Euthanasia Policy

Purpose: The purpose of this policy is to outline acceptable methods of euthanasia for laboratory animals at the University of Oregon.

Scope: This policy applies to all research investigators and animal care staff at the University of Oregon whose IACUC approved Animal Use Protocols (AUP) require euthanasia of animal subjects.

 

Background/Definitions: 

Euthanasia is defined as a pain-free or stress-free death. Animals are normally euthanized at the end of a study for the purpose of sample collection or post-mortem examination.  Animals may be euthanized because they are experiencing pain or distress. The IACUC has approved certain methods for humanely killing animals that meet the definition of euthanasia.  The appropriateness of the method may vary from species to species. These guidelines are adapted from AVMA Guidelines for the Euthanasia of Animals: 2013 Edition. All methods of euthansia must be in accordance with this document or justified and approved in the AUP.

You may only use a euthanasia method that is approved in your IACUC approved AUP. A change in euthanasia method, including dose or route of administration, is a significant change in protocol and must be reviewed and approved by the IACUC before implementation.

Only trained individuals may perform euthanasia. Individuals who receive IACUC approval for euthanasia must have their technical competence certified by authorized AWS staff in advance of performing the procedure independently.

Policy: 

Below are descriptions of standard acceptable euthanasia methods:

RODENTS

Chemical Methods

A. Pentobarbital and Phenytoin (Euthasol)

  1. Chemical euthanasia with an overdose of intravenous pentobarbital is the preferred euthanasia method for mammals with practical vascular access (e.g., rabbits).
  2. Intraperitoneal pentobarbital is acceptable for smaller mammals when intravenous access is not practical.
  3. Pentobarbital-based euthanasia solution (Euthasol: 390mg Pentobarbital/50mg Phenytoin / ml) can be used for immediate euthanasia.
Species Dose Route Needle size
Mouse 200-500mg/kg BW IP 22-25g
Rat 200-500mg/kg BW IP 18-22g

B. Isoflurane Inhalation Overdose

  1. The preferred method of delivering isoflurane is via precision vaporizer.
  2. Under certain circumstances, the open drop method of delivering isoflurane may be appropriate for euthanasia (not anesthesia). If using an open-drop system (application of isoflurane to an absorbent material then placed into the bottom of the chamber), the animal must be physically separated from the absorbent material by a physical barrier (e.g., screen or platform). The procedure must take place in a biosafety acbinet or hood to avoid personnel exposure. The open drop method requires justification for approval by the IACUC.
  3. Euthanasia must be confirmed; a secondary method of euthanasia is recommended (see below).

C. Carbon Dioxide (CO2) Inhalation

  1. Compressed CO2 gas in cylinders is the only allowable source of carbon dioxide because the inflow to the chamber can be regulated precisely. CO2 delivery must be monitored to ensure CO2 does not displace air by more than 10-30 % of the chamber volume per minute. CO2 generated by other methods (e.g., dry ice) is unacceptable.
  2. To ensure compliance with the AVMA CO2 guidelines; our institution recommends the use of the Euthanex Smartbox Automated Euthanasia chambers. See the Carbon Dioxide (CO2) Euthanasia of Mice and Rats SOP.
  3. Euthanasia must be confirmed; a secondary method of euthanasia is recommended (see below). According to the AVMA Guidelines on CO2 euthanasia: "Death may be confirmed by physical examination, ensured by an adjunctive physical method, or obviated by calibration and validation of the euthanasia chamber and process.If an animal is not dead, CO2 narcosis must be followed with another method of euthanasia." (p. 49)

D. Avertin

  1. Avertin is approved in some AUPs for acute, nonsurvival procedures as an anesthetic agent. Once a surgical plane of anesthesia is acheived with Avertin, a secondary method of euthanasia is employed such as cardiac perfusion or a physical method listed below.

Physical Methods

A. General Consideration

Physical methods of euthanasia can be used as secondary methods of euthanasia or, following careful training and assessment, as a primary method. From the 2013 AVMA Guidelines:

"When properly used by skilled personnel, with well-maintained equipment, physical methods of euthanasia may result in less fear and anxiety and be more rapid, painless, humane, and practical than other forms of euthanasia." (pg. 34)

"Personnel using physical methods of euthanasia must be well trained and monitored for each type of physical method performed to ensure euthanasia is conducted appropriately. Inexperienced persons should be trained by experienced persons and should practice on euthanized animals or anesthetized animals to be euthanized until they are proficient in performing the method properly and humanely." (pg. 35)

B. Cervical Dislocation

  1. When properly performed by trained personnel, manual cervical dislocation is a humane technique for euthanasia of mice, other small rodents, and rats weighing less than 200 grams.
  2. As indicated above, when personnel are being trained in this technique, animals should be anesthetize until proficiency is demonstrated.

