Arginine participates in multiple pathways with enormous nutritional and physiological importance, including the synthesis of protein, NO, creatine, proline, glutamate, polyamines and agmatine as well as the secretion of hormones(Reference Yao, Yin and Chu1–Reference Tan, Yin and Liu6). Increasing evidence shows that l-arginine is an essential amino acid for young mammals, particularly under conditions of severe stress(Reference Wakabayashi, Yamada and Yoshida7, Reference Wu, Ruan and Gao8). For instance, our previous studies have shown that dietary supplementation with arginine, which is 50–95 % greater than National Research Council-recommended intake, stimulates muscle growth(Reference Yao, Yin and Chu1, Reference Frank, Escobar and Hguyen9), enhances the immune status(Reference Tan, Li and Kong5) and improves microvascular development(Reference Zhan, Ou and Piao10) in neonatal pigs weaned at 7–21 d of age. Particularly, NO (a metabolite of l-arginine) is a major vasodilator that regulates vascular tone and haemodynamics. Proline (another product of arginine catabolism) is a key component of extracellular matrix collagen that is crucial for angiogenesis and vascular remodelling(Reference Mussini, Hutton and Udenfriend11, Reference Adams and Frank12). Systemic administration of l-arginine has been proposed as a safe and effective method to enhance the synthesis of NO, proline and polyamines in animals, therefore improving wound healing and microcirculation(Reference Wu and Meininger13–Reference Barbul, Lazarou and Efron15).
Weaning stress is associated with reductions in food consumption, weight gain and growth, as well as increases in the incidence of diarrhoea and disease, intestinal dysfunction and atrophy, and deaths in piglets(Reference Yuan, Zhang and Wu16–Reference Lalles, Bosi and Smidt22). In response to weaning, the digestive system of piglets must adapt to a dry diet, which is often based primarily on plant sources of ingredients (e.g. maize and soyabean meal)(Reference Kelly, Smyth and McCracken23, Reference Ou, Li and Cao24). In the piglet small intestine, microvessels are present mainly in the mucosa and submucosa(Reference Gore and Bohlen25, Reference Sieber, Beglinger and Jaeger26), and the optimal development of villi depends on an adequate supply of nutrients from both blood via the intestinal microvessel and enteral feeding(Reference Matheson, Wilson and Garrison27). Changes in gut morphology may result in microvessel injury and disorders of microcirculation(Reference Kalia, Pockley and Wood28, Reference Nakajima, Baudry and Duranteau29). Microvascular endothelial dysfunction may be a major factor contributing to impaired absorption and transport of nutrients in animals(Reference Granger and Kubes30).
Of particular interest, weanling piglets have a particularly high requirement for dietary arginine(Reference Wu, Knabe and Kim31). Due to depressed feed intake in the first week post-weaning, low arginine intake may be one of the reasons for increased intestinal epithelial damage in early-weaned pigs. Adverse effects of an l-arginine deficiency may also include abnormal gene expression in the vasculature(Reference Zhan, Ou and Piao10, Reference Mussini, Hutton and Udenfriend11). Therefore, we hypothesised that dietary l-arginine supplementation might prevent or alleviate intestinal atrophy and microcirculation disorder by increasing the expression of vascular endothelial growth factor (VEGF) in the small intestine of weanling pigs. The present study was conducted to test this hypothesis using 21- to 28-d-old piglets.
Materials and methods
Animals and feeding
We conducted the experiment in accordance with the Chinese guidelines for animal welfare, and it was approved by the Animal Welfare Committee of the Institute of Subtropical Agriculture, Chinese Academy of Sciences(Reference Tang, Yin and Zhang32).
Basal diets were formulated to meet National Research Council-recommended nutrient requirements for weanling piglets(33). Two diets were formulated by supplementing the basal diet with 0 and 1 % l-arginine (free base). The diets were made isonitrogenous by the addition of l-alanine, as described by Kim & Wu(Reference Kim and Wu34).
