Voluntary Oral Ingestion of a Sedative Prior to Euthanasia with CO(2): Behavioural Responses of Mice

SIMPLE SUMMARY: Carbon dioxide (CO(2)) inhalation is the most common euthanasia method for laboratory mice despite causing distress, pain and suffering. Since many laboratories will continue to use CO(2) until a suitable alternative is identified, there is merit in exploring options for reducing its aversiveness. We evaluated the potential of using a voluntarily ingested sedative (tiletamine-zolazepam; Zoletil(®)) prior to euthanasia. Male and female C57BL/6 mice were offered cream cheese mixed with Zoletil(®) in one of the following doses: 0, 10, 20, 40, 80, or 100 mg/kg. Behaviour during the sedation and euthanasia periods was recorded. A dose of 20 mg/kg was found to achieve mild sedation and was likely to reduce the aversiveness of euthanasia with CO(2). Higher doses also produced sedation, but these resulted in an incomplete intake of the dosed cream cheese in some mice. The voluntary ingestion of a sedative (20 mg/kg) prior to CO(2) deployment could be a valid option for reducing the stress of this euthanasia method for both mice and staff. Additionally, we suggest that since mice readily ingested dosed cream cheese, this could also be an easy, effective, non-invasive, and low-cost means of reducing stress in other applications (e.g., repeated handling or sampling). ABSTRACT: Laboratory mice are commonly euthanised with carbon dioxide (CO(2)); however, there is ample evidence that this gas is aversive. Previous work suggests that sedation achieved via injection with benzodiazepines prior to CO(2) 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 CO(2). 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 CO(2) difficult. Voluntary oral administration of a sedative is an effective, affordable, and easy way to minimize the stress of mice to euthanasia with CO(2).