CO₂, CO, and N₂ Euthanasia in Laboratory Mice: a multisystem welfare assessment

Millions of laboratory mice are euthanized worldwide every year, most often with carbon dioxide (CO2). However, CO2 raises major animal welfare concerns, because it is aversive and induces strong respiratory distress, which may cause anxiety and pain. Currently, insufficient data are available to assess and classify the impact of CO2 and other gases proposed as alternatives. In this context, it is crucial to accurately determine when animals lose consciousness as the humaneness of an euthanasia method depends on the experience of pain or distress before loss of consciousness (LOC). Here, we analyzed and compared the effect of CO2, N2, and CO euthanasia in female and male C57Bl/6N mice, one of the most widely used mouse strains. Mice were implanted with transmitters to record neurological and cardiovascular signals while assessing also respiration, behavior by video-tracking, and measuring biochemical stress parameters. This multi-modal assessment enabled us to determine the time points of loss of posture (LOP) and LOC. CO2 induced gasping and muscle fasciculations before and after LOP whereas CO and N2 caused severe seizures and strong agitation, and delayed cessation of electrical brain activity. LOC, induced by the anesthetic properties of CO2, was the fastest with a high-volume displacement rate of CO2. Multi-modal assessment revealed that CO and N2 led to a hypoxia-induced death that is less humane than with CO2 euthanasia. CO and N2 cannot be recommended for a more humane euthanasia of laboratory mice.