Euthanasia of Rodents
PHS policy requires Institutional Animal Care & Use Committees (IACUCs) to determine that methods of euthanasia utilized in research proposals are consistent with the report of the American Veterinary Medical Association Panel on Euthanasia (AVMA Guidelines on Euthanasia) unless a deviation is justified for scientific reasons in writing by the investigator. IACUC approval of such deviations must be project-specific and include critical review of assertions of scientific necessity.
- Euthanasia techniques must be consistent with the AVMA Guidelines on Euthanasia, June 2007. Methods are chosen to minimize animal pain and distress consistent with needs of the research protocol.
- The method of euthanasia must be specified in the approved IACUC protocol. Animal death must be verified after euthanasia and prior to disposal (for specific details, please refer to the section Verifying Death below).
- Use of anesthetic for euthanasia must be an overdose, not an anesthetic dose. Regardless of amount of chemical administered, animal must be completely nonresponsive to noxious stimuli (hind paw pinch) before any physical means are applied.
- Sodium pentobarbital and ketamine are controlled substances and must be maintained according to the BU Controlled Substances Program.
- Physical methods of euthanasia such as decapitation or cervical dislocation of unanesthetized animals require demonstration of competence, and may be approved with proper justification in the IACUC protocol and documentation of training.
- Whenever possible, euthanasia should not be performed in the presence of other animals.
The procedures listed below are suggested common methods for euthanasia of rodents. Other methods outlined in the AVMA Guidelines on Euthanasia are acceptable with proper justification in the approved IACUC protocol.
- Intraperitoneal injection of at least 200 mg/kg sodium pentobarbital euthanasia solution, or other anesthetic at an overdose.
- Inhalation of CO2 from a pressurized tank in an uncrowded chamber (a standard-sized mouse cage may contain no more than five mice) followed by cervical dislocation, decapitation, or bilateral thoracotomy.
- Intraperitoneal injection of at least 200 mg/kg sodium pentobarbital or pentobarbital euthanasia solution.
- Inhalation of CO2 from a pressurized tank in an uncrowded chamber.
- Avoid overcrowding. Standard animal facility guidelines for cage density must be maintained.
- Whenever possible, euthanize rodents in their home cage to minimize the stress of being handled and placed into an unfamiliar enclosure.
- Set the regulator so that the CO2 gas flows slowly and does not hiss or overpower the animals and frighten them. A rule of thumb is 20% displacement per minute, and to not let the psi go above 15.
- The regulator for the CO2 tank should be adjusted so that the flow of CO2 is slow enough not to make a noise or frighten the animals. Slower flow also allows the animals a more gradual induction of unconsciousness and is thus less stressful. Animals must be left in the chamber for a sufficient time (four or five minutes) so that complete asphyxia has been attained.
- When chemical fixation or rapid freezing of the whole fetus is required, anesthesia must be used prior to immersion in, or perfusion with, fixative solutions.
- Anesthesia may be induced by hypothermia of the fetus or by injection of the fetus with a chemical anesthetic.
- Euthanasia of individual fetal animals may be induced by the skillful injection of chemical anesthetics.
- Decapitation with surgical scissors is an acceptable method of euthanasia.
- Inhalation of CO2 is an approved method for guinea pigs.
- Intraperitoneal injection of at least 200 mg/kg sodium pentobarbital or pentobarbital euthanasia solution. If necessary, one may sedate the animal first using ketamine 50 mg/kg IM plus xylazine 2 mg/kg IM. Once deeply sedated (5–10 minutes after injection), inject at least 200 mg/kg sodium pentobarbital euthanasia solution intraperitoneally.
Fetal mice, rats, and hamsters up to 15 days
Fetal guinea pigs up to 34 days’ gestation
Neural development at this stage is minimal and pain perception is considered unlikely. Due to nonviability of fetuses at this stage of development, euthanasia of the mother or the removal of the fetus should ensure rapid death of the fetus. No additional action is needed.
Fetal mice, rats, and hamsters 15 days’ gestation to birth
Fetal guinea pigs 35 days’ gestation to birth
Pain perception at this stage of development is likely. Fetuses at this stage are less susceptible to inhalant anesthetics.
When fetuses are not required for study, the method chosen for euthanasia of a pregnant dam should ensure rapid cerebral anoxia to the fetus with minimal disturbance to the uterine milieu minimizing fetal arousal. The recommended method consists of CO2 exposure of the dam. Death of the dam must be verified after euthanasia and prior to disposal (see Verifying Death guidelines below).
Alternatively, an overdose of pentobarbital (200 mg/kg) IP may be administered. Injection must be made carefully to avoid intrauterine administration; however, this will be absorbed and will euthanize the fetuses as well. Death of dam must be verified.
Neonatal mice, rats, gerbils, and hamsters ≤10 days
At this stage of development, neonates are highly tolerant to inhalant anesthetics, including CO2. Maturation of nociceptors and the development of excitatory and inhibitory receptor systems occur during the period just prior to birth and into the second week of postnatal life. Resistance to hypoxia at this age results in a prolonged time to unconsciousness and death when CO2 is used as a euthanasia agent and must be followed by a second, physical method.
Place neonates in a CO2 chamber for four to five minutes to begin the euthanasia process, then follow with decapitation. Neonates may instead be anesthetized with isoflurane in an induction chamber or jar, followed by decapitation. Alternatively, for neonatal rats and mice, rapid decapitation with sharp scissors may be performed. However, the use of decapitation without prior anesthesia with isoflurane or carbon dioxide must be justified.
Intraperitoneal injection of at least 200 mg/kg sodium pentobarbital or a euthanasia solution such as Euthasol® (390 mg/ml sodium pentobarbital + 50 mg/ml phenytoin) or Fatal Plus® is also acceptable.
Neonatal guinea pigs
Follow guidelines for adult guinea pigs.
Mice, rats, and hamsters >10 days
Adult (post-weaning) mice, rats, and hamsters
Guinea pigs are born so mature and precocious that even the neonates and pre-weaning animals are euthanized by the same procedures as adults.
CO2 euthanasia procedures
In order to minimize stress on the animals, the following procedures are followed.
Procedures for Administration of Pentobarbital or Other Euthanasia Agents Containing Pentobarbital
Pentobarbital, because of its basic pH (pH ~11) and concentration, is best diluted to a concentration of no more than 60 mg/ml. Also, due to the small size of most rodents, this allows for better calculation of the dose and, in addition, facilitates absorption.
Death must be verified after euthanasia and prior to disposal. Unintended recovery must be prevented by the use of appropriate CO2 concentrations and exposure times or by other means as noted below.
Euthanasia by CO2 or inhalation of other gaseous agents or an injectable agent requires the use of a secondary physical method to assure death. Examples of secondary methods include:
- Cervical dislocation
- Exsanguination—i.e., great vessels severed (cardiac puncture does not result in consistent exsanguination and may not be used as a secondary method)
- Cardiac perfusion
- Removal of vital organs
- Incision of the chest cavity to produce a bilateral pneumothorax (collapsed lung) and cessation of respiration
Unintended recovery of animals after apparent death from CO2 (e.g., in necropsy coolers) is a documented occurrence. Such incidents constitute serious noncompliance with the PHS policy and a serious deviation from the provisions of the Guide for the Care and Use of Laboratory Animals and must be reported by the institution to OLAW. Researchers must make sure the animal is dead before disposal, tissue collection, or leaving the animal unattended.
- IACUC Guidelines, Boston University School of Medicine, 2007
- IACUC Guidelines, New Jersey Medical School, UMDNJ Newark Campus, 2005
- IACUC Guidelines, University of California at San Francisco, 2007