Minimal Exposure Times for Irreversible Euthanasia with Carbon Dioxide in Mice and Rats

When using an anesthetic overdose to euthanize laboratory rodents, a secondary method of euthanasia is recommended to ensure that the apparent death is irreversible. This secondary method usually is accomplished through the collection of tissues that are required to complete the research project. However, frequently laboratory rodents must be euthanized because they cannot be used for studies; in these cases, caretakers must perform a secondary method of euthanasia. Performing physical methods of euthanasia, even on unconscious rodents, can contribute to compassion fatigue in these persons. The current study was designed based on existing literature regarding minimal exposure times for preweanling rats and mice euthanized with carbon dioxide. The study evaluated the minimal time that adult rats and mice must remain in 100% carbon dioxide for death to be irreversible on removal. Adult rats (14 stocks and strains) and mice (more than 40 stocks and strains) were euthanized using a 50% volume per minute displacement rate of carbon dioxide for 2 min. The cages were then left undisturbed for predetermined times, ranging from 0 to almost 12 min. Upon removal from the cage, the animals were stimulated to determine whether they could be resuscitated. If an animal recovered, it was euthanized by using a physical method of euthanasia, and a duration that was 30 s longer than the previous predetermined time was assessed using other animals. The study demonstrated that exposure times of at least 3 min in carbon dioxide reliably result in irreversible euthanasia of mice but that exposure times of at least 10.5 min in carbon dioxide were required to ensure irreversible euthanasia of rats. Although an irreversible death can be attained with carbon dioxide, the use of appropriate species-specific exposure times is critical.