Welfare Impact of Carbon Dioxide Euthanasia on Laboratory Mice and Rats: A Systematic Review

Abstract

Background: There has been increased concern about the suitability of CO 2 as a method for euthanasia of laboratory mice and rats, including the potential discomfort, pain or distress that animals may experience prior to loss of consciousness; time to loss of consciousness; best methods for use of CO 2 ; and the availability of better alternatives. These discussions have been useful in providing new information, but have resulted in significant confusion regarding the acceptability of CO 2 for rodent euthanasia. In some cases, researchers and veterinarians have become uncertain as to which techniques to recommend or use for euthanasia of laboratory mice and rats. Methods: The International Association of Colleges of Laboratory Animal Medicine (IACLAM) convened a taskforce to examine the evidence for adverse welfare indicators in laboratory rats and mice undergoing CO 2 euthanasia using a SYRCLE-registered systematic review protocol. Of 3,772 papers identified through a database search (PubMed, Web of Science, CAB Direct, Agricola, and grey literature) from 1900 to 2017, 37 studies were identified for detailed review (some including more than one species or age group), including 15 in adult mice, 21 in adult rats, and 5 in neonates of both species. Experiments or reports were excluded if they only assessed parameters other than those directly affecting animal welfare during CO 2 induction and/or euthanasia. Results: Study design and outcome measures were highly variable and there was an unclear to high risk of bias in many of the published studies. Changes in the outcome measures evaluated were inconsistent or poorly differentiated. It is likely that repeated exposures to carbon dioxide inhalation are aversive to adult rats and mice, based on Turner et al. CO 2 Euthanasia of Laboratory Rodents avoidance behavior studies; however, this effect is largely indistinguishable from aversion induced by repeated exposures to other inhalant anesthetic gasses. Conclusion: There is insufficient evidence to permit an unbiased assessment of the effect of CO 2 inhalation during euthanasia on welfare indicators in laboratory mice and rats. Additional well-designed, unbiased, and adequately powered studies are needed to accurately assess the welfare of laboratory mice and rats undergoing euthanasia via CO 2 gas.

Carbon dioxide tolerability and toxicity in rat and man: A translational study

“…Additionally, CO 2 at high concentrations activates nociceptors in the nasal cavity which is associated with severe pain sensations (Thürauf et al, 1991;Green and Hummel, 2013;Hickman et al, 2016). Hence, from an animal welfare perspective it is questionable whether this form of euthanasia is harmless as our findings as well as those of others indicate that a CO 2 overdose is highly distressful and will cause damage to lung tissue (Conlee et al, 2005;Boivin et al, 2017;Turner et al, 2020).…”

Attitudes of laboratory animal professionals and researchers towards carbon dioxide euthanasia for rodents and perceived barriers to change

“…These results are consistent with the ongoing debate within the scientific community where some authors argue that CO 2 euthanasia causes suffering in rodents 11 and others argue there is insufficient evidence to draw conclusions regarding the method. 10 The current study did not assess if the information used by participants to interpret the animals’ experiences came from their knowledge of the scientific literature, personal experience, or information provided by regulatory bodies. Future studies should consider what and how information is used by laboratory animal professionals to inform their views.…”

“…Blackshaw et al 8 and Britt 9 ), its use remains controversial. For example, two recently published literature reviews arrived at contrasting conclusions; Turner and colleagues concluded that there was not enough evidence to determine whether CO 2 killing compromises rodent welfare, 10 while Améndola and Weary concluded that CO 2 inhalation induces negative emotions in rats likely corresponding to fear, anxiety, dyspnea, distress, and panic. 11 Conflict also surrounds alternative methods such as inhalant anaesthetics, with some scholars arguing that that use of CO 2 should continue, 12 and others concluding that alternative methods are more humane.…”

Determining Candidate Hypobaric Hypoxia Profiles for Humane Killing of Laboratory Mice

“…During CO 2 exposure, rodents show behaviors indicative of fear and anxiety at concentrations as low as ~10% ( 9 , 10 ), which is significantly lower than the concentrations required for loss of consciousness (around 30%) ( 11 , 12 ). While there is some controversy surrounding the severity of the affective state consequences of exposure to CO 2 ( 3 ), there is general agreement that CO 2 inhalation is aversive and there is a requirement for the development of high-throughput alternatives ( 3 , 4 , 13 , 14 ). As a result, its inclusion (and indirect endorsement) in existing legislation and guidance is of significant concern, not only from the perspective of animal welfare, but also risks damaging public acceptance of animal research.…”