Chronic Heat Stress Damages Small Intestinal Epithelium Cells

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Agricultural and Environmental Chemistry

Chronic Heat Stress Damages Small Intestinal Epithelium Associated with AMPK Pathway in Broilers he xiaofang, Zhuang Lu, Bingbing Ma, Lin Zhang, Jiaolong Li, yun jiang, Guanghong Zhou, and Feng Gao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02145 • Publication Date (Web): 28 Jun 2018 Downloaded from http://pubs.acs.org on July 1, 2018

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Journal of Agricultural and Food Chemistry

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Chronic Heat Stress Damages Small Intestinal Epithelium Associated

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with AMPK Pathway in Broilers

3 †

†

†

†

†

‡

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Xiaofang He , Zhuang Lu , Bingbing Ma , Lin Zhang , Jiaolong Li , Yun Jiang , Guanghong

5

Zhou and Feng Gao*

†

†

6 7

†

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and Safety Guarantee of Jiangsu Province; Jiangsu Collaborative Innovation Center of Meat

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Production and Processing, Quality and Safety Control, Nanjing Agricultural University, Nanjing

College of Animal Science and Technology; Key Laboratory of Animal Origin Food Production

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210095, P. R. China.

11

‡

Ginling College, Nanjing Normal University, Nanjing 210097, P.R. China

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* Corresponding author: Feng Gao

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Full address: College of Animal Scie nce and Technology, Nanjing Agricultural University. No. 1

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Weigang, Nanjing, 210095, P.R. China

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Tel.: +86-25-84399007

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Fax: +86-25-84395314

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E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT: Heat-stressed broilers usually reduce their feed intake, leading to energy imbalance

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and disturbing the homeostasis in the small intestine. This study was aimed to explore heat

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stress-mediated physiological features that may ascribe to impairments in intestinal tract of

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broilers. The results revealed that heat exposure increased the activities of trypsin and

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Na /K -ATPase, while decreased the activities of amylase, lipase and maltase, as well as the

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proliferating cell nuclear antigen cells in the jejunum after 14 d of heat exposure. Meanwhile, heat

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stress upregulated the mRNA expression of AMPK α1, LKB1 and HIF-1α, and protein expression

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of p-AMPKα

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conclusion, chronic heat exposure impeded the development of digestive organs, disordered the

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activities of intestinal digestive enzymes, impaired the intestinal epithelial cells by increasing the

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cell apoptosis and declining cell proliferation, which might be correlated with the AMPK signaling

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pathway. Additionally, heat stress upregulated the genes expression of HIF-1α, which indicated

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that heat stress may disturb the homeostasis in the intestine.

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KEYWORDS: broiler; chronic heat stress; small intestine; epithelial cells

+

+

Thr172

and p-LKB1

Thr189

in the small intestine after 7 or 14 d of heat exposure. In


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INTRODUCTION

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The climate warming is unequivocal, and will greatly impact animal health, either directly or

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secondarily. The disadvantages of heat stress are readily apparent on the poultry industry,

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including compromised growth performance, lower feed efficiency, upregulated or dysregulated

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expression of heat stress-related genes, as well as some other abnormal physiological reactions.1-3

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In the commercial poultry industry, chickens are more susceptible to high temperature currently

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because of sustainable selection of fast-growing traits in the past few decades. When the ambient

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temperature exceeds the upper limit of the broiler’s heat neutral zone, it will induce heat stress, the

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body temperature will increase, result in asthmatic and anorectic. Moreover, the intestine is the

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most important organ to absorb nutrients and defense pathogens in animal body.4 Heat stress

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impairs the epithelial cells via shortening the length of villus and deepening the depth of crypt, 5-7

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then reduces average daily feed intake, increase average daily feed to gain ratio, which finally

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declines weight gain.8

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The intestine, as a gatekeeper, plays an important role in secreting signaling molecules,

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modulating the digestion and absorption to control the homeostatic environment.9,10 About one

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third of blood flows through broiler’s intestine under normal condition. However, more blood

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flows to broiler’s skin to accelerate the heat dissipation when the broiler suffered heat stress. At

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the same time, blood supply to the intestinal tissue is reduced to maintain the necessary blood

