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Bioactive Constituents, Metabolites, and Functions

Effects of dietary L-arginine and N-carbamylglutamate supplementation on intestinal integrity, immune function and oxidative status in intrauterine growth retarded suckling lambs Hao Zhang, Li Dong, Mengzhi Wang, Lihuai Yu, and Hongrong Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00726 • Publication Date (Web): 29 Mar 2018 Downloaded from http://pubs.acs.org on March 30, 2018

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Journal of Agricultural and Food Chemistry

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Effects of dietary L-arginine and N-carbamylglutamate supplementation on

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intestinal integrity, immune function and oxidative status in intrauterine growth

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retarded suckling lambs

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Hao Zhang, †, ‡ Fangfang Zhao, †, ‡ Along Peng, †, ‡ Li Dong,†, ‡ Mengzhi Wang, †, ‡

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Lihuai Yu, †, ‡ Juan J. Loor,¥ and Hongrong Wang*,†, ‡

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†

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Animal Science and Technology, Yangzhou University, Yangzhou 225009, P. R.

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China.

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‡

Laboratory of Metabolic Manipulation of Herbivorous Animal Nutrition, College of

Joint International Research Laboratory of Agriculture ＆ Agri-product Safety,

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Yangzhou University, Yangzhou 225009, P. R. China.

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¥

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Illinois, Urbana, USA

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*Correspondence Hong Rong Wang, College of Animal Science and Technology,

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Yangzhou University, Yangzhou 225009, P.R. China. Tel.: +86-514-87979196; Fax:

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+86-514-8735044; E-mail: [email protected].

Department of Animal Sciences and Division of Nutritional Sciences, University of

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1


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ABSTRACT: This study investigated the effects of dietary L-arginine (Arg) and

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N-carbamylglutamate (NCG) supplementation on intestinal integrity, immune

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function and oxidative status in intrauterine growth retarded (IUGR) suckling lambs.

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A total of 48 newborn Hu lambs of normal birth weight (CON) and IUGR were

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allocated randomly into four groups of 12 animals each: CON, IUGR, IUGR+1% Arg,

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or IUGR+0.1% NCG. All lambs were raised for a period of 21 days from 7 to 28 days

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after birth. The Arg or NCG group exhibited improved (P < 0.05) final body weights

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compared to that of the IUGR group. Compared with the IUGR lambs, the apoptotic

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percentage was lower (P < 0.05) in the ileum of IUGR lambs supplemented with Arg

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and NCG. In addition, compared with IUGR, the concentrations of protein carbonyl

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and MDA were lower (P < 0.05) and the GSH concentration and ratio of GSH/GSSG

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greater (P < 0.05) in the jejunum, duodenum and ileum of IUGR+1%Arg or 0.1%

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NCG lambs. Compared with the IUGR group, the mRNA abundance of MyD88,

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TLR-9, TLR-4, IL-6 and NF-κB was lower (P < 0.05) and the mRNA SOD1, Bcl-2,

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ZO-1, and Occludin greater in the ileum of the IUGR lambs supplemented with Arg

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or NCG. Furthermore, the protein abundance of ZO-1 and claudin-1 in the ileum was

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greater (P < 0.05) in the IUGR+1% Arg or 0.1% NCG lambs. The results show that

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Arg or NCG supplementation improve the growth, intestinal integrity, immune

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function and oxidative status in IUGR Hu suckling lambs.

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KEYWORDS: l-arginine, suckling lambs, intrauterine growth restriction, immune

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function, intestinal integrity, oxidative stress, N-carbamylglutamate.

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INTRODUCTION

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Insufficient delivery and/or availability of nutrients to the conceptus during pregnancy

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can lead to intrauterine growth restriction (IUGR) in animals and human infants. In

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animals experiencing IUGR, neonatal morbidity is greater and the metabolic

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efficiency, meat quality, postnatal growth rate and reproductive performance are

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severely compromised.1 The risk of intestinal disorders also increases in surviving

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infants with IUGR2 , and partly explains the impairment of development and function

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of the intestinal immune response.3,4 All these physiologic consequences of IUGR

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contribute to delayed lamb growth after birth.5 Therefore, research on novel strategies

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to help mitigate IUGR in livestock during the suckling period may have practical

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value.

