研究简报 Short Communications

山羊瘤胃与粪便微生物多样性

  • 王继文 ,
  • 王立志 ,
  • 闫天海 ,
  • 郭伟 ,
  • 徐琴
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  • 四川农业大学动物营养研究所, 雅安 625014

收稿日期: 2015-02-02

  网络出版日期: 2015-08-13

基金资助

四川省科技厅国际合作项目"畜禽温室气体排放量检测及减排技术研究"(2013HH0043)

Diversity of Ruminal and Fecal Microbiota of Goat

  • WANG Jiwen ,
  • WANG Lizhi ,
  • YAN Tianhai ,
  • GUO Wei ,
  • XU Qin
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  • Animal Nutrition Institute, Sichuan Agricultural University, Ya'an 625014, China

Received date: 2015-02-02

  Online published: 2015-08-13

摘要

本试验旨在应用高通量测序技术比较山羊瘤胃和粪便微生物的结构与组成。选取6只10月龄波尔山羊,饲喂精粗比为3:7的饲粮14 d后采集瘤胃液(R组)和粪便样品(F组)(每组6个重复,每个样品为1个重复)。提取总DNA后用古菌/细菌16S rRNA通用引物扩增V4~V5区,并用Illumina MiSeq平台测序。结果表明:1)共获得有效序列227 729条,聚类后得13 601个运算分类单位(OTU)。2)所得OTU经物种注释99.337%被归类为细菌界,F组相对丰度最高优势菌门为厚壁菌门(65.400%),R组相对丰度最高优势菌门为拟杆菌门(60.188%)。3)从所有样品中共检测到129个科,F组中相对丰度最高为瘤胃球菌科(37.705%),极显著高于R组(P<0.01),而R组中,相对丰度最高的为普雷沃氏菌科(29.959%),极显著高于F组(P<0.01)。4)在粪便样品和瘤胃液样品种共检测到258个属,F组相对丰度最高的属为瘤胃球菌科未分类的属(26.914%),R组相对丰度最高为普雷沃菌属(28.621%)。5)所有12个样品间共发现了14个共享属,其中相对丰度最高的为厚壁菌门下的梭菌属4(Clostridium_Ⅳ,1.748%)。本试验结果表明瘤胃和粪便中微生物组成存在着较大差异,瘤胃中还有许多未被分类鉴定且相对丰度较高的微生物,需要进一步研究。

本文引用格式

王继文 , 王立志 , 闫天海 , 郭伟 , 徐琴 . 山羊瘤胃与粪便微生物多样性[J]. 动物营养学报, 2015 , 27(8) : 2559 -2571 . DOI: 10.3969/j.issn.1006-267x.2015.08.030

Abstract

This experiment was conducted to explore structure and composition of ruminal and fecal microbiota of goat. Six ten-month-old Boer goats were fed a diet consisting of 30% concentrate and 70% roughage. After 14-day experiment, the rumen fluid (R group) and feces (F group) were sampled [each group had 3 replicates (samples)]. Total DNA of rumen content and feces samples were extracted, and universal prokaryote primers were used to target the V4 to V5 hypervariable region of 16S rRNA, finally the products were sequenced on MiSeq Illumina sequencing platform. The results showed as follows: 1) a total of 227 729 sequences across all rumen content and fecal samples were generated, and the total number of operational taxonomic units (OTUs) detected by the cluster analysis reached 13 601. 2) The 99.337% of all OTUs belonged to bacteria. The most abundant phyla in F and R groups was Firmicutes(65.400%) and Bacteroidetes (60.188%), respectively. 3) One hundred and twenty nine families were detected in all samples. The most abundant families found in F group was Ruminococcaceae (37.705%), which was extremely significantly higher than that in R group (P<0.01); the most abundant families found in R group was Prevotellaceae (29.959%), which was extremely significantly higher than that in F group (P<0.01). 4) Two hundred and twenty nine genera were detected in all samples. The most abundant genus found in F and R groups was unclassified Ruminococcaceae (26.914%) and Prevotella (28.621%), respectively. 5) The analysis revealed 14 genera shared by all 12 samples. Among them, the most abundant genus was Clostridium_Ⅳ(1.748%). It is concluded that there is a significant difference between ruminal and fecal microbiota, and there are many unclassified microbiomes that have high relative abundance in the rumen, which need further investigations.

