郑琛¹, 李发弟^2,3, 李飞², 周巨旺¹, 段鹏伟¹, 刘绘汇¹, 樊海苗¹, 朱威力¹, 刘婷^,11 甘肃农业大学动物科学技术学院,兰州 730070
2 兰州大学草地农业生态系统国家重点实验室/兰州大学农业农村部草牧业创新重点实验室/ 兰州大学草地农业科技学院,兰州 730020
3 甘肃省肉羊繁育生物技术工程实验室,甘肃民勤 733300

Effects of Tannic Acid Addition in Milk Replacer on Development of Gastrointestinal Tract of 7 to 28 Days Old Hu Lambs

ZHENG Chen¹, LI FaDi^2,3, LI Fei², ZHOU JuWang¹, DUAN PengWei¹, LIU HuiHui¹, FAN HaiMiao¹, ZHU WeiLi¹, LIU Ting^,1 1 College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070
2 State Key Laboratory of Grassland Agro-ecosystems, Lanzhou University/Key laboratory of Grassland Livestock Industry Innovation, Ministry of Agriculture and Rural Affairs, Lanzhou University/College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020
3 Engineering Laboratory of Mutton Sheep Breeding and Reproduction Biotechnology in Gansu Province, Minqin 733300, Gansu

通讯作者: 刘婷,E-mail： liuting@gsau.edu.cn

收稿日期:2019-06-24接受日期:2019-08-6网络出版日期:2019-11-08

基金资助:

甘肃农业大学青年研究生指导教师扶持基金项目.GAU-QNDS-201603

Received:2019-06-24Accepted:2019-08-6Online:2019-11-08
作者简介 About authors
郑琛,E-mail： zhengc@gsau.edu.cn。











摘要
目的 观察代乳粉中添加单宁酸对7—28日龄湖羊羔羊胃肠道生长发育的影响。方法 选择同质性好的7日龄湖羊公羔（双羔）30只,随机分为2组,每组15只,每只1个重复,分别饲喂对照代乳粉或单宁酸含量为0.2%的代乳粉,试验期21 d。羔羊28日龄时饲养试验结束,每组随机选择8只羔羊进行屠宰,分离胃室和肠段,分别称量各胃室和各肠段净质量以及含内容物质量,并量取各肠段的长度,计算各胃室和各肠段的相对质量和相对长度及内容物分布。肠道组织形态学和上皮细胞凋亡率用皱胃胃底腺区及十二指肠、空肠和回肠中段的组织样品测定。肠道屏障功能相关的闭锁蛋白（occludin）、闭锁小带1（zonula occludens-1,ZO-1）和紧密连接蛋白1（claudin 1）mRNA表达量用采集的十二指肠、空肠和回肠黏膜测定。结果 除十二指肠指数（%活体质量,P=0.012）和相对质量（%全肠质量,P=0.034;%全胃肠质量,P=0.017）、空肠和结肠相对长度（%全肠长度,P=0.030,P=0.004）及结肠内容物分布（%活体质量,P=0.039）外,单宁酸对羔羊胃肠指数（%活体质量）、胃肠相对质量（%全胃质量、%全肠质量和%全胃肠质量）、肠道相对长度（%全肠长度）、内容物相对质量（%活体质量）、胃肠内容物相对总胃/肠内容物及总胃肠内容物相对质量（%总胃内容物、总肠内容物、总胃肠内容物）以及小肠claudin 1蛋白mRNA表达量均没有产生显著影响（P＞0.05）,但显著提高羔羊十二指肠肌层厚度并降低绒毛宽度（P=0.013,P=0.001）,显著上调空肠ZO-1蛋白mRNA表达量（P=0.003）,此外,单宁酸有上调羔羊空肠occludin蛋白mRNA表达量并降低空肠绒毛宽度和隐窝深度以及空肠和回肠上皮细胞凋亡率的趋势（P=0.077,P=0.073,P=0.062,P=0.097,P=0.052）。结论 单宁酸显著降低7—28日龄湖羊羔羊十二指肠相对质量和空肠相对长度,但可通过提高十二指肠肌层厚度、上调空肠ZO-1蛋白和occludin蛋白mRNA表达量并降低空肠隐窝深度及空肠和回肠上皮细胞凋亡率来改善肠屏障功能。
关键词： 单宁酸;羔羊;代乳粉;胃肠道;发育

