不同纤维源日粮下东北梅花鹿瘤胃微生物功能基因的宏基因组分析

为了研究不同粗纤维源日粮对梅花鹿瘤胃微生物区系的影响,选择10头健康公鹿分成JG、ZS 2组,每组5头,分别以玉米秸叶和柞树叶作为粗纤维源,试验时期为2个月,提取梅花鹿瘤胃液中微生物总基因,构建文库并测序及基因功能注释分析。结果表明:JG和ZS 2组样本的总碱基数量和总序列数量分别为11150909160和12540742633、75018396和84518090。在JG和ZS 2组样品中约1/2以上的纤维素糖苷水解酶(glycoside hydrolases,GHs)来自于瘤胃球菌属(Ruminococcus)(JG和ZS 2组样品分别为52.79%和51.07%)和纤维杆菌属(Fibrobacter)(JG和ZS 2组样品分别为14.22%和15.49%)。而很大一部分半纤维素酶由Ruminococcus(分别为39.20和37.55%)、普雷沃菌属(Prevotella)(分别为22.68%和20.66%)、Fibrobacter(8.86%,9.48%)、梭状芽孢杆菌属(Clostridium)(2.27%,1.94%)分泌。此外,共有24个GHs家族参与植物细胞壁多糖的降解,其中GH5、GH9、GH45和GH48为降解纤维素的优势酶类,GH8、GH10、GH11、GH26、GH28、GH51、GH53、GH78对于半纤维素降解起重要作用,而对于寡糖降解起主要作用的有GH1、GH2、GH3、GH43和GH94。结论:当饲喂粗纤维较多玉米叶时,以降解纤维素为主的纤维降解酶的比例升高,而寡糖降解酶在瘤胃中的比例降低;当饲喂含丰富可发酵碳水化合物的柞树叶时,结果相反。     

基于16S rRNA高通量测序技术分析草原红牛瘤胃微生物多样性和功能预测的研究

为系统探讨草原红牛瘤胃内的微生物多样性及其功能,本试验利用16S rRNA基因高通量测序技术检测分析草原红牛(20月龄左右,均重为577.5 kg)瘤胃液样本菌群结构并进行PICRUSt功能预测。结果显示:通过Illumina Miseq测序平台共获得35 848条优质序列,聚类分析得到387个操作分类单元(OTU),经分类学鉴定分属15个门、20个纲、25个目、41个科及110个属;厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes)为优势菌群,所占比例分别为50.09%和41.11%;基于属的组成,依次为普雷沃菌属(Prevotella)15.20%、未知属f型拟杆菌目(norankfBacteroidales)BS11菌群8.66%、瘤胃菌科(Ruminococcaceae)NK4A214菌群6.96%、理研菌科(Rikenellaceae)RC9菌群5.56%、未知属(Christensenellaceae)R-7菌群4.01%、瘤胃球菌属2(Ruminococcus2)3.33%等;16S rRNA基因组的PICRUSt功能预测结果显示,瘤胃内菌群功能主要集中在碳水化合物转运及代谢,表面草原红牛体内含有大量的纤维素和木质素降解酶基因。综上,基于16S rRNA基因的高通量测序技术全面揭示了草原红牛瘤胃菌群的多样性,且预测其含有丰富的蛋白质分解、木质纤维素降解酶系,为探索草原红牛瘤胃微生物的认知提供了基础,也为挖掘其他重要营养生理功能相关的瘤胃微生物功能基因提供了参考。     

云南大额牛瘤胃宏基因组Fosmid文库的构建与分析

为研究云南大额牛瘤胃中丰富的降解纤维素类物质的基因,采用包埋法和脉冲场电泳技术直接从云南大额牛瘤胃内容物提取总DNA,经不完全酶切获得DNA片段,大小为23~48 kb,与pcc2 FOS vector连接,转化至E.coli EPI 300宿主细胞,构建瘤胃微生物宏基因组Fosmid文库。该文库共保存38400个克隆,平均插入片段大小约35.5 kb,空载率小于2%,库容达1363 Mb。通过对该文库进行部分纤维素酶活力筛选,获得具有羧甲基纤维素酶活性的克隆95个,木聚糖酶活性的克隆52个,同时具有这2种酶活性的克隆23个,这为进一步研究大额牛瘤胃内纤维素酶基因奠定了基础。     

