




































In ternationa l
Scholars
Journa ls

 

African Journal of Pig Farming ISSN 2375-0731 Vol. 6 (5), pp. 001-011, May, 2018. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 

 

Full Length Research Paper 

 

Effects of L-arginine on intestinal development and 
endogenous arginine-synthesizing enzymes in 

neonatal pigs 

 
Lin Huang1,2, Zong-yong Jiang2*, Yin-cai Lin2, Chun-tian Zheng2, Shi-kui Wang2, Xue-feng 

Yang1,2 and Guo-yao Wu3
 

 
1
College of Animal Science, South China Agricultural University, Guangzhou 510642, P. R. China. 

2
Key Laboratory of Animal Nutrition and Feed (South China), Ministry of Agriculture of P. R. China, Institute of Animal 
Science, State Key Laboratory of Livestock and Poultry Breeding, Guangdong Academy of Agricultural Sciences, 

Guangzhou 510640, P. R. China. 
3
Department of Animal Science, Texas A&M University, College Station 77843, USA. 

 
Accepted 07 November, 2017 

 
This study aimed to investigate the effects of dietary L-arginine supplementation on the intestinal development of 
neonatal piglets and the underlying mechanisms. 36 neonatal piglets were randomly allocated into three diet groups: 
control group (supplemented with 0% L-arginine), 0.4 and 0.8% L-arginine groups. When compared with the control, 
dietary supplementation with L-arginine decreased (P<0.05) blood urea nitrogen (BUN), and improved (P<0.05) serum 
T3 and insulin level of the piglets on day 11. Arginine and its metabolites (citrulline and ornithine) were elevated, 
additionally, dietary supplementation with 0.8% L-arginine markedly enhanced jejunal villus height, villus area on day 
11 and D-xylose absorption rate on day 19. Dietary supplementation with 0.8% L-arginine increased (P<0.05) activities 
of maltose and lactose on day 18, respectively. This effect correlated with profound change in enzyme activities as 
inducible nitric oxide synthetase (iNOS), glutamine synthetase (GS) and ornithine decarboxylase (ODC) were elevated 
on day 18. The concentrations of spermine was increased (P<0.05) by L-arginine supplementation on day 18. These 
results collectively suggest that dietary L-arginine supplementation improves protein synthesis and intestinal 
development of the neonatal pigs, the underlying mechanism includes dietary L-arginine supplementation which 
regulated the productions of intestinal polyamine in jejunum, and stimulated endogenous arginine-synthesizing 
enzymes in neonatal piglets. 

 

Key words: Neonatal pig, L-arginine, intestinal development, arginine-synthetases. 
 
 
INTRODUCTION 

 
Early weaning has been reported to correlate with villus 
atrophy, which depresses feed intake and growth 
performance in piglets (Kelly et al., 1991; Ou et al., 2007). 
Adequate supply of nutrients from blood and intes-tine 
ensured the optimal development of villus (Matheson et 
al., 2000). The small intestine was suggested to be the 
major organ to synthesize arginine in neonatal animals 
(Wu et al., 1995). Low arginine intake associated with  
 
 
 
*
Corresponding author. E-mail: jiangz38@gmail.com Tel: +86 

020 87596262. Fax: +86 020 87503358. 

 
 
 
 

 
depresssed feed intake may be a major reason for 
increased intestinal epithelial damage in early-weaned 
pigs.  

Previous studies indicated that arginine was strikingly 
deficient in milk-fed piglets, as the relatively low contents 
of arginine were found in sow’s milk (Wu et al., 2000; 
O’Quinn et al., 2002). Arginine was therefore considered 
an essential amino acid for the optimal growth of neonatal 
pigs (Wu and Knabe, 1994, 1995; Flynn et al., 2000; Wu 
et al., 2004). Dietary supplementation of L-arginine 
improved the growth of neonatal pigs (Kim et al., 2004), 
mainly due to its beneficial effects in enhancing wound 
healing and angiogenesis, improving protein 



 
 
 

 
Table 1. Composition and nutrient contents of the 
basal diet (on an as-fed basis).  