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supply to heart and brain in heat stress mediated broilers. Such reduced blood supply causes

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ischemia and hypoxia in the intestinal tissue, leading to abnormal digestion and absorption,

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causing intestinal epithelium injuries and energy imbalance.11 As a critical transcriptional factor,

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hypoxia-inducible factor-1α (HIF-1α) involves with body hypoxia, which is regulated by cellular



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O2 concentration. When the epithelium is in hypoxic status, it will upregulate the expression level

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of HIF-1α.12,13

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The AMP-activated protein kinase (AMPK) is generally considered regulating energy

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metabolism in the body, which is a major kinase to maintain the balance of ATP in vivo. The

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activation of AMPK is controlled by the ratio of AMP/ATP in vivo. In addition, the AMPK could

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promote mitochondrial health and homeostasis, associated with cell growth and apoptosis.14-16

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Because of the decreased feed intake together with increased maintenance costs, broilers subjected

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to heat stress would experience energy stress.17 When organs are in stress state, the AMPK will be

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activated and enhanced by phosphorylating multiple substrates, which may be involved in many

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metabolism pathways and processes.18-20 What’s more, tumor suppressor liver kinase B1 (LKB1;

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also known as serine/threonine kinase 11, STK11) as an AMPK upstream kinase, has greatly effect

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on activation of AMPK, when the AMPK is respond to the increased AMP:ATP ratio in the

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intracellular.21 The LKB1-AMPK pathway is capable of sensing the nutrients and energy status,

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which is eventually maintains energy homeostasis. However, the role of APMK pathway in the gut

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is associated with heat stress remains obscure.

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Our previous study found that chronic heat exposure impairs the intestinal health via

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shortening the height of villus and deepening the depth of crypt.7 Therefore, it is necessary and

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urgently to explore the biological mechanisms by how heat stress impairs the intestinal epithelial.

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The objective of this study was to reveal the molecular mechanisms of chronic heat exposure

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effects on small intestinal epithelium cells, nutrients digestibility and development of digestive

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organs, activities of digestive enzymes, as well as the mRNA and protein expression of AMPKα1,

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LKB1 and HIF-1α.


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MATERIALS AND METHODS

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Broilers’ Management and Sample Collection. All experimental trials including the usage of

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broilers were consult to the Animal Care and Use Committee of Nanjing Agricultural University,

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Nanjing, P. R. China. Two hundred newly-hatched male Arbor Acres (AA) broilers were

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purchased from a commercial hatchery and kept in battery brooders. From 1 d to 27 d of age, the

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broilers were acquired commercial standard feeding and raising managements.

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At 28 d of age, a total of 144 chicks with an average initial body weight of 1.28 ± 0.05 kg were

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picked out from the flock and then were allocated into three groups. Each group includes 6 cages,

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each cage consists of 8 chickens. The three groups containing normal control (NC, 22°C), heat

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stress (HS, 32°C), and pair-fed (PF, 22°C) group. Chickens in NC and HS groups were provided

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ad libitum access to diet. In pair-fed (PF) group, birds were kept at 22°C, received the amount of

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diet same with the diet consumed in the previous day of the HS group each day. In order to better

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apprehend the heat stress-mediated impairment on small intestinal epithelium cells obviate the

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factor of reduction in feed intake, we set the PF group. All broilers were fed with common

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commercial grower diet, and allowed free access to water, provided 24 h light throughout the

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experimental period. All groups were kept at 55 ± 5% of relative humidity.

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Sample Collection and Preparation. After 7 d and 14 d of heat exposure, two broilers nearly

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the average weight were randomly taken out from each cage and slaughtered via exsanguination.

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Collected the blood samples, separated the serum samples, stored the serum samples at -20°C for

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next step analysis.

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Dissection Procedure. First, isolated and weighed the liver and pancreas. After getting rid of all

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the fillings and attached materials of the proventriculus and gizzard, we recorded the weights, and



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then gingerly separated the intestine into the duodenum, jejunum, and ileum on the ice-cold. The

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chyme of the duodenum, jejunum and ileum were squeezed out, kept at -20°C for analysis the

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activities of digestive enzymes. Then, we measured the weights and lengths of the duodenum,

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jejunum, and ileum. Later on, cut open the duodenum, jejunum and ileum, washed the mucosa

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with ice-cold saline, then collected the mucosa by scraping gently with a clean microscope slide,

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stored at -80°C for subsequent analysis.

