分子营养 Molecular Nutrition

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

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  • 南京农业大学动物科技学院, 南京 210095
李烨青(1992-),男,山西晋中人,硕士研究生,研究方向为牛生产学。E-mail:931815482@qq.com

收稿日期: 2018-03-15

  网络出版日期: 2018-10-20

基金资助

江苏省产学研前瞻性联合研究项目(205071-07)

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

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  • College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China

Received date: 2018-03-15

  Online published: 2018-10-20

摘要

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。

本文引用格式

李烨青, 奚雨萌, 曾涵芳, 张林, 陈孟姣, 韩兆玉 . 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[J]. 动物营养学报, 2018 , 30(10) : 4059 -4070 . DOI: 10.3969/j.issn.1006-267x.2018.10.031

Abstract

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.

参考文献

[1] MUYZER G,DE WAAL E C,UITTERLINDEN A G.Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA[J].Applied and Environmental Microbiology,1993,59(3):695-700.

[2] GETACHEW G,BLVMMEL M,MAKKAR H P S,et al.In vitro gas measuring techniques for assessment of nutritional quality of feeds:a review[J].Animal Feed Science and Technology,1998,72(3/4):261-281.

[3] 朱智,朱伟云.不同精粗比底物时添加大蒜油对体外瘤胃微生物发酵的影响[J].南京农业大学学报,2012,35(2):125-130.

[4] 陈亚迎,刘壮,吕朋安,等.溶菌酶对瘤胃体外发酵、甲烷生成及微生物菌群结构的影响[J].微生物学报,2017,57(5):758-768.

[5] HUMER E,ADITYA S,KALTENEGGER A,et al.Graded substitution of grains with bakery by-products modulates ruminal fermentation,nutrient degradation,and microbial community composition in vitro[J].Journal of Dairy Science,2018,101(4):3085-3098.  

[6] JOHNSON R R,DEHORITY B A,MCCLURE K E,et al.A comparison of in vitro fermentation and chemical solubility methods in estimating forage nutritive value[J].Journal of Animal Science,1964,23(4):1124-1128.  

[7] CUNHA I S,BARRETO C C,COSTA O Y A,et al.Bacteria and archaea community structure in the rumen microbiome of goats (Capra hircus) from the semiarid region of Brazil[J].Anaerobe,2011,17(3):118-124.  

[8] THEODOROU M K,WILLIAMS B A,DHANOA M S,et al.A simple gas production method using a pressure transducer to determine the fermentation kinetics of ruminant feeds[J].Animal Feed Science and Technology,1994,48(3/4):185-197.

[9] 朱伟云,毛胜勇,王全军,等.厌氧真菌体外发酵筛选技术的研究[J].南京农业大学学报,2001,24(3):44-48.

[10] WANG Q,GARRITY G M,TIEDJE J M,et al.Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy[J].Applied and Environmental Microbiology,2007,73(16):5261-5267.  

[11] PRUESSE E,QUAST C,KNITTEL K,et al.SILVA:a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB[J].Nucleic Acids Research,2007,35(21):7188-7196.  

[12] GENTLEMAN R C,CAREY V J,BATES D M,et al.Bioconductor:open software development for computational biology and bioinformatics[J].Genome Biology,2004,5(10):R80.

[13] EDGAR R C.UPARSE:highly accurate OTU sequences from microbial amplicon reads[J].Nature Methods,2013,10(10):996-998.  

[14] BHATT V D,DANDE S S,PATIL N V,et al.Molecular analysis of the bacterial microbiome in the forestomach fluid from the dromedary camel (Camelus dromedarius)[J].Molecular Biology Reports,2013,40(4):3363-3371.  

[15] LI Z P,LIU H L,LI G Y,et al.Molecular diversity of rumen bacterial communities from tannin-rich and fiber-rich forage fed domestic Sika deer (Cervus nippon) in China[J].BMC Microbiology,2013,13:151.

[16] PITTA D W,KUMAR S,VEICCHARELLI B,et al.Bacterial diversity associated with feeding dry forage at different dietary concentrations in the rumen contents of Mehshana buffalo (Bubalus bubalis) using 16S pyrotags[J].Anaerobe,2014,25:31-41.

[17] QIN J J,LI R Q,RAES J,et al.A human gut microbial gene catalogue established by metagenomic sequencing[J].Nature,2010,464(7285):59-65.  

