不同饲粮条件下湖北黑头羊瘤胃细菌多样性及群落结构分析
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摘要
旨在研究不同饲粮条件下湖北黑头羊瘤胃细菌多样性及群落结构。选取15只健康的湖北黑头山羊公羊,随机分为5组,每组3只,分别饲喂花生藤(A组)、苜蓿(B组)、苜蓿+花生藤(C组)为粗饲料来源的颗粒型全混合日粮以及进口苜蓿草颗粒(D组)和新疆苜蓿草颗粒(E组),试验期共45 d,其中预试期15 d。在试验结束后第1天,经口腔抽取瘤胃液抽提基因组DNA,采用Illumina Miseq PE300平台对瘤胃细菌进行分析。结果表明:1)在97%相似性水平下,5组样品共产生851个操作分类单元(OTU),共享295个OTU,占瘤胃液细菌总OTU数目的34.67%,D组在各组中α多样性指数指标(Ace指数、Chao1指数和Shannnon指数)最高,而3组颗粒型全混合日粮中A、C组高于B组(P<0.05,P<0.01);2)瘤胃中拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、无壁菌门(Tenericutes)为各组优势菌门,D组中的拟杆菌门显著高于B组(P<0.05),而厚壁菌门显著低于D组(P<0.05),而A、B、C组间拟杆菌门、厚壁菌门含量差异不显著(P>0.05);3)普雷沃氏菌属1(Prevotella_1)、1种未鉴定的属(Unidentified genus)、理研菌属RC9(Rikenellaceae_RC9_gut_group)为优势菌属,其中D组普雷沃氏菌属含量较高,A组与C组,D组与E组的遗传距离更接近。综合得出,苜蓿草颗粒品质和颗粒型全混合日粮中精料含量能影响湖北黑头羊瘤胃细菌的多样性;全苜蓿饲粮条件下湖北黑头羊瘤胃存在独特的细菌群落结构,细菌多样性最高。
This experiment was conducted to investigate the diversity of bacteria in rumen fluid and their community structure in Hubei blackhead goats under different dietary conditions. Fifteen healthy Hubei blackhead goat(rams) were used and randomly divided into five groups(n=3 per group). The animals were fed with granulated TMR, in which the roughages derived from peanut vine(Group A), alfalfa(Group B), and alfalfa and peanut vine(Group C) were used in roughage-sourced total mixed ration, in imported alfalfa hay pellet(Group D) and in Xinjiang alfalfa hay pellet(Group E). The trial period was 45 days, of which the pre-test period was 15 days. On the first day after the end of the experiment, the genomic DNA was extracted from the rumen fluid by oral extraction, and the bacteria in rumen fluid were analyzed by the Illumina Miseq PE300 platform. The results showed that: 1) at the level of 97% similarity, 851 operational taxonomic units(OTU) were generated in the five groups, sharing 295 OTUs, accounting for 34.67% of the total OTU of the bacteria in the rumen fluid. Group D had the highest alpha indexes(Ace, Chao1, Shannon) of all the groups. Groups A and C had higher Alpha indexes(Ace, Chao1, Shannon) than Group B(P<0.05, and P<0.01); 2) the Bacteroidetes, Firmicutes and Tenericumes in rumen were the dominant bacteria in each group; Bacteroides in Group D were significantly higher than in Group B(P<0.05), while the Firmicutes were significantly lower in Group B than in Group D(P<0.05); and there was no significant difference in the contents of Bacteroidetes and Firmicutes between groups A,B and C( P > 0. 05); 3) Prevotella_1,the Unidentified genus,and the Rikenellaceae RC9_gut_group were dominant genus. Among them,the content of Prevotella_1 in Group D was higher( P<0. 05),the genetic distances between Groups A and C,and between Groups D and group E were closer. To sum up,the quality of alfalfa hay pellet and the concentrate content of the total mixed ration might have effect on the diversity of bacteria in rumen of Hubei blackhead goats. Under the condition of whole alfalfa diet,there was a unique bacterial community structure in the rumen of Hubei blackhead goat,with the highest bacterial diversity.