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Proliferating Cell Nuclear Antigen Immunohistochemistry. Proliferating cell nuclear antigen

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(PCNA) positive cells were determined by utilize the anti-PCNA antibody of mouse monoclonal

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(BM0104; Boster, Wuhan, China),22 employed a diaminobenzidine (DAB) staining kit (K5007;

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Angle Gene Bioengineering Co., Ltd., Nanjing, China), referred to the manufacturer’s instructions.

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Once immunohistochemical stained, captured the duodenal and jejunal sections by employing

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DP12 CCD digital camera (Scope.A1, Carl Zeiss, Germany) with a final magnification of × 400.

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Then analyzed these images through the Image-Pro Plus 6.0 software (Media Cybernetics Inc.,

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MD, USA). We chose well-oriented villus in the duodenal and jejunal section and estimated cells

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in the middle portion. The number of PCNA positive cells divided by the total number of cells

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counted took as the percentage of PCNA positive cells on each villus (multiplied by 10000).

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Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay.

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TUNEL assay was conducted referring to the manufacturer’s instruction of the Apoptosis

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Detection Kit (Boster, Wuhan, China). Briefly, dewaxed the jejunal paraffin sections with 100%

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xylene, and rehydrated in following changes of 100%, 95%, 85% and 75% ethanol. Then

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quenched the activity of endogenous peroxidase in 3% H2O2 with distilled water at 37°C for 10

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min, incubated the sections with proteinase K diluted 1:200 in TBS at 37°C for 5-10 min in a


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humid chamber. Added a labeling mixture including digoxin-dUTP in Terminaldeoxynucleotidyl

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Transferase (TdT) enzyme buffer to the sections and incubated at 37°C for 2 h. After washings

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with TBS three continuous times for 2 min, enclosed the sections with anti-digoxin-biotin

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conjugate diluted 1:100 in blocking regent and incubated for 30 min at 37°C. Subsequently,

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incubated the tissues for 1 h at 37°C with streptavidin- biotin-complex (SABC) diluted 1:100 in

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TBS. Labeling was visualized with 3’3’-diaminobenzidene. After that, counterstained the sections

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with haematoxylin. The negative control was done in the same way, excepting omit TdT enzyme

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buffer for incubation.

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Analyzing Activities of Digestive Enzymes. Referring to the approach of Gao et al,23 frozen

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intestinal digesta, mucosa and pancreas (0.50 g) was homogenized in 4.5-mL ice-cold saline, and

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kept the supernatant preparing for measurement of the activities of digestive enzymes. Employing

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the reagent box of Nanjing Jiancheng Biochemical Institute (Nanjing, China) to determine the

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activities of digestive enzymes, containing intestinal lipase (Enzyme Commission [EC]3.1.1.3),

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Na+/K+-ATPase (EC 3.6.3.9), amylase (EC 3.2.1.1), trypsin (EC 3.4.21.4), surcrase (EC 3.2.1.48),

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maltase (EC 3.2.1.20), were analyzed as described by previous study.44 The concentrations of

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protein were detected via employing the commercial kit (G-250, Jiancheng Bioengineering Co.,

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Ltd., Nanjing, China), standardized with BSA.

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RNA Extraction and Real-Time Quantitative PCR. Utilizing the TRIzol reagent (TaKaRa,

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Dalian, China) to obtain the Total RNA from frozen samples of intestinal mucosa. We identified

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the purity and quality of total RNA spectrophotometrically via usage of OD260 and OD280

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measurements (Thermo Scientific, Wilmington, DE, USA). We chose the samples with the

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260/280 ratios of 1.8 to 2.0 and the 260/230 ratios of 2.0 to 2.2 for succeeding PCR reactions. And



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then handled the total RNA with DNase I (TaKaRa, Dalian, China) for getting rid of DNA. The

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500 ng RNA was reverse transcribed to complementary deoxyribonucleic acid (cDNA) through

