宏基因组学技术在反刍动物瘤胃微生态系统上的应用研究进展

微生物在自然界中广泛存在,除土壤和水中的微生物最多外,其次在农业畜牧业中反刍动物的瘤胃微生态系统中的微生物中所占比例最多。反刍动物瘤胃微生物由于种类繁多,数量巨大,微生物区系之间关系非常复杂,它们之间寄生共生,共同影响宿主的生长发育和机体代谢,所以是反刍动物营养学研究的热点之一。随着分子生物学技术的发展,宏基因组学技术帮助我们揭开了瘤胃微生物群落的真实面貌,有助于挖掘瘤胃微生物基因库并筛选其中的功能基因,使人们对反刍动物瘤胃微生物群落的研究更加方便、透彻。本文综述了近些年应用宏基因组学技术在反刍动物瘤胃微生态群落系统的应用,旨在对瘤胃微生物功能特征进行更深入的研究,为畜牧科学生产及微生物发酵等相关领域的研究提供科学指导。     

为动物类专业本科生讲授瘤胃微生物知识点的体会

瘤胃微生物与反刍动物的共生关系是动物类专业本科生必须掌握的重要知识。我们采取问题导向教学方法,使学生较好地掌握瘤胃微生物在反刍动物生长发育中的作用,积极培养学生在科学饲养反刍动物、正确防治瘤胃疾病以及未来开展反刍动物相关研究的素质和能力。     

奶牛瘤胃微生物研究进展和趋势

近年来在奶牛试验中,对瘤胃微生物的研究引起了人们越来越多的兴趣。这些研究的目的多是将微生物组成变化与日粮组成、宿主生产性能(如饲料效率,产奶量,乳脂等)、健康(如瘤胃酸中毒和亚急性酸中毒)以及环境(如甲烷排放)联系起来,另外还有一些研究则强调了微生物在多种反刍动物瘤胃发育中的作用。关于奶牛瘤胃微生物的大部分发现都是基于扩增子测序,可以揭示瘤胃微生物的分类组成,以及在不同处理条件下瘤胃菌群的变化。尽管新兴的宏基因组学和宏转录组学能够深入探索瘤胃微生物的功能,但在数据分析和解释方面也带来了更多的挑战,如目前大多数论文都严重依赖于相关性和推测分析。综述了奶牛瘤胃微生物研究的进展和局限,包括瘤胃微生物与产奶效率、甲烷排放以及瘤胃发育的关系,以及奶牛瘤胃微生物未来的研究趋势。     

试论微生态制剂对反刍动物的作用机制

要了解微生态制剂对反刍动物的作用 ,首先要了解反刍动物瘤胃内的微生态环境和瘤胃微生物对机体的生理效应 ,如同了解自然 ,才能改造自然一样 ,为此 ,许多畜牧专家、微生物和微生态专家都一直从事瘤胃微生物生态系及其生理效应的研究。企图通过复制或调控瘤胃微生物区系来增加产量 ,提高效益。1　反刍动物消化道器官的特殊性反刍动物 (牛、羊、鹿、驼等 )在其长期的历史进化过程中形成了独特的消化系统。其瘤胃分为四个室 ,即瘤胃、网胃、瓣胃和皱胃。前三个胃称为前胃 ,没有胃腺 ,主要靠微生物的发酵作用消化饲料 ,其中瘤胃最大 ,占动物总…     

动物胃肠道微生物元基因组学研究进展

动物胃肠道中寄居着庞大复杂的微生物,它们对宿主营养、健康和生产有着重要的作用.随着分子生物学的发展,未培养微生物的研究越来越被重视,宏基因组学方法研究胃肠道微生物不仅能了解未培养微生物多样性,还能获得微生物的遗传、代谢和生理等方面的信息.探讨了元基因组文库的构建和分析方法,并重点介绍了元基因组学在动物胃肠道尤其是反刍动物瘤胃微生物研究中的应用.     

近10年瘤胃微生物分离培养研究进展

瘤胃微生物的研究一直是反刍动物营养研究的重点领域,自20世纪中期以来,大量参与营养代谢的微生物被从瘤胃中分离和认识.当代,尽管分子生物学方法越来越广泛的应用于瘤胃微生物的研究,但传统的分离纯培养的研究方法,因能够获得新的微生物菌种,便于微生物生理功能与代谢的研究,仍具有不可或缺的作用.本文综述了2001年至2011年间中国知网和PubMed数据库中通过纯培养方法从瘤胃中分离微生物菌株(包括新属、种或首次从瘤胃中分离)的相关文献,并展示了这些微生物的形态和发酵特征,同时总结了瘤胃微生物的全基因组信息,旨在为今后瘤胃微生物的研究提供参考菌种和信息.     

