1. Euthanasia Training

2. Introduction
   • Most animals used today involve euthanasia of the animals at the end of their use in research
• In most cases, the animal is just euthanatized and tissue collected for further tissue study
• This training module is designed to meet the requirements that all personnel euthanatizing animals be properly trained
• While most personnel work with only 1 or 2 species, it is stated that everyone will have knowledge of
• The meaning of euthanasia
• Ethics involved in euthanatizing an animal
• Esthetics of the procedure
• Etc.
• As well as be specifically trained to perform proper euthanasia on a species that they will use
• This module will cover
  • General information about euthanasia
  • Different methods that are acceptable and unacceptable
  • And species specific methods
• This is now a required part of your General Training
• Review the General Training
• Then review the Euthanasia Training
• Then review for the species you will be euthanatizing
• Only required if your role on the project states euthanasia
• Then meet with the veterinarian for more specific training in relation to the species you will be using
3. The AVMA Report
• The report is intended for use by members of the veterinary profession
  • It is to serve as a guideline for veterinarians
1. Many situations and many species require a veterinarian's professional judgment to select the most appropriate method using the "guidelines" in this report
• Therefore, a consultation is required with a veterinarian
• All personnel should be trained
• Clearly stated in the report that all personnel performing euthanasia should be trained in general and in specific procedures for the species
1. In most cases, this will involve actual hands on using an animal
4. Definition
   • Euthanasia
     • EU meaning "good"
     • Thanatos meaning "death"
   • Note: For you purist, "euthanasia" is a word in the Oxford English Dictionary.  Words like euthanatize, euthanitize, euthanitization, euthanatization are all newer words that are now being seen in publication but a few years ago, did not exist in the English Dictionary.  So, any form of euthanasia could be considered to mean a "good death", quick and painless.
5. Euthanasia
   • Should be done with the highest degree of respect and with an emphasis on making the death as painless and distress free as possible
• This is primarily accomplished by 
  • Rapid loss of consciousness 
  • Followed by cardiac or respiratory arrest 
  • And the ultimate loss of brain function
6. Anatomical & Physiological Aspects
• Pain is that sensation (perception) that results from nerve impulses reaching the cerebral cortex via ascending neural pathways
• Noxious stimuli activate nociceptive receptors which sends signals to the spinal cord or brain stem
• Two general sets of neural networks
1. Nociceptive reflexes
• Withdrawal and flexion reflexes
• Occurs at the spinal level
2. Ascending pathways to the reticular formation, hypothalamus, thalamus, and cerebral cortex
7. Pain
• Term implies perception
  • Stimuli, receptors, reflexes or pathways do not imply perception
• Term "Pain", therefore, should only be used when an action is perceived
• To perceive pain, the cerebral cortex and subcortical structures must be functional
• Pain perception
• If the spinal cord is cut, squeezing the toe may cause a movement but the pain is not perceived
• If the toe is squeezed and the spinal cord is intact, then the "pain" could be perceived.
• If part of the brain is damaged, pain may be perceived but have no consequence (not matter or have meaning)
  1. Note: This example is taken from a source other than the "Report"
8. Distress
   • Results when an animal's response to stimuli interferes with its well-being and comfort
9. Proper Handling
   • Is vital to minimize pain and distress in animals
   • Ensures the safety of the person performing euthanasia
   • Protects other people and animals
10. Criteria Used for Acceptable, Conditionally Acceptable and Unacceptable Methods
    • Acceptable
      • Methods that consistently produce a humane death when used as the sole means of euthanasia
    • Conditionally Acceptable
    • Methods that, by 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
    • Unacceptable
    • Methods that are deemed inhumane under any conditions or that the panel found posed a substantial risk to the human applying the technique
11. General Considerations
    • Ability to induce loss of consciousness and death without causing pain, distress, anxiety, or apprehension
    • Time required to induce loss of consciousness
    • Reliability
    • Safety of personnel
    • Irreversibility
    • Compatibility with requirement and purpose
    • Emotional effect on observers or operators
    • Compatibility with subsequent evaluations, examination, or use of tissue
    • Drug availability and human abuse potential
    • Compatibility with species, age and health status
    • Ability to maintain equipment in proper working order
    • Safety for predators/scavengers should the carcass be consumed
12. Confirmation of Death
    • Imperative that death be verified after euthanasia and before disposal of the animal
    • Death must be confirmed by examining the animal for cessation of vital signs
    • Consideration must be given to the animal species and method of euthanasia when determining the criteria for confirming death
    • Use a secondary method to assure death
    1. i.e. Remove heart, brain, all the blood, etc.
