Diversity Analysis And Isolation Identification Of Fecal Flora Of White Feathered Broilers At Different Age

White feather broilers occupies an important position in my country's meat supply.They have excellent characteristics such as short growth cycle,high nutritional value and high feed conversion rate.These characteristics are largely due to the digestion and absorption of nutrients by the intestinal flora.It plays an important role in barrier immunity and other aspects.In recent years,the rapid development of high-throughput sequencing and omics technology has greatly improved people's understanding of intestinal flora.In order to explore the influence of age factors on the composition and function of the intestinal flora of broilers,this study chose 16 S amplicon sequencing technology to longitudinally analyze the structural diversity and composition characteristics of the bacterial flora,which is a normal intestine in the growth cycle of healthy broilers.The distribution law of the flora provides baseline information,which has certain reference value for the prevention of broiler related diseases.On the other hand,because many of the sequences obtained by sequencing cannot be annotated to the genus and species level,the strains obtained through culturomics can make up for the shortcomings in the sequencing results,which is indispensable to fully understand the interaction between the bacterial community and the host.At the same time,the isolation and identification of new strains not only enriches the broiler bacterial strain resource library,but also provides basic materials for subsequent research on diseases related to intestinal flora.Among them,the potential probiotic strains also provide an excellent source of strains for disease prevention and the development of probiotic preparations for poultry.The main research results of this experiment are as follows:1.Diversity analysis of fecal floraThe 16 S amplicon sequencing technology was used to analyze the composition and distribution of the flora in the fecal flora of white feather broilers of different ages.Six time points covering the entire growth cycle were selected for day age,namely 1,7,14,21,35 and 42 d.Based on the results of the relative abundance of species composition relationships in the sequencing results being greater than 1%,this study has annotated a total of 5 phyla,20 families,25 genera and 5 species.Species mainly include Firmicutes,Bacteroidetes,Proteobacteria,Actinobacteria and Cyanobacteria,but these 5 dominant bacteria phyla are There are differences in the proportion of each group of samples.At the phylum level,Firmicutes is the main dominant flora,and the proportion of the six groups is higher than 60%,but its proportion in the flora gradually decreases as the age increases.The phylum Bacteroides belongs to the second most dominant flora(except for the 7d group),and its proportion in the flora gradually increases as the age increases.In this experiment,in the early growth stage of broilers,the ratio of Firmicutes to Bacteroides in the flora is the largest,which means that the body has a higher level of heat absorption,which is the rapid growth period for broilers;At the family level,newborns Chicks are mainly composed of Clostridiaceae(78.24%),but the ratio of Lachnospiraceae and Ruminococcaceae increases with the increase of age;At the genus level,Clostridium is the main difference bacteria,and the abundance of Clostridium in the 1 dgroup(78.23%)is about the 42 d group(16.4%)5 times;At the species level,the sequence obtained by sequencing only annotated 5 species of bacteria.The analysis of ? and ? diversity of the flora showed that there was no significant difference in the diversity of the broiler fecal flora between the 7d and 42 d groups,but there were significant differences before the 7d of age.Function prediction found that functional genes are mostly enriched in carbohydrate metabolism pathway,amino acid metabolism pathway,cofactor and vitamin metabolism pathway,terpenoid and polyketone metabolism and lipid compound metabolism pathway.These metabolic pathways are mostly related to the production performance,feed conversion rate and disease resistance of broilers.In addition,there are significant differences in the metabolic pathways between the 1d and otherd groups,and most of these pathways are important pathways in the process of bacterial genetic and environmental information processing.2.Separation and cultivation of fecal floraIn this study,the characteristics of the flora were obtained through high-throughput sequencing analysis,combined with culture omics,and a variety of media and culture conditions were used to separate and culture broiler manure samples at 35 and 42 d.A total of 899 were identified through 16 S r RNA gene sequencing.Strains of bacteria represent 5 phyla,7 classes,10 orders,15 families,19 genera and 82 species.At the phylum level,Firmicutes(78.31%)accounted for most of the proportion,followed by Proteobacteria(14.91%),Bacteroides(5.34%),Actinomycetes(1.22%)and Fusobacteria.(Fusobacteria,0.22%);At the family level,Bacillaceae(33.82%)and Lactobacillaceae(28.7%)account for about half of the total number of cultured bacteria;At the genus level,Bacillus(33.82%)has the most bacteria,followed by Lactobacillus(27.81%),Enterococcus(11.12%)and Shigella(10.46%),etc.;At the species level,a total of 82 species of bacteria were obtained,including 11 potential probiotics and 25 potential new strains,which require follow-up Further identification and analysis.In addition,the study found that the diversity of 42 d fecal flora was higher than that of 35 d.Only Firmicutes(87.86%)and Proteobacteria(12.14%)bacteria were cultivated in the 35 d samples.At 42 d of age,a total of five phylum of Firmicutes(67.1%),Proteobacteria(18.16%),Bacteroides(11.62%),Actinomycetes(2.67%)and Fusobacteria(0.48%)were cultivated.3.Comparative analysis of high-throughput sequencing combined with culture resultsComparing the results of the culture with the results detected by the sequencing,it is found that most of the dominant bacterial populations in the phylum level in the sequencing results can be cultivated through culturomics,but at the family level,only 5 of the same families obtained by the two methods are obtained.They are Bacteroidaceae,Bifidobacteriaceae,Lactobacillaceae,Micrococcaceae and Tannerellaceae.At the species level,only B.dorei and B.fragilis were obtained by the two methods.There are different preferences between the results of bacterial community structure obtained between the two methods.