Euthanasia of Research and Teaching Animals

The methods listed below primarily apply to mice and rats, although most are applicable to many other rodent species.  Please consult with Veterinary Services for questions regarding species differences.

General Considerations

• Animals must be transported and euthanized in a method that minimizes stress.
• Inhaled agents should be administered in the home cage when possible.
• If animals must be combined into a single cage, they should be combined only with compatible cohorts of the same species.
• Chambers must not be overloaded: each animal should be able to place all four paws on the floor of the chamber and have enough room to turn around and move freely.
• Chambers must be kept clean, including cleaning between animals.
• Live animals must never be placed in the chamber with dead animals.

Confirming Death and Secondary Methods of Euthanasia

• For all types of euthanasia, it is the responsibility of the individual performing euthanasia to confirm death has occurred by utilizing reliable indicators such as the lack of heartbeat and respirations. Lack of corneal reflex may be used as an indicator of death but must be used in combination with another method of confirmation.
• For euthanasia by CO₂, a secondary method of euthanasia is required and must be listed in the IACUC-approved protocol. Appropriate secondary methods following CO₂ euthanasia include:
  • placing the animal in a tightly knotted bag filled with CO₂
  • a physical method such as cervical dislocation (only for animals <200g), exsanguination, thoracotomy, decapitation
• For euthanasia by overdose of inhalant anesthesia other than CO₂, a secondary method of euthanasia is required and must be listed in the IACUC-approved protocol. Appropriate secondary methods following euthanasia by inhalants other than CO₂ include:
  • a physical method such as cervical dislocation (only for animals <200g), exsanguination, thoracotomy, decapitation
  • overdose of injectable anesthetic (e.g., sodium pentobarbital, etc.)

Euthanasia by Overdose of Injectable Anesthetics

• Pentobarbital or Pentobarbital/Phenytoin combinations (e.g. Euthasol®) in a dose at least three times the accepted anesthetic dose may be used.
  • For mice, the recommended dose is >270 mg/kg given IP
  • For rats, the recommended dose is >150 mg/kg given IP

Note: dosages above are determined by pentobarbital component of the solution

• Ketamine/xylazine combinations can be utilized for euthanasia of mice and rats at a dose at least 4 times the accepted anesthetic dose.

Methods above are preferred, but with scientific justification Avertin (tribromoethanol) may be used in mice only at a dose greater than 500 mg/kg IP and must be followed by a secondary physical method (cervical dislocation, exsanguination, thoracotomy, or decapitation) to ensure death.

Euthanasia by Overdose of Inhalation Agents

• Carbon Dioxide (CO₂)
  • CO₂ must be administered from compressed commercial cylinders utilizing a flow meter to deliver 30-70% of the chamber volume/minute.
  • Pre-filling of the chamber with CO₂ is not acceptable and CO₂ within the chamber must be removed between groups.
  • Animals must never be left unattended during CO₂ exposure.
  • Total gas exposure time must be as follows;
    • at least 5 minutes for mice
    • at least 10 minutes for rats
  • A timer must be used to ensure adequate length of exposure.
  • Gas flow must be maintained for at least 1 minute after apparent clinical death.
  • Death must be confirmed after removing animals from the chamber and secondary methods performed as outlined above in the section on Confirming Death and Secondary Methods.
  • Neonates: The time period for euthanasia utilizing CO₂ is substantially prolonged in neonatal rodents that are 10 days of age or younger. Due to their inherent resistance to hypoxia, CO₂ narcosis in neonates must be followed by decapitation.
• Inhalant Agents other than CO₂
  • The order of preference of commercially available inhalant anesthetics from most preferable to least preferable is isoflurane, sevoflurane, enflurane, desflurane.
  • All machinery used must be in good working order with no leaks and noise and cold drafts minimized.
  • In some cases, euthanasia by anesthetic exposure via open drop method may be approved when justified. If utilizing this method, the animal must never come into direct contact with anesthetic agent (e.g. apply liquid anesthetic to gauze contained within a tissue cassette).

Euthanasia by Physical Methods

Physical methods of euthanasia usually require certification of competency (see below).  Following training, competency must be demonstrated utilizing the same species and conditions in which the method will be performed. Granting of certification is at the discretion of the Attending Veterinarian or designee. In some cases (e.g. remote field locations), proficiency may be assessed by other means, such as interviews or acknowledgement of training completed at other institutions. These exceptions must be reviewed and approved by the Attending Veterinarian, or designee.