参考文献

[1] MACKIE R I,WHITE B A.Recent advances in rumen microbial ecology and metabolism:potential impact on nutrient output[J].Journal of Dairy Science,1990,73(10):2971-2995.  

[2] RUSSELL J B,RYCHLIK J L.Factors that alter rumen microbial ecology[J].Science,2001,292(5519):1119-1122.  

[3] EL-MEADAWAY A,MIR Z,MIR P S,et al.Relative efficacy of inocula from rumen fluid or faecal solution for determining in vitro digestibility and gas production[J].Canadian Journal of Animal Science,1998,78(4):673-679.  

[4] HESPELL R B,AKIN D E,DEHORITY B A.Bacteria,fungi,and protozoa of the rumen[C]//MACKIE R I,WHITE B A,ISAACSON R E.Gastrointestinal microbiology.New York:Chapman and Hall,1997.

[5] YANG L Y,CHEN J,CHENG X L,et al.Phylogenetic analysis of 16S rRNA gene sequences reveals rumen bacterial diversity in Yaks(Bos grunniens)[J].Molecular Biology Reports,2010,37(1):553-562.  

[6] PACE N R.A molecular view of microbial diversity and the biosphere[J].Science,1997,276(5313):734-740.  

[7] EDWARDS J E,HUWS S A,KIM E J,et al.Characterization of the dynamics of initial bacterial colonization of nonconserved forage in the bovine rumen[J].FEMS Microbiology Ecology,2007,62(3):323-335.  

[8] HIGHLANDER S K.High throughput sequencing methods for microbiome profiling:application to food animal systems[J].Animal Health Research Reviews,2012,13(01):40-53.  

[9] SANDRI M,MANFRIN C,PALLAVICINI A,et al.Microbial biodiversity of the liquid fraction of rumen content from lactating cows[J].Animal,2014,8(04):572-579.  

[10] REY M,ENJALBERT F,COMBES S,et al.Establishment of ruminal bacterial community in dairy calves from birth to weaning is sequential[J].Journal of Applied Microbiology,2014,116(2):245-257.  

[11] DE OLIVEIRA M N V,JEWELL K A,FREITAS F S,et al.Characterizing the microbiota across the gastrointestinal tract of a Brazilian Nelore steer[J].Veterinary Microbiology,2013,164(3):307-314.

[12] 张丽英.饲料分析及饲料质量检测技术[M].北京:中国农业大学出版社,2003.

[13] CAPORASO J G,LAUBER C L,WALTERS W A,et al.Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample[J].Proceedings of the National Academy of Sciences,2011,108(Suppl.1):4516-4522.  

[14] CAPORASO J G,KUCZYNSKI J,STOMBAUGH J,et al.QIIME allows analysis of high-throughput community sequencing data[J].Nature Methods,2010,7(5):335-336.  

[15] SCHLOSS P D,WESTCOTT S L,RYABIN T,et al.Introducing mothur:open-source,platform-independent,community-supported software for describing and comparing microbial communities[J].Applied and Environmental Microbiology,2009,75(23):7537-7541.  

[16] EDGAR R C.Search and clustering orders of magnitude faster than BLAST[J].Bioinformatics,2010,26(19):2460-2461.  

[17] CAPORASO J G,BITTINGER K,BUSHMAN F D,et al.PyNAST:a flexible tool for aligning sequences to a template alignment[J].Bioinformatics,2010,26(2):266-267.  

[18] COLE J R,WANG Q,CARDENAS E,et al.The Ribosomal Database Project:improved alignments and new tools for rRNA analysis[J].Nucleic Acids Research,2009,37(suppl.1):D141-D145.  

[19] DOWD S E,CALLAWAY T R,WOLCOTT R D,et al.Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing(bTEFAP)[J].BMC Microbiology,2008,8(1):125.  

[20] LETTAT A,NOZIÈRE P,SILBERBERG M,et al.Rumen microbial and fermentation characteristics are affected differently by bacterial probiotic supplementation during induced lactic and subacute acidosis in sheep[J].BMC Microbiology,2012,12(1):142.  

