Analysis Of Gut Microbiota Diversity In Blue Fox And Comparative Study On Its Enrichment Methods In Vitro

In order to explore the intestinal microorgannisms diversity of blue foxes and the enrichment methods on intestinal microorganisms of blue fox in vitro cultrue,the fresh faeces and colon contents were collected and proformed by high-throughput sequencing.The three different media were chose on the fresh faeces and colon contents of blue foxes in enrichment culture and subculture.And then the bacterial cultures were proformed by high-throughput sequencing.According to the high-throughput sequencing results,the diversity of gut microbiota were analyzed and the best culture was selected,which provided a microbiological basis for further studies on the functions andnurtrient metabolism of intestinal microorganisms of blue fox.The paper consists of three parts.part 1: Analysis of faeces microbiota diversity in blue foxThe composition and diversity of gut microbiota in faeces,which were from eight females of healthy growing(5-6 month)blue fox and were analyzed by the high-throughput sequencing.The results showed as follows:A total of 654 716 valid sequences were identified in faecal samples from 8 healthy blue fox.The numbers of OTU ranged from 468 to 574,representing 16 phylum and 209 genus bacterial phylotypes(operational taxonomical units,OTUs).The bacterial phylum of higher abundance included Firmicutes,Bacteroidtes,Actinobacteria,Proteobacteria and Fusobacteria.The bacterial genus of higher abundance included Streptococcus,Lactobacillus,Prevotella,Megasphaera,Collinsella,Blautia,Bacteroides.Conclusion: The present study represented that there was a complex intestinal flora of blue fox by the high-throughput sequencing.And there were significant difference among individuals.Part 2: Comparative study on culturemethods of blue fox faeces microbiota invitro.Six males blue foxes of healthy growing period(4-5 months old)were randomly selected.Then the fresh feces were collected.The fresh faeces of blue fox were enriched and cultured in three media of A,B and C.After three generations of cultivation,the composition and diversity of gut microbiota was analyzed by Illumina HiSeq sequencing technology.The results showed as follows:After three generations of cultivation,there were significant difference in the microbial communities of there media.Through Alpha index analysis,there were significant difference(P>0.05).The microbial diversity of A medium is greater than that of medium C.Then the microbial diversity of medium C is greater than that of medium B.The analysis of Beta diversity showed that the dominant microflora of three enriched medium changed.The dominant microbiota of medium A were Proteobacteria,Firmicutes and Fusobacteria.The dominant microbiota of medium B were Firmicutesand Proteobacteria.The dominant microbiota of medium C were Fusobacteria,Firmicutes and Proteobacteria.Compared with the microbial communities of media A and C.And medium B had a single microbial communities.conclusion:The results showed that the microbial diversity obtained by medium A was abundant.Therefore,medium A could be used as the enrichment medium for blue fox colonic microbiota in vitro.Part 3:Analysis of blue fox colonic microbiota diversity and comparative study on enrichment methods o in vitro.Seven males blue foxes of healthy growing period(4-5 months old)were randomly selected.Then the colonic contents were collected.The composition and diversity of faeces microbiota were analyzed by the high-throughput sequencing.The colonic contents of the blue fox were enriched and cultured in three media(A,B and C).After nine consecutive generations of cultivation,the composition and diversity of colonic microbiota were analyzed by high-throughtput sequencing technology.The results showed as follows:With the increase generations of cultivation,the microbial communities of three media changed significantly.Alpha index analysis showed that the microbial diversity of medium A was greater than that of medium C.And the microbial diversity of medium C was greater than that of medium B.The analysis of Beta diversity showed that the microbial species richness in medium A was relatively high,but the composition of microbial species in media B and C were relatively simple.The dominant microflora accounting for more than 1% of medium A was Firmicutes,Bacteroidetes,Actinobacteria,Proteobacteria on the level of phylum classification.The dominant microflora accounting for more than1% of medium B was Firmicutes,Oxyphotobacteria on the level of phylum classification.The dominant microflora accounting for more than 1% of medium C was Firmicutes on the level of phylum classification.At the level of genus classification,the dominant microflora of medium A was Haloimpatiens(26.07%),Lactobacillus(20.21%),Acidaminococcus(16.05%),Peptostreptococcus(15.38%),accounting for 77.71% in total.And the dominant microflora of medium B was Lactobacillus(91.99%).And the dominant microflora of medium C was Lactobacillus(57.26%),Megamonas(17.35%),Peptostreptococcus(10.18%),accounting for 84.79% in total.With the increase generations of cultivation,the composition of the microbiota in the media A and C tended to be stable,but the microbiota in the medium B gradually disappeared.The analysis of PICRUSt gene prediction showed that the functional genes of the microbiota community in medium A were highly similar to the functional genes of the blue fox colonic microbiota,while the functional genes of the bacterial community in media B and C were significantly different from the functional genes of the blue fox colonic microbiota.Conclusion: The results showed that there was relatively rich microbial diversity of medium A,followed by medium C.And medium B was the lowest.Therefore,medium A could be used as the enrichment medium for blue fox colonic microbiota in vitro.