表面活性剂调控瘤胃营养功能研究进展

表面活性剂分为化学表面活性剂和生物表面活性剂两大类,非离子表面活性剂和生物表面活性剂作为新型反刍动物饲料添加剂,可通过改变瘤胃液乳化特性、瘤胃微生物种群数量、分泌酶活性、酶吸附能力和瘤胃发酵模式,来增强瘤胃微生物对粗饲料的降解能力,进而提高反刍动物生产性能。综述提出了表面活性剂在反刍动物瘤胃营养调控领域的研究重点。     

微生物介导反刍动物瘤胃氨生成及其对瘤胃功能的影响

反刍动物瘤胃中栖息着丰富多样的微生物,其在瘤胃内氨生成过程中发挥了重要的作用。微生物介导的氨基酸脱氨基作用和非蛋白氮水解作用是瘤胃内氨生成的主要途径。微生物介导了瘤胃内氨的生成,同时瘤胃内产生的氨也会反馈影响微生物菌群结构及瘤胃上皮功能,进而影响瘤胃发酵及宿主健康。本文主要综述了瘤胃微生物在介导氨生成中的作用和氨对瘤胃消化及瘤胃上皮功能的影响,以期对后续研究有所启发。     

未培养技术在瘤胃产甲烷菌群研究中的应用

瘤胃中的产甲烷菌为严格厌氧微生物,很难通过分离培养的方法进行研究。现已经培养并公布的瘤胃产甲烷菌只有4种,成千上万种类的瘤胃产甲烷菌不能培养出来。未培养技术的发展与应用突破了传统分离培养检测方法的限制,使瘤胃产甲烷菌的研究有了较大的进展。综述了实时定量PCR、16S rDNA文库、DGGE和高通量测序技术等6项具有代表性的未培养技术及其在瘤胃产甲烷菌研究中的应用,为以后瘤胃产甲烷菌的研究提供方法上的参考。     

丁酸调控幼龄反刍动物瘤胃上皮发育研究进展

瘤胃是反刍动物营养物质消化吸收和代谢的重要器官,其发育状态直接影响反刍动物生产性能和健康。初生犊牛和羔羊,瘤胃功能尚未发育完全,不能够充分消化和吸收固体饲料。因此,在幼龄时期,通过营养调控手段促进反刍动物的瘤胃发育对维持动物健康及提高生产性能具有重要意义。丁酸是瘤胃微生物降解植物性饲料的主要产物,也是瘤胃上皮及宿主的重要能量来源。丁酸调控幼龄反刍动物瘤胃上皮发育是一个历久弥新的话题。主要介绍了幼龄反刍动物瘤胃上皮形态及功能的发育以及丁酸调控幼龄反刍动物瘤胃上皮发育的研究进展。     

Ruminant feces harbor diverse uncultured symbiotic actinobacteria

To isolate actinobacteria from ruminant feces and elucidate their correlations with ruminants, the actinobacterial community in sheep (Ovis aries) and cattle (Bos taurus) feces was determined by cultivation and clone library methods. Most of actinobacteria isolated belonged to Streptomyces, Amycolatopsis, Micromonospora, and Cellulosimicrobium genera. The strains showed above 99 % similarity with the type strains, respectively. All the strains isolated could grow on media containing pectin, cellulose, or xylan as the sole carbon sources. However, most antibacterial and antifungal activities were found in Streptomyces species. Clone library analysis revealed that the genera Mycobacterium, Aeromicrobium, Rhodococcus, Cellulomonas were present in cattle and sheep feces. In contrast, the 16S rRNA genes showed less than 98 % similarity with the type strains. The analysis of actinobacterial community in ruminant feces by clone library and cultivation yielded a total of 10 actinobacterial genera and three uncultured actinobacterial taxa. The ruminant feces harbored diverse actinobacterial community. Ruminants may represent an underexplored reservoir of novel actinomycetes of potential interest for probiotics and drug discovery.     

Comparison of Ileum Microflora of Pigs Fed Corn-, Wheat-, or Barley-Based Diets by Chaperonin-60 Sequencing and Quantitative PCR

We have combined the culture-independent methods of high-throughput sequencing of chaperonin-60 PCR product libraries and quantitative PCR to profile and quantify the small-intestinal microflora of pigs fed diets based on corn, wheat, or barley. A total of 2,751 chaperonin-60 PCR product clones produced from samples of ileum digesta were examined. The majority (81%) of these clones contained sequences independently recovered from all three libraries; 372 different nucleotide sequences were identified, but only 14% of the 372 different sequences were recovered from all three libraries. Taxonomic assignments of the library sequences were made by comparison to a reference database of chaperonin-60 sequences combined with phylogenetic analysis. The taxa identified are consistent with previous reports of pig ileum microflora. Frequencies of each sequence in each library were calculated to identify taxa that varied in frequency between the corn, barley, and wheat libraries. The chaperonin-60 sequence inventory was used as a basis for designing PCR primer sets for taxon-specific quantitative PCR. Results of quantitative PCR analysis of ileum digesta confirmed the relative abundances of targeted taxa identified with the library sequencing approach. The results of this study indicate that chaperonin-60 clone libraries can be valid profiles of complex microbial communities and can be used as the basis for producing quantitative PCR assays to measure the abundance of taxa of interest during experimentally induced or natural changes in a community.     