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The role of L-arginine (Arg) on intestinal mucosal physiology has attracted much

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interest.6 Dietary Arg supplementation can improve function in the intestine of

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weaned pigs,7 enhance immune response in different rat models,8,9 and stimulate the

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growth of intestinal mucosa in newborn piglets.10 Because the activity of

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mitochondrial

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endogenously is markedly low during the suckling period, supplementation of Arg

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during this physiologic state may be a valuable strategy to reduce morbidity.6

N-acetylglutamate

synthase

(NAG)

that

produces

arginine

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As a metabolically stable analogue of NAG, N-carbamylglutamate (NCG) has been

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demonstrated to increase plasma arginine concentration and endogenous arginine

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synthesis by activating carbamylphosphate synthase-1 and pyrroline-5-carboxylate

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synthase in the intestine.6 In addition, NCG supplementation increased serum arginine 3


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concentration, pregnancy outcome in rats11 and sheep,12,13and muscle protein synthesis

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in sow-reared piglets14.

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Although Arg plays an important role in neonatal growth and nutrition, there is a

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lack of data about the effects of dietary Arg and NCG treatment during the suckling

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period on intestinal development in lambs that experienced IUGR. The Hu sheep was

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chosen as a model to investigate the role of dietary Arg and NCG due to its

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prolificacy and precociousness.15-17

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MATERIAL AND METHODS

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All trials were conducted in accordance with the law of animal protection approved

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by the Guide for the Care and Use of Laboratory Animals prepared by the Ethics

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Committee of Yangzhou University (SXXY 2015-0054).

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Milk Replacer Diets. The nutrient content of the milk replacer diets is shown in

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Table 1. Suckling lambs were fed with either one of the following three

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iso-nitrogenous diets from 7 to 28 days after birth: basal diet, basal diet + 1% Arg,

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and basal diet + 0.1% NCG. The doses of supplemental Arg (1%) and NCG (0.1%)

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were chosen according to previous studies with piglets and rats.18-20 The diets were

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formulated to meet the National Research Council (NRC, 2007) nutrient requirements

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for sheep. All diets were energy amounts and equivalent nitrogen. L-alanine was used

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to equilibrate nitrogen. The NCG (purity, 97%) was purchased from Sigma-Aldrich

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Corporate (Louis, Missouri, US). L-alanine and Arg were purchased from Ajinomoto

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(Beijing, China) Company Limited.

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Animal Handling and Treatments. Suckling lambs with birth weight at least 1.5 4



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SD below the average and those with birth weight within 0.5 SD were defined as

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IUGR and normal birth weight (NBW) lambs.21 A total of 48 newborn NBW Hu

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lambs (Jiangyan Experimental Station, Taizhou, Jiangsu, P.R. China) with body

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weight (BW) at 4.09 (SD 0.12) kg and IUGR with BW at 3.51 (SD 0.10) kg

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were used. All lambs were weaned at 7 d of age, and allocated randomly into four

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groups according to their initial body weight: CON (n=12), IUGR (n=12), IUGR+1%

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Arg (n=12), or IUGR+0.1% NCG (n=12). There were 6 males and 6 female lambs in

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each group. Three replications were in each treatment, and four lambs were in each

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replication. Lambs were housed in a 4 × 1 m2 indoor pen in each replicate from 7 to

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28 days after birth.

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The milk replacer (MR) feeding level was adjusted to 2% of the lambs’ live weight

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every 10 days. From 7 to 28 days of age, the MR was fed thrice daily via bottle at

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0700 h, 1300 h, and 1900 h, respectively. The MR was dissolved in warm water

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before feeding to obtain a 40℃ solution containing 16.67 % DM, and the solution was

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then offered to each animal. Lambs had free access to clean water throughout the

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study. Skilled farm staff were in charge of administering MR to exclude daily

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management and handling as a confounding factor. Milk replacer intake was recorded

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daily, and calculated as the difference between the amount offered and refused. The

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average daily DM intake (ADMI) was calculated via multiplying the average daily

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milk intake by its DM content (%).

