Merenick, D. R., Munro, B. A., Gee, J. M. et al. 2024. Assessing susceptibility to carbon dioxide gas in three rat strains using the loss of righting reflex. JAALAS 63(3), 310–315.

Overdose of carbon dioxide gas (CO2) is a common euthanasia method for rodents; however, CO2 exposure activates nociceptors in rats at concentrations equal to or greater than 37% and is reported to be painful in humans at concentrations equal to or greater than 32.5%. Exposure of rats to CO2 could cause pain before loss of consciousness. We used 2 standardized loss of righting reflex (LORR) methods to identify CO2 concentrations associated with unconsciousness in Wistar, Long–Evans, and Sprague–Dawley rats (n = 28 animals per strain). A rotating, motorized cylinder was used to test LORR while the rat was being exposed to increasing concentrations of CO2. LORR was defined based on a 15-second observation period. The 2 methods were 1) a 1-Paw assessment (the righting reflex was considered to be present if one or more paws contacted the cylinder after the rat was positioned in dorsal recumbency), and 2) a 4-Paw assessment (the righting reflex was considered to be present if all 4 paws contacted the cylinder after the rat was positioned in dorsal recumbency). 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 Wistar, 27.2%; Long–Evans, 29.2%; and Sprague–Dawley, 35.0%. 4-Paw EC95 values were Wistar, 26.2%; Long–Evans, 25.9%, and Sprague–Dawley, 31.1%. Sprague–Dawley EC95 values were significantly higher in both 1- and 4-Paw tests as compared with Wistar and Long–Evans rats. No differences were detected between sexes for any strain. The 1-Paw EC95 was significantly higher than the 4-Paw EC95 only for Sprague-Dawley rats. These results suggest that a low number of individual rats from the strains studied may experience pain during CO2 euthanasia.