# Effects of Dietary Arginine Level on Growth Performance and Immune Response inMonopterus albus

Authors: Huanhuan Huo; Mo Peng; Mingjin Yu; Yazhou Zhang; Liufeng Xiong; Qiubai Zhou
Journal: Aquaculture Research
 (2023)

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## Abstract

To assess the dietary arginine requirement ofMonopterus albus (21 ± 0.03 g), we compared six diets containing different arginine levels (2.03%, 2.58%, 3.16%, 3.63%, 4.24%, and 4.88%). The results revealed that dietary arginine content had a significant effect on weight gain (WG), specific growth rate (SGR), IL-1β, IL-10, lysozyme, and complement C3 activity in Monopterus albus (p<0.05). The WG and SGR increased with increasing dietary arginine levels, and the regression analysis found that the optimum dietary arginine level for growth of rice field eel was 4.28–4.35% of the dry diet. However, the C3 and LZM activities peaked in the 2.58% arginine diet group (p<0.05). The dietary arginine level had no significant effect on the viscerosomatic index, hepatosomatic index, condition factor, survival rate, nitric oxide, total nitric oxide synthase, insulin, growth hormone, trypsin, lipase, or amylase activities (p>0.05). Arginine can significantly promote Monopterus albus growth; however, the immunity of Monopterus albus decreases as the arginine level exceeds 2.58%. A comprehensive assessment of growth and immunity recommends the optimum arginine level for Monopterus albus was 3.16–3.63% of the dry diet, corresponding to 6.58–7.70% of the dietary protein.


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## Body

## 1. Introduction

Amino acids are important molecules in fish nutrition as they play critical roles in protein synthesis, immune function, and health. Among the essential amino acids, arginine is a functional amino acid in animals. In fish, arginine is an essential amino acid with different physiological roles in growth performance and health [1]. Diet is the main source of arginine for successfully culturing farmed fish species. The arginine requirement in fish ranges from 3.0% to 8.1% of the dietary protein. The growth promoting effect of arginine may induce a somatotropic axis in fish and is characterized by an increase in insulin, growth hormone (GH), and insulin-like growth factor 1 (IGF-1). In addition, evidence suggests arginine may contribute to stress and cortisol responses in fish [2, 3]. Fish immunomodulation caused by arginine has been suggested in several species, as the amino acid is involved in nitric oxide (NO) production, polyamine synthesis, inflammation, and innate immune responses [4, 5]. Arginine deficiency can lead to severe immune-related issues in different fish species, including fewer leukocytoses and haemagglutination [6]; as well as decreased resistance against pathogen challenges. Nevertheless, surplus levels of dietary arginine can decrease resistance in some teleost species [3, 4, 7, 8]. Therefore, it is necessary to study the optimal requirement of arginine for each fish.Rice field eel (Monopterus albus, M. albus) is a subtropical freshwater benthic fish inhabiting caves, muddy ponds, swamps, rice fields, and burrows within moist Earth [9]. This fish is an important economic species distributed in central and eastern areas with a high value in food production. Currently, research on the nutritional requirements of swamp eel has involved identifying optimum quantities of protein and fat, protein-energy ratios, and methionine [9–11]. However, the dietary arginine requirements for swamp eels have not been investigated. This experiment examines the effects of dietary arginine on growth performance, serum biochemical indices, and the nonspecific immunity index in swamp eels, to determine the optimum dietary arginine requirement for M. albus.


## 2. Materials and Methods

### 2.1. Diet Preparation

Six isonitrogenous and isolipidic diets (named D1, D2, D3, D4, D5, and D6) were formulated containing graded levels of L-arginine with approximately 0.6% increments (Table1). The final levels of arginine in the diets were 2.03%, 2.58%, 3.16%, 3.63%, 4.24%, and 4.88% (dry weight), respectively. Arginine was added to crystalline L-arginine which was analyzed by reverse phase high-performance liquid chromatography (HPLC, HP1100, USA). Arginine was purchased from Henan Wanbang Industrial Co., Ltd (China). All dietary amino acids were consistent in all diets except for arginine and glycine (Table 2). Dietary arginine was quantitatively increased at the expense of glycine. All ingredients were ground into a powder using 80-mesh. The resulting powder was thoroughly mixed with oil and water before being forced through a pelletizer. Pellets were manufactured by passing the moistened mixture through a 1-mm die using a pelletizer (Product model 40, Jindouyun Industrial Co., Ltd, China). The temperature is 120∼130°C and pH 7.0. Pellets were air-dried to approximately 10% moisture. After drying, all diets were sealed in bags and stored at −20°C until required.Table 1
Composition and nutrient levels of experimental diets % used for determining the effects of dietary arginine on the growth and biochemical activities ofM.albus.
IngredientsD1D2D3D4D5D6Fish meal30.0030.0030.0030.0030.0030.00Casein10.0010.0010.0010.0010.0010.00Soybean meal16.0016.0016.0016.0016.0016.00Corn gluten meal10.0010.0010.0010.0010.0010.00Wheat flour8.468.468.468.468.468.46α starch12.0012.0012.0012.0012.0012.00Fish oil3.003.003.003.003.003.00Ca (H2PO4)21.501.501.501.501.501.50Choline chloride (50%)0.500.500.500.500.500.50Premix11.001.001.001.001.001.00Amino acid mixture24.504.504.504.504.504.50Crystalline glycine3.002.401.801.200.60Crystalline arginine0.601.201.802.403.00Calcium propanoate0.030.030.030.030.030.03Ethoxyquin0.010.010.010.010.010.01Total100.00100.00100.00100.00100.00100.00Nutrient levels3Moisture8.018.428.888.498.578.24Crude protein47.6447.3248.0647.1747.4548.13Crude fat5.545.495.145.375.525.36Ash8.077.878.047.867.808.30Carbohydrates30.7430.9029.8830.8930.6629.97Nitrogen free extract29.4829.6328.6129.6329.3928.71Gross energy415.5815.4815.5315.4015.5315.631Provided by MGO Ter bio-tech (Qingdao, Shandong, China). Vitamin and mineral premix composition (mg/kg diet): KCl, 200; KI (1%), 60; CoCl2·6H2O (1%), 50; CuSO4·5H2O, 30; FeSO4·H2O, 400; ZnSO4·H2O, 400; MnSO4·H2O, 150; Na2SeO3·5H2O (1%), 65; MgSO4·H2O, 2000; zeolite power, 3645.85; VB1, 12; riboflavin, 12; VB6, 8; VB12, 0.05; VK3, 8; inositol, 100; pantothenic acid, 40; nicotinic acid, 50; folic acid, 5; biotin, 0.8; VA, 25; VD3, 5; VE, 50; VC, 100; ethoxyquin, 150; wheat meal, 2 434.15. 2Amino acid mixture provides the following per kg of diet: Asp 4.89 g, Thr 2.38 g, Ser, 2.13 g, Glu 7.63 g, Pro 2.74 g, Ala 3.27 g, Cys 0.59 g, Val 2.51 g, Met 1.76 g, Ile 2.45 g, Leu 4.09 g, Tyr 1.83 g, Phe 2.24 g, His 1.26 g, Lys 4.74 g, Trp 0.47 g. 3Measured values. 4Calculated as protein 24 kJ/g, fat 38 kJ/g, starch 17 kJ/g.Table 2
Fifteen amino acids composition of experiment diets %.
Amino acidsDietsD1D2D3D4D5D6Threonine1.901.901.921.871.901.94Valine2.372.382.412.352.402.44Isoleucine1.871.881.891.861.901.93Leucine4.234.214.234.184.294.37Phenylalanine2.152.122.162.132.192.22Arginine2.032.583.163.634.244.88Histidine1.121.111.141.111.121.14Lysine3.053.063.123.033.053.06Aspartic acid3.983.984.023.913.984.04Serine2.062.062.082.022.072.11Glutamic acid7.807.777.857.647.857.96Proline2.922.963.042.912.933.04Glycine4.724.083.602.902.391.83Alanine2.732.632.732.672.732.77Tyrptophan1.621.611.601.581.601.71


