Euthanasia of Research and Teaching Animals
Number: IACUC-POL-015
Responsible Office: Office of Research and Creative Scholarship (ORCS)
Applies to: Principal Investigators Conducting Animal Research or Teaching Protocols Involving Live Vertebrate Animals
1. Purpose
To provide guidance and establish institutional requirements for the humane euthanasia of animals used in research and teaching activities at the 91次元.
2. Policy
All euthanasia methods must be described in the applicable Animal Use Protocol (AUP) and approved by the IACUC prior to being performed. Unless a deviation is scientifically or medically justified and approved by the IACUC, euthanasia methods must be consistent with the most current edition of the AVMA Guidelines on Euthanasia.
- When specific AVMA guidance is absent for a species or situation, proposed methods must be fully described and justified. Justification may include peerreviewed literature or published guidance from professional societies.
- Protocols involving housing of animals or field studies that do not routinely require euthanasia must still describe an emergency euthanasia method.
- Protocols proposing physical methods of euthanasia without anesthesia (e.g., cervical dislocation without anesthesia) must include specific justification in the AUP and meet training and proficiency requirements outlined in this policy.
Emergency Euthanasia and Depopulation
In the event that standard euthanasia guidelines cannot be met due to emergency conditions, euthanasia or depopulation of large numbers of animals will be managed in accordance with the AVMA Guidelines for the Depopulation of Animals, consistent with institutional emergency and disaster plans.
Confirmation of Death
All euthanasia procedures require confirmation of death and/or application of a secondary physical method as appropriate for the species. Unintended recovery of animals after apparent death constitutes serious noncompliance and must be reported to the IACUC.
3. Training
All personnel performing animal euthanasia must be trained, knowledgeable, and proficient in the selected techniques and confirmation of death. Principal Investigators are responsible for ensuring that personnel listed on their protocols are appropriately trained.
Physical Methods of Euthanasia Without Anesthesia
Personnel using physical methods of euthanasia without anesthesia must have their proficiency evaluated and verified by the Attending Veterinarian (AV) or an AVapproved designated trainer. Verification must be documented prior to independent performance.
Methods requiring verification include, but are not limited to:
- Cervical dislocation without anesthesia
- Decapitation without anesthesia
- Penetrating captive bolt without sedation/anesthesia
- Firearms (where legally permitted and only by highly trained personnel)
- Pithing without anesthesia
- Exemptions from inperson verification may be granted by the AV on a casebycase basis based on documented experience or credentials.
4. Minimizing Pain and Distress
Pain and distress prior to and during euthanasia must be minimized.
- Animals must be handled calmly and gently and restrained only as necessary for safe and effective euthanasia.
- Euthanasia should generally not be performed in animal housing rooms to avoid distress to other animals, unless scientifically justified, required for disease control, or necessary in emergency situations.
- Chambers used for inhalant euthanasia must not be overcrowded or go beyond the allowed maximum number approved for caging size.
- Animals prone to aggression or fighting must not be mixed during transport or euthanasia.
5. Procedure Guidelines
The following summarizes commonly used and accepted euthanasia methods by species. This list is not exhaustive. Methods must be consistent with AVMA Guidelines and IACUC approval.
ALL METHODS OF EUTHANASIA MUST BE FOLLOWED BY A SECONDARY METHOD
Rodents
Acceptable Methods
- Injected barbiturates
- Injected anesthetic overdose
- Inhaled anesthetic overdose
- Carbon dioxide (CO₂)
Acceptable With Conditions
- Cervical dislocation
- Decapitation
*Confirmation of death must include a secondary physical method such as cervical dislocation, decapitation, cardiac perfusion, removal of vital organs, pneumothorax, or exsanguination.
CO₂ Conditions
- Source must be compressed CO₂ gas (dry ice or prefilled chambers are not acceptable).
- Flow rate must displace 30–70% of the chamber volume per minute.
- CO₂ must be maintained for at least one minute after respiratory arrest.
- Animals should be euthanized in their home cage whenever practical.
- Chambers must be cleaned after each use.
Rodent Fetuses
Up to 14 days gestation: Neural development is minimal and pain perception is considered unlikely. Euthanasia of the pregnant dam or removal of the fetus will result in rapid fetal death due to loss of blood supply and non-viability at this stage.
