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代乳粉添加甘露寡糖对7—28日龄湖羊羔羊胃肠道发育的影响代乳粉添加甘露寡糖对7—28日龄湖羊羔羊胃肠道发育的影响 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1 （1甘肃农业大学动物科学技术学院，兰州 730070；2兰州大学草地农业科技学院，草地农业生态系统国家重点实验室/ 农业农村部草牧业创新重点实验室，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） 摘要：【目的】探讨代乳粉中添加甘露寡糖（mannan oligosaccharides，MOS）对7—28日龄湖羊羔羊胃肠道生长发育的影响。【方法】选择同质性良好的7日龄湖羊公羔（双羔）30只，随机分为2组，每组15只，每只为1个重复，对照组羔羊饲喂不含MOS的代乳粉，试验组羔羊饲喂含0.2 % MOS的代乳粉，试验期21d。羔羊28日龄时，两个试验组各随机选择8只羔羊屠宰，取出消化道，称量各胃室和肠段包含内容物的质量和净质量，量取各肠段长度，用以计算各部位的相对质量和内容物分布，以及各肠段的相对长度。多聚甲醛固定皱胃胃底腺区及十二指肠、空肠和回肠中段的组织样品，测定组织形态和小肠上皮细胞凋亡率。采集十二指肠、空肠和回肠的黏膜样品，测定紧密连接蛋白1 (claudin 1)、闭锁小带1（zonula occludens-1，ZO-1）和闭锁蛋白（occludin）的mRNA表达量。【结果】除空肠相对长度外（%全肠长度，P=0.040），MOS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）、内容物相对活体质量（%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）、小肠上皮细胞凋亡率和小肠黏膜claudin 1蛋白mRNA的表达量均没有产生显著影响（P＞0.05），但MOS显著提高羔羊十二指肠绒毛高度和肌层厚度并显著降低绒毛宽度（P=0.033，P=0.047，P=0.015），显著上调空肠ZO-1蛋白mRNA表达量（P=0.028），此外，MOS有提高羔羊回肠绒毛高度、绒毛宽度和隐窝深度、皱胃肌层厚度及回肠occludin蛋白mRNA表达量的趋势（P=0.075，P=0.078，P=0.085，P=0.084，P=0.052）。【结论】MOS对7—28日龄湖羊羔羊胃肠道相对质量、长度和内容物分布基本无显著影响，但可改善十二指肠和回肠绒毛及肌层的组织形态，维持小肠屏障功能，有利于提高养分消化率。 关键词：羔羊；甘露寡糖；代乳粉；胃肠道；发育 0 引言【研究意义】胃肠道的结构是保障消化功能的前提，当组织形态发育正常及功能完善时，胃肠道中的营养物质才会被充分消化吸收[1]。反刍动物胃肠道发育受多种因素影响和调节，如年龄[2]、断奶[3-4]、饲粮类型[5-7]、肠营养素、激素和生长因子[8]等。饲喂幼畜时，除了需要提供高浓度的能量和养分以满足幼畜快速生长发育和器官发育的需要[9-11]，还需要强化一些微量营养性和非营养性饲料添加剂以提高幼畜成活率和机体免疫力，益生菌和化学益生素就是目前幼龄动物养殖中常用的免疫增强剂[12]。化学益生素被定义为营养活性物质（Nutricine），是一种非药品类功能食品，虽然不具备直接营养功能，但可以维持肠道消化吸收功能，因而增强动物健康和生长发育[13]。【前人研究进展】甘露寡糖（mannan oligosaccharides, MOS）是化学益生素的一种，来自于酵母（Saccharomyces cerevisiae）细胞壁，富含甘露蛋白和β-葡聚糖等复杂碳水化合物等[14]，广泛应用于养殖业以提高动物机体免疫机能并消除肠道病原菌[15-16]。在单胃动物和水产动物养殖中，添加MOS具有提高生产性能和促进动物健康的作用[17-20]，也促进肠道发育，如前期和后期饲粮中分别添加0.2 %和0.1 % MOS可提高肉仔鸡小肠绒毛高度并降低隐窝深度[21]，添加0.1 % MOS可显著提高仔猪小肠黏膜绒毛高度/隐窝深度值（V/C）[22]，添加0.1 %、0.15 %和0.2 % MOS均可显著提高兔小肠绒毛高度[23]。【本研究切入点】MOS在反刍动物上的应用研究较少，主要是因为很多学者认为瘤胃微生物能够降解MOS，从而消除其保健功能。然而，有限的资料仍然显示MOS在反刍动物养殖中起到了一定的有益作用，如改善绵羊瘤胃健康[24]和提高抗氧化能力[25]，提高羔羊血液免疫球蛋白水平[26]等，但MOS对幼龄反刍动物消化道生长发育的影响鲜见报道。鉴于MOS在单胃动物胃肠道发育中所表现出的积极作用，本研究提出假设，添加MOS对羔羊胃肠道发育也具有一定的影响，可为幼龄反刍动物的健康养殖提供帮助。【拟解决的关键问题】本试验以7日龄湖羊公双羔作为试验对象，研究代乳粉中添加MOS对羔羊消化道生长发育的影响，为幼龄反刍动物养殖中化学益生素的使用提供基础数据。 