反刍动物瘤胃纤维降解微生物的研究进展

反刍动物瘤胃纤维降解微生物能分泌大量的纤维素酶和半纤维素酶,在瘤胃纤维降解过程中发挥着重要作用,是反刍动物高效利用粗饲料资源的基础。该文简要介绍了纤维素酶组成、瘤胃纤维素酶基因多样性研究进展,综述了纤维素酶降解纤维素的作用机制以及厌氧真菌对纤维物质的降解过程,概述了近年来瘤胃纤维降解微生物的分离筛选研究进展,以及碳水化合物、饲料加工和添加剂对瘤胃纤维降解微生物的调控作用,以期为相关研究提供参考。     

瘤胃微生物在木质纤维素价值化利用的研究进展

瘤胃是反刍动物消化的第一个腔室，各种微生物（细菌、真菌和原生动物）相互作用将木质纤维素植物生物降解为易于代谢的化合物。瘤胃也是目前自然界公认的木质纤维素高效降解和利用的天然反应器，其真菌和细菌可分泌多种木质纤维素降解酶，在木质纤维素生产生物燃料和化学用品方面具有潜在的价值。因此，本研究在瘤胃内木质纤维降解的微生物及其降解木质纤维素相关酶的基础上，重点综述了瘤胃微生物在木质纤维素生物转化为乙醇、生物化学（有机酸）及沼气等方面的研究进展，旨在为瘤胃微生物和瘤胃酶在木质纤维素价值化利用方面的研究和应用提供新的方法和思路。     

基于16S rRNA高通量测序技术分析中国西门塔尔牛瘤胃微生物多样性和功能预测的研究

为系统探讨中国西门塔尔牛瘤胃内的微生物多样性及其功能,本试验利用16S rRNA基因高通量测序技术检测分析西门塔尔牛(20月龄,平均体重约为550 kg)瘤胃液样本菌群结构并进行PICRUSt功能预测。结果显示,两组样本通过Illumina Miseq测序平台共获得66 712条优质序列,聚类分析得到1 797个操作分类单元(OTU),经分类学鉴定分属13个门20个纲22个目33个科及59个属;厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes)为优势菌群,所占比例分别为62.46%和22.45%;基于属的组成,依次为粪杆菌真核菌群([Eubacterium]_coprostanoligenes_group,9.16%)、瘤胃球菌属_2 (Ruminococcus_2,7.90%)、未知属f型拟杆菌目RF16菌群(norank_f__Bacteroidales_RF16_group,6.23%)、未知属f型瘤胃菌科(norank_f__Ruminococcaceae,4.79%)、理研菌科_RC9菌群(Rikenellaceae_RC9_gut_group,4.72%)、未知属f型拟杆菌目BS11菌群(norank_f_Bacteroidales_BS11_gut_group,4.49%)、瘤胃球菌属_1(Ruminococcus_1,4.43%)等;16S rRNA基因组的PICRUSt功能预测发现,瘤胃菌群功能主要集中在碳水化合物转运及代谢上,可能含有大量的纤维素和木质素降解酶基因。综上,基于16S rRNA基因的高通量测序技术全面揭示了西门塔尔牛瘤胃菌群的多样性,并预测其含有丰富的蛋白质分解、木质纤维素降解酶系,为探索西门塔尔牛瘤胃微生物奠定基础,也为进一步挖掘与某些重要营养生理功能密切相关的瘤胃微生物功能基因提供参考。     

瘤胃微生物在木质纤维素生物质资源可再生生物转化中的应用研究进展

木质纤维素生物质是最丰富的可再生资源之一,将其高效转化为生物能源是缓解能源短缺和回收废弃物的有效途径。然而,因为木质纤维素复杂的网状结构,需要多种微生物和酶系的协同作用使其有效降解。反刍动物瘤胃作为一种天然的厌氧发酵体系,具有高效转化木质纤维素的功能,主要依赖于其复杂且协同的瘤胃微生物群落及分泌的酶。本文在总结瘤胃内与木质纤维素降解相关的微生物群落结构的基础上,重点综述了瘤胃微生物及其分泌的酶类在木质纤维素降解中的相关机理、人工瘤胃发酵系统的构建和提升降解效率的相关调控手段等方面的研究进展,旨在为利用瘤胃微生物实现木质纤维素生物质的价值化应用提供新的思路和方法。     

瘤胃内纤维素酶活性的测定

反刍动物依靠瘤胃微生物分泌的纤维素酶来分解利用纤维物质.近年来人们研究发现,只有与饲料颗粒紧密结合的微生物才直接负责纤维物质的降解,即分泌纤维素酶,而瘤胃液内纤维素酶活性很小或检测不到.这里介绍与饲料颗粒相粘附的纤维素酶(CMCase)活性的测定方法.     