A total of twenty large White × Landrace castrated piglets from five litters were weaned at 21 d of age (5·3 (sem 0·13) kg) and assigned randomly to one of the two treatment groups (ten pigs/group), representing dietary supplementation with 0·0 % control or 1 % l-arginine (Table 1). Piglets were individually housed in 1·3 × 1·2 m pens with slotted stainless steel floors(Reference Kang, Yin and Ruan35). Each pen was equipped with a feeder and a nipple waterer. This facility allows pigs to have free access to feed and water. The room temperature was maintained at 25–27°C(Reference Yin, Li and Huang36). All the piglets were fed four times per day at 07.00, 11.00, 15.00 and 19.00 hours. The feed intake was measured daily. Fresh manure samples were collected daily for the determination of moisture. Samples with moisture content higher than 70 % were considered as diarrhoea. The incidence of diarrhoea for the piglets was calculated as(Reference Hampson18):
Composition of the basal diet (as-fed basis)
* Supplied per kg diet: Cu, 10 mg; Fe, 100 mg; Na, 0·30 mg; Zn, 100 mg; Mn, 10 mg; cholecalciferol, 386 IU; retinyl acetate, 3086 IU; all-rac-α-tocopheryl acetate, 15·4 IU; menadione, 2·3 mg; riboflavin, 3·9 mg; d-pantothenic acid, 15·4 mg; niacin, 23 mg; choline, 77 mg; cyanocobalamin, 15·4 μg.
† Analysed values.
Sample collection
Following a 7 d period of arginine supplementation, all piglets were weighed, and at 1 h after the last meal, jugular venous blood samples were obtained from heparinised tubes for the analysis of hormones and metabolites, whereas six pigs from each group were randomly selected and humanely killed by a lethal intraperitoneal injection of sodium pentobarbital(Reference He, Kong and Wu4). Samples of duodenum, jejunum, ileum, liver, kidney, heart, spleen and lung were obtained immediately after the abdomen was opened(Reference Wang, Chen and Li37).
Analysis of metabolites and hormones
Amino acids in plasma were analysed using a HPLC method involving precolumn derivatisation with ortho-phthaldialdehyde(Reference Yin, Liu and Yin38). l-Norvaline was used as an internal standard. An automated biochemistry analyzer (Synchron CX Pro; Beckman Coulter, Fullerton, CA, USA) was used to determine concentrations of urea, NH3, glucose, total protein and immune globulins in plasma, according to commercial kits and the manufacturer's instructions(Reference Li, Wu and Peng39, Reference Kong, Yin and He40). Cortisol, insulin, growth hormone, insulin-like growth factor-1, triiodothyronine and thyroxine in plasma were determined by RIA according to reagent kits and the manufacturer's instructions (China Institute of Atomic Energy, Beijing, China)(Reference Deng, Wu and Bin41–Reference Kong, Wu and Liao43).
Histological analysis
Intestinal tissue samples were fixed as described previously(Reference Mussini, Hutton and Udenfriend11). Briefly, samples were placed in 10 % neutral buffered formalin and embedded in paraffin for subsequent histological measurement. Six cross-sections were obtained from each formalin-fixed segment and processed for histological examination using the standard haematoxylin and eosin method(Reference Mussini, Hutton and Udenfriend11). Villus height and crypt depth were measured according to Wu et al. (Reference Wu, Bazer and Davis44, Reference Wu, Meier and Knabe45). The histological analysis was performed by an investigator who was unaware of the origin of tissue sections.
Immunohistochemistry
Protein levels for VEGF were measured as described previously(Reference Mussini, Hutton and Udenfriend11). Briefly, sections of formalin-fixed paraffin-embedded tissues were digested with 3 % H2O2 for 20 min at room temperature and incubated sequentially with 10 % normal rabbit serum for 20 min after microwave antigen recovery, with VEGF (1:50) at 4°C overnight and then with corresponding biotinylated secondary antibodies against rabbit and streptavidin peroxidase. Subsequently, binding of the primary antibody was detected with diaminobenzidine. Sections were counterstained with haematoxylin. In the negative control, the antibodies were substituted by PBS. Immunochemical staining sections were photographed using a Leica DFC 320 digital camera (Leica Microsystems, Cambridge, UK). The optical density for tissues was integrated by computer-assisted image analysis (Image-Pro Plus; Media Cybernetics, Bethesda, MD, USA) in each 400 × magnified field(Reference Zhou, Li and Yin46). Eight microscopic fields for each section were quantified.
