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definitive metabolite identification was performed. Spectral data were analysed as an untargeted metabolomics dataset.</p>MetaboLightsPublicNuclear Magnetic Resonance (NMR) -Four Haylages, One Metabonome: Defining Diet Driven Metabolic Shifts in Ponies. 10.1111/jpn.70099. PMID:42561131Forage chemical composition drives early metabolic shifts in ponies fed different haylages.<p>1H NMR spectra were acquired using a Bruker spectrometer operating at 600 MHz with water suppression. Spectra were collected under standardised acquisition conditions for all samples.</p>Simon DanielsRoyal Agricultural UniversityurinefecesNMR spectroscopy assay<p>Faecal and urine samples were collected and prepared for NMR analysis. Samples were homogenised and extracted using phosphate buffer prepared in D2O prior to spectral acquisition.</p>Equidaehttps://www.ebi.ac.uk/metabolights/MTBLS15865Simon Daniels. Royal Agricultural University, Stroud Road, Cirencester, GL7 6JS. simon.daniels@rau.ac.uk.Simon Daniels. Royal Agricultural University. simon.daniels@rau.ac.uk.<p>One-dimensional 1H NMR spectra were phased, baseline corrected and referenced prior to analysis. Spectral regions corresponding to residual water were excluded and processed spectra were used for downstream statistical analysis.</p>TreatmentTimesimon.daniels@rau.ac.uk<p>Faecal and urine samples were collected free catch as described in https://onlinelibrary.wiley.com/doi/full/10.1111/jpn.70099 </p>MetabolomicsMetabolomicsurineuntargeted analysisFecalBruker 600 MHz spectrometerHorsenuclear magnetic resonance spectroscopyEquidaefecesexperimental sampleuntargeted metabolite profilingforage materialMetabolomicsurineuntargeted analysisFecalBruker 600 MHz spectrometerHorsenuclear magnetic resonance spectroscopyEquidaefecesexperimental sampleuntargeted metabolite profilingforage material<p>Samples were prepared in phosphate buffer prepared in D2O containing TSP as a chemical shift reference and transferred to 5 mm NMR tubes prior to analysis.</p>LactateAcetateHaylage 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.Four Haylages, One Metabonome: Defining Diet Driven Metabolic Shifts in Ponies.Moore-Colyer Meriel M, Harris Pat P, Daniels Simon SfalseThe effect of changing haylages of differing chemical composition on the equid metabonomeRapid changes between feeds can cause dysbiosis and colic. The metabolic consequences of changing between haylages is currently unknown. Objectives Characterise urinary and faecal metabonomics in ponies transitioned between haylages varying in botanical composition and physiological age and to determine the speed of metabolic adaptation. Study Design: Repeated Latin Square with 8 Welsh Mountain ponies Methods: Ponies fed four haylages in four x 15-day periods; structured three-day changeover old: new 70: 30; 50.50; 30:70; on days 13,14,15. Day 1 was 100% new haylage. Apparent digestibility determined by multivatiate mixed effects model, from total faecal collections on days 10,11,12. Urinary and faecal collections on day 0, 5, 12 and 14. Metabolic profiling using 1H NMR spectroscopy in urine and faeces with PCA and OPLS-DA to identify diet-induced metabolic shifts. Alignment and normalisation (total quotient method) by Mathworks Matlab R2014a.. Results Ponies achieved metabolic adaptation within five days. Metabonomics were functionally stable at mid changeover (day 14) for most haylage changes. Metabolic responses were driven by forage chemical composition rather than haylage category. Physiologically young haylage R1 produced the greatest shifts, including elevated urinary glycine and altered faecal short-chain fatty acid and aromatic amino acid metabolites, reflecting higher protein bioavailability and rapid microbial reconfiguration. Main limitations Ponies were on all forage diets with no complementary feed. Results may not reflect more abrupt changes between haylages or between different forage types. Conclusions. Metabolic adaptation to new haylages occurred within the first 5 days, were shaped by forage composition, and low fibre haylage altered outputs compared with fibrous mature forages. Metabolism was stable during diet mixing. Metabonomics provides a sensitive, non invasive tool and is discriminatory between forages for nutrient availability and hindgut function. Vigilance should be recommended in the first 5 days of introducing a new forage for horses at risk of diet related gastrointestinal disturbance.2026-09-292026-09-29MTBLS15865HMDB0000190HMDB000018242561131