C. Decapitation

  1. Decapitation when performed properly is virtually instantaneous and is considered humane. This method must be justified by the experimental design and approved by the IACUC.
  2. Guillotines that are designed for decapitation of rodents are commercially available. It is critical to follow manufacturer guidelines to ensure equipment used is appropriate for the size of animals being euthanized. The equipment used to perform decapitation must be maintained in good working order and serviced on a regular basis to ensure sharpness of blades.
  3. Alternatively, sharp scissors may be used for smaller animals, such as neonates and mice, if described in an AUP and approved by the IACUC.

LIFE STAGE CONSIDERATIONS
Rodent Neonates and Fetuses

A. The AVMA Guidelines for the Euthanasia of Animals, 2013 Edition states that “Scientific data indicate that mammalian embryos and fetuses are in a state of unconsciousness throughout pregnancy and birth.” It also states that “The precocious young of guinea pigs remain insentient and unconscious until 75% to 80% of the way through pregnancy and remain unconscious until after birth due to chemical inhibitors" and “embryos and fetuses cannot consciously experience feelings such as breathlessness or pain. Therefore, they also cannot suffer while dying in utero after the death of the dam, whatever the cause." (Pg. 46-47)  Based on that guidance, we recommend the following:

B. Neonates (up to 10 days of age): Acceptable methods for euthanasia include: injection of chemical anesthetics (e.g., pentobarbital), decapitation or cervical dislocation. Additionally, these animals are sensitive to inhalant anesthetics; e.g., CO2 or isoflurane from a vaporizer (used with appropriate safety considerations) although prolonged exposure, up to 50 minutes, may be necessary. A secondary physical method of euthanasia is recommended to ensure death (e.g. cervical dislocation, decapitation, bilateral pneumothorax). From the AVMA Guidelines: “Fetuses that are believed to be unconscious and altricial neonates < 5 days of age may be quickly killed by rapidly freezing in liquid N2.” (Pg. 50) For neonates 5 days or greater, immersion in liquid nitrogen may be used only if preceded by anesthesia. Anesthesia may be induced by inhalant or injectable anesthetics; the Attending Veterinarian should be consulted for appropriate agents and dosages. Alternatively, when adequately justified, hypothermia may be used to induce anesthesia in pups six days of age or less (however 3-4 days of age is more typical).

C. Fetuses

  1. For mouse and rat fetuses up to 15 days of gestation, euthanasia of the mother or removal of the fetus should ensure rapid death of the fetus due to hypoxia, blood loss and non-viability, thus no additional method of euthanasia is required.
  2. For mouse and rat fetuses >15 days of gestation after general anesthesia or euthanasia of the mother, decapitation or cervical dislocation of the fetus must be performed.

 

FOR ALL EUTHANASIA METHODS, VERIFICATION OF COMPLETE EUTHANASIA IS MANDATORY.  CONFIRM THAT THE ANIMAL IS DEAD AS FOLLOWS:

Verification of death is especially important when CO2 or anesthetic gases are used and the animal is discarded intact, i.e., it is not used for tissue harvest or other invasive postmortem procedures

  1. Visual inspection to assure no signs of respiration.
  2. Ensure that the heart is not beating by feeling the chest between your thumb and forefinger.
  3. Ensure that there is no blink reflex by touching the eyeball.
  4. If there is a heartbeat or blink reflex, repeat the euthanasia process as described above.
  5. Alternatively, use a secondary physical method (following a nonresponsive toe-pinch to ensure a deep level of anesthesia):
    1. Cervical dislocation
    2. Decapitation
    3. Pnemothorax
    4. Exsanguination
    5. Perfusion with fixative

FISH

Chemical Methods

A. MS-222

  1. The dosage required for euthanasia is higher than the anesthetic dose, but varies greatly with species, size, and water temperature. In addition, a longer time of exposure to the chemical agent (relative to anesthesia) is required to ensure that death occurs. Fish should be removed only after ten minutes have passed since their last observed opercular movements (respiration) have occurred.
  2. Prepare MS-222 solution and follow steps according to the Zebrafish Facility Euthanasia of Fish via Tricaine methanesulfonate (MS-222) SOP.

