异位酸调节断奶前后犊牛瘤胃及小肠黏膜发育机制研究

The molecular basis and mechanism of the development of rumen and small intestine mucosa in pre- and post-weaning calves is not clarified by researches at home and abroad, in particular, the response mechanism of intestinal metabolic function during rumen development is not yet clarified. The objective of this work is to investigate the effects of the isoacids on the regulation of the development of rumen and small intestine mucosa. One hundred and seventeen pre-weaning calves with similar body weight and 45 d after birth will be selected from three dairy cow farms, 39 calves per farm. Thirty-six calves in each farm will be allocated into four treatments at random. Graded levels (0, 3, 6, and 9 g/d) of isoacids (isobutyrate, isovalerate or 2-methylbutyrate) will be supplemented in the four treatments from day 45 to 120 after birth. Calves will be weaned at day 67 after birth. During the experiment period, calves will be slaughtered at 4 time intervals i.e. at day 45, 67, 90 and 120 after birth. Samples of ruminal liquid, tissue of rumen and small intestine mucosa will be collected at the same time. Rumen microbial diversity will be determined by using DGGE, 16S and 18S rRNA gene library. Rumen fiber-degrading bacteria will be quantitated by using quantitative 16S rRNA hybridization probes. Histological studies of the various parts of the rumen and small intestine will be conducted by using biological microscopy techniques. The expression of gene related to growth axis in rumen and small intestine mucosa, ketogenic enzyme in rumen mucosa and glucose transporter in small intestine mucosa will be measured by using RT-PCR. Protein expression of ketogenic enzyme in rumen mucosa and glucose transporter in small intestine mucosa will be measured by using Western Blotting. The results can be applied to reveal the regulation mechanism of different level BCVFA on the the development of rumen and small intestine in calves, and provide a theoretical basis for the regulation of the development of rumen and small intestine and enhancement of the health in dairy cattle by using isoacids.

国内外研究尚不能阐明断奶前后犊牛瘤胃及小肠黏膜发育的分子基础及其机制，尤其未能阐明瘤胃发育期间小肠代谢功能的应答机制。项目以断奶前后犊牛为研究对象，在日粮中添加不同水平异位酸（异丁酸、异戊酸或2-甲基丁酸），于断奶前、断奶时、断奶后屠宰采集样品，采用变性梯度凝胶电泳、16S和18S rRNA基因克隆文库、16SrRNA特异性探针定量杂交方法、生物显微技术、RT-PCR和Western blot技术，从分析瘤胃微生物区系、瘤胃及小肠黏膜组织形态学变化以及与瘤胃生酮作用和小肠葡萄糖吸收功能相关基因及蛋白质表达入手，揭示断奶前后犊牛瘤胃和小肠黏膜组织形态和代谢功能变化及支链挥发性脂肪酸调控机制，初步建立断奶前后犊牛瘤胃发育期间小肠葡萄糖吸收功能应答机制，为深入研究犊牛断奶前后瘤胃发育期间小肠应答机制奠定理论基础。

国内外研究尚不能阐明断奶前后犊牛瘤胃及小肠黏膜发育的分子基础及其机制，尤其未能阐明瘤胃发育期间小肠代谢功能的应答机制。项目以断奶前后犊牛为研究对象，在日粮中添加不同水平异位酸（异丁酸、异戊酸或2-甲基丁酸），于断奶前、断奶时、断奶后屠宰采集样品，采用变性梯度凝胶电泳、16S 和18S rRNA 基因克隆文库、16SrRNA 特异性探针定量杂交方法、生物显微技术、RT-PCR 和Western blot 技术，分析瘤胃微生物区系、瘤胃及小肠黏膜组织形态学变化以及与瘤胃生酮作用和小肠葡萄糖吸收功能相关基因及蛋白质表达。结果表明：在断奶前后犊牛奶及日粮中添加异丁酸、异戊酸和2-甲基丁酸，促进犊牛瘤胃微生物生长繁殖，尤其是纤维分解菌的增殖，从而使瘤胃酶活得以显著改善，使瘤胃发酵趋向于乙酸发酵类型，瘤胃发酵产物乙酸和丁酸显著增加，瘤胃上皮和小肠粘膜组织形态发生明显变化，上调了瘤胃上皮组织生长激素受体、胰岛素受体和生酮作用酶mRNA以及生酮作用酶蛋白质的表达，上调了小肠粘膜生长激素受体、胰岛素受体和钠-葡萄糖转运载体1mRNA及钠-葡萄糖转运载体1的蛋白质表达，从而刺激瘤胃上皮和小肠粘膜的发育，促使瘤胃和小肠粘膜代谢功能发生显著改善。此项研究结果揭示了犊牛断奶前后瘤胃上皮和小肠黏膜组织形态和代谢功能变化及异丁酸、异戊酸和2-甲基丁酸的调控机制，与此同时，发现补充6 g/d的异丁酸、异戊酸和2-甲基丁酸，效果最佳，犊牛增重提高了27.3%，饲料效率提高14.5%。该项成果不仅对研究犊牛胃肠道发育调控及其在生产中应用有重要指导作用，而且可为深入开展犊牛断奶前后和瘤胃发育期间小肠应答的研究奠定理论基础。