Combining Nitrous Oxide with Carbon Dioxide Decreases the Time to Loss of Consciousness during Euthanasia in Mice — Refinement of Animal Welfare?

Abstract

Carbon dioxide (CO2) is the most commonly used euthanasia agent for rodents despite potentially causing pain and distress. Nitrous oxide is used in man to speed induction of anaesthesia with volatile anaesthetics, via a mechanism referred to as the “second gas” effect. We therefore evaluated the addition of Nitrous Oxide (N2O) to a rising CO2 concentration could be used as a welfare refinement of the euthanasia process in mice, by shortening the duration of conscious exposure to CO2. Firstly, to assess the effect of N2O on the induction of anaesthesia in mice, 12 female C57Bl/6 mice were anaesthetized in a crossover protocol with the following combinations: Isoflurane (5%)+O2 (95%); Isoflurane (5%)+N2O (75%)+O2 (25%) and N2O (75%)+O2 (25%) with a total flow rate of 3l/min (into a 7l induction chamber). The addition of N2O to isoflurane reduced the time to loss of the righting reflex by 17.6%. Secondly, 18 C57Bl/6 and 18 CD1 mice were individually euthanized by gradually filling the induction chamber with either: CO2 (20% of the chamber volume.min−1); CO2+N2O (20 and 60% of the chamber volume.min−1 respectively); or CO2+Nitrogen (N2) (20 and 60% of the chamber volume.min−1). Arterial partial pressure (Pa) of O2 and CO2 were measured as well as blood pH and lactate. When compared to the gradually rising CO2 euthanasia, addition of a high concentration of N2O to CO2 lowered the time to loss of righting reflex by 10.3% (P<0.001), lead to a lower PaO2 (12.55±3.67 mmHg, P<0.001), a higher lactataemia (4.64±1.04 mmol.l−1, P = 0.026), without any behaviour indicative of distress. Nitrous oxide reduces the time of conscious exposure to gradually rising CO2 during euthanasia and hence may reduce the duration of any stress or distress to which mice are exposed during euthanasia.

Euthanasia using gaseous agents in laboratory rodents

“…Excitation is also observed when injectable anaesthesia is administered, but not jumping, indicating a behaviour that is more in line with the distress induced by volatile agents. 30 Isoflurane is an anaesthetic that is commonly used in most laboratories, is less soluble than halothane and generally induces anaesthesia more rapidly. However, it has a slightly pungent odour 37 and animals often hold their breath, thus delaying the onset of LOC and increasing levels of distress.…”

Preanesthesia, Anesthesia, Analgesia, and Euthanasia

“…Additionally, altricial neonates require an extended period of CO 2 exposure, which may need to be followed by a physical adjunctive method of euthanasia (Klaunberg et al, 2004;Pritchett-Corning, 2009). There remains controversy regarding the recommended administration rate of CO 2 and the potential benefit for administration of isoflurane anesthesia or other gases or even injectable tranquilizers, sedatives, or anesthetics prior to, or in combination with, CO 2 (Hackbarth et al, 2000;Weary, 2006, 2007;Makowska et al, 2008Makowska et al, , 2009Chisholm et al, 2013;Wong et al, 2013;Leach et al, 2002aLeach et al, ,b, 2009Valentine et al, 2012;Makowska and Weary, 2009a,b;Makowska et al, 2012;Niel et al, 2008a,b;Thomas et al, 2012a). There remains controversy regarding the recommended administration rate of CO 2 and the potential benefit for administration of isoflurane anesthesia or other gases or even injectable tranquilizers, sedatives, or anesthetics prior to, or in combination with, CO 2 (Hackbarth et al, 2000;Weary, 2006, 2007;Makowska et al, 2008Makowska et al, , 2009Chisholm et al, 2013;Wong et al, 2013;Leach et al, 2002aLeach et al, ,b, 2009Valentine et al, 2012;Makowska and Weary, 2009a,b;Makowska et al, 2012;Niel et al, 2008a,b;Thomas et al, 2012a).…”

Comparison of pentobarbital‐phenytoin alone vs propofol prior to pentobarbital‐phenytoin for euthanasia in 436 client‐owned dogs

“…Euthanasia with an IV barbiturate such as pentobarbital‐phenytoin (PP) is the preferred method of euthanasia for companion animals . Sedation or anesthesia prior to euthanasia is common in shelter and laboratory environments to minimize stress and pain, and has been objectively evaluated . Current companion animal consensus guidelines suggest that “pre‐euthanasia sedation or anesthesia should be provided whenever practicable” during owner‐attended euthanasia, but these guidelines offer no supporting scientific data .…”