 
 Ingredient (%) Ratio (%) 

 Whole milk power (23% CP) 50.84 

 Whey protein concentrate (34% CP) 22.00 

 Plasma protein powder (78% CP) 5.00 

 Whey power (4% CP) 14.32 

 Coconut oil 5.00 

 L-alanine (98%) 1.67 

 DL-methionine (98%) 0.08 

 L-threonine (98%) 0.14 

 L-tryptophan (98%) 0.02 

 liquid chloride choline (70%) 0.12 

 Vitamin-mineral premix
1
 0.5 

 emulsifying agent 0.11 

 Calculated composition  
 Crude protein (%) 26.34 

 Digestible energy (MJ/kg) 19.25 

 Fat (%) 15.80 

 Lactose (%) 32.20 

 Calcium (%) 1.10 

 Total phosphorus (%) 0.70 

 Lysine (%) 2.20 

 Methionine (%) 0.50 

 Arginine (%) 0.72 
 

1
Providing the following (mg/kg powder diet): Fe (as 

FeSO4.7H2O), 105; Cu (as CuSO4.5H2O), 10; Mn (as 

MnSO4.H2O), 5; Zn( as ZnSO4.7H2O), 110; retinyl 
palmitate, 13.2; cholecalciferol, 1.32; all-rac-α-tocopheryl 
acetate, 96; menadione sodium bisulfite, 1.50; biotin, 
0.24; folic acid, 0.90; nicotinic acid, 60.0; calcium 
pantothenate, 36; riboflavin, 12; thiamin, 4.5; pyridoxine, 
6; cyanocobalamin, 0.06. 

 

 

anabolism and immune functions as suggested by study 
in mice (Cremades et al., 2004) and human (Barbul et al., 
1990). Biochemically, arginine plays an important role as 
a substrate for protein synthesis, intermediates in the he-
patic urea cycle, as well as precursors for the synthesisof 
various important metabolic molecules, including nitric 
oxide (NO), polyamines and creatinine (Wu and Morris 
1998; Kim et al., 2007; Flynn et al., 2002; Cynober et al., 
1995). Wu and colleague suggested that L-arginine 
supplementation may boost the growth of young piglet 
possibly by enhancing the synthesis of NO, proline and 
polyamines in animals (Wu et al., 2004; Kim et al., 2004). 
However, it remains unclear whether L-arginine supple-
mentation has other effects besides these metabolic 
pathways and higher dosage of L-arginine may lead to 
beneficial effect or detrimental effect.  

In an attempt to unveil the underlying mechanisms of L-
arginine supplementation and young piglet growth, we 
carried out this study with wider range of dosages and 
started during early neonatal period. We found that 

 
 
 
 

 

dietary arginine supplementation can prevent intestinal 
atrophy in early-weaned pigs, in addition to the impact on 
nitrogen metabolism as reported previously. The dosage-
dependent data suggested that higher arginine 
supplementation has more substantial effect on some but 
not all aspects of analysis, which may be of potential 
interest in applying such practice in livestock industry of 
China and the world. 
 

 
MATERIALS AND METHODS 

 
Experimental procedures in this study were approved by Animal 
Experimental Committee of Guangdong Institute of Animal Science. 
36, 4-day-old healthy male neonatal piglets (Duroc, Landrace x 
Largininee White) were randomly assigned to three treatment 
groups, with four replicates of three piglets each. Every replicate of 
piglets were housed in a pen in an air-conditioned room with 
ambient temperature of 32 ± 1°C and constant humidity. The basal 
milk-powder diets were formulated to meet required NRC 1998 
minimal levels for 3 to 5 kg piglets (Table 1). Levels of exogenous 
L-arginine supplemented in diets of the three treatments were set at 
0 (control group), 0.4 and 0.8% (on the basis of milk replacer 
powder). Appropriate amounts of alanine were added to formulate 
isonitrogenous diets. The diets were mixed with water at a ratio of 
1:4 freshly before feeding, and provided to the piglets every 3 h 
from 06:00 a.m and 12:00 p.m. Piglets had easy access to water 
supplied by the semiautomatic device. Experimental piglets were 
observed to become accustomed to the feeding method quickly and 
consumed each meal with no spillage. Feed intake (as-fed basis) 
was calculated in accordance with the weight difference in feed 
trough before and after feeding. Body weights (BW) of all piglets 
were individually measured on day 1, 11 and 18 of the experiment. 
Average daily gain (ADG), average daily feed intake (ADFI) and 
feed : gain (F : G) ratio were calculated. 

 

Sample collection 

 
Blood samples of all piglets were obtained from anterior vena cava 
and both serum and plasma were separately kept for further 
determination on day 11 and 18 of the experiment. Respectively, on 
day 11 and 18 of the experiment, one pig out of each experimental 
replicate was randomly selected and slaughtered after 
intraperitoneal injection of sodium pentobarbital (50 mg/kg BW). 
The small intestine from the pyloric sphincter to the ileocolonic 
junction was rapidly removed by cutting along the mesenteric 
border on an ice-cold metal plate and divided into three segments. 
The segment of small intestine proximal to the ligament of Treitz 
was designated as the duodenum, with the stomach being 
removed. The remainder of the small intestine was divided into 2 
equal portions; the proximal half was designated the jejunum and 
the distal half, the ileum (Burrin et al., 2000). From the midline of 
each region, a 10 cm piece was slit along its length and mucosa 
was removed by gentle scraping with a glass slide. These samples 
were rapidly frozen in liquid nitrogen for analysis of the activities of 
disaccharidases (maltase, lactase and sucrase). On day 19 of the 
experiment, a D-xylose test was conducted on the remaining 
piglets. D-Xylose was orally administrated to the piglets at dose of 1 
ml/kg BW, blood samples were collected from anterior vena cava 2 
h later for the determination of D-xylose. 

