• Brittany Munro: Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine, University of Calgary
• Dexter Merenick: Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine, University of Calgary
• Julia Gee: College of Engineering and Physical Sciences, Faculty of Engineering, University of Guelph
• Daniel Pang: Faculty of Veterinary Medicine, University of Calgary | Université de Montréal

Overdose with CO2 gas is a common technique for euthanizing laboratory rats. However, rats show aversion to CO2 at low concentrations, and it is unclear if pain occurs before loss of consciousness (LOC). Reported methods for determining LOC vary considerably resulting in a wide range of CO2 concentrations associated with LOC (21-39%). One outcome measure, loss of righting reflex (LORR), is strongly positively correlated with LOC in humans. This blinded, randomized study aimed to determine the CO2 concentration at which LORR occurs in adult rats using a standardized assessment method. Animals: 28 adult Sprague-Dawley rats (226-455g, 11 males, 17 females). A 3D-printed apparatus was designed and built to house a motor-driven rotating cylinder (3 rpm). CO2 in-flow was controlled with a flowmeter and calibrated CO2 analyzer, with concentrations increased stepwise. Dose response curves for LORR were generated with probit regression for the study population: EC50 = 30.2%, EC95 = 34.4% (n = 28). Separate curves were generated for each sex: males; EC50 29.3%, females; EC50 30.9% (p = 0.003, 95%CI 0.6 to 2.6%). Males; EC95 32.2%, females; EC95 35.1% (p = 0.01, 95%CI 0.7 to 5.0%). The EC95 for the study population (34.4%) falls within the CO2 concentration range associated with pain in humans. These data suggest a small difference in CO2 sensitivity between sexes and the potential for pain before LORR.