Homemade Nucleic Acid Preservation Buffer Proves Effective in Preserving the Equine Faecal Microbiota over Time at Ambient Temperatures

SIMPLE SUMMARY: The microbial community in horse faeces can be assessed to make inferences about the gut bacteria, which is linked to the animals’ health. However, faecal bacterial communities can shift over time if not preserved between the points of sampling and processing, which could cause misleading results. This study stored equine faecal samples under four preservation treatments at room temperature for up to 150 h and assessed the resulting impact on the samples’ bacterial communities. Treatments included “COLD” (samples packaged with a cool pack), “CLX” (2% chlorhexidine digluconate solution), “NAP” (nucleic acid preservation buffer), and “FTA” (Whatman FTA™ cards). Samples were assessed after storage for 0, 24, 72, and 150 h at room temperature under the different treatments. The results showed that NAP buffer was effective in preserving the most prominent features of the equine faecal bacterial community for up to 150 h at room temperature, but the processing of FTA cards was inadequate to capture the full bacterial profile present. The cold preservation, CLX, and NAP treatments were equally effective in maintaining the bacterial community in equine faecal samples for up to 24 h. These findings demonstrate the effectiveness of NAP buffer and the potential of using COLD and CLX treatments for sample preservation at room temperature. This study also showed changes in the bacteria found in equine faeces that occur under preservation for up to 150 h. ABSTRACT: The equine faecal microbiota is often assessed as a proxy of the microbial community in the distal colon, where the microbiome has been linked to states of health and disease in the horse. However, the microbial community structure may change over time if samples are not adequately preserved. This study stored equine faecal samples from n = 10 horses in four preservation treatments at room temperature for up to 150 h and assessed the resulting impact on microbial diversity and the differential abundance of taxa. Treatments included “COLD” (samples packaged with a cool pack), “CLX” (2% chlorhexidine digluconate solution), “NAP” (nucleic acid preservation buffer), and “FTA” (Whatman FTA™ cards). The samples were assessed using 16S rRNA gene sequencing after storage for 0, 24, 72, and 150 h at room temperature under the different treatments. The results showed effective preservation of diversity and community structure with NAP buffer but lower diversity (p = 0.001) and the under-representation of Fibrobacterota in the FTA card samples. The NAP treatment inhibited the overgrowth of bloom taxa that occurred by 72 h at room temperature. The COLD, CLX, and NAP treatments were effective in preserving the faecal microbiota for up to 24 h at room temperature, and the CLX and NAP treatments improved the yield of Patescibacteria and Fibrobacterota in some cases. The cold and CLX treatments were ineffective in preventing community shifts that occurred by 72 h at room temperature. These findings demonstrate the suitability of the COLD, NAP, and CLX treatments for the room temperature storage of equine faeces for up to 24 h and of NAP buffer for up to 150 h prior to processing.