 

Analysis procedures 
 

Blood hormones (insulin, IGF-1, T3 and T4) were measured through 



  
 
 

 
Table 2. Effect of dietary L-arginine supplementation on growth performance of 
piglets*.  

 
 Item Control 0.4% Arginine 0.8% Arginine 

 Body weight (kg)    

 Day 1 2.07±0.01 1.99±0.07 2.08±0.08 

 Day 11 3.28±0.12 3.36±0.04 3.37±0.03 

 Day 18 5.54±0.32 5.79±0.09 5.76±0.19 

 Daily weight gain (kg/d)    
 Days 1 to 11 0.12±0.01 0.14±0.01 0.13±0.01 

 Days 12 to 18 0.32±0.02 0.34±0.01 0.34±0.11 

 Days 1 to 18 0.22±0.01 0.24±0.01 0.24±0.04 

 Daily feed intake (kg/d)    
 Days 1 to 11 0.12±0.01 0.12±0.01 0.12±0.01 

 Days 12 to 18 0.26±0.01 0.26±0.02 0.27±0.04 

 Days 1 to 18 0.19±0.01 0.19±0.01 0.19±0.01 

 Feed : gain ratio    
 Days 1 to 11 0.99±0.12 0.86±0.03 0.91±0.02 

 Days 12 to 18 0.82±0.05 0.75±0.05 0.79±0.02 

 Days 1 to 18 0.86±0.04 0.78±0.04 0.82±0.01 
 

*Data are means ± SD (n = 4). Values in a row with different superscripts differ 
(P<0.05). 

 

 

Radio-immuno method using reagent kits (Tianjin JiuDing 
Bioengineering Co. Ltd). Blood urea nitrogen (BUN) was 
determined by automatic biochemistry analyzer (Beckman 
Instruments, Fullerton, CA) at 340 nm using the assay kits from 
Beckman Coulter Inc (Fullerton, CA). Plasma concentrations of 
arginine, glutamate, proline, glutamine, citrulline, ornithine, lysine, 
threonine, valine, histidine and alanine were analyzed by amino-
acid autoanalyzer (L-8900, Hitachi). Nitric oxide synthetase (NOS), 
NO, disaccharidases and serum D-xylose were determined using 
colorimetric methods with a spectrophotometer (Biomate 5, Thermo 
Electron Corporation, Rochester, NY). The Assays were conducted 
using the assay kits purchased from Nanjing Jiancheng Institute of 
Bioengineering (Nanjing, Jiangsu, China) and the procedures were 
followed accordingly. Activities of argininosuccinate synthetase 
(ASS), pyrroline-5-carboxylate synthetase (P5CS) and spermine 
were determined using swine enzyme-linked immunosorbent assay 
kits (Adlitteram Diagnostic Laboratories, USA). 
 

 
Small intestinal morphology 

 
 

 
Statistical analysis 
 
Data were presented as means ± SD. Statistical analyses were 
performed using the general linear model procedures of SPSS 
(version 11.5, SPSS Institute). Statistical comparisons were done 
by ANOVA followed by the post-hoc Newman–Keuls multiple range 
test. Difference was considered significant when P value <0.05. 

 

RESULTS 
 
Growth performance 

 
The effect of dietary L-arginine supplementation on growth 
performance is summarized in Table 2. When compared 
with the control group, arginine supplementation did not 
improve BW, ADG and ADFI, but slightly decreased F : G 
ratio of the piglets throughout the experiment (P>0.05). 

 

 
Two centimeters segment of the jejunum was collected immediately 
after slaughter and processed as previously described (Jensen et 
al., 2001). In brief, the samples were fixed with 10% neutral 
buffered formalin for 24 h, and trimmed to prepare paraffin-
embedded block for histological slides. Three cross sections (5 µm 
thick) of each intestinal segment were stained with hematoxylin and 
eosin following standard protocol. The 10 straightest villus and their 
associated crypts from each segment were quantified. The villus 
height was measured from the tip to the base, and then the crypt 
depth was measured from the base of the villus to the base of the 
crypt. The villus area was calculated according to the published 
method (Frankel et al., 1993). 

