Research Article: Evidence for consistent individual differences in rat sensitivity to carbon dioxide

Date Published: April 24, 2019

Publisher: Public Library of Science

Author(s): Lucía Améndola, Daniel M. Weary, Edna Hillmann.


Carbon dioxide (CO2) gradual-fill is commonly used to kill laboratory rats, but this use remains controversial due to a lack of agreement between studies. Inconsistencies may arise from differences in behaviors measured (e.g. active versus passive behaviors), in how rats cope with threats, or in rat sensitivity to CO2. The aims of the current study were to 1) describe active and passive responses during CO2 forced exposure, 2) determine if these responses are consistent within individuals and across aversive stimuli, 3) assess individual differences in aversion to CO2 in aversion-avoidance and approach-avoidance tests and 4) determine how responses in aversion tests relate to individual differences in behavior during forced exposure. Twelve Sprague Dawley female rats were exposed twice to three treatments: CO2, oxygen (O2), and fox scent, and were exposed to CO2 twice in each aversion test. The change in behavior from baseline was higher for rearing and locomotion when rats were exposed to CO2 than when exposed to O2 and fox scent. Responses varied among rats but were consistent across multiple tests within rats. For example, rearing was consistent within individuals between two exposures to CO2. Similarly, the strength of aversion was consistent within individuals across multiple exposures to CO2 in aversion-avoidance and approach-avoidance testing. Latency to avoid CO2 in aversion-avoidance tests was negatively correlated with rearing during CO2 forced exposure. Collectively, these results indicate that rat responses to CO2 vary between (but are consistent within) individuals, suggesting that rats vary in CO2 sensitivity. However, even the less responsive rats avoided CO2 concentrations far below those necessary to achieve unconsciousness, indicating that all rats likely experience negative states when euthanized with CO2.

Partial Text

Carbon dioxide (CO2) is a widely used but controversial method of killing laboratory rodents [1]. Guidelines and regulations commonly accept this agent as a ‘humane’ killing method (e.g. [2–4]), implying that animals should not experience high arousal negative emotions during exposure, including pain, fear, distress, or anxiety.

All procedures were approved by The University of British Columbia Animal Care Committee (protocol number: A15-0071) and were performed in accordance with the guidelines on care and use of rodents in research, established by the Canadian Council on Animal Care.

Rats varied consistently in their responsiveness to CO2 exposure. If these responses relate to the animal’s affective states, then the emotional experience when killed with CO2 may also vary among rats. These results reinforce the importance of assessing affective states at the level of the individual, rather than relying on measurements of central tendency (discussed by [65]). In addition, accounting for individual differences may allow for of a better understanding experimental results, perhaps especially when assessing animal welfare [66]. Overall, our results indicate that variation in rat responses to CO2 exposure is situation-specific and relate to variation in CO2 sensitivity. CO2 concentrations well below those necessary to induce unconsciousness were aversive to all rats, indicating that CO2 exposure compromises rat welfare even for the least sensitive rats.