### 2.2. Fish and Experimental Conditions

WildM. albus was sourced from Chaohu (Anhui province, China). All specimens were cultured in a floating cage (2.0 × 1.5 × 1.5 m) and fed for two weeks to acclimate to the environment and diet. After fasting for 24 h, 900 similar-sized individuals (21 ± 0.03 g) were randomly distributed into 18 floating cages (2.0 × 1.5 × 1.5 m) at a density of 50 fish per cage. To simulate culture conditions, each cage was 95% filled with fresh grass, Eichhornia crassipes. Monopterus albus was fed once daily at 18:00 pm for 56 days. The feed was crushed and 80% tap water was added to the crumbs to make ball-shaped dough before feeding. Fish were fed by hand to satiation at a feeding rate of 3% of the body weight and was accordingly adjusted for their weight gain during the feeding trial. The environmental conditions throughout the feeding trial were: temperature, 26∼29°C; pH, 7.0 ± 0.5; dissolved oxygen >5.0 mg/L; NH4+-N <0.3 mg/L; and NO2−N <0.1 mg/L.


### 2.3. Sample Collection

After 24 h of fasting, the caged fish were anaesthetized using 100µl/L MS222. Fish were then weighed, counted, and sampled. Blood samples were collected following the method outlined in Hu et al. [12] and stored at −80°C in preparation for analyzing the physiological and biochemical parameters. Visceral mass, liver, body length, and total weight were individually measured or weighed. The liver and head-kidney from three fish were pooled into 1.5 ml (liver) and 1.5 ml (head-kidney) tubes and stored at −20°C in preparation for determining the digestive and immune system parameters.The experiment was performed in strict accordance with the guidelines and ethical principles of the Experiment Animal Welfare Ethics Committee of China. The experimental design was approved by the Committee on Research Ethics of the Department of Laboratory Animal Science, Jingxi Agricultural University. All efforts were made to minimize fish suffering.


### 2.4. Biochemical Analysis

Plasma complement component 3 (C3), lysozyme (LZM), nitric oxide (NO), total nitric oxide synthase (TNOS), growth hormone (GH), insulin (INS), LPS (lipase), AMS (amylase), and typsin (TPS) were assayed using commercial kits (Nanjing Jiancheng Biotechnic Institute, China).


### 2.5. Real-Time Quantitative PCR

Sampling the total RNA isolation of the head-kidney used Trizol Reagent (Invitrogen) following the instructions provided by the manufacturer. Quantitative real-time PCR (qPCR) analyses used a quantitative thermal cycler (Mastercycler ep realplex; Eppendorf). The amplification was performed in a total volume of 20µl and contained 10 µl of power SYBR Green PCR Master Mix (Applied Biosystems), 1 µl of each primer (10 µmol/l), 6 µl of nuclease-free water and 2 µl of cDNA mix. The qPCR program was as follows: 95°C for 90 s, 40 cycles of 95°C for 5 s, 60°C for 15 s, and 72°C for 20 s. The real-time RT-PCR primer pairs for IL-1β and IL-10 were designed by Primer Premier 5.0 based on the published nucleotide sequences listed in Table 3. The target gene mRNA concentration was normalized to the mRNA concentration of the reference gene 18S, a housekeeping gene of M. albus.Table 3
Real-time PCR primer sequences were used to amplify IL-1β and IL-10 gene of M. albushead-kidney.
GenePrimer sequence (5′--3′)Annealing temperatureFragment sizeIL-1βFAGT​TGT​TTG​CGG​ACC​GAG​AT58.4127RGCA​ATC​TGG​CAT​TTC​CTC​ACA64.2IL-10FGGA​CAA​AGA​TGG​GAA​ACG​AAA56.4158RGCC​AGG​TAT​TTG​GTC​TGT​TCA56.418SFGTG​GAG​CGA​TTT​GTC​TGG​TTA57.8162RCGG​ACA​TCT​AAG​GGC​ATC​AC57.7Note. The reference gene 18s is a housekeeping gene of M. albus.


### 2.6. Evaluation of Growth Parameters

The growth performance ofM. albus in response to varying levels of dietary arginine was measured as a function of weight gain by calculating the following parameters:Weight gain (WG, %) = 100 × (final total weight–initial total weight)/initial total weight; special growth rate (SGR, %/day) = 100 × (Ln final individual weight–Ln initial individual weight)/number of days; condition factor (CF, g/cm3) = 100 × (body weight, g)/(body length, cm)3; viscerosomatic index (VSL, %) = 100 × (viscera weight, g)/(whole body weight, g); hepatosomatic index (HSI, %) = 100 × (liver weight, g)/(whole body weight, g); survival rate (SR, %) = final number of fish × 100/initial number of fish.


### 2.7. Statistical Analysis

The results were expressed as mean ± SD. (standard deviation of the mean) and were analyzed using one-way ANOVA in SPSS 19.0 software. Any significant differences were further investigated by comparing the group means using Turkey’s test. Statistical significance was considered ifp<0.05.


