Isolation, Identification And Diversity Analysis Of Rumen Microorganisms In Longjiang And Cattle

Changes in the abundance of ruminant microbial communities in ruminants are related to many phenotypes of the body,including feed efficiency,disease status,methane emissions and the body's immune function.Research on the relationship between rumen microorganisms and body phenotype is a hot topic in ruminants.In this study,the composition of rumen microorganisms in Longjiang Wagyu was explored through high-throughput sequencing technology.Cultureomics technology was used to isolate and identify rumen microbial flora.Combining cultureomics technology with high-throughput sequencing technology to analyze the composition of the Longjiang Wagyu bacterial community.A Longjiang Wagyu rumen flora bank was initially established and more beneficial bacteria were screened.The aim is to provide material for future research on rumen microorganism.The rumen contents used in the experiment were collected from a commercial Longjiang Wagyu slaughterhouse in Longjiang County,Qiqihar City,Heilongjiang Province.A total of 5 male Longjiang Wagyu rumen contents were collected.Aseptically,the total DNA of the rumen contents flora were extracted,and the V3-V4 region sequencing analysis of the 16 S total DNA sequence was performed.A total of 37 bacterial families were identified,7 species of bacterial families that accounted for more than 90% of the entire DNA sequence,including Bacteroidia(Bacteroidia 38.22%-45.39%),Clostridia(Clostridia30.31%-34.88%),(Negativicutes 7.21%-10.06%),Unclassified mycelia(Unclassified4.72%-10.72%),Erysipelotrichia(Erysipelotrichia 0.42%-2.52%),Gammaproteobacteria(Gammaproteobacteria 0.49%-2.57%),Spirochaetia(Spirochaetia 0.73%-2.73%).A total of 207 bacterial genera were identified,49 accounted for more than 0.1%.There are 8 dominant species that accounted for more than 90% of the total 16 S total DNA sequence V3-V4 region,including unclassified bacteria(Unclassified 37.37%-51.61%),Prevotella(Prevotella 20.15%-26.18%),Succiniclasticum(Succiniclasticum 6.39%-8.59%),Barnesiella(Barnesiella 0.09%-4.41%),Rumenococcus(Ruminococcus 0.79%-5.91%),(Saccharofermentans 1.14%-2.13%),Lachnospiracea incertae sedis(Lachnospiracea incertae sedis 1.31%-1.61%),(Sporobacte0.28%-1.67%).Unclassified bacteria is the highest proportion of bacteria in this rumen contents.The collected rumen contents were separated and cultured by culturing technology.The isolated and cultured bacteria were amplified and sequenced by 16 S rDNA gene.The sequencingresults were compared in GenBank.A total of 520 strains of bacteria were obtained,and 314 strains were completely tested,including 161 Enterococcus strains and it accounts for 51.27% of the total genus;28 strains of bacillus(8.92%);26 strains of Acinetobacter(8.28%);23 strains of Proteus(7.32%);22 strains of Staphylococcus(7.01%);19 strains of Clostridium(6.05%);2strains of Clostridium sensu stricto(0.64%);6 strains of Acidaminococcus(1.91%);5 strains of Escherichia / Shigella(1.59%);3 strains of Bravibacterium(0.96%);4 strains of Streptococcus(1.27%);2 strains of Aerococcus(0.64%);2 strains of Citrobacter(0.64%);1 strain of Corynebacterium(0.32%);1 strain of Fusobacterium(0.32%);1 strain of Kocuria(0.32%);1strain of Microbacterium(0.32%);2 strains of Mitsuokella(0.64%);2 strains of Propionibacterium(0.64%);1 strain of Serratia(0.32%);1 strain of Unclassified_Bacillaceae 1(0.32%).In the results of the isolation and culture,314 strains of bacteria were detected,wit h a total of 52 strains,and the homology was between 99% and 100%.The top three b acteria in the results included Enterococcus,bacillus and Acinetobacter.Many beneficial bacteria were also isolated in this test,including Bacillus subtilis,Bacillus thuringiensis a nd Bacillus licheniformis.In this study,the high-throughput sequencing technology was used to gain a full-scal e view of the dominant bacteria genus in rumen of Longjiang Wagyu beef,which includ ing 21 phyla,37 classes,58 orders and 207 genera.which including Unclassified(37.37%-51.61%),Prevotella(20.15%-26.18%),Succiniclasticum(6.39%-8.59%),Barnesiella(0.09%-4.41%),Ruminococcus(0.79%-5.91%),Saccharofermentans(1.14%-2.13%),Lachnospi racea incertae sedis(1.31%-1.61%),Sporobacte(0.28%-1.67%).In parallel the 520 strain s of bacteria were isolated and cultured from the contents of rumen of Longjiang bovine by laboratory culture group technology,including 314 strains in 4 phylum,6 classes,9orders,20 families,21 genera after completely sequencing,among which 159 were of dif ferent genotypes.All in all,a preliminary library of rumen flora of Longjiang bovine wa s established successfully.