Mapping the bacterial ecology on the phyllosphere of dry and post soaked grass hay for horses

Soaking hay fodder to reduce dust and soluble carbohydrate (WSC) contents prior to feeding is common practice among horse owners. Soaking can increase bacteria load in hay but no information exists on how this process alters the bacteria profile, which could pose a health risk or digestive challenge, to horses by introducing foreign bacteria into the gastrointestinal tract and so altering the normal profile. The current objectives were to map the bacterial profile of 3 different hays and determine how soaking alters this with the aim of improving best practice when feeding stabled horses. A Perennial Rye grass hay and two meadow s hays were soaked for 0, 1.5, 9 or 16 hours. Pre and post treatment, hays were analysed for WSC and total aerobic bacteria (CFU/g), and differences in bacteria family profiles were determined using ANOVA with significance set at P<0.05. Bacteria were identified via genomic DNA extraction and 16S library preparation (V3 and V4 variable region of 16S rRNA) according to the Illumina protocol. Differences in family operational taxonomic units (OTUs) between individual dry hays and different soaking times 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 and actual g/kg lost on DM basis (+/- SE) increased with soaking time being 18% = 30 (10.7), 38% = 72 (43.7), and 42% = 80 (38.8) for 1.5, 9 and 16 hours soak respectively. No relationship existed between WSC leaching and bacteria growth or profile. Grass type influenced bacterial profiles and their responses to soaking, but no differences were seen in richness or Shannon diversity indices. PCA analyses showed clustering of bacteria between meadow hays which differed from the perennial rye grass hay and this difference increased post soaking. Soaking hay pre-feeding causes inconsistent WSC leaching, bacteria growth and alterations in bacterial profiles which are unpredictable but may decrease the hygienic quality of the fodder.