Combining Nitrous Oxide with Carbon Dioxide Decreases the Time to Loss of Consciousness during Euthanasia in Mice - Refinement of Animal Welfare?

Carbon dioxide (CO2) is the most commonly used euthanasia agent for rodents despite potentially causing pain and distress. Nitrous oxide is used in man to speed induction of anaesthesia with volatile anaesthetics, via a mechanism referred to as the second gas effect. We therefore evaluated the addition of Nitrous Oxide (N2O) to a rising CO2 concentration could be used as a welfare refinement of the euthanasia process in mice, by shortening the duration of conscious exposure to CO2. Firstly, to assess the effect of N2O on the induction of anaesthesia in mice, 12 female C57Bl/6 mice were anaesthetized in a crossover protocol with the following combinations: Isoflurane (5%)+O-2 (95%); Isoflurane (5%)+ N2O (75%)+O-2 (25%) and N2O (75%)+O-2 (25%) with a total flow rate of 3l/min (into a 7l induction chamber). The addition of N2O to isoflurane reduced the time to loss of the righting reflex by 17.6%. Secondly, 18 C57Bl/6 and 18 CD1 mice were individually euthanized by gradually filling the induction chamber with either: CO2 (20% of the chamber volume. min(-1)); CO2 + N2O (20 and 60% of the chamber volume. min(-1) respectively); or CO2 + Nitrogen (N-2) (20 and 60% of the chamber volume. min(-1)). Arterial partial pressure (P-a) of O-2 and CO2 were measured as well as blood pH and lactate. When compared to the gradually rising CO2 euthanasia, addition of a high concentration of N2O to CO2 lowered the time to loss of righting reflex by 10.3% (P < 0.001), lead to a lower PaO2 (12.55+/-3.67 mmHg, P < 0.001), a higher lactataemia (4.64+/-1.04 mmol.l(-1), P = 0.026), without any behaviour indicative of distress. Nitrous oxide reduces the time of conscious exposure to gradually rising CO2 during euthanasia and hence may reduce the duration of any stress or distress to which mice are exposed during euthanasia.