## 2.1. Diet Preparation

Six isonitrogenous and isolipidic diets (named D1, D2, D3, D4, D5, and D6) were formulated containing graded levels of L-arginine with approximately 0.6% increments (Table1). The final levels of arginine in the diets were 2.03%, 2.58%, 3.16%, 3.63%, 4.24%, and 4.88% (dry weight), respectively. Arginine was added to crystalline L-arginine which was analyzed by reverse phase high-performance liquid chromatography (HPLC, HP1100, USA). Arginine was purchased from Henan Wanbang Industrial Co., Ltd (China). All dietary amino acids were consistent in all diets except for arginine and glycine (Table 2). Dietary arginine was quantitatively increased at the expense of glycine. All ingredients were ground into a powder using 80-mesh. The resulting powder was thoroughly mixed with oil and water before being forced through a pelletizer. Pellets were manufactured by passing the moistened mixture through a 1-mm die using a pelletizer (Product model 40, Jindouyun Industrial Co., Ltd, China). The temperature is 120∼130°C and pH 7.0. Pellets were air-dried to approximately 10% moisture. After drying, all diets were sealed in bags and stored at −20°C until required.Table 1
Composition and nutrient levels of experimental diets % used for determining the effects of dietary arginine on the growth and biochemical activities ofM.albus.
IngredientsD1D2D3D4D5D6Fish meal30.0030.0030.0030.0030.0030.00Casein10.0010.0010.0010.0010.0010.00Soybean meal16.0016.0016.0016.0016.0016.00Corn gluten meal10.0010.0010.0010.0010.0010.00Wheat flour8.468.468.468.468.468.46α starch12.0012.0012.0012.0012.0012.00Fish oil3.003.003.003.003.003.00Ca (H2PO4)21.501.501.501.501.501.50Choline chloride (50%)0.500.500.500.500.500.50Premix11.001.001.001.001.001.00Amino acid mixture24.504.504.504.504.504.50Crystalline glycine3.002.401.801.200.60Crystalline arginine0.601.201.802.403.00Calcium propanoate0.030.030.030.030.030.03Ethoxyquin0.010.010.010.010.010.01Total100.00100.00100.00100.00100.00100.00Nutrient levels3Moisture8.018.428.888.498.578.24Crude protein47.6447.3248.0647.1747.4548.13Crude fat5.545.495.145.375.525.36Ash8.077.878.047.867.808.30Carbohydrates30.7430.9029.8830.8930.6629.97Nitrogen free extract29.4829.6328.6129.6329.3928.71Gross energy415.5815.4815.5315.4015.5315.631Provided by MGO Ter bio-tech (Qingdao, Shandong, China). Vitamin and mineral premix composition (mg/kg diet): KCl, 200; KI (1%), 60; CoCl2·6H2O (1%), 50; CuSO4·5H2O, 30; FeSO4·H2O, 400; ZnSO4·H2O, 400; MnSO4·H2O, 150; Na2SeO3·5H2O (1%), 65; MgSO4·H2O, 2000; zeolite power, 3645.85; VB1, 12; riboflavin, 12; VB6, 8; VB12, 0.05; VK3, 8; inositol, 100; pantothenic acid, 40; nicotinic acid, 50; folic acid, 5; biotin, 0.8; VA, 25; VD3, 5; VE, 50; VC, 100; ethoxyquin, 150; wheat meal, 2 434.15. 2Amino acid mixture provides the following per kg of diet: Asp 4.89 g, Thr 2.38 g, Ser, 2.13 g, Glu 7.63 g, Pro 2.74 g, Ala 3.27 g, Cys 0.59 g, Val 2.51 g, Met 1.76 g, Ile 2.45 g, Leu 4.09 g, Tyr 1.83 g, Phe 2.24 g, His 1.26 g, Lys 4.74 g, Trp 0.47 g. 3Measured values. 4Calculated as protein 24 kJ/g, fat 38 kJ/g, starch 17 kJ/g.Table 2
Fifteen amino acids composition of experiment diets %.
Amino acidsDietsD1D2D3D4D5D6Threonine1.901.901.921.871.901.94Valine2.372.382.412.352.402.44Isoleucine1.871.881.891.861.901.93Leucine4.234.214.234.184.294.37Phenylalanine2.152.122.162.132.192.22Arginine2.032.583.163.634.244.88Histidine1.121.111.141.111.121.14Lysine3.053.063.123.033.053.06Aspartic acid3.983.984.023.913.984.04Serine2.062.062.082.022.072.11Glutamic acid7.807.777.857.647.857.96Proline2.922.963.042.912.933.04Glycine4.724.083.602.902.391.83Alanine2.732.632.732.672.732.77Tyrptophan1.621.611.601.581.601.71


## 2.2. Fish and Experimental Conditions

WildM. albus was sourced from Chaohu (Anhui province, China). All specimens were cultured in a floating cage (2.0 × 1.5 × 1.5 m) and fed for two weeks to acclimate to the environment and diet. After fasting for 24 h, 900 similar-sized individuals (21 ± 0.03 g) were randomly distributed into 18 floating cages (2.0 × 1.5 × 1.5 m) at a density of 50 fish per cage. To simulate culture conditions, each cage was 95% filled with fresh grass, Eichhornia crassipes. Monopterus albus was fed once daily at 18:00 pm for 56 days. The feed was crushed and 80% tap water was added to the crumbs to make ball-shaped dough before feeding. Fish were fed by hand to satiation at a feeding rate of 3% of the body weight and was accordingly adjusted for their weight gain during the feeding trial. The environmental conditions throughout the feeding trial were: temperature, 26∼29°C; pH, 7.0 ± 0.5; dissolved oxygen >5.0 mg/L; NH4+-N <0.3 mg/L; and NO2−N <0.1 mg/L.


## 2.3. Sample Collection

After 24 h of fasting, the caged fish were anaesthetized using 100µl/L MS222. Fish were then weighed, counted, and sampled. Blood samples were collected following the method outlined in Hu et al. [12] and stored at −80°C in preparation for analyzing the physiological and biochemical parameters. Visceral mass, liver, body length, and total weight were individually measured or weighed. The liver and head-kidney from three fish were pooled into 1.5 ml (liver) and 1.5 ml (head-kidney) tubes and stored at −20°C in preparation for determining the digestive and immune system parameters.The experiment was performed in strict accordance with the guidelines and ethical principles of the Experiment Animal Welfare Ethics Committee of China. The experimental design was approved by the Committee on Research Ethics of the Department of Laboratory Animal Science, Jingxi Agricultural University. All efforts were made to minimize fish suffering.


## 2.4. Biochemical Analysis

Plasma complement component 3 (C3), lysozyme (LZM), nitric oxide (NO), total nitric oxide synthase (TNOS), growth hormone (GH), insulin (INS), LPS (lipase), AMS (amylase), and typsin (TPS) were assayed using commercial kits (Nanjing Jiancheng Biotechnic Institute, China).