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Blood Sampling and Analyses. After an overnight fast, samples of blood were

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collected by venipuncture into heparinized tubes at 08:00h on day 28, and placed on 5


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ice until flow cytometry analysis to determine leucocyte concentrations (within 2 h of

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collection). An ADVIA 2120 Hematology System (Bayer HealthCare, Tarrytown, NY)

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was used to obtain the differential leucocyte count. A 200 µL sample of blood from

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each lamb was taken and mixed with 2 mL erythrocytelysate; erythrocyte-depleted

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lymphocytes were suspended in phosphate-buffered saline (PBS, pH=7.4), then

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centrifuged at 1500 × g for 7 min at 4℃. Pure lymphocytes were resuspended at a

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final concentration of 1×106 cells per mL. The lymphocytes were stained with

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fluorescein isothiocyanate (FITC)-conjugated mouse anti-sheep CD4 monoclonal

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antibody (mAb, MCA2213F, IgG2a) and phycoerythrin (PE)-conjugated mouse

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anti-sheep CD8 mAb (MCA2216PE, IgG2a), respectively. All antibodies (AbD

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Serotec, Raleigh, NC, USA) were used at recommended dilutions. The of PE or

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FITC-conjugated isotype antibodies (4ABIO, Beijing, China) served as controls,

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including mouse IgG2a-PE (FMCP002-100) and mouse IgG2a-FITC (FMCF002-100).

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Cells were incubated at 4°C for 30 min, and then resuspended in staining buffer and

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analyzed by flow cytometer (FACS-Calibur; Becton Dickinson, Franklin Lakes, NJ,

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USA) after washing. For each sample, 10,000 data events were collected, and the Cell

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Quest software (Becton Dickinson) used to analyse the data.

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Tissue Sample Collection. Suckling lambs were weighed at day 7 and 28. On day

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28, an intravenous injection of 15 mg/kg BW sodium pentobarbital was used to

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anaesthetize all lambs prior to sacrifice. The kidney, liver, heart and spleen were from

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each lamb were removed and weighed immediately. After the intestinal lumen

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contents were removed, the small and large intestine weights were recorded. A sample 6



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of approximately 2 cm of duodenum, jejunum and ileum was stored in 4%

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paraformaldehyde solution for histological analyses. The remaining ileal tissue was

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rapidly frozen and stored at -80 ℃until further analysis.

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Intestinal Morphology Analysis and Goblet Cell Counting. A graded series of

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ethanol and xylene washings were used to dry the samples of duodenum, jejunum and

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ileum fixed in 4% paraformaldehyde. The dried samples were then embedded in

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paraffin. Five slides with three sections each (5 µm thickness) were prepared from the

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duodenum, jejunum and ileum. A rehydration with graded dilutions of ethanol and

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xylene was used to deparaffinize the samples (5 µm). For intestinal morphology, the

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slides were stained with hematoxylin and eosin by 20 well-oriented villi and crypts

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each section (Optimus software version 6.5; Media Cybergenetics). The villi crypt

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ratio (VCR) was calculated and the goblet cell number per villi measured

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(NIS-Elements BR 2.3; Nikon France SAS). Values from 10 villi from each segment

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of small intestine were obtained and averaged.

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Plasma Cytokine and Immunoglobulin Subset Analyses. The following

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commercial kits were used: TNF-α (R&D Systems, Oxford, UK), IgA (Bethyl Lab.

