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

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.

Keywords: CO2; anxiety; distress; euthanasia; hypercapnia; panic.

Figure 1

Line graphs in (a–f) respectively represent ambient concentrations of CO₂ and O₂; mean arterial blood pressure (mmHg); respiration rate (breaths per minute); general locomotor activity; and heart rate (beats per minute) at baseline during 10 min atm. air infusion (all rats), then also during either a 5 min atm. air challenge or 20% CO₂ challenge (gray shaded region) following by all rats receiving another 5 min atm. * represents between-subject significance p < 0.05 with a Fisher’s LSD post hoc test protected by a significant one way ANOVA with gas treatment as the main factor and time as a repeated measure. ^# symbol in a-b represent significant p <0 .05 within subject over time effects (against t-1 min) from a Dunnett’s post hoc test. * symbol in (e) represents between subjects significance p < 0.05 with a two-tailed Dunnett’s post hoc test protected by a significant one-way ANOVA with gas treatment as the main factor. * in (f) represents between-subject significance with a two-tailed paired t-test. Behavior testing immediately following gas challenges were (g) social interaction duration, and (h) open field behaviors.

Figure 2

Line graphs in (a–f) respectively represent ambient concentrations of CO₂ and O₂; core body temperature (Celsius); general locomotor activity (counts/min); mean arterial blood pressure (mmHg); and heart rate (beats per minute) when placed in volume displacement cage for 5 min prior to either infusions of atmospheric air (Atm air), or 10%, 30%, or 100% CO₂ volume displacement per minute procedures (gray shaded region). * represents between-subject significance p < 0.05 with a Fisher’s LSD post hoc test protected by a significant one-way ANOVA with gas treatment as the main factor and time as a repeated measure.

Figure 3

Line graphs in (a,b), respectively, represent the mean duration of gasping behaviors and running behaviors just prior to and following gas VP (gray shaded region). * represents between-subject significance p < 0.05 with a Fisher’s LSD post hoc test protected by a significant one-way ANOVA with gas treatment as the main factor and time as a repeated measure; (c) bar graph represents the mean time before the rat stopped moving; and (d) bar graph represents the mean time before breathing became non apparent. Individual points in c-d represent actual values of each animal. * represents a Fisher’s LSD post hoc test protected by a significant ANOVA.