Construction Of An Intestinal Microbial Metabase For Domestic Animals And Isolation And Culture Of Intestinal Bacteria

Objective:Livestock gut microbes play an important role in a variety of host physiological functions(e.g.,metabolism,immunity,inflammation,etc.).With the flood of rapid development of high-throughput sequencing technology,the coverage of macrogenomic data in public databases has become more and more extensive,resulting in unprecedented large and scattered data generated,causing great distress and inconvenience to scientific researchers in their studies,and researchers still find it difficult to accurately mine,compare and analyze metadata of livestock gut microbes.In addition,Xinjiang,one of the five major pastoral regions,is very suitable for the development of horse industry due to its unique geographical conditions,but the gut microbial resources of horses have not been fully exploited,and the isolation and culture of equine gut microorganisms are of great significance for the subsequent research on the development of equine-related probiotics.Methods:Firstly,we used modern information technology and literature to search NCBI and other databases for animal gut microbial metadata,downloaded the corresponding data,found the information used to build the database after preliminary screening,and further analyzed and organized the data to build the livestock gut microbial metadatabase.The information obtained from the database collection was combined with relevant literature to isolate and culture the gut microorganisms of Xinjiang horses using traditional microbiological research methods,analyze the structural composition of the gut microorganisms of Xinjiang horses,and finally test the functions of the gut bacteria using mouse experiments to provide an experimental basis for the possible functions of the gut flora of horses.1.According to the literature and web search,the databases recording the metadata of animal gut microbial macrogenomes were examined,clicked into them,checked whether the information contained in the databases was what we needed,marked and recorded them,and when all the databases were viewed,entered the marked databases,first checked whether there were duplicates between the databases,and discarded the included databases if there was an inclusion relationship between them.Then we found the link to store the metadata,used the download tool to download all the metadata to the local area,and used the EXCEL table tool to organize the downloaded data.We chose to first download and organize all the metadata of one animal in different databases,and then proceed to collect information for the next animal.Finally,all the attributes were standardized and the organized metadata were unified and uploaded to the server.2.The equine gut microorganisms were precultured using four methods:aerobic blood bottle preculture,aerobic blood bottle plus sheep blood preculture,aerobic blood bottle plus rumen fluid preculture,and BHI preculture,and were purified by delineation using sheep blood agar plates,and the purified bacteria were amplified by PCR and sequenced using bacterial universal primers.At the NCBI website,the BLAST tool was used to determine the genus of the isolates.The results with the highest sequence similarity to the isolates were downloaded and evolutionary trees were constructed using MEGA software.3.Obesity model mice were constructed using mice fed high-fat chow.The intestinal bacteria to be functionally verified were counted in CFU,diluted into a suspension of appropriate concentration(2.4×10~4CFU)for gavage on the mice,and the changes in body weight of the mice were recorded.After the gavage,the mice body weight data were analyzed by SPSS software and the experimental results were analyzed.Results:1.The database ultimately collected a total of 82,097 metadata including from four common domestic animals and 540 wildlife species,covering 15 years of experiments from May 2006 to May 2021,with a total of 1044 items and 69,302 samples,and we also classified the item types into health traits,production traits,microbial diversity,and life history traits.The classification was as follows:health traits were classified into disease,immunocompetence,pathogens and parasites;life history traits included weaning;and production traits included growth,meat quality,methane emission,metabolism,feed efficiency and feed intake.The browsing,searching and downloading functions of the information in the database were implemented through the WEB terminal.2.A total of 946 bacterial strains were obtained by isolating and culturing equine intestinal microorganisms using four different preculture methods,and were identified to include 13 genera:Escherichia,Shigella,Acinetobacter,Bacillus,Photobacterium,Streptococcus,Enterobacter,Hafnia,Lacto Bacillus,Enterococcus,Citrobacter,Klebsiella and Prolinoborus.3.The slower weight gain in mice gavaged with Hafnia compared to mice gavaged with saline may have a function in inhibiting animal growth.Conclusion:1.By constructing a database of animal gut microorganisms,it can help researchers to obtain information on relevant microbial metadata more rapidly and accurately,and provide a favorable tool and platform for animal gut microbial research.2.Aerobic microorganisms in the equine intestine have a very rich species diversity,and Escherichia spp.,Bacillus and Shigella occupy a large proportion of the equine intestinal microorganisms and are the main core flora of the equine intestine.3.Hafnia has a significant regulatory effect on animal growth,and the exact mechanism of action remains to be analyzed.