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. Supporting Information