## 2.5. Real-Time Quantitative PCR

Sampling the total RNA isolation of the head-kidney used Trizol Reagent (Invitrogen) following the instructions provided by the manufacturer. Quantitative real-time PCR (qPCR) analyses used a quantitative thermal cycler (Mastercycler ep realplex; Eppendorf). The amplification was performed in a total volume of 20µl and contained 10 µl of power SYBR Green PCR Master Mix (Applied Biosystems), 1 µl of each primer (10 µmol/l), 6 µl of nuclease-free water and 2 µl of cDNA mix. The qPCR program was as follows: 95°C for 90 s, 40 cycles of 95°C for 5 s, 60°C for 15 s, and 72°C for 20 s. The real-time RT-PCR primer pairs for IL-1β and IL-10 were designed by Primer Premier 5.0 based on the published nucleotide sequences listed in Table 3. The target gene mRNA concentration was normalized to the mRNA concentration of the reference gene 18S, a housekeeping gene of M. albus.Table 3
Real-time PCR primer sequences were used to amplify IL-1β and IL-10 gene of M. albushead-kidney.
GenePrimer sequence (5′--3′)Annealing temperatureFragment sizeIL-1βFAGT​TGT​TTG​CGG​ACC​GAG​AT58.4127RGCA​ATC​TGG​CAT​TTC​CTC​ACA64.2IL-10FGGA​CAA​AGA​TGG​GAA​ACG​AAA56.4158RGCC​AGG​TAT​TTG​GTC​TGT​TCA56.418SFGTG​GAG​CGA​TTT​GTC​TGG​TTA57.8162RCGG​ACA​TCT​AAG​GGC​ATC​AC57.7Note. The reference gene 18s is a housekeeping gene of M. albus.


## 2.6. Evaluation of Growth Parameters

The growth performance ofM. albus in response to varying levels of dietary arginine was measured as a function of weight gain by calculating the following parameters:Weight gain (WG, %) = 100 × (final total weight–initial total weight)/initial total weight; special growth rate (SGR, %/day) = 100 × (Ln final individual weight–Ln initial individual weight)/number of days; condition factor (CF, g/cm3) = 100 × (body weight, g)/(body length, cm)3; viscerosomatic index (VSL, %) = 100 × (viscera weight, g)/(whole body weight, g); hepatosomatic index (HSI, %) = 100 × (liver weight, g)/(whole body weight, g); survival rate (SR, %) = final number of fish × 100/initial number of fish.


## 2.7. Statistical Analysis

The results were expressed as mean ± SD. (standard deviation of the mean) and were analyzed using one-way ANOVA in SPSS 19.0 software. Any significant differences were further investigated by comparing the group means using Turkey’s test. Statistical significance was considered ifp<0.05.


## 3. Results

### 3.1. Growth Performance

The results revealed that dietary arginine levels had a significant effect on final body weight, WG, and SGR inM. albus (p<0.05) (Table 4). WG and SGR increased with increasing dietary arginine levels without decreasing until the arginine level reached 4.88%. The polynomial regression analysis of WG (Figure 1) and SGR (Figure 2) indicated that the optimum dietary arginine level for growth of rice field eel was 4.28–4.35% of the dry diet, corresponding to 9.02–9.17% of dietary protein. There was no significant variation in CF, VSI, HSI, and SR among all treatments (p>0.05). Increasing levels of arginine caused the SR to peak at low arginine levels and decreased with higher doses. The highest SR (98%) occurred with 2.58% dietary arginine levels and then declined to 90.67% with increasing arginine levels in the diet. The minimum CF occurred in D1, indicating 2.03% arginine was insufficient for M. albus. This suggestion is supported by the WG and SGR data analyses. The VSI and HSI varied irregularly.Table 4
Effects of dietary arginine level on growth performance ofM.albus fed with diets containing graded levels of dietary arginine.
Diets (Arg%)Diet 1Diet 2Diet 3Diet 4Diet 5Diet 6P2.032.583.163.634.244.88Initial weight (g)20.98 ± 0.0321.06 ± 0.0220.99 ± 0.0320.99 ± 0.5320.99 ± 0.6221.01 ± 0.020.204Final weight (g)26.67 ± 1.07b33.10 ± 1.56ab33.43 ± 3.07ab34.1 ± 0.46a34.2 ± 1.23a35.97 ± 0.99a0.001WG (%)27.06 ± 5.08b57.17 ± 7.55ab59.37 ± 14.8ab62.46 ± 2.2a63.05 ± 5.29a71.16 ± 4.81a0.001SGR (%/d)0.43 ± 0.07b0.81 ± 0.08a0.83 ± 0.17ab0.87 ± 0.02a0.87 ± 0.06a0.96 ± 0.05a0.001CF (g/cm3)24.58 ± 3.7326.86 ± 2.4025.22 ± 2.9527.87 ± 1.9526.48 ± 2.8626.55 ± 3.280.166VSI (%)11.11 ± 1.6311.17 ± 2.3110.79 ± 1.8711.17 ± 1.1910.52 ± 1.7611.00 ± 1.460.951HSI (%)6.48 ± 0.876.75 ± 1.436.90 ± 0.536.80 ± 0.846.33 ± 1.356.33 ± 1.550.796SR (%)97.33 ± 4.6298.67 ± 2.3195.33 ± 5.0392.00 ± 3.4695.33 ± 4.1690.67 ± 9.450.465Note. Values are means ± SD of three replications. Means in the same raw with different superscripts are significantly different (p<0.05). WG: weight gain; SGR: specific growth rate; CF: condition factor; VSI: viscerosomatic index; HSI: hepatosomatic index; SR: survival rate.Figure 1
Polynomial regression the effects of dietary arginine level on weight gain ofM. albus. Each point represents a data and individual data were removed due to excessive bias.Figure 2
Polynomial regression the effects of dietary arginine level on specific growth rate ofM. albus. Each point represents a data and individual data were removed due to excessive bias.


### 3.2. Digestive Enzymes in Liver

Table5 demonstrates the dietary arginine level significantly affected liver trypsin activity (p<0.05). The highest trypsin levels occurred in the 3.16% dietary arginine group, with the variations in trypsin levels showing an irregular pattern. There was no variation in lipase and amylase activities in the liver (p>0.05).Table 5
Effects of dietary arginine level on liver enzyme in rice filed eel (Monopterus albus).
Diets (Arg%)Diet 1Diet 2Diet 3Diet 4Diet 5Diet 6P2.032.583.163.634.244.88TPS (U/mgprot)957.13 ± 144.85a830.71 ± 115.39ab1082.01 ± 181.52a600.02 ± 95.42b819.19 ± 103.97ab939.86 ± 194.02a0.004LPS (U/gprot)1.32 ± 0.411.28 ± 0.561.43 ± 0.321.56 ± 0.311.43 ± 0.421.61 ± 0.190.938AMS (U/mgprot)1.47 ± 0.351.87 ± 0.741.5 ± 0.312.66 ± 0.541.88 ± 0.791.89 ± 0.420.104Note. Values are means ± SD of three replications. Means in the same raw with different superscripts are significantly different (p<0.05). TPS: trypsin; LPS: lipase; AMS: amylase.


