采用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
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摘要
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.
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.
引文
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[3]MOSS A R,JOUANY J P,NEWBOLD J.Methane production by rum inants:its contribution to global warm ing[J].Anim al de Zootechnie,2000,49(3):231-253.
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[6]CAO Y,TAKAHASHI T,HORIGUCHI K I,et al.Methane em issions from sheep fed ferm ented or nonferm ented total m ixed ration containing whole-crop rice and rice bran[J].Anim al Feed Science and Technology,2010,157(1/2):72-78.
[7]严淑红,蒋林树.茶皂素对反刍动物瘤胃发酵的影响研究进展[J].中国农学通报,2015,31(5):20-24.
[8]胡伟莲.皂甙对瘤胃发酵与甲烷产量及动物生产性能影响的研究[D].博士学位论文.杭州:浙江大学,2005.
[9]ODONGO N E,BAGG R,VESSIE G,et al.Longterm effects of feeding m onensin on m ethane production in lactating dairy cow s[J].Journal of Dairy Science,2007,90(4):1781-1788.
[10]WANG J K,YE J A,LIU J X.Effects of tea saponins on rum en m icrobiota,rum en ferm entation,m ethane production and grow th perform ance-a review[J].Tropical Anim al Health and Production,2012,44(4):697-706.
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[12]BAYATLI F,AKKU爦D,KILIC E,et al.The protective effects of grape seed extract on MDA,AOPP,apoptosis and eNOS expression in testicular torsion:an experim ental study[J].W orld Journal of Urology,2013,31(3):615-622.
[13]GESSNER D K,WINKLER A,KOCH C,et al.Analysis of hepatic transcript profile and plasm a lipid profile in early lactating dairy cow s fed grape seed and grape m arc m eal extract[J].BMC Genom ics,2017,18(1):253.
[14]高巍,卜登攀,李永刚,等.葡萄籽粕对反刍动物营养价值的综合评定研究[C]//中国畜牧兽医学会动物营养学分会第七届中国饲料营养学术研讨会论文集.郑州:中国畜牧兽医学会动物营养学分会,2014.
[15]周敏,叶子弘,蒋林树.瘤胃氢化多不饱和脂肪酸的影响因素[J].中国农学通报,2010,26(8):38-44.
[16]杨德莲,童津津,张婕,等.葡萄籽原花青素对奶牛瘤胃体外发酵参数及微生物区系的影响[J].动物营养学报,2018,30(2):717-725.
[17]BRGMANN H,PESARO M,WIDMER F,et al.A strategy for optim izing quality and quantity of DNA extracted from soil[J].Journal of Microbiological Methods,2001,45(1):7-20.
[18]CAPORASO J G,KUCZYNSKI J,STOMBAUGH J,et al.QIIME allow s analysis of high-throughput com m unity sequencing data[J].Nature Methods,2010,7(5):335-336.
[19]DRAY S,DUFOUR A,LEEUW J D,et al.The ade4package:im plem enting the duality diagram for ecologists[J].Journal of Statistical Software,2007,22(4):1-20.
[20]邸凌峰,曹雪,秦炜赜,等.两种含单宁饲草对反刍动物应用的价值研究[J].饲料工业,2017,38(7):43-50.
[21]李晓鹏.缩合单宁对体外发酵特性的影响[D].硕士学位论文.兰州:甘肃农业大学,2009.
[22]王慧玲,王小平,尕藏桑智,等.单宁酸对绵羊瘤胃体外发酵和甲烷产量的影响[J].中国草食动物科学,2013,33(6):46-48.
[23]ANANTASOOK N.日粮添加含单宁和皂苷的雨树荚对奶牛瘤胃发酵和甲烷产量调控的影响[J].王聪译.中国畜牧兽医,2014,41(2):152.
[24]HOOK S E,STEELE MA,NORTHWOOD K S,et al.Im pact of high-concentrate feeding and low rum inal pH on m ethanogens and protozoa in the rum en of dairy cow s[J].Microbial Ecology,2011,62(1):94-105.
[25]李志鹏,刘晗璐,司华哲,等.不同粗饲料条件下梅花鹿瘤胃甲烷菌结构的比较分析[J].动物营养学报,2016,28(9):2911-2919.
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[27]TAJIMA K,NAGAMINE T,MATSUI H,et al.Phylogenetic analysis of archaeal 16S rRNA libraries from the rum en suggests the existence of a novel group of archaea not associated with know n m ethanogens[J].FEMS Microbiology Letters,2001,200(1):67-72.
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