Testing three measures of mouse insensibility following induction with isoflurane or carbon dioxide gas for a more humane euthanasia

Laboratory mice are commonly killed via exposure to gradually increasing concentrations of isoflurane and carbon dioxide (CO2) gas. Once rendered insensible using isoflurane or CO2, a high concentration of CO2 is used to decrease time to death. When the switch from isoflurane to a high flow rate of CO2 is made, it is important that the mice are insensible (complete loss of sensation) to prevent conscious exposure to potentially painful and distressing CO2 concentrations (>40%). Currently, no science-based methods exist for users to know when insensibility occurs during isoflurane or CO2 euthanasia protocols. The objective of our study was to examine three measures of insensibility: recumbency onset, loss of the righting reflex and loss of the pedal withdrawal reflex, in mice during euthanasia with 20% gradual-fill CO2 (n = 6) or 5% isoflurane using 2 L/min oxygen as the carrier gas, followed by CO2 (n = 7). In addition to testing for the three insensibility indicators, an 'escape response' was recorded if a mouse exhibited leg paddling and forward or lateral movement in response to the experimenter's approaching hand, when about to test for loss of the righting reflex. A 'purposeful movement' response was recorded if the animal showed the inability to self-right, but exhibited leg paddling while on its back. The results of this study demonstrated that all isoflurane treatment mice showed an escape response, purposeful movement and a pedal withdrawal response, in comparison to only two, zero and one CO2 mice, respectively. Thus, even after recumbency and loss of the righting reflex, mice showed indications of sensibility, suggesting that these are not reliable indicators of insensibility when using the isoflurane method of euthanasia as outlined in this study. On average (+/- S.D.) the interval between recumbency onset and loss of the pedal withdrawal reflex was 40 (+/- 13)s for isoflurane versus 16 (+/- 11)s for the CO2 method tested. When using isoflurane induction as described here, we recommend that users wait a minimum of 79s (mean + 3 S.D.) after the appearance of recumbency before switching to a high flow rate of CO2. If using the gradual-fill CO2 method as described here, users should wait a minimum of 49 s (mean +3 S.D.) before increasing the flow rate of CO2. (C) 2014 Elsevier B.V. All rights reserved.