Mapping the bacterial ecology on the phyllosphere of grass hay and the potential hazards of soaking fodder for horse gut health

Globally hay is the preferred forage for stabled horses. Variable nutritional and hygienic quality stimulates pre-feeding soaking to reduce dust and nutrients to reduce respiratory and metabolic disorders in horses. However, this practice has potential negative impacts on horse health. The objectives of this study were to map the bacterial profile of different hays and determine how soaking alters this with the aim of recommending best practice when feeding fodder to stabled horses. Two meadow and one Perennial Ryegrass hays were soaked for 0, 1.5, 9 or 16 hours. Post treatment, hays were analysed for water-soluble carbohydrate (WSC) and total aerobic bacteria (TVC), with differences determined using ANOVA and least significant difference. Bacteria were identified via genomic DNA extraction (V3 and V4 variable region of the 16S rRNA gene) and 16S library preparation according to the Illumina protocol. Differences in phyla and family operational taxonomic units within hay types were identified via paired t-tests on the DESeq2 normalised data and false discovery rates accounted for using Padj (P