Voluntary Oral Ingestion of a Sedative Prior to Euthanasia with CO₂: Behavioural Responses of Mice

Laboratory mice are commonly euthanised with carbon dioxide (CO₂); however, there is ample evidence that this gas is aversive. Previous work suggests that sedation achieved via injection with benzodiazepines prior to CO₂ administration could reduce aversive behaviours during euthanasia. We explored the potential of using a voluntarily ingested sedative (tiletamine-zolazepam, Zoletil^®) prior to euthanasia. Male and female C57BL/6 mice were allocated into one of the five experimental groups, which differed in the dose of Zoletil: 0, 10, 20, 40, 80 or 100 mg/kg. A dose of 20 mg/kg was found to achieve mild sedation prior to euthanasia; mice which received this dose numerically reared and walked on the cage lid less, and showed ataxia, immobility and recumbency for longer than mice that received a lower dose. During euthanasia, mice that received 20 mg/kg showed fewer aversive responses to CO₂. Doses of 40 to 100 mg/kg were associated with signs of moderate to severe sedation, but resulted in an incomplete intake of the sedative, which made the interpretation of the aversiveness to CO₂ difficult. Voluntary oral administration of a sedative is an effective, affordable, and easy way to minimize the stress of mice to euthanasia with CO₂.