Guidelines for Euthanasia of Rodents
The Animal Welfare Act and PHS policy mandate that when experimental animals are
euthanized, death must be achieved quickly and painlessly.i,ii The AVMA has published
guidelines for acceptable methods of euthanasia that comply with the AWA and PHS
policy when performed on experimental animals.iii Several methods are approved by the
AVMA for euthanasia of rodents, including chemical methods and physical methods. The
2013 AVMA guidelines for euthanasia consider the use of lethal doses of injectable
barbiturates and certain barbiturate combinations or lethal doses of dissociative anesthetic
combinations as the only two methods that are acceptable without conditions. Other
methods of euthanasia are acceptable assuming certain conditions are met (Inhaled
anesthetics, CO2, CO, Tribromethanol/Avertin, Ethanol, Cervical Dislocation,
Decapitation and Focused Beam Microwave).iii The conditions that must be met for those
methods can be found at: https://www.avma.org/KB/Policies/Documents/euthanasia.pdf.
Carbon dioxide (CO2) overexposure is a widely used method for euthanasia of rodents
and other species for many reasons including ease of use, high safety margin for
researchers, and lack of residues in rodent tissues that may interfere with research results.
The 2013 AVMA panel on Euthanasia indicates that CO2 exposure alone is only an
acceptable method of euthanasia for appropriate species when certain conditions are met.
These conditions include using a gradual fill method at a rate of 10-30% chamber
volume/minute, and maintained for at least 1 minute beyond apparent clinical death
and that death is verified before removing the animal from the CO2 chamber.
Immersion of a rodent into a pre-filled container of CO2 is unacceptable.iii
Studies have shown that rodents, particularly pre-weaned rodents, that have stopped
breathing during CO2 exposure can recover when removed to room air.iv,v,vi Therefore,
when using CO2 exposure to euthanize animals it is imperative that death is assured prior
to carcass disposal. Failure to ensure death of animals after euthanasia procedures is
a reportable instance of non-compliancevii and violates the PHS policyii and the
AWA.i To minimize the risk of failed euthanasia, it is recommended that a secondary
measure is used to ensure death after the animal is rendered unconscious from CO2
exposure. Acceptable secondary measures include decapitation, cervical dislocation, and
thoracotomy.iii
Neonatal Rodents:
As with adult rodents, the preferred methods of euthanasia are lethal overdose of
barbiturates and certain barbiturate combinations as well as lethal overdose of certain
dissociative anesthetic combinations.iii Due to the high tolerance of many neonatal
species to elevated CO2 levels, death by hypoxia is prolonged compared to post weaning
animals. Therefore the 2013 AVMA Guidelines for Euthanasia recommends that
neonates are exposed to CO2 for upwards of 50 minutes OR adjunctive methods
performed after the neonate is non-responsive to painful stimuli.iii However, it is well
known in the animal research community that pre-weaned rodent pups are
relatively resistant to successful euthanasia when CO2 exposure is the sole means of
euthanasiaviii. In one study, neonatal mouse pups up to 7 days of age experienced cardiac
arrest after 4:41 minutes exposure to CO2, however some pups were able to revive once
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Guidelines for Euthanasia of Rodents
they were removed to room air.iv A second study confirmed that 5 minutes of exposure to
CO2 was insufficient to achieve 100% euthanasia in mouse pups up to 20 days of age in a
variety of inbred mouse strains.v
Due to the inconsistency of achieving 100% euthanasia in neonatal rodents after only 5
minutes of exposure to CO2, secondary measures must be used to ensure death after the
pups are rendered unconscious by CO2 exposure. Acceptable secondary measures include
decapitation, cervical dislocation, and thoracotomy.iii In some cases, physical secondary
methods cannot be used because they will interfere with research goals. If CO2 exposure
must be the sole means of euthanasia for rodent pups less than 21 days, prolonged
exposure to 100% CO2 according to times established by Pritchett et al should be
used.v
Other methods for the euthanasia of neonatal rodents that are acceptable with conditions
include hypothermia (altricial neonates <5d of age), decapitation (altricial neonates <7d
of age), and cervical dislocation. The conditions for these methods can be found at:
https://www.avma.org/KB/Policies/Documents/euthanasia.pdf.
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Guidelines for Euthanasia of Rodents
References
i
http://edocket.access.gpo.gov/cfr_2008/janqtr/pdf/9cfr1.1.pdf
ii
http://grants.nih.gov/grants/olaw/references/phspol.htm#ReviewofPHSConductedorSupportedResearchProjects
iii
https://www.avma.org/KB/Policies/Documents/euthanasia.pdf
Klaunberg B.A., O’Malley J., Clark T., Davis .JA. 2004. Euthanasia of Mouse Fetuses and Neonates.
Contemp. Top. Lab. Anim. Sc. 43:(5) 29-34.
iv
v
Pritchett K, et al. Euthanasia of neonatal mice with carbon dioxide. Comparative Med, 55(3):275-281,
2005
vi
Artwohl, J., P. Brown, B. Corning, and S. Stein (2006). Report of the ACLAM Task Force on Rodent
Euthanasia. Journal of the American Association for Laboratory Animal Science 45(1): 98-105
vii
http://grants.nih.gov/grants/guide/notice-files/NOT-OD-05-034.html
viii
The Laboratory Mouse. Mark A. Suckow, Peggy Denneman, Cory Brayton. CRC Press, Washington,
DC 2001. pp. 109-112.
Drafted August 2010 KMJ; Updated August, 2013 KTG