导　　师： 沈赞明

学科专业： 090601

授予学位： 硕士

作　　者： ;

机构地区： 南京农业大学

摘　　要： 本研究包括三个部分：1、日粮精粗比对奶牛瘤胃上皮屏障结构的影响；2、日粮精粗比对山羊瘤胃上皮屏障结构的影响；3、低pH和SCFA对山羊瘤胃上皮细胞连接蛋白mRNA表达的影响。 1日粮精粗比对奶牛瘤胃上皮屏障结构的影响 试验一：4头装有永久性瘤胃瘘管的初产荷尔斯坦泌乳奶牛分成高精粗比组/(7：3,HC组/)和低精粗比组/(6：4,HF组/),采用2×2重复交叉试验设计。两组每天8：00和20：00分两次饲喂全混日粮,自由饮水,饲喂21天后采样,研究了高精粗比组与低精粗比组奶牛瘤胃上皮屏障的结构变化。结果显示高精粗比组pH降低,SCFA含量升高；光学显微镜观察发现瘤胃上皮细胞间隙变大；扫描电镜下看到高精粗比组瘤胃上皮乳头有过度角化现象；透射电镜观察结果显示,瘤胃上皮紧密连接、桥粒连接和半桥粒连接减少；基因检测发现高精粗比组紧密连接跨膜蛋白基因claudin-1、claudin-4和occludin,膜内连接蛋白ZO-1,间隙连接蛋白基因connxin43和桥粒连接蛋白基因DSG1mRNA的表达量都趋于减少,差异不显著；ZO-1mRNA表达量显著降低/(P<0.05/)。 试验二：11头装有永久性瘤胃瘘管的荷尔斯坦泌乳奶牛按照日粮组分分成单一秸秆组/(SC组,精粗比6：4/)和混合粗饲料组/(MF组,精粗比4：6/),两组饲料等氮、等能。饲喂方式和试验期限同于试验一。检测指标同于试验一,试验结果与试验一一致。 两项结果均显示,饲喂高精粗比日粮时奶牛瘤胃上皮屏障结构遭到破坏。 2日粮精粗比对山羊瘤胃上皮屏障结构的影响 13只装有永久瘘管的的山羊分成高精粗比组/(6：4,HC/)和低精粗比组/(4：6,HF/),自由饮水,饲喂21天后屠宰采样。研究了高精粗比组与低精粗比组山羊瘤胃上皮屏障的结构变化。结果显示高精粗比组pH降低,SCFA含量升高；光学显微镜观察发现瘤胃上皮细胞间隙减小；扫描电镜下发现瘤胃上皮乳头有过度角化现象；透射电镜观察结果显示,瘤胃上皮紧密连接、桥粒连接和半桥粒连接增加；基因检测发现紧密连接跨膜蛋白基因claudin-1、claudin-4和膜内连接蛋白ZO-1mRNA的表达量增加,且差异显著/(P<0.05/)；桥粒连接蛋白基因DSG1mRNA的表达量都减少/(P<0.05/)；紧密连接跨膜蛋白occludin和间隙连接蛋白connxin43mRNA的表达量都趋于增加,差异不显著。 结果显示,饲喂高精粗比日粮时山羊瘤胃上皮屏障结构得到加强。与奶牛试验结果相反。 3pH和SCFA对山羊瘤胃上皮细胞连接蛋白基因表达的影响 体外培养的奶牛瘤胃上皮细胞,经低pH和高SCFA处理后细胞连接基因claudin-4和occludin mRNA表达主要受pH影响,ZO-1和DSG1主要受SCFA影响,而claudin-1和connxin43即受pH影响又受SCFA的影响。 In this study, three experiments are included:1. Effect of different forage to concentrate ratio dietary on cows rumen epithelial barrier function.2. Effect of forage to concentrate ratio dietary on goat rumen epithelial barrier function.3. Effect of pH and SCFA on mRNA abundance of cell junction protein in rumen epithelial. 1. Effect of different forage to concentrate ratio dietary on cow rumen epithelial barrier function Experiment1:Four ruminally cannulated Holstein dairy cows in their first lactation were used for this study. The cows were assigned to two dietary treatments. In the first group the cow get a high forage to concentrate ratio /(7:3, HC/) dietary and in the second group get a low forage to concentrate ratio /(6:4, HF/) dietary. Using a2x2repeated crossover design. Cows were fed in2equal portions at08:00and18:00h daily, and had free access to water. Every group was taken samples after21days feeding. Rumen epithelial barrier structural changes were studied between the two groups. It was found that in the high forage to concentrate ratio group the concentration of propionate, acetate and butyrate of rumen fluid were high, and pH was low; By using scanning electron microscope, hyperkeratosis phenomenon was found in stratum corneum; Using optical microscope, the intercellular space of the rumen epithelium was became big; Using transmission electron microscope, it was found that tight junction, desmosome junction and hemidesmosome junction were decreased and not clearly. Using real time qPCR method, it was found that the claudin-1, claudin-4, occludin, ZO-1, connxin43and DSG1mRNA expression