Abstract
【Objective】 This study was conducted to investigate the effects of tannic acid supplementation to milk replacer on the development of gastrointestinal tract of 7 to 28 day-old Hu lambs. 【Method】 Thirty 7 day-old Hu male lambs were chosen and divided into 2 groups, fifteen lambs in each group and each lamb as a duplication. Lambs were fed milk replacer with or without 0.2% tannic acid, respectively. The test lasted 21 days. Eight lambs were selected from each group randomly and slaughtered at 28 day-old. The weights of the compound stomach and the intestinal tract with and without content, and lengths of the intestinal tract were measured, then the relative quality and length were calculated. While the tissue samples from fundus gland region of the abomasum, the middle part of duodenum, jejunum and ileum were fixed in paraformaldehyde to analyse the histomorphology, and the apoptotic rate of intestinal epithelial cells as well. And the mRNA expression of occludin, ZO-1 and claudin 1 protein of duodenum, jejunum and ileum mucosa were measured.【Result】 The results showed that except relative weights of duodenum (% body weight, P=0.012; % intestinal tract weight, P=0.034; % gastrointestinal tract weight, P=0.017), relative length of jejunum and colon (% intestinal tract length, P=0.030, P=0.004), and content of colon (% body weight, P=0.039), the relative weights (% body weight, % stomach weight, % intestinal tract weight, and % gastrointestinal tract weight), relative lengths (% intestinal tract length), content of stomach and intestinal tract (% body weight, % stomach content weight, % intestinal tract content weight, and % gastrointestinal tract content weight), and mRNA expression of claudin 1 protein in intestinal tract of lambs were not affected by tannic acid (P＞0.05). However, tannic acid elongated the muscular thickness of duodenum and decreased the villus width of lamb duodenum significantly (P=0.013,P=0.001), and up-regulated mRNA expression of ZO-1 protein of jejunum (P=0.003). And there was a tendency that tannic acid up-regulated mRNA expression of occludin protein of jejunum (P=0.077), and decreased villus width and crypt depth of jejunum and the apoptotic rate of jejunum and ileum cells (P=0.073, P=0.062, P=0.097, P=0.052). 【Conclusion】 In conclusion, tannic acid decreased the relative weights of duodenum and relative length of jejunum of 7-28 day-old Hu lambs, but improved the barrier function of intestinal tract via elongating the muscular thickness of duodenum, up-regulating the mRNA expression of ZO-1 and occludin protein of jejunum, and decreasing the crypt depth of jejunum and the apoptotic rate of jejunum and ileum cells.
Keywords：tannic acid;lamb;milk replacer;gastrointestinal tract;development


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本文引用格式
郑琛, 李发弟, 李飞, 周巨旺, 段鹏伟, 刘绘汇, 樊海苗, 朱威力, 刘婷. 代乳粉添加单宁酸对7—28日龄湖羊羔羊胃肠道发育的影响[J]. 中国农业科学, 2019, 52(21): 3924-3933 doi:10.3864/j.issn.0578-1752.2019.21.019
ZHENG Chen, LI FaDi, LI Fei, ZHOU JuWang, DUAN PengWei, LIU HuiHui, FAN HaiMiao, ZHU WeiLi, LIU Ting. Effects of Tannic Acid Addition in Milk Replacer on Development of Gastrointestinal Tract of 7 to 28 Days Old Hu Lambs[J]. Scientia Agricultura Sinica, 2019, 52(21): 3924-3933 doi:10.3864/j.issn.0578-1752.2019.21.019

