单宁与饲用纤维素酶对湖羊瘤胃微生物菌群的影响
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摘要
试验旨在利用高通量测序技术研究添加单宁与饲用纤维素酶对湖羊生长育肥期瘤胃微生物菌群结构的影响,为单宁和饲用纤维素酶在反刍动物生产中更好地利用提供理论依据。选用3月龄生长发育良好、平均体重(19.85±1.45)kg的肉用湖羊36只,随机分成对照组和3个处理组,对照组饲喂基础日粮,处理组分别在基础日粮中加入0.1%单宁(单宁组)、0.1%饲用纤维素酶(纤维素酶组)和0.1%单宁+0.1%饲用纤维素酶(混合组),每组3个重复,每个重复3只湖羊。试验期70d,其中过渡期7d,预试期7d,正饲期56d。试验结束后,采集湖羊瘤胃液,提取细菌总DNA,进行PCR扩增后用Illumina Hi Seq 2500测序平台进行高通量测序。结果表明:(1)12个湖羊瘤胃微生物样品测序共获得957 440对序列,平均每个样品产生55 997条clean tags,过滤嵌合体后共产生502 965条effective tags;各组湖羊瘤胃微生物样品的AvgLen均在419～420之间;(2)各组间湖羊瘤胃微生物的Ace指数和Chao1指数差异均不显著(P>0.05);与对照组相比,单宁组、纤维素酶组和混合组湖羊瘤胃微生物的香农指数显著提高(P<0.05);纤维素酶组湖羊瘤胃微生物的辛普森指数较对照组显著降低(P<0.05);(3)在门水平上,单宁组湖羊瘤胃内拟杆菌门(Bacteroidetes)丰度比对照组显著降低(P<0.05);单宁组、纤维素酶组和混合组湖羊瘤胃内厚壁菌门(Firmicutes)丰度较对照组均呈下降趋势,但差异不显著(P>0.05);(4)在属水平上,单宁组湖羊瘤胃内普雷沃菌属-1(Prevotella_1)丰度降低,与对照组、纤维素酶组差异均显著(P<0.05),但与混合组差异不显著(P>0.05);单宁组和纤维素酶组湖羊瘤胃内瘤胃菌属(Rumen_bacterium)丰度较对照组和混合组提高,且与混合组的差异显著(P<0.05)。综合试验结果,在湖羊日粮中同时加入单宁和饲用纤维素酶可以提高湖羊瘤胃内菌群的多样性和丰度,影响瘤胃菌群结构,缓解单宁对拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes)菌落的抑制;同时混合添加可以缓解单独添加单宁对纤维素分解的抑制作用;在门水平上各组湖羊瘤胃内优势菌群均为拟杆菌门和厚壁菌门;在属水平上各组湖羊瘤胃内优势菌群均为理研菌科-RC9(Rikenellaceae_RC9_gut_group)、细菌(Bacterium)、普雷沃菌属-1和瘤胃菌属。
The purpose of this study was to investigate the effects of adding tannins and feeding cellulase on rumen microbes in the growth and fattening period of Hu sheep using high-throughput sequencing technology,and provide useful data for the better utilization of tannins and cellulase in ruminant production.Thirty-six meat Hu sheep with good growth and average body weight(19.85±1.45)kg at 3 months old were randomly divided into control group and 3treatmentgroups.The control group was fed with basal diet,and three treatment groups were added 0.1%tannin(tannin group),0.1%feeding cellulase(cellulase group)and 0.1%tannin+0.1%feeding cellulase(mixed group)in the basal diet respectively,3replicates per group,each repeat 3sheep.The test period was 70 days,in which the transition period was 7days,the pre-test period was 7days,and the positive feeding period was 56 days.At the end of the experiment,the rumen fluid of Hu sheep was collected,and the total DNA of the bacteria was extracted and PCR amplification was conducted.The amplified products were sequenced by Illumina Hi Seq 2500 for high-throughput sequencing.The results showed that:(1) A total of 957 440 pairs of microbial samples were obtained from 12 rumen samples,with an average of 55 997 clean tags,after filtering chimeras,a total of 502 965 effective tags were generated.AvgLen of rumen microbial samples from each group of sheep were between 419 and 420;(2)The Ace index and Chao1 index of rumen microorganisms in each group had no significant difference(P>0.05);Compared with the control group,the Shannon index of rumen microorganisms in tannin,cellulose and mixed groups were significantly increased(P<0.05).The Simpson index of rumen microorganisms in cellulase group was significantly lower than that in the control group(P<0.05).(3)At the level of phylum:The abundance of Bacteroidetes of tannin group was significantly lower than that of the control group(P<0.05);The abundance of the Firmicutes in tannin,cellulose and mixed group were lower than that of the control group,but the difference was not significant(P>0.05).(4)On the genus level,the abundance of Prevotella_1in tannin group was droped,and there was significant difference compared with the control and cellulase groups(P<0.05),but there was no significant difference with mixed groups(P>0.05).The abundance of Rumen-bacteriumin tannin and cellulase groups were higher than that of the control and mixed group,and the difference with mixed group was significant(P<0.05).In conclusion,adding tannin and cellulase at the same time could improve the diversity and abundance of rumen flora,affect the structure of rumen flora,and alleviate the inhibition of tannin on Bacteroidetes and Firmicutes.Mixed addition could alleviate the inhibition of cellulose decomposition by adding tannin alone.The dominant bacteria in the rumen of each group of Hu sheep were Bacteroidetes and Firmicutes.On the genus level,the dominant bacteria in the rumen of each group were studied Rikenellaceae_RC9_gut_group,Bacterium,Prevotella_1and Rumen_bacterium.
