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Sample storage conditions are an important factor in fecal microbiota analyses in general. The objective of this study was to investigate the effect of sample storage at room temperature on the equine fecal microbiota composition. Fecal samples were collected from 11 healthy horses. Each sample was divided into 7 sealed aliquots. One aliquot was immediately frozen at -80 °C; the remaining aliquots were stored at room temperature (21 to 22 °C) with one transferred to the freezer at each of the following time points: 6, 12, 24, 48, 72 and 96 h. The Illumina MiSeq sequencer was used for high-throughput sequencing of the V4 region of the 16S rRNA gene. Fibrobacteraceae ( Fibrobacter ) and Ruminococcaceae ( Ruminococcus ) were enriched in samples from 0 h and 6 h, whereas taxa from the families Bacillaceae, Planococcaceae, Enterobacteriaceae and Moraxellaceae were enriched in samples stored at room temperature for 24 h or greater. Samples frozen within the first 12 h after collection shared similar community membership. The community structure was similar for samples collected at 0 h and 6 h, but it was significantly different between samples frozen at 0 h and 12 h or greater. In conclusion, storage of equine fecal samples at ambient temperature for up to 6 h before freezing following sample collection had minimal effect on the microbial composition. Longer-term storage at ambient temperature resulted in alterations in alpha-diversity, community membership and structure and the enrichment of different taxa when compared to fecal samples immediately frozen at -80 °C.

Impact of Ambient Temperature Sample Storage on the Equine Fecal Microbiota