0 引言

【研究意义】胃肠道是哺乳动物最重要的消化器官,承担着为机体供给养分的重任,同时也是机体最大的免疫器官,起到保障机体健康的作用^[1]。幼龄哺乳动物出生前,胃肠道的发育与内部信号分子有关^[2],出生后则取决于胃肠道内养分及各种生物活性因子^[3]。因此,研究幼龄动物胃肠道的发育规律及调控效果,对提高动物养分消化利用率有重要意义。【前人研究进展】单宁酸是一种植物次级代谢物,广泛存在于豆科植物和高粱、五倍子、石榴等^[4],可溶于水和乙醇,化学结构较为复杂^[5],而其复杂的多元酚羟基结构,决定了单宁酸具有抗泻、抗菌、抗氧化和抗病毒等生物学功能^[6]。目前,欧盟已批准单宁酸可作为饲料添加剂,在猪、肉鸡和奶牛的养殖中已开始使用^[7]。REZAR等^[8]发现肉仔鸡饲粮中添加0.2%栗木单宁酸能显著增加粪干物质含量,减少腹泻的发生。隋慧等^[9]和孙展英等^[10]也均发现,饲粮中添加0.05 %和0.1%单宁酸可降低仔猪腹泻率。GIRARD等^[11]也指出,在产肠毒素大肠杆菌诱发腹泻的断奶仔猪模型中,添加1%栗木单宁提取物（54%水解单宁）可显著降低腹泻率。此外,单宁酸也在抑制产气荚膜梭菌^[12]、大肠杆菌^[13]和金黄色葡萄球菌^[14]等病原菌、提高总抗氧化活力和抑制丙二醛^[15,16]、抑制甲型流感病毒受体结合^[17]和丙型肝炎病毒进入细胞^[18]、以及通过损伤寄生虫表皮蛋白而抑制寄生虫^[19]等方面发挥着重要作用。【本研究切入点】通常情况下,肠黏膜的屏障作用和养分吸收作用与上皮细胞的抗氧化活性密切相关,因此,很多****认为具有抗氧化活性的植物提取物或次级代谢物,如单宁酸等,可通过改善肠黏膜上皮细胞组织形态而提高养分消化利用率^[20]。JAMROZ等^[21]报道,饲粮添加0.025%和0.05% 甜栗水解单宁对肉仔鸡肠道无显著影响,而添加量为0.1%时对空肠上皮细胞有负面作用。但LIU等^[22]报道,0.2%栗木单宁可提高热应激肉鸡空肠黏膜绒毛高度,ZHAO等^[23]指出,饲粮中添加0.1%单宁可提高湖羊空肠和回肠黏膜绒毛高度和绒毛高度/隐窝深度值（V/C）,刘洋等^[24]也指出,0.05%栗木水解单宁可修复肉鸡肠道损伤。鉴于此,本研究提出假设,适宜剂量的单宁酸可通过改善羔羊胃肠道内环境而影响胃肠道生长发育。【拟解决的关键问题】因此,本试验选择7日龄湖羊公双羔作为试验对象,研究代乳粉中添加单宁酸对胃肠道生长发育的影响,为羔羊生产提供基础数据。

1 材料与方法

1.1 试验设计及动物

试验选取同质性好的7日龄湖羊公羔（双羔）30只（甘肃金昌中天羊业有限公司）,采用对照试验设计,羔羊随机分入2个处理组（对照组和0.2%单宁酸组）,每组15只,每只1个重复,保证羔羊7日龄体重组间无差异（对照组（4.09 ± 0.66）kg,单宁酸组（3.89 ± 0.62）kg）。试验期21 d,羔羊28日龄结束试验,并从每组随机选择8只羔羊进行屠宰。

1.2 羔羊饲养管理

羔羊在1—3日龄时吸吮初乳,4日龄时与母羊分离,开始用奶瓶训饲代乳粉,7日龄晨饲前空腹称重并分组。8日龄开始,对照组羔羊饲喂羔羊专用代乳粉（北京精准动物营养研究中心,养分浓度见表1）,单宁酸组羔羊则饲喂在对照组代乳粉基础上添加0.2%单宁酸（MACKLIN^?,上海麦克林生化科技有限公司,AR,纯度98%）的代乳粉。饲喂时,用温水溶解代乳粉,比例为5﹕1,每天分别在6：00、12：00、18：00和24：00饲喂4次,喂量为羔羊体重的2 %。羔羊单笼饲养,自由饮水。

Table 1
表1
表1羔羊代乳粉养分浓度（风干基础,%）
Table 1The nutrients concentration in lamb milk replacer (air-dry basis, %)

营养成分 Ingredient	含量 Concentration
干物质 Dry matter	95.68
粗蛋白质 Crude protein	24.89
粗脂肪 Ether extract	17.50
粗纤维 Crude fiber	2.16
粗灰分 Crude ash	5.81
钙 Calcium	1.02
总磷 Total phosphorus	0.59

