CO2 Euthanasia of Rodents including Guinea Pigs
Guidelines for the Euthanasia of Adult Rodents
Background: Public Health Service (PHS) policy mandates that it is the Institutional Animal Care and Use Committee's (IACUCs) responsibility to assure that every effort has been made to ensure that death is as painless and distress-free as possible. Euthanasia techniques should result in rapid loss of consciousness followed by cardiac or respiratory arrest and ultimately loss of brain function. The selected method of euthanasia must be consistent with the recommendations of the 2007 AVMA Guidelines on Euthanasia unless scientifically justified (AVMA 2007 or later editions).
The IACUC may approve request for deviations from the AVMA guidelines if the deviations are scientifically justified.
Improper euthanasia of small laboratory animal rodents is considered noncompliance with PHS policy and the Guide for the Care and Use of Laboratory Animals and is reportable to the Office of Laboratory Animal Welfare (OLAW) (http://grants.nih.gov/grants/guide/notice-files/NOT-OD-02-062.html).
Personnel must be appropriately trained to perform euthanasia. If unfamiliar with a procedure, personnel should practice on anesthetized or euthanatized animals. Training is available free of charge by contacting University Laboratory Animal Resources (http://ular.osu.edu/training/). The Institutional Animal Care and Use Committee (IACUC) may require demonstration of competency as a contingency of protocol approval.
CO2 euthanasia, the IACUC expects the following recommendation to be followed when animals are euthanized using CO2. Compressed CO2 gas in cylinders is the only recommended source of carbon dioxide because the inflow to the chamber can be regulated precisely. Carbon dioxide generated by other methods such as from dry ice, fire extinguishers, or chemical means (e.g. antacids) is unacceptable.
Animals must be observed during euthanasia to ensure that death is humane. If chambers are used to contain animals during administration of inhalants, the chambers should be clear or have a viewing port.
Confirmation of death is required prior to disposal. Unintended recovery of animals (e.g. in refrigerators and freezers) after apparent death from inhalant anesthetics is a reportable incidence to OLAW.
The Ohio State University IACUC requires a secondary physical method of euthanasia such as creation of a pneumothorax, removal of a vital organ, decapitation, cervical dislocation or exsanguination. Absence of cardiovascular function is not considered an appropriate method of confirmation of death for rodents and guinea pigs.
All secondary methods listed above are considered approved by the IACUC and may be used in this capacity without listing in the Animal Use Protocol. Please note that decapitation and cervical dislocation without prior anesthesia is conditionally acceptable as a method of euthanasia when required by the experimental design and approved by the IACUC.
To minimize stress: animals being held in a cage while awaiting euthanasia must be compatible (e.g. multiple foreign males, or males and females of sexual maturity should not be placed together for euthanasia); animals must be of the same species; and the total number of animals in a single cage should not exceed 8 in a small rodent cage or 15 in a large rodent cage for mice, or 4 rats or guinea pigs (< 250 gm) or 2 (>250 gm) in a large rodent cage. Rodents other than rats, mice or guinea pigs should follow the appropriate size recommendations for numbers of animals within a cage.
Disposal bags must be labeled. Bags containing animals euthanized by CO2 gas must be labeled with the date, initials of person performing the procedure and name of the Principal Investigator (PI) prior to disposal in University Laboratory Animal (ULAR) facilities (freezers or refrigerators). Labels are available within ULAR vivariums adjacent to CO2 euthanasia chambers or the PI may provide their own labeling system (e.g. tape).
Guidelines for the Euthanasia of Rodent Fetuses and Neonates
Background: The Report of the AVMA Panel on Euthanasia provides limited recommendations for the euthanasia of prenatal or neonatal animals. The 2000 report states: “When ovarian hysterectomies are performed, euthanasia of fetuses should be accomplished as soon as possible after removal from the dam.” It also states “Neonatal animals appear to be resistant to hypoxia, and because all inhalant agents ultimately cause hypoxia, neonatal animals take longer to die than adults.” (http://www.avma.org/resources/euthanasia.pdf)
The following guidelines are to ensure the appropriate euthanasia of rodent fetuses or neonates when they are required in the Animal Use Protocol. In all cases, the person performing the euthanasia must be fully trained in the appropriate procedures.
Fetuses
At approximately 60 percent of the gestation period, the neural tube has developed into a functional brain and the likelihood that a fetus may perceive pain should be considered (1, 2).
Reflexive behavior in response to painful stimuli has been observed in fetuses and correlates with adult behaviors (http://oacu.od.nih.gov/GdeMammNeuro.pdf). However, fetal behavioral arousal and awareness may be suppressed by low arterial oxygen limiting higher cortical processing.
