Arginine Promotes Testicular Development in Boars through Nitric Oxide and Putrescine

doi:10.21203/rs.3.rs-40835/v1

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Arginine Promotes Testicular Development in Boars through Nitric Oxide and Putrescine

https://doi.org/10.21203/rs.3.rs-40835/v1

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published 01 Jul, 2021

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Background: The present work aimed to explore the influence of arginine on testicular development in boars and the underlying mechanism.

Methods: To this end, thirty 30-day-old male Duroc piglets (7.00 ± 0.30 kg) were randomly sorted into two groups maintained on either a basal diet (CON, n = 15) or a diet supplemented with 0.8% arginine (ARG, n = 15). The average daily gain, average daily feed intake, and testicular volume were determined periodically, and serum samples were collected once every 30 days. The amino acid composition and arginine metabolite levels were estimated in testes samples, collected by castration of three 150-day-old boars randomly selected from each group. Boars semen was collected at 240 days of age to estimate sperm production and quality.

Results: The results showed that dietary supplementation of arginine had no effect on boar growth performance (P > 0.05) but increased the testicular volume and weight of 120- and 150-day-old boars, respectively (P < 0.05). Quantitative analysis of testicular histology showed a higher value for the number of spermatogonia and height of the seminiferous epithelium in the ARG group (P < 0.05). The sperm density, total number of sperm, and effective number of sperm of the boars in the ARG group increased significantly compared with the CON group (P < 0.05). Although arginine supplementation did not affect plasma amino acid levels, the testicular arginine levels in 150-day-old boars showed a significant increase (P < 0.05). The level of serum nitric oxide (NO) and activity of nitric oxide synthase (NOS) also increased in 150-day-old boars in the ARG group (P < 0.05). Interestingly, dietary supplementation of arginine increased the testicular levels of putrescine in 150-day-old boars (P < 0.05).

Conclusions: These results indicate that arginine supplementation increases serum NO levels and testicular arginine and putrescine levels, thereby improving testicular development and semen quality in boars.

Animal Science

arginine supplementation

testicular development

spermatogonia proliferation

Testicular development in boars determines the onset of sexual maturity and subsequent quality of semen [1]. Therefore, maximizing the testicular development in boars would lead to an appreciable increase in the economic returns associated with pig farming. Arginine has been reported to play an important role in male reproductive performance [2]. Studies involving human males have shown that the intake of arginine-deficient food leads to decreased sperm count and sperm viability [3]. Moreover, an infusion of 0.5 g/day Arg-HCL administered orally to infertile men for a period of 6-8 weeks was found to improve sperm motility [4]. Diets supplemented with 0.8 % L-arginine has led to significant improvements in the quality and antioxidant capacity of boar semen [5]. Ren et al. have reported higher total sperm number, acrosome integrity ratio, and effective total sperm number in boars raised on an arginine-supplemented diet [6]. These results indicate that arginine has a positive influence on semen quality; however, the role of arginine in the testicular development process remains unclear.

Arginine can be metabolized to yield several functional products, such as polyamines, creatine, and NO [7]. Research on mouse models has established that L-arginine can catalyze the production of NO by NOS , which could lead to reduction in oxidative stress induced damage in the testes [8]. Early experiments on Leydig cells cultured in vitro demonstrated inhibition of testosterone secretion by NO [9]. However, NO synthesized from arginine has been shown to promote the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from cultured pituitary cells [10]. The NO diffuses into the luteinizing hormone-releasing hormone (LHRH) and follicle-stimulating hormone-releasing factor (FSHRF) terminals and induces the release of the latter by guanylate cyclase (GUCY) and cyclooxygenase activation. Gonadotropin secretion by the pituitary gland is under the control of LHRH and FSHRF [11]. NO regulates the synthesis of steroid hormones mainly via the GUCY / cGMP / protein kinase G (PKG) signaling pathway in the pituitary-gonadal axis [12]. On the other hand, studies have shown that NO likely regulates tight junction (TJ) dynamics in the testis via the cGMP / PKG signaling pathway [13]. This indicates that NO could regulate the formation of the blood-testis barrier and influence testicular development. Pig semen also contains polyamines at an approximate concentration of 90 μmol/L [7], which include spermine and putrescine. These polyamines prevent premature capacitation and acrosome reaction and ensure successful fertilization [14]. For example, testicular maturation and germ cell differentiation in rats are known to be related to polyamine concentrations. High levels of polyamines can promote spermatogenesis [15]. However, the role of arginine in promoting testicular development is not well-understood and needs further research.