营养水平均为实测值 The nutrient levels are measured values

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1.3 屠宰和指标测定

屠宰前羔羊不禁水禁食。羔羊28日龄时,先称量宰前活重,然后切断颈静脉放血致死。迅速打开羔羊腹腔,按马仲华^[25]的方法分出4个胃室和6段肠段,结扎各胃室和肠段的连接处并分离。称量各胃室和各肠段含内容物的质量后,排空内容物并称量各胃室和各肠段的净质量,计算各胃室和各肠段的相对质量及其内容物分布情况。同时量取各肠段的长度以计算相对长度。

1.4 组织样品采集及形态学和上皮细胞凋亡率测定

确定采样部位,用手术剪从皱胃胃底腺区以及十二指肠、空肠和回肠中部采集约1 cm²组织块,转移至10 mL带盖离心管中,加入10 mL多聚甲醛以固定样品。然后将样品送至成都里来生物科技有限公司,用苏木精-伊红染色法进行形态学测定,用TUNEL法测定上皮细胞凋亡率。

1.5 小肠黏膜紧密连接蛋白mRNA表达量测定

截取十二指肠、空肠和回肠中段组织样品,用载玻片刮取黏膜后转入2 mL冻存管并迅速转入液氮罐冷冻,带回实验室后转入-80℃冰箱冷冻保存,用于测定小肠黏膜闭锁蛋白（occludin）、闭锁小带1（zonula occludens-1,ZO-1）和紧密连接蛋白1（claudin 1）mRNA表达量。提取十二指肠、空肠和回肠黏膜中的总RNA,然后将各样品RNA反转录为cDNA。Oligo 7.0设计引物（occludin引物长度93 bp,GenBank号NC_040267;ZO-1和claudin 1引物长度分别为163 bp和216 bp,参考LIU等^[26]）,以β-Actin为内参基因（长度97 bp,GenBank号NC_040362）。RT-PCR使用20 μL扩增体系：10 μL 2×Biogold qPCR SuperMix（2×Biogold qPCR Mixture,浙江博而金科技股份有限公司）,0.4 μL上下游引物,1 μL cDNA,8.2 μL ddH₂O。反应条件为：95℃预变性3 min,95℃变性10 s,60℃退火20 s,72℃延伸10 s,40个循环,72℃延伸10 min。目的基因的相对表达量用2^-ΔΔCt法计算。

1.6 数据统计分析

试验数据用SPSS 22.0统计分析软件对数据进行独立样本t检验,以P≤0.05表示为差异显著,以0.05＜P≤0.10表示差异具有显著趋势。

2 结果

2.1 单宁酸对羔羊胃肠道相对质量和相对长度的影响

从表2可以看出,单宁酸显著降低羔羊十二指肠相对质量（%活体质量,P=0.012;全肠质量,P=0.034;%全胃肠质量,P=0.017）,并有降低羔羊盲肠相对质量的趋势（%活体质量,P=0.072）,对羔羊胃室和其他肠段指数没有产生显著影响（P＞0.05）。

Table 2
表2
表2单宁酸对羔羊胃肠相对质量的影响
Table 2Effects of tannic acid on relative quality of gastrointestinal tract of lamb

