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

SIMPLE SUMMARY: This study aimed to evaluate the pain associated with six methods of euthanasia: pentobarbital, CO(2), decapitation, isoflurane, ketamine + xylazine, and ketamine + CO(2) in Wistar rats, by applying the Rat Grimace Scale (RGS), comparing the scores, and determining the method with the highest score that might indicate pain in laboratory rodents. According to the results, during the application of the euthanasia method, the intraperitoneal administration of ketamine + xylazine and decapitation caused the highest RGS scores (0.6 ± 0.26 and 0.6 ± 0.16, respectively) (p < 0.0001), while after the application of the euthanasia methods, CO(2) and isoflurane recorded the highest scores (p < 0.0001) (0.9 ± 0.18 and 1.2 ± 0.20, respectively). The results might indicate that injection and guillotine use could cause short-term pain in rodents, while high isoflurane scores could be associated with nociception/pain or the myorelaxant properties of the drug. Further research is needed to establish a comprehensive study of pain during euthanasia, where the RGS could be used minding the limitations that anesthetics might have on facial expression. ABSTRACT: 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, CO(2), decapitation, isoflurane, ketamine + xylazine, and ketamine + CO(2). Video recording to assess the RGS scores was performed in four events: basal: 24 h before the procedure; Ti(1): three minutes before the procedure; Ti(2): during the application of the euthanasia method; and Ti(3): immediately after the application until LORR. The main findings of this study showed that, during Ti(2), decapitation and ketamine + xylazine had the highest scores (0.6 ± 0.26 and 0.6 ± 0.16, respectively) (p < 0.0001), while at Ti(3), CO(2) (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 Ti(3) 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.