本研究旨在基于Illumina MiSeq测序技术分析葡萄籽原花青素对奶牛体外瘤胃发酵产甲烷菌区系的影响.试验分为6组, 以500 g精粗比为40:60的全混合日粮为发酵底物, 分别添加0 (对照) 、0.1、0.2、0.3、0.4、0.5 g/kg的葡萄籽原花青素.体外发酵24 h后提取各发酵液总DNA, 采用Illumina MiSeq PE300高通量测序平台进行测序.结果表明:1) 与对照组相比, 添加0.1、0.2 g/kg葡萄籽原花青素降低了产甲烷菌的分类操作单元 (OTUs) 数量, 添加0.3、0.4、0.5 g/kg葡萄籽原花青素增加了产甲烷菌的OTUs数量, 但差异并不显著 (P>0.05) ;葡萄籽原花青素添加量对Chao1指数和Shannon指数均无显著影响 (P>0.05) , 即对产甲烷菌的多样性没有显著影响.2) 在门水平上, 与对照组相比, 添加0.3、0.4、0.5 g/kg葡萄籽原花青素显著降低了广古菌门的丰度 (P<0.05) .在科水平上, 与对照组相比, 甲烷杆菌科的丰度随着葡萄籽原花青素添加量的增加有降低的趋势, 且在添加量为0.4 g/kg时丰度显著降低 (P<0.05) ; Methanomassiliicoccaceae的丰度随着葡萄籽原花青素添加量的增加而增加, 且当添加量为0.3、0.4 g/kg时显著增加 (P<0.05) .在属水平上, 与对照组相比, 甲烷短杆菌属的丰度随着葡萄籽原花青素添加量的增加而降低, 且当添加量为0.4 g/kg时显著降低 (P<0.05) , 当添加量为0.5 g/kg时有回升的趋势, 但是仍低于对照组;甲烷杆菌属的丰度随着葡萄籽原花青素添加量的增加而降低, 且当添加量为0.4、0.5 g/kg时显著降低了该属的丰度 (P<0.05) ; Candidatus_Methanoplasma的丰度随着葡萄籽原花青素添加量的增加有上升趋势, 且当添加量为0.3、0.4 g/kg时显著增加了该属的丰度 (P<0.05) ;甲烷球形菌属的丰度随着葡萄籽原花青素添加量的增加而降低, 且当添加量为0.3、0.4、0.5 g/kg时显著降低了该属的丰度 (P<0.05) .由此可知, 添加葡萄籽原花青素在门、科、属水平上均对奶牛体外瘤胃产甲烷菌区系有一定的影响.%The present study was designed to explore effects of grape seed procyanidine on Methanogens flora of in vitro rumen fermentation of dairy cows using Illumina MiSeq sequencing technology.The trial was divided into 6 groups.Total mixed ration with the concentrate to forage ratio at 40:60 was used as the fermentation substrate and added grape seed procyanidin at levels of 0 (control) , 0.1, 0.2, 0.3, 0.4 and 0.5 g/kg, respectively.Total DNA was extracted from fermentation liquor after 24 h fermentation, and sequenced by Illumina MiSeq PE300 high-throughput sequencing platform.The results showed as follows:1) compared with the control group, the supplementation of grape seed procyanidins at 0.1, 0.2 g/kg significantly decreased the operational taxonomic units (OTUs) , while the supplementation of grape seed procyanidins at 0.3, 0.4 and 0.5 g/kg increased the OTUs, but did not have significant difference (P>0.05) ;the supplementation of grape seed procyanidins did not significantly affect Chao1 index and Shannon index (P>0.05) , namely did not significantly affect the diversity of Methanogens.2) At phyla level, compared with the control group, the supplementation of grape seed procyanidine at 0.3, 0.4 and 0.5 g/kg significantly decreased the abundance of Euryarchaeota (P<0.05) .At family level, compared with the control group, the abundance of Methanobacteriaceae had a decreased trend with the supplementation of grape seed procyanidine increasing, and when the supplementation was at 0.4 g/kg, the abundance of it significantly decreased (P<0.05) ;the abundance of Methanomassiliicoccaceae increased with the supplementation of grape seed procyanidine increasing, and when the supplementation was 0.3 and 0.4 g/kg, the abundance of it significantly increased (P<0.05) .At genus level, compared with the control group, the abundance of Methanobrevibacter decreased with the supplementation of grape seed procyanidine increasing, and when the supplementation was 0.4 g/kg, the abundance of it significantly decreased (P<0.05) , when the supplementation was 0.5 g/kg, the abundance of it had an increasing trend, but was lower than that in control group;the abundance of Methanobacterium decreased with the supplementation of grape seed procyanidine increasing, and when the supplementation was 0.4 and 0.5 g/kg, the abundance of it significantly decreased (P<0.05) ;the abundance of Candidatus_Methanoplasma had an increasing trend with the supplementation of grape seed procyanidine increasing, and when supplementation was 0.3 and 0.4 g/kg, the abundance of it significantly increased (P<0.05) ;the abundance of Methanosphaera decreased with the supplementation of grape seed procyanidine increasing, and when the supplementation was 0.3, 0.4 and 0.5 g/kg, the abundance of it significantly decreased (P< 0.05) .Therefore, the supplementation of grape seed procyanidine has significant effects on Methanogens at phyla, family and genus levels.
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