[21] VAN BAALE M J,SARGEANT J M,Gnad D P,et al.Effect of forage or grain diets with or without monensin on ruminal persistence and fecal Escherichia coli O157:H7 in cattle[J].Applied and Environmental Microbiology,2004,70(9):5336-5342.  

[22] VAN DONKERSGOED J,GRAHAM T,GANNON V.The prevalence of verotoxins,Escherichia coli O157:H7,and Salmonella in the feces and rumen of cattle at processing[J].The Canadian Veterinary Journal,1999,40(5):332.

[23] HUO W,ZHU W,MAO S.Impact of subacute ruminal acidosis on the diversity of liquid and solid-associated bacteria in the rumen of goats[J].World Journal of Microbiology and Biotechnology,2014,30(2):669-680.  

[24] MIN B R,SOLAIMAN S,SHANGE R,et al.Gastrointestinal bacterial and methanogenic archaea diversity dynamics associated with condensed tannin-containing pine bark diet in goats using 16S rDNA amplicon pyrosequencing[J/OL].International Journal of Microbiology,2014.http://dx.doi.org/10.1155/2014/141909.

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

[26] JAMI E,MIZRAHI I.Composition and similarity of bovine rumen microbiota across individual animals[J].PloS One,2012,7(3):e33306.  

[27] STEVENSON D M,WEIMER P J.Dominance of Prevotella and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR[J].Applied Microbiology and Biotechnology,2007,75(1):165-174.  

[28] BEKELE A Z,KOIKE S,KOBAYASHI Y.Genetic diversity and diet specificity of ruminal Prevotella revealed by 16S rRNA gene-based analysis[J].FEMS Microbiology Letters,2010,305(1):49-57.  

[29] AVGUTIN 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.

[30] 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.  

[31] WOOD T M,WILSON C A,STEWART C S.Preparation of the cellulase from the cellulolytic anaerobic rumen bacterium Ruminococcus albus and its release from the bacterial cell wall[J].Biochemical Journal,1982,205:129-137.  

[32] DOERNER K C,WHITE B A.Assessment of the endo-1,4-beta-glucanase components of Ruminococcus flavefaciens FD-1[J].Applied and Environmental Microbiology,1990,56(6):1844-1850.

[33] GIRIJA D,DEEPA K,XAVIER F,et al.Analysis of cow dung microbiota-a metagenomic approach[J].Indian Journal of Biochemistry,2013,12:372-378.

[34] DE JESÙS-LABOY K M,GODOY-VITORINO F,PICENO Y M,et al.Comparison of the fecal microbiota in feral and domestic goats[J].Genes,2011,3(1):1-18.

[35] GU S,CHEN D,ZHANG J N,et al.Bacterial community mapping of the mouse gastrointestinal tract[J].PloS One,2013,8(10):e74957.  

[36] SUEN G,STEVENSON D M,BRUCE D C,et al.Complete genome of the cellulolytic ruminal bacterium Ruminococcus albus 7[J].Journal of Bacteriology,2011,193(19):5574-5575.  

[37] FRANK D N,AMAND A L S,FELDMAN R A,et al.Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases[J].Proceedings of the National Academy of Sciences,2007,104(34):13780-13785.  

[38] HUWS S A,KIM E J,LEE M R F,et al.As yet uncultured bacteria phylogenetically classified as Prevotella,Lachnospiraceae incertae sedis and unclassified Bacteroidales,Clostridiales and Ruminococcaceae may play a predominant role in ruminal biohydrogenation[J].Environmental Microbiology,2011,13(6):1500-1512.  

[39] DEFNOUN S,AMBROSIO M,GARCIA J L,et al.Degradation of cinnamate via β-oxidation to benzoate by a defined,syntrophic consortium of anaerobic bacteria[J].Current Microbiology,2003,46(1):0047-0052.  

[40] FREY J C,PELL A N,Berthiaume R,et al.Comparative studies of microbial populations in the rumen,duodenum,ileum and faeces of lactating dairy cows[J].Journal of Applied Microbiology,2010,108(6):1982-1993.

[41] 冯仰廉.反刍动物营养学[M].北京:科学出版社,2004.

[42] WILLIAMS A G,COLEMAN G S.The rumen protozoa[M].New York:Springer-Verlag; 1992.
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