• Cervical dislocation:
  • Cervical dislocation is an acceptable method of euthanasia for mice and for rats that are <200g.
  • Certification is required for utilization of cervical dislocation as a method of euthanasia in awake or anesthetized rodents.
• Decapitation
  • Decapitation of neonatal mice or rats less than 14 days of age does not require certification.
  • Decapitation of non-anesthetized older neonates (≥ 14 days of age), juvenile, and adult rodents requires certification of the individual performing the procedure and scientific justification for its use.
  • Decapitation of anesthetized rodents does not require certification.
  • The equipment used to perform decapitation should be dedicated for this purpose and must be maintained in good working order (e.g. to ensure sharpness). Records demonstrating maintenance/service of the equipment on a regular basis must be maintained.
  • Additional requirements for use and maintenance of guillotines for decapitation:
    • Guillotine must be appropriate for the size of the animal.
    • Before use, it must be checked for rust, lack of visible nicks or damage to cutting edge, and cleanliness. Action must be smooth with no perceptible binding or resistance, there must be no (or minimal) lateral gap between upper and lower blades, and the handle/mechanism must not be loose or wobble.
    • Guillotine must be tested for sharpness on suitable materials before use on live animals. Polyethylene tubing 5/16^th inch OD x 3/16^th inch ID is a suitable test material; test lengths are available from the Office of Animal Welfare (OAW). During semi-annual IACUC site visits, lab members must be prepared to demonstrate cutting the test material with the guillotine.
    • If equipment is found to NOT be in good working condition, alternative IACUC-approved euthanasia methods or guillotines must be used. Do NOT use equipment that is not in good working condition and not sharp.
    • Guillotines must be sharpened according to their usage in order to maintain proper function. When used for rats, generally, sharpening is required after every 15-20 animals.
    • A log must be maintained and stored in close proximity to the device which identifies the date of last servicing.

The methods listed below primarily apply to the fish most commonly used in research such as zebrafish, medaka, fathead minnow, goldfish, swordtail, and platyfish as well as larger fish species that may include salmon, trout, tilapia, catfish, hybrid-striped bass, bass, bluegill, sturgeon and others. When methods differentiate between small and large fish, large fish are considered any fish >6 cm in length.

General Considerations

• Chemical agents such as buffered tricaine methanesulfonate (MS-222) or buffered benzocaine hydrochloride are generally the method of choice for the euthanasia all fish species.
• Dosages required for euthanasia are higher than the anesthetic dose, but vary greatly with species, life stage, size, and water temperature.  Prolonged exposure times are required to ensure that death occurs.

Confirming Death and Secondary Methods

• For all types of euthanasia, it is the responsibility of the individual performing euthanasia to confirm death has occurred. Given the diversity in fish species, reliable indicators of death may vary. For most fish species, death can be confirmed by lack of opercular movement for 30 minutes.
• The heart may continue to beat beyond clinical death and is not a reliable indicator of death for many fish.
• Due to the difficulty in confirming death in some fish, the use of a secondary method of euthanasia (e.g. severing a large vessel or freezing) after loss of opercular movement should be performed unless otherwise justified.
• Proposed euthanasia methods not listed below will be reviewed on a case-by-case basis, based on the AVMA Guidelines and the needs of the proposed work.

Euthanasia by Immersion

For all methods utilizing an immersion bath, fish must be placed in an appropriate volume of water to provide free movement. Early life stages (larvae and fry) typically require higher concentrations and longer contact times compared to adults.

Note: Zebrafish are considered live vertebrate animals at 3 days post-fertilization (dpf), and are considered adults at 3 months of age. Embryos <3 dpf are not covered under this policy, and methods described in this policy may not be appropriate for embryos. . Investigators are encouraged to consult with veterinary services for additional information on embryo management.