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Inc., Montgomery, USA), IL-10 (Bio Source/Med Probe, Camarillo, CA), and IL-1β

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(R&D Systems, Oxford, UK). A Bio-Tek synergy HT microplate reader (Bio-Tek

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Instruments, VT) was used to determine the absorbance (450 nm). For IL-1β, IL-10,

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IgA, and TNF-α, the detection limits were 30.0 pg/mL, 8.0 pg/mL, 12.5 ng/mL, and

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7.0 pg/mL, respectively. The inter- and intra-assay variation coefficients were no

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greater than 10 %. 7


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Intestinal Enzyme Activity Measurement. Commercial kits (Jiancheng

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Bioengineering Institute, Nanjing, Jiangsu, China) were used to determine the

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concentrations of protein carbonyls, malondialdehyde (MDA), oxidized glutathione

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(GSSG) and GSH and the activities of the maltase, sucrase, lactase, glutathione

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reductase (GR), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD).

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All results for each sample were normalized to concentration of total protein. The

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bicinchoninic acid protein assay kit (Nanjing Jiancheng Bioengineering Institute,

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Nanjing, Jiangsu, China) was used to quantify the protein concentrations.

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TUNEL Staining. A terminal deoxynucleotidyl transferase-mediated deoxyuridine

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triphosphate nick end labeling (TUNEL) assay with the TUNEL Bright-Green

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Apoptosis Detection Kit (Vazyme Biotech, Nanjing, Jiangsu, China) was used to

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evaluate apoptosis. The ileal specimens were de-waxed and incubated at room

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temperature with 20 µg/mL Proteinase K for 20 min. The terminal deoxynucleotidyl

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transferase (TdT) buffer was used to treat the specimens followed by a thorough

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washing with PBS to stop the reaction. The 4,6-diamidino-2-phenylindole solution

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(Beyotime Institute of Biotechnology, Nantong, Jiangsu, China) was used to stain the

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specimens for 5 min to detect cell nuclei. An LSM 700 confocal laser scanning

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microscope (Carl Zeiss, Oberkochen, Germany) was used to count the number of

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positive cells. The index of apoptosis was calculated as the ratio of apoptotic cells to

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total cells.

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Total RNA Extraction and Real-Time PCR. The TRIZOL Reagent (Invitrogen

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Life Technologies, Gaithersburg, USA.) was used to extract total RNA from frozen 8



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intestinal mucosae. The electrophoresis (A260/A280, Beckman DU-800; Beckman

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Coulter, Inc, Fullerton, CA, U.S.A.) was used to assess the purity and quality of RNA

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samples, respectively. Subsequently, cDNA was synthesized using 1 µg RNA in a

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20-µL reaction system. For real-time PCR, a 1 µL amount of the RT products was

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used. ABI-7900HT instrument (Applied Biosystems, Foster City, CA, USA) was used

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to perform Real-time PCR. The target gene and β-actin expression was detected using

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the SYBR green system (TaKaRa Biotechnology Co. Ltd., Dalian, China). The

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sequence of primers are included in Table 2. A total of 0.2 µL ROX Reference Dye II

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(50×), 5 µL of fresh SYBR® Premix Ex Taq II (Tli RNaseH Plus), 1 µL of RT

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products, 0.8 µL of the primers and 3 µL of diethylpyrocarbonate-treated water were

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in the reaction mixture (10 µL). The PCR protocol used was as follows: 1 cycle (95℃

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30 s); 40 cycles (95℃ 5s, 60℃ 31 s); 1 cycle (95℃ 15s, 60℃ 1 min and 95℃ 15 s).

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The amplification efficiency values were obtained as reported previously.22 All the

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correlation coefficients (r) of the standard curves were no less than 0.99, and the

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values of the amplification efficiency range from 90 to 110%. The amplification

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specificity was identified by performing a melting curve analysis at the end of

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amplification. β-actin was used to normalize the results. Expression of each target

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mRNA was expressed relative to β-actin mRNA. The 2-△△Ct method was used to

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analyse relative gene expression.23 Lastly, each target gene mRNA level was set to 1.0

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in the CON suckling lamb group.

