Euthanasia - Confirmation of Death (Policy)

IACUC Policy:

Confirmation of Death

Policy: The IACUC has provided a set of guidance documents (Policies, Guidelines, and Informational Sheets) for use when planning animal procedures at the University of Iowa. An exception to a Policy must be described and justified in the Animal Protocol and approved by the full IACUC at a convened monthly meeting.

Purpose: This policy lists the IACUC’s requirements of confirmation of death.  The purpose of confirming death is to ensure that the animal cannot recover or reach consciousness.

The policy requires that any animal euthanized at the University of Iowa must be subject to a confirmatory method of death immediately after the method of euthanasia and preceding any other procedure.  Any animal found dead must also go through the confirmation of death procedure unless there is secondary physical indication of death (i.e. rigor, autolysis, or desiccation)  in addition to cardiac and/or respiratory arrest.

USDA-regulated Species (e.g ferrets, Guinea pigs, swine, hamsters, etc.)

• A physical confirmation of death is required immediately following a method of euthanasia and preceding all other proceduresConfirmatory methods include, but are not limited to:
  • Bilateral thoracotomy
  • Sternotomy
  • Vital tissue harvest (inclusive of heart, lungs, and/or brain)
  • Decapitation
  • Exsanguination
  • Perfusion

Mice and Rats

• Confirmation of death is required immediately following a method of chemical (e.g. gas or injection) euthanasia
• Confirmatory methods for animals older than 10 days of age include, but are not limited to:
  • Cervical dislocation
    • Not acceptable for rats > 200 grams of body weight
  • Decapitation
  • Thoracotomy
  • Vital tissue harvest (inclusive of heart, lungs, and/or brain)
  • Continued exposure to CO₂ for at least 15 minutes after respiratory arrest in the original container
• Confirmatory methods for neonates through 10 days of age or younger include, but are not limited to:
  • Decapitation
    • Preferred confirmatory method
  • Thoracotomy
  • Vital tissue harvest (inclusive of heart, lungs, and/or brain)
  • Continued exposure to CO2
    • This method is discouraged as neonates are more resistant to CO₂ hypoxia because it requires extended exposure up to 90 minutes.
• For isoflurane euthanasia:
  • If using a vaporizer: a physical method or continued exposure to isoflurane for at least 15 minutes after respiratory arrest is acceptable
  • If using a drop jar: a physical method (cervical dislocation, decapitation, thoracotomy or vital tissue harvest) must be used for confirmation   

Xenopus

• A physical confirmation of death is required following a method of euthanasia
• Confirmatory methods of euthanasia following an overdose of anesthesia:
  • Double pithing
  • Removal of heart

Zebrafish         

• Confirmation of death is required following a primary method of euthanasia
• Confirmatory methods for Zebrafish 8 days post fertilization (dpf) and older include, but are not limited to:
  • • Observation of no opercular movements for at least 10 minutes following anesthetic overdose or chilling
    • Decapitation

• Confirmatory methods for Zebrafish 4-7 dpf include, but are not limited to:
  • • Observation of no opercular movements for at least 10 minutes following anesthetic overdose or chilling
    • Decapitation
  • Confirmatory methods for Zebrafish 0-3 dpf include, but are not limited to:
    • Exposure to a dilute (1-10%) sodium hypochlorite or calcium hypochlorite solution

References

1. " AVMA Guidelines for the Euthanasia of Animals: 2020 Edition" AVMA.org., 2020.

Last Reviewed by the IACUC 6/11/2025