【作者】 刘玉洁；

【导师】 毛胜勇；

【作者基本信息】 南京农业大学 ， 动物营养与饲料科学， 2015， 硕士

【摘要】 反刍动物生产中,长期饲喂高谷物日粮易导致瘤胃微生物区系失调和代谢紊乱,引发一系列动物健康问题,给反刍动物生产带来巨大经济损失。本文在已发现长期饲喂高谷物日粮导致瘤胃上皮受损与瘤胃内环境改变的基础上,进一步研究了高谷物曰粮对山羊瘤胃微生物区系和瘤胃、肝脏、血液中代谢产物的影响,以期更全面的阐明高谷物日粮危害反刍动物健康的微生物学与代谢组学相关机制,拟为反当动物健康养殖提供理论依据。试验分以下三部分进行。1高谷物日粮对山羊瘤胃液中微生物区系的影响本试验旨在研究高谷物日粮对山羊瘤胃液微生物多样性和组成的影响。试验选用10头装有永久性瘤胃瘘管的去势公山羊,随机分为两组,对照组为干草日粮(Hay;0%谷物;n = 5),处理组为高谷物日粮(HG;65%谷物;n = 5),在试验期的第50天,采集瘤胃内容物,四层纱布过滤,采用高通量测序技术手段来评定了瘤胃液中微生物区系的变化情况。结果显示,饲喂高谷物日粮显著降低了瘤胃液微生物菌群多样性(P<0.05);导致瘤胃液中部分淀粉分解菌包括Selenomonas,Succiniclasticum,Ruminococcus和 unclassified Veillonellaceae 的相对丰度显著升高(P<0.05);相反,干草组中Butyrivibrio,unclassified Lachnospiraceae,unclassified Rikenellaceae,unclassified Erysipelotrichaceae,unclassified Ruminococcaceae 和 unclassified Mollicutes 相对丰度显著高于(P<0.05)高谷物组;同时PCA分析结果表明,干草组与高谷物组间的微生物区系存在显著差异。结果表明,高谷物日粮降低了山羊瘤胃细菌菌群的多样性和组成,说明高谷物日粮会导致瘤胃微生物区系紊乱,进而影响反刍动物瘤胃健康。2高谷物日粮对山羊瘤胃上皮微生物区系的影响本试验拟利用高通量测序技术手段,研究高谷物日粮对山羊瘤胃上皮微生物组成的影响。利用试验1中的山羊,在实验期的第50天屠宰时采取瘤胃上皮组织,用于高通量测序分析,比较高谷物组和干草组瘤胃上皮微生物区系的差异。结果显示:饲喂高谷物日粮导致山羊瘤胃上皮微生物组成发生剧烈改变,在属水平上,高谷物日粮显著增加了(P<0.05)Butyrivibrio,unclassified Clostridiales,Mogibacterium,unclassified Anaerolineaceae,Succiniclasticum 和Ruminococcus 的相对丰度,而 unclassified Ruminococcaceae,unclassified Rikenellaceae,unclassified Erysipelotrichaceae,Howardella和unclassified Neisseriaceae的相对丰度在高谷物组中显著降低(P<0.05)。同时,主成分分析结果表明,两组间瘤胃上皮细菌菌群结构与组成存在显著差异。结果说明,饲喂高谷物日粮引起山羊瘤胃内pH值下降,致栖息在瘤胃上皮上的菌落结构发生紊乱。3高谷物日粮对山羊瘤胃液、肝脏和血清中代谢物的影响本试验利用基于GC-MS的代谢组学技术,研究了高谷物日粮对山羊瘤胃、肝脏、血液中代谢物组成的变化。利用试验1的山羊,试验期的第50天取瘤胃液、颈静脉外周血和肝脏样品用于GC-MS代谢组分析。结果表明,在瘤胃液、肝脏和血清中,分别检测到84、83和69种代谢物;PCA和PLS-DA分析结果表明,高谷物日粮组和干草组的代谢物可被明显分开;统计分析表明,瘤胃液、肝脏和血清中分别有12、10和9种代谢物的含量差异显著(VIP>1.3;P<0.05),通路分析表明,瘤胃中这些差异代谢物主要参与氨基酸代谢和糖代谢;同时,高谷物组山羊瘤胃液中腐胺和LPS浓度增加;对肝脏炎症因子的定量结果表明,高谷物组IL-10的相对表达量有升高的趋势(0.05<P<0.10)。结果说明,高谷物日粮导致瘤胃中氨基酸和糖代谢改变,机体有毒或抗炎物质含量升高,肝脏中炎症因子上调。结果初步说明饲喂高谷物日粮导致山羊机体代谢改变,并对瘤胃与宿主健康造成了危害。更多还原

