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Current recommendations for assessing animal wellbeing during euthanasia suggest that measuring neuroendocrine hormones-such as ACTH, noradrenaline, and adrenaline-is preferable to measuring corticosterone and blood glucose because of the sensitivity of neuroendocrine hormones to the acute stress associated with rapid methods of euthanasia. However, theseneuroendocrine hormones can be stimulated in ways that confound interpretation of welfare assessment in euthanasia studies.Although this property does not negate the usefulness of neuroendocrine hormones as tools of assessment, it is importantto differentiate the stress associated with the induction of anesthesia before the loss of consciousness (an animal wellbeingconcern) with the physiologic responses that occur after the loss of consciousness (not an animal wellbeing concern). In thisstudy, rats were anesthetized by using a ketamine-xylazine combination. Once the rats achieved a surgical plane of anesthesia,they were exposed to O2, CO2, or isoflurane, followed by terminal blood collection to assess concentrations of ACTH,noradrenaline, corticosterone, and blood glucose. Compared with animals exposed to O2 or isoflurane, rats exposed to CO2had significant increases in their serum concentrations of ACTH and noradrenaline, but blood glucose and corticosteronedid not differ between groups. These findings indicate that noradrenaline and ACTH should be used with caution to assessanimal wellbeing when the method of euthanasia might confound that assessment.

Interpreting Neuroendocrine Hormones, Corticosterone, and Blood Glucose to Assess the Wellbeing of Anesthetized Rats during Euthanasia