The purpose of this study was to investigate the effects of adding tannins and feeding cellulase on rumen microbes in the growth and fattening period of Hu sheep using high-throughput sequencing technology,and provide useful data for the better utilization of tannins and cellulase in ruminant production.Thirty-six meat Hu sheep with good growth and average body weight(19.85±1.45)kg at 3 months old were randomly divided into control group and 3treatmentgroups.The control group was fed with basal diet,and three treatment groups were added 0.1%tannin(tannin group),0.1%feeding cellulase(cellulase group)and 0.1%tannin+0.1%feeding cellulase(mixed group)in the basal diet respectively,3replicates per group,each repeat 3sheep.The test period was 70 days,in which the transition period was 7days,the pre-test period was 7days,and the positive feeding period was 56 days.At the end of the experiment,the rumen fluid of Hu sheep was collected,and the total DNA of the bacteria was extracted and PCR amplification was conducted.The amplified products were sequenced by Illumina Hi Seq 2500 for high-throughput sequencing.The results showed that:(1) A total of 957 440 pairs of microbial samples were obtained from 12 rumen samples,with an average of 55 997 clean tags,after filtering chimeras,a total of 502 965 effective tags were generated.AvgLen of rumen microbial samples from each group of sheep were between 419 and 420;(2)The Ace index and Chao1 index of rumen microorganisms in each group had no significant difference(P>0.05);Compared with the control group,the Shannon index of rumen microorganisms in tannin,cellulose and mixed groups were significantly increased(P<0.05).The Simpson index of rumen microorganisms in cellulase group was significantly lower than that in the control group(P<0.05).(3)At the level of phylum:The abundance of Bacteroidetes of tannin group was significantly lower than that of the control group(P<0.05);The abundance of the Firmicutes in tannin,cellulose and mixed group were lower than that of the control group,but the difference was not significant(P>0.05).(4)On the genus level,the abundance of Prevotella_1in tannin group was droped,and there was significant difference compared with the control and cellulase groups(P<0.05),but there was no significant difference with mixed groups(P>0.05).The abundance of Rumen-bacteriumin tannin and cellulase groups were higher than that of the control and mixed group,and the difference with mixed group was significant(P<0.05).In conclusion,adding tannin and cellulase at the same time could improve the diversity and abundance of rumen flora,affect the structure of rumen flora,and alleviate the inhibition of tannin on Bacteroidetes and Firmicutes.Mixed addition could alleviate the inhibition of cellulose decomposition by adding tannin alone.The dominant bacteria in the rumen of each group of Hu sheep were Bacteroidetes and Firmicutes.On the genus level,the dominant bacteria in the rumen of each group were studied Rikenellaceae_RC9_gut_group,Bacterium,Prevotella_1and Rumen_bacterium.
引文
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[22]KOIKE S,PAN J,SUZUKI T,et al.Ruminal distribution of the cellulolytic bacteriumFibrobacter succinogenes in relation to its phylogenetic grouping[J].Animal Science Journal,2015,75(5):417-422.
[23]李大彪,张梅梅,于永强,等.单宁和聚乙二醇对绵羊和山羊瘤胃纤维降解菌数量的影响[J].动物营养学报,2015,27(2):596-605.LI D B,ZHANG M M,YU Y Q,et al.Effects of tannin and polyethylene glycol on the number of rumen fiber-degrading bacteria in sheep and goats[J].Chinese Journal of Animal Nutrition,2015,27(2):596-605.(in Chinese)
[24]金龙.紫色达利菊提取缩合单宁对大肠杆菌和瘤胃氮代谢以及瘤胃微生物的影响[D].哈尔滨:东北农业大学,2011.JIN L.Effects of condensed tannins from purple prairie clover on fecal shedding of Escherichia coli by beef cattle and on rumen fermentation and rumen bacteria[D].Haerbin:Northeast Agricultural University,2011.(in Chinese)
[25]PATRA A K,SAXENA J.Dietary phytochemicals as rumen modifiers:A review of the effects on microbial populations[J].Antonie Van Leeuwenhoek,2009,96(4):363-375.