13. Knowledge of Euthanasia
    • A person should be knowledgeable about what to expect
    • Misinterpretation of movement
    1. Lack of movement could be misinterpreted as being a loss of consciousness
    2. Movement could be misinterpreted as having consciousness
14. Mechanisms of Action of Euthanatizing Agents
    • Hypoxia, direct or indirect
    • Direct depression of neurons necessary for life function
    • Physical disruption of brain activity and destruction of neurons necessary for life

Agents and Methods

14. Inhalants
    • Induce hypoxia
    • Younger animals tolerate hypoxia better than older animals
    1. Not to be used alone for younger animals (less than 16 weeks)
    • Rapid gas flows can produce a noise which could frighten the animal
    • Reptiles, amphibians, and diving birds and mammals have a great capacity for holding their breath and for anaerobic metabolism
    • Halothane is the preferred agent
      • Isoflurane is acceptable
    • Nitrous oxide can not be used as a sole agent since it will not normally lead to anesthesia by itself
    • Advantages for an inhalant
      • Better for smaller animals where veins are hard to hit
    • Halothane and isoflurane are not flammable or explosive
    • Disadvantages for an inhalant
    • Animals may struggle
    • Nitrous oxide can not be used alone and is subject to abuse
    • Biohazard to personnel
15. Carbon Dioxide
    • Heavier than air and nearly odorless
    • Gradual filling of tank is preferable
    • A lower concentration (30%) is less distressful for animals
    • In some species, higher concentrations failed to cause death
    • Can only use a gas cylinder, not dry ice or other means
    • Gas flow should be maintained for at least one minute after signs of clinical death
    • A second method of death should always be used to ensure death such as open chest, heart removed, exsanguination, etc.
    • Advantages
    • Rapid depressant, analgesic and anesthetic effects
    • Inexpensive, nonflammable, nonexplosive and not a biohazard
    • No accumulation of tissue residues
    • Do not distort murine cholinergic markers and corticosterone concentrations
    1. Can cause acidosis
    • Disadvantages
      • Incomplete filling of a chamber because heavier than air
      • Some species such as fish and burrowing and diving mammals, may have extraordinary tolerance for carbon dioxide
      • Reptiles and amphibians may breathe too slowly
16. Non-Inhalant Pharmaceutical Agents
    • Intracardiac injection is acceptable only when performed on heavily sedated, anesthetized, or comatose animals
17. Pentobarbital and Pentobarbital Combinations
    • Produces respiratory arrest followed by cardiac arrest
    • Intravenous injection is the acceptable method
    • Intraperitoneal injection may be used in situations when an intravenous injection would be distressful or even dangerous
    • Advantages
      • Rapid, smooth action depending on concentration and route
    • Disadvantages
      • Intravenous is best route but not possible in some animals
      • Animal has to be restrained
      • Controlled substance
      • Leave residue in animal tissues
18. Chloral Hydrate
    • Conditionally acceptable
      • For use in large animals
      • Only intravenously
      • Only after sedation to reduce side effects
      • Depresses the cerebrum slowly
    • Advantages
      • None
    • Disadvantages
      • Slow
      • Requires long restraint
      • May see gasping, muscle spasms, and vocalization
      • Conditionally acceptable
19. Tricaine Methane Sulfonate
    • (MS 222, TMS)
    • Used for amphibians and fish
    • Store stock solution made from a concentrate in a dark brown bottle and refrigerate
      • Label with date mixed
        1. Good for one (1) month
    • Leave animal in solution for 10 minutes after cessation of opercular movement
    • Drug is retained in the tissues for up to 21 day, so is not appropriate for euthanasia of animals intended for food 
20. Potassium Chloride
    • Always has to be used in a fully anesthetized animal (Adjunctive Method)
    • Preferred injectable technique for euthanasia of livestock or wildlife species
      • Reduces risk for predators and scavengers
    • Advantages
      • Not a controlled substance
      • Less toxic
    • Disadvantages
      • Animal has to be anesthetized
      • Rippling of muscle tissue and clonic spasms
21. Unacceptable Injectable Agents
    • When used alone these are unacceptable
      • Strychnine
      • Nicotine
      • Caffeine
      • Magnesium sulfate
      • Potassium chloride
      • Cleaning agents
      • Solvents
      • Disinfectants
      • Neuro-blocking agents
22. Physical Methods
    • Require specific training by experienced person
      • Require practice on carcasses or anesthetized animals until proficient
    • Most of the time it is considered conditionally acceptable for euthanasia
    • Penetrating Captive Bolt
      • Used on ruminants, horses, swine, laboratory rabbits and dogs
      • Advantage
        • Effective method when use of drugs is inappropriate
      • Disadvantage
        • Aesthetically displeasing
        • Death may not occur if equipment is not maintained and used properly
    • Blow to the Head
      • Used in neonatal animals with thin craniums such as young pigs
      • Not used in calves
      • Personnel must be
        1. Properly trained
        2. Monitored for proficiency
        3. Aware of its aesthetic implications
    • Gunshot
      • Must be aimed so that the projectile enters the brain, causing instant loss of consciousness
      • Gunshot to the heart or neck does not immediately render animals unconscious and thus is not considered acceptable
