Effects of Dietary Arginine Level on Growth Performance and Immune Response in Monopterus albus

To assess the dietary arginine requirement of Monopterus albus (21 ± 0.03g), we compared six diets containing diferent arginine levels (2.03%, 2.58%, 3.16%, 3.63%, 4.24%, and 4.88%). Te results revealed that dietary arginine content had a signifcant efect on weight gain (WG), specifc growth rate (SGR), IL-1 β , IL-10, lysozyme, and complement C3 activity in Monopterus albus ( p < 0 . 05). Te WG and SGR increased with increasing dietary arginine levels, and the regression analysis found that the optimum dietary arginine level for growth of rice feld 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). Te dietary arginine level had no signifcant efect 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 signifcantly 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.


Introduction
Amino acids are important molecules in fsh 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 fsh, arginine is an essential amino acid with diferent physiological roles in growth performance and health [1].Diet is the main source of arginine for successfully culturing farmed fsh species.Te arginine requirement in fsh ranges from 3.0% to 8.1% of the dietary protein.Te growth promoting efect of arginine may induce a somatotropic axis in fsh 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 fsh [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, infammation, and innate immune responses [4,5].Arginine defciency can lead to severe immune-related issues in diferent fsh 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].Terefore, it is necessary to study the optimal requirement of arginine for each fsh.
Rice feld eel (Monopterus albus, M. albus) is a subtropical freshwater benthic fsh inhabiting caves, muddy ponds, swamps, rice felds, and burrows within moist Earth [9].Tis fsh 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][10][11].However, the dietary arginine requirements for swamp eels have not been investigated.Tis experiment examines the efects of dietary arginine on growth performance, serum biochemical indices, and the nonspecifc immunity index in swamp eels, to determine the optimum dietary arginine requirement for M. albus.

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 (Table 1).Te fnal 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 highperformance 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.Te 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).Te 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.

Fish and Experimental
Conditions.Wild M. albus was sourced from Chaohu (Anhui province, China).All specimens were cultured in a foating 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 foating cages (2.0 × 1.5 × 1.5 m) at a density of 50 fsh per cage.To simulate culture conditions, each cage was 95% flled with fresh grass, Eichhornia crassipes.Monopterus albus was fed once daily at 18:00 pm for 56 days.Te 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.Te 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 NO 2 − N <0.1 mg/L.

Sample Collection.
After 24 h of fasting, the caged fsh were anaesthetized using 100 µl/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.Te liver and head-kidney from three fsh 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.Te experiment was performed in strict accordance with the guidelines and ethical principles of the Experiment Animal Welfare Ethics Committee of China.Te experimental design was approved by the Committee on Research Ethics of the Department of Laboratory Animal Science, Jingxi Agricultural University.All eforts were made to minimize fsh sufering.
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 ).Te amplifcation was performed in a total volume of 20 µl 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.Te 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.Te 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. Te target gene mRNA concentration was normalized to the mRNA concentration of the reference gene 18S, a housekeeping gene of M. albus.2.6.Evaluation of Growth Parameters.Te growth performance of M. 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 × (fnal total weight-initial total weight)/initial total weight; special growth rate (SGR, %/day) � 100 × (Ln fnal individual weight-Ln initial individual weight)/number of days; condition factor (CF, g/cm 3 ) � 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, %) � fnal number of fsh × 100/initial number of fsh.

Statistical Analysis.
Te results were expressed as mean ± SD. (standard deviation of the mean) and were analyzed using one-way ANOVA in SPSS 19.0 software.Any signifcant diferences were further investigated by comparing the group means using Turkey's test.Statistical signifcance was considered if p < 0.05.

Growth Performance.
Te results revealed that dietary arginine levels had a signifcant efect on fnal body weight, WG, and SGR in M. albus (p < 0.05) (Table 4).WG and SGR increased with increasing dietary arginine levels without decreasing until the arginine level reached 4.88%.Te polynomial regression analysis of WG (Figure 1) and SGR (Figure 2) indicated that the optimum dietary arginine level for growth of rice feld eel was 4.28-4.35% of the dry diet, corresponding to 9.02-9.17% of dietary protein.Tere was no signifcant 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.Te highest SR (98%) occurred with 2.58% dietary arginine levels and then declined to 90.67% with

Expression of Infammatory
Cytokines.Dietary arginine levels had signifcant efects on the infammatory 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.Te lowest IL-1β occurred in the 3.16% dietary arginine group and was higher in groups fed higher doses.Te 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).Te IL-10 of the 2.58% and 3.16% arginine groups were lower than the other four groups.Te IL-1β and IL-10 trends followed an almost negative correlation with SR.