This experiment was conducted to investigate the diversity of bacteria in rumen fluid and their community structure in Hubei blackhead goats under different dietary conditions. Fifteen healthy Hubei blackhead goat(rams) were used and randomly divided into five groups(n=3 per group). The animals were fed with granulated TMR, in which the roughages derived from peanut vine(Group A), alfalfa(Group B), and alfalfa and peanut vine(Group C) were used in roughage-sourced total mixed ration, in imported alfalfa hay pellet(Group D) and in Xinjiang alfalfa hay pellet(Group E). The trial period was 45 days, of which the pre-test period was 15 days. On the first day after the end of the experiment, the genomic DNA was extracted from the rumen fluid by oral extraction, and the bacteria in rumen fluid were analyzed by the Illumina Miseq PE300 platform. The results showed that: 1) at the level of 97% similarity, 851 operational taxonomic units(OTU) were generated in the five groups, sharing 295 OTUs, accounting for 34.67% of the total OTU of the bacteria in the rumen fluid. Group D had the highest alpha indexes(Ace, Chao1, Shannon) of all the groups. Groups A and C had higher Alpha indexes(Ace, Chao1, Shannon) than Group B(P<0.05, and P<0.01); 2) the Bacteroidetes, Firmicutes and Tenericumes in rumen were the dominant bacteria in each group; Bacteroides in Group D were significantly higher than in Group B(P<0.05), while the Firmicutes were significantly lower in Group B than in Group D(P<0.05); and there was no significant difference in the contents of Bacteroidetes and Firmicutes between groups A,B and C( P > 0. 05); 3) Prevotella_1,the Unidentified genus,and the Rikenellaceae RC9_gut_group were dominant genus. Among them,the content of Prevotella_1 in Group D was higher( P<0. 05),the genetic distances between Groups A and C,and between Groups D and group E were closer. To sum up,the quality of alfalfa hay pellet and the concentrate content of the total mixed ration might have effect on the diversity of bacteria in rumen of Hubei blackhead goats. Under the condition of whole alfalfa diet,there was a unique bacterial community structure in the rumen of Hubei blackhead goat,with the highest bacterial diversity.
引文
[1] 曾燕,简平,倪学勤,等.Illumina MiSeq测序平台测定蒙古羊瘤胃液相和固相菌群多样性[J].动物营养学报,2015,27(10):3256-3262.
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[14] 林波,梁辛,李丽莉,等.饲粮精粗比对泌乳水牛瘤胃细菌和甲烷菌区系的影响[J].动物营养学报,2016,28(10):3101-3109.
[15] Belanche A,Doreau M,Edwards J E,et al.Shifts in the rumen microbiota due to the type of carbohydrate and level of protein ingested by dairy cattle are associated with changes in rumen fermentation[J].J Nutr,2012,142(9):1684-1692.
[16] Petri R M,Forster R J,Yang W,et al.Characterization of rumen bacterial diversity and fermentation parameters in concentrate fed cattle with and without forage[J].J Appl Microbiol,2012,112(6):1152-1162.
[17] 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.
[18] Kittelmann S,Seedorf H,Walters W A,et al.Simultaneous amplicon sequencing to explore co-occurrence patterns of bacterial,archaeal and eukaryotic microorganisms in rumen microbial communities[J].PLoS One,2013,8(2):e47879.
[19] 王继文,王立志,闫天海,等.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.
[20] 金磊,周美丽,王禄禄,等.不同能量代谢率的山羊瘤胃微生物结构与组成的差异性[J].微生物学通报,2018,45(01):91-101.
[21] 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 J Microbiol Biotechnol.,2014,30(2):669-680.
[22] Chen Y H,Penner G B,Li M J,et al.Changes in bacterial diversity associated with epithelial tissue in the beef cow rumen during the transition to a high-grain diet[J].Appl Environ Microbiol.,2011,77(16):5770-5781.
[23] Mao S Y,Zhang R Y,Wang D S,et al.Impact of subacute ruminal acidosis (SARA) adaptation on rumen microbiota in dairy cattle using pyrosequencing[J].Anaerobe,2013,24:12-19.
[24] 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.
[25] 徐俊,胡丽芳,侯玉洁,等.不同牧草来源饲粮对奶牛瘤胃液中细菌群落结构多样性的影响[J].动物营养学报,2015,27(11):3549-3557.
[26] 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.
[27] Zened A,Combes S,Cauquil L,et al.Microbial ecology of the rumen evaluated by 454 GS FLX pyrosequencing is affected by starch and oil supplementation of diets[J].FEMS Microbiol Ecol,2013,83(2):504-514.