项目 Item		对照组 Control	单宁酸组 Tannic acid	SEM	P值 P value
胃室 Stomach
瘤胃 Rumen	%活体质量 % body weight	0.54	0.55	0.02	0.856
	%全胃质量 % the quality of total stomach	29.70	31.03	1.10	0.564
	%全胃肠质量 % the quality of total gastrointestinal tract	6.69	7.50	0.34	0.247
网胃 Reticulum	%活体质量 % body weight	0.16	0.16	0.01	0.864
	%全胃质量 % the quality of total stomach	9.16	9.03	0.35	0.859
	%全胃肠质量 % the quality of total gastrointestinal tract	2.04	2.19	0.09	0.426
瓣胃 Omasum	%活体质量 % body weight	0.08	0.08	0.01	0.659
	%全胃质量 % the quality of total stomach	4.08	4.59	0.32	0.450
	%全胃肠质量 % the quality of total gastrointestinal tract	0.92	1.14	0.10	0.304
皱胃 Abomasum	%活体质量 % body weight	1.04	1.00	0.05	0.683
	%全胃质量 % the quality of total stomach	57.05	55.34	1.23	0.506
	%全胃肠质量 % the quality of total gastrointestinal tract	12.77	13.40	0.40	0.448
肠道 Intestinal tract
十二指肠 Duodenum	%活体质量 % body weight	0.20a	0.15b	0.01	0.012
	%全肠质量 % the quality of total intestinal tract	3.21a	2.69b	0.12	0.034
	%全胃肠质量 % the quality of total gastrointestinal tract	2.48a	2.04b	0.10	0.017
空肠Jejunum	%活体质量 % body weight	4.00	3.59	0.15	0.162
	%全肠质量 % the quality of total intestinal tract	63.93	63.29	0.73	0.675
	%全胃肠质量 % the quality of total gastrointestinal tract	49.63	47.98	0.79	0.317
回肠 Ileum	%活体质量 % body weight	0.15	0.14	0.01	0.878
	%全肠质量 % the quality of total intestinal tract	2.35	2.52	0.17	0.642
	%全胃肠质量 % the quality of total gastrointestinal tract	1.83	1.91	0.14	0.772
盲肠 Cecum	%活体质量 % body weight	0.35	0.29	0.02	0.072
	%全肠质量 % the quality of total intestinal tract	5.54	5.16	0.25	0.456
	%全胃肠质量 % the quality of total gastrointestinal tract	4.30	3.90	0.19	0.321
结肠 Colon	%活体质量 % body weight	1.18	1.11	0.05	0.522
	%全肠质量 % the quality of total intestinal tract	18.85	19.64	0.53	0.477
	%全胃肠质量 % the quality of total gastrointestinal tract	14.61	14.88	0.40	0.750
直肠 Rectum	%活体质量 % body weight	0.38	0.37	0.02	0.755
	%全肠质量 % the quality of total intestinal tract	6.11	6.70	0.34	0.407
	%全胃肠质量 % the quality of total gastrointestinal tract	4.73	5.06	0.24	0.517

同行数据后所标字母相异表示差异显著（P＜0.05）,所标字母相同表示差异不显著（P＞0.05）。下同
Different letters in the same row means significant difference between the treatments (P＜0.05), same letter in the same row means not significant difference between treatments (P＞0.05). The same as below

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从表3可以看出,单宁酸显著降低羔羊空肠相对长度并显著增加结肠相对长度（%全肠长度,P=0.030,P=0.004）,对羔羊其他肠段相对长度无显著影响（P＞0.05）。

Table 3
表3
表3单宁酸对羔羊肠道相对长度的影响
Table 3Effects of tannic acid on relative length of intestinal tract of lambs (% intestinal tract length)

项目Item	对照组Control	单宁酸组 Tannic acid	SEM	P值 P value
十二指肠 Duodenum	2.28	2.08	0.11	0.361
空肠Jejunum	81.90a	80.33b	0.37	0.030
回肠 Ileum	0.84	0.84	0.06	0.990
盲肠 Cecum	0.90	0.93	0.05	0.848
结肠 Colon	11.84b	13.56a	0.33	0.004
直肠 Rectum	2.23	2.26	0.13	0.886

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2.2 单宁酸对羔羊胃肠道内容物分布的影响

从表4可以看出,单宁酸显著降低羔羊结肠内容物相对活体质量（%活体质量,P=0.039）,并有降低皱胃内容物相对活体质量的趋势（%活体质量,P=0.053）,对羔羊胃室及其他肠段内容物相对质量（%总胃内容物、%总肠内容物和%总胃肠内容物）没有产生显著影响（P＞0.05）。

Table 4
表4
表4单宁酸对羔羊胃肠内容物相对质量的影响
Table 4Effects of tannic acid on relative quality of gastrointestinal tract content of lambs