 
Blood urea nitrogen (BUN) 
 

BUN levels of the piglets of 0.4 and 0.8% arginine groups 
were 39.4 (P<0.05) and 18.3% (P<0.05), both lower than 
that of the control group on day 11 (Figure 1). No obvious 
difference was observed among BUN contents of the 
three groups at day 18 (Figure 1). 

 

Blood hormones 
 

Blood T3   levels of piglets obviously increased with 



 
 
 
 
 
 
 
 

 

B
U

N
 (

m
m

o
l/

L
) 

 
 
 

 
 

4.5 
  

a 
 Control  

 

    
 

4 

          

ab 

 0.4% Arginine 
 

 

           
 

           

0.8% Arginine 

 
 

3.5 

            
 

            
 

3   

     

b 

                  
 

                       
 

                         
 

2.5 

      

 

                   

                        
 

                            
 

                            
 

2   

                          
 

                          
 

                            
 

1.5                             
 

1                             
 

                            
 

0.5 
                            

 

                            
 

0 

                            
 

                            
 

     Day 11       
Day 18 

 
 

             
 

                      
  

  
Figure 1. Effect of dietary arginine supplementation on serum urea  
nitrogen levels of piglets. Bars represent the means ± SD. Within  
an  experimental phase,  means  without  a common  letter  differ  
(P<0.05). 

 

 
Table 3. Effect of dietary L-arginine supplementation on serum hormone levels of piglets*.  

 
 Item Control 0.4% Arginine 0.8% Arginine 

 T3 (ng/ml)    

 Day 11 0.43±0.02
a
 0.81±0.06

b
 0.90±0.13

b
 

 Day 18 0.64±0.08
a
 0.97±0.05

b
 0.87±0.02

b
 

 T4 (ng/ml)    
 Day 11 55.03±2.23 53.95±2.84 64.16±3.91 

 Day 18 50.44±3.57 44.35±3.60 49.78±2.76 

 IGF-1 (ng/ml)    
 Day 11 8723.2±1254.1 9511.6±2703.1 9494.8±957.3 

 Day 18 12858.4±548.8 14166.6±752.0 13158.9±593.8 

 Insulin    

 Day 11 8.46±1.46
a
 21.24±3.28

ab
 25.79±7.04

b
 

 Day 18 6.93±0.43 7.51±0.76 6.73±1.53 
 

*Data are means ± SD (n = 4). Values in a row with different superscripts differ (P<0.05). 
 

 

arginine supplementation in contrast to those of control 
piglets on both days 11 and 18 (P<0.05; Table 3). No 

significant difference in blood T4 or IGF-1 content was 
seen among groups at either experimental phase, 
although, arginine supplementation did tend to increase 
blood IGF-1 at both experimental periods. 0.8% arginine 
group possessed prominently higher level of blood insulin 
than the control on day 11 (P<0.05). 

 

Small intestinal morphology 
 
Jejunal villus height (P<0.05) and villus areal (P<0.05) of 

 
 

 

piglets were significantly higher in 0.8% arginine group 
than the control group on day 11. Jejunal crypt depth was 
notably higher in 0.4% arginine group than the other two 
groups on day 18 (P<0.05, Table 4). 
 

 

D-xylose absorption 

 

Two hours after oral gavage of D-xylose, contents of 
plasma D-xylose of 0.4 and 0.8% arginine groups 
increased by 200 (P<0.05) and 140% (P<0.05), 
respectively than the control group (Figure 2). 



  
 
 

 
Table 4. Effect of dietary L-arginine supplementation on jejunum histomorphology of piglets*.  

 
 Item control 0.4% arginine 0.8% arginine 

 Villus height (µm)    

 Day 11 364.9±19.3
a
 414.1±21.6

ab
 464.7±11.6

b
 

 Day 18 452.7±20.3 501.0±33.3 479.2±41.5 

 Crypt depth (µm)    
 Day 11 160.9±16.8 181.9±8.5 184.5±3.0 

 Day 18 133.6±4.6
a
 181.4±6.3 

b
 154.0±9.7

a
 

 Villus areal (µm
2
)    

 Day 11 138702±7244
a
 180169±14027

ab
 205545±15023

b
 

 Day 18 184193±12304 187220±11190 167949±8587 
 

*Data are means ± SD (n = 4). Values in a row with different superscripts differ (P<0.05). 
 