1 材料与方法1.1 试验设计及动物试验选用同质性良好的30只7日龄湖羊公双羔（选自甘肃省金昌中天羊业有限公司）作为试验动物，采用对照试验设计，将羔羊随机分为2个处理组，每组15只，每只为1个重复。羔羊分别饲喂对照代乳粉（北京精准动物营养研究中心，营养物质浓度见表1）或添加0.2 % MOS（SCIPHAR®，陕西森弗天然制品有限公司，纯度＞90 %）的代乳粉。饲养试验持续21 d。 我国地表水资源丰富，近几十年来人口增长和经济快速发展对河流水体造成了严重的污染。河流水体是工业废水和生活污水的直接收纳水体，河流水体污染已成为一个事关公共安全和人类健康的重要社会问题，而水环境中对人类和生态系统威胁最大的是有机污染物。河流水体中的持久性有机污染物已在全国范围内得到充分证明，对城市水资源保护构成了巨大的挑战［13-14］。有机污染物可能会默默地吞噬中国有限的水资源，并对生态系统和人类健康造成持续的潜在危害［15］。 表1 羔羊代乳粉营养物质浓度（风干基础） Table 1 The chemical composition of lamb milk replacer (air- dry basis, %)  营养成分Ingredients1}含量Concentration 干物质Dry matter95.68 蛋白质Protein24.89 粗脂肪Ether extract17.50 粗纤维Crude fiber 2.16 粗灰分Crude ash 5.81 钙Calcium 1.02 总磷Total phosphorus 0.59
1)营养水平均为实测值The nutrient levels are measured values 1.2 羔羊饲养管理羔羊出生的1—3 d内采食母乳，4日龄与母羊分离后奶瓶训饲代乳粉。7日龄清晨空腹称重，按组间体重无差异（对照组4.09 ± 0.66 kg，MOS组4.07 ± 0.61 kg）的原则将羔羊随机分为2组。8日龄时按试验设计饲喂羔羊，喂量为羔羊体重的2 %，每日饲喂4次，分别为6:00、12:00、18:00和24:00，代乳粉与水的比例为1:5。羔羊单笼饲养，自由饮水。 1.3 羔羊屠宰及胃肠道相关指标测定羔羊28日龄时屠宰，宰前不禁食禁水，称量活重后立即颈静脉放血致死。打开腹腔后按照马仲华[27]的方法分离瘤胃、网胃、瓣胃、皱胃、十二指肠、空肠、回肠、盲肠、结肠和直肠。称量胃肠道各部位净质量和含内容物的质量，计算各部位的相对质量及其内容物分布。测量肠道各段的长度，计算相对长度。 选择2016年3月—2017年2月入科且无中心静脉穿刺经验的进修医生40名作为研究对象。按照数字表法，将其随机分为两组，实验组20名，对照组20名。实验组中，男性12名，女性8名，年龄24～32岁；对照组中，男性13名，女性7名，年龄25～34岁。实验组和对照组进修医生中心静脉穿刺相关理论基础知识摸底考核平均成绩分别为（62.7±10.1）分、（63.3±9.8）分。 1.4 组织样品采集及组织形态和小肠上皮细胞凋亡率测定采集皱胃胃底腺区以及十二指肠、空肠和回肠中部约1 cm2的组织样品，转入多聚甲醛固定液中，在成都里来生物科技有限公司用H.E染色法和Tunel法观察胃肠道组织形态和测定小肠上皮细胞凋亡率。 1.5 小肠黏膜紧密连接蛋白mRNA表达量测定采集十二指肠、空肠和回肠中段黏膜样品测定紧密连接蛋白1（claudin 1）、闭锁小带1（zonula occludens-1，ZO-1）和小肠黏膜闭锁蛋白（occludin）的mRNA表达量。提取样品总RNA，检测RNA浓度和纯度合格后将各样品RNA反转录为cDNA。使用Oligo 7.0设计引物，其中claudin 1和ZO-1参考LIU等[28]方法设计，引物长度分别为216和163 bp，occludin参考GenBank设计（NC_040267），引物长度为93 bp，以β-Actin为内参基因（NC_040362，长度97 bp）。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。RT-PCR在Roche LightCycler® 480II进行，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 数据统计分析使用SPSS 22.0对试验数据进行独立样本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。 是什么时候开始吵架的呢？买回豆浆机是第一次。吵过以后，温简坐在床边看杂志，她紧绷着脸一下一下地从杂志上抬眼偷望顾青，每一次都被他的目光逮到。到最后好像成了两个赌气孩子的游戏，她和他都没有忍住扑哧地笑了。 2 结果2.1 MOS对羔羊胃肠道相对质量的影响2.1.1 羔羊胃肠指数 从表2可以看出，MOS对羔羊胃肠指数（%活体质量）并未产生显著影响（P＞0.05），仅有降低羔羊十二指肠指数的趋势（P=0.066）。 （1）通过问卷调查了解实验班对标准化沟通训练的看法。结果显示，护生对该教学方法满意度较高，认为能够提高沟通能力和学习积极性（见表1）。 2.1.2 羔羊胃肠相对质量 从表3可以看出，MOS对羔羊胃肠相对质量（%全胃质量、%全肠质量和%全胃肠质量）均未产生显著影响（P＞0.05），但饲喂含MOS代乳粉羔羊的大肠相对质量略高于对照组羔羊（P＞0.05）。 2.1.3 羔羊肠道相对长度 从表4可以看出，MOS显著降低了羔羊空肠相对长度（%全肠长度，P=0.040），此外，MOS有增加盲肠相对长度的趋势（P=0.094）。 2.2 MOS对羔羊胃肠道内容物分布的影响2.2.1 羔羊胃肠内容物相对活体质量（%活体质量） 从表5可以看出，MOS对羔羊胃肠内容物相对活体质量（%活体质量）没有产生显著影响（P＞0.05）。 2.2.2 羔羊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） 从表6可以看出，MOS对羔羊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、%总肠内容物和%总胃肠内容物）均没有产生显著影响（P＞0.05），但采食含MOS代乳粉羔羊瘤胃内容物相对质量略高于对照组羔羊，而皱胃、盲肠和结肠内容物相对质量略低于对照组羔羊（P＞0.05）。 表2 MOS对羔羊胃肠指数的影响 Table 2 Effects of MOS on gastrointestinal index of lambs (% body weight)  项目Item对照组ControlMOS组MOSSEMP值P value 胃室Stomach 瘤胃Rumen0.540.500.020.386 网胃Reticulum0.160.150.010.555 瓣胃Omasum0.080.070.010.488 皱胃Abomasum1.040.920.040.138 肠道Intestinal tract 十二指肠Duodenum0.200.170.010.066 空肠Jejunum4.003.710.140.316 回肠Ileum0.130.140.010.697 盲肠Cecum0.350.410.020.201 结肠Colon1.181.150.040.675 直肠Rectum0.380.390.010.663
表3 MOS对羔羊胃肠相对质量的影响 Table 3 Effects of MOS on relative quality of gastrointestinal tract of lambs  项目Item对照组ControlMOS组MOSSEMP值P value 胃室Stomach 瘤胃Rumen%全胃质量% the quality of total stomach29.7030.450.580.533 %全胃肠质量% the quality of total gastrointestinal tract6.716.620.270.869 网胃Reticulum%全胃质量% the quality of total stomach9.169.460.400.724 %全胃肠质量% the quality of total gastrointestinal tract2.052.060.100.939 瓣胃Omasum%全胃质量% the quality of total stomach4.084.150.210.883 %全胃肠质量% the quality of total gastrointestinal tract0.920.890.050.795 皱胃Abomasum%全胃质量% the quality of total stomach57.0555.940.740.471 %全胃肠质量% the quality of total gastrointestinal tract12.8112.140.350.356 肠道Intestinal tract 十二指肠Duodenum%全肠质量% the quality of total intestinal tract3.222.930.140.297 %全胃肠质量% the quality of total gastrointestinal tract2.492.280.090.292 空肠Jejunum%全肠质量% the quality of total intestinal tract64.2161.691.070.254 %全胃肠质量% the quality of total gastrointestinal tract49.8048.361.060.517 回肠Ileum%全肠质量% the quality of total intestinal tract1.912.410.210.258 %全胃肠质量% the quality of total gastrointestinal tract1.691.880.130.477 盲肠Cecum%全肠质量% the