项目 Item		对照组 Control	单宁酸组 Tannic acid	SEM	P值 P value
胃室内容物 Stomach content
瘤胃内容物 Rumen content	%活体质量 % body weight	1.44	1.75	0.32	0.648
	%全胃内容物质量 % the quality of total stomach content	26.96	37.02	4.58	0.287
	%全胃肠内容物质量 % the quality of total gastrointestinal tract content	17.32	22.73	3.15	0.410
网胃内容物 Reticulum content	%活体质量 % body weight	0.27	0.20	0.06	0.553
	%全胃内容物质量 % the quality of total stomach content	4.99	3.39	0.91	0.405
	%全胃肠内容物质量 % the quality of total gastrointestinal tract content	3.51	2.13	0.71	0.358
瓣胃内容物 Omasum content	%活体质量 % body weight	0.01	0.01	0.0005	0.776
	%全胃内容物质量 % the quality of total stomach content	0.23	0.31	0.03	0.191
	%全胃肠内容物质量 % the quality of total gastrointestinal tract content	0.14	0.17	0.01	0.333
皱胃内容物 Abomasum content	%活体质量 % body weight	3.43	2.27	0.30	0.053
	%全胃内容物质量 % the quality of total stomach content	68.58	60.65	4.65	0.412
	%全胃肠内容物质量 % the quality of total gastrointestinal tract content	43.32	34.89	3.00	0.168
肠道内容物 Intestinal tract content
十二指肠内容物 Duodenum content	%活体质量 % body weight	0.15	0.09	0.02	0.111
	%全肠内容物质量 % the quality of total intestinal tract content	7.25	3.46	1.44	0.198
	%全胃肠内容物质量 % the quality of total gastrointestinal tract content	1.99	1.47	0.29	0.392
空肠内容物 Jejunum content	%活体质量 % body weight	1.26	1.10	0.18	0.670
	%全肠内容物质量 % the quality of total intestinal tract content	39.64	44.17	2.75	0.429
	%全胃肠内容物质量 % the quality of total gastrointestinal tract content	14.33	18.16	1.91	0.332
回肠内容物 Ileum content	%活体质量 % body weight	0.08	0.04	0.02	0.218
	%全肠内容物质量 % the quality of total intestinal tract content	1.75	1.77	0.32	0.978
	%全胃肠内容物质量 % the quality of total gastrointestinal tract content	0.80	0.75	0.16	0.904
盲肠内容物 Cecum content	%活体质量 % body weight	0.43	0.33	0.08	0.554
	%全肠内容物质量 % the quality of total intestinal tract content	12.85	13.20	1.82	0.928
	%全胃肠内容物质量 % the quality of total gastrointestinal tract content	4.90	4.89	0.64	0.991
结肠内容物 Colon content	%活体质量 % body weight	1.08a	0.57b	0.13	0.039
	%全肠内容物质量 % the quality of total intestinal tract content	33.28	25.72	2.57	0.148
	%全胃肠内容物质量 % the quality of total gastrointestinal tract content	12.53	10.96	1.42	0.596
直肠内容物 Rectum content	%活体质量 % body weight	0.17	0.23	0.04	0.449
	%全肠内容物质量 % the quality of total intestinal tract content	6.10	11.68	2.09	0.192
	%全胃肠内容物质量 % the quality of total gastrointestinal tract content	2.08	4.70	0.87	0.137

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2.3 单宁酸对羔羊皱胃和小肠组织形态的影响

从表5可以看出,单宁酸显著提高羔羊十二指肠肌层厚度并显著降低绒毛宽度（P=0.013,P=0.001）,此外,单宁酸有降低空肠绒毛宽度和隐窝深度的趋势（P=0.073,P=0.062）。单宁酸对羔羊皱胃和小肠其他形态学指标未产生显著影响（P＞0.05）。

Table 5
表5
表5单宁酸对羔羊皱胃和小肠组织形态的影响
Table 5Effects of tannic acid on abomasum and intestinal tract histomorphology of lambs

项目Item	对照组Control	单宁酸组 Tannic acid	SEM	P值 P value
皱胃 Abomasum
黏膜厚度 Mucosa thickness (μm)	333.36	343.82	8.54	0.605
肌层厚度 Muscular thickness (μm)	548.48	278.36	52.15	0.810
十二指肠 Duodenum
绒毛高度 Villus height (μm)	361.99	425.00	21.54	0.150
绒毛宽度 Villus width (μm)	80.04a	54.80b	4.43	0.001
隐窝深度 Crypt depth (μm)	148.87	156.30	8.10	0.665
肌层厚度 Muscular thickness (μm)	161.93b	208.41a	10.01	0.013
绒隐比 V / C	2.57	2.76	0.13	0.505
空肠Jejunum
绒毛高度 Villus height (μm)	431.59	378.90	19.85	0.194
绒毛宽度 Villus width (μm)	71.13	64.74	1.79	0.073
隐窝深度 Crypt depth (μm)	173.48	143.65	8.05	0.062
肌层厚度 Muscular thickness (μm)	157.73	146.10	7.41	0.451
绒隐比 V / C	2.59	2.79	0.07	0.154
回肠 Ileum
绒毛高度 Villus height (μm)	373.81	383.77	29.23	0.883
绒毛宽度 Villus width (μm)	68.91	78.12	2.76	0.108
隐窝深度 Crypt depth (μm)	176.96	207.92	11.78	0.228
肌层厚度 Muscular thickness (μm)	274.31	302.91	46.72	0.792
绒隐比 V / C	2.19	1.85	0.12	0.194

