Impact of Ambient Temperature Sample Storage on the Equine Fecal Microbiota.
Authors: Martin de Bustamante Michelle, Plummer Caryn, MacNicol Jennifer, Gomez Diego
Journal: Animals : an open access journal from MDPI
Summary
# Editorial Summary: Ambient Temperature Storage and Equine Fecal Microbiota When submitting faecal samples for microbiota analysis, the handling and storage protocol significantly influences which bacterial populations you'll detect—a critical consideration for anyone investigating equine digestive health, probiotics, or feed interventions. De Bustamante and colleagues examined this directly by collecting faecal samples from 11 healthy horses, freezing one aliquot immediately at −80°C whilst holding the remaining samples at room temperature (21–22°C) and freezing them at 6, 12, 24, 48, 72 and 96-hour intervals, then sequencing the bacterial 16S rRNA gene using the Illumina MiSeq platform. Samples frozen within 12 hours of collection showed minimal compositional differences, with the key cellulose-digesting families Fibrobacteraceae and Ruminococcaceae preserved; however, samples stored at ambient temperature for 24 hours or longer demonstrated significant shifts in alpha-diversity, community structure and membership, alongside enrichment of spore-forming and opportunistic taxa (Bacillaceae, Enterobacteriaceae, Moraxellaceae) that don't reflect the live horse's microbiota. For practitioners collecting field samples, this work validates a practical 6–12-hour window for ambient storage before freezing without compromising data integrity, but emphasises that delays beyond 24 hours risk artefactual results that could mislead dietary or therapeutic decisions.
Read the full abstract on PubMed
Practical Takeaways
- •If equine fecal samples cannot be immediately frozen at -80°C, storing them at room temperature for up to 6 hours is acceptable for microbiota analysis with minimal artifacts
- •Samples must be frozen within 12 hours of collection to preserve accurate microbial community structure; delays beyond this point introduce significant composition alterations
- •For accurate representation of the equine hindgut microbiota, establish a protocol to freeze samples within 6 hours of collection rather than relying on room temperature storage
Key Findings
- •Storage of equine fecal samples at room temperature (21-22°C) for up to 6 hours before freezing had minimal effect on microbial composition compared to immediate freezing at -80°C
- •Samples frozen within 12 hours shared similar community membership, but samples frozen at 12 hours or later showed significantly different community structure compared to 0-hour controls
- •Room temperature storage for 24 hours or longer resulted in enrichment of Bacillaceae, Planococcaceae, Enterobacteriaceae and Moraxellaceae taxa, indicating microbial overgrowth
- •Fibrobacteraceae and Ruminococcaceae were preferentially preserved in samples frozen within 6 hours, while longer storage selected for different bacterial families