The contribution of arginine to pre-puberty testicular development and the possible mechanisms underlying the regulation have been explored in a limited number of studies. In this backdrop, the present study aimed to explore the effect of arginine on testicular development and metabolism in boars. This study, therefore, represents a valuable framework for future research on arginine-mediated enhancements in male reproductive behavior.

2.1 Animals and diets

Thirty 30-day-old male Duroc piglets (7.00 ± 0.30 kg) were randomly allocated to two groups maintained on a basal diet (CON, n = 15) or a diet supplemented with 0.8 % L-arginine (ARG, n = 15). Details of the basal diets are shown in Table 1. In the diet of the ARG group, arginine was used to replace the alanine content of the basic diet to reach the expected 0.8% increment in arginine ingestion by the members of this group compared to those from the CON group. The diets of the two groups were formulated to ensure that these were isonitrogenous and isocaloric, in compliance with guidelines from the NRC (2012). Boars were fed with the test diet during their 30- to 150-day growth period. Boars from the ARG and CON groups were fed basic diets after 150 days of age.

2.2 Feeding management

The experiments were carried out at the Teaching and Research Base of the Animal Nutrition Institute at the Sichuan Agricultural University. Boars were fed ad libitum three times a day and had free access to water. Animals were housed in individual pens in a well-ventilated room with a temperature of 20-26 ℃ and 50-70 % humidity. During the entire duration of the experiments, the boars were cleaned daily, and immunization was performed periodically.

2.3 Estimation of growth performance indices

The total amounts of feed, surplus, and waste per day for each boar were recorded during the trial. The daily feed intake was defined as the total amount of daily feed minus surplus and waste. The body weight of each animal was recorded every 30 days. The feed conversion rate (FCR) was the total amount of feed consumed (kg) per unit weight gain (kg). The distances between the longitudinal and lateral apices of the testis, i.e., the long and short diameters, respectively, of the testis were measured using Vernier calipers every 30 days. Volume was calculated using Lambert's formula[16], as described below:

Testicular volume (cm³) = 0.5236 * (long diameter) * (short diameter)²

2.4 Sample collection

Blood samples (10 mL) were collected from the jugular vein of 30-, 90-, and 150-days-old boars. Samples were centrifuged at 2550 ×g for 10 min at 4 °C. Plasma samples were then transferred to 200 μL microcentrifuge tubes and stored at −20 °C until further analysis.

Three 150-day-old boars, randomly selected from each group, were subjected to surgical castration under anesthesia, and their testicles were collected. Weight of the testicular samples were recorded. Testicular index was defined as testicular weight per unit body weight. The samples were fixed in 4 % formalin and stored for further histological examinations. The remaining testicular samples were stored at -80 °C until further analysis.

At 240-day-old, boars were trained to mount an artificial sow, and semen was collected by gloved-hand technique twice weekly. The gelatinous portion was separated through double layers of sterile gauze, and only the sperm-rich fraction of the ejaculate was collected. To minimize the effects of individual differences, 8 ejaculations per boar were used to estimate sperm production and quality.

2.5 Sample analysis

The fixed testicular samples were sliced into sections of 5 µm thickness with a microtome and stained with hematoxylin and eosin. Quantitative histological analysis was performed with 20 fields per testicular sample. For each field of view, the diameter of the seminiferous tubules, height of the spermatogenic epithelium, number of spermatogonia, and the number of Sertoli cells were estimated [17, 18].

Semen volume and gel weight: Semen volume was measured after straining through two layers of disposable filter membranes to remove gelatinous fraction by weighing each ejaculate and converted to volume as described by Lovercamp et al. [19]. The gelatinous fraction was weighed.

Sperm viability, sperm density, and sperm motility characteristics: The filtered semen was determined using a computer-assisted sperm analysis (CASA) system. The detection method was as follows: 5ml of the mixed semen and the isothermal dilution (BTS) were mixed in a ratio of 1:5. After mixing, 10 μL of semen sample was pipetted onto a glass slide, covered with a coverslip, and incubated at 37 °C for 30 s, and then transferred to a microscope to analyze the sperm viability, density and movement characteristics using the CASA system. Total sperm count and effective sperm count per ejaculation: Refer to the calculation formula of Ren et al. [6].

