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Equine veterinary journal2026; doi: 10.1002/evj.70351

Association of facial expression, biochemistry, and microRNAs with lameness in endurance horses.

Abstract: Lameness is common in endurance horses. Conventional gait-based assessment may not fully capture its various aspects. Facial expression scales can provide complementary, non-locomotor indicators of lameness-related pain. Objective: To evaluate the performance of facial expression scales Horse Grimace Scale (HGS) and Equine Utrecht University Scale for Facial Assessment of Pain (EQUUS-FAP) for assessing pain-related lameness in endurance horses and their associations with the American Association of Equine Practitioners (AAEP) lameness grading system, biochemical, and molecular markers. Methods: Case-control study. Methods: Forty-four Arabian horses (24 stallions, 20 mares) were enrolled as lame (n = 23), non-lame (n = 11), and control (n = 10). Field-based grouping was determined by veterinary-gate assessment: horses eliminated for irregular gait were classified as lame, and finishers passing veterinary inspection as non-lame. Blinded trot-up videos were reviewed to verify grouping and assign AAEP lameness grades; facial expression videos recorded at the veterinary gate were used to assess facial action units. Blood analyses measured biochemical markers. Real-time PCR quantified serum miR-21 and miR-155. Results: Facial expression scales had high area under the curve values for distinguishing lame from non-lame horses (HGS: 0.947, 95% CI: 0.850-1.000; EQUUS-FAP: 0.917, 95% CI: 0.817-1.000). Facial scores were strongly correlated with AAEP lameness grades (r ≈ 0.78, p ≤ 0.001). Lame horses had higher oxidative stress index, lactate, lactate dehydrogenase, cortisol, aspartate aminotransferase concentrations, and miR-21 expression than other groups (p ≤ 0.015). Conclusions: The small sample size warrants confirmation of the results in larger populations. Conclusions: Facial expression scores were associated with AAEP lameness grades and selected biochemical and molecular parameters in endurance horses. Facial expression assessment may provide complementary information alongside established lameness evaluation methods. Further studies are needed to confirm its role in pain-related lameness assessment.
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Publication Date: 2026-11-03 PubMed ID: 42974908DOI: 10.1002/evj.70351Google 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 whether facial expression scales can be used to assess pain caused by lameness in endurance horses and how these assessments relate to established lameness grading, as well as biochemical and molecular markers.
  • The research aimed to determine if facial expressions provide complementary information to conventional gait assessments and associated physiological changes.

Introduction and Objective

  • Lameness, or difficulty in movement due to pain or injury, is common in endurance horses and typically evaluated by observing gait.
  • Conventional gait-based assessments may not fully capture all aspects of lameness-related pain.
  • Facial expression scales, such as the Horse Grimace Scale (HGS) and EQUUS-FAP (Equine Utrecht University Scale for Facial Assessment of Pain), have potential as non-locomotor pain indicators.
  • The study’s objective was to evaluate how effective these facial expression scales are in detecting pain-related lameness and how they correlate with:
    • Veterinarian-assigned lameness grades (AAEP scale)
    • Biochemical markers in blood related to stress and muscle damage
    • Molecular markers like circulating microRNAs (miR-21 and miR-155)

Methods

  • The study was a case-control design involving 44 Arabian horses (24 stallions and 20 mares).
  • Horses were classified into three groups based on veterinary gate assessments during endurance competitions:
    • Lame: horses eliminated for irregular gait (n=23)
    • Non-lame: horses that passed veterinary inspection and completed the race (n=11)
    • Control: horses not competing or baseline controls (n=10)
  • Blinded evaluations of trot-up videos were used to confirm lameness status and assign AAEP lameness grades.
  • Facial expression videos taken at the veterinary gate were analyzed using the HGS and EQUUS-FAP scales, which score specific facial action units indicative of pain.
  • Blood samples were collected for biochemical analyses measuring:
    • Oxidative stress index
    • Lactate & lactate dehydrogenase (LDH) – markers of muscle metabolism and damage
    • Cortisol – a stress hormone
    • Aspartate aminotransferase (AST) – an enzyme elevated in muscle injury
  • Real-time PCR was used to quantify expression levels of serum microRNAs miR-21 and miR-155, which are associated with inflammatory and stress responses.

Key Results

  • Both facial expression scales demonstrated high accuracy in distinguishing lame from non-lame horses:
    • HGS Area Under the Curve (AUC): 0.947 (very high accuracy)
    • EQUUS-FAP AUC: 0.917 (also high accuracy)
  • Facial expression scores correlated strongly with the AAEP lameness grades (correlation coefficient approx. 0.78), supporting the idea that facial cues reflect lameness severity.
  • Lame horses showed significantly elevated physiological markers relative to non-lame and control groups:
    • Higher oxidative stress index indicating increased oxidative damage
    • Elevated lactate and lactate dehydrogenase levels, consistent with muscle exertion or injury
    • Increased cortisol, indicating heightened stress
    • Elevated aspartate aminotransferase pointing to muscle cell damage
    • Upregulated miR-21 expression, a microRNA involved in inflammation and tissue repair
  • These biochemical and molecular findings support the facial expression data, linking pain-related lameness with systemic physiological alterations.

Conclusions and Implications

  • The study suggests that facial expression assessments using HGS and EQUUS-FAP are promising complementary tools for evaluating pain in lame endurance horses alongside traditional gait-based methods.
  • The observed associations with biochemical markers and microRNA expression reinforce the validity of facial expressions as indicators of underlying physiological stress and tissue damage.
  • However, due to the relatively small sample size, the authors recommend that these preliminary findings be validated in larger populations before clinical adoption.
  • If confirmed, integrating facial expression scoring could enhance welfare monitoring and clinical assessment accuracy by providing a rapid, non-invasive, and sensitive measure of pain in horses.
  • Future research should focus on refining facial scoring methods, exploring their sensitivity and specificity in diverse equine populations, and understanding how they can best complement other assessment techniques.

Cite This Article

APA
Mehrafrouz P, Imani Rastabi H, Borujeni MP, Tabandeh MR. (2026). Association of facial expression, biochemistry, and microRNAs with lameness in endurance horses. Equine Vet J. https://doi.org/10.1002/evj.70351

Publication

Equine veterinary journal
ISSN: 2042-3306
NlmUniqueID: 0173320
Country: United States
Language: English

Researcher Affiliations

Mehrafrouz, Paria
  • Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran.
Imani Rastabi, Hadi
  • Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran.
Borujeni, Mahdi Pourmahdi
  • Department of Food Hygiene, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran.
Tabandeh, Mohammad Reza
  • Department of Basic Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran.

Grant Funding

  • SCU.VC1402.291 / Shahid Chamran University of Ahvaz, Ahvaz, Iran

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