EUTHANASIA For additional information, click on the blue underlined text throughout this presentation.

1. EUTHANASIA For additional information, click on the blue underlined text throughout this presentation.

2. What is Euthanasia?

3. Because it is necessary to euthanize most animals as part of experimental protocols, it is very important to use appropriate euthanasia techniques. Members of the Laboratory Animal Facilities (LAF) veterinary staff are willing to assist laboratories with issues related to animal euthanasia. This assistance may include training personnel in the actual process or our veterinary group actively conducting the euthanasia procedure. Investigators are encouraged to consult with a veterinarian for euthanasia techniques specific to the age and species of animals. Assistance may be arranged by contacting the LAF office at 4-1385or visiting the offices on the 2nd floor of the Guyton Building (G201).

5. PHS Policy and the Guide state that methods of euthanasia should be consistent with the recommendations of a panel sponsored by the American Veterinary Medical Association (AVMA), unless the IACUC approves deviations for scientific reasons. The Report of the AVMA Panel on Euthanasia contains many guidelines used by the IACUC to evaluate methods of euthanasia.

7. Acceptable The AVMA Panel on Euthanasia states that "acceptable methods are those that consistently produce a humane death when used as the sole means of euthanasia." Such methods are considered humane and are preferred for euthanizing laboratory animals.

8. Conditionally acceptable The AVMA Panel on Euthanasia states that, "conditionally acceptable methods are those techniques that by the nature of the technique or because of greater potential for operator error or safety hazards might not consistently produce humane death or are methods not well documented in the scientific literature." The inclusion of conditionally acceptable methods in your protocol require scientific justification before your IACUC will approve their use. Such methods may be humane under controlled circumstances and may be necessary to avoid adverse effects by the euthanasia agent (e.g., an effect on tissues ante mortem) on the research study.

9. Unacceptable The AVMA Panel on Euthanasia states that, "unacceptable techniques are those methods deemed inhumane under any conditions or that the panel found posed a substantial risk to the human applying the technique." Unacceptable methods are considered inhumane and are absolutely condemned for use as euthanasia agents. These methods cannot be used on laboratory animals.

10. Secondary(Adjunctive)Methods of Euthanasia

11. Secondary (Adjunctive) Methods Secondary methods of euthanasia must be used as an adjunctive method to assure death of the animal. All methods must be documented in the IACUC-approved Animal Activity Protocol form. Take the opportunity to review your protocol with the lab personnel and specifically note question # 20, identifying both the primary and secondary (adjunct) methods of euthanasia.

12. What does that mean? If primary euthanasia method is: CO2 inhalation Inhalation anesthesia Injectable anesthesia …..then you MUST use a secondary method: Exsanguination or Decapitation or Cervical dislocation or Tissue harvest or Bilateral pneumothorax Training in euthanasia techniques is available by contacting the LAF office at 4-1385.

13. In their report, the AVMA Panel on Euthanasia also include a discussion of adjunctive methods of euthanasia which cannot be used as a sole methodbut can be used in conjunction with other methods to produce a humane death. The following methods of euthanasia should not be used alone without special justification and IACUC approval:

14. Euthanasia by CO2

15. Advantages • CO2 provides a rapid depression and anesthesia (narcosis). • CO2 is non-flammable and non-explosive. • CO2 does not introduce chemical residues into tissues. • CO2 does not result in distortion of cellular architecture.

16. Disadvantages • CO2 is heavier than air, so incomplete filling of the chamber can induce some animals to avoid exposure by climbing or jumping. This may be a sign of distress. • 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 on Euthanasia considers the degree of distress to be mild and unlikely that it is more unpleasant than inhalation of volatile anesthetics.

17. Basement of the Guyton Building G044 8th floor of the Research Wing R820 The LAF has two (2) CO2 euthanasia stations.