Discussion
4.1.Growth Performance.In fsh, arginine is an essential amino acid with diferent 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 fsh species.Dietary arginine requirements have been determined for several fsh species using growth response curves; some fsh had broken-line responses to graded dietary arginine levels, others showed polynomial responses.Aquaculture Research Te dietary arginine requirements of fsh 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 feld eel was 9.02-9.17% of dietary protein.Te 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].Te reasons for the signifcant variation may be due to diferent 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 fsh [17].M. albus is a carnivorous fsh; thus, higher dietary arginine requirements are reasonable.
In this experiment, the CF, HSI, and VSI were not signifcantly diferent between the diferent dietary groups (p > 0.05), like the largemouth bass (Micropterus salmoides) [18] and golden pompano (Trachinotus ovatus) [4].Conversely, dietary arginine content had signifcant efects on CF in yellow grouper (Epinephelus awoara) [19] and stinging catfsh (Heteropneustes fossilis) [20].In our research, the CF of fsh fed 2.03% dietary arginine was the lowest of all groups.Tis suggests that 2.03% dietary arginine does not satisfy the basic demand in the rice feld eel.
Te SR in all groups did not vary signifcantly (p > 0.05) with difering 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.Tis suggests the 4.88% dietary arginine level was not optimum for the rice feld eel diet.Except for SR, LZM, and C3 negatively correlated with the infammatory cytokines.Tis suggests insufcient or excess arginine is disadvantageous for the rice feld eel.Surplus dietary arginine can inhibit fsh immune responses, as demonstrated in European sea bass (Dicentrarchus labrax) [7] and common carp (Cyprinus carpio L.) [3].Terefore, immunity should be considered when identifying the optimal arginine level for the rice feld eel.

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].Te liver is a key metabolic organ.Chen et al. [23] identifed trypsin, lipase, and amylase activities in the hepatopancreas signifcantly increased with higher levels of dietary arginine in Jian carp (Cyprinus carpio var.Jian).In the present experiment, only trypsin varied signifcantly with diferent dietary levels of arginine (p < 0.05).Arginine has diferent efects on the digestive enzyme activities in diferent fsh species.Tere are many factors afecting the digestive enzyme activities in fshes, 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].

Serum Growth and Immune
Parameters.Te somatotropic axis plays an important role in fsh growth, and GH is a main factor in the axis, which depresses growth in fsh exposed to catabolism and malnutrition [25].Fish research has identifed that dietary arginine administration can lead to increased GH levels and gene expression; mostly accompanied with increased fsh growth performance [26,27].Nevertheless, in our experiment, GH did not signifcantly increase with increased growth performance.Te mechanisms by which arginine stimulates the somatotropic axis remains unknown in fsh, 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 fsh [28].Arginine is known as a strong insulinotropic agent in animals, including fsh.Te efect 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 signifcantly vary between the diferent dietary groups.
Tis lack of variation may be due to the fsh 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.Te efect of arginine intake on insulin requires further research.
Arginine is an important immunostimulant in fsh.Arginine defciency leads to several immune-related issues in various fsh 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 signifcantly vary between the diferent dietary groups.Te results support previous studies on Scophthalmus maximus [2] and Trachinotus ovatus [4].However, other fsh studies have revealed NOS and growth performance respond similarly, in a dose-dependent manner to dietary arginine levels [5,14,26,31].Tese diferent results indicate that the responses may be species specifc.
Humoral immunity is an important part of the fsh innate immune system.Tere is a lack of information available on arginine efects 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) signifcantly increased lysozyme activity [32].Both in vivo and in vitro experiments with channel catfsh showed that arginine supplementation improved lysozyme activity [33,34].Our research supports these previous fndings, lysozyme activity increased signifcantly 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 fndings, the C3 activity increased signifcantly with increasing dietary arginine levels up to 2.58%    Aquaculture Research and then declined.However, other studies did not observe any efects of dietary arginine levels on complement activity [6,19,35].Further research is required to assess the efects of arginine administration on the complement system in fsh.

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

Conclusions
Te experiment revealed that dietary arginine levels had a signifcant efect on the growth, immunity, and infammation responses in the rice feld eel.Te decreased survival rate may be related to decreases in immune factors and increases in infammatory factors.Our comprehensive analysis of growth, immunity, and infammation indicated the optimal recommended dietary arginine level for the growth of M. albus was 3.16-3.63% of the dry diet, corresponding to 6.58-7.70% of the dietary protein.

Figure 1 :
Figure 1: Polynomial regression the efects of dietary arginine level on weight gain of M. albus.Each point represents a data and individual data were removed due to excessive bias.

Figure 2 :
Figure 2: Polynomial regression the efects of dietary arginine level on specifc growth rate of M. albus.Each point represents a data and individual data were removed due to excessive bias.

Figure 3 :Figure 4 :
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 fsh per treatment, with standard deviations represented by vertical bars.a, b Mean values with unlike letters were signifcantly diferent (p < 0.05).

Table 1 :
Composition and nutrient levels of experimental diets % used for determining the efects of dietary arginine on the growth and biochemical activities of M.albus.

Table 2 :
Fifteen amino acids composition of experiment diets %.
increasing arginine levels in the diet.Te minimum CF occurred in D1, indicating 2.03% arginine was insufcient for M. albus.Tis suggestion is supported by the WG and SGR data analyses.Te VSI and HSI varied irregularly.3.2.Digestive Enzymes in Liver.Table5demonstrates the dietary arginine level signifcantly afected liver trypsin activity (p < 0.05).Te highest trypsin levels occurred in the 3.16% dietary arginine group, with the variations in trypsin levels showing an irregular pattern.Tere was no variation in lipase and amylase activities in the liver (p > 0.05).

Table 3 :
Real-time PCR primer sequences were used to amplify IL-1β and IL-10 gene of M. albushead-kidney.Note.Te reference gene 18s is a housekeeping gene of M. albus.

Table 4 :
Efects of dietary arginine level on growth performance of M.albus fed with diets containing graded levels of dietary arginine.