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employing the Prime Script RT Master Mix kit (TaKaRa, Dalian, China). Estimated the mRNA

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expression of target genes via real-time PCR. We carried out Real-time fluorescence quantitative

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PCR utilizing the SYBR Green dye approach and the SYBR premix Ex Taq reagent kit (Dalian,

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China)

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glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as a housekeeping gene to normalize

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the expression data of the target gene, which was described by Azad et al.24 The target genes and

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the housekeeping gene were synthesized by Sangon and applied for real-time PCR (Table 1). The

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reaction system of 20 µL comprised 10 µL SYBR Premix Ex Taq, 0.4 µL ROX Reference, 1.0 µL

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cDNA templates, 0.25 µL of each primer (10 µM) and 8.1 µL of RNA enzyme-free water. The

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PCR program was guided as follows: 95°C for 30 s, 40 cycles of 95°C for 5 s and 60°C for 34 s.

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all genes were repeated trice. We calculated the relative gene expression (arbitrary units) utilizing

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the 2-∆∆Ct method,25 and normalized the relative abundance to the control (as 1).

on

a

7500

Real-time

PCR

instrument

(ABI,

USA).

We

Applied

the

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Western Blot Analysis. We Ground the frozen samples of jejunal mucosa into powder in liquid

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nitrogen with a mortar and pestle, and gathered the supernatants via centrifuging the homogenates.

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Determined the concentration of protein via utilizing the BCA protein assay kit (Sangon Biotec.,

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Shanghai, China). Total cellular protein (20 µg) was exposed to a sodium dodecyl

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sulfate−polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. Membrane

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blocking was carried out through applying 5.0% fat-free milk for 1 h. After washing, we incubated

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the membrane with primary antibodies overnight at 4°C with gentle shaking. Then, incubated the

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membrane with a second horseradish peroxidase-conjugated antibody (1:3000, Cell Signaling


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Technology Inc., Beverly, MA, USA) at room temperature for 3 h. We purchased the specific

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primary antibodies of anti-pLKB1Thr189, anti-p-AMPKαThr172 and β-actin (1:1500) from Cell

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Signaling Technology (Beverly, MA, U.S.A.), and validated formerly for practice on chicken

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samples.26 The membrane was developed utilizing Super Signal West Pico chemiluminescent

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substrate (Thermo-Scientific, Waltham, MA, USA) and exposed to Kodak film. We quantified the

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band densities by means of the Scion Image software (Scion Corporation, Frederick, MD, USA).

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Statistical Analyses. We employed One-way analysis of variance for the data applying the

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statistical software SPSS 19.0 (SPSS Inc., Chicago, USA). Differences among the means were

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verified utilizing Duncan’s multiple-range tests. Descripted the data as means ± SEs, and set

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significance at P < 0.05. Analyzed the data with the cage as the experimental unit (n=6).

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RESULTS

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Development of Digestive Organs. As Table 2 shows, after 7 d of heat exposure, the relative

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weights of the duodenum and ileum were significantly decreased in the HS group than those in the

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other two groups (P < 0.05). Compared with those of the NC group, the relative weights of

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jejunum showed a decreased trend (P = 0.062). After 14 d of heat exposure, the relative weights of

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proventriculus were obviously amplified both in the HS and PF group than that in the NC group

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(P < 0.05). Additionally, the relative weights of duodenum and jejunum in the PF group were

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significantly higher in comparison with the other two groups.

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There was no significant difference among all three groups in the relative length of intestinal

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tract after 7 d of heat exposure, whereas the relative length of duodenum, jejunum and ileum in the

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HS group was notably lengthened compared with the NC and PF groups after 14 d of heat

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exposure ((P < 0.001, Table 3).



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Activities of Digestive Enzymes in Digesta and Brush Border. Table 4 displays that after 7 d

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of heat exposure, the activities of trypsin in the pancreas and duodenum of the HS group showed a

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significant lower compared with those of the NC group. Additionally, the activities of lipase in the

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duodenum and jejunum of the HS group were significantly lower (P < 0.05) than those of the

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other two groups. However, the activities of trypsin in the jejunum of the HS group and PF group

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were higher than that of the NC group (P < 0.001). 14 days of heat exposure lowered (P < 0.05)

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the activities of lipase and amylase in the pancreas of the HS group, whereas the activities of

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trypsin in the duodenum and jejunum of the HS group were significantly increased (P < 0.05)

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compared with those of the other two groups.