18. Carbon Dioxide Euthanasia Procedures • The animal(s) should remain in their “home cage” to reduce stress. • Remove water bottles from cages, leaving the wire bar lid on the cage top. [note: place red cap over the water lixit hole on rat cages] • Attach the appropriate size stainless steel top to the clear CO2 gas line. Place stainless steel top on top of the cage. • Turn silver knob at the top of the CO2 tank counterclockwise (ON); the pressure gauge farthest from the tank should now indicate the tank pressure. • Turn the white knob on the regulator clockwise (ON) until the flow meter (pressure gauge closest to the tank) registers about 5 – 10 psi. • Observe the animal(s) and maintain gas flow for at least 1 minute after breathing has stopped. • Turn silver knob on top of the CO2 tank clockwise (OFF). • Turn black knob on the regulator counterclockwise (OFF); the gauge closest to the tank should now read “0 psi”. • Remove cage from the box and complete secondary (adjunct) method of euthanasia. These procedures are posted at each CO2 Euthanasia station.

19. Procedural Guidelines from OLAW Source: PHS Policy on Humane Care and Use of Laboratory Animals Clarification Regarding Use of Carbon Dioxide for Euthanasia of Small Laboratory Animals

20. CO2 is generally considered an acceptable euthanasia agent for small laboratory animals when properly administered, its acceptability is predicated on the following conditions: 1. Pre-filling the chamber may be recommended only under circumstances in which such use has not been shown to cause distress. 2. Death must be verified after euthanasia and prior to disposal. Unintended recovery must be obviated by the use of appropriate CO2 concentrations and exposure times or by other means. OLAW notes that thoracotomy after apparent death from CO2 is one way to ensure the irreversibility of the procedure. 3. Institutions must ensure that all individuals responsible for administering CO2 euthanasia are appropriately qualified and monitored, and that they adhere to IACUC-approved protocols and institutional policies.

21. 4. Chambers must not be overcrowded. In this regard, it is important to also consider that mixing unfamiliar or incompatible animals in the same container may be distressful. 5. Compressed CO2 in cylinders is the only AVMA Panel-recommended source of CO2 for euthanasia purposes. 6. Unintended recovery of animals after apparent death from CO2 (e.g., in necropsy coolers) is a documented occurrence. Institutions are reminded that such incidents constitute serious noncompliance with the PHS Policy and serious deviation from the provisions of the Guide for the Care and Use of Laboratory Animals. As such, the IACUC, through the Institutional Official, must promptly provide OLAW with a full explanation of the circumstances and actions taken. Prompt reporting in accordance with PHS is an essential component of the formal relationship between OLAW and PHS-Assured institutions. If you notice any such occurrence, you should report it immediately to a member of the LAF.

22. 7.OLAW advises IACUCs to review their policies and practices regarding CO2 euthanasia of small laboratory animals and take appropriate action as needed to conform to this guidance. 8. CO2 should be administered for several minutes after the animal has stopped breathing. A clear chamber helps in monitoring the animal. 9. Death of the animal must be confirmed following euthanasia by CO2. Some methods to confirm death: • Cervical dislocation • Bilateral thoracotomy • Exsanguination • Decapitation • Potassium chloride injection

23. The key objectives in using CO2 for euthanasia of small laboratory animals are that the animals should have a minimum of distress and should become unconscious quickly. The following considerations will help you achieve these objectives: • You should use a clear chamber so that you can monitor the animals' behavior while they remain conscious and to monitor respiration. • It is normal to observe a small degree of agitation as animals explore and sniff about the chamber. • The animals should not appear frantic, e.g., attempting to escape by upward leaps as though to flee a CO2-rich stratum in the bottom of the cage. • If you observe such behavior, please consult the veterinary staff for assistance in modifying your procedure and equipment set-up. • After the animals show no respiration for ay least one minute, remove them from the chamber and perform a method to ensure death, such as listed above in the OLAW guidelines.

24. For additional information, please consult The AVMA Guidelines on Euthanasia, 2007 at http://www.avma.org/issues/animal_welfare/euthanasia.pdf

25. To document training, please complete a Training Documentation form and return it to the IACUC office or the LAF office. Thank you.