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Dietary l -arginine supplementation enhances porcine β-defensins gene expression in some tissues of weaned pigs
Livestock Science 148 (2012) 103–108
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Dietary L-arginine supplementation enhances porcine b-defensins gene expression in some tissues of weaned pigs Xiangbing Mao 1, Sharina Qi 1, Bing Yu, Zhiqing Huang, Hong Chen, Qian Mao, Guoquan Han, Daiwen Chen n Key Laboratory of Animal Disease-Resistance Nutrition, Animal Nutrition Institute, Sichuan Agricultural University, Ministry of Education, China, Ya’an 625014, People’s Republic of China
a r t i c l e i n f o
abstract
Article history: Received 21 August 2011 Received in revised form 21 March 2012 Accepted 21 May 2012
The present study was mainly conducted to test the hypothesis that dietary L-arginine supplementation regulates gene expression of b-defensins in some tissues of weaned pigs. Thirty crossbred piglets weaned at 21 days of age were allotted to 1 of 3 diets supplemented with 0.0, 0.5 and 1.0% L-arginine (10 pigs/treatment). Pigs consumed the diets for 7 d. On d 7, 6 pigs from each treatment were randomly killed, and the tissue and blood samples were collected. Compared with control pigs, dietary L-arginine supplementation did not affect the growth performance and serum concentrations of IgA, IgG, IgM, IL-1b, IL-2 and TNF-a (P 4 0.05), but dietary supplementation with 0.5% L-arginine reduced serum urea concentration (P o 0.05). Additionally, dietary L-arginine supplementation significantly increased porcine b-defensin-1 gene expression of tongue, porcine b-defensin-3 gene expression of ileum and inguinal lymph node, and porcine b-defensin-2 gene expression of oral epithelium, tongue, ileum and inguinal lymph node (P o 0.05). These results suggest that dietary supplementation with L-arginine enhances gene expressions of porcine b-defensins in some tissues of weaned pigs. Crown Copyright & 2012 Published by Elsevier B.V. All rights reserved.
Keywords: L-arginine Porcine b-defensins Gene expression Weaned pigs
1. Introduction The mammalian immunity, including the innate and adaptive immunity, plays an important role for preventing from the invasion of pathogens (Yuan and Walker, 2004). The secretion of antimicrobial peptides, such as cathelicidins and defensins, is a part of the innate immunity in host,
Abbreviations: mTOR, Mammalian target of rapamycin; Ig, Immunoglobulin; IL, Interleukin; TNF, Tumor necrosis factor; pBDs, Porcine b-defensins n Correspondence to: Key Laboratory of Animal Disease-Resistance Nutrition, Animal Nutrition Institute, Sichuan Agricultural University, Xingkang Road 46#, Ya’an, Sichuan Province 625014, China. Tel./fax: þ86 835 288 5106. E-mail address: [email protected] (D. Chen). 1 Xiangbing Mao and Sharina Qi contributed equally to this work.
especially in immature animals (Froy and Gurevitz, 2003; Ganz, 2003). Defensins are the small cationic polypeptides, which are composed of 20–40 amino acids in length and three intramolecular disulfide bonds. Based on the relative positions of intramolecular disulfide bonds, they could be classified as a-, b-, y-defensins (Kaiser and Diamond, 2000). Current investigations about them mainly focus on the b-defensin. Recent studies have shown that some nutrients, such as L-isoleucine, Ca2 þ , arginine and glucose, may regulate the expressions of human b-defensin-1 in cell-culture experiments (Fehlbaum et al., 2000; Harder et al., 2004; Sherman et al., 2006). The investigations of porcine b-defensins (pBDs) mainly are about the effect of pathogens on their expression (Liu et al., 2001; Tarver et al., 1998; Veldhuizen et al., 2006), but the role for the nutrients in regulation of pBDs’ expressions has not been investigated.
1871-1413/$ - see front matter Crown Copyright & 2012 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.livsci.2012.05.016
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Arginine, which is a functional amino acid, may regulate protein metabolism, reproduction, intestinal physiology and immunity (Flynn et al., 2002; Mateo et al., 2007; Tan et al., 2009; Yao et al., 2008; Zeng et al., 2008; Zhan et al., 2008). Therefore, it plays a critical role for the fetal and neonatal pigs (Wu et al., 1999, 2004). The studies of its immunomodulatory effect have shown that L-arginine administration could enhance thymus size, improve lymphocyte proliferation, affect the constitution of blood leukocytes, augment the concentration of some immunoglobulins and cytokines, and increase the activity of macrophage and natural killer cell (Kwak et al., 1999; Popovic et al., 2007; Rodriguez et al., 2003; Yeh et al., 2002). Furthermore, dietary arginine supplementation increases immunity of some animals under immunological challenges (Li et al., 2007; Liu et al., 2008). However, it is unknown whether arginine can regulate the expressions of pBDs in pigs. On the basis of the foregoing, the major objective of the present study was to evaluate whether dietary arginine supplementation might stimulate gene expressions of pBDs in some tissues of weaned pigs. 2. Materials and methods 2.1. Animals and diets The Animal Care Advisory Committee of Sichuan Agricultural University approved the protocol used in this experiment. A total of 30 crossbred (Duroc Large White Landrace) barrows, weaned at 21 d of age, were housed individually in 1.20 0.45 m2 pens over plastic-coated and expanded-metal floors. The temperature of room was maintained at 25–28 1C, and lighting was natural. After a 3-d adaptation period, on the basis of the initial body weights and origin of litters, all pigs were weighed (7.16 70.22 kg) and allotted randomly to 1 of 3 treatments (n¼10/treatment), representing supplementation with 0.0, 0.5 and 1.0% L-arginine–HCl (Ajinomoto, Tokyo, Japan) to a basal diet (Table 1), which was formulated to meet National Research Council-recommended nutrient requirements for weanling pigs (National Research Council (NRC), 1998). All diets were made isonitrogenous with addition of appropriate levels of L-alanine, as described by Kim et al. (2004). Therefore, the measured levels of arginine in these diets were 1.21, 1.65 and 1.99%, respectively. The pigs were fed their respective diets 4 times daily at 0800, 1200, 1600 and 2000 h. Among meals, all pigs had free access to drinking water. At 0800 h of d 0 and 7, the body weight and feed intake of all pigs were measured. These values were used to determine average daily weight gain (ADG), average daily feed intake (ADFI), and feed efficiency. 2.2. Sample collection Following weighed on d 7, the pigs received their feed. The 6 pigs in each treatment were selected randomly for obtaining blood samples by the jugular vein at 2 h after feeding. The blood samples were centrifuged at 3500 g for 10 min, and serum was stored at 80 1C until analysis.
