Rat Grimace Scale as a Method to Evaluate Animal Welfare, Nociception, and Quality of the Euthanasia Method of Wistar...

Refinement of experimental procedures in animal research has the objective of preventing and minimizing pain/distress in animals, including the euthanasia period. This study aimed to evaluate pain associated with six methods of euthanasia in Wistar rats (injectable, inhalational, and physical), by applying the Rat Grimace Scale (RGS), comparing the scores, and determining the method with the highest score that might indicate pain for laboratory rodents. Sixty adult male and female Wistar rats were used and assigned to six treatments: pentobarbital, CO₂, decapitation, isoflurane, ketamine + xylazine, and ketamine + CO₂. Video recording to assess the RGS scores was performed in four events: basal: 24 h before the procedure; Ti₁: three minutes before the procedure; Ti₂: during the application of the euthanasia method; and Ti₃: immediately after the application until LORR. The main findings of this study showed that, during Ti₂, decapitation and ketamine + xylazine had the highest scores (0.6 ± 0.26 and 0.6 ± 0.16, respectively) (p < 0.0001), while at Ti₃, CO₂ (0.9 ± 0.18) and isoflurane (1.2 ± 0.20) recorded the highest scores (p < 0.0001). According to the present results, decapitation and ketamine + xylazine elicited short-term acute pain, possibly due to tissue damage caused by both methods (injection and guillotine). In contrast, isoflurane's RGS scores recorded during Ti₃ might be associated with nociception/pain due to the pungency of the drug or to the pharmacological muscle relaxant effect of isoflurane. Further research is needed to establish a comprehensive study of pain during euthanasia, where RGS could be used minding the limitations that anesthetics might have on facial expression.