 
 
 
 

 

P
la

s
m

a
 D

-x
y
lo

s
e

 (
m

m
o

l/
L
) 

 
 
 

 

1.6 

 

1.4 

 

1.2 

 

1 

 

0.8 

 

0.6 

 

0.4 

 

0.2 

 

0 

 

 

b  Control 
 0.4% Arginine   

b  0.8% Arginine 

 
 
 
 
 

 

a 
 
 
 
 
 
 
 
 

 

Day 19 
 

 
Figure 2. Effect of dietary arginine supplementation on intestinal 
permeability of piglets. Bars represent the means ± SD. Within an 
experimental phase, means without a common letter differ (P<0.05). 

 

 

Levels of jejunal disaccharidases 

 

In contrast to the control and 0.4% arginine groups, the 
activity of maltases in jejunal mucosa was notably 
elevated (P<0.05) in 0.8% arginine treatment on day 18 
(Table 5). Both 0.4 (P<0.05) and 0.8% (P<0.05) arginine 
groups showed at least 2-fold higher contents of lactase 
in jejunal mucosa than the control group on day 11. 
Strikingly, the enhancing effect of arginine decreased on 
day 18, although, there was still significant effect between 
two arginine supplementation groups and the control 
group. Additionally, 0.8% arginine group also showed 

 
 

 

significant increase as compared to 0.4% arginine group. 
Concentrations of jejunal mucosa sucrase did not differ 
among the three groups on day 11 as well as day 18. 
Sucrase level significantly increased from 0.88 on day 11 
to 3.55 on day 18 in the control group, while this change 
was not observed in arginine group with almost the same 
level of sucrase from day 11 to 18. 
 

 

Concentration of plasma amino acids 

 

In comparison with the control group, 0.4 and 0.8% 



 
 
 

 
Table 5. Effect of dietary L-arginine supplementation on disaccharidases activities in jejunal 
mucosa of piglets (U/mg prot)*.  

 
Item Control 0.4% Arginine 0.8% Arginine 

Maltases    

d 11 5.21±2.14 4.81±1.25 7.50±2.27 

d 18 9.77±0.75
a
 9.78±1.29 

a
 13.36±1.29

b
 

Lactase    

d 11 5.26±1.49
a
 22.67±7.02

b
 20.20±4.97

b
 

d 18 3.41±1.12
a
 4.66±0.84

a
 6.94±1.2

b
 

Sucrase    
d 11 0.88±0.38 3.70±1.52 4.43±1.56 

d 18 3.55±0.19 2.49±0.74 2.42±0.83 
 

* Data are means ± SD (n = 4). Values in a row with different superscripts differ (P<0.05). 
 

 
Table 6. Effect of dietary L-arginine supplementation on concentrations of plasma amino acid in piglets*.  

 
 Item Control 0.4% Arginine 0.8% Arginine 

 Arginine    

 Day 11 57.44±5.06
a
 89.08±6.96

b
 92.17±10.05

b
 

 Day 18 72.75±4.66
a
 129.00±21.59

b
 137.67±11.48

b
 

 Aspartate    
 Day 11 21.75±1.11 25.75±5.12 29.75±5.54 

 Day 18 18.50±2.66
a
 24.75±2.7

ab
 30.50±2.25

b
 

 Citrulline    

 Day 11 76.25±8.64
a
 115.25±8.14

b
 77.75±8.19

a
 

 Day 18 95.25±7.43 99.25±6.93 91.00±18.87 

 Glutamate    

 Day 11 163.00±13.68
a
 305.00±40.14

b
 300.75±28.25

b
 

 Day 18 247.00±49.79 306.75±46.96 350.50±32.72 

 Ornithine    
 Day 11 54.25±7.73 52.50±4.73 58.00±8.75 

 Day 18 56.75±6.34
a
 95.75±14.94

b
 111.00±11.1

b
 

 Proline    
 Day 11 55.25±2.32 43.00±2.27 51.75±13.36 

 Day 18 38.25±5.91
a
 72.50±14.23

b
 78.50±3.20

b
 

 
*Data are means ± SD (n = 4). Values in a row with different superscripts differ (P<0.05). 

 

 

arginine addition improved plasma concentrations of 
arginine respectively, by 29.53 (P<0.05) and 59.84% 
(P<0.05) on day 11, and 77.32 (P<0.05) and 89.24%  
(P<0.05) on day 18 (Table 6). Plasma citrulline level was 
higher in 0.4% arginine group than in the other two on 
day 11 (P<0.05), while it reduced to the similar level of 
the other two groups on day 18, suggesting some change 

 
 

 

of citrulline metabolism at early stage. Contents of 
plasma ornithine (P<0.05) were notably raised by both 
0.4 and 0.8% arginine supplementation than the control 
treatment only on day 18, but not on day 11. In com-
parison with the control group, both 0.4 and 0.8% 
arginine increased the level of plasma glutamate (P<0.05) 
on day 11, and those of plasma ornithine 



