NC State IACUC Guideline on Euthanasia for Mice and Rats
This guideline describes primary and secondary forms of euthanaisa for mice and rats used in research, testing, or teaching. Definitions, conditions, proper use of equipment, and training are all outlined. All persons involved with the care and/or use of animals in research and/or teaching at the University including, but not limited to Principal Investigators, researchers, animal handlers, and students.
Standard
All euthanasia in mice or rats must follow the AVMA Guidelines on Euthanasia 2020 edition. Per the AVMA Guidelines, “Methods of euthanasia likely to elicit distress vocalizations or pheromones that other animals in the room could hear or smell may be best performed in another location, if transportation distress can be minimized.” Euthanasia should be conducted in a dedicated procedure room outside of the housing space or in a fume hood that ventilates outside. All methods used must be described in appropriate protocol.
Definitions
AVMA defines the following methods:
- Acceptable: Consistently produce humane death when used as the sole method
- Acceptable with Conditions: Certain conditions must be met to produce humane death. These methods have a greater potential for operator error or safety hazard, are not well documented in literature, or may require a secondary method.
- Unacceptable: Methods that are deemed inhumane under any condition or pose a risk to humans applying technique.
Animal ages:
- Adult: Weaned animals, older than 23 days
- Fetus: Animals not born yet, younger than day 14 of gestation
- Neonates: Newly-born animals until age 5-7 days old
- Preweanling: Pups that are still with dam but are no longer considered neonates
Methods
Gaseous Carbon Dioxide (CO2)
Euthanasia by CO2 is acceptable with conditions in species where aversion or distress can be minimized and must be conducted using appropriately designed and maintained equipment by trained personnel. A compressed gas tank with a gradual-fill method and displacement rate from 30% to 70% of the chamber volume/minute to minimize pain and distress should be used. Methods include manual CO2 chambers and automated CO2 units.
Inhalant Anesthetics
The use of inhaled anesthetics delivered as a volatile gas to the respiratory tract to induce anesthesia is acceptable with conditions for euthanasia. Animals must be rendered unconscious through inhaled gas and then subsequently euthanized by a secondary method. The use of a vaporizer and induction chamber are recommended. Animals must not be able to come into contact with the anesthesia liquid.
Cervical Dislocation
Manual cervical dislocation is acceptable with conditions for euthanasia in mice and rats weighing < 200g when performed by individuals with a demonstrated high degree of technical proficiency. In lieu of demonstrated technical competency, animals must be unconscious or anesthetized prior to cervical dislocation.
Decapitation
This method is acceptable with conditions if performed correctly, and it may be used in research settings when its use is required by the experimental design and approved by the IACUC. Decapitation is justified for studies where undamaged and uncontaminated brain tissue is required. Animals must be unconscious or anesthetized prior to decapitation.
Hypothermia
The gradual cooling of fetuses and altricial neonates (mice and rats)to induce deep anesthesia and subsequent euthanasia is not a preferred method of euthanasia but is acceptable with conditions per the AVMA Guidelines.
Special Cases
When animals to be euthanized are fully anesthetized, adjunctive methods such as bilateral thoracotomy, exsanguination, perfusion, and IV or intracardiac injection of potassium chloride are acceptable.
Training
Training in all methods is required and must be documented. Training should include primary and secondary methods, proper use and disinfection of equipment, and proficiency assessment. Decapitation and live cervical dislocation must be trained and signed off by the institutional trainer or an appointed verified trainer.
Equipment
Equipment that may be used include commercially available guillotine or scissors. Equipment must be maintained in good working order and serviced on a regular basis to ensure sharpness of blades. An updated log should be kept to document such maintenance. Equipment must also be appropriately sized for the animal. Clean and disinfection of all instruments should occur immediately after use. The use of plastic cones to restrain animals may be to reduce distress from handling, minimize the chance of injury to personnel, and improve positioning of the animal
When using a guillotine, the blade must be sharpened and maintained according to the manufacturer’s instructions. In instances where no manufacturer recommendations are available, blade maintenance should be carried out based on blade performance. An example of a method for testing sharpness is to use a freshly euthanized rodent carcass. If the blade is noted to be dull, it should be either replaced or sharpened by a professional sharpening service. Blades that are replaced should be disposed of in a dedicated sharps container.
Conditions for Methods Reference Table
| Method | Age | Condition |
| Carbon Dioxide (CO2) | Adult and preweanlings | Utilize a chamber, cylinder/tank with a flow meter. Must be followed by a secondary method. |
| Neonates | Require extended exposure time ~30-50 mins. Not recommended | |
| Fetuses | Effective for fetuses in utero | |
| Inhaled anesthetic agents (isoflurane, sevoflurane, etc.) | Adult | Maintain for 1 minute following respiratory arrest. Must be followed by a secondary/physical method. |
| Neonates and preweanlings | Animals must be under deep anesthesia (nonresponsive to painful stimuli) immediately followed by a secondary/physical method. | |
| Fetuses | Effective for fetuses in utero | |
| Cervical Dislocation | Adult | Animals must weigh less than 200g, must be unconscious or anesthetized unless there is justification in protocol. |
| Preweanlings | May be performed in animals >10 days of age | |
| Fetuses | Not performed. | |
| Decapitation | Adult | Must be justified in protocol and equipment must be managed appropriately. Requires additional training |
| Preweanlings | May be performed with or without anesthesia and must be justified in protocol | |
| Neonates/fetuses | Can be performed with or without anesthesia. | |
| Hypothermia | Fetuses/neonates | Animals should not come into direct contact with ice or precooled surfaces and must be followed by a secondary/physical method. Not acceptable after 7 days of age. |