IV. Specific Experimental Procedures

A. Rodent Euthanasia

CARBON DIOXIDE (CO2)

1.   Considered an acceptable euthanasia agent for laboratory rodents when properly administered.

2.   The NIH Office of Laboratory Animal Welfare (OLAW) has issued a PHS Policy on Humane Care and Use of Laboratory Animals Clarification Regarding Use of Carbon Dioxide for Euthanasia of Small Laboratory Animals. The acceptability of CO2 as a euthanasia agent is predicated on the following:

  • High concentrations of CO2 may be distressful to some species. Accordingly, pre-filling of the CO2 chamber is recommended only under circumstances in which such use has not been shown to cause distress.
  • Death must be verified after euthanasia and prior to disposal.
  • All individuals administering CO2 euthanasia must be appropriately qualified and monitored. IACUC-approved protocols and institutional policies regarding CO2 euthanasia must be followed.
  • Euthanasia chambers must not be overcrowded. Mixing unfamiliar or incompatible animals in the same container may be distressful.
  • Compressed CO2 in cylinders is the only AVMA Panel-recommended source of CO2 for euthanasia purposes.

3.   Advantages:

  • Provides rapid depression and anesthesia (CO2 narcosis).
  • Non-flammable and non-explosive.
  • No chemical residues are introduced into tissues.
  • It does not result in distortion of cellular architecture.

4.   Disadvantages:

  • CO2 is heavier than air. Incomplete filling of the chamber can induce some animals to avoid exposure by climbing or jumping which appears distressful.
  • CO2 may be distressful to some animals due to irritation of the mucous membranes of the respiratory tract and stimulation of respiratory centers in the brain. The AVMA panel considers the degree of distress to be mild and unlikely that it is more unpleasant then inhalation of volatile anesthetics.
  • Neonatal rodents are resistant to CO2 induced euthanasia. Euthanasia of neonatal rodents is discussed further in the next section.

5.   Recommendations for CO2: 

  • At the College of Charleston, CO2 is the preferred method of euthanasia for rodents. CO2 tanks with euthanasia chambers are located in the surgery suite of the RITA.
  • Gas flow should be maintained for at least one minute after apparent clinical death (cessation of respiration). If an animal is not dead, CO2 narcosis must be followed with another method of euthanasia.
  • To insure that unintended recovery does not occur after CO2 exposure, cervical dislocation may be performed or a stab incision may be made (between the ribs) into the chest cavity on both sides of the animal.

VOLATILE INHALANT ANESTHETICS

  • Allowable Inhalants: Sevoflurane and Isoflurane
  • Animals are placed in a sealed container such as a bell jar containing gauze soaked with the anesthetic agent.
  • Animals should be separated from the anesthetic soaked gauze by a false bottom or other method to prevent direct animal contact with the liquid anesthetic.
  • A number of volatile inhalant anesthetics may be used for anesthesia. Contact the veterinarian for information regarding the use of volatile inhalant anesthetics for euthanasia.
  • All volatile inhalant anesthetics require some method of scavenging the waste anesthetic vapors (i.e., working in a biosafety cabinet).

CERVICAL DISLOCATION

 1.   Acceptable under the following conditions:

  • Cervical dislocation should be performed on sedated or unconscious rodents.
  • Investigators are responsible to determine that personnel using cervical dislocation are properly trained to do so. The IACUP will provide training to those who request it.
  • Cervical dislocation on conscious rodents requires scientific justification and prior approval by the IACUC.

2    Advantages:

  • Induces rapid unconsciousness.
  • Does not chemically contaminate tissue.
  • A quick procedure.

3.   Disadvantages:

  • Aesthetically displeasing to personnel.
  • Physical damage to tissues and structures in the cervical area along with bleeding may interfere with sample collection.
  • When performed incorrectly it may induce or prolong animal suffering.

BARBITURATES

  1. Acceptable
  2. Barbiturates may be injected intraperitoneally to euthanize rodents.
  3. Agents available for use include sodium pentobarbital and pentobarbital combinations.
  4. Barbiturate drugs must be used under the supervision of personnel registered with the United States Drug Enforcement Administration (DEA).
  5. Requires strict accounting of quantities used.
  6. Requires locked storage.

DISPOSAL OF CARCASSES

  1. Rodent carcasses that have NOT been subjected to the introduction of any chemicals, hormones, or radioactive material may be disposed of in the black freezer in the vivarium closet CL41.
  2. All other carcasses must be disposed of in Biohazard bags in Biohazard bins in the cold room RITA.  The bag must be tied and bin closed. 

REFERNCES 

AVMA Guidelines on Euthanasia

NIH Guidelines for the Euthanasia of Rodent Feti and Neonates

Approved: April 5, 2001

Revised:  April 19, 2001

Revised:  September 11, 2001

Reviewed and revised:  August 24, 2012, September 27, 2019