Animal Feed Science and Technology 220 (2016) 8392

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Animal Feed Science and Technology

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N-acetylcysteine improves the growth performance and

intestinal function in the heat-stressed broilers
Dan Yi a , Yongqing Hou a , Linglin Tan a , Man Liao a , Jiaqian Xie a , Lei Wang a ,
Binying Ding a, , Ying Yang b , Joshua Gong a,c
a
Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative Innovation Center for Animal Nutrition and Feed
Safety, Wuhan Polytechnic University, Wuhan 430023, China
b
State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, 100193,
China
c
Guelph Research and Development Centre, Agriculture and Agri-Food Canada, Guelph, Ontario, N1G 5C9, Canada

a r t i c l e

i n f o

Article history:
Received 29 April 2016
Received in revised form 26 July 2016
Accepted 28 July 2016
Keywords:
N-acetylcysteine
Intestinal function
Broiler
Heat stress

a b s t r a c t
This study was carried out to investigate the effects of N-acetylcysteine (NAC) on growth
performance and intestinal function of heat-stressed broilers. A total of two hundred 1d-old Cobb male chicks were allocated to 1 of 4 treatments, with 5 replicated pens per
treatment and 10 birds per pen. The experiment consisted of 4 treatments in a 2 2 factorial arrangements with two diets (basal diet or 1 g/kg NAC diet) and two temperatures
(thermoneutral or heat stress). From day 835 of age, broilers were raised at thermoneutral
(26 1 C) or exposed to cyclic heat stress (36 1 C from 0800 to 1800 and 26 1 C from
1800 to 0800). The results showed that heat stress reduced ADFI, ADG, plasma concentrations of triiodothyronine (T3 ) and thyroxine (T4 ), intestinal villus height (VH), ratio of VH to
crypt depth (CD), intestinal mucosal ATP level, adenylate energy charge (AEC), activities of
alkaline phosphatase (AKP), antioxidative and digestive enzymes, and mRNA level for Bcl2, whereas increased the feed/gain, mortality rate, plasma corticosterone level, intestinal
CD, intestinal mucosal AMP and malondialdehyde (MDA) levels, and mRNA levels of heat
shock protein (HSP70), caspase-3, AMP-activated protein kinase (AMPK), heme-oxigenase
(HMOX), and xanthine oxidoreductase (XOR). Dietary supplementation with NAC decreased
the feed/gain, mortality rate, plasma corticosterone level, MDA concentration and intestinal
mucosal mRNA levels of HSP70, AMPK and HMOX, while elevated the ratio of VH to CD, ATP,
catalase (CAT) and trypsine activity in the small intestine of heat-stressed broilers. Taken
together, these results suggest that dietary supplementation of 1 g/kg NAC could improve
growth performance and intestinal function of broilers exposed to heat stress.
2016 Elsevier B.V. All rights reserved.

Abbreviations: ADFI, average daily feed intake; ADG, average daily gain; ADP, adenosine diphosphate; AEC, adenylate energy charge; AKP, activities
of alkaline phosphatase; AMP, adenosine monophosphate; AMPK, AMP-activated protein kinase; ATP, adenosine triphosphate; CAT, catalase; CD, crypt
depth; HMOX, heme-oxigenase; HSP70, heat shock protein; MDA, malondialdehyde; NAC, N-acetylcysteine; T3 , triiodothyronine; T4 , thyroxine; TAN, total
adenine nucleotide; VH, intestinal villus height; XOR, xanthine oxidoreductase.
Corresponding author. Hubei Key Laboratory of Animal Nutrition and Feed Science, Wuhan Polytechnic University, Wuhan 430023, China.
E-mail address: dbying7471@126.com (B. Ding).
http://dx.doi.org/10.1016/j.anifeedsci.2016.07.014
0377-8401/ 2016 Elsevier B.V. All rights reserved.

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D. Yi et al. / Animal Feed Science and Technology 220 (2016) 8392

1. Introduction
Animal farming, particular poultry production, is facing the challenge of global warming. Hyperpyrexia condition causes
high mortality and growth depression of poultry and consequently results in huge economic losses (Loyau et al., 2015). Due to
lack of sweat glands, birds under thermal stress spend less time feeding, more time drinking and panting, as well as more time
apping wings, less time moving or walking, and more time resting for dissipating heat (Lara and Rostagno, 2013). In addition,
the physiological and metabolic responses such as modications of blood parameters, plasma hormone concentrations,
oxidative stress and meat acidity were also reported in poultry challenged with high temperature (Loyau et al., 2015).
Specically, heat stress can induce intestinal dysfunction showing the impaired intestinal morphology (Mitchell and Carlisle,
1992; Lambert et al., 2002), increased intestinal permeability (Quinteiro-Filho et al., 2010; AI-Fataftah and Abdelqader, 2014),
decreased absorptive function (Ruan and Niu, 2001), and enhanced oxidative and immune injury (Bouchama and Knochel,
2002; AI-Fataftah and Abdelqader 2014). To date, in addition to temperature-reducing equipments, nutritional manipulation
techniques, such as increasing diet nutrient concentration, maintaining amino acids balance and dietary electrolyte balance
(Ahmad et al., 2008), and supplementing vitamin C, selenium, chromium (Rao et al., 2016), betaine (Sayed and Downing,
2011), probiotics (AI-Fataftah and Abdelqader, 2014), or plant extract (Song et al., 2013, 2014), are increasingly developed
and used in poultry to enhance their capacity against high temperature environment.
Based on our previous studies on N-acetylcysteine (NAC) (Hou et al., 2012, 2013; Yi et al., 2014, 2016a), we suppose that
NAC may exert benecial effects on growth performance and capacity against heat stress of broilers. NAC is a precursor of
l-cysteine, and can be rapidly metabolized by the small intestine to produce reduced glutathione (GSH) (Wu et al., 2004).
Indeed, NAC can not be detected in animals without supplementation. By providing sulfhydryl groups, NAC was reported
to play critical roles in the rodents and pigs, including detoxication and protecting cells and cellular components against
oxidative stress (Wu et al., 2004). Our previous study also showed that dietary NAC could increase average daily gain and
attenuate the intestinal injury in piglets induced by lipopolysaccharide (Hou et al., 2012, 2013). However, up to now, there
is only one study by Valdivia et al. (2001) on the application of NAC in poultry production. They found that NAC attenuated
negative effects on growth performance, liver function, and biochemical parameters induced by aatoxin B1 in broiler
chickens (Valdivia et al., 2001).
In order to clarify the efcacy of NAC application in poultry production, further study are warranted to investigate the
effects of NAC on growth performance and intestine function of broilers under heat stress.