【Abstract】 In ruminants industry,long-term high-grain diet feeding results in rumen microbial flora imbalance,metabolic disorders,impairs animal health,and causes economic loss.We have found that long-term diet feeding resulted in the damage of rumen epithelial and changes of the rumen environment,and the objectives of this paper was to investigate the impact of high-grain diet feeding on bacterial composition associated with rumen fluid and rumen epithelium,and was to explore the changes in metabolites of rumen fluid,liver and blood during high-grain feeding which allows us to make sure the mechanisms of metabolomics relating to host health during high-grain feeding in goats,and provides a theoretical basis for the research of ruminant health.1 Effect of high-grain diet on rumen microbiota in goatsThis study was designed to study the influence of high-grain diet feeding on bacterial diversity and composition in rumen fluid of goats.Ten ruminally fistulated,castrated male goats were randomly assigned to either a hay diet(0%grain;n=5)or high-grain(HG)diet group(65%grain;n = 5)with continuous feeding of 7 weeks.On day 50,a representative sample of rumen digesta(at least 200 ml)was used to determine the pH value and was then strained through 4 layers of cheese cloth to assess changes in bacterial composition in rumen fluid using bar-coded 454-pyrosequencing techniques.The results showed that HG diet feeding decreased the diversity of bacterial community in rumen fluid significantly(P<0.05);HG feeding increased percentage of amylolytic bacteria,such as Selenomonas,Succiniclasticum,Ruminococcus and unclassified Veillonellaceae(P<0.05),while decreased the relative abundance of Butyrivibrio,unclassified Lachnospiraceae,unclassified Rikenellaceae,unclassified Erysipelotrichaceae,unclassified Ruminococcaceae and unclassified Mollicutes(P<0.05).Furthermore,Principal component analysis showed that ruminal bacterial composition of HG group were significant different from that of hay group.The results indicated that HG feeding affected diversity and composition of bacterial community in rumen fluid,caused disorder of bacterial flora in rumen,and impaired the health of ruminants.2 Effect of high-grain diet on rumen epithelial bacterial in goatsThe high-throughput sequencing technique was used to study the effect of HG diet feeding on rumen epithelial bacterial composition in this experiment.On day 50,ten goats were slaughtered,then rumen epithelial tissue was collected immediately and used for high-throughput sequencing analysis to evaluate the difference of rumen epithelial microflora composition between hay and HG group.The results showed that HG diet feeding caused dramatic changes in rumen epithelial bacterial composition;at the genus level,HG diet feeding increased the relative abundance of Butyrivibrio,unclassified Clostri diales,Mogibacterium,unclassified Anaerolineaceae,Succiniclasticum and Ruminococcus significantly(P<0.05),however,the relative abundance of Howardella,unclassified Ruminococcaceae,unclassified Neisseriaceae,unclassified Rikenellaceae and unclassified Erysipelotrichaceae showed a lower percentage in HG group(P<0.05).The research indicated that HG diet feeding caused a drop in pH in the rumen,perturbing the bacteria associated with ruminal epithelium.3 GC-MS metabolomics analysis of metabolites in rumen fluid,liver,and serum following high-grain diets in goatsThe purpose of this experiment was to observe the changes of metabolites in rumen,liver and serum during HG feeding in goats.On day 50,rumen fluid,liver and peripheral blood samples were collected for GC-MS analysis.The results showed that there were 84,83 and 69 kinds of metabolites detected in the rumen fluid,liver and serum;according to the results of PCA and PLS-DA score plot,the metabolites in HG group were separated from hay group;combining PLS-DA analysis with independent sample T-test,we found that there were 12,10 and 9 kinds of metabolites separated significantly between hay and HG group in rumen fluid,liver and serum(VIP>1.3;P<0.05),these significant different metabolites mainly involved in amino acid metabolism and glucose metabolism;HG group occupied a higher concentrations of abnormal metabolites,such as putrescine and LPS;The quantitative real-time PCR results showed that the relative expression of IL-10 in the HG group tended to increase(0.05<P<0.10).These results indicated that HG diet feeding impaired rumen metabolism,increased the concentrations of toxic or anti-inflammatory cytokines,and finally affected amino acids and glucose metabolism.These findings indicated that high-grain diet feeding changed the contents of metabolites in goats,causing metabolism disorder,eventually,imposing a threat on ruminants health.更多还原