Evaluating methods of gas euthanasia for laboratory mice

Laboratory mice are most commonly killed with carbon dioxide (CO2), a gas they may find aversive. The aim of this study was to use approach-avoidance testing to record mouse responses to CO2 and to four alternatives: argon, carbon monoxide (CO), and the inhalant anaesthetics halothane and isoflurane. Mice were trained to enter the bottom cage of a two-cage apparatus for a reward of sweetened shredded coconut. Treatment gases were turned on at a pre-determined flow rate as soon as mice started eating, and we recorded the latency to leave the test cage and the gas concentration when mice left the test cage during the CO2, argon and CO experiments. For the CO and inhalant anaesthetics experiments, we also conducted preliminary testing to determine the time to recumbency with the chosen flow rates and concentrations, and calculated the amount of time between when mice chose to leave the test cage and when they would have been recumbent. When exposed to CO2, argon, and CO, mice never remained in the test cage long enough to lose consciousness. Mice left the test cage when CO2 concentrations were 13.5–18.2%, when argon reduced oxygen concentration to 8.3–9.3%, and when CO concentration was approximately 2.5%. When exposed to inhalant anaesthetics, mice never remained until recumbency with halothane but two mice did so during exposure to isoflurane. Mice also remained in the test cage longer with isoflurane than halothane. We conclude that CO2, argon, CO, and the two inhalant anaesthetics are all aversive to mice, so the search for a non-aversive agent should continue. However, aversion to the inhalant anaesthetic isoflurane appears to be weaker than aversion to the other agents, suggesting that this is a preferred alternative to CO2.