Which of the following methods is considered acceptable for euthanizing mice?

BU IACUCInstitutional Animal Care and Use Committee IACUC oversee... Approved October 2008, Revision 1: February 2012, Revised March 2019, Approved April 2019, Revised February 2021.

Federal Policy

PHS Policy requires Institutional Animal Care and Use Committees (IACUCs) to determine that methods of euthanasia utilized in research proposals are consistent with the Report of the American Veterinary Medical Association Panel on Euthanasia (AVMA American Veterinary Medical Association Guidelines on Euthanasia) unless a deviation is justified for scientific reasons in writing by the investigator. IACUC approval of such deviations must be project-specific and include critical review of assertions of scientific necessity.

• Euthanasia techniques must be consistent with the AVMAAmerican Veterinary Medical Association The American Vete... Guidelines on Euthanasia, 2020 Edition. Methods are chosen to minimize animal pain and distress consistent with needs of the research protocol.
• The method of euthanasia must be specified in the approved Institutional Animal Care and Use (IACUC) protocol. Animal death must be verified after euthanasia and prior to disposal (for specific details please refer to the section “Verifying Euthanasia” below).
• Use of anesthetic as a primary means of euthanasia must be an overdose, not an anesthetic dose. Regardless of amount of chemical administered, animal must be completely non-responsive to noxious stimuli (hind paw pinch) before any physical means are applied.
• Sodium pentobarbital, including derivative euthanasia solutions, and ketamine are controlled substances and must be maintained according to the BU Controlled Substances Program.
• Physical methods of euthanasia such as decapitation or cervical dislocation of unanesthetized animals require demonstration of competence, and may be approved with proper scientific justification in the IACUC protocol and documentation of training and equipment maintenance.
• Euthanasia should not be performed in the presence of other live animals whenever possible.

Suggested Common Methods for Euthanasia

• The procedures listed below are suggested common methods for euthanasia of rodents. Other methods outlined in the AVMA Guidelines on Euthanasia are acceptable with proper justification in the approved IACUC protocol.
• Intraperitoneal injection of at least 200 mg/kg sodium pentobarbital euthanasia solution. For barbiturates, 3x the anesthetic dose is often appropriate for euthanasia.
• Dissociative agents, such as ketamine, should be used in combination with alpha-2 adrenergic agonists (e.g. xylazine, dexmedetomidine) or benzodiazepines for euthanasia. In order to euthanize, an anesthetic overdose must be administered.
• Inhalation of CO2 from a pressurized tank in an uncrowded chamber (a standard size rodent cage may contain no more than standard occupancy: up to 5 adults, or parent(s)+litter) followed by cervical dislocation, decapitation or bilateral thoracotomy. Rodents may be euthanized by CO2 inhalation in their home cage or a clean induction chamber or appropriate size.
  • Set the regulator so that the CO2 gas flows slowly and dose not hiss or overpower the animals and frighten them. The acceptable range is 30-70% displacement per minute. The flow rate will depend on chamber size.
  • Refer to signage available near euthanasia stations for common cage sizes.
  • Chambers should not be pre-filled to avoid discomfort related to high CO2 concentrations prior to unconsciousness. CO­2 flow should be maintained for at least one minute following respiratory arrest.
  • A secondary physical method, such as decapitation, bilateral thoracotomy, or decapitation, should be performed once animals have been exposed to CO2.
• Other methods considered “Acceptable with Conditions” may be found in S2.2.2 of the AVMA Guidelines for Euthanasia, 2020 Edition. These should be described and justified appropriately in the IACUC protocol.
  • Physical methods, such as exsanguination, terminal tissue collection, or perfusion may be performed under appropriate anesthesia.
• Fetuses are in a state of unconsciousness during pregnancy, so euthanasia of the dam is considered sufficient to euthanize fetuses if they remain in the uterus. However, if fetuses must be removed from the uterus for tissue collection or experimentation, they should be treated as altricial neonates.
• Altricial neonates, such as mice and rats, are highly resistant to hypoxia and have underdeveloped pain pathways up to 5-7 days of age. Age specific guidelines for these animals are different than they are for adults. Precocial neonatal rodents, such as guinea pigs, should be treated as adults.
  • Anesthetic combination or barbiturate overdose is acceptable for fetuses, neonates, and adults.
  • Anesthesia may be induced by hypothermia of the neonate up to 10 days of age. This should not be used as a sole method, but may be performed prior to a physical method.
  • Rapid freezing of neonates up to 5 days of age in liquid N2 is acceptable.
  • CO2 may be used to euthanize altricial neonates, but it may take up to 50 minutes as a sole method and is not acceptable as a sole method. An adjunctive method (e.g. decapitation with sharp scissors, cervical dislocation) must be performed after neonate has become unresponsive to stimuli.
  • Decapitation with sharp scissors or blades is acceptable for neonates as long as they are small enough to do so quickly and cleanly.
  • Cervical dislocation by disrupting the spinal cord is acceptable with conditions for fetal and neonatal mice and rats.

Verifying Death

Death must be verified after euthanasia and prior to disposal. A combination of cessation of heartbeat and respiration, loss of corneal and toe pinch reflexes, graying of mucous membranes, or rigor mortis alone may be used to verify death. Many of these signs may be difficult to evaluate in small rodent species, especially if they’ve been euthanized via CO2, so secondary, physical methods are recommended. An adjunctive method is recommended for CO2 euthanasia. Unintended recovery must be prevented by use of appropriate exposure times and concentrations of CO2 or by physical means outlined below.

• Decapitation
• Cervical dislocation
• Exsanguination (great vessels severed, cardiac puncture may not be adequate)
• Cardiac perfusion
• Removal of vital organs
• Incision of chest cavity to produce bilateral pneumothorax, precluding respiration

Unintended recovery of animals after apparent death from CO2 (e.g. in necropsy coolers) is a documented occurrence. Such incidents constitute serious noncompliance with PHS policy and the Guide for the Care and Use of Laboratory Animals, so they must be reported to OLAW. Researchers must ensure the animal is dead before disposal, tissue collection, or leaving the animal unattended.

1. AVMA Panel on Euthanasia, AVMA Guidelines for Euthanasia of Animals, 2020 Edition https://www.avma.org/sites/default/files/2020-01/2020-Euthanasia-Final-1-17-20.pdfEuthanasia of Rodents BU IACUC 20192. Boivin, G. P., Bottomley, M. A., & Grobe, N. (2016). Responses of Male C57BL/6N Mice to Observing the Euthanasia of Other Mice. Journal of the American Association for Laboratory Animal Science : JAALAS, 55(4), 406–411.3. Boivin, G. P., Hickman, D. L., Creamer-Hente, M. A., Pritchett-Corning, K. R., & Bratcher, N. A. (2017). Review of CO₂ as a Euthanasia Agent for Laboratory Rats and Mice. Journal of the American Association for Laboratory Animal Science : JAALAS, 56(5), 491–499.4. Hickman D. L. (2018). Home Cage Compared with Induction Chamber for Euthanasia of Laboratory Rats. Journal of the American Association for Laboratory Animal Science : JAALAS, 57(6), 729–733. Advance online publication. https://doi.org/10.30802/AALAS-JAALAS-17-000160.5. Moffitt, A. D., Brignolo, L. L., Ardeshir, A., & Creamer-Hente, M. A. (2019). The Role of Emotional Contagion in the Distress Exhibited by Grouped Mice Exposed to CO₂. Journal of the American Association for Laboratory Animal Science : JAALAS, 58(4), 430–437. https://doi.org/10.30802/AALAS-JAALAS-18-000098