level of rumen epithelial were tend to decreased, and the expression level of ZO-1mRNA was significantly decreased /(P<0.05/). Experiment2:Eleven ruminally cannulated Holstein dairy cows were used for this study. Accordance with the dietary component the cows were separated to mixed forage /(MF/) group /(n=6/) and com straw /(CS/) group /(n=5/). Nitrogen and energy in the dietary of the two group were same. Feeding mode, experiment period and detection indicators were the same with experiment one. Test results were consistented to experiment one. But, the forage to concentrate ratio in the two group is different. The forage to concentrate ratio in mixed forage /(MF/) group was4:6, and in mixed forage /(MF/) group was6:4. Cows were fed in2equal portions and had free access to water. The results showed in this experiment were consistent with experiment1. The results of the two experiment were that rumen epithelium barrier structures were damaged when the cows feed a high forage to concentrate ratio dietary. 2. Effect of forage to concentrate ratio dietary on goat rumen epithelial barrier function Thirteen ruminally cannulated goats were used for this study. The goats were separated to high forage to concentrate ratio dietary /(HC,6:4/) group and low forage to concentrate ratio dietary /(HF,4:6/) group. Goats were fed in2equal portions at08:00and18:00h daily, and had free access to water. Every group were taken samples after21days feeding. Rumen epithelial barrier structural changes were studied between the two groups. It was found that in the high forage to concentrate ratio group the concentration of propionate, acetate and butyrate of rumen fluid were high, and pH were low; Hyperkeratosis phenomenon was found in stratum corneum; The intercellular space of the rumen epithelium was became small; Tight junction, desmosome junction and hemidesmosome junction were increased and more clear. Using real time qPCR method, it was found that the claudin-1, claudin-4and ZO-1mRNA expression level of rumen epithelial were significantly increased /(P<0.05/). Occludin and connxin43mRNA expression level tend to increased, and the expression level of DSG1mRNA was significantly decreased /(P<0.05/). The results of the goat experiment were that rumen epithelium barrier structures were Strengthened when the goats feed a high forage to concentrate ratio dietary, and this was contrasted with cow's experiment. 3. Effect of pH and SCFA on mRNA abundance of cell junction protein in rumen epithelial The attached cells were treated by low pH and high SCFA. It was found that claudin-4and occludin mRNA expression level were mainly influenced by pH. ZO-1and DSG1mRNA expression level were mainly influenced by SCFA. Claudin-1and connxin43mRNA expression level were influenced by pH and SCFA.

关 键 词： 牛和羊 瘤胃上皮 日粮组成 紧密连接 屏障作用

分 类 号： [S823.5 S827.5]

领　　域： [农业科学] [农业科学] [农业科学] [农业科学]