Table 1 Composition and nutrient levels of the basal diet (on an as-fed basis). Ingredients
g/kg
Corn Soybean meal Fish meal Corn protein powder Whey Extruded full-fat soybean Sucrose L-Tryptophan L-Lysine HCl, 78% DL-Methionine L-Threonine Calcium carbonate Dicalcium phosphate Sodium Chloride Vitamin–mineral premixa
554.2 60 67.2 8 80 180 17.6 0.6 3.5 1.7 1.5 8 5.2 2.5 10
Nutrients Digestible energy (kJ/g)b Crude proteinc Calciumc Total phosphorusc Argininec Lysinec Methioninec
13.49 184.7 8.0 6.5 12.4 16.3 5.2
a Supplied per kg diet: Mn (as MnSO4 H2O), 4 mg; Fe (as FeSO4 7H2O), 100 mg; Zn (as ZnSO4 7H2O), 100 mg; Cu (as CuSO4 5H2O), 6 mg; I (KI), 0.14 mg; Se (as Na2SeO3 5H2O), 0.3 mg; vitamin A, 2800 IU; vitamin D3, 320 IU; vitamin E, 20 IU; vitamin K3, 1 mg; vitamin B12, 0.03 mg; riboflavin, 5 mg; pantothenic acid, 15 mg; nicotinic acid, 20 mg; thiamin, 1.5 mg; pyridoxine, 2 mg; folic acid, 0.5 mg; biotin, 0.1 mg; and choline chloride, 500 mg. b Calculated nutrient levels. c Measured nutrient levels.
When sampling blood was completed, the selected pigs were killed with an intracardial injection of sodium pentobarbital (50 mg/kg body wt.) and jugular exsanguinations. Then, the liver, respiratory tract, oral epithelium, longissimus muscle, tongue, jejunum, ileum, inguinal lymph node and mesenteric lymph node were immediately isolated, frozen in liquid nitrogen, and stored at 80 1C for the extraction of total RNA. 2.3. Chemical analyses Samples of the diets were analyzed according to the methods of the Association of Official Analytical Chemists (Association of Official Analytical Chemists (AOAC), 1995). Analyses were conducted for calcium (AOAC method 935.13), phosphorus (AOAC method 965.17), and crude protein (AOAC method 988.05). Serum free amino acids were analyzed using a L-8800 Amino Acid Analyzer (Hitachi), as previously described (Zhan et al., 2008). Serum urea was measured using an assay kit from Nanjing Jiancheng Biochemistry. 2.4. Analysis of serum immunoglobulins and cytokines Serum concentrations of IgA, IgG, IgM, IL-1b, IL-2 and TNF-a were measured by using the commercially available pig enzyme-linked immunosorbent assay kits from
X. Mao et al. / Livestock Science 148 (2012) 103–108
Table 2 Primer sequences used for real-time PCR. Gene
Primer
b-actin
Forward Reverse
50 -TCTGGCACCACACCTTCT-30 50 -TGATCTGGGTCATCTTCTCAC-30
pBD-1
Forward Reverse
50 -TCCTTGTATTCCTCCTCA-30 50 -ACACGCCTTTATTCCTTA-30
pBD-2
Forward Reverse
50 -ACCTGCTTACGGGTCTTG-30 50 -CTCTGCTGTGGCTTCTGG-30
pBD-3
Forward Reverse
50 -GAAGTCTACAGAAGCCAAAT-30 50 -GGTAACAAATAGCACCATAA-30
Table 3 Effects of dietary L-arginine supplementation on growth performance of weaned pigsa Item
2.5. Determination of pBDs gene expressions in some tissues
2.6. Statistical analysis All data, expressed as mean7SEM, were analyzed using one-way ANOVA, followed by Duncan’s Multiple Range test. All analyses were performed using SAS (Version 8.1; SAS Institute, Gary, NC). Po0.05 was taken to indicate statistical significance. 3. Results 3.1. Effects of dietary L-arginine supplementation on growth performance of weaned pigs The growth performance data of weaned pigs are shown in Table 3. The initial body weight of weaned pigs among 3 treatments did not differ (P40.05). During the experimental period, dietary L-arginine supplementation
0.5
1.0
Initial weight (kg) 7.127 0.24 7.247 0.20 7.14 7 0.21 Final weight (kg) 8.157 0.20 8.387 0.21 8.52 7 0.30 ADG (g/d) 147.147 11.23 162.867 17.52 197.147 20.27 ADFI (g/d) 242.637 20.44 265.30 7 21.88 262.197 18.82 Feed efficiency 1.817 0.19 1.437 0.14 1.46 7 0.15 (g feed/g gain) Values are mean 7 SEM, n ¼6.