quality of total intestinal tract5.576.890.440.135 %全胃肠质量% the quality of total gastrointestinal tract4.325.380.340.116 结肠Colon%全肠质量% the quality of total intestinal tract18.9519.430.570.686 %全胃肠质量% the quality of total gastrointestinal tract14.6715.170.410.558 直肠Rectum%全肠质量% the quality of total intestinal tract6.146.650.230.275 %全胃肠质量% the quality of total gastrointestinal tract4.755.190.160.162
表4 MOS对羔羊肠道相对长度的影响 Table 4 Effects of MOS on relative length of intestinal tract of lambs (% total intestinal tract length)  项目Item对照组ControlMOS组MOSSEMP值P value 十二指肠Duodenum 2.29 2.010.090.107 空肠Jejunum 81.84a 80.53b0.330.040 回肠Ileum 0.83 0.950.010.292 盲肠Cecum 0.91 1.120.060.094 结肠Colon11.9612.890.290.115 直肠Rectum 2.17 2.490.110.151
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 表5 MOS对羔羊胃肠内容物相对活体质量的影响 Table 5 Effects of MOS on relative quality of gastrointestinal content to body weight of lambs (% body weight)  项目Item对照组ControlMOS组MOSSEMP值P value 胃室内容物Stomach content 瘤胃内容物Rumen content1.441.670.200.587 网胃内容物Reticulum content0.240.340.070.473 瓣胃内容物Omasum content0.010.010.0010.946 皱胃内容物Abomasum content3.433.180.240.623 肠道内容物Intestinal tract content 十二指肠内容物Duodenum content0.150.140.020.868 空肠内容物Jejunum content1.261.350.200.833 回肠内容物Ileum content0.110.050.030.378 盲肠内容物Cecum content0.430.300.080.422 结肠内容物Colon content1.080.810.130.298 直肠内容物Rectum content0.170.210.030.503
表6 MOS对羔羊胃肠内容物相对总胃/肠内容物及总胃肠内容物相对质量的影响 Table 6 Effects of MOS on relative quality of each part of stomach and intestinal content to total stomach and intestinal tract content, and gastrointestinal tract content of lambs  项目Item对照组ControlMOS组MOSSEMP值P value 胃室内容物Stomach content 瘤胃内容物Rumen content%全胃内容物质量% the quality of total stomach content26.9633.152.560.239 %全胃肠内容物质量% the quality of total gastrointestinal tract content17.3021.121.800.306 网胃内容物Reticulum content%全胃内容物质量% the quality of total stomach content4.996.681.280.532 %全胃肠内容物质量% the quality of total gastrointestinal tract content3.504.060.030.742 瓣胃内容物Omasum content%全胃内容物质量% the quality of total stomach content0.230.240.030.988 %全胃肠内容物质量% the quality of total gastrointestinal tract