[26]JONES G A,MCALLISTER T A,MUIR A D,et al.Effects of Sainfoin(Onobrychis viciifolia Scop.)condensed tannins on growth and proteolysis by four strains of ruminal bacteria[J].Applied&Environmental Microbiology,1994,60(4):1374-1378.
[27]MOLAN A L,ATTWOOD G T,MIN B R,et al.The effect of condensed tannins from Lotus pedunculatus and Lotus corniculatus on the growth of proteolytic rumen bacteria in vitro and their possible mode of action[J].Canadian Journal of Microbiology,2001,47(7):626-633.
[28]MIN B R,ATTWOOD G T W C,MOLAN A L,et al.The effect of condensed tannins fromLotus corniculatus on the proteolytic activities and growth of rumen bacteria[J].Animal Feed Science&Technology,2005,121(1):45-58.
[29]BAE H D,MCALLISTER T A,YANKE J,et al.Effects of condensed tannins on endoglucanase activity and filter paper digestion by Fibrobacter succinogenes S85[J].Applied and Environmental Microbiology,1993,59(7):2132-2138.
[30]MCSWEENEY C S,PALMER B,BUNCH R,et al.Effect of the tropical forage calliandra on microbial protein synthesis and ecology in the rumen[J].Journal of Applied Microbiology,2001,90(1):78.
[31]杨琦玥,黄勇,陈亚冰,等.16SrRNA高通量测序技术筛选牦牛瘤胃细菌基因组DNA提取方法及菌群结构[J].中国农业科学,2017,50(5):932-941.YANG Q Y,HUANG Y,CHEN Y B,et al.Screening of optimal DNA extraction methods and the bacterial community composition of yak rumen revealed by high 16SrRNA throughput sequencing[J].Scientia Agricultura Sinica,2017,50(5):932-941.(in Chinese)
[32]张雪娇,王立志.饲粮中性洗涤纤维水平对山羊瘤胃细菌结构及组成的影响[J].动物营养学报,2018,30(4):1377-1386.ZHANG X J,WANG L Z.Effects of dietary neutral detergent fibre level on structure and composition of rumen bacteria in goats[J].Chinese Journal of Animal Nutrition,2018,30(4):1377-1386.(in Chinese)
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[2]RITTNER U,REED J D.Phenolics and in vitro degradability of protein and fibre in West African Browse[J].Journal of the Science of Food&Agriculture,1992,58(1):21-28.
[3]TEFEREDEGNE B.New perspectives on the use of tropical plants to improve ruminant nutrition[J].Proceedings of the Nutrition Society,2000,59(2):209.
[4]MIN B R,BARRY T N,ATTWOOD G T,et al.The effect of condensed tannins on the nutrition and health of ruminants fed fresh temperate forages:A review[J].Animal Feed Science&Technology,2003,106(1):3-19.
[5]MCSWEENEY C S,PALMER B,BUNCH R,et al.Effect of the tropical forage calliandra on microbial protein synthesis and ecology in the rumen[J].Journal of Applied Microbiology,2001,90(1):78.
[6]徐奇友,秦江帆,关湛铭,等.纤维素酶在蛋鸡日粮的应用研究[J].饲料工业,1998,1:32-33.XU Q Y,QIN J F,GUAN Z M,et al.Study on the application of cellulase in laying hens diets[J].Feed Industry,1998,1:32-33.(in Chinese)
[7]BEAUCHEMIN K A,RODE L M,VJH S.Fibrolytic enzymes increase fiber digestibility and growth rate of steers fed dry forages[J].Canadian Journal of Animal Science,1995,75(4):641-644.
[8]陈鑫珠,张文昌,张建国,等.纤维素酶对象草玉米秸秆混合青贮品质的影响[J].家畜生态学报,2011,32(6):46-50.CHEN X Z,ZHANG W C,ZHANG J G,et al.Effect of cellulase on quality of napier grass and corn straw mixed silage[J].Journal of Domestic Animal Ecology,2011,32(6):46-50.(in Chinese)
[9]BURGMANM H,PESARO M F,ZEYER J.A strategy for optimizing quality and quantity of DNA extracted from soil[J].Journal of microbiological methods,2001,45(1):7-20.
[10]金迪,王加启,卜登攀,等.应用DGGE分析秸秆日粮对奶牛瘤胃细菌群落的影响[J].中国农业科学,2013,46(5):1025-1035.JIN D,WANG J Q,BU D P,et al.Effect of corn straw diet on diversity of the bacterial community in the rumen of dairy cows analyzed by DGGE[J].Scientia Agricultura Sinica,2013,46(5):1025-1035.(in Chinese)
[11]MAGOC T,SALZBERG S L.Flash:Fast length adjustment of short reads to improve genome assemblies[J].Bioinformatics,2011,27(21):2957-2963.