2. Material and methods

2.1. Birds, diets, and experimental design
The animal protocol used in the present study was approved by the Animal Care and Use Committee of Hubei Province,
China. Two hundred healthy one-day-old male Cobb chicks (46.2 0.4 g; from a commercial source) were housed in stainlesssteel cages in a temperature-controlled room with relative humidity of 60% and a 24-h photoperiod (Li et al., 2015). The
experiment consisted of 4 treatment groups in a 2 2 factorial arrangements with two diets (basal diet or 1 g/kg NAC
diet) and two temperatures (thermoneutral or heat stress). Birds were randomly divided into 4 treatment groups, each of
which included 5 replicates with 10 broiler chickens per replicate. The thermoneutral and heat treatment were divided into
separate rooms (Song et al., 2014). The basal diet was a maize- and soybean meal-based diet (Table 1) and was formulated to
meet National Research Council (NRC, 1994)-recommended requirements for all nutrients, whereas NAC diet was prepared
by basal diet supplemented with 1 g/kg NAC (Cat. A7250, Sigma Chemical, Inc.). Hou et al. (2012) reported that 0.5 g/kg
NAC in diet (about 20 mg/kg BW, daily) improved the intestinal function of lipopolysaccharide-challenged piglets. Given
the depression of feed intake caused by heat stress in broilers, we increased the NAC level (1 g/kg, about 60 mg/kg BW,
daily) in diet to ensure that adequate NAC can be absorbed by chicks. In addition, Valdivia et al. (2001) reported that
the high level of NAC (800 mg/kg BW, daily) in diet was safe and did not change the production parameters of broilers.
For preparation of NAC diet, NAC and other minor ingredients (sodium chloride, lysine, methionine, and vitamin-mineral
premix) were accurately weighted and then hand-mixed (Teo and Tan, 2007). The mixture were then divided into 4 portions
and blended in a small mixer with soybean meal using the quartering technique (AI-Fataftah and Abdelqader, 2014). Finally,
the resulting mixture was added to the basal diet and mixed for 5 min. Because the supplementation of 1 g/kg NAC resulted
in only an increase of 0.0084% nitrogen, we deemed it not necessary to use a non-essential amino acid as an isonitrogenous
control. The dietary contents of crude protein and total phosphorus were determined according to the Weende method of
the feed proximate analysis as described by Henneberg and Stohmann, (1864). The levels of methionione, cystine, lysine,
threonine, and tryptophan in the basal diet were determined by automatic amino acids analyser (S433D, Sykam GmbH,
Eresing, Germany). All chicks were given ad libitum access to feed and water.
From day 17 of age, all chicks were raised at 32 1 C. However, from day 835 of age, two groups, including one basal
diet group and one NAC-diet group, were exposed to 36 1 C (from 0800 to 1800) and 26 1 C (from 1800 to 0800) as
cyclic heat stress groups. The other two groups (one basal diet group and one NAC-diet group) were raised at 30 1 C (from
day 814 of age) and 26 1 C (from day 1535 of age) as thermoneutral groups. Average daily gain (ADG), average daily
feed intake (ADFI), and feed to gain ratio were calculated. Mortality was recorded by daily visual observation.

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85

Table 1
Ingredients and chemical composition of the basal diet (air-dried basis).
Item
Ingredients (g/kg)
Maize
Soybean meal (440 g/kg CP)
Corn gluten meal (600 g/kg CP)
Soybean oil
Dicalcium phosphate
Limestone
Sodium chloride
L-Lysine hydrochloride
DL-Methionine
Vitamin-mineral Premixa
Nutritional Composition
Metabolizable energy (MJ/kg)b
Crude protein (g/kg)c
Methionine (g/kg)c
Methionine + Cysteine (g/kg)c
Lysine (g/kg)c
Threonine (g/kg)c
Tryptophan (g/kg)c
Calcium (g/kg)b
Total phosphorus (g/kg) c
Non-phytate phosphorus (g/kg)b

