Fecal microbiome composition and diversity of cryopreserved canine stool at different duration and storage conditions

Fresh-frozen stool banks intended for human patients with gastrointestinal and metabolic disorders have been recently established and there is an ongoing effort to establish the first veterinary fresh-frozen stool bank. Fresh frozen stored feces provide an advantage of increased availability and accessibility to high-quality optimal donor fecal material. The stability of frozen canine feces regarding fecal microbiome composition and diversity has not been reported in dogs, providing the basis for this study. We hypothesized that fecal microbial composition and diversity of healthy dogs would remain stable when stored at -20 deg C and -80 deg for up to 12 months compared to baseline samples evaluated before freezing. Stool samples were collected from 20 apparently healthy dogs, manually homogenized, cryopreserved in 20% glycerol and aliquoted, snapped frozen in liquid nitrogen and stored at -20 deg C or -80 deg C for 3, 6, 9, and 12 months. Baseline samples and thawed aliquots were treated with propidium monoazide before fecal DNA extraction and following long-read 16S-rRNA amplicon sequencing, the community composition and diversity were compared across treatment groups. We demonstrated that 20% glycerol cryopreserved fresh-frozen canine stools collected from 20 apparently healthy dogs could be stored for up to 12 months at -80 deg C with minimal change in microbial community composition and diversity and that storage at -80 deg C is superior to storage at -20 deg C. We also found that differences between dogs had the largest effect on community composition and diversity. Certain bacterial taxa, such as short-chain fatty acid fermenters, had positive and negative survival correlations with specific storage conditions and duration. It remains to be determined if fecal donor material that differs in bacterial community composition and diversity across storage conditions and duration could lead to differences in clinical efficacy for specific clinical indications of fecal microbiota transplantation.