These guidelines have been written to assist Protocol Directors in complying with AVMA recommendations as well as to apprise them of effective techniques for rodent euthanasia with CO2.
KEY POINTS TO REMEMBER
- CO2 must come from a controlled source (i.e. compressed gas cylinder). Dry ice is NOT an acceptable source of CO2.
- Euthanasia chambers must be clean and of appropriate size for the rodent species being euthanized. Rodents must not be overcrowded in the euthanasia chamber.
- Because of the sedative/anesthetic effect of CO2 exposure, animals may appear dead when they are not. Animals must not be disposed of until death has been confirmed by a secondary physical method (i.e., cervical dislocation or thoracotomy), or until they have been outside the chamber for a period of 5-10 minutes and have been carefully observed for multiple indicators that signify death (loss of bladder control, absence of heart rate, lack of a toe-pinch response, cessation of respiration). Confirmation of death should not be based solely on a single sign, for example, cessation of respiration.
- CO2 is not recommended for neonates. Alternative methods, such as decapitation, should be considered for euthanasia. If CO2 is used, prolonged (e.g. up to 50 minutes) exposure to the gas is required.
- Animals must be disposed of in a biohazard bag labeled with the protocol number.
- If you have any questions or concerns about use of CO2 for rodent euthanasia contact the VSC (725-9901).
I. Characteristics
Carbon dioxide (CO2) is a safe and humane method of euthanasia that is a preferred technique for use with adult rodents. The gas is inexpensive, nonflammable, and nonexplosive. Furthermore, several rodents can be rapidly euthanized simultaneously. It causes no accumulation of exogenous chemical residues in tissues nor does it produce observable histological changes in non-pulmonary tissues (however there may be slight pulmonary effects). It can be administered using fairly simple equipment that can be located anywhere in a facility or fixed to a mobile platform for portable use. Exposure to high concentrations of CO2 has an initial rapid depressant and anesthetic effect, which is followed by death through asphyxiation. However, high concentrations of CO2 may be distressful to some species. Accordingly, the chamber should not be pre-filled when euthanizing rats and mice. The filling rate (typically 20% of the chamber volume per minute) of the euthanasia chamber should be based on the time required to rapidly and successfully render the animals unconscious, which is typically induced by exposing animals to a CO2 concentration of 70% or more. It is recommended that 100% CO2 be used. Under these conditions, an exposure time of 5 minutes (at least 3 minutes at 100% CO2) appears generally adequate to euthanize adult animals. To shorten the time needed to perform euthanasia, sublethal exposure to CO2 can be followed by exsanguination, cervical dislocation or decapitation.
Carbon dioxide should be purchased in compressed gas cylinders. CO2 generated from other sources, such as dry ice, fire extinguishers, or from Alka-Seltzer, are unacceptable because gas flow cannot be regulated precisely.
II. Humane Considerations
The most common errors when using CO2 for euthanasia are: (1) not using equipment and methods which assure that the air in the chamber is totally displaced by CO2; (2) overcrowding animals; and (3) not maintaining and replenishing the carbon dioxide within the chamber during sequential use. Since carbon dioxide is 50 percent heavier than air, chambers should be designed so that they fill rapidly with gas from bottom to top. This allows the air to exit at the top and be completely replaced by carbon dioxide. Lethal levels of carbon dioxide are then easily maintained with a minimum of additional gas. Incomplete filling of a chamber may permit tall or climbing animals to avoid exposure to an optimal concentration of gas, which can lead to prolonged distress to the animals.
Animals placed together in chambers must be of the same species, and, if needed, should be restrained so that they will not hurt themselves or other animals. Chambers must not be overcrowded. Overcrowding of the euthanasia chamber has been noted to lead to inadequate narcosis and asphyxiation. The number of animals should be limited to allow free flow of CO2 to each animal. Animals must be able to keep all four feet on the floor of the euthanasia chamber, must be able to turn around and must not be so crowded that they must sit on top of each other.
In this regard, it is important to also consider that mixing unfamiliar or incompatible animals (i.e. animals from different cages, especially males) in the same container may be distressful. Chambers should be kept clean to minimize odors that might distress animals subsequently euthanized.
III. Methods
COMPRESSED GAS
Use of a compressed gas cylinder to administer CO2 should always include an appropriate two-stage regulator. The animal is placed inside the chamber and the lid secured. A fill rate of 20% of the chamber volume per minute with CO2, added to existing room air in the chamber should be appropriate to achieve the objective of rapid unconsciousness with minimal distress to the animals. However, animals should be closely observed during the filling process, as individual systems may require adjustment to achieve the desired effect. To assure bottom-to-top filling, the gas should enter either through a port located at the bottom of the chamber or through tubing which enters at the top and extends down into the chamber at least 3/4 of the way.
Examples of chambers:
1. A large plastic or glass dessicator jar with a tubulature in the top can be used if a two-holed rubber stopper is inserted. The hose from a CO2 regulator is connected to a six-to-eight-inch piece of rigid plastic or stainless steel tubing that passes through one hole of the stopper and allows the carbon dioxide to be admitted at the bottom of the chamber. A three-inch piece of plastic tubing is passed through the other hole and is connected to a short length of hose which has an adjustable screw-type clamp placed to regulate the escape of air and carbon dioxide from the chamber. Before administering gas, the ground glass surfaces should be sealed with silicone grease.