The amino acid composition was examined using an amino acid auto-analyzer (L-8800; Hitachi, Tokyo, Japan). The testicular samples were pretreated using ion exchange chromatography following hydrolysis in 6 N hydrochloric acid for 24 h at 110 °C under a nitrogen atmosphere, whereas the sulfur-containing amino acids were first oxidized using performic acid (AOAC, 2003). The plasma was pretreated according to a method described by Zhuo et al. [20], with suitable modifications. Blank solutions (0.1 M HCl and 160 μM norleucine) and external standards (acidic/neutral and basic amino acid; Sigma-Aldrich Corp. St. Louis, MO, USA) were prepared in addition to the samples [21].

The samples were homogenized by centrifugation, and the supernatant was collected. Pretreatment of the samples was done according to a method outlined in Kamei et al. [22]. with some modifications. NO and NOS activity of the samples were measured using a commercially available kit (Nanjing Jiancheng Institute of Biological Engineering, Nanjing, China) in an Microplate Reader (Varioskan^TM; Thermo Electron Corporation, Waltham, MA, USA) at wavelengths of 450 and 530 nm, respectively. The concentrations of spermine and putrescine in the samples were analyzed using high performance liquid chromatography (E2695; Waters, Milford, MA, USA). The spermine and putrescine standards were purchased from Sigma-Aldrich Corp.

2.6 Statistical analysis

The Kolmogorov–Smirnov tests were used to test the data for normality. The testicular volume, plasma amino acid level, serum NO level, and polyamine level data were subjected to ANOVA to determine the effects of the dietary arginine supplements. Replicates were analyzed by the PROC GLM program to check for cooperativity of the treatment (arginine supplementation) and duration of treatment. A multivariate model was used to analyze the differences between the CON and ARG groups at each time point. All other data from growth performance evaluations and testis samples were analyzed using the t-test for independent samples in SAS 9.4 (SAS Inst. Inc., Cary, NC, USA). Data are expressed as mean ± SEM. When P < 0.05, the difference was significant, and when P < 0.01, the difference was extremely significant.

3.1 Growth performance

The different indices representative of the growth performance of the boars under study are listed in Table 2. Dietary arginine supplementation was found to have insignificant influences on average daily weight gain, average daily feed intake, and FCR of boars (P > 0.05).

3.2 Testicular development

As shown in Table 3, there was no cooperativity for testicular volume between the arginine supplementation and duration of treatment (P > 0.05), and testicular volume gradually increased with age (P < 0.05). Dietary supplementation with arginine significantly increased testicular volume in 120- and 150-day-old boars (P < 0.05). Although the testicular weight was higher in the 150-day-old boars in the ARG group than in the CON group (Table 4, P < 0.05), there was no significant differences in the testicular index (P > 0.05). Quantitative histological analysis showed a significant increase in the number of spermatogonia and the height of the seminiferous epithelium in boars belonging to the ARG group (Table 4, P < 0.05). However, there was no significant difference in the number of Sertoli cells and tubular diameter (P > 0.05) among the two groups.

3.3 Semen quality

It can be seen from Table 5 that the sperm density, total number of sperm, and effective number of sperm of the boars in the ARG group increased significantly compared with the CON group (P < 0.05). Compared with the CON group, the boar semen volume, gel weight, VAP, VSL, VCL and sperm viability of the boars in the ARG group were not significantly different (P > 0.05).

3.4 Amino acid composition in blood plasma and testis

As shown in Table 6, free amino acids in plasma had no cooperativity between the arginine supplementation and duration of treatment (P > 0.05). The levels of threonine and tryptophan in the plasma did not change significantly with increasing age (P > 0.05); however, that of other free amino acids showed significant changes with age (P < 0.05). Dietary arginine exerted negligible effects on the plasma concentration of arginine and other amino acids across all the time points studied (P > 0.05). Importantly, testicular arginine and glutamate concentrations were significantly higher in boars of the ARG group than in boars of the CON group (P < 0.05, Table 6). Dietary arginine did not influence the testicular concentration of the other amino acids (P > 0.05, Table 7).

3.5 NO levels and NOS activity in boar serum and testis

Supplementation of arginine significantly increased serum NO levels in 150-day-old boars, along with a significant increase in TNOS-type activity (P < 0.05, Figures 1a and 1b). The NO level and NOS activity in the testis of boars in the ARG group did not show any significant differences on comparison to the same parameters in boars of the CON group (P > 0.05, Figures 1c and 1d).

