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

Fresh-frozen stool banks intended for humans with gastrointestinal and metabolic disorders have been recently established and there are ongoing efforts 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 -20̊C and -80̊C 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, frozen in liquid nitrogen and stored at -20̊C or -80̊C for 3, 6, 9, and 12 months. At baseline and after period of storage, aliquots were thawed and treated with propidium monoazide before fecal DNA extraction. Following long-read 16S-rRNA amplicon sequencing, bacterial community composition and diversity were compared among treatment groups. We demonstrated that fresh-frozen canine stools collected from 20 apparently healthy dogs could be stored for up to 12 months at -80̊C with minimal change in microbial community composition and diversity and that storage at -80̊C is superior to storage at -20̊C. We also found that differences between dogs had the largest effect on community composition and diversity. Relative abundances of certain bacterial taxa, including those known to be short-chain fatty acid producers, varied significantly with specific storage temperatures and duration. Further work is required to ascertain whether 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.