Table 4 Effects of dietary L-arginine supplementation on serum concentrations of amino acids and urea in weaned pigs1. Item
Total RNA extraction, reverse transcription reaction and quantitative real-time PCR of pBDs and b-actin genes were determined as previously described (Qi et al., 2009). Briefly, total RNA of tissues was extracted with RNAiso Reagent (TaKaRa, Japan), and reverse-transcribed with RT Reagents (TaKaRa, Japan) according to manufacturer’s instructions. Quantitative real-time PCR was performed using 96-well iCycler iQTM Real-Time PCR Detection System (BIO-RAD, USA). The gene-specific primers used were listed in Table 2, and purchased from TaKaRa (Japan). The PCR system consisted of 12.5 mL SYBR Green PCR Master Mix (TaKaRa, Japan), 2.0 mL of cDNA, 8.5 mL of PCR-grade water, and 2.0 mL of primer pairs (100 mM forward and 100 mM reverse) for a total volume of 25 mL. All samples were assayed in triplicate. Cycling conditions were as follows: 94 1C for 10 s, and 40 cycles involving a combination of 94 1C for 5 s, 55.5 1C for 20 s and 72 1C for 15 s. Relative gene expression to the housekeeping gene (b-actin) was performed in order to correct for the variance in amounts of RNA input in the reactions. However, the relative gene expressions compared to the housekeeping gene were calculated with the Pfaffl (2001) method.
L-Arginine supplementation (%) 0.0
a
R&D system (Minneapolis, MN) according to the manufacturer’s instructions.
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L-Arginine supplementation (%) 0.0
Amino acids (mmol/L) Arginine 67.17 7 2.28c Lysine 76.55 7 10.35a Citrulline 42.17 7 7.21b Ornithine 44.36 7 1.00b Serum urea, mg/L 82.21 7 1.15a 1
0.5
1.0
94.08 7 0.60b 115.3577.82a 54.13 7 0.55ab 36.89 72.90b 56.42 7 4.81b 82.39 73.84a 71.95 7 14.06ab 102.37 74.91a 73.85 7 2.98b 76.57 70.62ab
Values are means7 SEM; n¼ 6. Values with different letters are significantly different (P o0.05).
a–c
did not affect ADG, ADFI and feed efficiency of weaned pigs (P40.05).
3.2. Effects of dietary L-arginine supplementation on serum concentrations of free amino acids and urea in weaned pigs As shown in Table 4, dietary supplementation with 0.5 and 1.0% L-arginine significantly increased (Po0.05) serum concentration of arginine by 40 and 72%, respectively. Serum concentrations of citrulline and ornithine were significantly greater (Po0.05) in pigs supplemented with 1.0% L-arginine compared with control pigs. Supplementation with 1.0% L-arginine also decreased (P o0.05) lysine concentration in serum compared with control pigs. In addition, the serum concentrations of urea were 10% lower (Po0.05) in pigs of 0.5% L-arginine supplementation treatment compared with control pigs.
3.3. Effects of dietary L-arginine supplementation on serum concentrations of immunoglobulins and some cytokines in weaned pigs The serum concentrations of IgA, IgG, IgM, IL-1b, IL-2 and TNF-a were determined (Fig. 1). During experimental period, dietary supplementation with 0.5 and 1.0% L-arginine did not significantly affect (P40.05) serum concentrations of these immunoglobulins and cytokines in weaned pigs.
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Fig. 1. Effects of dietary L-arginine (L-Arg) supplementation on serum concentrations of IgA, IgG, IgM, IL-1b, IL-2 and TNF-a in weaned pigs on d 7. Values are mean 7 SEM, n¼ 6. Values with different letters are significantly different (P o 0.05).
