Isoflurane and Carbon Dioxide Elicit Similar Behavioral Responses in Rats

Euthanasia in rodents is an ongoing topic of debate due to concerns regarding the aversive nature of gases with anesthetic properties such as carbon dioxide (CO₂) and isoflurane. The aim of this study was to expand upon previously published work evaluating the aversiveness of CO₂ by introducing an isoflurane treatment group in parallel. Aversion was tested using a forced exposure setup and an aversion-avoidance setup. In the first part of the study, 12 naïve female Sprague-Dawley rats were exposed during four consecutive days, once to each of four treatments: isoflurane, fox urine, oxygen, and CO₂. In the second part of the study, 24 naïve female Sprague-Dawley rats and 12 rats from the first experiment were exposed to CO₂, isoflurane, or both gases. In the forced exposure study, there were no significant differences between CO₂ and isoflurane treatments except in line crosses. Overall, rats were more active in the isoflurane and CO₂ treatments compared to the control groups, suggesting that isoflurane and CO₂ are similarly aversive. In the aversion-avoidance study, rats previously exposed to isoflurane left the dark chamber significantly earlier compared to naïve rats during exposure to isoflurane. We also show that learned aversion to isoflurane is sustained for at least 15 days after initial exposure. Given this result, we suggest that CO₂ is superior to isoflurane when euthanizing rodents with prior exposure to isoflurane. Overall, these results confirm previous studies which suggest that care should be taken when considering the serial use of isoflurane as an anesthetic.