Stuckey, J. E., Makhija, S. D., Reimer, D. C. et al. 2023. Effects of different grades of carbon dioxide on euthanasia of mice (Mus musculus). JAALAS 62(5), 430–437.

Carbon dioxide (CO2) is a conditionally acceptable method for mouse euthanasia, according to the American Veterinary Medical Association Guidelines for the Euthanasia of Animals. In a 2012 Office of Laboratory Animal Welfare (OLAW) webinar, representatives of OLAW, the USDA and AAALACi stated that different grades of CO2, including nonpharmaceutical grades, are acceptable as long as the minimum purity is 99.0%. No systematic studies have been published on the effects of different grades of CO2 on animal welfare during euthanasia. In this study, mice were euthanized with one of 3 grades of CO2: USP medical (> 99.2% CO2), bone-dry (> 99.9% CO2), or industrial (> 99.0% CO2). Times to recumbency and cessation of breathing were measured, and escape and avoidance behaviors were noted. Our results show no difference in time to recumbency and time to cessation of breathing in mice euthanized with these different grades of CO2. The average time to recumbency for medical, bone-dry, and industrial grades of CO2 were 39±6s, 37±7s, and 37±6s, respectively. The average time to cessation of breathing for medical, bone-dry, and industrial grades of CO2 were 135±16 s, 131±12s, and 133±10s, respectively. Histologic assessment of the nasal cavity, pharynx, trachea, and lungs showed no difference between grades of CO2 used for euthanasia. According to survey results, responding facilities most commonly used industrial and pharmaceutical grades, with use of the pharmaceutical grade increasing with facility size. A small portion of respondents reported occasional difficulty in obtaining the desired grades of CO2. Given our demonstration that the CO2 grade used for euthanasia is not associated with welfare issues, institutions can have the flexibility to plan for disasters or supply chain problems. In addition, cost savings may be associated with the option of switching between medical, bone-dry, and industrial grades of CO2.

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