3.4. Effects of dietary L-arginine supplementation on pBDs gene expressions of some tissues in weaned pigs Dietary supplementation with 0.5 and 1.0% L-arginine significantly increased (Po0.05) pBD-1 gene expression of tongue (Fig. 2A), pBD-3 gene expression of ileum and inguinal lymph node (Fig. 2C), and pBD-2 gene expression of oral epithelium, tongue, ileum and inguinal lymph node (Fig. 2B). The pBD-1 gene expression of oral epithelium was dramatically greater (Po0.05) in pigs of 1.0% L-arginine supplementation groups than that of the control pigs (Fig. 2A). Furthermore, dietary supplementation with 0.5% L-arginine enhanced (Po0.05) pBD-2 gene expression in longissimus muscle, compared with control pigs (Fig. 2B). The pBDs gene expressions of other tissues did not differ among the 3 treatments. 4. Discussion The results of recent published studies are inconsistent about the effect of arginine administration on growth performance of piglets. Kim et al. (2004) and Tan et al. (2009) showed that dietary arginine supplementation could significantly increase the average daily gain of piglets weaned at 7 days of age. However, Liu et al. (2008) and Zhan et al. (2008) demonstrated that dietary arginine supplementation did not alter growth performance of piglets weaned at 14 or 21 days of age. In this study, dietary arginine supplementation did not affect the growth performance of weaning pigs, too (Table 3). Furthermore, some studies have already demonstrated that the regulation of nutrients was gradually decreased with the increasing age (Kimball et al., 2001; Suryawan et al., 2006). Therefore, the difference among the results of these studies could be due to the initial age of piglets in experiments. As shown by some investigators (Kim et al., 2004; Zhan et al., 2008), dietary L-arginine supplementation increased serum L-arginine concentration in weaned pigs
compared with the control (Table 4). The serum concentrations of citrulline and ornithine, which are correlated with metabolism of arginine, were also increased significantly by dietary supplementation with 1.0% L-arginine (Table 4). Due to the antagonism between arginine and lysine, the intestinal absorption of these 2 amino acids could be influenced by the change of dietary arginine:lysine ratio. Serum lysine concentration was decreased dramatically in weaned pigs supplemented with 1.0% L-arginine in this study (Table 4). Furthermore, dietary supplementation of 0.5% L-arginine substantially reduced serum urea level of weaned pigs (Table 4). The decreasing urea level suggests the reduction of ammonia, which is a result that the efficiency of absorbed amino acids for tissue growth is increased (Coma et al. 1995). Dietary arginine supplementation may increase protein synthesis in skeletal muscle of neonatal pigs by mTOR signaling (Yao et al., 2008). However, dietary supplementation of 1.0% L-arginine did not affect serum urea level of weaned pigs in this study (Table 4), which could be derived from the arginine–lysine antagonism that led to imbalance of amino acids. In previous studies, arginine is one of the most important nutrients that may regulate immune function in different animals (Evoy et al., 1998; Li et al., 2007; Peranzoni et al., 2008). Its immunomodulatory effect may improve the inhibited immune function derived from some diseases and stress conditions in laboratory animals and humans (Han et al., 2009; Li et al., 2007; Liu et al., 2008). Some recent studies of laboratory animals demonstrate that dietary L-arginine supplementation could augment thymus size, improve lymphocyte proliferation, affect the constitution of blood leukocytes, and increase the activity of macrophage and natural killer cell (Kwak et al., 1999; Popovic et al., 2007; Rodriguez et al., 2003; Yeh et al., 2002). In addition, the study with early-weaned pigs showed that dietary administration of L-arginine significantly increased the serum concentrations of some immunoglobulins and cytokines at day 14, but did not affect their concentrations at day 7 (Liu et al., 2008). In present study, the serum concentrations of immunoglobulins and some cytokines were not also influenced by the dietary L-arginine supplementation at day 7 (Fig. 1). These results demonstrated that the effect of dietary L-arginine supplementation on serum concentrations of immunoglobulins and cytokines in weanling pigs needed for the enough duration. As an important part of mammalian innate immunity, the broad-spectrum antimicrobial peptides, such as cathelicidins and defensins, play a critical role in protecting organisms from all kinds of microbial (Froy and Gurevitz, 2003; Ganz, 2003). These antimicrobial peptides are expressed in multiple tissues and organs of mammal. Our previous studies have shown that there were the expressions of pBDs in multiple tissues of the crossbred and Tibetan pigs, and there was the difference of pBDs expression among different tissues and pig breeds (Qi et al., 2009). Some studies demonstrated that human b-defensin-1 expression of various cells could be stimulated by some nutrients, such as L-isoleucine, Ca2 þ and glucose (Fehlbaum et al., 2000; Harder et al., 2004; Sherman et al., 2006).
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Fig. 2. Effects of dietary L-arginine (L-Arg) supplementation on gene expressions of pBD-1 (A), pBD-2 (B) and pBD-3 (C) in some tissues of weaned pigs. Values are mean 7 SEM, n¼ 6. Values with different letters are significantly different (P o0.05).
Additionally, L-arginine treatment could upregulate expression of human b-defensin-1 in HCT-116 cells (Sherman et al., 2006). In this study, we found that dietary L-arginine supplementation could stimulate pBDs expressions of some
tissues in weaned pigs (Fig. 2). However, further studies are required to determine the mechanism responsible for the different effect of arginine on regulating pBDs’ expressions in various tissues.
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5. Conclusions This study indicates that the different doses of supplemental L-arginine for 7 days did not affect the growth performance and serum concentrations of immunoglobulins and some cytokines, but significantly increased the expressions of pBDs in some tissues of weaned pigs. This finding provides further explanation whereby arginine improves immune function of animals. However, further studies are required to determine the intracellular signaling mechanism involving in the effect of arginine on regulating pBDs’ expressions. Additionally, because pigs are a efficiency animal model for investigating human nutrition (Wu et al., 2004), the results of this work may be an important implication for improving the immune function of human, especially infants. Conflicts of interest statement Mao, Qi, Yu, Huang, Chen, Mao, Han, and Chen declare that: we have no proprietary, financial, professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled: Dietary L-arginine supplementation enhances porcine b-defensins gene expression in some tissues of weaned pigs.