3.6 Spermine and putrescine levels in blood plasma and testis

The serum spermine and putrescine levels in boars belonging to the ARG group did not show any significant differences compared to those in the CON group (P > 0.05, Figures 2a and 2b). The testicular level of putrescine in 150-day-old boars in the ARG group was significantly higher than that in boars of the CON group (P < 0.01, Figure 2c). However, the spermine level in the testis of 150-day-old boars of the ARG group was similar to that in boars of the CON group (P > 0.05, Figure 2c).

Previous studies on milk-fed piglets [23-25] have demonstrated that dietary supplementation of arginine or activation of endogenous pathways of arginine synthesis could effectively improve their growth. On the contrary, Wu et al. have reported that dietary supplementation of arginine in adult pigs had no significant effect on their growth performance [26]. This could be explained by the fact that the arginine synthesis pathway is underdeveloped in newborns and therefore, arginine supplementation can effectively contribute to their growth and development (Wu et al., 2009). However, arginine is not considered to be an essential amino acid in adult animals and humans in the presence of nitrogen balance [27]. Therefore, the effects of arginine on growth performance depend on the physiological age of the animals under study. The present study shows that dietary supplementation of arginine had no effect on the growth performance of piglets after weaning.

A recent study of roosters [28] has shown that that arginine could increase the testicular weight and volume. In overall agreement to this report, the present study demonstrated that boars on a diet supplemented with arginine show increased testicular weight and volume. Further exploration of the mechanism indicates that the increase in testicular weight observed in this study was due to an increase in the number of spermatogonia in the boars. Previous studies on mice have shown significant promotion of germ cell proliferation in the testes upon L-arginine supplementation [29]. Spermatogonia are a group of cells that can produce sperm through mitosis and meiosis. Therefore, the increase in the number of spermatogonia could support spermatogenesis, thereby significantly improving the quality of semen [30]. Similarly, the results of this study show that the sperm density, total number of sperm, and effective number of sperm have increased significantly. The results of Chen et al. [5] showed that dietary supplementation with 0.8% to 1.0% L-arginine can significantly improve the boar sperm count and semen antioxidant capacity. In this context, the present study is the first demonstration that arginine can promote proliferation of boar spermatogonia, providing novel insights into the contribution of arginine toward enhancing semen quality.

This study found that there was no significant change in plasma arginine levels at any stage of growth. In general, the level of free amino acids in animals is relatively stable, whereas dietary composition and postprandial time are known to affect the plasma free amino acid (PFAA) levels [31]. Studies on sheep fed with an arginine-supplemented diet have demonstrated that serum arginine concentrations increased 1 to 2 hours after food intake and were restored to basal levels after 4 to 7 hours [32]. Therefore, PFAA levels may vary due to differences in blood collection times. However, the present study shows that the testicular concentration of arginine and glutamate increased upon arginine supplementation. This also indicates that dietary supplementation of arginine may promote male reproduction by increasing arginine levels in the testis. L-arginine is taken up by the Sertoli and peritubular cells in the testis, principally via system y(+)L (SLC3A2/SLC7A6) and system y(+) (SLC7A1 and SLC7A2) [33]. Increased testicular concentrations of arginine result in increased synthesis of Arg-rich proteins [26]. L-arginine could then be transported into the testis through the blood-testis barrier to promote protein synthesis and proliferation of spermatogenic cells.

The testis and semen have been reported to be rich in arginine, citrulline, and polyamines, which can promote spermatogenesis and improve semen quality [7]. The present study demonstrates that arginine supplementation increased serum NO levels and NOS activity. An earlier study by Knowles et al. [34] showed that the NOS enzyme catalyzes the formation of NO and citrulline from arginine. On the other hand, Lee et al. [35] confirmed L-arginine to be the only endogenous nitrogen-containing substrate of NOS enzyme. Therefore, arginine can regulate the activity of NOS enzyme, and consequently regulate NO production. NOS and NO are known to participate in diverse physiological functions and cellular signaling pathways. For example, NO can manipulate the tight junction of the blood-testis barrier to promote spermatogenesis, which is mediated via the sGUCY/cGMP/PKG pathway[36, 37]. Moreover, NO can induce proliferation and apoptosis of germ cells and promote the secretion of testosterone by Leydig cells (Dietrich et al., 2015; Welch et al., 1995). In a recent study, L-arginine has been shown to stimulate protein synthesis via the activation of the mTOR (Thr 2446)/p70S6K signaling pathway in an NO-dependent manner [38]. This suggests that arginine or NO could mediate the mTOR signaling pathway. The mTOR/p70S6K signaling pathway is reported to be an important pathway for regulating spermatogonia proliferation [39]. In that context, the present study reported an increase in the number of spermatogonia. The possibility of this being related to the regulation of cell proliferation by a crosstalk between NO and mTOR associated signaling pathways would be the focus of future investigations.

