The effect of light level, CO2 flow rate, and anesthesia on the stress response of mice during CO2 euthanasia

Euthanasia protocols are designed to mitigate the stress experienced by animals, and an environment that induces minimal stress helps achieve that goal. A protocol that is efficient and practical in a typical animal research facility is also important. Light intensity, isoflurane, and CO2 flow rate were studied for their impact on the stress response of mice during CO2 euthanasia. Behavior was observed and scored during euthanasia and serum corticosterone was measured immediately after death. Unsurprisingly, animals euthanized with a high-flow rate of CO2 became unconscious in the least amount of time, while animals euthanized with a low-flow rate required the most time to reach unconsciousness. There was a significant increase in anxious behaviors in animals in the isoflurane group (F1,12 = 6.67, P = 0.024), the high-flow rate CO2 group (F1,12 = 10.24, P = 0.007), and bright chamber group (F1,12 = 7.27, P = 0.019). Serum corticosterone was highest in the isoflurane group (124.72 ± 83.98 ng/ml), however there was no significant difference in corticosterone levels observed for the other study variables of light and flow-rate. A darkened chamber and low CO2 flow rates help to decrease stress experienced during CO2 euthanasia, while the use of isoflurane was observed to increase the stress response during euthanasia.