When researchers performed intestinal microbiota analysis using fecal samples, sample preparation processes are not standardized as of now. In particular, contrary to lab conditions, there are various factors hard to be controlled when sampling livestock feces on the farm. In this study, we wanted to know the influences of sample preparation conditions on fecal microbiota. We designed an experiment considering various sample preparation conditions (sample origin: rectum and ground; transporting condition: lysis buffer-treated, dry ice, ice, and room temperature; delayed times to extract DNA: 0, 1, 6, and 24 h) that can occur during microbiota analysis using cattle feces. First, we investigated the influences of sample origin, and microbial diversity (observed OTUs, p<0.001) was significantly higher when fecal samples were obtained from the ground than from the rectum and the principal coordinates analysis plot showed that samples were divided into two groups by origin. When we investigated the influences of transporting and storage conditions, observed OTUs (p<0.05) was significantly higher when samples were transported at room temperature than other conditions, and microbiota was affected by transporting and storage conditions. Finally, we investigated the effects of delayed time before DNA extraction from frozen fecal samples on microbial composition. Although this factor did not have a significant influence on microbial diversity, multidimensional scaling revealed its impact on microbial composition. Our findings will contribute to establishing an optimal procedure for microbiota analysis using farm-housing livestock feces.

This study was conducted to analyze the diversity census of fecal microbiome in horses using meta-analysis of equine 16S rRNA gene sequences that are available in the Ribosomal Database Project (RDP; Release 11, Update 5). The search terms used were “horse feces (or faeces)” and “equine feces (or faeces)”. A total of 842 sequences of equine feces origin were retrieved from the RDP database, where 744 sequences were assigned to 10 phyla placed within Domain Bacteria. Firmicutes (n = 391) and Bacteroidetes (n = 203) were the first and the second dominant phyla, respectively, followed by Verrucomicrobia (n = 58), Proteobacteria (n = 30) and Fibrobacteres (n = 24). Clostridia (n = 319) was the first dominant class placed within Bacteroidetes while Bacteroidia (n = 174) was the second dominant class placed within Bacteroidetes. The remaining 98 sequences were assigned to phylum Euryarchaeota placed within Domain Archaea, where 74 sequences were assigned to class Methanomicrobia. The current results will improve understanding of the diversity of fecal microbiome in horses and may be used to further analyze equine fecal microbiome in future studies.