13 wk

45 wk

593
287
50.0
19.4
18.8
11.9
3.6
3.7
2.6
10.0

621
253
50.0
29.9
17.9
11.7
3.1
2.1
1.3
10.0

12.33
205
5.9
9.2
12.0
7.9
2.3
10.0
6.8
4.7

12.75
190
4.4
7.6
10.0
7.3
2.1
9.5
6.5
4.5

a
Supplied per kg diet: Mn 75 mg, Zn 40 mg, Fe 80 mg, Cu 10 mg, iodine 0.3 mg, selenium 0.2 mg, retinol acetate 24 mg, DL--tocopheryl acetate 20 mg,
cholecalciferol 0.034 mg, menadione 1 mg, thiamine 1.1 mg, riboavin 3 mg, folic acid 1.2 mg, calcium pantothenate 5.5 mg, nicotinamide 30 mg, pyridoxine
2 mg, cobalamin 0.015 mg, biotin 0.2 mg, choline chloride 900 mg.
b
Calculated value.
c
Analysed value.

2.2. Sample collection

On day 36 of age, 10 broilers per group (2 per replicate) were randomly selected and blood was drawn from left wing
vein after 8-h of feed deprivation. Whole blood was used to collect the serum for triiodothyronine (T3 ), thyroxine (T4 ),
and corticosterone assays. After sampling of blood, birds were euthanized and their abdomens were opened immediately.
The small intestine was dissected and placed on a chilled stainless steel tray. The 3- and 5-cm segments were cut at midjejunum. The 3-cm segments were gently ushed with cold physiological saline, and then xed in 4% paraformaldehyde
over night at 4 C for histological examination (Li et al., 2015). The digesta of 5-cm segments were removed in 10 mL tubes
for measurements of digestive enzymes, and the segments were further opened longitudinally and ushed the residual
digesta with cold physiological saline for collecting mucosa. Jejunal mucosa were collected by scraping with a sterile glass
microscope slide, rapidly frozen in liquid nitrogen, and then stored at 80 C until analysis. All samples were collected within
10 min after killing of the animals (Li et al., 2015).

2.3. Serum hormones determination

Serum T3 (Cat. T140510), T4 (Cat. T140511), and corticosterone (Cat. T140513) levels were determined by commercially
available 125 I RIA kits (Beijing North Institute of Biological Technology, Beijing, China). The detection limit for corticosterone
was 2.5 ng/ml, and the coefcients of variation (CV) for intra- and inter-assays were less than 10% and 15%, respectively. The
detection limits for T3 and T4 analyses were 0.1 ng/ml and 5 ng/ml, respectively. The coefcients of variation for intra- and
inter-assays were less than 15% and 10% for T3 , and less than 15% and 10% for T4 , respectively.

2.4. Intestinal morphology

The 3-cm jejunal samples were dehydrated and embedded in parafn, and then sectioned into 4-m slices, which were
then stained with hematoxylin and eosin. Jejunal morphology was examined using a light microscope with a computerassisted morphometric system (BioScan Optimetric, BioScan Inc., Edmonds, WA, USA.). The villus height (VH) was measured
from the villus tip to the valley between individual villus. The crypt depth (CD) was measured from the valley between
individual villus to the basolateral membrane. The 10 longest and straightest villi and associated crypts were measured
from each segment (Xu et al., 2003; Li et al., 2015).

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D. Yi et al. / Animal Feed Science and Technology 220 (2016) 8392

2.5. Jejunal energy status

Concentrations of ATP, ADP, and AMP in jejunal mucosal samples were analysed by HPLC according to our previous study
(Yi et al., 2014). Briey, frozen mucosal samples (0.1 0.2 g) were homogenized with cold perchloric acid and centrifuged.
The supernatant (1 mL) was then neutralised with potassium carbonate on ice, and the solution was centrifuged to collect
the supernatant, which was stored at 80 C until analysis.
The chromatographic system consisted of the Waters Breeze HPLC system (Waters Corporation, Milford, MA, USA), including 1525 binary HPLC pumps, a 2487 Dual- Absorbance Detector, a 717 plus autosampler and Breeze system software, and a
chromatographic column (Waters XBridge C18; 5 m, 4.6 mm 150 mm). The mobile phase (50 mM-K2 HPO4 -KH2 PO4 buffer
solution and methanol; 77: 23, v/v; pH 7.0) was ltered through a 0.45 m lter membrane and degassed 15 min before
use. The detection wavelength was 260 nm, the column temperature was 35 C and the pump ow rate was 1.0 mL/min.
The frozen sample was ltered through a 0.20 m lter membrane after being thawed at 22 C, and the injection volume
was 20 L. Peaks were identied by their retention times using authentic standards (Sigma Chemical, Inc.). Total adenine
nucleotide (TAN) and adenylate energy charges (AEC) were calculated according to the following equation (Yi et al., 2014;
Li et al., 2015). TAN = ATP + ADP + AMP, while AEC = (ATP + 0.5 ADP)/(ATP + ADP + AMP).

2.6. Jejunal oxidative and antioxidative parameters

Frozen mucosal samples (0.1 g) were powdered under liquid nitrogen, homogenized with cold physiological saline, and
then centrifuged to collect the supernatant for analysis. The concentrations of malondialdehyde (MDA, Cat. A003-1) and
hydrogen peroxide (H2 O2 , Cat. A064-1) and the activities of catalase (CAT, Cat. A007-1), glutathione peroxidase (GSH-Px,
Cat. A005), and superoxidase (SOD, Cat. A001-1) were determined using commercial kits (Nanjing Jiancheng Bioengineering
Institute, Nanjing, China) following the instructions of the manufacturer. Total protein concentrations were determined
using the Coomassie Brilliant Blue G-250 reagent with BSA as a standard.