Statistical analysis
Results are expressed as means with pooled sem. Data were analysed statistically using the General Linear Model procedure of the Statistical Analysis System (version 2000; SAS Institute, Cary, NC, USA) for one-way ANOVA. Differences between the groups were determined by the Student–Newman–Keuls multiple comparison test. Relationship between intestinal villus height and VEGF protein levels was evaluated by the Pearson correlation analysis (SAS Institute). Probability values ≤ 0·05 were taken to indicate statistical significance.
Results
Feed intake and growth performance
Daily food intake did not differ between control and arginine-supplemented piglets during a 7 d experimental period (Table 2). The initial body weight (BW) at 21 d of age did not differ between the two groups (Table 2). However, compared with the control group, dietary supplementation with l-arginine increased (P < 0·05) the final BW by 9 % and enhanced daily weight gain of piglets by 56 %, respectively. Moreover, 1 % l-arginine supplementation decreased (P < 0·05) the ratio of DM intake:BW gain (g/g; an indicator of feed efficiency) by 28 %. Diarrhoea incidence did not differ between the control and arginine-supplemented groups.
(Mean values with their pooled standard errors, n 10)
ADG, average daily gain; ADFI, average daily feed intake.
a,b Mean values within a row with unlike superscript letters were significantly different (P < 0·05).
Plasma amino acids
Dietary supplementation with 1 % l-arginine increased (P < 0·05) plasma concentrations of arginine and ornithine by 33 and 30 %, respectively, but had no effect on plasma concentrations of citrulline or lysine (Table 3). Plasma concentrations of histidine, asparagine, aspartate, isoleucine, leucine, methionine, phenylalanine, serine, taurine, threonine, tryptophan, tyrosine or valine did not differ between the control and arginine-supplemented groups (data not shown).
(Mean values with their pooled standard errors, n 6)
GH, growth hormone; IGF-1, insulin-like growth factor-1; T3, triiodothyronine; T4, thyroxine.
a,b Mean values within a row with unlike superscript letters were significantly different (P < 0·05).
Plasma urea, ammonia and hormones
Dietary supplementation with 1 % l-arginine decreased (P < 0·05) plasma concentrations of NH3 and urea by 17 and 18 %, respectively (Table 3). Plasma concentrations of glucose, total protein and total albumin were not affected by l-arginine supplementation (Table 3). Compared with control pigs, dietary supplementation with 1 % l-arginine increased (P < 0·05) plasma concentrations of insulin by 27 % and decreased (P < 0·05) plasma concentrations of cortisol by 33 % (Table 3). Dietary supplementation with 1 % l-arginine did not affect plasma concentrations of growth hormone, insulin-like growth factor-1, triiodothyronine or thyroxine (Table 3).
Relative weight of internal organs
The relative weights (g/kg BW) of kidney, heart, spleen and lung were not affected by l-arginine supplementation (data not shown). The relative weight of the small intestine was 33 % heavier (P < 0·05) in arginine-supplemented pigs compared with that of the control group (73·1 v. 54·9 g/kg BW, pooled sem = 4·4 g/kg BW).
Small-intestinal morphology and vascular endothelial growth factor immunoreactive expression
To determine the effect of l-arginine supplementation on intestinal development, the piglet small intestine was collected at the end of the experiment, and the villus height and crypt depth were examined (Fig. 1). Supplementation with 1 % l-arginine increased (P < 0·05) the villus height throughout the small intestine, compared with the control group. Crypt depth was greater (P < 0·05) in the duodenum and jejunum of the control group than that of the arginine-supplemented pigs.
Effects of dietary l-arginine supplementation for 7 d on the morphology of the small intestine in early-weaned piglets. Values are means with pooled sem (n 6). a,b Mean values with unlike letters were significantly different (P < 0·05). ■, Control; □, 1 % l-arginine.
