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

2. Euthanasia literally means a "good death". A more appropriate simple definition is a "gentle death". The Guide for the Care and Use of Laboratory Animals defines euthanasia simply as "the act of killing animals by methods that induce rapid unconsciousness and death without pain or distress."Guide for the Care and Use of Laboratory Animals Specifically, this means that animals should be killed humanely with minimal pain and distress. Everyone has the responsibility to ensure that if an animals life is to be taken, it is done with the highest degree of respect, and with an emphasis on making the death as painless and distress-free as possible. Euthanasia techniques should result in a rapid loss of consciousness followed by cardiac or respiratory arrest and finally, the loss of brain function. The technique selected should minimize distress and anxiety experienced by the animal prior to its loss of consciousness. 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.Laboratory Animal Facilities (LAF) 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 4-1385 or visiting the offices on the 2 nd floor of the Guyton Building (G201).

4. Do not perform euthanasia, or any other procedure on an animal until a person experienced with the procedure has trained you. Because improper technique can cause pain and suffering to animals during euthanasia, you must be trained to properly and humanely perform euthanasia. The LAF veterinary staff offers training for all personnel working with animals. To arrange a training session, contact the LAF office at 4-1385. you must be trainedyou must be trained

5. PHS PolicyPHS 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.Guide The Report of the AVMA Panel on Euthanasia contains many guidelines used by the IACUC to evaluate methods of euthanasia.

6. The American Veterinary Medical Association (AVMA) classifies methods of euthanasia as follows:AVMA Acceptable Conditionally acceptable Unacceptable Both the United States Department of Agriculture (USDA) and the Office of Laboratory Animal Welfare (OLAW) endorse these AVMA standards. What do these standards mean?United States Department of Agriculture (USDA)Office of Laboratory Animal Welfare (OLAW) endorse

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: CO 2 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 method but 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:adjunctive methodsshould not be used alone MethodDisadvantage Exsanguination Loss of blood can cause anxiety associated with low blood pressure. Decompression Decompression has numerous disadvantages. Rapid freezing This method is not humane. Air embolism Injection of air intravenously can be accompanied by convulsions and other neurological signs Drowning This method is not humane. Strychnine It causes violent convulsions and muscle contractions. Nicotine, magnesium sulfate, potassium chloride These agents cause cardiac and/or respiratory arrest before unconsciousness. Chloroform Chloroform has known toxicity for animals and humans. Cyanide This agent poses extreme danger to humans and other animals. Stunning by a blow to the head This method may not cause death and is esthetically objectionable.

14. Euthanasia by CO 2

15. Advantages CO 2 provides a rapid depression and anesthesia (narcosis). CO 2 is non-flammable and non- explosive. CO 2 does not introduce chemical residues into tissues. CO 2 does not result in distortion of cellular architecture.

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

18. Carbon Dioxide Euthanasia Procedures 1. 1. The animal(s) should remain in their home cage to reduce stress. 2. 2. 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] 3. 3. Attach the appropriate size stainless steel top to the clear CO 2 gas line. Place stainless steel top on top of the cage. 4. 4. Turn silver knob at the top of the CO 2 tank counterclockwise (ON); the pressure gauge farthest from the tank should now indicate the tank pressure. 5. 5. Turn the white knob on the regulator clockwise (ON) until the flow meter (pressure gauge closest to the tank) registers about 5 – 10 psi. 6. 6. Observe the animal(s) and maintain gas flow for at least 1 minute after breathing has stopped. 7. 7. Turn silver knob on top of the CO 2 tank clockwise (OFF). 8. 8. Turn black knob on the regulator counterclockwise (OFF); the gauge closest to the tank should now read 0 psi. 9. 9. Remove cage from the box and complete secondary (adjunct) method of euthanasia. These procedures are posted at each CO 2 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 PHS Policy on Humane Care and Use of Laboratory Animals Clarification Regarding Use of Carbon Dioxide for Euthanasia of Small Laboratory AnimalsPHS Policy on Humane Care and Use of Laboratory Animals Clarification Regarding Use of Carbon Dioxide for Euthanasia of Small Laboratory Animals

20. CO 2 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 CO 2 concentrations and exposure times or by other means. OLAW notes that thoracotomy after apparent death from CO 2 is one way to ensure the irreversibility of the procedure. 3.Institutions must ensure that all individuals responsible for administering CO 2 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 CO 2 in cylinders is the only AVMA Panel- recommended source of CO 2 for euthanasia purposes. 6. Unintended recovery of animals after apparent death from CO 2 (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. 7. OLAW advises IACUCs to review their policies and practices regarding CO 2 euthanasia of small laboratory animals and take appropriate action as needed to conform to this guidance. 8.CO 2 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 CO 2. Some methods to confirm death: Cervical dislocation Bilateral thoracotomy Exsanguination Decapitation Potassium chloride injection

23. The key objectives in using CO 2 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 CO 2 -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 formTraining Documentation form and return it to the IACUC office or the LAF office. Thank you.