  
 
 
 
 
 
 
 

 

S
p

e
rm

in
e
 (

µ
g

/u
L

) 

 

 
 

20 

     Control              
 

                  
 

     0.4% arginine 
     

b 
    

 

18 
          

a 
 

     

0.8% arginine 
         

 

16 
                  

 

                             
 

14 
                             

 

                             
 

12    

                          
 

                          
 

10 
                

a 
         

 

                         
 

8 
                             

 

                             
 

6                              
 

4 
                             

 

                             
 

2 
                             

 

                             
 

0 

                             
 

 

  

                          
 

   

Day 11 
   

Day 18 
    

 

             
  

  
Figure 3. Effect of dietary L-arginine supplementation on spermine  
concentrations in jejunal mucosa of piglets. Bars represent the  
means  ± SD. Within an  experimental phase, means  without  a  
common letter differ (P<0.05). 

 

 
Table 7. Effect of dietary L-arginine supplementation on concentration of NO in small intestinal mucosa of 
piglets (U/mgprot)*.  

 
 Item Control 0.4% Arginine 0.8% Arginine 

 Day 11    

 Duodenum 0.36±0.02
b
 0.60±0.11

a
 0.34±0.05

b
 

 Jejunum 0.37±0.04 0.31±0.04 0.30±0.03 

 Ileum 0.40±0.06 0.45±0.07 0.29±0.04 

 Day 18    
 Duodenum 0.26±0.05 0.46±0.21 0.52±0.25 

 Jejunum 0.19±0.03 0.22±0.02 0.26±0.07 

 Ileum 0.25±0.02 0.22±0.03 0.21±0.03 
 

*Data are means ± SD (n = 4). Values in a row with different superscripts differ (P<0.05). 
 

 

(P<0.05) and proline (P<0.05) on day 18. 
 

 

Contents of spermine in jejunal mucosa 

 

As revealed in Figure 3, no noteworthy difference in 
jejunal mucosa spermine contents was observed among 
the three groups on day 11. As compared with the control 
and 0.8% arginine groups, concentration of spermine in 
jejunal mucosa of piglets was raised by 0.4% arginine 
supplementation at day 18 (P<0.05). 
 

 

Concentration of NO in small intestinal mucosa 

 

The level of NO in the duodenal mucosa of piglets in 

 
 

 

0.4% arginine group was higher than the other two on 
day 11 (P<0.05). Contents of duodenal NO in 0.4 and 
0.8% arginine groups was respectively, 81.25 (P>0.05) 
and 101.95% (P>0.05) higher than the control group on 
day 18. No obvious difference in NO concentration was 
observed in jejunum or ileum at either experimental 
phase (Table 7). 
 

 

Activities of NOS, ASS and P5CS in jejunal mucosa 

 

0.8 and 0.4% arginine treatment prominently increased 
jejunal NOS level (P<0.05) against the control group on 
day 18 (Table 8), but no difference was observed on day 
11 among three groups. In comparison with the control 
group, 0.4% arginine supplementation elevated jejunal 



 
 
 

 
Table 8. Effect of dietary L-arginine supplementation on enzyme activities in jejunal mucosa of piglets*.  

 
Item Control 0.4% Arginine 0.8% Arginine 

NOS (U/mgprot)    

Day 11 0.33±0.02 0.33±0.02 0.32±0.01 

Day 18 0.28±0.01
a
 0.38±0.04

ab
 0.44±0.05

b
 

ASS (mg/ml)    

Day 11 8.19±1.53
a
 20.43±0.85

b
 17.24±5.33

ab
 

Day 18 5.05±0.83 6.16±0.48 8.32±2.18 

P5CS (mg/ml)    
Day 11 330.10±13.15 325.57±6.48 396.52±43.41 

Day 18 395.39±20.42
a
 462.53±12.26

b
 402.00±24.36

ab
 

 
* Data are means ± SD (n = 4). Values in a row with different superscripts differ (P<0.05). NOS, Nitric oxide 
synthetase; ASS, argininosuccinate synthetase; P5CS, pyrroline-5-carboxylate synthetase. 

 

 

ASS activity on day 11 (P<0.05), while 0.8% arginine 
group showed no obvious difference from the other two 
groups. However, the effects of arginine supplementation 
seemed to be dampened on day 18 as similar level of 
ASS was found in three groups. Jejunal P5CS activity 
was improved by 0.4% arginine treatment on day 18 
(P<0.05) against the control group, while 0.8% arginine 
treatment had no significant effect at either stage. 
 