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Table 5 shows that 7 days of heat exposure significantly decreased the activities of maltase in

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the duodenum, as well as amylase in the jejunum compared with those of the other two groups.

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Compared with NC group, the activity of amylase in the duodenum (P = 0.080) and sucrose in the

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jejunum (P = 0.075) showed a decreasing trend in the HS group. No significant difference in the

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activities of Na+/K+-ATPase were observed among the three groups after 7 d of heat exposure.

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However, the activities of Na+/K+-ATPase in the jejunum were significantly higher (P < 0.05) in

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the HS group than that of the other two groups after 14 d of heat exposure.

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Immunohistochemical Assessment of PCNA. Figure 1A and 1B show that there were no

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significant differences of the PCNA-positive cells in the duodenum either after 7 d or 14 d of heat

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exposure among the three groups. 14 days of heat exposure significantly decreased (P < 0.05) the

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percentage of PCNA-positive cells of the jejunal villus in HS group compared with NC and PF

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groups. The pictures of PCNA-positive cells of intestinal tract after 7 d and 14 d heat exposure are

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shown in the Figure 2 and Figure 3, respectively.


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The Jejunal Cell Apoptosis by TUNEL assay. Figure 4 shows that the nuclei of

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TUNEL-positive cells were stained green in the three groups. These positive cells were mainly

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allocated in the apical region of villus. In comparison with the NC group, more TUNEL-positive

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cells were observed in the HS group (Figure 4B and 4D) after 7 d and 14 d of heat exposure.

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Gene Expressions. After 7 d of heat exposure, the mRNA expressions of AMPKα1 and LKB1

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in the duodenum of the PF group were significantly higher than those of the NC group (Figure 5A,

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P < 0.05). No significant difference was observed in the mRNA expressions of AMPKα1, LKB1

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and HIF-1α between the HS and NC groups in the jejunum and ileum after 7 d of heat exposure.

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After 14 d of heat exposure, the mRNA expressions of AMPKα1 and LKB1 in the jejunum, as well

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as HIF-1α in the intestinal tract of the HS group were significantly higher than those of the NC

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group (Figure 5D, 5E and 5F, P < 0.05). No significant difference in the mRNA expressions of

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AMPKα1 and LKB1 was detected among the three groups in the duodenum and ileum after 14 d of

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heat exposure.

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Protein Expressions. After 7 d of heat exposure, the phosphorylation levels of AMPKα1 and

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LKB1 in the HS group and PF group were significantly increased than those of the NC groups

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(Figure 6A,6B, P < 0.05). After 14 d of heat exposure, there was no significant difference in the

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phosphorylation levels of AMPKα1 between the NC and HS groups (Figure 6C), whereas the

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phosphorylation levels of AMPKα1 in the PF group were significantly amplified than those of the

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other two groups (Figure 6C, P < 0.05), and the phosphorylation levels of LKB1 in the HS group

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and PF group were notably elevated than those of the NC group (Figure 6D, P < 0.05).

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DISCUSSION

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Animal’s growth is considered particularly depending on the degree of development of the



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digestive organs. Pancreas, liver and small intestine as the main digestive organs, are closely

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related to various physiological responses. In this experiment, it is significant that chronic heat

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exposure obviously discouraged the relative weights of duodenum and ileum. Besides, the relative

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weight of jejunum in HS group has a tendency to decrease after 7 d of heat exposure, and these are

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consistent with previous researches.5,6 Moreover, the relative weight of some organs in PF group

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was significantly increased compared to the other two groups, and the relative length of intestinal

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tract in the HS group was increased after 14 d of heat exposure. It is suggested that heat stress

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could reduce the weight of digestive organs which contributed to the shorter villus of morphology

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even the length of intestinal was significantly higher. On the one hand, both the HS and PF group

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suffered the insufficient feed intake, the BW of birds after 14 d of heat exposure in the HS group

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were lower than that in NC group, which resulted in the relative length of intestine and relative

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weight of organs in the HS group were significantly increased. On the other hand, there may be a

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mechanism of compensation to cope with heat stress induced impairment to the body.

