Comparison Of DNA Extraction Methods For The Analysis Of Songliao Black Swine Gut Microbiota And Its Cecum Microbial Diversity

Gut microbes are closely related with host health, and its community structure directly affects the health and production efficiency of livestock husbandry. Piglets’ gut microbial is instability in the post weaning, gut microorganism disorders can lead to diarrhea and death. Low doses of antibiotics can reduce stress reaction and diarrhea, improve production efficiency in the post weaning. But the use of long-term antibiotics also has potential hazard in livestock husbandry,such as antibiotic residues, the effects of antibiotics on gut microorganism and host health.Therefore, it is of great significance to develop alternative antibiotics. The target of antibiotics or alternative antibiotics is mainly gut microorganism. The influence of alternative antibiotics on the gut microbes is one of the important indicators to evaluate the effect of alternative antibiotics. Gut microbial composition is very complex, gut microbial DNA extraction method is crucial for the study of gut microbial diversity. So, at first, we should find a suitable DNA extraction method when using molecular biological techniques to study gut microbial community structure.Massa Medicata Fermentata(MMF) is made from Artemisia annua, Polygonum hydropiper,Xanthium sibiricum, bitter almond, ted bean, wheat bran and flour by a certain proportion.Contains a variety of enzymes, such as protease, amylase and cellulose, with help digestion and reduce diarrhea. Probiotics have the function of maintaining microbial community structure balance, improving the immunity, and absorbing nutrients. MMF and probiotics mixture has the characteristics of probiotics, chinese herbal medicine and enzyme, its development potential is huge. In this study, we selected Songliao Black Swine as the research object, used PCR-DGGE and real-time PCR to compare the effectiveness of DNA extraction by five DNA extraction methods, and analysis the effects of different feed additive(bacitracin and colistin sulfate mixture,MMF and probiotics mixture) on growth performance and caecal microbial diversity of Songliao Black Swine. The main conclusions are as follows:(1) The effectiveness of the total DNA extracted by the five methods was significantly different in cecal contents and feces from Songliao Black SwineComparative analysis the effectiveness of five different DNA extraction methods, the results show that the highest DNA purity, yield and microbial diversity was extracted using QIAamp?DNA Stool Mini Kit, more suitable for the analysis of microbial DNA extract and microbialdiversity of cecum and feces from Songliao Black Swine. The low DNA purity, yield and microbial diversity was extracted using TIANamp Stool DNA Kit and SDS-based DNA extraction method. Solarbio Stool DNA Kit and Cell lysing solution TZ with the minimum purity of DNA,the lowest microbial diversity and the worst abundance, which was not suitable for the analysis on gut microbial diversity.(2) The structure and composition of microbial communities were obviously different in cecal contents and feces from Songliao Black SwineCompared the microbial diversity of cecal contents with feces from Songliao Black Swine,the results show cecal contents and fecal microbial community structure was difference, the microbial richness of feces was higher than cecal contents. Cecal contents and fecal microorganisms belong to Firmicutes, Bacteroidetes, Proteobacteria and Actinomycetes. Real time PCR results showed that cecal contents and fecal microbial population numbers were different.Therefore, to fully understand the differences of microbial community structure in cecum and feces was important to study the cecal content and fecal microbial diversity.(3) MMF and probiotics mixture can partly replace antibioticsThe effects of different feed additive on growth performance of piglets, research results show that the piglets with initial body weight were basically similar, but the body weight of different treatment groups had significant difference(p< 0.05) after 49 days, and compared to the control group, antibiotic group increased 40.63%, MMF group increased 21.73%; the average daily gain(ADG) of different treatment groups had significant difference(p< 0.05), the antibiotic group was2.09 times of the control group, MMF group was 1.59 times; the feed conversion rate of MMF group and antibiotic group were higher than control group; the serious diarrhea and mortality of MMF group and antibiotic group were lower than control group, and the MMF group had no deaths. Therefore, the MMF and probiotics mixture as feed additive can promote the growth of piglets, improve the ADG and feed conversion rate, reduce the serious diarrhea and mortality,which had the potential to replace antibiotics.(4) MMF and probiotics mixture can improve the microbial composition of pigletsThe effects of different feed additive on caecal microbial diversity of Songliao Black Swine,the results showed that there were different of microbial composition and relative contents in the contents and mucous layer, the MMF and probiotics mixture as feed additive can improveLactobacilli spp., Streptococcus spp., Enterococcus spp. population quantity and Firmicutes/Bacteroidetes ratio, reduced Enterobacteriaceae relative content and Escherichia coli population quantity. The MMF and probiotics mixture as feed additive can improving the gut microbial community structure and immunity of piglets.