Towards an improved pain assessment in castrated horses using facial expressions (HGS) and circulating miRNAs

BACKGROUND: Pain in horses is an emergent welfare concern, and its assessment represents a challenge for equine clinicians. This study aimed at improving pain assessment in horses through a convergent validation of existing tools: we investigated whether an effective analgesic treatment influences the horse grimace scale (HGS) and the concentration of specific circulating microRNAs (miRNAs). METHODS: Eleven stallions underwent routine surgical castration under general anaesthesia. They were divided into two analgesic treatment groups: castration with the administration of preoperative flunixin and castration with preoperative flunixin plus a local injection of mepivacaine into the spermatic cords. HGS and levels of seven circulating miRNAs were evaluated pre-, 8 and 20 hours post-procedure. RESULTS: Compared to pre-castration, HGS, miR-126-5p, miR-145 and miR-let7e increased significantly in horses receiving flunixin at 8 hours post-castration (Friedman test, p < 0.05). Both behavioural and molecular changes occurred in horses receiving flunixin only, confirming that the addition of local mepivacaine is an effective analgesic treatment. CONCLUSIONS: Combining the use of HGS and circulating miRNAs, particularly miR-145, could be meaningful to monitor acute pain conditions in horses. Our results further validate the HGS as a method to assess acute pain in horses and point out miR-145 as a promising biomarker to identify pain.