Journal of Integrative Agriculture 2016, 15(11): 25782587

Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Regulatory effects of dietary L-Arg supplementation on the innate

immunity and antioxidant ability in broiler chickens
HU Ya-di, TAN Jian-zhuang, QI Ji, ZHANG Hong-fu
State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081,
P.R.China

Abstract
Here, we investigated the effect of dietary arginine (Arg) supplementation on innate immunity and the antioxidant ability of
broiler chickens. The experiment was designed as a single-factorial arrangement (n=8 cages/treatment, six birds/cage),
and we used four dietary Arg concentrations (10.0, 15.0, 20.0 or 25.0 g kg1). On day 21, the birds were killed to obtain
spleen, cecal tonsil and liver samples to determine the gene expression and antioxidant characteristics. Increasing the
Arg concentration linearly decreased (P<0.05) the mRNA expression of splenic interleukin-18 (IL-18) and tumor necrosis
factor- (TNF-). Dietary Arg supplementation quadratically decreased (P<0.05) the expression of interleukin-1b (IL-1b) and
interferon- (IFN-) mRNA in the spleen. Increasing Arg concentrations linearly and quadratically reduced the expression
of IL-18 mRNA in the spleen. Meanwhile, increasing dietary Arg supplementation linearly and quadratically increased the
lymphotactin mRNA (P<0.05) expression, and linearly increased the macrophage inflammatory protein-1 (MIP-1) and
toll-like receptor 15 (TLR15) mRNA expression in the cecal tonsils. Dietary Arg supplementation linearly (P<0.05) increased
the glutathione peroxidase (GSH-Px), catalase (CAT), and lysozyme (LZM) activities in the liver. However, the malondialdehyde (MDA) activity in the liver was not influenced by the dietary Arg concentration (P>0.05). No significant (P>0.05) effect
was found on the activity of superoxide dismutase (SOD) in the liver. Thus high levels of Arg supplementation (>20.0 g
kg1) may potentially suppress the innate immunity of broiler chickens, and dietary Arg supplementation enhances the
antioxidant activity in broiler chickens.
Keywords: arginine, innate immunity, antioxidant ability, broiler

1. Introduction
Arginine (Arg) plays a key role in body metabolism during

Received 9 November, 2015 Accepted 12 April, 2016

HU Ya-di, E-mail: huyadi529@163.com; Correspondence ZHANG
Hong-fu, Tel: +86-10-62818910, E-mail: zhanghf6565@vip.sina.
com
2016, CAAS. All rights reserved. Published by Elsevier Ltd.
doi: 10.1016/S2095-3119(16)61404-1

health and illness (Nieves and Langkamp-Henken 2002).

It is classified as a semi-essential amino acid and conditionally as an essential nutrient for adults who are injured or
stressed (Stechmiller et al. 2004). Arg has many functions.
It is a precursor for nitric oxide (NO), urea, ornithine, proline,
creatine phosphate, polyamines, and other molecules that
have pharmacologic actions on multiple systems in the body
and specific effects on wound healing (Stechmiller et al.
2005). Among them, NO plays an important role in organism repair. Additionally, Arg acts as a substratein the urea
cycle and in NO synthesis and serves as a secretagogue
for growth hormone, prolactin, and insulin. Arg acts as a
carrier in human metabolism of biological agents to trigger

HU Ya-di et al. Journal of Integrative Agriculture 2016, 15(11): 25782587

the generation of adrenaline and reduce the generation

of fat, which is important to maintain normal blood sugar
levels. Meanwhile, Arg play a critical role in liver protection, wound healing, and immune system stimulation, and
it promotes the secretion of growth hormone to enhance
wound healing (Daly et al. 1988). However, current studies
have focused on the immune function of Arg (Popovic et al.
2007). Arg supplementation can increase the thymus weight
to improve immunity (Bronte and Zanovello 2005).
Arg is an immunologic modulator partially because of
its role as a substrate in the immune system (Wu et al.
2009). Birds are unable to synthesis Arg because they
have an incomplete urea cycle. Arg is considered to be an
immune-enhancing dietary supplement, which not only provides nourishment, but also exerts versatile immuno-modulating functions for avian (Kidd et al. 2001; Swaggerty et al.
2011). Thus, Arg is a conditionally essential amino acid for
birds. Arg enhances humoral and cell-mediated immune
responses in chickens against virus-specific hydropericardium syndrome (Munir et al. 2009). Several studies have
provided evidence that Arg is used in inflammation and
wound healing (Bronte and Zanovello 2005). Arg plays an
important role in the innate immune response. Studies in
mice showed that dietary Arg supplementation decreased
expression of the proinflammatory cytokine in ulcerative
colitis (Coburn et al. 2012). Recent studies indicate that Arg
stimulates functional activities of different cell types in the
immune system, including natural killer cells, macro-phages,
lymphokine-activated killer cells, and T and B cells (Rhoads
et al. 2004). Studies have also revealed that Arg enhances T
lymphocyte responses in surgical patients (Daly et al. 1988).
It is well known that immune-modulating nutrients can be
helpful and harmful if not utilized in moderation (Tan et al.
2014). Our previous studies reported that increasing dietary
Arg supplementation linearly suppressed the percentages of
circulating and splenic B cells; however, increasing dietary
Arg supplementation linearly reduced the jejunal mucosal
sIgG concentration (Tan et al. 2014), and high levels of Arg
supplementation (21.3 g kg1) reduced the serum anti-New
castle disease virus antibody titers (Tan et al. 2015).
In recent decades, many studies have focused on the
immune regulatory function of Arg in broiler chickens;
however, few studies determined the regulatory effects of
Arg on the antioxidant ability of broiler chickens. Previous
studies have shown that superoxide dismutase (SOD) activities in liver homogenates were significantly lower in the
Arg group than in the control group (Gonzales et al. 1984).
Arg is an essential amino acid for severely burned patients.
The results of this study reveal that SOD and glutathione
peroxidase (GSH-Px) activities in the liver, kidney, and lung
were significantly lower or did not differ in the Arg group at
different time points compared with the control group (Tsai

