Metadata about: SIMPLE SUMMARY: Current recommendations for the use of CO(2) as a euthanasia agent for rats require the use of gradual fill methods in order to render the animal insensible prior to their experience of pain. However, there is concern that the use of these gradual fill methods may increase the distress experienced by these animals. We evaluated social and anxiety behavior of rats that had been exposed to concentrations of CO(2) that did not cause a loss of consciousness. We also evaluated the physiologic changes of rats that were euthanized with gradual fill protocols as compared to rapid fill methods. We found that rats exposed to concentrations of CO(2) that did not cause a loss of consciousness did exhibit increased anxiety and decreased social behavior. We also found that the use of a 10% volume per minute displacement rate of CO(2) resulted in physiologic and behavioral changes suggestive of distress. ABSTRACT: Current recommendations for the use of CO(2) as a euthanasia agent for rats require the use of gradual fill protocols (such as 10% to 30% volume displacement per minute) in order to render the animal insensible prior to exposure to levels of CO(2) that are associated with pain. However, exposing rats to CO(2), concentrations as low as 7% CO(2) are reported to cause distress and 10%–20% CO(2) induces panic-associated behavior and physiology, but loss of consciousness does not occur until CO(2) concentrations are at least 40%. This suggests that the use of the currently recommended low flow volume per minute displacement rates create a situation where rats are exposed to concentrations of CO(2) that induce anxiety, panic, and distress for prolonged periods of time. This study first characterized the response of male rats exposed to normoxic 20% CO(2) for a prolonged period of time as compared to room air controls. It demonstrated that rats exposed to this experimental condition displayed clinical signs consistent with significantly increased panic-associated behavior and physiology during CO(2) exposure. When atmospheric air was then again delivered, there was a robust increase in respiration rate that coincided with rats moving to the air intake. The rats exposed to CO(2) also displayed behaviors consistent with increased anxiety in the behavioral testing that followed the exposure. Next, this study assessed the behavioral and physiologic responses of rats that were euthanized with 100% CO(2) infused at 10%, 30%, or 100% volume per minute displacement rates. Analysis of the concentrations of CO(2) and oxygen in the euthanasia chamber and the behavioral responses of the rats suggest that the use of the very low flow volume per minute displacement rate (10%) may prolong the duration of panicogenic ranges of ambient CO(2), while the use of the higher flow volume per minute displacement rate (100%) increases agitation. Therefore, of the volume displacement per minute rates evaluated, this study suggests that 30% minimizes the potential pain and distress experienced by the animal.

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