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从表6可以看出,单宁酸显著上调羔羊空肠ZO-1蛋白mRNA表达量（P=0.003）,且有上调空肠occludin蛋白mRNA表达量及降低空肠和回肠上皮细胞凋亡率的趋势（P=0.077,P=0.097,P=0.052）。单宁酸对羔羊十二指肠ZO-1蛋白和claudin 1蛋白mRNA表达量及上皮细胞凋亡率、空肠claudin 1蛋白mRNA表达量以及回肠occludin蛋白、ZO-1蛋白和claudin 1蛋白mRNA表达量均没有产生显著影响（P＞0.05）。

Table 6
表6
表6单宁酸对羔羊小肠黏膜紧密连接蛋白mRNA表达量及上皮细胞凋亡的影响
Table 6Effects of tannic acid on mRNA expression of intercellular tight junction protein of intestinal tract mucosa and apoptosis of intestinal epithelial cell of lambs

项目Item	对照组Control	单宁酸组 Tannic acid	SEM	P值 P value
十二指肠 Duodenum
Occludin	0.86	1.16	0.10	0.124
ZO-1	0.91	1.07	0.08	0.317
Claudin 1	0.91	0.94	0.09	0.850
细胞凋亡 Apoptosis (%)	11.27	7.90	2.97	0.595
空肠Jejunum
Occludin	0.82	1.39	0.16	0.077
ZO-1	0.71b	1.29a	0.11	0.003
Claudin 1	0.97	1.10	0.25	0.800
细胞凋亡 Apoptosis (%)	21.09	3.99	5.13	0.097
回肠 Ileum
Occludin	0.95	1.02	0.10	0.744
ZO-1	0.88	1.11	0.10	0.249
Claudin 1	0.80	1.00	0.16	0.544
细胞凋亡 Apoptosis (%)	47.29	20.76	7.00	0.052

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3 讨论

3.1 单宁酸对羔羊胃肠道相对质量的影响

反刍动物出生时皱胃占总容积的60%,瘤网胃占35%,而随着生长发育,前胃迅速发育,成年时瘤胃占整个胃重的80%,皱胃仅占8%^[27];同样,新生反刍动物肠管占消化道的70%—80%,而随日龄增长和饲粮变化,肠道比例逐渐下降,胃的比例大大上升,成年时肠管占消化道的比例降为30%—50%^[28]。初乳、乳源性促生长因子、营养水平、断奶等均会影响幼龄反刍动物胃肠道发育,饲粮也是影响胃肠道发育的重要因子^[29]。本次试验中,单宁酸显著降低羔羊十二指肠相对质量,对其他胃室和肠段均未产生显著影响,这是因为单宁酸与蛋白质可结合为紧密的络合物^[30],虽然皱胃中的强酸可分解该络合物,但羔羊皱胃功能发育不完善,pH较高,不能有效分解单宁酸和蛋白质的络合物,导致流入十二指肠的可消化蛋白质数量减少,对十二指肠的刺激作用减弱^[1]。此外,在试验全期,羔羊均饲喂液态代乳粉,胃肠道缺乏有效的物理刺激,导致羔羊胃肠道发育延滞,尤其是瘤胃^[28-29,31]。

3.2 单宁酸对羔羊胃肠道内容物分布的影响

本次试验中,除结肠内容物相对活体质量（%活体质量）外,单宁酸对羔羊胃室和肠道各段的内容物分布并未产生显著的影响,这是因为,胃肠道内容物的分布主要由饲粮的物理形态及胃肠道的蠕动所决定^[28]。本试验全期均给羔羊饲喂液态代乳粉,会缩短胃肠道中食糜的停留时间,而添加量为0.2%的单宁酸不足以改变饲粮物理特性,导致羔羊胃肠道内容物分布无显著差异^[28]。此外,胃肠道的节律运动由机体神经系统、内分泌系统、食糜压力差和物理化学性质等多种因素调控^[32],单宁酸作为外源添加的少量植物次级代谢物,不能对羔羊胃肠道蠕动和内容物分布产生显著影响。