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Dietary L-arginine supplementation enhances porcine b-defensins gene expression in some tissues of weaned pigs Xiangbing Mao 1, Sharina Qi 1, Bing Yu, Zhiqing Huang, Hong Chen, Qian Mao, Guoquan Han, Daiwen Chen n Key Laboratory of Animal Disease-Resistance Nutrition, Animal Nutrition Institute, Sichuan Agricultural University, Ministry of Education, China, Ya’an 625014, People’s Republic of China
a r t i c l e i n f o
abstract
Article history: Received 21 August 2011 Received in revised form 21 March 2012 Accepted 21 May 2012
The present study was mainly conducted to test the hypothesis that dietary L-arginine supplementation regulates gene expression of b-defensins in some tissues of weaned pigs. Thirty crossbred piglets weaned at 21 days of age were allotted to 1 of 3 diets supplemented with 0.0, 0.5 and 1.0% L-arginine (10 pigs/treatment). Pigs consumed the diets for 7 d. On d 7, 6 pigs from each treatment were randomly killed, and the tissue and blood samples were collected. Compared with control pigs, dietary L-arginine supplementation did not affect the growth performance and serum concentrations of IgA, IgG, IgM, IL-1b, IL-2 and TNF-a (P 4 0.05), but dietary supplementation with 0.5% L-arginine reduced serum urea concentration (P o 0.05). Additionally, dietary L-arginine supplementation significantly increased porcine b-defensin-1 gene expression of tongue, porcine b-defensin-3 gene expression of ileum and inguinal lymph node, and porcine b-defensin-2 gene expression of oral epithelium, tongue, ileum and inguinal lymph node (P o 0.05). These results suggest that dietary supplementation with L-arginine enhances gene expressions of porcine b-defensins in some tissues of weaned pigs. Crown Copyright & 2012 Published by Elsevier B.V. All rights reserved.
Keywords: L-arginine Porcine b-defensins Gene expression Weaned pigs
1. Introduction The mammalian immunity, including the innate and adaptive immunity, plays an important role for preventing from the invasion of pathogens (Yuan and Walker, 2004). The secretion of antimicrobial peptides, such as cathelicidins and defensins, is a part of the innate immunity in host,
Abbreviations: mTOR, Mammalian target of rapamycin; Ig, Immunoglobulin; IL, Interleukin; TNF, Tumor necrosis factor; pBDs, Porcine b-defensins n Correspondence to: Key Laboratory of Animal Disease-Resistance Nutrition, Animal Nutrition Institute, Sichuan Agricultural University, Xingkang Road 46#, Ya’an, Sichuan Province 625014, China. Tel./fax: þ86 835 288 5106. E-mail address: [email protected] (D. Chen). 1 Xiangbing Mao and Sharina Qi contributed equally to this work.
especially in immature animals (Froy and Gurevitz, 2003; Ganz, 2003). Defensins are the small cationic polypeptides, which are composed of 20–40 amino acids in length and three intramolecular disulfide bonds. Based on the relative positions of intramolecular disulfide bonds, they could be classified as a-, b-, y-defensins (Kaiser and Diamond, 2000). Current investigations about them mainly focus on the b-defensin. Recent studies have shown that some nutrients, such as L-isoleucine, Ca2 þ , arginine and glucose, may regulate the expressions of human b-defensin-1 in cell-culture experiments (Fehlbaum et al., 2000; Harder et al., 2004; Sherman et al., 2006). The investigations of porcine b-defensins (pBDs) mainly are about the effect of pathogens on their expression (Liu et al., 2001; Tarver et al., 1998; Veldhuizen et al., 2006), but the role for the nutrients in regulation of pBDs’ expressions has not been investigated.
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Arginine, which is a functional amino acid, may regulate protein metabolism, reproduction, intestinal physiology and immunity (Flynn et al., 2002; Mateo et al., 2007; Tan et al., 2009; Yao et al., 2008; Zeng et al., 2008; Zhan et al., 2008). Therefore, it plays a critical role for the fetal and neonatal pigs (Wu et al., 1999, 2004). The studies of its immunomodulatory effect have shown that L-arginine administration could enhance thymus size, improve lymphocyte proliferation, affect the constitution of blood leukocytes, augment the concentration of some immunoglobulins and cytokines, and increase the activity of macrophage and natural killer cell (Kwak et al., 1999; Popovic et al., 2007; Rodriguez et al., 2003; Yeh et al., 2002). Furthermore, dietary arginine supplementation increases immunity of some animals under immunological challenges (Li et al., 2007; Liu et al., 2008). However, it is unknown whether arginine can regulate the expressions of pBDs in pigs. On the basis of the foregoing, the major objective of the present study was to evaluate whether dietary arginine supplementation might stimulate gene expressions of pBDs in some tissues of weaned pigs. 2. Materials and methods 2.1. Animals and diets The Animal Care Advisory Committee of Sichuan Agricultural University approved the protocol used in this experiment. A total of 30 crossbred (Duroc Large White Landrace) barrows, weaned at 21 d of age, were housed individually in 1.20 0.45 m2 pens over plastic-coated and expanded-metal floors. The temperature of room was maintained at 25–28 1C, and lighting was natural. After a 3-d adaptation period, on the basis of the initial body weights and origin of litters, all pigs were weighed (7.16 70.22 kg) and allotted randomly to 1 of 3 treatments (n¼10/treatment), representing supplementation with 0.0, 0.5 and 1.0% L-arginine–HCl (Ajinomoto, Tokyo, Japan) to a basal diet (Table 1), which was formulated to meet National Research Council-recommended nutrient requirements for weanling pigs (National Research Council (NRC), 1998). All diets were made isonitrogenous with addition of appropriate levels of L-alanine, as described by Kim et al. (2004). Therefore, the measured levels of arginine in these diets were 1.21, 1.65 and 1.99%, respectively. The pigs were fed their respective diets 4 times daily at 0800, 1200, 1600 and 2000 h. Among meals, all pigs had free access to drinking water. At 0800 h of d 0 and 7, the body weight and feed intake of all pigs were measured. These values were used to determine average daily weight gain (ADG), average daily feed intake (ADFI), and feed efficiency. 2.2. Sample collection Following weighed on d 7, the pigs received their feed. The 6 pigs in each treatment were selected randomly for obtaining blood samples by the jugular vein at 2 h after feeding. The blood samples were centrifuged at 3500 g for 10 min, and serum was stored at 80 1C until analysis.
