Carbon dioxide, but not isoflurane, elicits ultrasonic vocalizations in female rats

Gradual filling of a chamber with carbon dioxide is currently listed by the Canadian Council on Animal Care guidelines as a conditionally acceptable method of euthanasia for rats. Behavioural evidence suggests, however, that exposure to carbon dioxide gas is aversive. Isoflurane is less aversive than carbon dioxide and may be a viable alternative, though objective data are lacking for the period leading up to loss of consciousness. It has been shown that during negative states, such as pain and distress, rats produce ultrasonic vocalizations. The objective of this study was to detect ultrasonic vocalizations during exposure to carbon dioxide gas or isoflurane as an indicator of a negative state. Specialized recording equipment, with a frequency detection range of 10 to 200 kHz, was used to register these calls during administration of each agent. Nine female Sprague-Dawley rats were exposed to either carbon dioxide or isoflurane on two different occasions. All rats vocalized in the ultrasonic range (30 to 70 kHz) during exposure to carbon dioxide. When exposed to isoflurane, no calls were detected from any of the animals. The frequent occurrence of ultrasonic vocalizations during carbon dioxide exposure suggests that the common practice of carbon dioxide euthanasia is aversive to rats and that isoflurane may be a preferable alternative.