3.3 单宁酸对羔羊皱胃和小肠组织形态的影响

幼龄反刍动物由于瘤胃功能发育不完善,大量养分会进入皱胃和小肠,皱胃和小肠也成为了养分最主要的消化吸收部位^[33,34,35],而养分的吸收取决于皱胃和小肠的组织形态。肠道肌层主要参与蠕动,以及通过紧张性收缩维持肠道正常形态^[36];肠道绒毛与养分吸收有关,绒毛越高表明吸收表面积越大^[37];肠道隐窝内的未分化细胞与肠绒毛形态和功能正常有关^[38];V/C值与肠黏膜上皮的成熟有关,比值越高表明成熟细胞所占比例越大,肠道吸收功能越强^[36]。本次试验中,单宁酸显著提高羔羊十二指肠肌层厚度并显著降低绒毛宽度,且有提高羔羊回肠绒毛宽度和降低空肠绒毛宽度和隐窝深度的趋势,这是因为十二指肠可消化蛋白质数量降低,肠绒毛的脱落减缓,导致绒毛高度较高而降低绒毛宽度,并使肌层厚度增加^[23]。而食糜快速通过十二指肠后,较多的蛋白质刺激空肠段绒毛脱落,导致空肠绒毛高度变低,宽度变窄,且由于空肠上皮细胞成熟度升高而使隐窝深度变浅^[39]。但本试验中,采食含单宁酸代乳粉羔羊的十二指肠和空肠V/C值均高于对照组羔羊,表明单宁酸有助于提高羔羊十二指肠和空肠的养分吸收能力^[23]。本试验中单宁酸显著上调羔羊空肠ZO-1蛋白mRNA表达量且有上调十二指肠和空肠occludin蛋白mRNA蛋白表达量的趋势,小肠上皮细胞凋亡率均低于对照组羔羊,也与羔羊小肠形态学结果相符合。肠道紧密连接蛋白是肠黏膜屏障中机械屏障的组成成分,对保障屏障正常功能、阻止病原微生物入侵、养分的选择性通透等起重要作用^[40]。幼龄哺乳动物出生后,肠道开始逐渐发育,上皮细胞凋亡和有丝分裂共同作用,使功能逐渐完善,而母乳或胃肠道中的胰高血糖素样肽（glucagon-like peptide）、胰岛素（insulin）、胰岛素样生长因子（insulin- like growth factors,IGFs）、瘦素（leptin）、肿瘤坏死因子α（tumor necrosis factor α,TNF-α）、表皮生长因子（epidermal growth factor,EGF）等,都会对上皮细胞的增殖、分化和凋亡产生重要作用^[39]。通常情况下,肠道炎症出现时,会使TNF-α等细胞因子的分泌量增加,而TNF-α也是诱导肠道上皮细胞凋亡的重要信号之一^[39]。但单宁酸有多种抗炎活性^[41,42],可以抑制炎症因子,从而保障肠道屏障功能并降低上皮细胞凋亡率,这也是本次试验中,采食含单宁酸代乳粉羔羊小肠紧密连接蛋白mRNA表达量上调且上皮细胞凋亡率降低的主要原因。LIU等^[43]研究发现,炎症小鼠注射从水柏枝（Myricaria bracteate）中提取的水解单宁后,抗炎作用明显。HOFFMANN等^[44]也报道,委陵菜（Potentilla erecta）提取物富含单宁,表现出很强的抗炎作用。此外,LIU^[43]等研究发现,饲粮添加0.2%栗木单宁可上调热应激肉鸡空肠黏膜ZO-1蛋白mRNA表达量,也与本试验中单宁酸改善羔羊肠道屏障功能的结果相似。

4 结论

7—28日龄湖羊羔羊代乳粉中添加0.2%单宁酸显著降低十二指肠相对质量和空肠相对长度,显著提高结肠相对长度,对胃肠道其他部位的相对质量和长度无显著影响。单宁酸显著提高羔羊十二指肠肌层厚度、降低绒毛宽度并上调空肠ZO-1蛋白mRNA表达量,且有上调羔羊空肠occludin蛋白mRNA表达量并降低空肠绒毛宽度和隐窝深度以及空肠和回肠上皮细胞凋亡率的趋势。表明单宁酸对7—28日龄湖羊羔羊胃肠道生长的影响较为微弱,但在调节小肠组织形态并维持正常屏障功能方面发挥一定的作用。

（责任编辑 林鉴非）

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