Table 1 Composition and nutrient levels of the basal diet (on an as-fed basis). Ingredients
g/kg
Corn Soybean meal Fish meal Corn protein powder Whey Extruded full-fat soybean Sucrose L-Tryptophan L-Lysine HCl, 78% DL-Methionine L-Threonine Calcium carbonate Dicalcium phosphate Sodium Chloride Vitamin–mineral premixa
554.2 60 67.2 8 80 180 17.6 0.6 3.5 1.7 1.5 8 5.2 2.5 10
Nutrients Digestible energy (kJ/g)b Crude proteinc Calciumc Total phosphorusc Argininec Lysinec Methioninec
13.49 184.7 8.0 6.5 12.4 16.3 5.2
a Supplied per kg diet: Mn (as MnSO4 H2O), 4 mg; Fe (as FeSO4 7H2O), 100 mg; Zn (as ZnSO4 7H2O), 100 mg; Cu (as CuSO4 5H2O), 6 mg; I (KI), 0.14 mg; Se (as Na2SeO3 5H2O), 0.3 mg; vitamin A, 2800 IU; vitamin D3, 320 IU; vitamin E, 20 IU; vitamin K3, 1 mg; vitamin B12, 0.03 mg; riboflavin, 5 mg; pantothenic acid, 15 mg; nicotinic acid, 20 mg; thiamin, 1.5 mg; pyridoxine, 2 mg; folic acid, 0.5 mg; biotin, 0.1 mg; and choline chloride, 500 mg. b Calculated nutrient levels. c Measured nutrient levels.
When sampling blood was completed, the selected pigs were killed with an intracardial injection of sodium pentobarbital (50 mg/kg body wt.) and jugular exsanguinations. Then, the liver, respiratory tract, oral epithelium, longissimus muscle, tongue, jejunum, ileum, inguinal lymph node and mesenteric lymph node were immediately isolated, frozen in liquid nitrogen, and stored at 80 1C for the extraction of total RNA. 2.3. Chemical analyses Samples of the diets were analyzed according to the methods of the Association of Official Analytical Chemists (Association of Official Analytical Chemists (AOAC), 1995). Analyses were conducted for calcium (AOAC method 935.13), phosphorus (AOAC method 965.17), and crude protein (AOAC method 988.05). Serum free amino acids were analyzed using a L-8800 Amino Acid Analyzer (Hitachi), as previously described (Zhan et al., 2008). Serum urea was measured using an assay kit from Nanjing Jiancheng Biochemistry. 2.4. Analysis of serum immunoglobulins and cytokines Serum concentrations of IgA, IgG, IgM, IL-1b, IL-2 and TNF-a were measured by using the commercially available pig enzyme-linked immunosorbent assay kits from
X. Mao et al. / Livestock Science 148 (2012) 103–108
Table 2 Primer sequences used for real-time PCR. Gene
Primer
b-actin
Forward Reverse
50 -TCTGGCACCACACCTTCT-30 50 -TGATCTGGGTCATCTTCTCAC-30
pBD-1
Forward Reverse
50 -TCCTTGTATTCCTCCTCA-30 50 -ACACGCCTTTATTCCTTA-30
pBD-2
Forward Reverse
50 -ACCTGCTTACGGGTCTTG-30 50 -CTCTGCTGTGGCTTCTGG-30
pBD-3
Forward Reverse
50 -GAAGTCTACAGAAGCCAAAT-30 50 -GGTAACAAATAGCACCATAA-30
Table 3 Effects of dietary L-arginine supplementation on growth performance of weaned pigsa Item
2.5. Determination of pBDs gene expressions in some tissues
2.6. Statistical analysis All data, expressed as mean7SEM, were analyzed using one-way ANOVA, followed by Duncan’s Multiple Range test. All analyses were performed using SAS (Version 8.1; SAS Institute, Gary, NC). Po0.05 was taken to indicate statistical significance. 3. Results 3.1. Effects of dietary L-arginine supplementation on growth performance of weaned pigs The growth performance data of weaned pigs are shown in Table 3. The initial body weight of weaned pigs among 3 treatments did not differ (P40.05). During the experimental period, dietary L-arginine supplementation
0.5
1.0
Initial weight (kg) 7.127 0.24 7.247 0.20 7.14 7 0.21 Final weight (kg) 8.157 0.20 8.387 0.21 8.52 7 0.30 ADG (g/d) 147.147 11.23 162.867 17.52 197.147 20.27 ADFI (g/d) 242.637 20.44 265.30 7 21.88 262.197 18.82 Feed efficiency 1.817 0.19 1.437 0.14 1.46 7 0.15 (g feed/g gain) Values are mean 7 SEM, n ¼6.