### 3.3. Serum Growth and Immune Parameters

No significant differences between the dietary groups were detected in the concentrations of serum GH, INS, NO, and TNOS (see Table6) (p>0.05). However, dietary arginine level had a significant effect on serum lysozyme and complement C3 activity (p<0.05). Serum lysozyme and complement C3 both increased with increasing dietary arginine levels, peaking in the 2.58% group before declining with larger arginine doses.Table 6
Effects of dietary arginine level on serum growth and immune indices in rice filed eel (Monopterus albus).
Diets (Arg%)Diet1Diet2Diet3Diet4Diet5Diet6P2.032.583.163.634.244.88GH (U/mgprot)2.93 ± 0.413.43 ± 0.653.28 ± 0.442.97 ± 0.343.04 ± 0.313.14 ± 0.400.565INS (mIU/L)19.59 ± 6.0214.4 ± 1.6218.59 ± 2.8617.82 ± 3.0121.67 ± 5.2118.62 ± 4.410.295NO (μmol/L)8.22 ± 3.97.05 ± 5.2810.5 ± 3.318.44 ± 3.317.84 ± 0.769.21 ± 2.420.760TNOS (μmol/L)16.91 ± 2.4616.26 ± 2.5717.67 ± 2.6517.32 ± 2.5815.75 ± 2.819.48 ± 3.880.545LZM (U/mL)758.93 ± 331.68b1625.00 ± 626.44a1187.5 ± 605.65ab794.64 ± 287.75b651.79 ± 60.99b821.43 ± 445.11b0.048C3 (μg/m)271.12 ± 44.08b402.91 ± 31.18a371.16 ± 50.73ab295.49 ± 27.15b253.97 ± 11.01b301.03 ± 44.78b0.02Note. Values are means ± SD of three replications. Means in the same raw with different superscripts are significantly different (p<0.05). GH: growth Hormone; INS: insulin; NO: nitric oxide; TNOS: total nitric oxide synthase; LZM: lysozyme; C3: complement C3.


### 3.4. Expression of Inflammatory Cytokines

Dietary arginine levels had significant effects on the inflammatory cytokines IL-1β (Figure 3) and IL-10 (Figure 4) in the head-kidney (p<0.05). Both IL-1β and IL-10 were lower with low arginine doses and higher with increased dietary arginine. The lowest IL-1β occurred in the 3.16% dietary arginine group and was higher in groups fed higher doses. The IL-1β in the 4.24% and 4.88% arginine diet groups were significantly higher than in the 2.03%, 2.58%, and 3.16% arginine groups (p<0.05). The IL-10 of the 2.58% and 3.16% arginine groups were lower than the other four groups. The IL-1β and IL-10 trends followed an almost negative correlation with SR.Figure 3
Relative expression of IL-1β in head kidney of M. albus fed with diets containing graded levels of arginine. Values are means for three fish per treatment, with standard deviations represented by vertical bars. a, bMean values with unlike letters were significantly different (p<0.05).Figure 4
Relative expression of IL-10 in head kidney ofM. albus fed with diets containing graded levels of arginine. Values are means for three fish per treatment, with standard deviations represented by vertical bars. a, bMean values with unlike letters were significantly different (p<0.05).


## 3.1. Growth Performance

The results revealed that dietary arginine levels had a significant effect on final body weight, WG, and SGR inM. albus (p<0.05) (Table 4). WG and SGR increased with increasing dietary arginine levels without decreasing until the arginine level reached 4.88%. The polynomial regression analysis of WG (Figure 1) and SGR (Figure 2) indicated that the optimum dietary arginine level for growth of rice field eel was 4.28–4.35% of the dry diet, corresponding to 9.02–9.17% of dietary protein. There was no significant variation in CF, VSI, HSI, and SR among all treatments (p>0.05). Increasing levels of arginine caused the SR to peak at low arginine levels and decreased with higher doses. The highest SR (98%) occurred with 2.58% dietary arginine levels and then declined to 90.67% with increasing arginine levels in the diet. The minimum CF occurred in D1, indicating 2.03% arginine was insufficient for M. albus. This suggestion is supported by the WG and SGR data analyses. The VSI and HSI varied irregularly.Table 4
Effects of dietary arginine level on growth performance ofM.albus fed with diets containing graded levels of dietary arginine.
Diets (Arg%)Diet 1Diet 2Diet 3Diet 4Diet 5Diet 6P2.032.583.163.634.244.88Initial weight (g)20.98 ± 0.0321.06 ± 0.0220.99 ± 0.0320.99 ± 0.5320.99 ± 0.6221.01 ± 0.020.204Final weight (g)26.67 ± 1.07b33.10 ± 1.56ab33.43 ± 3.07ab34.1 ± 0.46a34.2 ± 1.23a35.97 ± 0.99a0.001WG (%)27.06 ± 5.08b57.17 ± 7.55ab59.37 ± 14.8ab62.46 ± 2.2a63.05 ± 5.29a71.16 ± 4.81a0.001SGR (%/d)0.43 ± 0.07b0.81 ± 0.08a0.83 ± 0.17ab0.87 ± 0.02a0.87 ± 0.06a0.96 ± 0.05a0.001CF (g/cm3)24.58 ± 3.7326.86 ± 2.4025.22 ± 2.9527.87 ± 1.9526.48 ± 2.8626.55 ± 3.280.166VSI (%)11.11 ± 1.6311.17 ± 2.3110.79 ± 1.8711.17 ± 1.1910.52 ± 1.7611.00 ± 1.460.951HSI (%)6.48 ± 0.876.75 ± 1.436.90 ± 0.536.80 ± 0.846.33 ± 1.356.33 ± 1.550.796SR (%)97.33 ± 4.6298.67 ± 2.3195.33 ± 5.0392.00 ± 3.4695.33 ± 4.1690.67 ± 9.450.465Note. Values are means ± SD of three replications. Means in the same raw with different superscripts are significantly different (p<0.05). WG: weight gain; SGR: specific growth rate; CF: condition factor; VSI: viscerosomatic index; HSI: hepatosomatic index; SR: survival rate.Figure 1
Polynomial regression the effects of dietary arginine level on weight gain ofM. albus. Each point represents a data and individual data were removed due to excessive bias.Figure 2
Polynomial regression the effects of dietary arginine level on specific growth rate ofM. albus. Each point represents a data and individual data were removed due to excessive bias.


