Animal Care and Use Policies and Guidelines

JHU Policy for Euthanasia of Rodents Using Carbon Dioxide [1]

Policy:
Euthanasia of animals at Johns Hopkins University must be carried out according to the most recent guidance of the American Veterinary Medical Association (AVMA) [2].  Carbon dioxide (CO2) inhalation is a common method of euthanasia used for rats and mice.  It is the method that will be used by central facilities staff with rats and mice identified for euthanasia by researchers.  Use of CO2 euthanasia by researchers must be included in an ACUC-approved protocol.   Appropriate technique, equipment, and source of CO2  must be used.  Compressed CO2 gas in cylinders is the only approved source because the inflow of gas to the euthanasia chamber can be regulated precisely.  CO2 generated by other means such as dry ice, fire extinguishers, or chemical means (e.g., antacids) is unacceptable.

Upon completion of the procedure, death must be confirmed for each animal by one or more of the methods listed below.  CO2 is an anesthetic and failure of the animal to move or show a reflex response is not sufficient to confirm death.  Disposal of an anesthetized, rather than a euthanized, animal is a serious animal welfare concern and a significant breach of the conditions of JHU’s approved Public Health Service Animal Welfare Assurance.  Understanding how to avoid this is incumbent upon anyone carrying out euthanasia with CO2.

Guidelines:
Animals in the euthanasia chamber should be readily visible.  Do not overcrowd the chamber by combining multiple cages.  All animals in the chamber must be able to make normal postural adjustments. One rat or up to three mouse cages may be placed in the chamber at one time.

Sudden exposure of conscious animals to CO2 concentrations of 70% or greater has been shown to be distressful [3].  Yet death should be induced as quickly as possible.  Without pre-charging the chamber, place the animals in the chamber and introduce 100% CO2 at a slow rate so as to optimize reduction in distress.  Animals should be exposed to the CO2 for the duration listed in the table below.

Neonatal animals (up to 14 days of age) are resistant to the effects of CO2, therefore, a secondary physical  method must  follow the CO2 exposure. This may include decapitation using a sharp blade or cervical dislocation.

Death must be ensured for all animals prior to disposal.  One of the methods below must be used unless harvest of vital organs will occur right away.

1. Verification that the heart has stopped beating and the animal is not breathing; paleness of mucosal tissue.  (This typically is not very practical if many animals are being sacrificed at one time since it involves handling each one.)
2. Observation that the animal fails to recover within 10 minutes after CO2 exposure ends.
3. Cervical dislocation.
4. Thoracotomy.
5. Decapitation.

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**All the methods listed above are approved by the Animal Care and Use Committee, and may be used even if they were not  included in your approved protocol.**

Carcass bags must be identified with the carcass tags or cage cards. This should include the date and protocol number.