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 (F 1,12 = 6.67, P = 0.024), the high-flow rate CO₂ group (F 1,12 = 10.24, P = 0.007), and bright chamber group (F 1,12 = 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.