不同粗饲料条件下梅花鹿瘤胃甲烷菌结构的比较分析

本研究旨在基于高通量测序技术分析比较采食3种常见粗饲料梅花鹿瘤胃甲烷菌结构。选取3只2岁龄的装有永久性瘤胃瘘管的成年雄性梅花鹿为研究对象,采用3×3拉丁方设计,分别饲喂以柞树叶（OL组）、玉米秸秆（CS组）和玉米青贮（CI组）为主要粗饲料的饲粮。预试期为1周,正试期为4周。采用引物A519F、A976R扩增瘤胃甲烷菌16S rRNA基因V3-V4区,基于Illumina M iseq PE250平台进行测序。结果表明：9个样本共获得600 352条高质量的甲烷菌16S rRNA基因序列,基于97%相似性共归为111个分类操作单元（OTU）。覆盖度指数表明本试验样品覆盖了瘤胃99%的甲烷菌。分类分析结果表明甲烷短杆菌属（Methanobrevibacter spp.）[OL组：（97.80±6.00）%;CS组：（97.10±1.50）%;CI组：（87.50±5.50）%]是梅花鹿瘤胃优势甲烷菌,但采食3种粗饲料梅花鹿瘤胃甲烷菌在种水平的分布有差异。CI组Methanosphaera stadtmanae相对丰度显著高于OL组与CS组（P〈0.05）。OL组与CI组Methanobrevibacter millerae相对丰度高于CS组（P〉0.05）,OL组与CS组Methanobrevibacter boviskoreani相对丰度高于CI组（P〉0.05）,而CS组与CI组Methanobrevibacter olleyae相对丰度高于OL组（P〉0.05）。本研究发现Methanobrevibacter spp.是梅花鹿瘤胃优势甲烷菌。

Comparison of Fecal Methanogenic Archaeal Community Between Erhualian and Landrace Pigs Using Denaturing Gradient Gel Electrophoresis and Real-Time PCR Analysis

Erhualian and Landrace breeds are typical genetically obese and lean pigs, respectively. To compare the fecal methanogenic Archaeal community between these two pig breeds, fecal samples from different growth phase pigs were collected and used for PCR-denaturing gradient gel electrophoresis （DGGE） with two primer pairs （344fGC/519r and 519f/915rGC） and real-time PCR analysis. Results showed that a better separation and higher quality of bands pattern were obtained in DGGE proifles using primers 344fGC/519r as compared with primers 519f/915rGC. Sequencing of DGGE bands showed that the predominant methanogens in the feces of Erhualian and Landrace pigs belonged to Methanobrevibacter spp. and Methanosphaera spp. Real-time PCR analysis revealed that there was no signiifcant difference in the numbers of fecal total methanogens between Erhualian and Landrace pigs;however, pig growth phase affected the numbers of 16S rRNA genes of total methanogens and Methanobrevibacter smithii. Dissociation curves of methyl coenzyme-M reductase subunit A （mcrA） gene fragments ampliifed with real-time PCR showed all samples possessed a single peak at 82＆#176;C, which might be associated with M. smithii. Samples from the same growth phase of each breed showed good replicative dissociation curves. The results suggest that the growth phase （including diet factor） other than genotype of pig may affect the fecal methanogenic Archaeal community of pigs.

The structure of microbial populations in Nelore GIT reveals inter-dependency of methanogens in feces and rumen

Background:The success of different species of ruminants in the colonization of a diverse range of environments is due to their ability to digest and absorb nutrients from cellulose,a complex polysaccharide found in leaves and grass.Ruminants rely on a complex and diverse microbial community,or microbiota,in a unique compartment known as the rumen to break down this polysaccharide.Changes in microbial populations of the rumen can affect the host’s development,health,and productivity.However,accessing the rumen is stressful for the animal.Therefore,the development and use of alternative sampling methods are needed if this technique is to be routinely used in cattle breeding.To this end,we tested if the fecal microbiome could be used as a proxy for the rumen microbiome due to its accessibility.We investigated the taxonomic composition,diversity and inter-relations of two different GIT compartments,rumen and feces,of 26 Nelore(Bos indicus)bulls,using Next Generation Sequencing(NGS)metabarcoding of bacteria,archaea and ciliate protozoa.Results:We identified 4265 Amplicon Sequence Variants(ASVs)from bacteria,571 from archaea,and 107 from protozoa,of which 143(96 bacteria and 47 archaea)were found common between both microbiomes.The most prominent bacterial phyla identified were Bacteroidetes(41.48%)and Firmicutes(56.86%)in the ruminal and fecal microbiomes,respectively,with Prevotella and Ruminococcaceae UCG-005 the most relatively abundant genera identified in each microbiome.The most abundant archaeal phylum identified was Euryarchaeota,of which Methanobrevibacter gottschalkii,a methanogen,was the prevalent archaeal species identified in both microbiomes.Protozoa were found exclusively identified in the rumen with Bozasella/Triplumaria being the most frequent genus identified.Co-occurrence among ruminal and fecal ASVs reinforces the relationship of microorganisms within a biological niche.Furthermore,the co-occurrence of shared archaeal ASVs between microbiomes indicates a dependency of the predominant fecal methanogen population on the rumen population.Conclusions:Co-occurring microorganisms were identified within the rumen and fecal microbiomes,which revealed a strong association and inter-dependency between bacterial,archaeal and protozoan populations of the same microbiome.The archaeal ASVs identified as co-occurring between GIT compartments corresponded to the methanogenic genera Methanobrevibacter and Methanosphaera and represented 26.34%of the overall archaeal sequencesdiversity in the rumen and 42.73%in feces.Considering that these archaeal ASVs corresponded to a significant part of the overall diversity of both microbiomes,which is much higher if one includes the interactions of these co-occurring with other rumen archaea ASVs,we suggest that fecal methanogens could be used as a proxy of ruminal methanogens.