2579

et al. 2002). SOD and GSH-Px are enzymes that protect

tissues from the damags of free radicals and lipid peroxides,
and the activities of both SOD and GSH-Px increase after
free-radical-mediated injury and lipid peroxidation occurs
(Gonzales et al. 1984). A previous study suggested that
Arg supplementation may enhance humoral immunity and
attenuate the oxidative stress induced by burn injuries in
mice (Shang et al. 2003).
Therefore, the present study was designed to test the
the effect of dietary Arg supplementation on the innate
immunity andantioxidant ability of broiler chickens, and to
gain a better understanding of the application of dietary Arg
supplementation in the poutry industry for improving health
and preventing infectious diseases.

2. Materials and methods

2.1. Experimental design
The broiler management protocol for this study was approved by the Institutional Animal Care and Use Committee
of the State Key Laboratory of Animal Nutrition at the Chinese Academy of Agricultural Sciences.
A total of 192 1-d-old male Arbor Acres Plus broilers were
obtained from the Zhengda Broiler Commercial Company
(China). The broilers were weighed and allocated to groups
so that their initial weights were 45 g across all of the groups.
The experiment was designed with four treatments each
invovled eight replicate cages, and each cage contained
six birds. This experiment included four concentrations of
Arg (10.0, 15.0, 20.0, and 25.0 g kg1), with a single-factor
arrangement. All of the diets (except Arg) were formulated
to meet or exceed the National Research Council (NRC)
requirements. The birds were housed in wire cages and had
free access to feed and water with a 22-h light and 2-h dark
constant-light program. The temperature of the experimental
facility was maintained at approximately (351)C during the
first 3 d and was reduced gradually until it reached 27C at
14 d. The chickens were provided free access to drinking
water and feed.

2.2. Chemical analysis of the diet

The control diet included corn, soybean meal, corn gluten
meal, and poultry as the product meal, which was used as
the intact source of protein. Before beginning the experiment, these ingredients were analyzed for crude protein and
amino acid concentration. The experimental diets included
four concentrations of Arg (Sigma-Aldrich, St. Louis, MO,
catalog number A8094). To analyze the dietary amino acid
composition, diet samples were hydrolyzed with 6 mol L1
HCl at 100C for 24 h, and the amino acid concentrations

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HU Ya-di et al. Journal of Integrative Agriculture 2016, 15(11): 25782587

in the hydrolysate were determined using high performance

liquid chromatography (HPLC) (AOAC International, 2000;
method 982.30 E (a, b, c)). As a result of the formulation
method, many of the essential amino acids were at or near
the minimum recommended levels, and Arg was at the
minimum specified level in every diet. In this experiment,
all diets (except Arg) were formulated to meet or exceed the
NRC (1994) requirements. The composition and calculated
nutrient content of the diets is included in Table 1.

2.3. Sample collection

At 21 d of age, 32 birds from each replicate were selected
and, weighed randomly. Each bird was killed via exsanguination while under deep anesthesia via an intraperitoneal

injection of sodium pentobarbitone (30 mg kg1) of body

weight (BW), and samples were collected. For each bird,
we collected peripheral blood from the wing vein, and the
blood was centrifuged at 3 500g for 10 min at 4C. The
serum was stored at 20C until assay. The spleen from
each bird was aseptically extracted. Tissue samples were
collected for RNA extraction and were held overnight at
4C in RNAlater (Ambion, USA) and stored at 80C until
the RNA was extracted. In the same manner, segments
(23 cm) of the cecal tonsil from each bird were aseptically
extracted and rinsed with RNAlater.
The liver from each bird was aseptically extracted,
washed in PBS (flushed with 0.05 mol L1 PBS, pH 7.2) in
a freezing tube, and stored at 80C until the antioxidant
index was determined.

