Impact of fecal sample preservation and handling techniques on the canine fecal microbiota profile

Abstract

Canine fecal microbiota profiling provides insight into host health and disease. Standardization of methods for fecal sample storage for microbiomics is currently inconclusive, however. This study investigated the effects of homogenization, the preservative RNAlater, room temperature exposure duration, and short-term storage in the fridge prior to freezing on the canine fecal microbiota profile. Within 15 minutes after voiding, samples were left non-homogenized or homogenized and aliquoted, then kept at room temperature (20–22°C) for 0.5, 4, 8, or 24 hours. Homogenized aliquots then had RNAlater added or not. Following room temperature exposure, all aliquots were stored in the fridge (4°C) for 24 hours prior to storing in the freezer (-20°C), or stored directly in the freezer. DNA extraction, PCR amplification, then sequencing were completed on all samples. Alpha diversity (diversity, evenness, and richness), and beta diversity (community membership and structure), and relative abundances of bacterial genera were compared between treatments. Homogenization and RNAlater minimized changes in the microbial communities over time, although minor changes in relative abundances occurred. Non-homogenized samples had more inter-sample variability and greater changes in beta diversity than homogenized samples. Storage of canine fecal samples in the fridge for 24 h prior to storage in the freezer had little effect on the fecal microbiota profile. Our findings suggest that if immediate analysis of fecal samples is not possible, samples should at least be homogenized to preserve the existing microbiota profile.

Comparison of the fecal microbiota of adult healthy dogs fed a plant-based (vegan) or an animal-based diet

“…With the use of client-owned animals, despite instructions to freeze fecal samples immediately after defecation, there was no supervision by the research team to ensure the fecal collection and storage was performed properly by pet guardians prior to delivery of the samples. However, the authors do not expect this to influence the results based on previous research in companion animals showing limited changes in the microbiota from short term (2 weeks) exposure to ambient temperature and time (Weese and Jalali, 2014;Tal et al, 2020;Chui et al, 2023). A further limitation factor with respect to data collection and the research variables assessed in the study was the frequency of sampling.…”

“…Bridge amplification was completed on an Illumina MISeq system (Illumina, San Diego, CA, USA) at the University of Guelph Agri-Food Labs using terminator nucleotides that were incorporated into the amplified PCR products with the removal of the terminator group ( Tal et al, 2020 ; Chui et al, 2023 ).…”