All euthanasia procedures performed within animal facilities are to be consistent with the recommendations in the 2000 Report of the AVMA Panel on Euthanasia. Requests to perform euthanasia using techniques other than those in that document must be approved by the IACUC and may require monitoring by a veterinarian.

Euthanasia may be performed as required by protocol study endpoints, to relieve pain/distress from experimental manipulations or spontaneous conditions, and as appropriate in other situations deemed necessary by a veterinarian.  Animals that are experiencing undue pain/distress must be humanely euthanized unless these conditions are required for scientific objectives that have been documented in a study protocol, justified, and approved by the IACUC.

It is important for researchers to delineate criteria for euthanasia in the study protocol including measurable physiological parameters and observable signs indicative of pain and distress.  Euthanasia methods must also be described, including the persons who will be responsible for observations and euthanasia procedures. Veterinarians are available to demonstrate or discuss these techniques.

Distress vocalizations, fearful behavior and release of pheromones by a frightened animal can all cause anxiety and apprehension in other animals. Therefore animals must be euthanized in an area separated from other live animals, especially of their own species. The only exception to this rule is when animals do not share the same environment (e.g., housed in microisolator caging systems) and thus will not be exposed to the effects mentioned above.

Any animal that is moribund (i.e., “near death�?) must be immediately addressed by animal care staff or specific satellite facility staff. The Principal Investigator or specified contact person for that study will be contacted immediately and asked to respond as soon as possible.  If there is no response in a timely manner, the veterinarian reserves the right to euthanize that animal within an acceptable time period. No animal should be left in a moribund state at the end of the workday if there is no one left to continue to observe the animal and euthanize it if it does not recover. 

Euthanized animals will be bagged, labeled with the PI’s name, protocol number and date. They will be stored in a specially labeled bin inside the animal carcass refrigerator for that facility.  The carcass will be saved in the specific location for no more than 48 hours.

Regardless of the technique used, all animals must be evaluated following euthanasia to assure that they are dead. This includes observing or palpating the chest for absence of a heartbeat and respiration (animals can have absence of respiration but still have a heart beat and subsequently fully recover.)  For large animals, adjunct signs used to confirm death include fixed and dilated pupils; these are helpful indicators because most euthanasia agents may also induce deep anesthesia from which the animal can recover if not administered properly.  Euthanasia of multiple animals at once is strongly discouraged because of the inability to easily evaluate each animal for the absence of vital signs.

The 2000 Report of the AVMA Panel on Euthanasia does not provide specific recommendations for the euthanasia of prenatal or neonatal animals. Guidelines provided for these animals are based on recommendations followed by NIH Intramural Animal Care and Use Committees.

Euthanasia systems and agents are available in all animal facilities (NB: investigators who frequently use controlled substances for euthanasia should maintain their own DEA license and stocks in accordance with federal and state laws). Specific instructions are posted in designated areas where euthanasia should be conducted. These include portable Euthanex® systems that are located in procedure rooms throughout each rodent facility.  Persons responsible for euthanizing animals must be properly trained and should be aware of where the equipment and drugs are located. Research staff are encouraged to euthanize their own animals whenever possible. Animal care staff can also euthanize animals for research staff as a service at no extra charge. When research staffs want to request that specific animals be euthanized, they should place a EUTHANASIA card in front of the cage card (small animals) or document it in the animal’s record (large animals). This card/notation must be signed and dated before any action will be taken. Cages can be left in place on the rack or left in designated areas within the animal facility. Investigators are responsible for ensuring that rodent cages removed from the room and left in designated spaces meet the following criteria:

1. A completed EUTHANASIA CARD is attached
2. Cages are not overcrowded (<5 mice per cage or <2-3 rats per cage)
3. Cages have no more than one mother and one litter per cage
4. Pups less than 21 days must have the mother in the cage
5. Food and water are available for all animals in the cage
6. Static microisolator tops/filter tops remain on the cage at all times

If there are any questions or proper equipment/drugs cannot be found, the On-Call Veterinarian should be contacted immediately.