2. Various commercially-available or customized tops allow a glass aquarium to serve as a euthanasia chamber. Similar tops are available to euthanize animals inside plastic rodent cages. These tops seal the enclosure and create inlet/outlet holes that can be connected to the supply of CO2 (as mentioned in above paragraph). The simplest functional system is one that has a single hole through which a length of tubing connected to the regulator passes. The tubing extends down to the very bottom of the chamber and does not quite entirely block the hole in the lid. This system allows gas to enter at the bottom of the chamber while air is vented out through the small opening around the tubing at the top.
It is not acceptable to use a plastic bag for a euthanasia chamber. A biohazard bag may be used to line the euthanasia chamber to facilitate cleaning but animals should still be visible through the top of the chamber and the lid should create a tight seal to ensure the concentration of CO2 reaches and maintains an effective level.
IV. Removal from Chamber
Death must be verified after euthanasia and prior to disposal. Since the anesthetic effects of CO2 are reversible, animals that are prematurely removed from the chamber prior to death can recover. Unintended recovery after the procedure will be very rare if appropriate CO2 concentrations and exposure times are used. However, whenever an inhalant gas is used for euthanasia, it is imperative to be sure that all animals are dead when removed from the chamber. Before animals are removed, all visible movement (including breathing) should have stopped. After removal, check again to confirm the absence of multiple vital signs (loss of bladder control, absence of heart rate, lack of a toe-pinch response, cessation of respiration). Confirmation of death should not be based solely on a single sign, for example, cessation of respiration. Alternatively, CO2 narcosis can be followed with another method of euthanasia, such as cervical dislocation or thoracotomy. Thoracotomy (making an incision into the chest cavity) after apparent death from CO2 is widely used as a way to ensure the irreversibility of the procedure. If large numbers of animals are being euthanized and individual examination is not feasible, animals should not be disposed of until the animals have been outside of the chamber and exposed to room air for at least 5-10 minutes and have been carefully observed for multiple vital signs. Animals should be disposed of in a biohazard bag that is labeled with the protocol number.
V. Neonates
Time for euthanasia may be SUBSTANTIALLY PROLONGED in neonatal rodents due to their inherent resistance to hypoxia. For this reason, CO2 should not be used in neonates unless the animal can be exposed long enough to ensure death. The length of exposure can be variable (up to 50 minutes). An alternate method such as decapitation or cervical dislocation should be considered for euthanasia of newborn mice.
VI. Assistance with euthanasia
The Veterinary Service Center staff can perform euthanasia of all species at a reasonable cost on a fee-for-service basis. Persons interested in learning more about this option should contact the VSC at 723-3876.
To see the Report of the American Veterinary Medical Association on Euthanasia, go to:
http://www.avma.org/resources/euthanasia.pdf
Rodent CO2 Euthanasia References- Primary references
Public Statement: Report of the ACLAM Task Force on Rodent Euthanasia, 2005
AVMA Guidelines on Euthanasia, 2007
Rodent CO2 Euthanasia References- Secondary References
Anton F, Euchner I, Handwerker HO. Psycophysical examination of pain induced by defined CO2 pulses applied to nasal mucosa. Pain 1992; 49:53–60.
Britt DP. The humaneness of carbon dioxide as an agent of euthanasia for laboratory rodents. In: Euthanasia of unwanted, injured or diseased animals for educational or scientific purposes. Potters Bar, UK: UFAW, 1987; 19–31.
Carr, V.M., B. Menco, M.P. Yankova, R.I. Morimoto, A.I. Farbman. 2001. Odorants as cell-type specific activator of a heat shock response in the rat olfactory mucosa. J. Comp. Neurol., 432:425-439.
Danneman PJ, Stein S, Walshaw SO. Humane and practical implications of using carbon dioxide mixed with oxygen for anesthesia
or euthanasia of rats. Lab Anim Sci 1997; 47:376–385.
Sharp, J., T. Zammit, T. Azar, D. Lawson. 2003. Stress-like responses to common procedures in individually and group-housed female rats. Cont. Top. in Lab. An. Sci., 42:9-18.
Hornett, T.D., A.P. Haynes. 1984. Comparison of carbon dioxide/air mixture and nitrogen/air mixture for the euthanasia of rodents, design of a system for inhalation euthanasia. Anim. Technol., 35:93-99.
Mazurkiewica-Kwilecki. 1980. Single and repeated air blast stress and brain histamine. Pharmacol. Biochem. Beh., 12:35-39.
Methods of Behavioral Analysis in Neuroscience, J. J. Buccafusco (ed.). 2001. CRC Press.
Schnecko, A.K., K. Witte, B. Lemmer. 1998. Effects of routine procedures on cardiovascular parameters of Sprague-Dawley rats in periods of activity and rest. J. Exp. An. Sci., 38:181-190.
Sharp, J., T. Zammit, T. Azar, D. Lawson. 2003. Stress-like responses to common procedures in individually and group-housed female rats. Cont. Top. in Lab. An. Sci., 42:9-18.
Provider: Office of the Vice Provost and Dean of Research, Stanford University
Contact: A-PLAC Administrator
Last updated: March 2008