[18] TUN H M,BRAR M S,KHIN N,et al.Gene-centric metagenomics analysis of feline intestinal microbiome using 454 junior pyrosequencing[J].Journal of Microbiological Methods,2012,88(3):369-376.  

[19] TAJIMA K,ARAI S,OGATA K,et al.Rumen bacterial community transition during adaptation to high-grain diet[J].Anaerobe,2000,6(5):273-284.  

[20] SINGH K M,AHIR V B,TRIPATHI A K,et al.Metagenomic analysis of Surti buffalo (Bubalus bubalis) rumen:a preliminary study[J].Molecular Biology Reports,2012,39(4):4841-4848.  

[21] PENG S,YINJ G,LIU X L,et al.First insights into the microbial diversity in the omasum and reticulum of bovine using Illumina sequencing[J].Journal of Applied Genetics,2015,56(3):393-401.  

[22] QUAISER A,OCHSENREITER T,LANZ C,et al.Acidobacteria form a coherent but highly diverse group within the bacterial domain:evidence from environmental genomics[J].Molecular Microbiology,2003,50(2):563-575.  

[23] RUSSELL J B,DIEZ-GONZALEZ F.The effects of fermentation acids on bacterial growth.[J].Advances in Microbial Physiology,1998,39:205-234.

[24] WRIGHT A D G,KLIEVE A V.Does the complexity of the rumen microbial ecology preclude methane mitigation?[J].Animal Feed Science and Technology,2011,166-167:248-253.

[25] LIU J H,ZHANG M L,XUE C X,et al.Characterization and comparison of the temporal dynamics of ruminal bacterial microbiota colonizing rice straw and alfalfa hay within ruminants[J].Journal of Dairy Science,2016,99(12):9668-9681.  

[26] NOCEK J E.Bovine acidosis:implications on laminitis[J].Journal of Dairy Science,1997,80(5):1005-1028.  

[27] BULYGINA E S,GALCHENKO V F,GOVORUKHINA N I,et al.Taxonomic studies on methylotrophic bacteria by 5S ribosomal RNA sequencing[J].Journal of General Microbiology,1990,136(3):441-446.  

[28] KIM M,MORRISON M,YU Z T.Status of the phylogenetic diversity census of ruminal microbiomes[J].FEMS Microbiology Ecology,2011,76(1):49-63.  

[29] EVANS N J,BROWN J M,MURRAY R D,et al.Characterization of novel bovine gastrointestinal tract Treponema isolates and comparison with bovine digital dermatitis treponemes[J].Applied and Environmental Microbiology,2011,77(1):138-147.  

[30] RAMŠAK A,PETERKA M,TAJIMA K,et al.Unravelling the genetic diversity of ruminal bacteria belonging to the CFB phylum[J].FEMS Microbiology Ecology,2000,33(1):69-79

[31] PURUSHE J,FOUTS D E,MORRISON M,et al.Comparative genome analysis of Prevotella ruminicola,and Prevotella bryantii:insights into their environmental niche[J].Microbial Ecology,2010,60(4):721-729.  

[32] AVGUŠTIN G,WALLACE R J,FLINT H J.Phenotypic diversity among ruminal isolates of Prevotella ruminicola:proposal of Prevotella brevis sp.nov.Prevotella bryantii sp.nov.and Prevotella albensis sp.nov.and redefinition of Prevotella ruminicola[J].International Journal of Systematic Bacteriology,1997,47(2):284-288.  

[33] FLINT H J,BAYER E A,RINCON M T,et al.Polysaccharide utilization by gut bacteria:potential for new insights from genomic analysis[J].Nature Reviews Microbiology,2008,6(2):121-131.  

[34] NEWBOLD C J,LÓPEZ S,NELSON N,et al.Propionate precursors and other metabolic intermediates as possible alternative electron acceptors to methanogenesis in ruminal fermentation in vitro[J].British Journal of Nutrition,2005,94(1):27-35.  

[35] 冯仰廉.反刍动物营养学[M].北京:中国农业出版社,2004:1-12.

[36] 雷冬至, 金曙光, 乌仁塔娜. 用体外产气法评价不同粗饲料与相同精料间的组合效应[J]. 饲料工业, 2009, 30(3):30-33.

[37] ZHOU X, ZEITZ J O, MEILE L, et al. Influence of pH and the degree of protonation on the inhibitory effect of fatty acids in the ruminal methanogen Methanobrevibacter ruminantium strain M1[J]. Journal of Applied Microbiology, 2016, 119(6):1482-1493.
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