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Abstract Background Currently, lack of standardization for fecal microbiota transplantation (FMT) in equine practice has resulted in highly variable techniques, and there is no data on the bacterial metabolic activity or viability of the administered product. The objectives of this study were to compare the total and potentially metabolically active bacterial populations in equine FMT, and assess the effect of different frozen storage times, buffers, and temperatures on an equine FMT product. Fresh feces collected from three healthy adult horses was subjected to different storage methods. This included different preservation solutions (saline plus glycerol or saline only), temperature (-20 °C or -80 °C), and time (fresh, 30, 60, or 90 days). Samples underwent DNA extraction to assess total bacterial populations (both live and dead combined) and RNA extraction followed by reverse transcription to cDNA as a proxy to assess viable bacteria, then 16s rRNA gene amplicon sequencing using the V1-V2 region. Results The largest difference in population indices and taxonomic composition at the genus level was seen when evaluating the results of DNA-based (total) and cDNA-based (potentially metabolically active) extraction method. At the community level, alpha diversity (observed species, Shannon diversity) was significantly decreased in frozen samples for DNA-based analysis (P

Abstract Background An association between equine gastrointestinal disease causing colic signs and changes in faecal bacterial microbiota has been identified. The reasons for these changes and their clinical relevance has not been investigated. Withholding feed, which is an integral part of managing horses with colic, may contribute to the observed changes in the microbiota and impact interpretation of findings in horses with colic. Study objectives were, therefore, to determine the effect of withholding feed for 24 h on equine faecal bacterial microbiota in healthy mares to differentiate the effects of withholding feed from the changes potentially associated with the disease. Results Species richness and Shannon diversity (alpha diversity) were significantly lower at the late withheld (10–24 h post withholding feed) and early refed (2–12 h post re-feeding) time points compared to samples from fed horses (P