Nitrogen gas produces less behavioural and neurophysiological excitation than carbon dioxide in mice undergoing euthanasia

Carbon dioxide (CO(2)) is one of the most commonly used gas euthanasia agents in mice, despite reports of aversion and nociception. Inert gases such as nitrogen (N(2)) may be a viable alternative to carbon dioxide. Here we compared behavioural and electrophysiological reactions to CO(2) or N(2) at either slow fill or rapid fill in C57Bl/6 mice undergoing gas euthanasia. We found that mice euthanised with CO(2) increased locomotor activity compared to baseline, whereas mice exposed to N(2) decreased locomotion. Furthermore, mice exposed to CO(2) showed significantly more vertical jumps and freezing episodes than mice exposed to N(2). We further found that CO(2) exposure resulted in increased theta:delta of the EEG, a measure of excitation, whereas the N(2) decreased theta:delta. Differences in responses were not oxygen-concentration dependent. Taken together, these results demonstrate that CO(2) increases both behavioural and electrophysiological excitation as well as producing a fear response, whereas N(2) reduces behavioural activity and central neurological depression and may be less aversive although still produces a fear response. Further studies are required to evaluate N(2) as a suitable euthanasia agent for mice.