1053–1063. Xie, S., Tian, L., Jin, Y., Yang, H., Liang, G., Liu, Y., 2014. Effect of glycine supplementation on growth performance, body composition and salinity stress of juvenile Pacific white shrimp, Litopenaeus vannamei fed low fishmeal diet. Aquaculture 418, 159–164. Yue, F., Pan, L., Xie, P., Zheng, D., Li, J., 2010. Immune responses and expression of immune-related genes in swimming crab Portunus trituberculatus exposed to elevated ambient ammonia-N stress. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 157, 246–251. Zhang, Y., Dabrowski, K., Hliwa, P., Gomulka, P., 2006. Indispensable amino acid concentrations decrease in tissues of stomachless fish, common carp in response to free amino acid-or peptide-based diets. Amino Acids 31, 165–172. Zhang, J., Liu, Y., Tian, L., Yang, H., Liang, G., Xu, D., 2012. Effects of dietary mannan oligosaccharide on growth performance, gut morphology and stress tolerance of juvenile Pacific white shrimp, Litopenaeus vannamei. Fish Shellfish Immunol. 33, 1027–1032.