The immunoreactive VEGF protein was readily detected in the piglet small intestine (Fig. 2). Quantitatively, dietary supplementation with 1 % l-arginine increased (P < 0·05) the integrated optical density in the duodenal submucosa, middle jejunal mucosa and submucosa, and the ileal mucosa by 14, 39 and 54 %, respectively (Table 4 and Fig. 2). Correlation coefficients (R 2) between villus height and VEGF protein intensity were 0·954, 0·958 and 0·956 (P < 0·01), respectively, for the duodenum, jejunum and ileum.
Immunostaining of the vascular endothelial growth factor protein in the duodenum (a), jejunum (b) and ileum (c) of early-weaned piglets.
(Mean values (arbitrary unit) with their pooled standard errors, n 6)
a,b Mean values within a row with unlike superscript letters were significantly different (P < 0·05).
Discussion
Intestinal development of the piglet was greatly suppressed in the first week after weaning in association with intestinal dysfunction and atrophy(Reference Lalles, Bosi and Smidt22), which is the major cause of reductions in nutrient absorption and utilisation as well as increases in diarrhoea and deaths. Available evidence shows that VEGF enhances intestinal vascular development, endothelial function and epithelial cell migration while inhibiting endothelin-1 release as well as platelet aggregation and adhesion(Reference Rhoads, Chen and Gookin47–Reference Cao, Eriksson and Kubo50). Results of the present study indicated that dietary supplementation with 1 % l-arginine had a positive effect on increasing villus height (Fig. 1) and the expression of the VEGF protein (Table 4 and Fig. 2). Notably, increases in both relative weight and villus height of the small intestine were positively correlated with the augmented expression of VEGF (see the Results section). The findings indicate that dietary supplementation with 1 % arginine can improve the vascular development of the small intestine in weanling pigs by stimulating VEGF expression.
Elevated levels of cortisol in plasma are considered to be biomarkers for weaning stress(Reference Kim and Wu34). Interestingly, in the present study, we found that dietary supplementation with 1 % l-arginine decreased the circulating level of cortisol, but underlying mechanisms are unknown. Cortisol is the major glucocorticoid in pigs(Reference Breinekova, Svoboda and Smutna51). It is synthesised in the adrenal cortex and released into the circulation in response to external and internal factors acting on the hypothalamus and pituitary glands(Reference Flynn, Bird and Guthrie52). It is possible that NO or l-arginine itself attenuates the release of the adrenocorticotropic hormone from corticotroph cells and/or interferes with adrenocorticotropic hormone's actions on the adrenal cortex via specific receptors (e.g. type 2 melanocortin receptors). Further research is warranted to test this novel hypothesis.
Arginine plays important roles in both growth and metabolic function in piglets(Reference Papadimitriou and Priftis53). In rodents and human subjects, l-arginine is a nutritionally essential amino acid under conditions associated with increased utilisation relative to endogenous synthesis, including growth, inflammation and tissue repair(Reference Wu, Bazer and Davis44). In such cases, dietary supply can become rate limiting for the arginine-metabolising pathways(Reference Wu, Knabe and Flynn54). As previously reported(Reference Li, Wu and Peng39), arginine is deficient in weanling piglets (Table 3). Interestingly, intestinal synthesis of citrulline and arginine from glutamine and glutamate decreases by 70–73 % in 7-d-old suckling pigs in comparison with newborn pigs and declines further in 14- to 21-d-old pigs(Reference Wu, Bazer and Davis55). Thus, dietary supplementation with 0·2 and 0·4 % arginine to 7- to 21-d-old pigs (artificially reared on a milk feeding system) dose dependently enhanced plasma arginine concentrations (30 and 61 %) and reduced plasma NH3 levels (20 and 35 %)(Reference Leibholz56).