 

DISCUSSION 

 

In this study, we systematically analyzed the effects of 
arginine supplementation on growth performance of 
young piglets, intestine morphology, enzymes related 
with arginine metabolism and some parameters related to 
amino acid metabolism. Our result demonstrates that 
dietary arginine supplementation did not significantly 
improve BW, ADG, ADFI and feed conversion rate of 
neonatal piglets, which is contradictory to previous 
studies showing that dietary arginine supplementation, 
improved feed conversion rate, daily gain and BW of the 
weaned or early-weaned piglets (Southern and Baker, 
1983). This discrepancy may be due to the different dose 
of arginine supplementation and the time course of piglet 
growth, in additional to different species of piglet studied. 
However, we did observe that blood urea nitrogen of both 
arginine groups were diminished through the experiment 
(Figure 1) on day 11, indicating the improvement of 
protein utilization efficiency in vivo at early stage (Sherry 
et al., 1978). Arginine was reported to be effective in 
stimulating the secretion of insulin and growth hormone 
(Flynn et al., 2000; Wu and Morris, 1998; Yao et al., 
2008). In this study, arginine treatment tended to increase 
blood IGF-1 at both experimental periods (no signi-
ficance, P>0.05), and 0.8% arginine administration 
significantly raised blood insulin on day 11. The results 

also reveal that blood T3 levels on both days 11 and 18 

 
 

 

were increased by arginine (Table 3). These findings 
indicate that hormone secretion might partly contribute to 
the enhancing tissue protein synthesis and the growth-
promoting effect of arginine supplementation.  

In early infancy, the small intestine undergoes a period 
of rapid growth and development associated with multiple 
alterations in intestinal structure and function (Butzner 
and Gall, 1990). Reduction of villus height was reported 
to retard the growth of weaned piglets (Cera et al., 1988). 
Our work reveal that jejunal villus height and villus area of 
piglets were improved by 0.8% arginine supplementation 
on day 11, and jejunal crypt depth was notably greater in 
0.4% arginine group on day 18 than the control group, 
which were consistent with previous findings that 0.7% 
dietary L-arginine supplementation enhanced intestinal 
villus height on day 6 and 10 of experiment (Zhan et al., 
2008).  

Absorption rate of D-xylose in both arginine groups 
were markedly higher than control group by the end of 
the experiment (Figure 2), indicating that arginine 
supplementation improved the intestinal capability in both 
digestion and absorption in neonatal piglets. Moreover, in 
contrast with the control group, both 0.4 and 0.8% 
arginine addition improved lactase level in jejunal mucosa 
on day 11, and 0.8% arginine supplementation elevated 
the activities of maltase and lactase in jejunal mucosa on 
day 18 (Table 5). Nevertheless, concentrations of small 
jejunum mucosa sucrase did not differ among the three 
groups on day 11 as well on day 18; it may change the 
pattern of sucrase level from day 11 to 18 period. These 
results show that the absorption efficiency of macro-
molecules and lactose of the intestinal mucosa was 
largely enhanced by arginine supplementation. As 
Shulman et al. (2005) reported that changes in lactose 
absorption related primarily to lactase activity, previous 
work in miniature pigs also suggested that disac-
charidase-specific activity together with changes in small-
intestinal mucosal growth contributed to disaccharide 



 
 
 

 

digestibility (Redel et al., 1997). Our results indi-cate that 
arginine contributed to the maturational change in 
disaccharides digestion and absorption by im-proving the 
activities of lactase and maltase in the intestine.  

Arginine family amino acids, including arginine, gluta-
mine, glutamate, proline, aspartate, asparagine, ornithine 
and citrulline, were reported to be interconvertible via 
complex interorgan metabolism in most mammals, 
including pigs (Wu et al., 2007). The small intestine is an 
essential organ for the inter-conversion of arginine family 
in pigs (Wu, 1997). Orni-thine and citrulline were two key 
amino acids involved in the endogenous synthesis of 
arginine in the enterocyte. Citrulline and arginine, which 
were derived from glutamine, glutamate and proline in the 
small intestine of pigs, could be utilized for arginine 
synthesis by extrahepatic tissues and cells (Wu et al., 
1995, 2007). Plasma arginine concentration was believed 
to be the most sensitive indicator of in vivo arginine status 
in neonates, including piglets (Batshaw et al., 1984; 
Johnson et al., 1972). In a research conducted in female 
mice, deficiency of dietary arginine reduced the 
concentration of plasma arginine, citrulline and ornithine 
(Cremades et al., 2004). On the contrary, it has been 
reported that 0.48% dietary arginine supplementation 
significantly increased plasma arginine of the 28-day-old 
weaning piglets (Southern et al., 1983). Our study 
demonstrates that plasma content of arginine was dose-
dependently elevated by arginine treatment on both days 
11 and 18 of the experiment (Table 6), which was 
consistent with the findings of Kim et al. (2004) and 
Wilkinson et al. (2004).  