content0.140.130.010.698 皱胃内容物Abomasum content%全胃内容物质量% the quality of total stomach content68.5860.092.710.119 %全胃肠内容物质量% the quality of total gastrointestinal tract content43.1439.302.350.434 肠道内容物Intestinal tract content 十二指肠内容物Duodenum content%全肠内容物质量% the quality of total intestinal tract content7.206.231.630.777 %全胃肠内容物质量% the quality of total gastrointestinal tract content1.982.220.390.765 空肠内容物Jejunum content%全肠内容物质量% the quality of total intestinal tract content39.3446.212.220.126 %全胃肠内容物质量% the quality of total gastrointestinal tract content14.2816.951.690.451 回肠内容物Ileum content%全肠内容物质量% the quality of total intestinal tract content1.751.450.310.666 %全胃肠内容物质量% the quality of total gastrointestinal tract content0.800.570.200.618 盲肠内容物Cecum content%全肠内容物质量% the quality of total intestinal tract content12.739.881.350.312 %全胃肠内容物质量% the quality of total gastrointestinal tract content4.883.510.700.345 结肠内容物Colon content%全肠内容物质量% the quality of total intestinal tract content33.0230.572.830.681 %全胃肠内容物质量% the quality of total gastrointestinal tract content12.4910.251.170.356 直肠内容物Rectum content%全肠内容物质量% the quality of total intestinal tract content5.997.801.050.405 %全胃肠内容物质量% the quality of total gastrointestinal tract content2.062.770.420.414
2.3 MOS对羔羊皱胃和小肠组织形态的影响从表7可以看出，MOS显著提高羔羊十二指肠绒毛高度和肌层厚度但显著降低绒毛宽度（P=0.033，P=0.047，P=0.015），此外，MOS有提高羔羊回肠绒毛高度、绒毛宽度和隐窝深度及皱胃肌层厚度的趋势（P=0.075，P=0.078，P=0.085，P=0.084）。MOS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）。 从表8可以看出，MOS显著上调了羔羊空肠ZO-1蛋白mRNA的表达量（P=0.028），且有上调回肠occludin蛋白mRNA表达量的趋势（P=0.052）。MOS对羔羊十二指肠3种紧密连接蛋白、空肠claudin 1和occludin蛋白、回肠claudin 1和ZO-1蛋白mRNA的表达量以及各肠段上皮细胞凋亡率均没有产生显著影响（P＞0.05）。 3 讨论3.1 MOS对羔羊胃肠道相对质量的影响幼龄反刍动物胃肠道发育中，胃肠道的相对质量（%活体质量、%全胃质量、%全肠质量和%全胃肠道质量）及相对长度（%全肠长度）等是反映机体消化道生长发育的重要指标[29]。幼龄反刍动物在生长发育中，体内组织器官会因机体不同的功能需要而表现出不同的生长发育速度[29]。本次试验中，对照组和MOS处理组羔羊胃肠道相对质量未出现显著差异，这是因为，整个试验期羔羊均饲喂液体代乳粉，尽管代乳粉营养均衡，但前胃得不到正常发育，单纯吃奶的动物，瘤胃缺乏粗糙物质的刺激[30]，而固体饲料的摄入会为胃肠道发育带来更好的物理刺激[31]。寇占英等[32]也报道采食粗饲料可以刺激瘤胃发育，而幼龄反刍动物仅喂乳汁或代乳品，会延滞前胃发育。但采食含MOS代乳粉的羔羊空肠相对长度显著低于对照组羔羊，盲肠相对长度有高于对照组羔羊的趋势且大肠各段的相对长度均高于对照组羔羊，主要是因为MOS作为低聚糖，在羔羊小肠不能被消化吸收，而在大肠段可作为有益菌的发酵底物并促进有害菌排出体外，促进大肠发育[33]，使大肠相对长度增加而小肠相对长度有所降低，但由于其作为添加量很低的饲料添加剂，不能显著改变羔羊胃肠道的生长发育，仅有微弱的作用。