## 3.2. Digestive Enzymes in Liver

Table5 demonstrates the dietary arginine level significantly affected liver trypsin activity (p<0.05). The highest trypsin levels occurred in the 3.16% dietary arginine group, with the variations in trypsin levels showing an irregular pattern. There was no variation in lipase and amylase activities in the liver (p>0.05).Table 5
Effects of dietary arginine level on liver enzyme in rice filed eel (Monopterus albus).
Diets (Arg%)Diet 1Diet 2Diet 3Diet 4Diet 5Diet 6P2.032.583.163.634.244.88TPS (U/mgprot)957.13 ± 144.85a830.71 ± 115.39ab1082.01 ± 181.52a600.02 ± 95.42b819.19 ± 103.97ab939.86 ± 194.02a0.004LPS (U/gprot)1.32 ± 0.411.28 ± 0.561.43 ± 0.321.56 ± 0.311.43 ± 0.421.61 ± 0.190.938AMS (U/mgprot)1.47 ± 0.351.87 ± 0.741.5 ± 0.312.66 ± 0.541.88 ± 0.791.89 ± 0.420.104Note. Values are means ± SD of three replications. Means in the same raw with different superscripts are significantly different (p<0.05). TPS: trypsin; LPS: lipase; AMS: amylase.


## 3.3. Serum Growth and Immune Parameters

No significant differences between the dietary groups were detected in the concentrations of serum GH, INS, NO, and TNOS (see Table6) (p>0.05). However, dietary arginine level had a significant effect on serum lysozyme and complement C3 activity (p<0.05). Serum lysozyme and complement C3 both increased with increasing dietary arginine levels, peaking in the 2.58% group before declining with larger arginine doses.Table 6
Effects of dietary arginine level on serum growth and immune indices in rice filed eel (Monopterus albus).
Diets (Arg%)Diet1Diet2Diet3Diet4Diet5Diet6P2.032.583.163.634.244.88GH (U/mgprot)2.93 ± 0.413.43 ± 0.653.28 ± 0.442.97 ± 0.343.04 ± 0.313.14 ± 0.400.565INS (mIU/L)19.59 ± 6.0214.4 ± 1.6218.59 ± 2.8617.82 ± 3.0121.67 ± 5.2118.62 ± 4.410.295NO (μmol/L)8.22 ± 3.97.05 ± 5.2810.5 ± 3.318.44 ± 3.317.84 ± 0.769.21 ± 2.420.760TNOS (μmol/L)16.91 ± 2.4616.26 ± 2.5717.67 ± 2.6517.32 ± 2.5815.75 ± 2.819.48 ± 3.880.545LZM (U/mL)758.93 ± 331.68b1625.00 ± 626.44a1187.5 ± 605.65ab794.64 ± 287.75b651.79 ± 60.99b821.43 ± 445.11b0.048C3 (μg/m)271.12 ± 44.08b402.91 ± 31.18a371.16 ± 50.73ab295.49 ± 27.15b253.97 ± 11.01b301.03 ± 44.78b0.02Note. Values are means ± SD of three replications. Means in the same raw with different superscripts are significantly different (p<0.05). GH: growth Hormone; INS: insulin; NO: nitric oxide; TNOS: total nitric oxide synthase; LZM: lysozyme; C3: complement C3.


## 3.4. Expression of Inflammatory Cytokines

Dietary arginine levels had significant effects on the inflammatory cytokines IL-1β (Figure 3) and IL-10 (Figure 4) in the head-kidney (p<0.05). Both IL-1β and IL-10 were lower with low arginine doses and higher with increased dietary arginine. The lowest IL-1β occurred in the 3.16% dietary arginine group and was higher in groups fed higher doses. The IL-1β in the 4.24% and 4.88% arginine diet groups were significantly higher than in the 2.03%, 2.58%, and 3.16% arginine groups (p<0.05). The IL-10 of the 2.58% and 3.16% arginine groups were lower than the other four groups. The IL-1β and IL-10 trends followed an almost negative correlation with SR.Figure 3
Relative expression of IL-1β in head kidney of M. albus fed with diets containing graded levels of arginine. Values are means for three fish per treatment, with standard deviations represented by vertical bars. a, bMean values with unlike letters were significantly different (p<0.05).Figure 4
Relative expression of IL-10 in head kidney ofM. albus fed with diets containing graded levels of arginine. Values are means for three fish per treatment, with standard deviations represented by vertical bars. a, bMean values with unlike letters were significantly different (p<0.05).


## 4. Discussion

### 4.1. Growth Performance

In fish, arginine is an essential amino acid with different physiological roles in growth and health [1]. Since arginine is an essential amino acid, diet is the main source of arginine for successfully culturing farmed fish species. Dietary arginine requirements have been determined for several fish species using growth response curves; some fish had broken-line responses to graded dietary arginine levels, others showed polynomial responses. The dietary arginine requirements of fish ranged from 3% to 8.1% of the dietary protein content [1]. However, our results revealed the optimum dietary arginine level for growth of rice field eel was 9.02–9.17% of dietary protein. The result was higher than previous studies on hybrid grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂, 3.65%) [13], blunt snout bream (Megalobrama amblycephala, 7.23%) [14], golden pompano (Trachinotus ovatus) [4], and tilapia (Oreochromis niloticus, 6.24%) [15]. The reasons for the significant variation may be due to different species, growth stages, dietary protein sources and contents, composition of dietary protein, amino acids, water temperature, salinity, and/or experimental assessment indices [16]. In addition, carnivorous species have higher dietary arginine requirements than omnivorous fish [17]. M. albus is a carnivorous fish; thus, higher dietary arginine requirements are reasonable.In this experiment, the CF, HSI, and VSI were not significantly different between the different dietary groups (p>0.05), like the largemouth bass (Micropterus salmoides) [18] and golden pompano (Trachinotus ovatus) [4]. Conversely, dietary arginine content had significant effects on CF in yellow grouper (Epinephelus awoara) [19] and stinging catfish (Heteropneustes fossilis) [20]. In our research, the CF of fish fed 2.03% dietary arginine was the lowest of all groups. This suggests that 2.03% dietary arginine does not satisfy the basic demand in the rice field eel.The SR in all groups did not vary significantly (p>0.05) with differing dietary arginine levels in this experiment. Interestingly, the 4.88% dietary arginine group had the lowest SR and simultaneously had the highest WG and SGR. This suggests the 4.88% dietary arginine level was not optimum for the rice field eel diet. Except for SR, LZM, and C3 negatively correlated with the inflammatory cytokines. This suggests insufficient or excess arginine is disadvantageous for the rice field eel. Surplus dietary arginine can inhibit fish immune responses, as demonstrated in European sea bass (Dicentrarchus labrax) [7] and common carp (Cyprinus carpio L.) [3]. Therefore, immunity should be considered when identifying the optimal arginine level for the rice field eel.


### 4.2. Digestive Enzymes in Liver

Fish growth is mainly associated with digestive and absorptive abilities [21]. Nutrient digestion and absorption depends on the activities of digestive enzymes and brush-border membrane enzymes, which are responsible for breaking down and assimilating food. Fish exocrine pancreases synthesize and secrete a variety of digestive enzymes into the intestinal lumen, such as trypsin, lipase, and amylase [22]. The liver is a key metabolic organ. Chen et al. [23] identified trypsin, lipase, and amylase activities in the hepatopancreas significantly increased with higher levels of dietary arginine in Jian carp (Cyprinus carpio var. Jian). In the present experiment, only trypsin varied significantly with different dietary levels of arginine (p<0.05). Arginine has different effects on the digestive enzyme activities in different fish species. There are many factors affecting the digestive enzyme activities in fishes, such as diet, growth stage, physical and chemical factors (such as water temperature, pH, and salinity), and feed and nutrition (including feed raw materials and feed additives) [24].