Table 4 Effects of dietary L-arginine supplementation on serum concentrations of amino acids and urea in weaned pigs1. Item
Total RNA extraction, reverse transcription reaction and quantitative real-time PCR of pBDs and b-actin genes were determined as previously described (Qi et al., 2009). Briefly, total RNA of tissues was extracted with RNAiso Reagent (TaKaRa, Japan), and reverse-transcribed with RT Reagents (TaKaRa, Japan) according to manufacturer’s instructions. Quantitative real-time PCR was performed using 96-well iCycler iQTM Real-Time PCR Detection System (BIO-RAD, USA). The gene-specific primers used were listed in Table 2, and purchased from TaKaRa (Japan). The PCR system consisted of 12.5 mL SYBR Green PCR Master Mix (TaKaRa, Japan), 2.0 mL of cDNA, 8.5 mL of PCR-grade water, and 2.0 mL of primer pairs (100 mM forward and 100 mM reverse) for a total volume of 25 mL. All samples were assayed in triplicate. Cycling conditions were as follows: 94 1C for 10 s, and 40 cycles involving a combination of 94 1C for 5 s, 55.5 1C for 20 s and 72 1C for 15 s. Relative gene expression to the housekeeping gene (b-actin) was performed in order to correct for the variance in amounts of RNA input in the reactions. However, the relative gene expressions compared to the housekeeping gene were calculated with the Pfaffl (2001) method.
L-Arginine supplementation (%) 0.0
a
R&D system (Minneapolis, MN) according to the manufacturer’s instructions.
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L-Arginine supplementation (%) 0.0
Amino acids (mmol/L) Arginine 67.17 7 2.28c Lysine 76.55 7 10.35a Citrulline 42.17 7 7.21b Ornithine 44.36 7 1.00b Serum urea, mg/L 82.21 7 1.15a 1
0.5
1.0
94.08 7 0.60b 115.3577.82a 54.13 7 0.55ab 36.89 72.90b 56.42 7 4.81b 82.39 73.84a 71.95 7 14.06ab 102.37 74.91a 73.85 7 2.98b 76.57 70.62ab
Values are means7 SEM; n¼ 6. Values with different letters are significantly different (P o0.05).
a–c
did not affect ADG, ADFI and feed efficiency of weaned pigs (P40.05).
3.2. Effects of dietary L-arginine supplementation on serum concentrations of free amino acids and urea in weaned pigs As shown in Table 4, dietary supplementation with 0.5 and 1.0% L-arginine significantly increased (Po0.05) serum concentration of arginine by 40 and 72%, respectively. Serum concentrations of citrulline and ornithine were significantly greater (Po0.05) in pigs supplemented with 1.0% L-arginine compared with control pigs. Supplementation with 1.0% L-arginine also decreased (P o0.05) lysine concentration in serum compared with control pigs. In addition, the serum concentrations of urea were 10% lower (Po0.05) in pigs of 0.5% L-arginine supplementation treatment compared with control pigs.
3.3. Effects of dietary L-arginine supplementation on serum concentrations of immunoglobulins and some cytokines in weaned pigs The serum concentrations of IgA, IgG, IgM, IL-1b, IL-2 and TNF-a were determined (Fig. 1). During experimental period, dietary supplementation with 0.5 and 1.0% L-arginine did not significantly affect (P40.05) serum concentrations of these immunoglobulins and cytokines in weaned pigs.
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Fig. 1. Effects of dietary L-arginine (L-Arg) supplementation on serum concentrations of IgA, IgG, IgM, IL-1b, IL-2 and TNF-a in weaned pigs on d 7. Values are mean 7 SEM, n¼ 6. Values with different letters are significantly different (P o 0.05).
