Pooled feces reduce the inter-sample variation of microbiota in pig models

For in vitro batch fermentation or fecal microbiota transplantation, preparation of inoculum was recommended by pooled feces to minimize inter-individual variation. However, the impact of pooling on microbiota variability remains unclear. The present study investigated the changes in inter-sample variation of microbial community in fecal samples after pooling from different numbers of donors, and verified the hypothesis that the variation of microbiota in feces affects the fermentation outcomes in vitro using fecal inoculum. The pooled feces from different pigs increased the microbial richness (Sobs and Ace indexes, P < 0.05). The PCoA indicated that the microbiota composition was not distinct among the treatments, however, the distance of the sample within group was reduced as the incremental number of feces in the mixed samples. Microbial composition analysis indicated the variation of microbiota abundance was reduced with the increasing number of feces in mixed samples. The in vitro fermentation results suggested that the coefficient variation (CV), especially inter-bath and total CV, the kinetic parameters of gas production, in vitro fermentability of dry matter (IVDMF), and SCFA production reduced as the number of mixture donors increased. Collectively, pooled feces originating from different donors can reduce the variation of microbiota between samples, and it is proposed that the fecal samples should be mixed, derived from 5 to 10 pigs, and then prepared as an inoculum to improve the reproducibility of in vitro fermentation.