2.7. Jejunal alkaline phosphatase (AKP) and digestive enzyme activity

The activity of AKP was determined according to the assay kits instructions, obtained from Nanjing Jiancheng Bioengineering Institute (Cat. A059-1, Nanjing, China) (Hao et al., 2012). For determination of mucosa AKP activity, mucosal samples
were diluted with saline and homogenized. The homogenate was then centrifuged to collect the supernatants for AKP determination. One unit of AKP activity was dened as the production of 1 mg of nitrophenol per gram of jejunal mucosal protein
(Hao et al., 2012).
For determination of trypsine and lipase, jejunal digesta was homogenized with saline and centrifuged to collect the
supernatant. Lipase (Cat. A080-2) and trypsine (Cat. A054) activities in jejunal digesta were determined using commercial
kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) (Hu and Guo, 2008).

2.8. Jejunal mRNA levels for genes associated with intestinal growth, oxidation and energy metabolism
A qRT-PCR method was used to determine the mRNA level for genes including B-cell lymphoma-2 (Bcl-2), caspase-3,
AMP-activated protein kinase (AMPK), adenine nucleotide translocator (ANT), peroxisome proliferator-activated receptor
coactivator-1 (PGC-1), cytochrome oxidase III (COXIII), heat shock protein (HSP70), hypoxia-inducible factor 1, subunit
alpha (HIF-1), heme-oxigenase (HMOX), and xanthine oxidoreductase (XOR). Total RNA of mucosal samples was extracted
and puried with the TRIzol Reagent (Invitrogen, Carlsbad, CA) according to our previous studies (Yi et al., 2016b). Total RNA
was quantied using the NanoDrop ND-2000 UVvis spectrophotometer (Thermo Scientic, Wilmington, DE, USA) at an
OD of 260 nm. The purity of RNA was assessed by determining OD260/OD280 ratios. All of the samples had an OD260/OD280
ratio above 1.8, corresponding to 90100% pure nucleic acids. Meanwhile, RNA integrity in each sample was determined
using 1% denatured agarose gel electrophoresis. RNA was used for RT-PCR analysis when it had a 28 S/18 S rRNA ratio 1.8
(Hou et al., 2013). Total RNA was reverse-transcribed using a PrimeScript RT reagent kit (Cat. DRR047A) with gDNA Eraser
(Takara, Dalian, China) according to the manufacturers instruction. cDNA was synthesized and stored at 20 C until use.
To amplify cDNA fragments, primer pairs (Table 2) were used for RT-PCR. To minimise amplication of potentially contaminating genomic DNA, the primers were designed to span introns and intron-exon boundaries. The RT-PCR was performed
using the SYBR Premix Ex TaqTM (Cat. RR420A, Takara, Dalian, China) on an Applied Biosystems 7500 Fast Real-Time PCR
System (Foster City, CA). The specicity of the RT-PCR reactions was assessed by analyzing the melting curves of the products
and size verication of the amplicons (Meurens et al., 2009). The delta delta cycle threshold (CT ) method was used to analyse
the relative expression of the target gene (Fu et al., 2010). To ensure the sensitivity and accuracy of the results obtained
by RT-PCR, data were normalised geometrically averaging of two internal reference genes: glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) (Nygard et al., 2007; Ojano-Dirain et al.,
2007).

D. Yi et al. / Animal Feed Science and Technology 220 (2016) 8392

87

Table 2
Sequences of the primers used for quantitative PCR analysis.
Genes

Forward

Reverse

References

HSP70
Bcl-2
Caspase-3
AMPK1
ANT
PGC-1
COXIII
HIF-1
HMOX
XOR
GAPDH
HPRT1

AGCGTAACACCACCATTCC
GATGACCGAGTACCTGAACC
GGAACACGCCAGGAAACTTG
AAGGTTGGCAAGCATGAGTT
TGTGGCTGGTGTGGTTTCCTA
CCAAAGGACACGCTCTAGATCA
AGGATTCATTTTCACAGCCCTACAAG
CACCATTACCATACTTCAGCAG
CTTGGCACAAGGAGTGTTAAC
GTGTCGGTGTACAGGATACAGAC
TGAAAGTCGGAGTCAACGGATT
CGTTGCTGTCTCTACTTAAGCAG

TGGCTCCCACCCTATCTC
CAGGAGAAATCGAACAAAGGC
TCTGCCACTCTGCGATTTACA
TTCTGGGCCTGCATATAACC
GCGTCCTGACTGCATCATCA
TCTCGATCGGGAATATGGAGAA
AGACGCTGTCAGCGATTGAGA
CTTCACATCATCCACACGTTC
CATCCTGCTTGTCCTCTCAC
CCTTACTATGACAGCATCCAGTG
CCACTTGGACTTTGCCAGAGA
GATATCCCACACTTCGAGGAG

Yu and Bao, 2008

Huang et al., 2013
Sporer et al., 2011
Proszkowiec-Weglarz et al., 2006
Ojano-Dirain et al., 2007
Ojano-Dirain et al., 2007
Ojano-Dirain et al., 2007
Osselaere et al., 2013
Osselaere et al., 2013
Osselaere et al., 2013
Ojano-Dirain et al., 2007
Osselaere et al., 2013

