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Carbon dioxide (CO2) is commonly used to euthanize laboratory mice but it is controversially discussed from an animal welfare’s point of view. Inhalent anaesthetics are proposed as an alternative but they have not been fully characterized yet.
The aim of this study was the comprehensive investigation of the induction of euthanasia with CO2 (100% CO2 with filling rates of 20, 60 and 100% of chamber volume per minute (KV%/min)) as well as with isoflurane (2% and 5% isoflurane with 71 KV%/min) and sevoflurane (4.8% and 8% sevoflurane with 71 KV%/min) in NMRI and C57Bl/6 mice. We compared stress reactions between the groups or in comparison to an air stream (71 KV%/min). We evaluated the effectiveness and reliability of the narcotic gas treatment, the time course of the phases of induction of narcosis, behaviour inclusive vocalisations, plasma adrenaline and noradrenaline concentrations, blood glucose concentrations as well as the effects of the gases on respiratory movements and on tissues of the respiratory tract. The period of investigation was form start of the gas exposure until surgical tolerance was reached or after maximum 300 s. Mice were decapitated and blood and tissue samples were collected.
In both strains only the CO2 treatments with 60 and 100 KV%/min and the treatments with 5% isoflurane as well as the treatment with 8% sevoflurane in NMRI mice induced surgical tolerance within 300 s in at least 93,8% of animals of a group and were considered as effective and reliable. The induction of narcosis can be divided into a normal phase, a phase of ataxia, of muscle relaxation and of loss of consciousness until the onset of surgical tolerance. CO2 with 100 KV%/min was the fasted treatment to induce surgical tolerance. The CO2 treatment with 60 KV%/min did not differ from the treatment with 5% isoflurane in regard to time to reach surgical tolerance. Plasma adrenaline and noradrenaline concentrations of the effective CO2 treated groups were ca. 10-fold higher than the concentrations of isoflurane and sevoflurane treated groups. In C57Bl/6 mice, blood glucose concentrations were partly higher in isoflurane and sevoflurane treated groups than in CO2 treated groups. In NMRI mice, we could not detect any differences between the groups. We could not find distinct stress or pain indicating behaviour, all changes in behaviour in relation to normal behaviour under air stream exposure (e.g. ataxia, hypotonic gait, forward movement, rearings, grooming, excitatory phenomena) could mainly be explained by the narcotic properties of the gases. We could not detect any vocalizations in the audible nor in the ultrasound range.
Respiratory movements were influenced by all narcotic gases. Except atelectasis, all macroscopic and microscopic pathological findings were detected in all treatment groups as well as in the air control group. They could not clearly be ascribed to as effects of the narcotic gas treatment but are rather due to decapitation or cardiac and circulatory failure in agony.
Regarded in context with the reviewed literature the stress reaction under CO2 exposure, which is reflected by elevated adrenaline and noradrenaline concentrations in plasma, could be explained by the massive physiological changes (as hypercapnic acidosis, dyspnea, aversion and fear behaviour) that are produced by the inhalation of CO2. Viewed from the outside, the induction of narcosis with CO2 mostly seemed to look nice and peaceful yet hypotonic gait, reduced forward movements and reduced rearings should not deceive the real (inner) stress reaction.
Strain differences need to be considered when choosing a narcotic gas and gas concentration.
In regard to our results, 5% isoflurane with 71 KV%/min is better than CO2 with 20 and 60 KV%/min for the induction of narcosis or euthanasia in NMRI- and C57Bl/6-mice. Occupational safety needs to be ensured. Exact doses and modes of application of inhalant anaesthetics which are compliant with animal welfare and occupational safety need to be investigated in the future.