• Helen Valentine, Wendy O Williams, and Kirk J Maurer.
• Cornell Center for Animal Resources and Education, School of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
• J Am Assoc Lab Anim. 2012 Jan 1; 51 (1): 50-7.

AbstractCO(2) administration is a common euthanasia method for research mice, yet questions remain regarding whether CO(2) euthanasia is associated with pain and stress. Here we assessed whether premedication with acepromazine, midazolam, or anesthetic induction with isoflurane altered behavioral and physiologic parameters that may reflect pain or stress during CO(2) euthanasia. Mice were assigned to 1 of 6 euthanasia groups: CO(2) only at a flow rate of 1.2 L/min which displaces 20% of the cage volume per minute (V/min; control group); premedication with acepromazine (5 mg/kg), midazolam (5 mg/kg), or saline followed by 20% V/min CO(2); induction with 5% isoflurane followed by greater than 100% V/min CO(2) (>6L/min); and 100% V/min CO(2) only (6 L/min). Measures included ultrasonic sound recordings, behavioral analysis of video record- ings, plasma ACTH and corticosterone levels immediately after euthanasia, and quantification of c-fos from brain tissue. Compared with 20% V/min CO(2) alone, premedication with acepromazine or midazolam did not significantly alter behavior but did induce significantly higher c-fos expression in the brain. Furthermore, the use of isoflurane induction prior to CO(2) euthanasia significantly increased both behavioral and neuromolecular signs of stress. The data indicate that compared with other modalities, 20% V/min CO(2) alone resulted in the least evidence of stress in mice and therefore was the most humane euthanasia method identified in the current study.

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