      • Conditionally acceptable if other methods can not be used
      • Advantages
        1. If done properly, rapid death occurs
        2. Lack of having to handle animal makes it desirable for wild or free-ranging species
      • Disadvantage
        1. Dangerous to personnel
        2. Aesthetically unpleasant
        3. Have to be a good shot
        4. Brain is destroyed and can not be used in most instances
    • Cervical Dislocation
      • Used on
        1. Poultry and other small birds
        2. Mice
        3. Immature rats (<200g)
        4. Rabbits (<1 kg)
      • Electrical activity in the brain persists for 13 seconds and unlike decapitation, rapid exsanguination from hemorrhage does not contribute to loss of consciousness
      • Either done by
        1. Personnel who have demonstrated high degree of technical proficiency
        2. Or on anesthetized animals
      • Used only when scientifically justified by the user and approved by the IACUC
      • Advantage
        1. May induce rapid loss of consciousness
        2. Does not chemically contaminate tissue
        3. Rapidly accomplished
      • Disadvantage
        1. Aesthetically displeasing
        2. Requires mastering of technical skills
        3. Use is limited to
           • Poultry and other small birds
           • Mice
           • Immature rats
           • Immature rabbits
    • Decapitation
      • Used on rodents and small rabbits
      • Electrical activity in the brain persists for 13 to 14 seconds
        1. Now thought that loss of consciousness develops rapidly and that pain is not perceived
      • Is conditionally acceptable
      • Used only when
        1. Required by experimental design
        2. Approved by the IACUC
      • Requires proper training
      • Advantage
        1. Appears to induce rapid loss of consciousness
        2. Does not chemically contaminate tissue
        3. Rapidly accomplished
      • Disadvantage
        1. Handling and restraint required
        2. Still debate over electrical activity
        3. Require caution to prevent personal injury
        4. May be aesthetically displeasing
    • Electrocution
      • Used in such species as
        1. Dog, Cattle, Sheep, Swine, Foxes, Mink
      • Prior to electrocution, the animal is required to be unconscious by some other means (including electro-stunning)
      • Techniques which apply current from head to tail, head to foot, etc. are  unacceptable
      • Is conditionally acceptable
      • Advantages
        1. Is humane if animal unconscious
        2. Does not chemically contaminate the tissue
        3. Economical
      • Disadvantages
        1. Requires special training and equipment
        2. Hazardous to personnel
        3. Not useful in dangerous, intractable animals
        4. Aesthetically objectionable
        5. May not result in death in small animals
    • Microwave Irradiation
      • Acceptable method
      • Can only use instruments specifically designed for the purpose
        1. Not microwaves for home use
      • Advantages
        1. Loss of consciousness is achieved in less than 100 ms
        2. Most effective method to fix brain tissue in vivo for subsequent assay of enzymatically labile chemicals
      • Disadvantages
        1. Instruments are expensive
        2. Only equipment designed for mice and rat available
    • Kill Traps
      • Do not always meet criteria for euthanasia
      • Used only when no other means are available
23. Adjunctive Methods
    • Must be used in combination with some other method
      • Not acceptable by themselves
• Stunning and Pithing
• Induce loss of consciousness
• Exsanguination
  • Prior to use, animal must be
    1. Sedated
    2. Stunned
    3. Anesthetized
• Stunning
  • Stunned by
    1. Blow to the head
    2. Use of a non-penetrating captive volt
    3. Use of electric current
    4. Must be followed immediately by some other method that ensures death
• Blow to the head
  • Primarily in small laboratory animals with thin craniums
  • Should be a single blow with sufficient force to produce immediate depression of the central nervous system
  • Must be followed immediately by some other method that ensures death
• Non-penetrating captive bolt
  • May be used with
    1. Horses, Ruminants, Swine
  • Signs
    • Immediate collapse
    • Tetanic spasms
    • Increasing frequency
    • Must be followed immediately by some other method that ensures death
• Electric stunning
  • Used in
  1. Dogs, Cattle, Sheep, Goats, Hogs, Fish, Chickens
  • Requires an alternating electrical current applied directly to opposite sides of the head
  • Should be followed promptly by other appropriate methods to ensure death
• Pithing
  • Used in animals as adjunctive procedure to ensure death in an animal that has been rendered unconscious by other means
  • Some species such as frogs, with anatomic features that facilitate easy access to the central nervous system, pithing may be used as a sole means of euthanasia
    1. Double pithing required
    2. Anesthetic overdose is a more suitable method
24. Equine Euthanasia
    • Because of their size and potential for causing harm in an excitable animal, the "Report" needs to be reviewed for specific details for several different circumstances
25. Animals Intended for Human or Animal Food
    • The only methods that can be used are
      • Carbon dioxide
      • Potassium chloride
      • Or a physical method
    • Other methods or chemicals require proper disposal to ensure no human or other animal can consume them
26. Non-Conventional Species
    • Zoo, wild, aquatic, ectodermic animals
    • Many differences exist including anatomically, size, behavior, captive or wild, etc.