2.4. Measurements and analyses

Table 1 Composition and calculated nutrient content of
the dry diets
Item

Dietary arginine concentration

(g kg1)
10.0
15.0
20.0
25.0

Ingredients
Maize
630.4
Soybean meal
148.7
Canola meal
30
Maize gluten meal
100
Soybean oil
20
Limestone
11.7
Monocalcium phosphate
22
L-Lysine-HCL
7.5
DL-Methionine
3
L-Threonine
2.1
L-Tryptophan
0.6
Choline chloride
2.5
Sodium chloride
3
2.5
Vitamin/Trace mineral
premix1)
L-Arginine
0
L-Alanine
16
Nutrient and energy concentration2)
ME (MJ kg1)3)
12.8
20.4
CP4)
10
Calcium3)
5
Non-phytate phosphorus3)
14
Lysine4)
5
Methionine4)
9.8
Arginine4)
1)

630.4
148.7
30
100
20
11.7
22
7.5
3
2.1
0.6
2.5
3
2.5

630.4
148.7
30
100
20
11.7
22
7.5
3
2.1
0.6
2.5
3
2.5

630.4
148.7
30
100
20
11.7
22
7.5
3
2.1
0.6
2.5
3
2.5

5.1
10.9

10.2
5.8

15.3
0.7

12.8
20.7
10
5
13.8
5.1
14.7

12.8
20.8
10
5
13.9
4.9
19.1

12.8
21.5
10
5
14.1
4.9
23.4

Supplied the following (per kg complete diet): Cu, 8 mg; Zn, 75 mg;
Fe, 80 mg; Mn, 100 mg; Se, 0.15 mg; I, 0.35 mg; vitamin A,
12 500 IU; vitamin D3, 2 500 IU; vitamin E, 30 IU; vitamin K3,
2.65 mg; thiamine, 2 mg; riboflavin, 6 mg; vitamin B12, 0.025 mg;
biotin, 0.0325 mg; folic acid, 1.25 mg; pantothenic acid, 12 mg;
niacin, 50 mg.
2)
ME (metabolizable energy), calcium, and non-phytate
phosphorus are calculated values; CP (crude protein), lysine,
methionine and arginine are analyzed concentrations.
3)
Calculated values.
4)
Analyzed concentrations.

RNA isolation and reverse transcription The gene expression levels of the spleens were analyzed using real-time
quantitative PCR. The total RNA was isolated using the
Trizol reagent (Invitrogen Canada Inc., USA) according to
the manufacturers instructions. The total RNA concentration was determined via the absorbance at 260/280 nm
using a NanoDrop spectrophotometer (ND-1000; NanoDrop
Products, USA) as described previously (Tan et al. 2014).
Reverse transcription (RT) was conducted using the cDNA
Reverse Transcription Kit (TaKaRa, Bio, Otsu City, Japan)
with 1 g of RNA. RNA reverse transcription includes two
steps: elimination of genomic DNA (gDNA) and the reverse
transcription reaction. Genomic DNA was eliminated by
treatment for 2 min at 42C with gDNA Eraser (TaKaRa,
Bio., Otsu City, Japan), which has a potent DNA degrading
activity. Then, a reverse -transcription reaction reagent was
added, which includes a component that completely inhibits
the DNA degradation activity, and the reverse-transcription reaction proceeded for 15 min at 37C. Synthesized
complementary DNA (cDNA) was diluted 1:5 and stored at
80C (Abe et al. 2003).
Real-time quantitative PCR Real-time PCR were conducted using an ABI 7500 Fast Real-time PCR System and
SYBR Green Supermix Kit (Applied Biosystems, Japan) as
previously described. The primer sequences for the target
and reference genes (GAPDH, interleukin (IL)-1b, IL-6,
IL-8, IL-18, interferon- (IFN-), tumor necrosis factor-
(TNF-), lymphotactin, macrophage inflammatory protein-1
(MIP-1), toll-like receptor 5 (TLR5), TLR15) were given in
the Tables 2 and 3. Each real-time PCR contained 10 L
of SYBR Premix Ex Taq, 0.4 L of ROX Reference Dye II,
and 0.4 L of each forward primer and reverse primer. The
relative expression of all genes was calculated based on the
expression of the housekeeping gene, GAPDH. Additionally,

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HU Ya-di et al. Journal of Integrative Agriculture 2016, 15(11): 25782587