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Western Blotting. Total protein from ileal tissue was extracted with a commercial

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kit (Beyotime Biotechnology, Jiangsu, China) and homogenized according to the 9


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manufacturer’s protocol. The bicinchoninic acid protein assay kit (Pierce, Rockford,

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IL, USA) was used to measure the protein content. The following antibodies were

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used: goat polyclonal anti-claudin-1, goat polyclonal anti-ZO-1 and mouse

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monoclonal anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Western

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blot analysis was performed as previously described.24 The ECL Plus™ Western

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Blotting Detection System (Amersham, Arlington Heights, IL, USA) was used to

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perform the chemiluminescence detection. β-actin was used as the internal protein to

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standardize the relative expression of target proteins. The standardized values were

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used for the comparison of the expression for target protein in groups.

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Statistical Analysis. The SPSS 16.0 statistical software (SPSS, Chicago, IL, USA)

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was used to analyse the data. One-way analysis of variance and Tukey’s post hoc test

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for multiple comparisons were used to determine statistical differences between

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different groups. Differences were considered as significant when P < 0.05.

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RESULTS

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Growth Performance. Compared with CON lambs, the average daily gain (ADG)

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and average daily DM intake (ADMI) were lower (P < 0.05) and the feed conversion

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ratio (FCR) greater (P < 0.05) in IUGR lambs (Table 3). Compared with the IUGR

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lambs, the ADG was greater (P < 0.05) and the FCR lower (P < 0.05) in the Arg- or

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NCG-treated IUGR lambs.

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Organ mass. Compared with the IUGR lambs, the weights of the spleen, liver,

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large intestine and small intestine were greater (P < 0.05) in the Arg- or NCG-treated

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IUGR lambs (Table 4). Compared with the CON lambs, the weights of the heart, liver, 10



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spleen, kidney, large intestine and small intestine were lower (P < 0.05) in the IUGR

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lambs. The weights of the kidney and heart were not significantly different (P > 0.05)

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in the Arg- or NCG-treated IUGR compared with the IUGR lambs.

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Apoptotic Index. Compared with the CON lambs, the apoptotic percentage in the

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ileum was greater (P < 0.05) in the IUGR suckling lambs (Figure 1). In contrast, the

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apoptotic percentage in the ileum was lower (P < 0.05) in the Arg- or NCG-treated

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IUGR lambs compared with IUGR lambs.

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Intestinal Morphology and Goblet Cell Density. Compared with the CON

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suckling lambs, the goblet cell number per villous, VCR and villous height were

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lower (P < 0.05) in the duodenum of IUGR suckling lambs (Table 5). In contrast,

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IUGR suckling lambs treated with Arg or NCG had greater (P < 0.05) goblet cell

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number per villous, VCR and villous height in the duodenum compared with IUGR

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suckling lambs.

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Disaccharidase Activity. Compared with the CON group, the levels of sucrase,

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maltase and lactase were lower (P < 0.05) in the duodenum of IUGR lambs. In

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contrast, compared with the non-supplemented IUGR lambs, the amounts of maltase

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and lactase were greater (P < 0.05) in the IUGR suckling lambs treated with Arg or

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NCG (Figure 2). The IUGR group had lower maltase concentration in the jejunum

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than the CON group (P < 0.05), while the IUGR suckling lambs treated with Arg or

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NCG groups had greater (P < 0.05) concentration of maltase than the IUGR lambs.

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Compared with the CON group, the IUGR group had lower (P < 0.05) sucrase

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concentration in the ileum, whereas the IUGR suckling lambs treated with Arg or 11


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NCG groups had greater (P < 0.05) sucrase compared with the IUGR lambs.

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Intestinal Oxidative Stress Status. Compared with the CON suckling lambs, the

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concentrations of protein carbonyl and MDA were greater (P < 0.05) and GSH/GSSG

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ratio and GSH were lower (P < 0.05) in the jejunum, duodenum and ileum of the

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IUGR suckling lambs (Table 6). In contrast, compared with the IUGR suckling lambs,

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the GSH/GSSG ratio and GSH concentration were greater (P < 0.05) and

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concentrations of protein carbonyl and MDA lower (P < 0.05) in the jejunum,

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duodenum and ileum of IUGR+1% Arg or 0.1% NCG suckling lambs.

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Plasma Immunoglobulin and Cytokines. Compared with the CON suckling

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lambs, the IUGR suckling lambs had lower (P

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