### 4.3. Serum Growth and Immune Parameters

The somatotropic axis plays an important role in fish growth, and GH is a main factor in the axis, which depresses growth in fish exposed to catabolism and malnutrition [25]. Fish research has identified that dietary arginine administration can lead to increased GH levels and gene expression; mostly accompanied with increased fish growth performance [26, 27]. Nevertheless, in our experiment, GH did not significantly increase with increased growth performance. The mechanisms by which arginine stimulates the somatotropic axis remains unknown in fish, and further research into the controlling factors in arginine-induced somatotropic axis activation are required [1]. Insulin is an important anabolic hormone and acts as a growth promoter in fish [28]. Arginine is known as a strong insulinotropic agent in animals, including fish. The effect may be related to the elevated circulating levels of arginine-induced insulin, which tends to disappear after a few hours, at the same time as the circulating levels of arginine are expected to decline [29, 30]. In this experiment, insulin levels did not significantly vary between the different dietary groups.This lack of variation may be due to the fish fasting for 24 hours before sampling, providing time for the insulin levels to fall. However, Han et al. [27] found increased blood insulin levels in Epinephelus coioides 24 h after feeding arginine-supplemented diets. The effect of arginine intake on insulin requires further research.Arginine is an important immunostimulant in fish. Arginine deficiency leads to several immune-related issues in various fish species, including a decreased number of leukocytes, lysozyme and complement activities, superoxide anion, nitric oxide, and immunoglobulin production, phagocytosis, and haemagglutination [1]. Nevertheless, compared with the dietary requirements, surplus levels of dietary arginine can decrease immune function and disease resistance in some teleost species [3, 4, 7, 8]. In this experiment, nitric oxide and nitric oxide synthase did not significantly vary between the different dietary groups. The results support previous studies on Scophthalmus maximus [2] and Trachinotus ovatus [4]. However, other fish studies have revealed NOS and growth performance respond similarly, in a dose-dependent manner to dietary arginine levels [5, 14, 26, 31]. These different results indicate that the responses may be species specific.Humoral immunity is an important part of the fish innate immune system. There is a lack of information available on arginine effects on other humoral immune factors, such as lysozyme and the complement system. A limited number of studies have suggested a positive role for arginine in lysozyme activity. Dietary arginine supplementation in red drum (Sciaenops ocellatus) significantly increased lysozyme activity [32]. Both in vivo and in vitro experiments with channel catfish showed that arginine supplementation improved lysozyme activity [33, 34]. Our research supports these previous findings, lysozyme activity increased significantly with increasing dietary arginine levels up to 2.58%. However, lysozyme activity declined rapidly when the dietary arginine level exceeded 2.58% of the diet.A limited number of studies have revealed that an increase in dietary arginine levels may initially lead to increased complement activity before decreasing. For instance, when 1.85% arginine was added to the diet of young Jian carp, the activities of C3 and C4 increased by 44.3% and 40%, respectively, but when the arginine content was 2.45%, their activities decreased [8]. In research into the interaction between arginine and carp density, complement (ACH50) activity initially increased before declining [3]. Our results support previous findings, the C3 activity increased significantly with increasing dietary arginine levels up to 2.58% and then declined. However, other studies did not observe any effects of dietary arginine levels on complement activity [6, 19, 35]. Further research is required to assess the effects of arginine administration on the complement system in fish.


### 4.4. Expression of Inflammatory Cytokines

Inflammation is an important response by fish to disease and stress [36]. Evidence suggests arginine administrates inflammatory responses in fish. For example, arginine augmented proinflammatory (IL-1β) and anti-inflammatory (IL-10) gene expression in Cyprinus carpio (Var.Jian) head-kidney, and spleen in normal rearing conditions [8]. Similarly, in our experiment, proinflammatory (IL-1β) and anti-inflammatory (IL-10) gene expression in head-kidney were both increased in fish submitted to higher levels of dietary arginine (≥3.16%). The study on Cyprinus carpio (mirror phenotype) also suggested that dietary arginine supplementation upregulated anti-inflammatory cytokine genes (TGF and IL-10) in the intestine [35]. Contrarily, Azeredo et al. [7] identified dietary arginine supplementation suppressed inflammatory responses in Dicentrarchus labrax in normal conditions. Similar results occurred in our experiment when the level of dietary arginine was below 3.16%. Both deficient and surplus levels of arginine led to a decreased expression of proinflammatory cytokine genes in Cyprinus carpio when subjected to either normal or stressful rearing conditions [3]. The results are the opposite of our observations. The molecular basis of the arginine effects on inflammatory responses may be partially explained by two different pathways: the mTOR signaling pathway and the NO signaling pathway [1]. Further research is required to identify a definitive conclusion on how arginine induces inflammation.


## 4.1. Growth Performance

In fish, arginine is an essential amino acid with different physiological roles in growth and health [1]. Since arginine is an essential amino acid, diet is the main source of arginine for successfully culturing farmed fish species. Dietary arginine requirements have been determined for several fish species using growth response curves; some fish had broken-line responses to graded dietary arginine levels, others showed polynomial responses. The dietary arginine requirements of fish ranged from 3% to 8.1% of the dietary protein content [1]. However, our results revealed the optimum dietary arginine level for growth of rice field eel was 9.02–9.17% of dietary protein. The result was higher than previous studies on hybrid grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂, 3.65%) [13], blunt snout bream (Megalobrama amblycephala, 7.23%) [14], golden pompano (Trachinotus ovatus) [4], and tilapia (Oreochromis niloticus, 6.24%) [15]. The reasons for the significant variation may be due to different species, growth stages, dietary protein sources and contents, composition of dietary protein, amino acids, water temperature, salinity, and/or experimental assessment indices [16]. In addition, carnivorous species have higher dietary arginine requirements than omnivorous fish [17]. M. albus is a carnivorous fish; thus, higher dietary arginine requirements are reasonable.In this experiment, the CF, HSI, and VSI were not significantly different between the different dietary groups (p>0.05), like the largemouth bass (Micropterus salmoides) [18] and golden pompano (Trachinotus ovatus) [4]. Conversely, dietary arginine content had significant effects on CF in yellow grouper (Epinephelus awoara) [19] and stinging catfish (Heteropneustes fossilis) [20]. In our research, the CF of fish fed 2.03% dietary arginine was the lowest of all groups. This suggests that 2.03% dietary arginine does not satisfy the basic demand in the rice field eel.The SR in all groups did not vary significantly (p>0.05) with differing dietary arginine levels in this experiment. Interestingly, the 4.88% dietary arginine group had the lowest SR and simultaneously had the highest WG and SGR. This suggests the 4.88% dietary arginine level was not optimum for the rice field eel diet. Except for SR, LZM, and C3 negatively correlated with the inflammatory cytokines. This suggests insufficient or excess arginine is disadvantageous for the rice field eel. Surplus dietary arginine can inhibit fish immune responses, as demonstrated in European sea bass (Dicentrarchus labrax) [7] and common carp (Cyprinus carpio L.) [3]. Therefore, immunity should be considered when identifying the optimal arginine level for the rice field eel.


