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

Patricia V Turner^{1

2}, Debra L Hickman³, Judith van Luijk⁴, Merel Ritskes-Hoitinga⁴, Jan M Sargeant^{5

6}, T Miki Kurosawa⁷, Takashi Agui⁸, Vera Baumans⁹, Woo Sung Choi¹⁰, Yang-Kyu Choi¹¹, Paul A Flecknell¹², Byeong H Lee¹³, Pedro J Otaegui¹⁴, Kathleen R Pritchett-Corning^{15

16}, Keisuke Shimada¹⁷

Affiliations

¹ Department of Pathobiology, University of Guelph, Guelph, ON, Canada.
² Global Animal Welfare and Training, Charles River, Wilmington, MA, United States.
³ Laboratory Animal Resource Center, School of Medicine, Indiana University, Indianapolis, IN, United States.
⁴ Department of Health Evidence, SYstematic Review Center for Laboratory Experimentation (SYRCLE), Radboud University, Nijmegen, Netherlands.
⁵ Department of Population Medicine, University of Guelph, Guelph, ON, Canada.
⁶ Centre for Public Health and Zoonoses, University of Guelph, Guelph, ON, Canada.
⁷ Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan.
⁸ Department of Applied Veterinary Science, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
⁹ Department of Animals, Science and Society, Utrecht University, Utrecht, Netherlands.
¹⁰ National New Drug Development Cluster, Woojung Bio, Suwon-si, South Korea.
¹¹ Department of Laboratory Animal Medicine, College of Veterinary Medicine, Konkuk University, Seoul, South Korea.
¹² Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, United Kingdom.
¹³ Osong Medical Innovation Foundation, Cheongju, South Korea.
¹⁴ Laboratory Animal Facilities, Autonomous University of Barcelona, Barcelona, Spain.
¹⁵ Office of Animal Resources, Harvard University Faculty of Arts and Sciences, Cambridge, MA, United States.
¹⁶ Department of Comparative Medicine, University of Washington, Seattle, WA, United States.
¹⁷ Animal Resource Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.

PMID: 32793645
PMCID: PMC7387666
DOI: 10.3389/fvets.2020.00411

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

Patricia V Turner et al. Front Vet Sci. 2020.

. 2020 Jul 22:7:411.

doi: 10.3389/fvets.2020.00411. eCollection 2020.

Authors

Affiliations

¹ Department of Pathobiology, University of Guelph, Guelph, ON, Canada.
² Global Animal Welfare and Training, Charles River, Wilmington, MA, United States.
³ Laboratory Animal Resource Center, School of Medicine, Indiana University, Indianapolis, IN, United States.
⁴ Department of Health Evidence, SYstematic Review Center for Laboratory Experimentation (SYRCLE), Radboud University, Nijmegen, Netherlands.
⁵ Department of Population Medicine, University of Guelph, Guelph, ON, Canada.
⁶ Centre for Public Health and Zoonoses, University of Guelph, Guelph, ON, Canada.
⁷ Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan.
⁸ Department of Applied Veterinary Science, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
⁹ Department of Animals, Science and Society, Utrecht University, Utrecht, Netherlands.
¹⁰ National New Drug Development Cluster, Woojung Bio, Suwon-si, South Korea.
¹¹ Department of Laboratory Animal Medicine, College of Veterinary Medicine, Konkuk University, Seoul, South Korea.
¹² Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, United Kingdom.
¹³ Osong Medical Innovation Foundation, Cheongju, South Korea.
¹⁴ Laboratory Animal Facilities, Autonomous University of Barcelona, Barcelona, Spain.
¹⁵ Office of Animal Resources, Harvard University Faculty of Arts and Sciences, Cambridge, MA, United States.
¹⁶ Department of Comparative Medicine, University of Washington, Seattle, WA, United States.
¹⁷ Animal Resource Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.

PMID: 32793645
PMCID: PMC7387666
DOI: 10.3389/fvets.2020.00411

Abstract

Background: There has been increased concern about the suitability of CO₂ 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 CO₂; 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 CO₂ 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 CO₂ 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 CO₂ 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 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 CO₂ 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 CO₂ gas.

Keywords: animal welfare; carbon dioxide; distress; euthanasia; mouse; pain; rat; systematic review.

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Figures

**Figure 1**
Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) study flow diagram [modified from (13)].

**Figure 2**
Risk of bias assessment summary for the 16 single trial studies in adult mice and rats, and rodent pups (–19, 21, 25, 27, 28, 30, 32, 34, 35, 37, 39, 47, 49).

**Figure 3**
Quality assessment summary for the 37 papers included in this systematic review. (a) Reporting of any randomization, (b) reporting of any blinding, and (c) sample size calculation performed.

See this image and copyright information in PMC

References

1. AVMA AVMA Guidelines for the Euthanasia of Animals. 2020 ed. American Veterinary Medical Association; (2020). Available online at: https://www.avma.org/kb/policies/documents/euthanasia.pdf (accessed January 25, 2020).
1. Boivin GP, Hickman DL, Creamer-Hente MA, Pritchett-Corning KR, Bratcher NA. Review of CO2 as a euthanasia agent for laboratory rats and mice. J Am Assoc Lab Anim Sci. (2017) 56:491–9. - PMC - PubMed
1. Merriman JE. The role of carbon dioxide in anaesthesia. Can Anaesth Soc J. (1995) 2:273–80. 10.1007/BF03016171 - DOI
1. Wilson SR. Carbon dioxide in anaesthesia. Br J Anaesth. (1926) 3:112–8. 10.1093/bja/3.3.112 - DOI
1. Bert P. La Pression Barometrique. Paris: G. Masson; (1978). 610 p.

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

Affiliations

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

Authors

Affiliations

Abstract

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References

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