2.5 L of complementary DNA and 6.3 L of DNase-free

water were used. The following protocol was used for all of
the genes: 95C for 30 s, 40 cycles of 95C for 5 s, and 60C
for 34 s. The specificity of the amplification was verified at
the end of the PCR run using melting curve analysis. The
relative expression quantity was calculated using the 2Ct
method (Livak et al. 2001). The value represents the n-fold
difference relative to the calibrator.
Liver superoxide dismutase activity The liver SOD
activity was determined by its ability to inhibit superoxide
radical-dependent reactions using the Jiancheng SOD
Kit (Jiancheng Bioengineering Institute, Jiangsu, China).
Homogenates of the liver were measured colorimetrically
using a high through-put refiner. according to the manufacturers instructions. Approximately 200 L of liver
homogenization buffer sample was added to an 1 800 L
suspension of xanthine and 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT), which was dissolved

in 3-(cyclohexylamino)-1-propanesuhinic acid (CAPS) and

ethylenediaminetetraacetic acid (EDTA). In the presence of
xanthine oxidize, superoxide was produced from xanthine.
The super oxide radical then reacted with INT to produce
a red formazan dye. The optical density (OD) value was
measured spectrophotometrically at 550 nm after 10 min
of reaction.
Liver glutathione peroxide activity The liver GSH-Px
activity was measured using spectrophotometric kits in
accordance with the manufacturers instructions (Jiancheng
Bioengineering Institute). This was performed using the
same method as for the assayed superoxide dismutase
activity. First, homogenates of the liver were measured
colorimetrically using a high-through put refiner. Then
according to the manufacturers instructions, we used a
similar approach for the superoxide dismutase activity. The
OD value was measured spectrophotometrically at 412 nm
after 15 min of reaction.

Table 2 Primers used in the real-time quantitative PCR of the spleen

Target genes1)
GAPDH
IL-1b
IL-6
IL-8
IL-18
IFN-
TNF-
1)

2)

Primer sequence (53)2)

F: CAACACAGTGCTGTCTGGTGGTA
R: ATCGTACTCCTGCTTGCTGATCC
F: CCTCCTCCAGAAAAGTG
R: CTTGTAGCCCTTGATGCCCA
F: TTTATGGAGAAGACCGTGAGG
R: TGTGGCAGATTGGTAACAGAG
F: GCGGCCCCCACTGCAAGAAT
R: TCACAGTGGTGCATCAGAATTGAGC
F: AGTTGCTTGTGGTTCGTCCA
R: TCTACCTGGACGCTGAATGC
F: CTGACAAGTCAAAGCCGCAC
R: ACCTTCTTCACGCCATCAGG
F: GAGCGTTGACTTGGCTGTC
R: AAGCAACAACCAGCTATGCAC

GenBank accession no.

X00182
NM_204524
NM_204628
NM_205498
NM_204608
NM_205149
NM_204267

GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL-1b, interleukin-1b; IL-6, interleukin-6; IL-8, interleukin-8; IL-18, interleukin-18;
IFN-, interferon-; TNF-, tumor necrosis factor-.
F, forward; R, reverse.

Table 3 Primers used in the real-time quantitative PCR of the cecal tonsil
Target genes1)
GAPDH
Lymphotactin
MIP-1
TLR5
TLR15
IFN-
TNF-
1)

Primer sequence (53)

F: CCTAGGATACACAGAGGACCAGGTT
R: GGTGGAGGAATGGCTGTCA
F: GGATTTAAGGGTGAACAGTAGATG
R: TAGAAATAGAAAGCCCGAGGAT
F: ATTGCCATCTGCTACCAGACCT
R: TCAGGTAGCTCTCCATGTCACA
F: AACTCCCTTCCTTCCCACAT
R: TTGCGAGCCAGTTTCTCTCT
F: TACGTCCAGAAAACCCCAGT
R: GAAGGCTTTCGATGGGTGTA
F: CTGACAAGTCAAAGCCGCAC
R: ACCTTCTTCACGCCATCAGG
F: GAGCGTTGACTTGGCTGTC
R: AAGCAACAACCAGCTATGCAC

MIP-1, macrophage inflammatory protein-1; TLR5, toll-like receptor 5; TLR15, toll-like receptor 15.

GenBank accession no.

NM_204305
M 85034
NM_205367
NM_204139
NM_205303
NM_205149
NM_204267

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Liver malondialdehyde content The liver malondialdehyde (MDA) content was measured using the same
method. The kit uses the same as the Jiancheng MDA Kit
(Jiancheng Bioengineering Institute). Then, according to the
manufacturers instructions, the OD value was measured
spectrophotometrically at 532 nm.
Liver catalase activity The liver catalase activity was
determined using a catalase kit (Jiancheng Bioengineering
Institute), as previously described (Wu et al. 2007).
Liver lysozyme activity The liver lysozyme activity was
determined using a lysozyme kit (Jiancheng Bioengineering Institute), as previously described (Wu et al. 2007).
We prepared the bacterial liquid, sealed it, and placed it
in a refrigerator at 28C for 1 wk. Then, according the
manufacturers instructions, the OD value was measured
spectrophotometrically at 530 nm after an ice water bath
reaction for 3 min.
Serum peroxidase activity The serum peroxidase (POD)
activity was measured using spectrophotometric kits in accordance with the manufacturers instructions (Jiancheng
Biotechnology Institute). We obtained the test solution, and
according to the manufacturers instructions, the OD value
was measured spectrophotometrically at 420 nm.