[12]BOLGER A M,LOHSE M,USADEL B.Trimmomatic:A flexible trimmer for Illumina sequence data[J].Bioinformatics,2014,30(15):2114-2120.
[13]EDGAR R C,HAAS B J,CLEMENTE J C,et al.UCHIME improves sensitivity and speed of chimera detection[J].Bioinformatics,2011,27(16):2194-2200.
[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]EDGAR R C.Search and clustering orders of magnitude faster than BLAST[J].Bioinformatics,2010,26(19):2460-2461.
[16]SEGATA N,IZARD J,WALDRON L,et al.Metagenomic biomarker discovery and explanation[J].Genome Biology,2011,12(6):R60.
[17]EVANS N J,BROWN J M,MURRAY R D,et al.Characterization of novel bovine gastrointestinal tract Treponemaisolates and comparison with bovine digital dermatitis treponemes[J].Applied and Environmental Microbiology,2011,77(1):138-147.
[18]SADET-BOURGETEAU S,MARTIN C,MORGAVI D P.Bacterial diversity dynamics in rumen epithelium of wethers fed forage and mixed concentrate forage diets[J].Veterinary Microbiology,2010,146(1-2):98-104.
[19]SAMINATHAN M,SIEO C C,GAN H M,et al.Modulatory effects of condensed tannin fractions of different molecular weights from a Leucaena leucocephala hybrid on the bovine rumen bacterial community in vitro[J].Journal of the Science of Food and Agriculture,2016,96(13):4565-4574.
[20]冯仰廉.反刍动物营养学[M].北京:科学出版社,2004.Feng Y L,Ruminant Nutrition[M].Beijing:The Science Publishing Press,2004.
[21]SHINKAI T,UEKI T,KOBAYASHI Y.Detection and identification of rumen bacteria constituting a fibrolytic consortium dominated by Fibrobacter succinogenes[J].Animal Science Journal,2010,81(1):72-79.
[22]KOIKE S,PAN J,SUZUKI T,et al.Ruminal distribution of the cellulolytic bacteriumFibrobacter succinogenes in relation to its phylogenetic grouping[J].Animal Science Journal,2015,75(5):417-422.
[23]李大彪,张梅梅,于永强,等.单宁和聚乙二醇对绵羊和山羊瘤胃纤维降解菌数量的影响[J].动物营养学报,2015,27(2):596-605.LI D B,ZHANG M M,YU Y Q,et al.Effects of tannin and polyethylene glycol on the number of rumen fiber-degrading bacteria in sheep and goats[J].Chinese Journal of Animal Nutrition,2015,27(2):596-605.(in Chinese)
[24]金龙.紫色达利菊提取缩合单宁对大肠杆菌和瘤胃氮代谢以及瘤胃微生物的影响[D].哈尔滨:东北农业大学,2011.JIN L.Effects of condensed tannins from purple prairie clover on fecal shedding of Escherichia coli by beef cattle and on rumen fermentation and rumen bacteria[D].Haerbin:Northeast Agricultural University,2011.(in Chinese)
[25]PATRA A K,SAXENA J.Dietary phytochemicals as rumen modifiers:A review of the effects on microbial populations[J].Antonie Van Leeuwenhoek,2009,96(4):363-375.
[26]JONES G A,MCALLISTER T A,MUIR A D,et al.Effects of Sainfoin(Onobrychis viciifolia Scop.)condensed tannins on growth and proteolysis by four strains of ruminal bacteria[J].Applied&Environmental Microbiology,1994,60(4):1374-1378.
[27]MOLAN A L,ATTWOOD G T,MIN B R,et al.The effect of condensed tannins from Lotus pedunculatus and Lotus corniculatus on the growth of proteolytic rumen bacteria in vitro and their possible mode of action[J].Canadian Journal of Microbiology,2001,47(7):626-633.
[28]MIN B R,ATTWOOD G T W C,MOLAN A L,et al.The effect of condensed tannins fromLotus corniculatus on the proteolytic activities and growth of rumen bacteria[J].Animal Feed Science&Technology,2005,121(1):45-58.
[29]BAE H D,MCALLISTER T A,YANKE J,et al.Effects of condensed tannins on endoglucanase activity and filter paper digestion by Fibrobacter succinogenes S85[J].Applied and Environmental Microbiology,1993,59(7):2132-2138.
[30]MCSWEENEY C S,PALMER B,BUNCH R,et al.Effect of the tropical forage calliandra on microbial protein synthesis and ecology in the rumen[J].Journal of Applied Microbiology,2001,90(1):78.
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