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<0.05). Mean WSC losses g/kg DM (+/-SE) increased with soaking time being 30 (10.7), 72 (43.7), 80 (38.8) for 1.5, 9 and 16 hours soak respectively. No relationship existed between WSC leaching and bacteria content or profile. Grass type influenced bacterial profiles. Soaking altered the epiphytic bacterial profile across all hays and 9 hours soaking increased richness and Shannon diversity indices. Clustering of bacteria was seen between meadow hays which differed from perennial rye grass and this difference increased post soaking. The normal industry practice of soaking hay for 9 hours pre-feeding cannot be recommended as it increases total bacteria content with noted increases of some potential pathogens. The alterations in bacteria profile and hygienic quality may explain why changing fodder or pre-feeding treatments can frequently precipitate colic in horses.