2.5. Statistical analyses

All statistical analyses were performed using SAS 9.2 (SAS
Institute Inc., Cary, NC, USA) software. Data were subjected to one-way ANOVA in a single-factorial arrangement.
Duncans multiple range tests were used to determine the
significance of differences between the treatment means.
Arg concentration was the main effect with P0.05 considered to be significant, and 0.05<P0.10 was considered
a trend towards significance. Polynomial contrasts were
used to determine the linear and quadratic responses of
the main effect means to the dietary Arg concentration.
Additionally, the differences among treatment group
means were determined using Duncans multiple range

test. Linear and quadratic polynomial contrasts were performed based on calculated dietary Arg supplementation.
The differences between group means were compared
using Tukeys multiple range tests, and the results were
expressed as the meansSE.

3. Results
3.1. Gene expression in the spleen
As shown in Table 4, there was a significant trend regarding
the dietary Arg concentration for the IL-1b mRNA expression in the spleen. Increasing the dietary Arg supplementation quadratically decreased (P<0.05) the expression
of pro-inflammatory cytokine IL-1b in the spleen. Dietary
Arg supplementation quadratically (P<0.05) decreased
the expression of the proinflammatory cytokine IFN- in
the spleen. Additionally, the trend was significant for the
pro-inflammatory cytokine IL-18 mRNA expression in the
spleen. With increasing dietary Arg concentrations, the
expression of IL-18 in the spleen decreased linearly (P<0.05)
and quadratically (P<0.05). Arg supplementation produced
the lowest expression of Arg at 25.0 g kg1 of supplementation, and the expression increased for the highest level
of Arg supplementation (15.0 g kg1). Expression of pro-inflammatory cytokine IL-6 in the spleen was not influenced
by the dietary Arg concentration (P>0.05). Increasing the
dietary Arg supplementation linearly (P<0.05) decreased
the expression of pro-inflammatory cytokine TNF- in the
spleen. Increasing the Arg concentration linearly (P<0.05)
decreased the expression of chemokine factor IL-8 in the
spleen. Arg supplementation of 25.0 g kg1 resulted in the
lowest expression, and the expression reached the highest
level at 15.0 g kg1 of Arg supplementation.

3.2. Gene expression in the cecal tonsil

The gene expression data for the cecal tonsil are presented

Table 4 Effect of graded supplementation of the arginine (Arg) concentrations (10.0, 15.0, 20.0, and 25.0 g kg1) on the relative
mRNA expression of spleen inflammation-related genes in the broiler chickens on day 21
Items
IL-1b
IFN-
IL-18
IL-6
IL-8
TNf-
1)

10.0
0.25
0.43
0.28
0.49
0.46
0.59

Dietary Arg treatment (g kg1)

15.0
20.0
0.36
0.28
0.61
0.47
0.41
0.31
0.71
0.39
0.52
0.37
0.53
0.45

25.0
0.18
0.24
0.11
0.61
0.12
0.38

SEM1)
0.04
0.08
0.06
0.07
0.09
0.05

Arg
0.040
0.035
0.002
0.146
0.010
0.016

P-value2)
Linear
0.132
0.067
0.011
0.942
0.003
0.002

Quadratic
0.015
0.022
0.003
0.985
0.058
0.935

SEM, standard errors.

Analyzed via ANOVA. Linear and quadratic polynomial contrasts were performed based on calculated dietary Arg concentrations.
The same as below.

2)

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HU Ya-di et al. Journal of Integrative Agriculture 2016, 15(11): 25782587

in Table 5. Increasing the dietary Arg supplementation quadratically increased (P=0.07) the cecal tonsil IFN- mRNA
expression. Upon increasing dietary Arg concentrations,
the expression of lymphatactin in the cecal tonsil increased
linearly (P=0.001) and quadratically (P<0.05). Increasing
the dietary Arg supplementation linearly increased (P<0.05)
the cecal tonsil MIP-1 and TLR-15 mRNA expression. No
significant effect of dietary Arg supplementation was observed on the TLR-5 or TNF- mRNA expression (P>0.05).