HSP70: heat shock protein 70; Bcl-2: B-cell lymphoma-2; AMPK: AMP-activated protein kinase; ANT: adenine nucleotide translocator; PGC-1: peroxisome proliferator-activated receptor coactivator-1; COXIII: cytochrome oxidase III; HIF-1: hypoxia-inducible factor 1, subunit alpha; HMOX:
heme-oxigenase; XOR: xanthine oxidoreductase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HPRT1: hypoxanthine phosphoribosyltransferase
1.
Table 3
Effects of NAC on growth performance of broilers (from day 135 of the age).
Thermoneutral

ADFI (g/d)
ADG (g/d)
Feed/gain

Heat stress

SEM

Basal diet

NAC

Basal diet

NAC

63.6
37.0
1.72b

69.7
40.1
1.74b

45.0
24.6
1.83a

47.4
26.8
1.76b

2.56
1.62
0.013

P-value
Temperature

Diet

Interaction

<0.001
<0.001
0.013

0.043
0.053
0.126

0.354
0.747
0.014

a,b
means with different letters in the same row differ signicantly at P < 0.05.
ADFI: average daily feed intake; ADG: average daily gain.

2.9. Statistical analysis

Results were expressed as mean values with pooled SEM. The data were analysed using the General Linear Model procedure in SPSS17.0 (SPSS Inc., Chicago, IL, USA) in a 2 2 factorial arrangement with diet and temperature as the main effects.
Mortality data were subjected to arcsine square root transformation before analysis (Loetscher et al., 2013). The differences
among treatments were evaluated by the least signicant difference Bonferronis multiple comparisons test. Probability
values 0.05 were taken to indicate signicance.
3. Results
3.1. Effect of NAC on the growth performance of broilers
The growth performance of broilers was summarised in Table 3. Broilers exposed to the heat stress had lower (P < 0.05)
ADFI and ADG than broilers under thermoneutral condition. NAC supplementation increased the ADFI (P < 0.05) and ADG
(P = 0.053) of broilers in comparison with the basal diet group. Moreover, a signicant temperature diet interaction was
observed in feed/gain (Table 3), showing that broilers exposed to heat stress and fed with NAC diet exhibited lower (P < 0.05)
feed/gain than those fed the basal diet and exposed to the same heat stress condition. Additionally, under the thermoneutral
condition, NAC supplementation did not affect the broilers mortality rate (4% vs. 4%). However, under heat stress, broilers
receiving NAC diet exhibited lower mortality rate than those receiving basal diet from the day 135 of the age (6% vs. 12%).
3.2. Effect of NAC on the serum hormones of broilers
As shown in Table 4, serum concentration of corticosterone was elevated (P < 0.05), while levels of T3 and T4 were
decreased (P < 0.05) in broilers under heat stress than broilers under thermoneutral condition. Supplementation of NAC had
no effects on the levels of T3 and T4 in broilers compared with the basal diet group. However, broilers fed with NAC diet
exhibited lower (P < 0.05) concentration of corticosterone than broilers fed with the basal diet.
3.3. Effect of NAC on the jejunal intestinal morphology of broilers
The data of jejunal morphology of broilers were presented in Table 4. Heat stress induced a reduction (P < 0.05) in the ratio
of VH to CD and an increase in jejunal crypt depth of broilers in comparison with the thermoneutral group. However, dietary
supplementation of NAC increased (P < 0.05) the ratio of VH to CD in broilers than those in the basal diet group. Additionally,

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D. Yi et al. / Animal Feed Science and Technology 220 (2016) 8392

Table 4
Effects of NAC on serum hormone and jejunal morphology and digestive enzyme activity in broilers.
Thermoneutral

Serum hormone
T3 (ng/ml)
T4 (ng/ml)
Corticosterone (ng/ml)
Jejunal morphology
Villus height (m)
Crypt depth (m)
VH/CD
Jejunal digestive enzyme
AKP (U/g prot)
Lipase (U/g prot)
Trypsine (U/g prot)

Heat stress

SEM

Basal diet

NAC

Basal diet

NAC

1.33
35.7
18.1

1.47
35.6
16.3

0.50
21.6
28.7

0.51
25.5
20.8

947a
194
4.77

880a
181
5.02

687b
221
3.03

172
4.13
4.20

166
4.00
4.48

126
2.96
2.05

P-value
Temperature

Diet

Interaction

0.09
1.49
1.31

<0.001
<0.001
0.001

0.293
0.239
0.017

0.359
0.219
0.114

790ab
210
3.83

28.01
6.49
0.18

<0.001
0.031
<0.001

0.696
0.353
0.038

0.074
0.930
0.250

146
3.32
3.05

5.66
0.15
0.22

0.001
0.001
<0.001

0.271
0.642
0.032

0.103
0.349
0.214

Temperature

Diet

Interaction

a,b
means with different letters in the same row differ signicantly at P < 0.05.
T3 : triiodothyronine; T4 : thyroxine; VH: villus heigh; CD: crypt depth; AKP: alkaline phosphatase.