体外法研究银杏叶提取物对山羊瘤胃纤维降解的影响

以3只安装永久性瘤胃瘘管的山羊为试验动物,以纯淀粉、纤维素和酪蛋白为底物,银杏叶提取物在瘤胃培养液底物的浓度设为0(对照组)、0.3%、0.6%、0.9%、1.2%5个水平,利用人工瘤胃体外培养法,通过对瘤胃液pH值、微生物蛋白、纤维素降解率及与纤维素降解相关的酶等指标的测定,探讨银杏叶提取物对纤维素降解的影响.结果表明:银杏叶提取物增加了微生物蛋白的产量,提高了纤维素的降解率,增加了滤纸纤维素酶、羧甲基纤维素酶和木聚糖酶的活性.     

白腐真菌对稻草木质纤维素的降解试验

农作物秸秆中的纤维素、半纤维素、木质素是世界上最丰富的碳水化合物资源,其中纤维素和半纤维素能够被草食动物瘤胃微生物降解利用,但由于木质素与纤维素和半纤维素紧密结合,镶嵌形成酯键,从而阻碍了瘤胃微生物及单胃动物对秸秆中纤维素和半纤维素的利用,因此提高秸秆消化率的关键是对木质素的降解。如何利用微生物降解木质纤维素,生物学家进行了大量的研究,尤其在瘤胃微生物方面的研究,取得了重大进展。1971年Kirk.T.K发现某些真菌有降解木质素的能力,尤其是白腐真菌能彻底降解天然植物中的纤维素和木质素复合物,氧化成二氧化碳和水,这引起国际科学界和工业界的极大兴趣。     

Analysis of Rumen Microbial Diversity and Functional Prediction of Chinese Simmental Cattle Based on 16S rRNA High-throughput Sequencing Technology

To systematically explore the microbial diversity and function in the rumen of Chinese Simmental cattle,this experiment used 16S rRNA gene high-throughput sequencing technology to detect and analyze the rumen fluid samples of Simmental cattle (about 20 months old,the average weight was 550 kg),and its function was predicted by PICRUSt.The results showed that a total of 66 712 high-quality sequences were obtained from the Illumina Miseq sequencing platform,and 1 797 operational classification units (OTU) were obtained by cluster analysis.The taxonomic identification was divided into 13 phylums,20 classes,22 orders,33 families and 59 genuses;Firmicutes and Bacteroidetes were dominant bacteria,accounting for 62.46% and 22.45%,respectively;Based on the genus composition,[Eubacterium]_coprostanoligenes_group (9.16%),Ruminococcus_2 (7.90%),norank_f__Bacteroidales_RF16_group(6.23%),norank_f__Ruminococcaceae(4.79%),Rikenellaceae_RC9_gut_group(4.72%),norank_f_Bacteroidales_BS11_gut_group(4.49%),Ruminococcus_1(4.43%),etc.The PICRUSt function of the 16S rRNA genome predicted that the rumen flora function was mainly concentrated in carbohydrate transport and metabolism,and might contain a large amount of cellulose and lignin degrading enzyme genes.In summary,the high-throughput sequencing technology based on 16S rRNA gene fully revealed the diversity of rumen bacteria in Simmental cattle,and predicted that it was rich in protein decomposition and lignocellulose degrading enzymes.The basis of microbial cognition provided a reference for further research on rumen microbial functional genes closely related to some important nutritional and physiological functions.     

瘤胃厌氧真菌对木质纤维素降解的研究进展

瘤胃厌氧真菌在木质纤维素的降解中起着重要作用,它不仅能分泌降解含有微晶纤维素的天然木质纤维素的酶系,而且能组装成具有高效催化活性的纤维小体类似复合体。主要围绕瘤胃厌氧真菌在木质纤维素降解中的作用、瘤胃厌氧真菌的木质纤维素降解系统、瘤胃厌氧真菌的纤维小体类似复合体作一综述。     

Microbial population in the rumen of swamp buffalo (Bubalus bubalis) as influenced by coconut oil and mangosteen peel supplementation