周怿[34]在犊牛代乳粉中添加75 mg·kg-1酵母β-葡聚糖后发现对胃肠道相对质量无显著影响，闫晓刚[35]在犊牛饲粮中添加20 g·d-1酵母培养物，对犊牛前胃相对质量无显著影响，也与本试验结果相同。 表7 MOS对羔羊皱胃和小肠组织形态的影响 Table 7 Effects of MOS on abomasum and intestinal tract histomorphology of lambs (μm)  项目Item对照组ControlMOS组 MOSSEMP值P value 皱胃 Abomasum 黏膜厚度 Mucosa thickness333.36331.469.740.927 肌层厚度 Muscular thickness548.48686.9940.110.084 十二指肠 Duodenum 绒毛高度 Villus height361.99b446.39a20.440.033 绒毛宽度 Villus width80.04a61.96b3.920.015 隐窝深度 Crypt depth148.87163.338.460.414 肌层厚度 Muscular thickness161.93b201.87a10.250.047 绒毛高度/隐窝深度值V/C2.572.820.110.258 空肠Jejunum 绒毛高度 Villus height431.59445.3020.290.748 绒毛宽度 Villus width71.1368.012.040.464 隐窝深度 Crypt depth173.48158.216.650.265 肌层厚度 Muscular thickness157.73153.018.270.786 绒毛高度/隐窝深度值V/C2.592.890.090.118 回肠 Ileum 绒毛高度 Villus height373.81428.3815.250.075 绒毛宽度 Villus width68.9178.872.810.078 隐窝深度 Crypt depth176.96213.1010.380.085 肌层厚度 Muscular thickness274.31230.8218.540.267 绒毛高度/隐窝深度值V/C2.192.150.060.775
表8 MOS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 8 Effects of MOS on mRNA expression of intercellular tight junction protein of intestinal tract mucosa and apoptotic rate of intestinal epithelial cells of lambs  项目Item对照组ControlMOS组 MOSSEMP值P value 十二指肠 Duodenum Claudin 10.980.940.100.863 ZO-10.921.050.080.403 Occludin0.861.180.100.112 细胞凋亡Apoptosis (%)11.275.042.900.302 空肠Jejunum Claudin 10.911.090.210.671 ZO-10.80b1.20a0.100.028 Occludin0.961.040.080.630 细胞凋亡Apoptosis (%)21.0918.085.400.791 回肠 Ileum Claudin 10.870.810.180.875 ZO-10.921.060.070.362 Occludin0.831.170.090.052 细胞凋亡Apoptosis (%)47.2937.626.540.487
3.2 MOS对羔羊胃肠道内容物分布的影响胃肠道内容物滞留时间决定养分的消化吸收率，而内容物的滞留时间由食糜类型和胃肠道运动所决定。本次试验中，所有羔羊胃肠道内容物含量均处于较低水平，主要是因为羔羊只饲喂液体代乳粉，因此，会缩短食糜在胃肠道中的滞留时间，且食糜流通量显著低于采食固体饲料的动物[30]。胃肠道的节律性运动以及食糜的推送和分布，由食糜压力差、体液因素、交感神经、迷走神经、平滑肌细胞兴奋性、激素以及食糜的物理化学性质等多种因素调控[36]。本次试验中，MOS对羔羊胃肠道内容物分布并未产生显著影响，也说明食物的物理形态是决定胃肠道内容物分布的主要因素，MOS作为外源添加的益生素，不能对胃肠道运动和内容物分布产生显著影响，仅由于对胃肠道益生菌有促进作用而改变食糜在胃肠道不同部位的滞留时间，导致采食含MOS代乳粉羔羊瘤胃内容物相对质量较高，而皱胃、盲肠和结肠内容物相对质量较低，也与前人在成年羊上的研究结果类似[33]。 3.3 MOS对羔羊皱胃和小肠组织形态的影响对前胃功能发育不完善的幼龄反刍动物来说，皱胃和小肠是养分最主要的消化吸收部位，而养分的吸收取决于皱胃和小肠的组织形态。