This study further shows that the levels of testicular putrescine in boars of the ARG group are significantly higher than those in boars of the CON group. Polyamines, including putrescine and spermine, are essential for spermatogenesis [7]. Arginine and putrescine have been shown to significantly improve sperm motility in patients with primary and diabetic azoospermia [40]. Some earlier studies have also shown that polyamines can promote spermatogenesis mediated by proliferation and differentiation of spermatogonia [41, 42]. Higher levels of putrescine in the semen could prevent premature capacitation and acrosome reaction of sperms, thereby improving the semen quality. Therefore, the increase in testicular weight and number of spermatogonia observed in this study could be associated with the regulation of NO and polyamine levels.

Dietary supplementation of arginine led to significant enhancements in testicular volume and weight and number of sperm in boars. The observed increase is possibly due to arginine-induced increases in serum NO and testicular arginine and putrescine levels, which, in turn, regulate testicular development.

CON: control group

ARG: arginine group

NO: nitric oxide

NOS: Nitric oxide synthase

FCR: The feed conversion rate

LHRH: Luteinizing hormone-releasing hormone

FSHRF: Follicle-stimulating hormone-releasing factor

PFAA: Plasma free amino acid

mTOR: mammalian target of rapamycin

Availability of data and materials

All data included in this study are available upon request by contact with the corresponding author.

Acknowledgements

Thanks to National 13th Five-Year Plan Key R&D Projects and The National Natural Science Foundation of China for funding this project.

Funding

The present study was funded by National 13th Five-Year Plan Key R&D Projects: The National Key R&D Program of China [2018YFD0501002], The National Key R&D Program of China [2017YFD0501902], and The National Natural Science Foundation of China [NO. 31702128].

Author information

Affiliations

Key Laboratory of Animal Disease-Resistance Nutrition and Feed Science, Ministry of Agriculture, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China

Dongqin Wei, De Wu, Lianqiang Che, Shengyu Xu, Zhengfeng Fang, Bin Feng, Jian Li, Yong Zhuo, Caimei Wu, Yan Lin

Key Laboratory of Animal Disease-Resistance Nutrition, Ministry of Education, Chengdu, 611130, Sichuan, China.

Dongqin Wei, De Wu, Lianqiang Che, Shengyu Xu, Zhengfeng Fang, Bin Feng, Jian Li, Yong Zhuo, Caimei Wu, Yan Lin

School of Life Sciences, Sichuan Agricultural University, Ya’an, Sichuan 625014, China

Junjie Zhang

College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, Shanxi, China

Wenxian Zeng

Contributions

Dongqin Wei, De Wu, Wenxian Zeng, and Yan Lin conceived, designed, and conducted the experiments. Dongqin Wei, Yan Lin, Lianqiang Che, Shengyu Xu, Zhengfeng Fang conducted the experiments. Dongqin Wei, Yan Lin, Bin Feng, Jian Li, Yong Zhuo, Caimei Wu, and Junjie Zhang acquired and analyzed the data. Dongqin Wei and Yan Lin drafted the manuscript.

None of the authors have any conflict of interest to declare.

Corresponding authors

Correspondence to Yan Lin.

Ethics declarations

Ethics approval and consent to participate

All the protocols used in the study were approved by the Animal Care and Use Committee of Animal Nutrition Institute, Sichuan Agriculture University, and were in compliance with the current laws of animal protection (Ethics Approval Code: SCAUAC201408-3).

Consent for publication

Not applicable.

Competing interests

None of the authors have any conflict of interest to declare.

