Sex- and Strain-related Differences in the Stress Response of Mice to CO₂ Euthanasia

The 2013 edition of the AVMA Guidelines for the Euthanasia of Animals recommends a 10% to 30% volume displacement rate (VDR) per minute for CO2 euthanasia of rodents. Here we sought to evaluate behavior and plasma catecholamine levels in multiple strains of male and female mice, euthanized individually or in a group, with CO2 VDR of 10% to 100%. Behavioral observations included ataxia, labored breathing, time to recumbency, time to surgical plane of anesthesia, and the number of jumps or paws at the face during the euthanasia process. Behaviors did not differ significantly between male and female mice at any of the VDR, but interstrain differences occurred. Slower VDR resulted in longer periods of ataxia and labored breathing regardless of euthanasia as a group or as an individual. In addition, mice jumped and pawed at the face more often with slower VDR than higher. At all VDR, mice euthanized as a group had lower catecholamine levels than mice euthanized individually, but there were no significant differences between VDR. Time to recumbency and time to surgical plane anesthesia were longer with slower displacement rates; in addition, these parameters were prolonged for mice euthanized as a group compared with individually. Overall, faster VDR do not appear to be more distressful than slower rates. In fact, faster VDR shorten the time during which mice might experience distress prior to recumbency.