Four, rumen fistulated swamp buffalo bulls were used to study microbial populations in the rumen when supplemented with coconut oil and mangosteen peel. Animals were randomly assigned to a 4 × 4 Latin square design. Four treatments were un‐supplemented (Control), supplementation with coconut oil at 50 g/kg (CO5), supplementation with mangosteen peel at 30 g/kg (MP3) and supplementation with CO5 and MP3 (COM), of total DM intake. Animals received concentrate at 10 g/kg of BW, and rice straw was given ad libitum. Abundance of total bacteria was increased by CO5 supplementation, whereas populations of protozoa and Fibrobacter succinogenes were reduced by CO5 and COM supplementation. Dietary supplementation did not affect methanogen, Ruminococcus flavefaciens or Ruminococcus albus abundances. Dietary treatments changed denaturing gradient gel electrophoresis (DGGE) band patterns of methanogens and protozoa when compared with the control group, especially when supplemented with MP3. Supplementation of COM resulted in the greatest difference in pattern of DGGE bands for total bacteria compared with the control. Coconut oil and mangosteen peel supplementation resulted in changing of rumen microbial abundances and communities; however, combination of them could be more benefit to improve rumen fermentation of swamp buffalo fed on rice straw.     

Effects of live yeast on differential genetic and functional attributes of rumen microbiota in beef cattle

Several studies have evaluated the effects of live yeast supplementation on rumen microbial population; however,its effect on differential microbial genes and their functional potential has not been described. Thus, this study applied shotgun metagenomic sequencing to evaluate the effects of live yeast supplementation on genetic and functional potential of the rumen microbiota in beef cattle. Eight rumen-cannulated Holstein steers were randomly assigned to two treatments in a cross-over design with two 25-day experimental periods and a 10-day wash-out between the two periods. The steers were housed in individual pens and fed 50% concentrate-mix and 50% red clover/orchard hay ad libitum. Treatments were(1) control(CON; basal diet without additive) and(2) yeast(YEA;basal diet plus 15 g/d of live yeast product). Rumen fluid samples were collected at 3, 6, and 9 h after feeding on the last d of each period. Sequencing was done on an Illumina Hi Seq 2500 platform. Dietary yeast supplementation increased the relative abundance of carbohydrate-fermenting bacteria(such as Ruminococcus albus, R.champanellensis, R. bromii, and R. obeum) and lactate-utilizing bacteria(such as Megasphaera elsdenii, Desulfovibrio desulfuricans, and D. vulgaris). A total of 154 differentially abundant genes(DEGs) were obtained(false discovery rate 0.01). Kyoto Encyclopedia of Genes and Genomes(KEGG) annotation analysis of the DEGs revealed that 10 pathways, including amino sugar and nucleotide sugar metabolism, oxidative phosphorylation, lipopolysaccharide biosynthesis, pantothenate and coenzyme A biosynthesis, glutathione metabolism, beta-alanine metabolism,polyketide sugar unit biosynthesis, protein export, ribosome, and bacterial secretory system, were enriched in steers fed YEA. Annotation analysis of the DEGs in the carbohydrate-active enzymes(CAZy) database revealed that the abundance of genes coding for enzymes belonging to glycoside hydrolases, glycosyltransferases, and carbohydrate binding modules were enriched in steers fed YEA. These results confirm the effectiveness of a live S. cerevisiae product for improving rumen function in beef steers by increasing the abundance of cellulolytic bacteria, lactic acid-utilizing bacteria, and carbohydrate-active enzymes in the rumen.     

羊草茎在奶牛瘤胃中降解特性及其对食糜纤维分解菌数量的影响

本试验旨在研究羊草茎降解特性和紧密吸附于茎的3种主要纤维分解菌的动态变化。选用羊草茎为试验材料,将其纵切6份后装入尼龙袋投入瘤胃中,分别在6,12,24,48和72 h取出,利用扫描电子显微镜观察超微结构变化;取粉碎后羊草茎进行尼龙袋试验,分别在0.5,2,6、12,24,48和72 h取出,测定不同时间点中性洗涤纤维(neutral detergent fiber,NDF)降解率和吸附在茎中3种主要纤维分解菌的数量变化。结果表明,薄壁组织和韧皮部可被瘤胃微生物降解,维管束会伴随薄壁组织的降解而发生脱落。羊草茎和食糜不同时间点纤维分解菌数量均为产琥珀酸丝状杆菌>白色瘤胃球菌>黄色瘤胃球菌,茎中产琥珀酸丝状杆菌和黄色瘤胃球菌数量在24 h达峰值,分别为10⁹和10⁵ copy/g羊草茎,白色瘤胃球菌在12 h达峰值,为10⁸ copy/g羊草茎。瘤胃食糜中3种纤维分解菌的数量在24 h内基本处在一个恒定的水平,而羊草茎NDF降解率在72 h内逐渐提高,羊草NDF降解率与瘤胃食糜中3种纤维分解菌数量不同步,这可能与纤维分解菌分泌的酶活力存在滞后有关。     