15 days gestation to birth: Development of pain pathways suggests the potential for pain perception. Fetuses at this stage are resistant to inhalant anesthetics (e.g., CO₂, isoflurane). Euthanasia must be performed by direct injection of a chemical anesthetic into the fetus or by an approved physical method (e.g., cervical dislocation or decapitation with surgical scissors).
When whole-fetus chemical fixation is required, fetuses must be anesthetized prior to immersion or perfusion with fixative. Anesthesia may be achieved by: (a) direct injection of a chemical anesthetic into the fetus; (b) deep anesthesia of the dam with an agent that crosses the placenta (e.g., pentobarbital); or (c) hypothermia of the fetus following removal from the dam and prior to fixation. Consultation with the Attending Veterinarian is required when selecting anesthetic agents. If fetuses are not required for study, euthanasia of the dam must ensure rapid fetal death (e.g., pentobarbital overdose).
Rodent Neonates
Neonatal mice and rats (postnatal day P1–P10) are more resistant to CO₂ euthanasia than adults and require prolonged exposure. Neonates over P10 must be euthanized using adult rodent methods.
CO₂ or Inhaled Anesthetic
If CO₂ or inhalant anesthetics are used for P1–P10 neonates, a secondary physical method (e.g., cervical dislocation, decapitation with sharp scissors, or bilateral pneumothorax) is required to ensure death. Secondary methods may only be applied after the neonate is completely unresponsive to painful stimuli.
Hypothermia
Rapid immersion in liquid nitrogen is permitted only for neonatal rodents less than five (5) days of age and must be used only in conjunction with a secondary, confirmatory method of euthanasia.
Hypothermia may be used only for neonatal rodents and only in conjunction with a secondary, confirmatory method of euthanasia. Animals must not come into direct contact with ice or metal surfaces during the procedure. Hypothermia is not an acceptable method of euthanasia for neonatal rodents greater than ten (10) days of age.
Physical methods performed without prior anesthesia or CO₂ exposure must be specifically described and justified in the AUP. Personnel performing physical euthanasia on unanesthetized neonatal mice and rats after P5 must have verified proficiency prior to independent work.
Use of chemical agents (e.g., pentobarbital IP at appropriate dose or high-concentration isoflurane) is preferred over CO₂ for neonates. Prolonged exposure may be required, and a secondary physical method is still required to confirm death.
Rabbits
Acceptable Methods
-
Intravenous barbiturates
Acceptable With Conditions
-
Inhaled anesthetic overdose, CO₂, cervical dislocation
*Confirmation of death requires multiple criteria (e.g., lack of heartbeat, respiration, corneal reflex) and/or a secondary physical method.
Fish
Acceptable Methods
Injected barbiturates; immersion in buffered MS222, benzocaine, ethanol; rapid chilling (speciesspecific)
Acceptable With Conditions
CO₂saturated water, decapitation, cervical transection, blunt force trauma followed by pithing
Fish must remain in anesthetic solutions for a minimum of 30 minutes after cessation of opercular movement unless otherwise specified by AVMA guidance.
Amphibians and Reptiles
Speciesappropriate euthanasia methods must follow AVMA Guidelines. Confirmation of death generally requires secondary physical methods and multiple physiological criteria. Due to variability among species, consultation with the AV is strongly recommended.
Avian Species
Acceptable Methods
-
Injected barbiturates
Acceptable With Conditions
- Inhaled anesthetics
- Cervical dislocation
- Decapitation
Confirmation of death must have a secondary method such as decapitation, exsanguination or cervical dislocation.
Eggs
Avian eggs at <80% incubation may be destroyed by prolonged exposure (>20 minutes) to CO2 , cooling (<4°C for 4 hours) or freezing. If the egg is over 80% incubation, the methods specified above for older birds must be used.
For finfish, amphibian, and reptile eggs, larvae and embryos, acceptable euthanasia methods vary based on age and life stage, consultation with the AV is strongly recommended.
6. References
- AVMA Guidelines for the Euthanasia of Animals (current edition).
- AVMA Guidelines for the Depopulation of Animals.
- Guide for the Care and Use of Laboratory Animals, NRC.
- Guide for the Care and Use of Agricultural Animals in Research and Teaching.
- AVMA Guidelines for the Humane Slaughter of Animals.
- NIH Guidelines for the Euthanasia of Rodent Fetuses and Neonates.
7. Review, Approval and Version History
|
Version |
Date |
Description of Changes |
Approved By |
|---|---|---|---|
|
1.0 |
January 13, 2026 |
Initial policy creation |
IACUC Committee |