Effect of sampling and storage conditions upon equine fecal microbial community

Equine feces offer a non-invasive sample which can provide an indication of the microbial community structure of part of the hindgut; however, microbial communities may be altered by technical artifacts introduced under field conditions. Four types of preservation method (preservation buffer (NAP), treatment with chlorhexidine digluconate, FTA card sample collection, cold (buffer-free) storage with insulated ice packs (COLD)) and time at ambient temperature (0, 24, 48, 72 h), were tested for their effect upon the microbial community structure of fecal samples from n = 5 mature, mixed breed horses, collected immediately post defecation. Sequencing of the 16S rRNA gene was performed using the Qiagen QIAseq 16S/ ITS screening panel and Illumina MiSeq sequencing platform. Alpha diversity was estimated using Shannon, Simpson, Chao1 and Observed diversity metrics, and compared using Kruskal-Wallis’ test (P < 0.05) and post hoc Dunn's test with Bonferroni adjustment. Bray Curtis distances and permutational multivariate ANOVA were used to assess β-diversity. There were significant differences in α-diversity with preservation method, but not with time at ambient storage or individual. Alpha diversity was significantly lower in FTA card samples compared with COLD samples for Shannon and Simpson, but not Chao1 or Observed metrics. All α-diversity metrics were significantly lower in FTA card samples compared with those in NAP (Table 1). No significant differences in α-diversity were identified for other comparisons. Regarding β-diversity, individual pony (P = 0.001), and time at ambient storage (P = 0.004) were significant and explained most of variance between samplesacross the study. Preservation method significantly influenced β-diversity as the second term in an additive model, after accounting for variance driven by individual pony (P = 0.011). These results highlight the strength of inter-individual variability and suggest that minimizing sample time at ambient temperature remains important for preserving the microbial composition of equine fecal samples. While preservation method may not have drastically affected the composition