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**Table 1**: Ingredients and nutrient composition of basal diet
Ingredients, %	7-11kg	11-25kg	25-50kg	50-100kg	100-130kg	130kg-
Corn	19.96	26.10	72.17	73.78	75.47	74.15
Soy oil	1.50	2.00	2.00	1.80	1.80	-
Expanded corn	19.00	19.00	-	-	-	-
Sucrose	2.00	-	-	-	-	-
Glucose	2.00	2.00	-	-	-	-
Whey powder	13.00	8.00	-	-	-	-
Dehulled soybean meal	7.10	10.50	-	-	-	-
Extruded soybean	12.00	12.00	-	-	-	-
Rice broken	10.00	11.00	-	-	-	-
Plasma protein	4.00	-	-	-	-	-
Soybean meal	-	-	18.88	18.60	18.60	18.00
Wheat bran	-	-	-	-	-	4.00
Fish meal（CP60.2%）	5.00	5.00	2.00	0.80	0.80	0.80
Lys-HCL	0.54	0.55	0.45	0.39	0.39	0.34
DL-Met	0.13	0.10	0.16	0.12	0.12	0.05
L-Thr	0.18	0.20	0.14	0.11	0.11	0.06
L-Trp	0.03	0.04	0.03	0.01	0.01	-
L-Val	0.10	0.11	0.04	0.01	0.01	-
L-Ile	0.09	0.03	-	-	-	-
Choline chloride（50%）	0.10	0.10	0.10	0.10	0.10	0.10
Limestone	1.03	0.88	0.86	0.90	0.90	0.96
Dicalcium phosphate	-	-	0.99	1.15	1.15	1.00
Salt	0.30	0.45	0.30	0.35	0.35	0.35
Vitamin premix ^a	0.05	0.05	0.04	0.04	0.04	0.04
Mineral premix ^b	0.20	0.20	0.15	0.15	0.15	0.15
L-alanine	1.69	1.69	1.69	1.69	-	-
Total	100.00	100.00	100.00	100.00	100.00	100.00
Nutrient level
DE (Mcal/kg)	3.58	3.51	3.42	3.41	3.39	3.25
CP (%) ^c	20.46	19.49	17.50	16.64	15.12	15.19
Ca (%) ^c	0.80	0.70	0.70	0.70	0.70	0.70
Available P (%) ^c	0.42	0.33	0.34	0.34	0.34	0.32
Lys (%)^d	1.18	1.12	1.11	1.02	0.80	0.77
Met (%) ^d	0.30	0.26	0.38	0.28	0.20	0.11
Thr (%) ^d	0.86	0.69	0.73	0.66	0.60	0.57
Val (%) ^d	0.83	0.75	0.72	0.69	0.67	0.58
Ile (%) ^d	0.70	0.64	0.62	0.62	0.61	0.59
Arg (%) ^d	1.08	1.06	1.24	0.93	0.91	0.86

^aAdditive of vitamins mixture provided per kg of diets: VD₃ 1500 IU, VK₃ 2 mg, VB₁ 2.7 mg, VB₂ 6.5 mg, VE 109.5 IU, VB₆ 3.9 mg, VB₁₂ 0.015 mg, VA 6000 IU, niacin 26 mg, Pantothenic acid 1.3 mg, folic acid 1250 mg.

^bAdditive of trace minerals mixture provided per kg of diets: Cu, 16 mg; Zn, 125 mg; Fe, 125 mg; Mn, 20 mg; Se, 0.3 mg; I, 0.3 mg.

^cCalculated.

^dAnalyzed.

**Table 2**: Effect of dietary supplementation with arginine on growth performance of boars
Items	CON	ARG	P-value
Average daily gain (g)
31-60 d	458.86±24.00	501.92±24.23	0.22
61-90 d	539.43±51.77	581.17±28.61	0.50
91-120 d	903.23±49.85	978.00±26.73	0.13
121-150 d	1010.20±61.34	1085.30±13.13	0.25
31-150 d	737.90±22.17	779.00±15.98	0.27
Average daily feed intake (g)
31-60 d	729.73±24.87	737.53±22.40	0.82
61-90 d	1081.10±36.74	1043.45±23.32	0.39
91-120 d	1724.26±68.02	1764.08±47.49	0.64
121-150 d	2420.77±22.46	2443.24±16.83	0.43
31-150 d	1502.46±22.50	1510.82±17.37	0.77
Feed conversion rate
31-60 d	1.64±0.06	1.49±0.05	0.06
61-90 d	1.98±0.14	1.83±0.08	0.31
91-120 d	1.99±0.10	1.83±0.04	0.16
121-150 d	2.57±0.23	2.26±0.04	0.20
31-150 d	2.07±0.06	1.97±0.39	0.24