Table 5
Effects of NAC on jejunal energy status and oxidation and antioxidation related parameters in broilers.
Thermoneutral
Basal diet

Heat stress
NAC

Energy status
47.0
53.6
ATP (g/g)
ADP (g/g)
118
121
157b
181ab
AMP (g/g)
TAN (g/g)
325
362
AEC
0.34
0.33
Oxidation and antioxidation related parameters
CAT (U/mg prot)
1.52
1.91
89.0
85.7
SOD (U/mg prot)
GSH-Px (U/mg prot)
8.18
8.14
0.68b
0.73b
MDA (nmol/mg prot)
H2 O2 (mmol/g prot)
10.67
8.72

SEM

P-value

Basal diet

NAC

37.6
115
205a
344
0.28

44.0
136
180ab
366
0.30

1.74
3.71
5.82
9.72
0.008

0.005
0.390
0.033
0.746
0.023

0.029
0.105
0.969
0.084
0.715

0.858
0.214
0.032
0.872
0.876

1.07
74.6
6.42
1.06a
10.03

1.40
80.6
7.28
0.81ab
9.49

0.08
1.95
0.28
0.04
0.37

0.001
0.011
0.018
0.002
0.933

0.008
0.698
0.435
0.149
0.100

0.828
0.208
0.401
0.029
0.344

a,b
means with different letters in the same row differ signicantly at P < 0.05.
ATP: adenosine triphosphate; ADP: adenosine diphosphate; AMP: adenosine monophosphate; TAN: total adenine nucleotide; AEC: adenylate energy
charges; CAT: catalase; SOD: superoxidase; GSH-Px: glutathione peroxidase; MDA: malonaldehyde; H2 O2 : hydrogen peroxide.
TAN = ATP + ADP + AMP, AEC = (ATP + 0.5 ADP)/(ATP + ADP + AMP)

there was a signicant trend (P = 0.074) of temperature diet interaction in villus height, indicating that supplementation
of NAC mitigated the decrease of jejunal villus height in heat-stressed broilers.

3.4. Effect of NAC on the energy status in the jejunal mucosa of broilers
The energy status in the jejunal mucosa of broilers was shown in Table 5. Heat stress decreased (P < 0.05) the concentrations of ATP and AEC, while increased (P < 0.05) the level of AMP in the jejunal mucosa of broilers compared with broilers
in the thermoneutral group. However, supplementation of NAC increased the ATP level (P < 0.05) and TAN content (P = 0.84)
in jejunum of broilers than those in the basal diet group. Additionally, there was an interaction (P < 0.05) between diet and
temperature in jejunal AMP concentration (Table 5). It was shown that dietary supplement of NAC inhibited the increase of
jejunal AMP in heat-stressed broilers.

3.5. Effect of NAC on the antioxidative capacity in the jejunum of broilers

Heat stress affected the intestinal antioxidative capacity of broilers (Table 5). Heat stress induced the reduction (P < 0.05)
in activities of antioxidative enzymes, such as CAT, SOD, and GSH-Px in the jejunum of broilers as compared with broilers in
the thermoneutral group. However, dietary supplementation with NAC increased (P < 0.05) the activity of CAT in the jejunum
of broilers as compared with broilers fed the basal diet. Additionally, there was a signicant temperature diet interaction
for MDA concentration in the jejunum, showing that dietary NAC inhibited the elevation of MDA level in heat-stressed
broilers.

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89

Table 6
Effects of NAC on gene expressions in jejunum of broilers.
Thermoneutral

HSP70
Bcl-2
Caspase-3
AMPK
ANT
PGC-1
COXIII
HIF-1
HMOX
XOR

Heat stress

SEM

Basal diet

NAC

Basal diet

NAC

1.00b
1.00
1.00
1.00b
1.00
1.00
1.00
1.00
1.00b
1.00b

0.58c
1.13
0.89
0.85b
1.17
0.98
1.03
1.01
1.07b
1.15ab

1.62a
0.83
1.30
1.39a
1.16
1.00
0.94
1.02
1.57a
1.57a

0.97b
1.00
1.19
0.95b
1.21
0.90
1.00
1.17
1.24b
1.25ab

0.08
0.04
0.06
0.06
0.04
0.04
0.02
0.05
0.05
0.08

P-value
Temperature

Diet

Interaction

<0.001
0.053
0.006
0.006
0.176
0.631
0.366
0.359
<0.001
0.021

<0.001
0.048
0.283
0.001
0.148
0.412
0.314
0.401
0.039
0.549

0.043
0.764
0.978
0.083
0.413
0.607
0.747
0.491
0.002
0.097

a,b,c
means with different letters in the same row differ signicantly at P < 0.05.
HSP70: heat shock protein 70; Bcl-2: B-cell lymphoma-2; AMPK: AMP-activated protein kinase; ANT: adenine nucleotide translocator; PGC-1: peroxisome proliferator-activated receptor coactivator-1; COXIII: cytochrome oxidase III; HIF-1: hypoxia-inducible factor 1, subunit alpha; HMOX:
heme-oxigenase; XOR: xanthine oxidoreductas.

