Supplementary material from Striving for humane deaths for laboratory mice: hypobaric hypoxia provides a potential alternative to carbon dioxide exposure

Killing is often an unavoidable and necessary procedure for laboratory mice involved in scientific research, and providing a humane death is vital for public acceptance. Exposure to carbon dioxide (CO₂) gas is the most widely used methodology despite well proven welfare concerns. Consequently, the continued use of CO₂ and its globally permitted status in legislation and guidelines presents an ethical dilemma for users. We investigated whether killing with hypobaric hypoxia via gradual decompression was associated with better welfare outcomes for killing laboratory mice. We compared the spontaneous behaviour of mice exposed to CO₂, decompression or sham conditions, and used analgesic or anxiolytic interventions to determine their relative welfare impact. Gradual decompression resulted in longer times to unconsciousness and death and the pharmacological interventions support the notion of a minimally negative animal experience, while providing further evidence for pain and anxiety associated with exposure to CO₂. Decompression resulted in moderate ear haemorrhage, but our welfare assessment suggests this may happen when mice are unconscious. Hence, gradual decompression could be the basis of significant refinement for killing laboratory mice. Future work should corroborate behaviour with neurobiological markers of loss of consciousness to verify the conscious phase of concern for animal welfare.