The equine microbiome can change in response to dietary alteration and might play a role in insulin dysregulation. The aim of this study was to determine the effect of adding pasture to a hay diet on the fecal bacterial microbiome of both healthy and insulin-dysregulated ponies. Fecal samples were collected from sixteen ponies before and after dietary change to enable bacterial 16S rRNA sequencing of the V3-V4 region. The dominant phyla in all samples were the Firmicutes and Bacteroidetes. The evenness of the bacterial populations decreased after grazing pasture, and when a pony was moderately insulin-dysregulated (P=0.001). Evenness scores negatively correlated with post-prandial glucagon-like peptide-1 concentrations after a hay-only diet (r2;=-0.7, P=0.001). A change in diet explained 3% of fecal microbiome variability. We conclude that metabolically healthy ponies have greater microbial stability when challenged with a subtle dietary change, compared to moderately insulin-dysregulated ponies.

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