    • Zoo
      • N/A for this training module
    • Wildlife
      • Size and temperament have to be considered in selecting a method
      • Safety of the animal, other animals, and humans is required
      • Wild animals suffer distress the longer captured, handled, or restrained so the best method should be one that is quick
      • Ideally, a two stage euthanasia is best but in most instances, any method to render the animal accessible for IV inject is preferred
      • Training and monitoring should be taken into consideration when selecting a method
    • Birds
      • Nets and live traps may be required for free ranging birds followed by euthanasia
      • Gun shot may be used
        1. Preferably with a shotgun
           • With a load appropriate for the species
      • Large birds should be anesthetized prior to euthanasia
    • Amphibians, fish and reptiles
      • Must take into consideration ectothermic animals differences in their
        1. Metabolism
        2. Respiration
        3. Tolerance to cerebral hypoxia
           • More difficult to tell when an animal is dead
      • Injectables
        1. Sodium pentobarbital (60-100 mg/kg of BW) IV, intra abdominally or intra pleuroperitoneally
           • Requires up to 30 minutes
           • No other barbiturate should be used
        2. Clove oil
           • Must not be used
      • External or topical
        1. MS-222
           • In water
           • Lymph sacs
           • Intra pleuroperitoneally
           • Chemically contaminates the tissues so can not be used for food
        2. Benzocaine hydrochloride
           • Used for
             • Fish
             • Amphibians
             • Requires animal to remain in solution for at least 10 minutes after all movement stops
        3. 2-phenoxyethanol
           • Requires animal to remain in solution for at least 10 minutes after all movement stops
      • Inhalants
        1. Many reptiles and amphibians are capable of holding their breath for long periods of time or converting to anaerobic metabolism and not breathing for up to 27 hours
        2. Lizards, snakes, and fish do not hold their breath to the same extent and can be euthanatized with inhalants
        3. Carbon dioxide
           • Amphibians, reptiles and fish may be euthanatized with CO2
           • May take some time
           • Has to be from gas cylinder, not dry ice or other means
      • Physical methods
        1. Captive bolt and firearm used to shot the brain in
           • Amphibians
           • Reptiles
        2. Decapitation
           • Used on
             • Amphibians
             • Fish
             • Reptiles
             • Tolerant to hypoxic and hypotensive conditions so decapitation must be followed by pithing
      • Two stage procedure
        1. Stunning
           1. Most amphibians, fishes, and reptiles
           2. Followed by decapitation, pithing or some other physical method
        2. Pithing
           • Severing the spinal cord behind the head
           • Both brain and spinal cord needs to be pithed (double pithed)
           • Requires special training
        3. Cooling
           • Not in reptiles
           • Fish and amphibians
             • Cooling to 4 C will decrease metabolism and facilitate handling but does not relieve pain perception
             • Quick freezing of deeply anesthetized animals is acceptable
27. Prenatal and Neonatal
    • Do as soon as possible
    • Neonatal animals are relatively resistant to hypoxia

28. Summary
    • This report is based on scientific literature available at the time of composition
    • No procedure could be recommended if there was not scientific evidence that a true euthanasia would occur in a species
    • In all cases, the report acts as a guide for veterinarians in determining what is the best method of euthanasia for a particular species in a particular situation, however, the intent is to follow the recommendation

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