3.3. Antioxidant ability in the liver

Upon increasing the dietary Arg concentrations, significant
increases in the activity of GSH-Px, catalase (CAT), and
lysozyme (LZM) appeared with a significant difference,
compared with the control group.
As shown in Table 6, the MDA activity in the liver was
not influenced by the dietary Arg concentration (P>0.05).
However, dietary Arg supplementation linearly (P<0.05)
increased the GSH-Px activity in the liver. Arg supplementation at 10.0 (g kg1) produced the lowest activity of GSH-Px,
and it increased at the highest Arg supplementation level
(25.0 g kg1). Additionally, no significant (P>0.05) effect
was found on the activity of SOD in the liver. The dietary
Arg supplementation significantly influenced the activity of
CAT in the liver, which increased linearly (P<0.05). Upon
increase in the dietary Arg concentrations, the activity of
LZM in the liver increased linearly (P=0.001). As shown in
Table 4, there was no significant effect by dietary Arg on the

POD activity in the spleen.

4. Discussion
Under normal animal-rearing conditions, the animal is inevitably confronted with various microbial challenges that
lead to nutrient utilization and decreased performance,
often resulting in economic losses (Klasing et al. 1988).
The main role of the innate immune response is the rapid
detection and elimination of invading pathogens. The
maintenance of their innate immune function is critical for
broiler chickens. Dietary Arg supplementation could enhance the innate immunity and adaptive immunity (Bronte
and Zanovello 2005). Arg is considered to be an important
immune-enhancing dietary supplement for both mammals
and avian (Evoy et al. 1998; Abdukalykova et al. 2006; Yeh
et al. 2010). Dietary Arg supplementation could relieve
inflammation in mammals (Coburn et al. 2012). Studies
of rodents and humans have demonstrated that Arg is an
important immunomodulatory nutrient (Li et al. 2007). However, poultry immune systems are different from mammals.
Few studies have been conducted to determine the effect of
dietary Arg supplementation on the inflammatory response
in chickens. L-Arg supplementation improves epithelial
wound repair. Polyamines, which are synthesized from
L-Arg, play a role in intestinal cell migration (Rhoads et al.
2004). One of the most important metabolites of Arg is NO
which plays an important role in regulating intestinal barrier
function (Alican and Kubes 1996).

Table 5 Effect of graded supplementation of the Arg concentrate ions (10.0, 15.0, 20.0, and 25.0 g kg1) on the mRNA expression
of spleen inflammatory genes in the broiler chickens on day 21
Items
IFN-
Lymphotactin
MIP-1
TLR5
TLR15
TNf-

10.0
0.34
0.17
0.11
0.65
0.44
0.48

Dietary Arg treatment (g kg1)

15.0
20.0
0.91
0.21
0.36
0.49
0.31
0.51
0.56
0.66
0.38
0.52
0.34
0.46

25.0
1.07
1.18
0.97
0.65
0.63
0.49

SEM
0.21
0.22
0.18
0.02
0.05
0.03

Arg
<0.001
0.001
<0.001
0.571
0.088
0.127

P-value
Linear
0.374
0.000
<0.001
0.724
0.030
0.186

Quadratic
0.071
0.035
0.192
0.505
0.277
0.383

Table 6 Effect of graded supplementation of the Arg concentrations (10.0, 15.0, 20.0, and 25.0 g kg1) on the relative antioxidant
ability of the broiler chickens on day 21
Items
MDA
GSH-Px
CAT
SOD
LZM
POD

10.0
4.32
1176.66
14.23
17.36
70.27
5.98

Dietary Arg treatment (g kg1)

15.0
20.0
25.0
5.47
4.35
8.04
1395.72
1558.31
1953.86
15.00
20.86
22.87
14.08
19.27
20.09
78.04
81.08
87.16
7.53
6.49
5.59

SEM
0.87
164.07
2.14
1.34
3.51
0.42

Arg
0.648
0.181
0.041
0.139
0.005
0.228

P-value
Linear
0.307
0.038
0.007
0.122
0.001
0.601

Quadratic
0.746
0.714
0.958
0.281
0.784
0.834

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Chemokines and cytokines play a key role in pathogen