Mouse, Rat and Hamster Fetuses up to 15 days’ and Guinea Pig Fetuses up to 34 days’ gestation: Neural development at this stage is minimal and pain perception is considered unlikely (3,4). Euthanasia of the mother or removal of the fetus should ensure rapid death of the fetus due to loss of blood supply and non-viability of fetuses at this stage of development (5).
Mouse, Rat and Hamster Fetuses 15 days’ gestation to birth and Guinea Pig Fetuses 35 days’ gestation to birth: The neural development at this stage supports the likelihood that pain may be perceived (2, 3, 4). When fetuses are required for study, euthanasia of individual fetuses may be induced by the skillful injection of chemical anesthetics. Decapitations with surgical scissors or cervical dislocation are acceptable physical methods of euthanasia.
Rapid freezing, without prior anesthesia, as a sole means of euthanasia is not considered to be humane (AVMA 2007). Animals should be anesthetized prior to freezing. When chemical fixation of the whole fetus is required, fetuses should be anesthetized prior to immersion in or perfusion with fixative solutions.
The ULAR veterinarian should be consulted for considerations of fetal sensitivity to specific anesthetic agents. Fetuses at this age are resistant to hypoxia (6) and require extended exposure to inhalant anesthetics, including CO2 (7).
When fetuses are not required for study, the method chosen for euthanasia of a pregnant mother should ensure rapid cerebral anoxia to the fetus with minimal disturbance to the uterine milieu minimizing fetal arousal (3). Recommended methods for euthanasia of the mother are CO2 exposure with cervical dislocation. Death of the mother must be verified after euthanasia and prior to disposal. The ULAR veterinarian should be consulted for considerations of other euthanasia agents.
Neonates
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 (7-10). Resistance to hypoxia at this age results in a prolonged time to unconsciousness when CO2 is used as a euthanasia agent.
Mouse, Rat and Hamster 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. halothane or isoflurane (used with appropriate safety considerations) although prolonged exposure may be necessary. Immersion in liquid nitrogen may be used only if preceded by anesthesia. Similarly, anesthesia should precede immersion or perfusion with chemical fixatives. Anesthesia may be induced by inhalant or injectable anesthetics; the ULAR veterinarian should be consulted for appropriate agents and dosages.
Guinea Pig Neonates: Follow guidelines for adults.
Mouse, Rat and Hamster Neonates over 10 days of age: Follow guidelines for adults.
Death of fetuses and neonates must be confirmed after euthanasia and prior to disposal as for adult rodents. Disposal bags must be labeled with the date, initials of person performing the procedure and PI name as for adult rodents (above).
References
1. Close, B., K. Banister, V. Baumans, E.M. Bernoth, N. Bromage, J. Bunyan, W. Erhardt, P. Flecknell, N.G.H. Hackbarth, D. Morton, and C. Warwick. 1997. Recommendation for euthanasia of experimental animals: Part 2. Lab. Anim. 31:14-15.
2. Himwich, W.A. 1962. Biochemical and neurophysiological development of the brain in the neonatal period. Int. Rev. Neurobiol. 4:117-159.
3. Kaufman, W. 2000. p. 227-242. In G.J. Krinke (ed.), The Laboratory Rat. Academic Press, Inc., San Diego, Calif.
4. Yi, D.K., and G.A. Barr. 1997. Formalin-induced c-fos expression in the spinal cord of fetal rats. Pain 73:347-354.
5. Klaunberg B.A., O’Malley J., Clark T., Davis .JA. 2004. Euthanasia of Mouse Fetuses and Neonates. Contemp. Top. Lab. Anim. Sc. 43:(5) 29-34.
6. Singer, D. 1999. Neonatal tolerance to hypoxia: a comparative-physiological approach. Comp. Biochem. Physiol. 123:221-234.
7. Fitzgerald, M., and S. Beggs. 2001. The neurobiology of pain: developmental aspects. Neuroscientist 7:246-257.
8. Gupta, A., J. Cheng, S. Wang, and G.A. Barr. 2001. Analgesic efficacy of ketorolac and morphine in neonatal rat pups. Pharmacol. Biochem. Behav. 68:635-640.
9. Robinson, S.E., and M.J. Wallace. 2001. Effect of perinatal buprenorphine exposure on development in the rat. J. Pharmacol. Exp. Ther. 298:797-804. p. 3.
10. Woodbury, C.J., A.M. Ritter, and H.R. Koerber. 2001. Central anatomy of individual rapidly adapting low-threshold mechanoreceptors innervating the “hairy” skin of newborn mice: early maturation of hair follicle afferents. J. Comp. Neurol. 436:304-323.