应用康奈尔净碳水化合物-蛋白质体系评定广西水牛常用粗饲料的营养价值

为深入地揭示水牛常用粗饲料的碳水化合物及蛋白质组分的特性,本试验采用康奈尔净碳水化合物-蛋白质体系(CNCPS)对广西水牛4类20种常用粗饲料进行评定.测定粗饲料的常规营养成分,并应用CNCPS的公式计算出碳水化合物组分中糖类(CA)、淀粉和果胶(CBl)、可利用纤维(CB2)和不可利用纤维(CC)的含量以及蛋白质组分...     

An investigation of carbohydrate and protein degradation ratios,nitrogen to energy synchronization and hourly effective rumen digestion of barley: effect of variety and growth year

The objective of this study was to investigate rumen available soluble, insoluble and total protein (CP), estimated structural (SC) and non‐structural carbohydrate (starch: ST), degradation characteristic ratios and hourly effective degradation of six barley varieties during three consecutive growth years (2003, 2004, 2005). The magnitude of the differences was determined between the varieties and growth years. Measured degradation kinetics included soluble fraction (S), undegradable fraction (U), lag time (T₀) and rate of degradation (K_d) of the insoluble but degradable fraction (D). Rumen available soluble, insoluble, and total N, SC and ST and the rumen degradation characteristic ratios were determined using the Tamminga rumen degradation ratio system. Characteristics of the hourly effective degradation between N and carbohydrate (CHO) among the six barley varieties for 3 years were also studied. The degradation ratios included were total rumen available N and carbohydrate ratio (FN/FCHO), rumen available soluble N and carbohydrate ratio (SN/SCHO), and rumen available insoluble N and carbohydrate ratio (EN/ECHO). Results show that both the barley variety and growth year had a significant effect on degradation kinetics (S, D, U, T₀ and/or K_d). Differences in the ratio of FN/FCHO among varieties ranged from 16.6 to 19.0 g/kg (p < 0.01). There was no difference in SN/SCHO (p > 0.05) with an average of 4.9 g/kg. The difference in the EN/ECHO ratio tended to be significant among the varieties (p = 0.069) ranging from 18.4 to 21.3 g/kg. Differences in the hourly effective degradation between N and CHO were relatively small at shorter incubation times (2–4 h) However, as the length of rumen incubation increased (12–24 h), much larger differences in the rate of effective hourly degradation were observed. In conclusion, both barley variety and growth year had significant effects on rumen degradation kinetics. The mean FN/FCHO ratio of 17.2 (16.6–19.0) was observed for the six barley varieties in this experiment. All barley varieties exhibited a less optimal rumen fermentation ratio (17.2 < optimum: FN/FCHO = 25 to 33 g N per kg CHO). The large differences in the degradation kinetics, characteristics ratios and hourly effective degradation among barley varieties and growth years may help to explain some of the large variations that are seen in cattle performance.     

瘤胃微生物对结构性碳水化合物的降解机制研究进展

反刍动物瘤胃内含有庞大的微生物种群,与瘤胃内结构性碳水化合物的降解有密切关系。参与结构性碳水化合物降解的瘤胃微生物主要包括细菌、真菌和原虫,尤以细菌和真菌的降解作用最大。瘤胃细菌和真菌通过与结构性碳水化合物的附着、粘连或穿透等一系列作用,使这些紧密连接的结构物质变得松散,再由它们分泌的各种酶类将这些松散的结构性碳水化合物逐步分解,转化成可以被反刍动物及其寄居的瘤胃微生物吸收利用的单糖。瘤胃微生物对结构性碳水化合物的降解也受到诸多因素的影响,主要包括植物的表皮、细胞壁的物理和化学结构特性、细胞壁的组成成分,以及瘤胃内环境和其他营养素等。