3.6. Effect of NAC on the jejunal AKP and digestive enzyme of broilers
As indicated in Table 4, broilers in the heat stress group showed lower (P < 0.05) activities of AKP, lipase and trypsine in
the jejunum than those in the thermoneutral group. However, supplementation of NAC increased (P < 0.05) the activity of
trypsine in the jejunum of broilers in comparison with the basal diet group.
3.7. Effect of NAC on the mRNA levels in the jejunum of broilers
Relative mRNA levels of genes associated with intestinal development, energy metabolism, and oxidation were shown in
Table 6. Heat stress induced the downregulation (P = 0.053) of Bcl-2 expression and the upregulation (P < 0.05) of caspase-3
in the jejunal mucosa of broilers in comparison with the broilers under the thermoneutral zone. However, dietary supplementation of NAC increased (P < 0.05) the Bcl-2 mRNA abundance in broilers than those in the basal diet group. In addition,
there were signicant interactions (P < 0.05) between diet and temperature in mRNA levels for HSP70 and HMOX (Table 6).
It was shown that broilers exposed to heat stress and fed with NAC diet exhibited lower mRNA levels of HSP70 and HMOX
than broilers fed the basal diet under the same condition. Similarly, there was a signicant trend of temperature diet interactions in mRNA levels for AMPK (P = 0.083) and XOR (P < 0.097), indicating that dietary NAC decreased the AMPK abundance
and mitigated the increase of XOR mRNA levels in the jejunum of broilers under heat stress.
4. Discussion
Our results showed that heat stress induced adverse effects on growth performance and intestinal function of broilers,
which was consistent with the previous studies (Garriga et al., 2006; Quinteiro-Filho et al., 2010; Sohail et al., 2010; Song
et al., 2013; AI-Fataftah and Abdelqader, 2014). To resist the heat stress in poultry production, nutritional manipulations
are suggested to apply in animal feeding, such as increasing dietary fat and vitamin C contents (Rao et al., 2016), and
maintaining dietary amino acid and electrolyte balance (Ahmad et al., 2008). Given the benecial effects of NAC (0.5 g/kg)
on piglet growth and intestinal function (Hou et al., 2012, 2013; Yi et al., 2016b), herein we investigated the effect of NAC on
alleviating the growth depression and intestine dysfunction induced by heat stress in broilers, which could provide a new
nutritional strategy in protecting birds against hyperpyrexia condition.
It is well known that high temperature condition increases the hypothalamic-pituitary-adrenal (HPA) axis activity and
thus increases the corticosterone level (Lara and Rostagno, 2013; Loyau et al., 2015) with a concomitant decrease in the
concentrations of T3 and T4 in poultry (Sohail et al., 2010). These endocrinological changes caused by chronic heat stress
could reduce growth performance and redistribute the nutrient metabolism toward lipid lipogenesis and proteolysis (Geraert
et al., 1996; Lin et al., 2004, 2006). Specically, the increased corticosterone was reported to act feedback on the hypothalamic
feeding control nuclei that regulated food intake and satisfaction, determining a reduction in food consumption and animals
body weight gain (Quinteiro-Filho et al., 2010). In the present study, dietary NAC reduced the feed/gain and mortality rate of
broilers compared to broilers fed with basal diet under the heat stress, while increased the broilers ADFI in the thermoneutral
group (Table 3), indicating that NAC potentially alleviated the growth depression of broilers reared in the hyperthermia
environment. Moreover, NAC decreased the plasma corticosterone despite no signicant elevations of plasma T3 and T4 of
heat-stressed broilers (Table 4). Though NAC are reported to transport across the blood-brain barrier (Samuni et al., 2013), it
is still unclear how NAC affects the HPA activity and thus inhibits the increase of corticosterone level. Similarly, our previous
study also demonstrated that NAC attenuated the increase in the cortisol concentrations induced by lipopolysaccharide
challenge in piglets (Hou et al., 2013). Furthermore, another indicator of heat or oxidative stress, HSP70 in the intestine of
heat-stressed broilers, was also downregulated by dietary NAC supplementation in comparison with broilers fed with basal

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D. Yi et al. / Animal Feed Science and Technology 220 (2016) 8392