皱胃的黏膜和肌层厚度、小肠的绒毛高度、隐窝深度、黏膜厚度及V/C值等，是评价动物消化道对养分消化吸收的重要指标[37-39]，如绒毛高度与肠道上皮细胞发育呈正相关，高度越高养分吸收能力越强[40]，而隐窝深度与肠道上皮细胞成熟率呈负相关，隐窝越浅表明细胞成熟率高且分泌功能越强[41]，此外，V/C值与肠道上皮细胞更新程度有关，也与肠道养分吸收能力呈正相关[42-43]。本次试验中，羔羊肠道绒毛均保持在较高水平，这是因为，试验羔羊全期只饲喂液体代乳粉，而固体饲料会加大对肠绒毛的刺激而导致肠绒毛脱落速度加快[44]，液体饲料可维持肠绒毛高度[45]。本试验中，MOS显著提高了羔羊十二指肠绒毛高度和肌层厚度，并有提高回肠绒毛高度、绒毛宽度和皱胃肌层厚度的趋势，表明MOS能提高羔羊皱胃和小肠段的养分消化吸收能力。周怿[34]报道饲粮添加75 mg·kg-1酵母β-葡聚糖可提高犊牛小肠绒毛高度和V/C值。在仔猪和肉仔鸡的试验中，饲粮添加0.1 % MOS可显著提高小肠V/C值[21-22]。本试验中采食含MOS代乳粉羔羊空肠ZO-1蛋白mRNA表达量显著上调，回肠occludin蛋白mRNA表达量也有上调的趋势，且小肠上皮细胞凋亡率均低于对照组羔羊，也说明MOS有利于维持小肠正常屏障功能，并使肠绒毛维持在较高水平。occludin蛋白、claudin 1蛋白和ZO-1蛋白是肠上皮细胞间的紧密连接蛋白，构建肠道屏障，机械性阻止微生物入侵[46]。而外界刺激、生理和病理等会使肠道屏障发生改变，增加肠上皮细胞间隙通透性，导致病原菌侵入细胞引发感染性疾病。Puthenedam等[47]报道，肠道中的乳酸菌和双歧杆菌可上调ZO-1蛋白及occludin蛋白的表达，修复肠道损伤。杨俊等[48]也报道，肠上皮细胞被大肠杆菌（enteroinvasiveE.coli，EIEC）感染后，用乳酸菌处理时，紧密连接相关蛋白（claudin，occludin，junction adherensive molecular-1（JAM-1），ZO-1）表达量上调，肠道通透性得到改善。本试验中，MOS作为化学益生素可以促进肠道中乳酸杆菌和双歧杆菌等益生菌的增殖[16]，因此发挥出维护肠道屏障健康的作用。肠道黏膜的更新和上皮细胞的转型由上皮细胞凋亡和有丝分裂共同维持，凋亡一方面可以促进肠黏膜的适度发育和成熟，但另一方面，如细胞过度凋亡，将引起肠道功能紊乱[49]。尚沁沁[50]指出，益生菌可通过抑制病原菌在肠道的定植，调控细胞凋亡通路，降低肠上皮细胞凋亡率。Yan等[51]研究发现，乳酸菌可以抑制由肿瘤坏死因子α（tumor necrosis factor, TNF-α）诱导的肠道上皮细胞凋亡。本次试验中采食含MOS代乳粉羔羊小肠上皮细胞凋亡率低于对照组羔羊，也是由于MOS促进肠道有益菌增殖而引起的[33]。 借着月光，一根细长尖利的节足，猛地从崖下探了上来，似钢钎一般，杵在望天归的石面上，大力之下，竟插进一尺多深，登时碎石飞溅。 4 结论7—28日龄湖羊羔羊代乳粉中添加MOS对羔羊胃肠道相对质量和长度、内容物分布和组织形态基本无显著影响，但显著提高羔羊十二指肠绒毛高度和肌层厚度并显著降低绒毛宽度且上调空肠ZO-1蛋白mRNA表达量，还有提高羔羊回肠绒毛高度、绒毛宽度和隐窝深度、皱胃肌层厚度及回肠occludin蛋白mRNA表达量的趋势。表明代乳粉中添加0.2 % MOS对湖羊羔羊胃肠道发育的影响较为微弱，主要对小肠绒毛形态和屏障功能有一定促进作用。 References [1] 郭江鹏, 潘建忠, 李发弟, 张元兴, 杨宇泽, 郝正里. 不同早期断奶日龄对舍饲肉用羔羊胃组织形态发育变化的影响. 畜牧兽医学报, 2018, 49(5): 971-985. 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Effects of adding mannan oligosaccharides to milk replacer on the development of gastrointestinal tract of 7-28 days old Hu lambsZHENG Chen1, LI Fadi2,3, LI Fei2, ZHOU Juwang1, DUAN Pengwei1, LIU Huihui1, FAN Haimiao1, ZHU Weili1, LIU Ting1 (1College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070; 2State Key Laboratory of Grassland Agro-ecosystems, Key laboratory of Grassland Livestock Industry Innovation/Ministry of Agriculture and Rural Affairs, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020; 