Munro, B. A., Merenick, D. R., Gee, J. M. et al. 2023. Use of loss of righting reflex to assess susceptibility to carbon dioxide gas in three mouse strains. JAALAS 62(6), 553–558.

Exposure to CO2 gas is a common rodent euthanasia method. CO2 activates nociceptors in rats and is painful to humans at concentrations equal to or greater than 32.5% The concentration of CO2 at which rodents become unconsciousness is inadequately defined. We used loss of righting reflex (LORR) to identify the concentration at which CO2 caused loss of consciousness in C57Bl/6, CD1 and 129P3J mice (16 females and 16 males per strain). We used a custom built, rotating, motorized cylinder to determine LORR as CO2 concentrations were increased. Two LORR assessment methods were used: 1) a 1-Paw assessment in which the righting reflex was considered to be present if one or more paws contacted the cylinder after rotation into dorsal recumbency and 2) a 4-Paw assessment in which the righting reflex was considered to be present only if all 4 paws contacted the cylinder. LORR test data were analyzed with Probit regression and dose response curves were plotted. 1-Paw EC95 values (CO2 concentration at which LORR occurred for 95% of the population) were: C57Bl/6; 30.7%, CD1; 26.2%, 129P3J; 20.1%. The EC95 for C57Bl/6 was significantly higher than that of the 129P3J mice, with no significant differences between other strains. Four-Paw EC95 values were: C57Bl/6; 22.8%, CD1; 25.3%, 129P3J; 20.1%. Values for 129P3J mice were significantly lower than those of CD1 mice), with no significant difference between other strains. The EC95 varied significantly between 1-Paw and 4-Paw methods only for C57Bl/6 mice. These results suggest a potential for nociception and pain to occur in some individuals of some mouse strains during CO2 euthanasia.