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, 14 (1), e0210818

Nitrogen Gas Produces Less Behavioural and Neurophysiological Excitation Than Carbon Dioxide in Mice Undergoing Euthanasia


Nitrogen Gas Produces Less Behavioural and Neurophysiological Excitation Than Carbon Dioxide in Mice Undergoing Euthanasia

Carlotta Detotto et al. PLoS One.


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.

Conflict of interest statement

The authors have declared that no competing interests exist.


Fig 1
Fig 1. Experimental design.
(A) Graphic representation of the experimental apparatus. The gas flow controller was calibrated to deliver a precise amount of each gas used and to switch from 21% oxygen to the treatment gas at the end of the baseline period. (B) Timeline of the experimental procedure. Mice were randomly assigned to one of four treatment groups: CO2 or N2 and either rapid (R—80% volume fill minute-1) or slow fill (S—30% volume fill minute-1). The experiment started with a 5-minute baseline recording at 21% O2. Either CO2 or N2 was then infused into the chamber as a 100% concentration of total gas inflow until cessation of breathing. The experiment was terminated three minutes after cessation of breathing and animals exsanguinated.
Fig 2
Fig 2. Effects of CO2 and N2 on locomotion, jumping and freezing in grouped mice.
(A) Average (mean ± s.e.m) oxygen concentration at loss of motion (LOM). CO2R resulted in LOM at higher oxygen concentrations than other groups. CO2S resulted in LOM at oxygen concentrations higher than nitrogen groups (**P < 0.01). (B) Average speed (mean ± s.e.m), normalised to the last 30 seconds of baseline. Horizontal line indicates the normalisation value for baseline measurements. CO2 resulted in increased locomotion compared to nitrogen (**P < 0.01). (C-F) Normalised speed in relation to oxygen concentration from the start of gas exposure until LOM for CO2R (C), CO2S (D), N2R (E), and N2S (F). (G) Aversive jumps shown as median (± range) of total jumps and inter-quartile range per cage. Both CO2R and CO2S resulted in significantly more jumps compared to N2S (*P < 0.05) (H) Cumulative curves indicating the relative number of jumps in relation with oxygen concentration of the four treatment groups. (I) Freezing shown as mean (± s.e.m) number of freezing episodes per minute per animal. Mice in CO2S froze significantly more times than in the other three groups (*P < 0.05; ***P < 0.001). (J) Cumulative curves indicating the relative freezing episodes in relation with the oxygen concentration of the four treatments groups.
Fig 3
Fig 3. CO2 causes central neurological excitation whereas N2 causes central neurological depression.
(A) Schematic of recording. From bottom going clockwise: Single instrumented mice were used and underwent euthanasia with CO2 or N2 at either rapid fill (R—80% volume fill minute-1) or slow fill (G—30% volume fill minute-1). EEG and EMG electrodes were chronically implanted and animals allowed to recover for seven days. Data were acquired on a portable wireless Neurologger at 200 Hz and data downloaded after the experiment onto a computer. (B) Representative sample of a 20 s EEG recording from a mouse recorded during baseline. Filtered delta (1–4 Hz; blue) and theta (5–9 Hz; green) signals are shown below. Note the difference in amplitude between the delta and theta signals changing over time. The theta:delta power ratio (TD) shown in red on the bottom line. Note the high TD when theta amplitude is high and low TD when delta amplitude is high. (C, D, E, F) TD values with respect to O2 concentration for CO2R (C), CO2S (D), N2R (E) and N2S (F). Note the initial increase in TD for CO2 groups compared to the slow reduction in N2 groups. (G, H, I, J) Linear regressions of O2 concentration at isoelectric EEG (ISO-EEG) against O2 concentration at LOM for CO2R (G), CO2S (H), N2R (I) and N2S (J). Note the increased scatter of points for CO2S.

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Funding from this work was provided by a grant from the Swiss Federal Veterinary Office (Bundesamt für Lebensmittelsicherheit und Veterinärwesen). Grant number: 2.16.01 “Tierschuztgerechte Euthanasie von Nagern mittels inerten Gasen”.