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Animal exposed in heat stress involves many disadvantages, such as reducing energy intake,

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decreasing digestibility of nutrients, disordering enzyme activity or endocrine status, and elevating

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maintenance requirements,8 which would lead to a net decrease in nutrient/energy availability for

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production.27,28 Intestinal enzymes mainly secreted by the pancreas, undertake the final phase of

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digestion. These enzymes exert the critical function on digesting starch, sucrose, fats and protein.

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In the present study, we found that the activity of amylase, lipase and trypsin enzymes in the

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intestinal tract of HS group broilers were irregular, especially the activities of trypsin and amylase

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enzyme were enhanced in the jejunum, which is coincide with previous reports and indicates that

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the altered activities of digestive enzymes in the intestine may contributed to chronic heat


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exposure.29 Our previous study suggested that chronic heat exposure induced excessive secretion

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of intestinal hormones such as cholecystokinin and ghrelin,7 both of which can stimulate the

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pancreas releasing more digestive enzymes to the intestine.

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In order for maintaining the structure of intestine to guarantee its function, it is necessary to

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keep the rapid regeneration of intestinal mucosa, and cell proliferation.30 The cell proliferation was

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considered happening in the crypt and along the villus of chicken intestinal epithelium,31 after that,

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these cells differentiate in apical microvilli.32 As a coenzyme protein of polymerase A, PCNA is

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necessary for cell synthesis, mainly expressed in the intestinal epithelium area.33,34 In this study,

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chronic heat exposure significantly lowered the percentage of PCNA positive cells of the villus in

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the jejunum of broilers. This PCNA result combined with the augmented apoptosis cells in the HS

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group indicated that chronic heat exposure led to a diminished degree of mitosis and enterocyte

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proliferation. Histologically, structural changes of the intestinal epithelium were observed with a

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decline in cellular proliferation, sloughing of the stratum corneum and apoptosis of underlying

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epithelial layers. Chronic heat exposure might lead to a lengthening of the intestine at the expense

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of thinning the stratum granulosum, suggesting profound alterations in intestinal epithelial

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structure in this situation.

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The structure of AMPK is a trimeric complex, including three subunits, showing differential

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tissue-specific expression and activation. It has been shown that AMPKα1 is distributed

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ubiquitously in the animals. And AMPK signaling pathway carries the important function in

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maintaining energy homeostasis.14 Under energy stress, AMPK is phosphorylated to coping with

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an adverse housing environment and protecting cells from death, and the energy metabolism

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changes from lipids to protein.8,35,36 Some studies have indicated that HIF-1α displayed critical



275

importance in regulation of epithelial integrity and repair processes.37,38 Long-term heat exposure

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often increases the body temperature, and the blood accelerated flows to the surface to dissipate

277

heat, induce ischemia and hypoxia in the intestinal tract.8,39 In the present study, High temperature

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upregulated the mRNA expression of HIF-1α, which is consistent with previous study.40

279

Meanwhile, both HS group and PF group increased the phosphorylation levels of AMPKα1 and

280

LKB1, and upregulated the mRNA expression of AMPKα1 and LKB1 after 7 days or 14 days heat

281

exposure, which suggested that either heat stress or feed restriction, both might result in nutrient

282

deficient and energy imbalance. Besides, previous study demonstrated that HIF-1α may be

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activated by apoptosis which partially dependent on AMPK.41 What’s more, due to PF broilers

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suffering feed restriction, although in thermal neutral, the energy demand could not be fulfill, thus

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the increased levels of phosphorylated AMPK and LKB1 were as an adaptation to conserve energy

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for survival. Numerous researches demonstrated that animals suffered severe feed restriction

287

might induce energy imbalance and cell apoptosis.42,43

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In conclusion, chronic heat exposure attenuated the development of digestive organs, disordered

289

the activities of digestive enzymes, impaired the intestinal epithelial cells by increasing the cell

290

apoptosis and declining cell proliferation. What’s more, heat stress upregulated the genes

291

expression of AMPKα1, LKB1 and HIF-1α, as well as protein expression of p-LKB1 and

292

p-AMPKα, which might be attributable to the energy insufficient in the intestinal tract.