[2] Shi P J,Meng K,Zhou Z G,et al.The host species affects the microbial community in the goat rumen[J].Lett Appl Microbiol,2008,46(1):132-135.
[3] Kocherginskaya S A,Aminov R I,White B A,et al.Analysis of the rumen bacterial diversity under two different diet conditions using denaturing gradient gel electrophoresis,random sequencing,and statistical ecology approaches[J].Anaerobe,2001,7(3):119-134.
[4] Mackie R I,White B A.Recent advances in rumen microbial ecology and metabolism:potential impact on nutrient output[J].J.Dairy Sci.,1990,73(10):2971-2995.
[5] 于萍,王加启,卜登攀.反刍动物胃肠道微生物多样性研究进展[J].中国微生态学杂志,2009,21(7):655-658.
[6] 陈芸,刘旗,邓俊良,等.基于MiSeq分析川中黑山羊瘤胃细菌的多样性及群落结构[J].湖南农业大学学报(自然科学版),2017,43(3):286-291.
[7] 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(04):1377-1386.
[8] Grilli D J,Fliegerová K,Kope,et al.Analysis of the rumen bacterial diversity of goats during shift from forage to concentrate diet[J].Anaerobe,2016,42:17-26.
[9] 张宏福.动物营养参数与饲养标准[M].4版.北京:中国农业出版社,2016:5-6.
[10] Caporaso J G,Kuczynski J,Stombaugh J,et al.QIIME allows analysis of high-throughput community sequencing data[J].Nat Methods,2010,7(5):335-336.
[11] Edgar R C.Search and clustering orders of magnitude faster than blast[J].Bioinformatics,2010,26(19):2460-2461.
[12] Cole J R,Wang Q,Cardenas E,et al.The ribosomal database project:improved alignments and new tools for rRNA analysis[J].Nucleic Acids Res,2009,37(S1):D141-D145.
[13] 姚文,朱伟云,韩正康.应用变性梯度凝胶电泳和16S rDNA序列分析对山羊瘤胃细菌多样性的研究[J].中国农业科学,2004,37(9):1374-1378.
[14] 林波,梁辛,李丽莉,等.饲粮精粗比对泌乳水牛瘤胃细菌和甲烷菌区系的影响[J].动物营养学报,2016,28(10):3101-3109.
[15] Belanche A,Doreau M,Edwards J E,et al.Shifts in the rumen microbiota due to the type of carbohydrate and level of protein ingested by dairy cattle are associated with changes in rumen fermentation[J].J Nutr,2012,142(9):1684-1692.
[16] Petri R M,Forster R J,Yang W,et al.Characterization of rumen bacterial diversity and fermentation parameters in concentrate fed cattle with and without forage[J].J Appl Microbiol,2012,112(6):1152-1162.
[17] 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.
[18] Kittelmann S,Seedorf H,Walters W A,et al.Simultaneous amplicon sequencing to explore co-occurrence patterns of bacterial,archaeal and eukaryotic microorganisms in rumen microbial communities[J].PLoS One,2013,8(2):e47879.
[19] 王继文,王立志,闫天海,等.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.
[20] 金磊,周美丽,王禄禄,等.不同能量代谢率的山羊瘤胃微生物结构与组成的差异性[J].微生物学通报,2018,45(01):91-101.
[21] 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 J Microbiol Biotechnol.,2014,30(2):669-680.
[22] Chen Y H,Penner G B,Li M J,et al.Changes in bacterial diversity associated with epithelial tissue in the beef cow rumen during the transition to a high-grain diet[J].Appl Environ Microbiol.,2011,77(16):5770-5781.
[23] Mao S Y,Zhang R Y,Wang D S,et al.Impact of subacute ruminal acidosis (SARA) adaptation on rumen microbiota in dairy cattle using pyrosequencing[J].Anaerobe,2013,24:12-19.
[24] 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.
[25] 徐俊,胡丽芳,侯玉洁,等.不同牧草来源饲粮对奶牛瘤胃液中细菌群落结构多样性的影响[J].动物营养学报,2015,27(11):3549-3557.
[26] 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.
[27] Zened A,Combes S,Cauquil L,et al.Microbial ecology of the rumen evaluated by 454 GS FLX pyrosequencing is affected by starch and oil supplementation of diets[J].FEMS Microbiol Ecol,2013,83(2):504-514.