3Engineering Laboratory of Mutton Sheep Breeding and Reproduction Biotechnology in Gansu Province, Minqin 733300, Gansu) Abstract:【Objective】This study was conducted to investigate the effects of mannan oligosaccharides (MOS) supplementation to milk replacer on the development of gastrointestinal tract of 7-28 day-old Hu lambs. 【Method】Thirty 7 day-old Hu male lambs were chosen and divided into 2 groups randomly, fifteen lambs in each group and each lamb as a repeat. Lambs were fed milk replacer with or without 0.2 % MOS, respectively. The test lasted for 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, and 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 claudin 1, zonula occludens-1 (ZO-1), and occludin protein of duodenum, jejunum and ileum mucosa were measured.【Result】The results showed that except relative length of jejunum (P=0.040), 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), the apoptotic rate of intestinal epithelial cells and mRNA expression of claudin 1 protein in intestinal tract of lambs were not affected by MOS (P＞0.05). However, MOS elongated the villus height and the muscular thickness, and decreased the villus width of lamb duodenum significantly (P=0.033, P=0.047, P=0.015). MOS also up-regulated the mRNA expression of ZO-1 protein of lamb jejunum significantly (P=0.028). And there was a tendency that MOS elongated villus height, width and crypt depth of ileum, muscular thickness of abomasum, and mRNA expression of occludin protein of ileum (P=0.075, P=0.078, P=0.085, P=0.084, P=0.052).【Conclusion】MOS almost did not affect the relative weights, lengths, and content distribution of gastrointestinal tract of 7-28 days old Hu lambs, but improved the histomorphology of duodenum and ileum, indicating it could maintain barrier function of intestinal tract and benefit to nutrients digestibility. Key words: lamb; mannan oligosaccharides; milk replacer; gastrointestinal tract; development doi: 10.3864/j.issn.0578-1752.2020.02.014 开放科学（资源服务）标识码（OSID）： 收稿日期：2019-06-24； 接受日期：2019-08-14 基金项目：国家自然科学基金(31560646，31860657) （责任编辑 林鉴非）
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