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In this study, we comprehensively investigated the effect of dietary protein sources on the gut microbiome of weaned piglets with diets comprising different protein source using High-throughput 16SrRNA gene-based Illumina Miseq. A total of 48 healthy weaned piglets were allocated randomly to four treatments with 12 piglets in each group. The weaned piglets were fed with diets containing soybean meal (SBM), cottonseed meal (CSM), SBM and CSM (SC) or fish meal (FM). The intestinal content samples were taken from five segments of the small intestine. DNA was extracted from the samples and the V3-V4 regions of the 16SrRNA gene were amplified. The microbiota of the contents of the small intestine were very complex, including more than 4000 operational taxonomic units belonging to 32 different phyla. Four bacterial populations (i.e. Firmicutes, Proteobacteria, Bacteroidetes and Acidobacteria) were the most abundant bacterial groups. The genera Lactobacillus and Clostridium were found in slightly higher proportions in the groups with added CSM compared to the other groups. The proportion of reads assigned to the genus Escherichia/Shigella was much higher in the FM group. In conclusion, dietary protein source had significant effects on the small microbiome of weaned piglets.Dietary protein source have the potential to affect the small intestine microbiome of weaned piglets that will have a large impact on its metabolic capabilities and intestinal health. In this study, we successfully identified the microbiomes in the contents of the small intestine in the weaned piglets that were fed different protein source diets using high-throughput sequencing. The finding provided an evidence for the option of the appropriate protein source in the actual production.