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

Carbon dioxide (CO2) inhalation is a common method of euthanasia used for rodents and small mammals. The animals must be euthanized by trained personnel using appropriate technique, equipment and agents. This is necessary to ensure a painless death that satisfies research requirements. Upon completion of the procedure, death must be confirmed by an appropriate method, such as ascertaining cardiac and respiratory arrest or noting an animal's fixed and dilated pupils. Euthanasia should not be performed in the animal room. The euthanasia method must be approved by the IACUC in the protocol and conform to the most recent report of the AVMA Panel on Euthanasia [2].

A few important aspects of this procedure are:

1. Compressed CO2 gas in cylinders is the only approved source of carbon dioxide because the inflow of gas to the chamber can be regulated precisely. CO2 generated by other means such as dry ice, fire extinguishers, or chemical means (e.g. antacids) is unacceptable. 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. Sudden exposure of conscious animals to CO2 concentrations of 70% or greater has been shown to be distressful [3] .
2. The euthanasia chamber should allow ready visibility of the animals. Do not overcrowd the chamber: all animals in the chamber must be able to make normal postural adjustments.
3. Animals should be left in the container until clinical death has been ensured (ascertaining cardiac arrest or noting fixed and dilated pupils).
4. Neonatal animals (up to 14 days of age) are resistant to the effects of CO2, therefore, alternative methods are recommended [4]. CO2 may be used for narcosis of neonatal animals provided it is followed by another method of euthanasia (e.g., decapitation using sharp blades).
5. To ensure death prior to disposal, another method of euthanasia (e.g., cervical dislocation, decapitation, exsanguination, thoracotomy) must be performed while the animal is still under CO2 narcosis. Refer to the report of the AVMA Panel on Euthanasia for additional methods.
   

1. Approved by the JHU Animal Care and Use Committee on: April 17, 2003—revised 9/28/2006
2. 2000 Report of the AVMA Panel on Euthanasia. JAVMA 218: 669-696; 2001
3. Dannenman, P. J., Stein, S., Walshaw, S. O. Humane and practical implications of using carbon dioxide mixed with oxygen for anesthesia or euthanasia of rats. Laboratory Animal Science, 1997, 47: 376-385.
   
4. Guidelines for Euthanasia of Mouse and Rat Fetuses and Neonates. NIH Animal Research Advisory Committee, 2001. http://oacu.od.nih.gov/arac/euthmous.htm
   
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