Carcasses must be disposed of immediately after euthanasia has been confirmed. Carcass bags are located in all animal and procedure rooms for small numbers of animals. Carcasses should be placed in a chest specified for this purpose.

The research staff should record the removal of animals from the animal room using the census sheet for that room. Date of removal and number of animals and cages must be recorded in order to ensure they are deducted from the next census.

EUTHANASIA GUIDELINES

Acceptable methods include:

1. Carbon Dioxide (CO2) Asphyxiation using Compressed Gas
            a) Dry ice is not an acceptable source of CO2
            b) Chambers should be pre-charged with gas whenever possible to minimize
                 animal distress
            c) Recommended for rodents, small birds, and neonatal/weanling rabbits
            d) >70% CO2 should be used with appropriate exposure lengths
            e) Must confirm death

2. Barbiturates/Sodium pentobarbital
            a) DEA Schedule II Controlled Substance (double-locked cabinet)
            b) Dosage should be 2-3X that acceptable for anesthesia (i.e., 100-200 mg/kg)
            c) Route of administration should ensure rapid circulation
                        1. Intraperitoneal: rodents
                        2. Intravenous: rabbits

3. Gas Inhalants (Isoflurane, Halothane, etc.)
            a) Recommended for rodents
            b) Adequate ventilation and scavenging must be provided for safety reasons
            c) Expensive method
            d) Must confirm death

4. Cervical Dislocation
            a) Only applicable with anesthetized animals
            b) Acceptable for animals weighing less than 200 grams
            c) Must be scientifically justified that no other method can be done
            d) Aesthetically unpleasant; personnel must demonstrate competency

5. Decapitation with Guillotine or Scissors
            a) Only applicable with anesthetized small animals
            b) Conditionally Acceptable (AVMA Report)
            c) Must be scientifically justified that no other method can be done
            d) Aesthetically unpleasant; personnel must demonstrate competency

6. Potassium Chloride (KCL)
            a) Overdose only permissible in anesthetized animals
            b) Animal must demonstrate Stage III anesthesia before administration
            c) Leads to cardiac arrest

7. Exsanguination and Vital Organ Removal
            a) Only applicable with anesthetized animals
            b) Exsanguination is only appropriate when large volumes of blood are to be collected (i.e., >50% of total blood volume)
            c) Must confirm death

8. MS-222 (tricaine methanesulfonate)
            a) Used in amphibians and aquatics
            b) Dosage via dilution in water (pH 7.0)
                        1. 3 grams/L: Amphibians
                        2. 0.5 grams/L: Aquatics

9. Hypothermia
            a) Cooling to 4 degrees C
            b) Should be used only in anesthetized animals
            c) Best for small animals and young animals (less than one week old)
            d) Must confirm death

Fetuses (mice and rats):
Up to 14 days of gestation:       Because the neural development is minimal, pain perception is considered unlikely. Euthanasia of the mother or removal of the fetus should ensure rapid death due to loss of blood supply and non-viability at this stage of development.

15 days gestation - birth:           possibility of pain perception documented; perinates are not sensitive to inhalants therefore chemical anesthesia (intraperitoneal) is recommended. Decapitation, cervical dislocation, or rapid freezing are acceptable but may be aesthetically unpleasant. For fetuses that need to be chemically fixed, anesthesia such as hypothermia or a deep anesthesia of the mother with pentobarbital (crosses the placenta) should be done prior to immersion or perfusion.

Neonates (mice and rats):
Birth – 14 days old:                  Acceptable methods include intraperitoneal pentobarbital, decapitation (aesthetically unpleasant), or cervical dislocation (aesthetically unpleasant); animals can be exposed to inhalant gases or CO2 but exposure times should be 2-3x that for adults and all animals must be assessed for the absence of a heart beat; animals can be anesthetized via inhalant or injectable anesthetics prior to chemical immersion or perfusion; CO2 or gas anesthesia followed by hypothermia may be used for pups under 6 days of age.

Older than 14 days:                  Follow guidelines for adults.