Evaluation of low versus high volume per minute displacement CO2 methods of euthanasia in the induction and duration of panic-associated behavior and physiology

Debra Hickman, Stephanie D. Fitz, Cristian S. Bernabe, Izabela F. Caliman, Melissa M. Haulcomb, Lauren M. Federici, Anantha Shekhar, Philip Johnson

Research output: Contribution to journalArticle

10 Scopus citations


Current recommendations for the use of CO2 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 CO2 that are associated with pain. However, exposing rats to CO2, concentrations as low as 7% CO2 are reported to cause distress and 10%-20% CO2 induces panic-associated behavior and physiology, but loss of consciousness does not occur until CO2 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 CO2 that induce anxiety, panic, and distress for prolonged periods of time. This study first characterized the response of male rats exposed to normoxic 20% CO2 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 CO2 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 CO2 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% CO2 infused at 10%, 30%, or 100% volume per minute displacement rates. Analysis of the concentrations of CO2 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 CO2, 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.

Original languageEnglish (US)
Article number45
Issue number8
StatePublished - Aug 1 2016


  • Anxiety
  • CO
  • Distress
  • Euthanasia
  • Hypercapnia
  • Panic

ASJC Scopus subject areas

  • Animal Science and Zoology
  • veterinary(all)

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