B. Eugenol (Isoeugenol or Clove Oil)

  1. Prepare eugenol solution and follow steps according to the Zebrafish Facility Euthanasia of Fish via Eugenol (Isoeugenol or Clove Oil) SOP.

C. Anesthesia induced using MS-222 followed by rapid immersion in liquid nitrogen

  1. Acknowledging that rapid freezing of tissue in liquid nitrogen will inhibit RNA degradation and noting that in the AVMA Guidelines on Euthanasia, Amphibians, Fish, and Reptiles section, two-stage euthanasia procedures sub-section states "Pithing, freezing, and boiling are acceptable as the second step (adjunctive methods) of a 2-step euthanasia procedure," (Pg. 74) deeply anesthetizing fish using MS-222 and then immersing them into liquid nitrogen is an acceptable method of euthanasia.
  2. See Zebrafish Facility Euthanasia of Fish via Anesthesia Followed by Liquid Nitrogen SOP.
     

Physical Methods

A. Rapid Chilling (Hypothermal Shock)

  1. Because tropical fish species, (i.e. zebrafish, medaka, and platyfish), have minimal to no physiologic adaptation mechanism for adjusting to cold (4 °C) water, cooling to 4 °C is an acceptable method of euthanasia since the rapid decrease in temperature from 26 °C (or higher) to 4 °C induces rapid loss of consciousness and is lethal to these species. Fish euthanized by this method should not come in direct contact with the ice, because this may cause thermal burns and induce pain. 
  2. See Euthanasia of Fish Using Hypothermal Shock for procedures specifics.

LIFE STAGE CONSIDERATIONS

Zebrafish Embryos and Larvae
For zebrafish life stage definitions, see ZFIN Anatomy Terms.

A. The AVMA Guidelines for the Euthanasia of Animals, 2013 Edition states, " Use of rapid chilling and use of buffered MS 222 alone have been shown to be unreliable euthanasia methods for zebrafish embryos < 3 dpf. To ensure embryonic lethality these methods should be followed with an adjunctive method such as use of diluate sodium or calcium hypochlorite solution at 500 mg/L."

B. Embryos and larvae (up through 7 days post fertilization [dpf] reared at 28 °C):  Acceptable methods for euthanasia include: rapid chilling (hypothermal shock) followed by exposure to sodium hypochlorite (bleach); cranial concussion; immersion in fixative; immersion in lysis buffer; and freezing in liquid N2.

C. Larvae (8 dpf through 29 dpf reared at 28 °C):  Acceptable methods for euthanasia include: rapid chilling (hypothermal shock) for at least 20 minutes following loss of operculum movement; overdose via immersion in MS-222; overdose via immersion in eugenol (isoeugenol or clove oil); anesthesia followed by freezing in liquid N2.

FROGS / AMPHIBIANS

Chemical Methods

A. MS-222

Frogs are immersed in a 1% (10mg/ml) tricane (MS222) waterbath for up to 60 minutes will result in deep anesthesia if not death. Signs of an anesthetized frog are: body limp, eyes half to three quarter closed, not blinking, and no resistance when gently touched on the body or head. Once the animal is fully anesthetized, a secondary method can be applied to ensure death. Once the animal is deeply anesthetized as described above, decapitation with a guillotine followed by pithing of the brain matter is necessary.

B. EUTHASOL

Euthasol at a dose of  60-100 mglkg will be injected into the heart or caudal vein using a 22 guage needle on a 6cc syringe.

OWLS

Chemical Methods

A. Euthasol (390mg Pentobarbital/50mg Phenytoin / ml)

  1. Euthasol can be administered intravenously or intramuscularly. 1ml (500-800mg/kg) adminstered either route is typically adequate.
  2. Confirm death as above.

B. Isoflurane Inhalation Overdose

  1. Isoflurane delivered from a precision vaporizer via a face mask.
  2. Can use as primary method or follow with Euthasol injection or secondary method such as perfusion.
Approved by date
IACUC Committee December 10, 2015, 2019-Apr-04
 Appendices: 

References:

  • American Veterinary Medical Association (AVMA) (2013). AVMA Guidelines for the Euthanasia of Animals: 2013 Edition. (https://www.avma.org/kb/policies/documents/euthanasia.pdf).
  • Davis, Daniel J et al. 2015. Effects of Clove Oil as a Euthanasia Agent on the Blood Collection Efficiency and Serum Cortisol Levels in Danio rerio. JAALAS 54:564-567.
  • Fox, J. (2015). Laboratory animal medicine, 3rd Edition. San Diego: Academic Press limited.

 

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