3.4. Effects of dietary L-arginine supplementation on pBDs gene expressions of some tissues in weaned pigs Dietary supplementation with 0.5 and 1.0% L-arginine significantly increased (Po0.05) pBD-1 gene expression of tongue (Fig. 2A), pBD-3 gene expression of ileum and inguinal lymph node (Fig. 2C), and pBD-2 gene expression of oral epithelium, tongue, ileum and inguinal lymph node (Fig. 2B). The pBD-1 gene expression of oral epithelium was dramatically greater (Po0.05) in pigs of 1.0% L-arginine supplementation groups than that of the control pigs (Fig. 2A). Furthermore, dietary supplementation with 0.5% L-arginine enhanced (Po0.05) pBD-2 gene expression in longissimus muscle, compared with control pigs (Fig. 2B). The pBDs gene expressions of other tissues did not differ among the 3 treatments. 4. Discussion The results of recent published studies are inconsistent about the effect of arginine administration on growth performance of piglets. Kim et al. (2004) and Tan et al. (2009) showed that dietary arginine supplementation could significantly increase the average daily gain of piglets weaned at 7 days of age. However, Liu et al. (2008) and Zhan et al. (2008) demonstrated that dietary arginine supplementation did not alter growth performance of piglets weaned at 14 or 21 days of age. In this study, dietary arginine supplementation did not affect the growth performance of weaning pigs, too (Table 3). Furthermore, some studies have already demonstrated that the regulation of nutrients was gradually decreased with the increasing age (Kimball et al., 2001; Suryawan et al., 2006). Therefore, the difference among the results of these studies could be due to the initial age of piglets in experiments. As shown by some investigators (Kim et al., 2004; Zhan et al., 2008), dietary L-arginine supplementation increased serum L-arginine concentration in weaned pigs
compared with the control (Table 4). The serum concentrations of citrulline and ornithine, which are correlated with metabolism of arginine, were also increased significantly by dietary supplementation with 1.0% L-arginine (Table 4). Due to the antagonism between arginine and lysine, the intestinal absorption of these 2 amino acids could be influenced by the change of dietary arginine:lysine ratio. Serum lysine concentration was decreased dramatically in weaned pigs supplemented with 1.0% L-arginine in this study (Table 4). Furthermore, dietary supplementation of 0.5% L-arginine substantially reduced serum urea level of weaned pigs (Table 4). The decreasing urea level suggests the reduction of ammonia, which is a result that the efficiency of absorbed amino acids for tissue growth is increased (Coma et al. 1995). Dietary arginine supplementation may increase protein synthesis in skeletal muscle of neonatal pigs by mTOR signaling (Yao et al., 2008). However, dietary supplementation of 1.0% L-arginine did not affect serum urea level of weaned pigs in this study (Table 4), which could be derived from the arginine–lysine antagonism that led to imbalance of amino acids. In previous studies, arginine is one of the most important nutrients that may regulate immune function in different animals (Evoy et al., 1998; Li et al., 2007; Peranzoni et al., 2008). Its immunomodulatory effect may improve the inhibited immune function derived from some diseases and stress conditions in laboratory animals and humans (Han et al., 2009; Li et al., 2007; Liu et al., 2008). Some recent studies of laboratory animals demonstrate that dietary L-arginine supplementation could augment thymus size, improve lymphocyte proliferation, affect the constitution of blood leukocytes, and increase the activity of macrophage and natural killer cell (Kwak et al., 1999; Popovic et al., 2007; Rodriguez et al., 2003; Yeh et al., 2002). In addition, the study with early-weaned pigs showed that dietary administration of L-arginine significantly increased the serum concentrations of some immunoglobulins and cytokines at day 14, but did not affect their concentrations at day 7 (Liu et al., 2008). In present study, the serum concentrations of immunoglobulins and some cytokines were not also influenced by the dietary L-arginine supplementation at day 7 (Fig. 1). These results demonstrated that the effect of dietary L-arginine supplementation on serum concentrations of immunoglobulins and cytokines in weanling pigs needed for the enough duration. As an important part of mammalian innate immunity, the broad-spectrum antimicrobial peptides, such as cathelicidins and defensins, play a critical role in protecting organisms from all kinds of microbial (Froy and Gurevitz, 2003; Ganz, 2003). These antimicrobial peptides are expressed in multiple tissues and organs of mammal. Our previous studies have shown that there were the expressions of pBDs in multiple tissues of the crossbred and Tibetan pigs, and there was the difference of pBDs expression among different tissues and pig breeds (Qi et al., 2009). Some studies demonstrated that human b-defensin-1 expression of various cells could be stimulated by some nutrients, such as L-isoleucine, Ca2 þ and glucose (Fehlbaum et al., 2000; Harder et al., 2004; Sherman et al., 2006).
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Fig. 2. Effects of dietary L-arginine (L-Arg) supplementation on gene expressions of pBD-1 (A), pBD-2 (B) and pBD-3 (C) in some tissues of weaned pigs. Values are mean 7 SEM, n¼ 6. Values with different letters are significantly different (P o0.05).
Additionally, L-arginine treatment could upregulate expression of human b-defensin-1 in HCT-116 cells (Sherman et al., 2006). In this study, we found that dietary L-arginine supplementation could stimulate pBDs expressions of some
tissues in weaned pigs (Fig. 2). However, further studies are required to determine the mechanism responsible for the different effect of arginine on regulating pBDs’ expressions in various tissues.
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5. Conclusions This study indicates that the different doses of supplemental L-arginine for 7 days did not affect the growth performance and serum concentrations of immunoglobulins and some cytokines, but significantly increased the expressions of pBDs in some tissues of weaned pigs. This finding provides further explanation whereby arginine improves immune function of animals. However, further studies are required to determine the intracellular signaling mechanism involving in the effect of arginine on regulating pBDs’ expressions. Additionally, because pigs are a efficiency animal model for investigating human nutrition (Wu et al., 2004), the results of this work may be an important implication for improving the immune function of human, especially infants. Conflicts of interest statement Mao, Qi, Yu, Huang, Chen, Mao, Han, and Chen declare that: we have no proprietary, financial, professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled: Dietary L-arginine supplementation enhances porcine b-defensins gene expression in some tissues of weaned pigs.
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