Evaluation of Low versus High Volume per Minute Displacement CO2 Methods of Euthanasia in the Induction and Duration of Panic-Associated Behavior and Physiology

Abstract

Simple SummaryCurrent recommendations for the use of CO2 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 CO2 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 CO2 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 CO2 resulted in physiologic and behavioral changes suggestive of distress.AbstractCurrent 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.

Wellbeing of Alcohol-preferring Rats Euthanized with Carbon Dioxide at Very Low and Low Volume Displacement Rates

“…The current study supports other work that suggests that the 10% VD/min (very low flow rate) of 100% carbon dioxide is distressing to rats. 5,14,21 The data further suggest that distress is experienced by rats euthanized with 30% VD/min of 100% carbon dioxide, because many of the measures of animal wellbeing did not differ between these 2 groups. In contrast, 70% VD/min of 100% carbon dioxide appears to meet the expectations of minimizing pain (consciousness is lost prior to concentrations associated with the experience of pain) and distress (fewer behavioral changes in these rats as compared with those in the lower flow treatment groups).…”

“…17,18 These results were not unexpected, given that the exposure of rats to 20% carbon dioxide induces air hunger and dyspnea with associated anxiety. 14,15,16 If 10% VD/min of 100% carbon dioxide causes rats to experience distress, perhaps the low flow rate of 30% VD/min of 100% carbon dioxide is preferable. 4,20 However, the current study suggests that this flow rate is also associated with significant distress for rats.…”

“…4 Inhaling concentrations of 20% carbon dioxide is a proven model of anxiety in laboratory rats through the induction of a sensation of air hunger, associated with dyspnea. 14,15,16 This model suggests that exposure to 1 to 3 min of carbon dioxide prior to the loss of consciousness has a high likelihood of distress.…”

Euthanasia of laboratory mice: Are isoflurane and sevoflurane real alternatives to carbon dioxide?

“…From a practical point of view, we cannot recommend the induction of narcosis with CO 2 at the low filling rate of 20% of chamber volume per min as well as with lower concentrations than 5% for isoflurane and 8% for sevoflurane using our lid construction with turbulence device. This contradicts current euthanasia recommendations implying a filling rate of 20% CO 2 or gradual filling sufficient to induce LORR and LOPR and, hence, as appropriate methods for euthanizing adult mice [ 61 ] and rats [ 63 ]. Our recommendation is also in contrast to a recent study by Biovin et al [ 64 ].…”

Welfare Impact of Carbon Dioxide Euthanasia on Laboratory Mice and Rats: A Systematic Review

“…Of the 108 full text articles reviewed, 71 articles were excluded as they did not meet eligibility criteria and 37 articles were included. In total, 15 papers were reviewed for mice (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30), 21 papers for rats (16,22,23,(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48), and 5 papers were reviewed for neonatal rodents (21,(49)(50)(51)(52), with three studies reporting results for both adult mice and rats (16,22,23) and one study reporting results for both adult female mice and their pups (21,(49)(50)(51)…”