diet (Table 6). Therefore, dietary NAC supplementation could relieve heat stress and thus mitigated the growth depression
of broilers under heat stress partially via reducing the corticosterone level.
Another putative mechanism responsible for the NACs action on heat stress may involve the improvement of intestinal
function, such as intestinal morphology, energy status, antioxidative capacity, and intestinal digestion. A large body of
evidence showed that heat exposure can cause intestinal dysfunction (Mitchell and Carlisle, 1992; Quinteiro-Filho et al.,
2010; AI-Fataftah and Abdelqader, 2014). Findings of study by Lambert et al. (2002) showed that heat stress could impair
intestinal morphology by damaging the intestinal epithelial cells and increasing villus tips sloughing rate. Heat stress also
impaired intestinal villus-crypt system and decreased villus height and villus surface area (AI-Fataftah and Abdelqader, 2014).
There are some explanations for harmful effects of heat stress on broilers intestines. One is that chronic hot conditions
induces intestinal inammatory response and oxidative stress that generates pro-inammatory cytokines and reactive
oxygen species (ROS), and thus results in intestinal injury (Bouchama and Knochel, 2002; AI-Fataftah and Abdelqader, 2014).
However, our results indicated that dietary NAC could attenuate the adverse effect of heat stress on intestinal morphology
(Table 4). Given the antioxidative and anti-inammatory effects of NAC on animals (Hou et al., 2013; Samuni et al., 2013; Yi
et al., 2014), we suggest that NACs improvement on intestinal morphology of heat-stressed broilers may be attributed to
enhancing intestinal immune function and antioxidative capacity. Additionally, NAC also regulated the expression of Bcl-2,
a biomarker of enterocyte proliferation, and thus it is reasonable to suggest that enterocyte growth may be involved in NACs
action on intestinal integrity.
In addition to damaging intestinal morphology, heat stress also altered the intestinal absorption function by affecting the
activities of digestive enzymes, such as trypsine, lipase, and amylase. However, results are not consistent on the alterations
of digestive enzymes by different stress. Hu and Guo (2008) found that corticosterone treatment increased the activities of
intestinal digestive enzymes in broilers, which was similar with the study of Pinheiro et al. (2004) using a feed restriction
model. On the contrary, Ruan and Niu (2001) reported that total proteolytic enzyme, lipase, and amylase activity were
decreased in heat-stressed broilers. The divergence among these studies may be attributed to the stress model and duration.
Our results, which indicated the activities of trypsine and lipase were decreased in the jejunum of broilers after 4 weeks of
cyclic heat exposure (Table 4), are in line with the results of Ruan and Niu (2001). Moreover, dietary NAC supplementation
increased the activity of trypsine, whereas did not affect activities of the lipase and AKP in broilers under heat stress. To our
best knowledge, the current study is the rst report regarding the benecial effects of NAC on intestinal enzyme activities. The
possible explanation for increased trypsine activity may be due to the improvement of intestinal morphology and integrity
by NAC supplementation.
Furthermore, heat stress was shown to cause an increase in oxidative stress and an imbalance in antioxidant status (Lara
and Rostagno, 2013). It is known that the excessive ROS and related peroxides induced by heat stress can cause intestinal
oxidative injury, including lipid oxidation and oxidative damage to protein and DNA (AI-Fataftah and Abdelqader, 2014;
Mujahid et al., 2007). However, tissues and cells possess defence mechanisms to detoxify ROS by radical scavengers, such
as SOD, CAT, and GSH-Px (Wu et al., 2004). These antioxidant enzymes can cooperatively convert ROS into water and O2 .
In the present study, the activities of SOD, CAT, and GSH-Px in the jejunum were substantially lower in broilers under heat
stress, indicating that oxidative stress occurred in the intestine. This result was also substantiated by the evidence that the
concentration of MDA, an indirect parameter of lipid peroxidation and overproduction of ROS, was increased in the jejunum of
broilers in the heat stress group than broilers in the thermoneutral group. The other convincing evidence was that heat stress
also induced the upregulation of HMOX and XOR (Table 6), which are a sensitive marker of oxidative injury and an enzyme
associated with the synthesis of ROS, respectively (Osselaere et al., 2013). It is noteworthy that dietary NAC supplementation
increased the activity of CAT, reduced mRNA level for HMOX, and inhibited the increases of MDA concentration and XOR
mRNA level in the jejunum of broilers under heat stress. NAC was reported to not only directly react with oxidants and
protect cell from oxidative damage, but also indirectly exert antioxidative effects by increasing the synthesis of glutathione
(Hou et al., 2013). Thus, NAC can protect the intestine from oxidative damage by sparing antioxidative enzymes and possibly
by increasing glutathione content and regulating HMOX and XOR expression.
The last but important nding of the present study is that dietary NAC supplementation improves intestinal energy status
in heat-stressed broilers. Previous studies showed that stressors could cause mitochondrial dysfunction and impairs energy
metabolism in tissues (Hou et al., 2011; Kikusato and Toyomizu, 2013). Our results also showed that heat stress induced
the reduction of ATP content and elevation of AMP content in the jejunum (Table 5). Given that the energy charge of the
adenyl pool is considered as a more sensitive index of the energy state in a tissue than the level of a single nucleotide (Hou
et al., 2011), we measured the energy charge in the jejunum and observed that jejunal AEC was decreased in the heatstressed broilers. Additionally, mRNA for genes associated with energy metabolism, such as AMPK, PGC-1, ANT, and COXIII
were also determined. AMPK is an energy sensor and is upregulated as elevating ratio of AMP/ATP. PGC-1 can upregulate
mitochondrial transcription factor A, whereas ANT is reported to exert a key metabolic control over mitochondrial energy
production, and COXIII is responsible for modulating proton pumping and electron transport through the redox centers
(Ojano-Dirain et al., 2007). In the present study, NAC supplementation increased the concentrations of ATP and TAN, whereas
inhibited the increases of AMP level and AMPK mRNA abundance in the jejunum of heat-stressed broilers, indicating that NAC
could modulate the adenine nucleotide pool via regulating AMPK expression. Alternatively, NAC could act as an antioxidant
by scavenging enterocyte ROS induced by heat stress and inhibit the enzyme complexes of mitochondrial electron transport

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91

chain (Zhang et al., 1990). Therefore, these benecial effects of NAC on intestinal energy metabolism may also be related to
its capacity of scavenging ROS.
Conclusions
Dietary supplementation of 1 g/kg NAC improved the growth performance, intestinal morphology and absorptive function, maintained intestinal energy metabolism, and mitigated intestinal oxidative stress in the heat-stressed broilers.
Improving the intestinal function may be an effective approach to partially attenuate the detrimental effects of heat stress
on birds health and performance.
Conict of interest
The authors declare that they have no conict of interest.
Acknowledgements
This work was jointly supported by National Key Technology R&D Program of China (2012BAD39B04) and Hubei Provincial
Key Project for Scientic and Technical Innovation (2014ABA022).
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