Comparison of ileum microflora of pigs fed corn-, wheat-, or barley-based diets by chaperonin-60 sequencing and quantitative PCR

We have combined the culture-independent methods of high-throughput sequencing of chaperonin-60 PCR product libraries and quantitative PCR to profile and quantify the small-intestinal microflora of pigs fed diets based on corn, wheat, or barley. A total of 2,751 chaperonin-60 PCR product clones produced from samples of ileum digesta were examined. The majority (81%) of these clones contained sequences independently recovered from all three libraries; 372 different nucleotide sequences were identified, but only 14% of the 372 different sequences were recovered from all three libraries. Taxonomic assignments of the library sequences were made by comparison to a reference database of chaperonin-60 sequences combined with phylogenetic analysis. The taxa identified are consistent with previous reports of pig ileum microflora. Frequencies of each sequence in each library were calculated to identify taxa that varied in frequency between the corn, barley, and wheat libraries. The chaperonin-60 sequence inventory was used as a basis for designing PCR primer sets for taxon-specific quantitative PCR. Results of quantitative PCR analysis of ileum digesta confirmed the relative abundances of targeted taxa identified with the library sequencing approach. The results of this study indicate that chaperonin-60 clone libraries can be valid profiles of complex microbial communities and can be used as the basis for producing quantitative PCR assays to measure the abundance of taxa of interest during experimentally induced or natural changes in a community.     

Community analysis of hydrogen-producing extreme thermophilic anaerobic microflora enriched from cow manure with five substrates

The present study analyzed the community structures of anaerobic microflora producing hydrogen under extreme thermophilic conditions by two culture-independent methods: denaturing gradient gel electrophoresis (DGGE) and clone library analyses. Extreme thermophilic microflora (ETM) was enriched from cow manure by repeated batch cultures at 75 degrees C, using a substrate of xylose, glucose, lactose, cellobiose, or soluble starch, and produced hydrogen at yields of 0.56, 2.65, 2.17, 2.68, and 1.73 mol/mol-monosaccharide degraded, respectively. The results from the DGGE and clone library analyses were consistent and demonstrated that the community structures of ETM enriched with the four hexose-based substrates (glucose, lactose, cellobiose, and soluble starch) consisted of a single species, closely related to a hydrogen-producing extreme thermophile, Caldoanaerobacter subterraneus, with diversity at subspecies levels. The ETM enriched with xylose was more diverse than those enriched with the other substrates, and contained the bacterium related to C. subterraneus and an unclassified bacterium, distantly related to a xylan-degrading and hydrogen-producing extreme thermophile, Caloramator fervidus.     

From plants to animals; the role of plant cell death in ruminant herbivores

Plant cell death occurring as a result of adverse environmentalconditions is known to limit crop production. It is less wellrecognized that plant cell death processes can also contributeto the poor environmental footprint of ruminant livestock production.Although the forage cells ingested by grazing ruminant herbivoreswill ultimately die, the lack of oxygen, elevated temperature,and challenge by microflora experienced in the rumen induceregulated plant stress responses resulting in DNA fragmentationand autolytic protein breakdown during the cell death process.Excessive ruminal proteolysis contributes to the inefficientconversion of plant to microbial and animal protein which resultsin up to 70% of the ingested nitrogen being returned to theland as the nitrogenous pollutants ammonia and urea. This constitutesa significant challenge for sustainable livestock production.As it is estimated that 25% of cultivated land worldwide isassigned to livestock production, it is clear that understandingthe fundamental biology underlying cell death in ingested foragewill have a highly significant role in minimizing the impactof human activities. This review examines our current understandingof plant metabolism in the rumen and explores opportunitiesfor exploitation of plant genetics to advance sustainable landuse. Key words: Anoxia, cell death, environment, heat, plant–microbe interactions, proteolysis Received 7 September 2007; Revised 21 November 2007 Accepted 23 November 2007     

Biotic and abiotic factors influencing in vitro growth of Escherichia coli O157:H7 in ruminant digestive contents

The gastrointestinal tract (GIT) of ruminants is the main reservoir of enterohemorrhagic Escherichia coli, which is responsible for food-borne infections in humans that can lead to severe kidney disease. Characterization of biotic and abiotic factors that influence the carriage of these pathogens by the ruminant would help in the development of ecological strategies to reduce their survival in the GIT and to decrease the risk of contamination of animal products. We found that growth of E. coli O157:H7 in rumen fluid was inhibited by the autochthonous microflora. Growth was also reduced when rumen fluid came from sheep fed a mixed diet composed of 50% wheat and 50% hay, as opposed to a 100% hay diet. In fecal suspensions, E. coli O157:H7 growth was not suppressed by the autochthonous flora. However, a probiotic strain of Lactobacillus acidophilus inhibited E. coli O157:H7 growth in fecal suspensions. The inhibitory effect was dose dependent. These lactic acid bacteria could be a relevant tool for controlling O157:H7 development in the terminal part of the ruminant GIT, which has been shown to be the main site of colonization by these pathogenic bacteria.     

