基于16S rRNA测序分析奶牛体外发酵中瘤胃细菌多样性的变化

Diversity Changes of Bacterial Community in Rumen of Dairy Cows in Vitro Fermentation Analyzed by 16S rRNA Sequencing Technology

本试验旨在通过16S rRNA测序技术,分析奶牛体外发酵瘤胃微生物区系随时间推移的变化规律,为探究瘤胃微生物繁殖规律及完善体外发酵分析方法提供支持。试验使用体外产气法进行发酵。采集3头健康奶牛的新鲜瘤胃液,与人工唾液混合后,注入盛有发酵底物的发酵瓶,39℃水浴振荡培养,分别于0、6、12、24、48 h采集发酵液,用16S rRNA测序技术分析瘤胃细菌变化规律。结果表明:1)所有的序列被鉴定为14个门和59个属。随着时间的变化,在门水平上,纤维杆菌门(Fibrobacteres)、拟杆菌门(Bacteroidetes)和黏胶球形菌门(Lentisphaerae)的相对丰度减少,纤维杆菌门、拟杆菌门在48 h时显著低于其他时间点(P<0.05),黏胶球形菌门在24 h时显著低于其他时间点(P<0.05)。而螺旋体门(Spirochaetae)和变形菌门(Proteobacteria)的相对丰度增加,螺旋体门在48 h时显著高于其他时间点(P <0.05),变形菌门在12、24、48 h时显著高于其他时间点(P<0.05);随着时间的变化,在属水平上,丁酸弧菌属(Butyrivibrio)和瘤胃球菌属(Ruminococcus)的相对丰度增加,均在48 h时显著高于其他时间点(P<0.05);而解琥珀酸菌属(Succiniclasticum)和普雷沃氏菌属(Prevotella)的相对丰度则减少,解琥珀酸菌属在24、48 h显著低于其他时间点(P<0.05),普雷沃氏菌属在48 h时显著低于其他时间点(P<0.05)。2)试验获得15 792个操作分类单位(OTU)并进行多样性分析,稀释曲线达到平台期;Simpson指数和Shannon指数均表明菌群丰度具有先降低后升高的趋势。综上所述,奶牛瘤胃体外发酵过程中,瘤胃细菌主要的门以及属的相对丰度以及多样性均随时间推移发生改变,且因瘤胃细菌功能差异在发酵过程的变化更为显著。因此,在使用体外发酵法进行研究时,应充分考虑瘤胃细菌的功能差异与底物以及人工唾液的营养物质减少引起的发酵环境变化的关系。

This study was aimed to assess the dynamics of microbial bacterial community in the rumen of dairy cows with time in vitro fermentation analyzed by 16S rRNA sequencing technology,which provided a support for exploring the rumen microbial propagation and improving the in vitro fermentation method.The fermentation was carried out using an in vitro gas production method.The rumen liquid from 3 healthy Holstein dairy cows were collected,mixed with artificial saliva,and then injected into fermentation flasks containing fermentation substrates,and incubated at 39℃.Samples were collected at 0,6,12,24,48 h.The dynamics of microbial bacterial community in the rumen was analyzed by 16S rRNA sequencing technology.The results showed as follows:1)a total of 14 phyla and 59 genera were identified from all samples.With the change of time,at the phylum level,the relative abundance of Fibrobacteres,Bacteroidetes and Lentisphaerae declined,that of Fibrobacteres and Bacteroidetes at 48 h was significantly lower than that at other time points(P<0.05),and that of Lentisphaerae at 24 h was significantly lower than that at other time points(P<0.05).The relative abundance of Spirochaetae and Proteobacteria increased,that of Spirochaetae at 48 h was significantly higher than that at other time points(P<0.05),and that of Proteobacteria at 12,24,48 h was significantly higher than that at other time points(P<0.05).At the genus level,the relative abundance of Butyrivibrio and Ruminococcus increased,that of both them at 48 h was significantly higher than that at other time points(P<0.05);the relative abundance of Succiniclasticum and Prevotella decreased,that of Succiniclasticum at 24,48 h was significantly lower than that at other time points(P<0.05),and that of Prevotella at 48 h was significantly lower than that at other time points(P<0.05).2)The experiments were obtained 15 792 operational taxonomic units(OTU),diversity analysis was carried out,and the dilution curve reached the plateau stage.Simpson index and Shannon index showed that the abundance of bacteria first decreased and then increased.The results showed that the relative abundance and diversity of the main phylum and genus of rumen bacteria changed with time and the changes in fermentation process were more significant due to differences in rumen bacterial function.Therefore,when using the in vitro fermentation method for research,the relationship between the functional difference of rumen bacteria and the fermentation environment changes caused by the reduction of nutrients of substrate and artificial saliva should be fully considered.