clearance (Withanage et al. 2005). Cytokines are an important immunomodulatory molecule, which are activated via
immune cells and innate immune responses. Additionally,
cytokines are crucial protein mediators in humoral immunity
(Ma et al. 2007). They are small, non-structural proteins with
molecular weights that range from 8 to 40 000 Da. Pro-inflammatory cytokines, such as IL-1b, IL-6, IL-18, and TNF-,
promote inflammation, whereas anti-inflammatory cytokines,
such as IL-4, IL-10, and IL-13, suppress inflammation (Opal
and DePalo 2000). IL-6 is a hormone-like protein that plays
a central role in inflammation. It is produced by various cells,
such as monocytes, endothelial cells, and fibroblasts (Nijsten et al. 1991). IL-6 is a pro-inflammatory cytokine, and it
is well known that Arg plays an important role in the immunie
system. In the present study, dietary Arg supplementation
increased the expression of pro-inflammatory cytokine IL-6
in the spleen; thus, dietary Arg supplementation may inhibit
the inflammatory response. Moreover, lipopolysaccharide
(LPS) injection significantly increased the expression of
pro-inflammatory cytokine IL-6 in the spleen. IL-6 is only
one of many cytokines involved in inflammation, and in the
context of acute phase responses, TNF and IL-1 are important mediators. Dietary Arg supplementation increased
the expression of pro-inflammatory cytokine IL-1b, IFN-,
IL-18, and TNF- in the spleen. When we added 15.0 g
kg1 of dietary Arg, the expressions of the pro-inflammatory
cytokines, IL-1b, IFN-, IL-18, and TNF-, were the highest.
IFN-, a pleiotropic cytokine, which is produced principally
by CD4+ cells, CD8+ T cells, and natural killer (NK) cells, is
essential for both innate and adaptive immunity. Previous
studies showed that mice lacking IFN- display a disruption
in their innate and adaptive immunity, resulting in death via
infections (Durbin et al. 1996; Meraz et al. 1996). In the
current study, dietary Arg supplementation quadratically decreased the expression of pro-inflammatory cytokine IFN-
in the spleen, which suggested there are certain requirements regarding the amount of Arg for use in broiler chicken
diets. An excessive amount potentially affects the chicken
humoral immune function and increase dietary costs. With
an increase in the dietary Arg concentration, the mRNA
expression of splenic pro-inflammatory cytokine increased
IL-8 is a member of the chemokine family, which has selective chemotactic activity for neutrophils and lymphocytes
at nanomolar and picomolar concentrations, respectively
(Koch et al. 1992). Tumor necrosis factor, TNF-, is a pleiotropic cytokine that induces a variety of cellular responses,
including inflammatory cytokine production, cell survival,
cell proliferation, and unexpectedly, cell death (Ashkenazi
and Dixit 1998). Previous studies have indicated that the
inflammatory response of mammalian cells to TNF- can be

switched to apoptosis either via co-treatment with a protein

synthesis inhibitor, cycloheximide, or with smac mimutic,
a small molecule that mimics the Smac/Diablo protein
(Wang et al. 2008). As shown in this study, increasing the
dietary Arg supplementation linearly (P<0.05) decreased
the expression of pro-inflammatory cytokine TNF- in
the spleen. However, IL-8 has been identified previously
as a chemotactic factor that was secreted by activated
monocytes and macrophages that promote the directional
migration of neutrophils, basophils, and T lymphocytes
(Baggiolini et al. 1989). In that study, increasing the Arg
concentration decreased the expression of chemokine factor
IL-8 in the spleen, and the results suggested that dietary Arg
supplementation may have a potential regulatory effect that
alleviaties either humoral or cellular immunosuppression.
Additionally, high levels of Arg supplementation (>20.0 g
kg1) suppressed the innate immunity of broiler chickens.
Therefore, the study indicated that Arg acts as a valuable
immunoregulator of humoral immunity in chickens. Another
study suggested that lymphotactin serves as a bridge between the innate signals of the mucosa and the adaptive
mucosal immune system by enhancing the immune function.
Lymphotactin is chemotactic for NK and T cells and plays an
important role in maintaining the integrity of the epithelium
and in mucosal immune responses (Lillard et al. 1999). In
our study, increasing the dietary Arg concentrations linearly
and quadratically increased, the expression of Lymphatactin
in the cecal tonsil. Thus, dietary Arg supplementation enhances broiler immune function, demonstrating the ability
of high Arg supplementation to decrease the expression of
Lymphotactin. TLR15 was identified in chicken and zebra
fish genomes (Roach et al. 2005). It activates the innate
immunity (Boyd et al. 2012). In the present study, our results
showed that the expression of the TLR15 mRNA tended
to increase linearly as a function of increased dietary Arg
supplementation. MIP-1 plays a key role in the induction
and modulation of the inflammatory response (Maurer and
Von Stebut 2004). Increasing the Arg concentration linearly increased the expression of chemokine factor MIP-1
in the cecal tonsil. These results suggested that dietary
Arg supplementation may enhance the innate immunity in
broiler chickens.
Mammals are sensitive to LPS within a few hours after
injection, producing an increased rectal temperature, catering waste (Xie et al. 2000). The present study shows that
dietary Arg supplementation may attenuate LPS-induced
inflammation in broiler chickens via the suppression of the
LPS/TLR4 pathway (Tan et al. 2014). In myeloid cells, Arg is
mainly metabolized either by Inducible NO synthase (iNOS)
or by arginase 1 (ARG1) (Popovic et al. 2007). Dietary Arg
supplementation causes an inflammatory response. Arg

