Four Haylages, One Metabonome: Defining Diet Driven Metabolic Shifts in Ponies

Haylage is widely fed to all categories of stabled horses across Europe and New Zealand. Haylage can vary from high energy nutrient dense forage to low energy highly fibrous fodder, yet little is known about the impact different haylages have on metabolism in horses. This study provides the first direct comparison of feeding four different haylages: early cut (Lolium multiflorum), later cut (Lolium perenne) rye grass (R1 and R2); early and late cut meadow grass (M1 and M2), on urinary and faecal metabonomics, in ponies at maintenance. Using replicated Latin Square design, eight individually housed ponies were fed each haylage for 15 days in four periods. Faeces and urine were collected on day 12 for metabonomic profiling. Apparent digestibility was evaluated on 3 days of total faecal collection (days 10-12) before adaptation to the new diet (days 13-15). Metabolic profiles (¹H NMR Spectroscopy), were phased and baseline corrected via TSP singlet δ 0.0. Principal Component Analysis (PCA) via Matlab, was used to identify metabolic profiles and Orthogonal Projections to Latent-Structures Discriminate Analysis (OPLS-DA) models to group specific metabolic changes. Digestibility was determined using multivariate mixed effects modelling +ANOVA. Pre-study urinary metabolic profiles were similar for all ponies. R1 haylage produced differentiating urinary clusters relative to R2 and M1 although no individual metabolites differed significantly. Some faecal R1 clustering was evident. Pairwise metabolites comparisons reflected underlying nutrient profiles. High inter-pony variation did not prevent dietary discrimination, with R1 > M1 > M2 = R2. This study provides the first evidence that haylages differing in maturity and botanical composition generate measurable metabolic shifts in ponies. These findings highlight the biological importance of haylage selection and indicate that metabonomic profiling is a sensitive informative tool for assessing how forage characteristics shape digestive physiology.