Insulin is a major regulatory hormone in glucose and fat metabolism, vascular function, inflammation, tissue remodelling and the somatotropic axis of growth(Reference Kong, Yin and He40, Reference Deng, Wu and Bin41). Arginine is a potent stimulator of the secretion of insulin by pancreatic β-cells and of growth hormone by the anterior pituitary gland in mammals(Reference Wu, Meier and Knabe45), including young pigs(Reference Zhou, Li and Yin46). This is consistent with our finding that dietary supplementation with 1 % l-arginine increased plasma levels of insulin. Through an increase in arginine availability and the concurrent increases in plasma concentrations of anabolic hormones (Table 3), dietary arginine supplementation improved the efficiency of nutrient utilisation for enhancing tissue protein synthesis and growth performance. In support of this view, plasma concentrations of urea (the major nitrogenous product of protein and amino acid catabolism) were markedly reduced in arginine-supplemented pigs compared with control pigs (Table 3). Growth hormone is another important hormone in growth regulation, which plays a role in the secretion of thyroid hormone (mainly produced in the liver). However, l-arginine supplementation did not affect circulating levels of growth hormone or thyroid hormones (Table 3).
In keeping with the previous report(Reference Mussini, Hutton and Udenfriend11), dietary supplementation with 1 % arginine markedly enhanced daily weight gain in weaning piglets (Table 2), indicating that arginine deficiency is a major factor limiting their maximum growth performance. It is noteworthy that experimental conditions differed greatly between the present study and the work of Zhan et al. (Reference Zhan, Ou and Piao10). For instance, the initial age (21 d) and initial mean BW (5·3 kg) of piglets in the present study were greater than those of the piglets used by Zhan et al. (14 d and 5·0 kg, respectively). Additionally, food intake of piglets was lower in the Zhan et al.'s experiment (140 g/d) compared with the present one (185 g/d) (Table 2). Furthermore, the doses of supplemental arginine (0·7 v. 1 %) differed between the two investigations. Regardless of the low feed intake, 0·7 % arginine supplementation may not be sufficient to meet the requirement of weanling piglets for maximal growth, whereas 1·2 % arginine supplementation caused severe diarrhoea(Reference Mussini, Hutton and Udenfriend11). Clearly, proper supplementation of arginine is critical for experiment design.
Another significant finding from the present study is that dietary supplementation with 1 % l-arginine increased the weight of the small intestine (see the Results section), which is consistent with the observation that arginine can increase protein synthesis(Reference Jobgen, Fried and Fu57), inhibit protein degradation and enhance the proliferation of intestinal epithelial cells(Reference Benjamin, Odle and Niu58). It is now known that arginine activates the mammalian target of rapamycin signalling in the intestine(Reference Rhoads and Wu59), therefore promoting the initiation of polypeptide formation. These findings provide a molecular basis for our observation that l-arginine supplementation can improve the feed efficiency and growth performance of weanling piglets. Because preterm infants are deficient in arginine and exhibit intestinal dysfunction(Reference Ziche, Morbidelli and Choudhuri48), the present results may have important implications for managing this compromised population of neonates.
In conclusion, dietary supplementation with 1 % l-arginine stimulates intestinal VEGF expression and development in weanling piglets, thereby contributing to improved vascular function and growth performance. Adequate provision of l-arginine in the weanling diet may be an effective means of ameliorating microvessel injury and enhancing the absorption of nutrients in the small intestine.
Acknowledgements
The present research was jointly supported by grants from the Chinese Academy of Sciences and Knowledge Innovation Project (Kscx2-Yw-N-051 and Y022042020) National 863 Program of China (2008AA10Z316), Research Program of State Key Laboratory of Food Science and Technology, Nanchang University (project no. SKLF-TS-200817), National Basic Research Program of China (2009CB118806), NSFC (30901040, 30901041, 30928018, 30828025 and 30771558), National Scientific and Technological Supporting Project (2006BAD12B02-5-2 and 2006BAD12B02-5-2), the Program for Ganjiang Scholars and Innovative Research Team in Nanchang University (IRT0540), the CAS/SAFEA International Partnership Program for Creative Research Teams, the Thousand-People-Talent program at China Agricultural University, National Research Initiative Competitive Grants from the Animal Growth & Nutrient Utilization Program (2008-35206-18764) of the USDA National Institute of Food and Agriculture and Texas AgriLife Research (H-8200). The authors have no conflicts of interest to declare. Y. Y. was in charge of the whole project. K. Y. conducted the animal trial and wrote the paper. S. G., T. L., R. H. and Z. R. assisted with tissue collection and chemical analyses. G. W. helped to design the experiment, interpret the data and write the paper.