## 4.2. Digestive Enzymes in Liver

Fish growth is mainly associated with digestive and absorptive abilities [21]. Nutrient digestion and absorption depends on the activities of digestive enzymes and brush-border membrane enzymes, which are responsible for breaking down and assimilating food. Fish exocrine pancreases synthesize and secrete a variety of digestive enzymes into the intestinal lumen, such as trypsin, lipase, and amylase [22]. The liver is a key metabolic organ. Chen et al. [23] identified trypsin, lipase, and amylase activities in the hepatopancreas significantly increased with higher levels of dietary arginine in Jian carp (Cyprinus carpio var. Jian). In the present experiment, only trypsin varied significantly with different dietary levels of arginine (p<0.05). Arginine has different effects on the digestive enzyme activities in different fish species. There are many factors affecting the digestive enzyme activities in fishes, such as diet, growth stage, physical and chemical factors (such as water temperature, pH, and salinity), and feed and nutrition (including feed raw materials and feed additives) [24].


## 4.3. Serum Growth and Immune Parameters

The somatotropic axis plays an important role in fish growth, and GH is a main factor in the axis, which depresses growth in fish exposed to catabolism and malnutrition [25]. Fish research has identified that dietary arginine administration can lead to increased GH levels and gene expression; mostly accompanied with increased fish growth performance [26, 27]. Nevertheless, in our experiment, GH did not significantly increase with increased growth performance. The mechanisms by which arginine stimulates the somatotropic axis remains unknown in fish, and further research into the controlling factors in arginine-induced somatotropic axis activation are required [1]. Insulin is an important anabolic hormone and acts as a growth promoter in fish [28]. Arginine is known as a strong insulinotropic agent in animals, including fish. The effect may be related to the elevated circulating levels of arginine-induced insulin, which tends to disappear after a few hours, at the same time as the circulating levels of arginine are expected to decline [29, 30]. In this experiment, insulin levels did not significantly vary between the different dietary groups.This lack of variation may be due to the fish fasting for 24 hours before sampling, providing time for the insulin levels to fall. However, Han et al. [27] found increased blood insulin levels in Epinephelus coioides 24 h after feeding arginine-supplemented diets. The effect of arginine intake on insulin requires further research.Arginine is an important immunostimulant in fish. Arginine deficiency leads to several immune-related issues in various fish species, including a decreased number of leukocytes, lysozyme and complement activities, superoxide anion, nitric oxide, and immunoglobulin production, phagocytosis, and haemagglutination [1]. Nevertheless, compared with the dietary requirements, surplus levels of dietary arginine can decrease immune function and disease resistance in some teleost species [3, 4, 7, 8]. In this experiment, nitric oxide and nitric oxide synthase did not significantly vary between the different dietary groups. The results support previous studies on Scophthalmus maximus [2] and Trachinotus ovatus [4]. However, other fish studies have revealed NOS and growth performance respond similarly, in a dose-dependent manner to dietary arginine levels [5, 14, 26, 31]. These different results indicate that the responses may be species specific.Humoral immunity is an important part of the fish innate immune system. There is a lack of information available on arginine effects on other humoral immune factors, such as lysozyme and the complement system. A limited number of studies have suggested a positive role for arginine in lysozyme activity. Dietary arginine supplementation in red drum (Sciaenops ocellatus) significantly increased lysozyme activity [32]. Both in vivo and in vitro experiments with channel catfish showed that arginine supplementation improved lysozyme activity [33, 34]. Our research supports these previous findings, lysozyme activity increased significantly with increasing dietary arginine levels up to 2.58%. However, lysozyme activity declined rapidly when the dietary arginine level exceeded 2.58% of the diet.A limited number of studies have revealed that an increase in dietary arginine levels may initially lead to increased complement activity before decreasing. For instance, when 1.85% arginine was added to the diet of young Jian carp, the activities of C3 and C4 increased by 44.3% and 40%, respectively, but when the arginine content was 2.45%, their activities decreased [8]. In research into the interaction between arginine and carp density, complement (ACH50) activity initially increased before declining [3]. Our results support previous findings, the C3 activity increased significantly with increasing dietary arginine levels up to 2.58% and then declined. However, other studies did not observe any effects of dietary arginine levels on complement activity [6, 19, 35]. Further research is required to assess the effects of arginine administration on the complement system in fish.


## 4.4. Expression of Inflammatory Cytokines

Inflammation is an important response by fish to disease and stress [36]. Evidence suggests arginine administrates inflammatory responses in fish. For example, arginine augmented proinflammatory (IL-1β) and anti-inflammatory (IL-10) gene expression in Cyprinus carpio (Var.Jian) head-kidney, and spleen in normal rearing conditions [8]. Similarly, in our experiment, proinflammatory (IL-1β) and anti-inflammatory (IL-10) gene expression in head-kidney were both increased in fish submitted to higher levels of dietary arginine (≥3.16%). The study on Cyprinus carpio (mirror phenotype) also suggested that dietary arginine supplementation upregulated anti-inflammatory cytokine genes (TGF and IL-10) in the intestine [35]. Contrarily, Azeredo et al. [7] identified dietary arginine supplementation suppressed inflammatory responses in Dicentrarchus labrax in normal conditions. Similar results occurred in our experiment when the level of dietary arginine was below 3.16%. Both deficient and surplus levels of arginine led to a decreased expression of proinflammatory cytokine genes in Cyprinus carpio when subjected to either normal or stressful rearing conditions [3]. The results are the opposite of our observations. The molecular basis of the arginine effects on inflammatory responses may be partially explained by two different pathways: the mTOR signaling pathway and the NO signaling pathway [1]. Further research is required to identify a definitive conclusion on how arginine induces inflammation.


## 5. Conclusions

The experiment revealed that dietary arginine levels had a significant effect on the growth, immunity, and inflammation responses in the rice field eel. The decreased survival rate may be related to decreases in immune factors and increases in inflammatory factors. Our comprehensive analysis of growth, immunity, and inflammation indicated the optimal recommended dietary arginine level for the growth ofM. albus was 3.16–3.63% of the dry diet, corresponding to 6.58–7.70% of the dietary protein.



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*Source: 8371801-2023-02-06.xml*