| Waterfowl are renowned for their ability to exploit a wide variety of food resources, due to their big gizzard and fermentation of geese ceca. But birds cannot excrete cellulose by themselves, so there are many cellulose-splitting bacteria within the ceca. It is very important to study the gut microbial flora ecology in geese. With the development of molecular biology, different molecular techniques have been used to evaluate the microbial diversity of different ecosystems. In recent years, denaturing gradient gel electrophoresis (DGGE). as a molecular fingerprint and the use of16S ribosomal RNA gene (16S rRNA) sequences to classify and identify microorganisms, have been widely used in microbial diversity. And Real-time PCR (Real time ploymerase chain reaction, RT-PCR) can be quantitative of bacterial numbers of geese intestinal. Using group-specific primer sets, the abundance of a particular gene marker for a defined group in the community can be estimated by comparison to a standard curve. Unlike other avian species, the goose can take advantage of fibrous plant materials partly. So we must focus on the study about the effect of dietary fibre on geese. The purpose of this study was to understand the intestinal microbiota composition of geese, to know what kind of bacteria colonize the ceca with different age, to know the effect of dietary fibre on community of geese intestinal. So we start to make some experiments.Trial6Study on isolation and identifation of cellulose decomposing bacterial in geese ceca by16S rRNA genes analysisThe study also included a correlative research work developing cellulolytic microbiology resources. In order to know the function of geese ceca, and digestion mechanism of fiber, we select six geese in the experiment. Geese were slaughtered and sterilized then ceca were collected immediately. Using the technology of anaerobic culture, culture mixture were streaked onto CMC-Na culture medium. All15strains were selected from ceca content. Colonies were stained with Cong-red. Diameters of stain circles were measured and relative enzyme activity was calculated. Three of which was selected for the biggest diameters of stain circles. The clones were identified bby comparing16S rRNA partial gene sequence of strains with BLAST analysis. The results showed that the strain (M2) had a identity (96%) with the cultured species Olsenella sp.. The strain (M5) had a high identity (99%) with the species Uncultured Streptococcus sp..The strain (M5) had a high identity (99%) with the rumen bacterium enrichment culture clone for utilizing fumarate.Trial2Molecular profiling of bacterial species in the caecum of geeseThe purpose of this study was to analyse the microbial diversity in the caecum of geese using a16S rRNA clone library approach. A total of160clones and124clones were sequenced and phylogenetically analysed from the contents and mucosa of the caecum of Yang Zhou geese, respectively. The result indicated that there was a rich variety of bacteria in the caecum contents. Forty-six operational taxonomic units (OTUs) based on a98%similarity criterion were classified in the contents of goose caecum, as compared to29OTUs based on a97%similarity criterion in the mucosa of goose caecum. Contents of goose caecum were dominantly occupied by Clostridia-related species (58.7%) with other abundant sequences being related to Bacteroidetes (26.9%) and Erysipelotrichi (11.2%). There are four phylum in the content of geese caecum, such as Firmicutes (69.9%), Bacteroidetes (26.9), Elusimicrobia (2.6%), Proteobacteria (0.6%). Thirty-four clones had a high identity (99%) with the cultured species, Pseudomonas sp. and nine clones had a high identity (99%) with the cultured species, Stenotrophomonas rhizophila.in the mucosa of goose caecum. Gammaproteobacteria (59.6%) and Clostridia (20.1%) were predominant in the mucosa of goose caecum. There are four phylum in the content of geese caecum, such as Proteobacteria (71.89%), Firmicutes (24.26%), Actinobacteria (2.42%)Bacteroidetes (1.61).Trial3Molecular profiling of bacterial species in the ileum of geeseThe purpose of this study was to analyse the microbial diversity in the ileum of geese using a16S rRNA clone library approach. A total of86clones were sequenced and phylogenetically analysed from the contents of the ileum of Yang Zhou geese. The result indicated that eight operational taxonomic units (OTUs) based on a97%similarity criterion were classified in the contents of goose ileum. Contents of goose ileum were dominantly occupied by Turicibacter sanguinis (41.8%) and with other abundant sequences being related to Peptostreptococcaceae (32.5%).Trial4The bacterial community and diversity in the intestinal tract of geese analyzed by PCR-DGGEIn the present paper, it is presented and discussed how age affected the bacterial community in the gastrointestinal tract of geese. PCR-DGGE of bacterial16S rRNA gene fragments was applied. DGGE analyses revealed that there were the fewest bands in ileum and the most bands in cecum, respectively. At Week2, bands numbers in duodenum and jejunum were significantly more than other ages. Then, the bands number decreased with the increasing of age. The bands numbers in jejunum were relatively stable. Most of the sequences retrieved from the DGGE bands were closely related to uncultured bacteria. Six sequences were related to Pseudomonas, with98-100%similarity. The special bacterial in ceacal digesta of geese were Pseudomonas, Bacteroides sp., and plenty of uncultured bacterial. There are uncultured bacterial in the duodenum, jejunum, ileum. The special bacterial in jejunum digesta of geese were Streptococcus pyogenes. Pseudomonas sp. and Uncultured Turicibacter were predominant in the intestinal tract.Trial5Analysis of geese intestinal community shifts in response to dietary fibreThis study was carried out to investigate the effects of dietary fibre on intestinal microbial flora ecology in geese. Ninety six140-day old adult ganders were randomly assigned into4dietary treatments, each treatment allocated to4replicates and per replicate with6birds. The control (A group) was fed basal diet, and experimental B, C, D group were fed basal diets insteaded by20%,40%and60%rice husk. Food and water were offered ad libitum for3weeks. After3weeks, two birds from each replicate were randomly selected and killed. Duodenum, jejunum and cecal contents were aseptically collected, and the total genomic DNA was extracted, then the intestinal bacterial community was analyzed by denaturing gradient gel electrophoresis (DGGE) analysis of universal16S rDNA after amplication with PCR, and the populations of total bacteria, Clostridium genus cluster IV, Clostridium cluster XIVa and XIVb, Bifidobacterium, Lactobacillus, Enterobacteriaceae, Enterococcus spp., Veillonella spp., E.coil, butyryl-coenzyme A (CoA) CoA transferase gene copies, butyrate kinase gene were detected by real-time PCR. Results showed that:With the increase of dietary crude fibre level, the band numbers of DGGE profiles of the V3region gene amplication of16S rRNA of cecal microflora increased. Gene sequences analysis indicated that the specific bands were mainly uncultured bacteria in cecum of geese. Genomic DNA in the dominant band in the group B and group C confirmed to be closely related to DNA from Uncultured Clostridiaceae bacterium. Uncultured Treponema sp., Cellulomonas sp.,Uncultured Bacteroides sp., Uncultured Eubucteriaceae bacterium. Numbers of Clostridium genus cluster Ⅳ in group C and D was significant higeher than that in group A by Real-time PCR. There are no difference every group for populations of total bacteria and Bifidobacterium, but dietary fibre increased the populations of Lactobacillus. Numbers of Veillonella spp. in group C was significant higher than that in group A. and dietary fibre decreased the populations of E. coil. There were no significant differences in numbers of butyryl-coenzyme A (CoA) CoA transferase gene copies, butyrate kinase gene among every group. |
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