Arginine, as well as citrulline, which were synthesized 
from glutamine, glutamate and proline in enterocytes (Wu 
and Knabe, 1994; Wu et al., 1995), could be utilized for 
arginine synthesis by extrahepatic tissues and cells (Wu 
et al., 1995, 2007). Like the arginine, plasma content of 
citrulline was elevated by arginine treatment on day 11, 
indicating the possible improvement of de novo arginine 
synthesis (Wu and Knabe, 1995). ASS was found to be a 
key enzyme which converted citrulline to arginine (Morris, 
2004). Our work reveals that 0.4% arginine supple-
mentation elevated jejunal ASS activity on day 11 as well 
as P5CS level on day 18 against the control group, which 
was consistent with the changing trend of plasma 
citrulline of the piglets.  

As an important precursor of arginine synthesis in the 
enterocyte of neonatal piglets (Wu and Knabe, 1994; 
Wilkinson et al., 2004; Murphy et al., 1996), plasma gluta-
mate was concordantly raised in the arginine groups on 
day 11, meanwhile, plasma ornithine, proline and aspar-
tate levels were dose-dependently raised by arginine on 
day 18, which was consistent with the previous findings 
(Southern et al., 1983). Taken together, arginine supple-
mentation improved the absorption of arginine, as well as 
the endogenous synthesis of arginine in the neonatal 
pigs. In addition, the longer period of arginine treatment 
(18 day) offered, the better availability of arginine was 

  
  

 
 

 

observed.  
Apart from being an important precursor of arginine 

synthesis, ornithine also played a key role in synthesizing 
polyamines. As widely distributed organic cations, 
polyamines were involved in macromolecular synthesis 
as well as cell proliferation and differentiation in 
mammalian cell systems (Hoshiai et al., 1981; Morrison 
and Seidel, 1995; Wu et al., 2000; Teixeira et al., 2002). 
Polyamines were considered essential for endothelial cell 
proliferation (Morrison and Seidel, 1995). Spermine is  
a polyamine involved in cellular metabolism found in all 
eukaryotic cells; it plays a significant role in cell 
proliferation and differentiation, especially in mucosal 
epithelial cells of the small intestine (Tapiero and Mathé, 
2002). This study illustrates that jejunal spermine 
concentration was raised by 0.4% arginine treatment on 
day 18 in contrast to the other two groups (Figure 3), 
which was believed to further improve the structural 
development and function of the intestine (Hampson and 
Kidder, 1986).  

NO is a major endothelium-derived relaxing factor that 
plays an important role in regulating and maintaining 
vascular function (Tangphaoa et al., 1999). As a lipophilic 
molecule, NO diffuses easily into adjacent smooth muscle 
cells and activates soluble guanylate cyclase signaling 
pathway, which lead to vasodilation (Furchgott and 
Zawadzki, 1980; Stuehr, 2004). It has been reported that 
physiological variations of plasma arginine could 
influence endothelial NO production, thereby modifying 
vascular tone and platelet function (Tangphaoa et al., 
1999). Arginine was catalyzed by NOS to form 
endothelial NO in the small intestine (Moncada and 
Higgs, 1993; Berkowitz et al., 2003). In our study, 0.4% 
arginine supplementation improved the level of NO in the 
duodenal mucosa of piglets on day 11, and both 0.4 and 
0.8% arginine addition tended to improve duodenal NO 
content on day 18. These findings were in line with those 
of Clarkson et al. (1996), Kharitonov et al. (1995) and 
Urschel et al. (2007). Likewise, the content of jejunal 
NOS was obviously increased by 0.8% arginine 
supplementation on day 18 (Table 8).  

In conclusion, our study demonstrates that dietary 
arginine supplementation was capable of improving 
protein synthesis and intestinal development, stimulating 
enzymes synthesis of intestinal mucosa, thereby 
promoting growth performance of the piglets. In all factors 
considered, the 0.8% arginine administration exhibited a 
slightly better effect in promoting the growth of piglets as 
compared to 0.4% arginine group, which has already 
showed many beneficial effects. 
 

 

ACKNOWLEDGEMENTS 

 

This was supported by National Science Foundation of 
China (u0731002), International Science and Technology 
Cooperation Program of China (2009DFA31570) and 



 
 
 

 

National Basic Research Program of China 
(2010CB35701). We thank the staff of Institute of Animal 
Science, Guangdong Academy of Agricultural Science 
and Guangdong Public Laboratory of Animal Breeding 
and Nutrition for technical assistance. 
 

 
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