Date Published: January 31, 2019
Publisher: Public Library of Science
Author(s): Carlotta Detotto, Sarah Isler, Martin Wehrle, Alexei L. Vyssotski, Regula Bettschart-Wolfensberger, Thomas C. Gent, Kathleen R. Pritchett-Corning.
Carbon dioxide (CO2) is one of the most commonly used gas euthanasia agents in mice, despite reports of aversion and nociception. Inert gases such as nitrogen (N2) may be a viable alternative to carbon dioxide. Here we compared behavioural and electrophysiological reactions to CO2 or N2 at either slow fill or rapid fill in C57Bl/6 mice undergoing gas euthanasia. We found that mice euthanised with CO2 increased locomotor activity compared to baseline, whereas mice exposed to N2 decreased locomotion. Furthermore, mice exposed to CO2 showed significantly more vertical jumps and freezing episodes than mice exposed to N2. We further found that CO2 exposure resulted in increased theta:delta of the EEG, a measure of excitation, whereas the N2 decreased theta:delta. Differences in responses were not oxygen-concentration dependent. Taken together, these results demonstrate that CO2 increases both behavioural and electrophysiological excitation as well as producing a fear response, whereas N2 reduces behavioural activity and central neurological depression and may be less aversive although still produces a fear response. Further studies are required to evaluate N2 as a suitable euthanasia agent for mice.
Carbon dioxide (CO2) is the most commonly used gas euthanasia agent in mice due to rapid action, low cost and easy availability [1, 2]. However, multiple studies have demonstrated that carbon dioxide exposure may cause pain in humans as well as fear responses and aversion in rodents [3–6] therefore its use for euthanasia is controversial [7–12]. Alternative agents are sought to improve the welfare conditions of mice . Inert gases induce unconsciousness and death by hypoxia due to the displacement of oxygen and have been investigated for humane euthanasia of rodents [13–15]. The formation of carbonic acid in the mucous membranes following CO2 absorption is considered to cause pain and discomfort as reported in humans [3, 16], however inert gas absorption does not result in carbonic acid formation. Furthermore, increasing acidity in the blood from CO2 is detected in the amygdala of mice and results in a fear response . Humans undergoing short term hypoxia in hypobaric conditions do not report pain or distress and have poor perception of changes in environmental conditions . Whether the same effects occur during normobaric hypoxia is uncertain. Argon (Ar) is considered to be aversive to rats [18, 19] and mice  presumably because loss of consciousness occurs due to hypoxia alone, and has also been documented to cause dyspnoea or “air gasping” at hypoxic conditions, which is likely to be aversive . Its use is considered unsatisfactory for euthanasia, as it is slow to induce death and it causes hyperreflexia in rats . Nitrogen is an inert gas which is colourless, odourless and non-irritant, but it has received little attention as a potential euthanasia agent compared to other inert gases. The proposed mechanism of N2-induced loss of consciousness and death is also by hypoxia . One study suggested that N2, may be less aversive than Ar since it does not increase heart rate and mean arterial blood pressure . Additionally, N2 gas is relatively cheap and abundant in the environment and therefore safe to the operator and non-polluting, in contrast to CO2, which has a significant environmental impact . However, there remains insufficient data on the behavioural effects of N2 on mice to determine its usefulness as a euthanasia agent [20, 22].
No animals were excluded from experimentation or analysis. To determine whether nitrogen is less aversive than carbon dioxide as a euthanasia agent for mice, we first assessed the behavioural parameters of groups of non-instrumented C57Bl/6 mice being euthanised in four different treatment groups (CO2R; n = 10 cages; female: n = 5; male: n = 5; N2R; n = 10 cages; female: n = 5; male: n = 5; CO2S; n = 10 cages; female: n = 5; male: n = 5; and N2S; n = 10 cages; female: n = 5; male: n = 5). There were no significant differences in the number of animals per cage between the four gas exposure groups (CO2R; n = 6 (4–7); N2R; n = 6 (5–8); CO2S; n = 6 (5–8); N2S; n = 5.5 (4–8); P = 0.41; F = 0.99; one-way ANOVA).
The AVMA guidelines state that the use of humane euthanasia techniques should induce the most rapid, painless and distress-free death as possible . Identifying pain during mice euthanasia is a critical end-point and currently, no objective measures have been validated for assessing pain associated with euthanasia . However previous studies have identified markers of aversion  and fear  in response to CO2 exposure. Since inert gases are colourless, odourless and non-irritant, we hypothesised that N2 would be less aversive than CO2 in a gas euthanasia paradigm.
Our study demonstrates that N2 produces reduced behavioural and electrophysiological excitation during euthanasia compared to CO2. However, N2 may still cause some degree of fear response. Furthermore, rapid fill CO2 might be less aversive than slow fill. Further work is required to assess other physiological and behavioural parameters, such as nociception, to determine if N2 is a suitable euthanasia agent for mice.