• Immersion in Tricaine methanesulfonate (MS-222) at 250-500mg/L or in Benzocaine Hydrochloride at >250 mg/L.
  • Solutions must be buffered to the pH of the system or source water with sodium bicarbonate (NaHCO3).
  • Fish should be removed from the euthanasia solution after at least ten minutes (adult fish) or 20 minutes (larval or fry fish) have passed since their last observed opercular movements (respiration).
  • To ensure death of fish either a large vessel should be severed (gill arch or tail vein) or the carcass should be frozen.
  • If a secondary method is not being utilized, fish must remain in solution for a minimum of 30 minutes following cessation of opercular movements.
  • Note: Fish euthanized with MS-222 or with benzocaine hydrochloride cannot be used for human or animal consumption. The use of MS-222 poses health risks to users. Prior to use, individuals should familiarize themselves with these risks as outlined on the UW Environmental Health & Safety SOP (ms222benzocainesop.pdf (uw.edu).
• Immersion in Eugenol
  • Concentrations required for euthanasia will vary depending on species and size of the finfish but should be at least 10 times the upper range needed for anesthesia.  Researchers should consult with Veterinary Services to determine the appropriate euthanasia dosage for a particular finfish.
  • Standardized and known concentrations of the essential oil must be used for accurate dosing.  Refer to Appendix 1 for additional information regarding preparation of Eugenol solution.
  • Fish should be removed from the euthanasia solution after at least ten minutes (adult fish) or 20 minutes (larval or fry fish) have passed since their last observed opercular movements (respiration).
  • To ensure death of fish either a large vessel should be severed (gill arch or tail vein) or the carcass should be frozen.
  • If a secondary method is not being utilized, fish must remain in solution for a minimum of 30 minutes following cessation of opercular movements.
  • Note: Fish euthanized with eugenol cannot be used for human or animal consumption, and must be disposed of according to FDA guidelines.
• Immersion in Water Saturated with Carbon Dioxide (CO2)
  • Fish are placed in source or system water, which is then saturated with carbon dioxide delivered by the use of an air stone submerged into the water with a moderate flow rate.
  • Carbon dioxide must be from a compressed gas cylinder.
  • Fish should remain in solution for an appropriate amount of time after the last observed opercular movements (respiration). The length of time varies with species and procedures but should be specified in the approved protocol.
  • Certification is required for utilization of CO2 immersion as a method of euthanasia.
  • Due to the technical expertise required for this procedure, justification should be provided in the IACUC-approved protocol for use of this method.

Euthanasia by Chilling

• Rapid Chilling is acceptable for some tropical finfish.
  • Zebrafish (Danio rerio) may be euthanized by rapid immersion in water at temperatures of 2-4°C water. Fish should be immersed for a minimum of 20 minutes after cessation of opercular movement.
  • Water used for the ice water bath should be system or source water.
  • Fish should not come in direct contact with the ice.
  • To ensure death of fish either a large vessel should be severed (gill arch or tail vein) or the carcass should be frozen.
  • This method is not appropriate for medium to large-bodied tropical finfish (>3.8cm) due to surface-to-volume considerations.

Euthanasia by Physical Methods

Physical methods of euthanasia usually require certification of competency (see below). Following training, competency must be demonstrated utilizing the same species and conditions in which the method will be performed. Granting of certification is at the discretion of the Attending Veterinarian or designee. In some cases (e.g. remote field locations), proficiency may be assessed by other means, such as interviews or acknowledgement of training completed at other institutions. These exceptions must be reviewed and approved by the Attending Veterinarian, or designee.

• Sharp Blow to the Head (Blunt Force Trauma):
  • Manually applied blunt force trauma (a rapid, accurately placed blow of sufficient energy to the cranium) is an acceptable method of euthanasia when performed correctly.
  • Certification is required for utilization of blunt force trauma as a method of euthanasia.
  • To ensure death of fish, a secondary method must be performed (e.g. pithing, exsanguination by severing of a large vessel, or freezing).
  • Due to the technical expertise required, justification should be provided in the IACUC-approved protocol for use of this method.
• Decapitation with pithing:
  • Decapitation followed immediately by pithing is an acceptable method of euthanasia for most fish.
  • Certification is required for utilization of decapitation and pithing as a method of euthanasia.

The University of Washington IACUC notes that larger mammals appear to be distressed by CO₂ euthanasia. Therefore, CO₂ must not be used in University of Washington protocols for the euthanasia of animals such as rabbits, cats, swine, dogs, or nonhuman primates. In most cases, overdose of injectable agents is the preferred method of euthanasia for non-rodent mammals, but proposed methods will be reviewed and approved on a case-by-case basis in the IACUC protocol.

For all other species not specifically addressed in this policy, refer to the AVMA Guidelines for Euthanasia of Animals: 2020 Edition to determine which euthanasia methods are considered acceptable, acceptable with conditions, or unacceptable.