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.