Behavioural responses of rats to gradual-fill carbon dioxide euthanasia and reduced oxygen concentrations

Carbon dioxide (CO₂) is widely used for euthanasia of laboratory rats, but little is known about the pain and distress caused by this procedure. Physiological and human self-report data suggest that CO₂ may cause pain and dyspnea, a sensation of breathlessness and increased respiratory effort, at the concentrations used for gradual-fill euthanasia. However, previous studies examining the behavioural responses of rats have reported conflicting results. In this study, detailed behavioural responses of rats were examined during gradual-fill CO₂ euthanasia and during gradual-fill with argon to produce a similar reduction in oxygen concentration. Animals were randomly allocated to the CO₂ or reduced oxygen treatment groups (n = 8 for both), and were first tested with air exposure and then with treatment gas exposure on the following day. Observations were taken for 105 s before and after gas flow began (baseline and exposure periods), as animals had ceased purposeful movement within 105 s of CO₂ flow starting. Behavioural changes from baseline during gas exposure were compared to changes during air exposure on control days. In comparison to air exposure, CO₂ resulted in increased activity, rearing, touching the nose to the chamber lid, escape behaviours and vocalizations. A small increase in touching the nose to the chamber lid was observed when oxygen concentrations were reduced with argon, but no other behavioural changes were observed. These results suggest that gradual-fill CO₂ euthanasia causes distress in rats, and that hypoxia alone is not a major cause of this distress.