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AUTHOR INFORMATION

294

Corresponding Author

295

*Tel: +86-25-84399007. Fax: +86-25-84395314. E-mail: gaofeng0629@ sina.com.

296

ORCID*


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Xiaofang He:

0000-0002-3062-5170

298

Funding

299

This study was financially supported by the National Key Research and Development Program of

300

China (2016YFD0500501) and Three Agricultural Projects of Jiangsu Province (SXGC2017281).

301

Notes

302

The authors declare no competing financial interest.

303

ABBREVIATIONS USED

304

BW, body weight; AMPK, AMP-activated protein kinase; LKB1, liver kinase B1; HIF-1α,

305

Hypoxia-inducible factor-1 α; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCNA,

306

Proliferating cell nuclear antigen; ATP, adenosine triphosphate; ADP, adenosine diphosphate.



307

REFERENCES

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426

Page 22 of 38

Table 1. Primers Sequences for Real-Time Quantitative PCR analysis genes1

LKB1

primer sequences (5′→3′)

forward: TGAGAGGGATGCTTGAATACGA

amplicon

GeneBank

accession

size (bp)

no.

138

NM_001045833.1

125

NM_001039603.1

117

NM_204297.1

113

NM_204305.1

reverse: ACTTGTCCTTTGTTTCTGGGC AMPKα1

forward: ATCTGTCTCGCCCTCATCCT reverse: CCACTTCGCTCTTCTTACACCTT

HIF-1α

forward: AACTCCTGGGTCGTTCAATCTATG reverse: GCATTCTGTATTGTCCCGTAGTCA

GAPDH

forward: GAGGGTAGTGAAGGCTGCTG reverse: CATCAAAGGTGGAGGAATGG

427

1

428

HIF-1α, Hypoxia-inducible factor-1 α; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

LKB1, liver kinase B1; AMPKα1, adenosine 5’-monophosphate-activated protein kinase α1;


Page 23 of 38


429

Table 2. Effects of Chronic Heat Stress on the Relative Weight of Digestive Organs in

430

Broilers Treatmentsa NC

HS

SEM

P-value

PF

after 7 d of heat exposure liver, g/kg

27.22

26.93

28.19

0.61

0.698

pancreas, g/kg

2.62

2.49

2.65

0.06

0.560

proventriculus, g/kg

4.90

4.53

4.97

0.46

0.454

gizzard, g/kg

8.56b

9.53ab

9.77a

0.21

0.050

duodenum, g/kg

7.54a

6.47b

6.29b

0.19

0.012

jejunum, g/kg

13.89

12.40

13.21

0.25

0.062

ileum, g/kg

10.50a

9.48b

10.26a

0.16

0.026

after 14 d of heat exposure liver, g/kg

23.90

26.15

22.62

1.39

0.184

pancreas, g/kg

2.62

2.44

2.72

0.06

0.871

proventriculus, g/kg

3.95b

4.84a

5.35a

0.46

0.009

gizzard, g/kg

8.20b

9.00ab

9.86a

0.54

0.006

duodenum, g/kg

4.98b

6.17a

5.43b

0.34

0.005

jejunum, g/kg

10.78

10.58

11.81

0.69

0.093

ileum, g/kg

9.25

9.69

9.73

0.64

0.733

431

The results are presented as the mean values and standard error (n=6). Mean values with different

432

letters were significantly different (P < 0.05). aNC, normal control group; HS, heat-stress group;



433

PF, pair-fed group.


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434

Table 3. Effects of Chronic Heat Stress on the Relative Length of Intestinal Tract in

435

Broilers Treatmentsa NC

HS

SEM

P-value

PF

after 7 d of heat exposure duodenum, cm/kg

17.23

17.92

17.93

0.27

0.477

jejunum, cm/kg

40.93

42.83

40.36

1.92

0.867

ileum, cm/kg

42.64

41.89

40.16

1.90

0.869

after 14 d of heat exposure duodenum, cm/kg

12.90b

16.37a

13.39b

0.34

Chronic Heat Stress Damages Small Intestinal Epithelium Cells

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