Dynamic role of single-celled fungi in ruminal microbial ecology and activities

In ruminants, high fermentation capacity is necessary to develop more efficient ruminant production systems. Greater level of production depends on the ability of the microbial ecosystem to convert organic matter into precursors of milk and meat. This has led to increased interest by animal nutritionists, biochemists and microbiologists in evaluating different strategies to manipulate the rumen biota to improve animal performance, production efficiency and animal health. One of such strategies is the use of natural feed additives such as single-celled fungi yeast. The main objectives of using yeasts as natural additives in ruminant diets include; (i) to prevent rumen microflora disorders, (ii) to improve and sustain higher production of milk and meat, (iii) to reduce rumen acidosis and bloat which adversely affect animal health and performance, (iv) to decrease the risk of ruminant-associated human pathogens and (v) to reduce the excretion of nitrogenous-based compounds, carbon dioxide and methane. Yeast, a natural feed additive, has the potential to enhance feed degradation by increasing the concentration of volatile fatty acids during fermentation processes. In addition, microbial growth in the rumen is enhanced in the presence of yeast leading to the delivery of a greater amount of microbial protein to the duodenum and high nitrogen retention. Single-celled fungi yeast has demonstrated its ability to increase fibre digestibility and lower faecal output of organic matter due to improved digestion of organic matter, which subsequently improves animal productivity. Yeast also has the ability to alter the fermentation process in the rumen in a way that reduces methane formation. Furthermore, yeast inclusion in ruminant diets has been reported to decrease toxins absorption such as mycotoxins and promote epithelial cell integrity. This review article provides information on the impact of single-celled fungi yeast as a feed supplement on ruminal microbiota and its function to improve the health and productive longevity of ruminants.     

Construction and characterization of a bovine BAC library with four genome-equivalent coverage

A bovine artificial chromosome (BAC) library of 105 984 clones has been constructed in the vector pBeloBAC11 and organized in 3-dimension pools and high density membranes for screening by PCR and hybridization. The average insert size, determined after analysis of 388 clones, was estimated at 120 kb corresponding to a four genome coverage. Given the fact that a male was used to construct the library, the probability of finding any given autosomal and X or Y locus is respectively 0.98 and 0.86. The library was screened for 164 microsatellite markers and an average of 3.9 superpools was positive for each PCR system. None of the 50 or so BAC clones analysed by FISH was chimeric. This BAC library increases the international genome coverage for cattle to around 28 genome equivalents and extends the coverage of the ruminant genomes available at the Inra resource center to 15 genome equivalents.     

Modern methods for the evaluation of qualitative and quantitative changes in the characteristics of intestinal and vaginal microflora

Disturbances in normal intestinal and vaginal microflora in women have recently become quite frequent. This accounts for the need of bacteriological laboratories for introduction of reliable methods for the diagnosis of such disturbances. Correct methodological approaches to objective evaluation of the state of intestinal and vaginal microflora are described. The methods used in the study of anaerobic microflora (lacto- and bifidobacteria, eubacteria, peptostreptococci, clostridia, bacteriods, fusobacteria) and facultative anaerobic microorganisms (enterobacteria, staphylococci, streptococci, Gardnerella, fungi of the genus Candida) have been analyzed. All stages of the study are described in consecutive order: the transportation of the material under study, its treatment in a laboratory, the spectrum of selective nutrient media for the isolation of microorganisms, methods of their identification.     

鸡肠道菌群总DNA三种提取方法的比较

目的比较不同方法提取鸡肠道菌群总DNA的差异,为分子方法分析肠道菌群组成提供质量较高的DNA模板。方法采用反复冻融法、酶裂解法和试剂盒法（E.N.Z.A Stool DNA Kit）来提取鸡肠道菌群的总DNA,并根据DNA浓度及纯度、16S DNA扩增产物和ERIC-PCR产物所反映的片段多态性4个指标,对这3种方法提取的DNA质量进行比较。结果3种方法均能提取DNA,所得DNA都可以用于16S DNA的扩增,但后2种方法所得DNA的ERIC-PCR结果能反映出更高的菌群多样性。结论试剂盒法和酶裂解法所提取的DNA质量好,适合用于肠道菌群的分子生态研究。