HU Ya-di et al. Journal of Integrative Agriculture 2016, 15(11): 25782587

primarily is metabolized by iNOS and ARG1, and it generates

NO and polyamines (Calkins et al. 2001). It is known that
the induction of iNOS upon increased production of NO is
a critical factor that triggers the expression of inflammatory
cytokines.
In the present study, dietary Arg supplementation not
only suppressed the innate immunity of broiler chickens,
but also, regulated the antioxidant ability. The antioxidant
system consists of intrinsic enzymes and extrinsic antioxidant nutrients, which, serve to reduce free radicals to less
toxic states (Vertuani et al. 2004).
Glutathione peroxidase comprises an enzyme family. It
is one of the most important components of the anti-peroxidation system of the body and has been implicated in some
diseases. It has the dual effect of removing peroxidase
and detoxification in the body. The physiological function
of GSH-Px in erythrocytes has been comprehensively reviewed. Within the relatively degenerated cells, catalase
and GSH-Px can substantially substitute for each other in
the removal of H2O2. GSH-Px plays an important role in
biology. It is dependent on the micronutrient selenium (Se)
and plays a critical role in the reduction of lipid and hydrogen
peroxides (Arthur 2001). If GSH-Px activity is decreased,
more hydrogen peroxide is present, which leads to direct
tissue damage and activation of nuclear factor-B-related
inflammatory pathways (Yu and Chung 2006). In this study,
dietary Arg supplementation significantly increased the
GSH-Px activity in the liver, which is very important for the
entire broiler chicken. It can remove hydrogen peroxide;
thus, hydrogen peroxide injury was not obvious. GSH-Px
catalyzes the reduction of hydrogen peroxide and an organic
hydroxide compound, which is involved in the mediation
of prostaglandins. MDA is an organic compound that has
the molecular formula CH2(CHO)2. It is the most abundant
reactive aldehyde resulting from LPO and has been widely
used as an index of oxidative deterioration of lipids in food
and biological samples (Garcia and Gaya 1984). In this
study, the MDA activity in the liver was not influenced by the
dietary Arg concentration. SOD, CAT, and GSH-Px were the
primary anti-oxidant enzymes. Among them, SOD converts
superoxide radical into hydrogen peroxide and oxygen (O2),
whereas catalases and peroxidases convert hydrogen peroxide into water and, in the case of catalase, into oxygen and
water (Weydert et al. 2010). In this study, the activity of the
SOD in the liver was not influenced by different treatments,
and no linear or quardratic trend was observed. However,
dietary Arg supplementation significantly influenced the activity of CAT in the liver, which increased linearly. Previous
research has shown that in normal LPS-responder mice a
dose of 40 g of LPS or tumor induction caused a decrease
in the CAT activity of approximately 50% (Garcia and Gaya
1984). LZM, a group of antimicrobial proteins, are enzymes

2585

that cleave the 1,4--glycosidic linkage between N-acetylgucosamine and N-acetylmuramic acid in Gran-positive
bacterial cell walls (Fiolka et al. 2012). The activity of LZM
is an important index that reflects the non-specific immunity
of the body. As one of the non-specific immune factors of an
organism, it acts in inflammatory processes, such as repair
and regeneration of the organism, and it plays an important
role in the regulation. To a certain extent, LZM reflects the
cellular immunity function and the bodys ability to fight infection (Fukawa et al. 2003). Similarly, LZM and CAT had
the same trend in the present study. Upon an increase in
dietary Arg concentrations, the activity of LZM in the liver
increased linearly, which suggests that Arg may increase the
activity of LZM in the liver of the broiler chickens. This may
be the mechanism of resistance to infection. The inflammatory response requires rapid activation of pro-inflammatory
genes in cells of the innate immune system. The chicken
LZM gene is a well-studied model to investigate the effects
of pro-inflammatory stimuli on gene expression (Lefevre
et al. 2008). However, the activity of POD in the liver was
not influenced by different treatments, and no linear or
quadratic trend was observed.

5. Conclusion
The present study demonstrated that high level of Arg supplementation (>20.0 g kg1) may potentially suppress the
innate immunity of broiler chickens. Additionally, dietary
Arg supplementation enhanced the antioxidant activity of
broiler chickens.

Acknowledgements
This work was supported by the Laboratory of Animal
Nutrition, Institute of Animal Sciences, Chinese Academy
of Agricultural Sciences (CAAS). The authors thank Tan
Jianzhuang, Liu Shasha, Gao Lixiang, Qi Ji, Zhong Ruqing,
Pang Min, Luan Sujun, Yang Xia, L Shuaibing, and Liao
Rui, CAAS, for their assistance in the experiments. This
study was supported by the National Key Technology R&D
Program of China (2012BAD39B01) and the Agricultural
Science and Technology Innovation Program of CAAS
(ASTIP-IAS07) in China.

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