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Journal of animal physiology and animal nutrition2026; doi: 10.1111/jpn.70099

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

Abstract: 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 (H 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.
© 2026 The Author(s). Journal of Animal Physiology and Animal Nutrition published by Wiley‐VCH GmbH.
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Publication Date: 2026-08-06 PubMed ID: 42561131DOI: 10.1111/jpn.70099Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study investigated the metabolic effects of feeding four different types of haylage to ponies.
  • It demonstrated that differences in haylage maturity and botanical composition lead to distinct metabolic shifts in pony digestion and health.

Introduction and Purpose

  • Haylage is a common forage fed to stabled horses, varying in energy and fiber content depending on factors like plant species and harvest timing.
  • Despite its widespread use in Europe and New Zealand, the effects of different haylages on equine metabolism remain poorly understood.
  • This study aimed to directly compare the metabolic impacts of four haylages differing in botanical type and maturity on ponies maintained at a stable weight.

Experimental Design

  • Four haylages were tested:
    • Early cut ryegrass (Lolium multiflorum, labeled R1)
    • Later cut ryegrass (Lolium perenne, labeled R2)
    • Early cut meadow grass (M1)
    • Late cut meadow grass (M2)
  • Eight ponies were housed individually and fed these four haylages in a replicated Latin Square design, meaning each pony received all haylages over four periods.
  • Each feeding period lasted 15 days, with sample collections and measurements occurring during and near the end of each period.

Sample Collection and Measurement

  • Urine and feces samples were collected on day 12 of each feeding period for metabonomic profiling.
  • Digestibility of the diets was determined from total fecal collections on days 10-12.
  • Adaptation to the new diet occurred from days 13-15.

Analytical Methods

  • Metabolic profiling was performed using proton nuclear magnetic resonance (¹H NMR) spectroscopy, a technique that identifies compounds present in biological samples.
  • Data processing included phase and baseline correction relative to a chemical shift standard.
  • Statistical analyses were conducted utilizing:
    • Principal Component Analysis (PCA) to visualize overall metabolic differences across diets.
    • Orthogonal Projections to Latent-Structures Discriminate Analysis (OPLS-DA) to identify specific metabolic changes linked to each haylage.
    • Multivariate mixed effects modeling and ANOVA were used for digestibility assessments.

Key Findings

  • Prior to the study, all ponies had similar urinary metabolic profiles, ensuring baseline consistency.
  • Urine from ponies fed early cut ryegrass (R1) exhibited distinct clustering patterns compared to those fed later cut ryegrass (R2) and early cut meadow grass (M1), indicating a different metabolic response.
  • No single urinary metabolite showed statistically significant difference, but patterns of metabolites differed collectively.
  • Fecal samples also showed some clustering patterns linked to R1, suggesting changes in gut metabolism or microbial activity.
  • Differences in metabolites reflected the underlying nutritional makeup of the haylages, demonstrating the sensitivity of metabonomic analysis.
  • Despite natural variability between individual ponies, significant dietary discrimination was detected with the order of metabolic effects being R1 > M1 > M2 = R2.

Conclusions and Implications

  • This research provides the first direct evidence that the maturity stage and botanical composition of haylage cause measurable shifts in pony metabolism.
  • Selecting haylage carefully can biologically impact digestive physiology in horses, influencing how forage is metabolized and utilized.
  • Metabonomic profiling emerges as a powerful and sensitive method to detect subtle changes in equine digestive metabolism in response to diet.
  • These insights can help improve feeding strategies for horses to optimize health and nutrient utilization by considering forage characteristics.

Cite This Article

APA
Moore-Colyer M, Harris P, Daniels S. (2026). Four Haylages, One Metabonome: Defining Diet Driven Metabolic Shifts in Ponies. J Anim Physiol Anim Nutr (Berl). https://doi.org/10.1111/jpn.70099

Publication

Journal of animal physiology and animal nutrition
ISSN: 1439-0396
NlmUniqueID: 101126979
Country: Germany
Language: English

Researcher Affiliations

Moore-Colyer, Meriel
  • Deparment of Animal Health, Hartpury University, Gloucester, Hartpury, UK.
Harris, Pat
  • Equine Studies group, Waltham Petcare Science Institute, Mars Horsecare, Waltham-on-the-Wolds, Leicestershire, UK.
Daniels, Simon
  • Royal Agricultural University, Stroud Road, Cirencester, UK.

Grant Funding

  • Mars Petcare UK

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