Euthanasia of Mouse Fetuses and Neonates BRENDA A. KLAUNBERG, MS, VMD,1 JAMES O’MALLEY, DVM, MPH,2 TERRI CLARK, DVM,3 AND JUDITH A. DAVIS, DVM, MS2* We sought to determine whether any of the common methods of euthanasia for adult rodents would lead to an acceptable death for fetuses or neonates. We wanted to identify a method that was rapid, free of signs of pain or distress, reliable, and minimally distressful to the person performing the procedure and that minimized the amount of handling required to perform the procedure. We evaluated six methods of euthanasia, with and without anesthesia, in three age groups of mice: gravid mice (E14-20) and neonatal pups (P1-P7 and P8- P14). Euthanasia methods included: halothane inhalation, carbon dioxide inhalation, intraperitoneal sodium pentobarbital, intravenous potassium chloride, and cervical dislocation with and without anesthesia. Noninvasive echocardiography was used to assess heartbeat during euthanasia. With cardiac arrest as the definition of death, no method of euthanasia killed fetal mice. Halothane inhalation (5% by vaporizer) was not an acceptable method of euthanasia for mice of the age groups tested. Intraperitoneal administration of sodium pentobarbital for euthanasia required a higher dose than the previously established dose, and there is a risk of reduced efficacy in pregnant animals due to potential intrauterine injection. Carbon dioxide asphyxiation was the most efficient method of euthanasia for neonatal mouse pups P1-14. For pregnant adult mice, intravenous potassium chloride under anesthesia, carbon dioxide asphyxiation, and cervical dislocation alone or under anesthesia were excellent methods of euthanasia. Timed-pregnant animals constitute a predominant percentage of methods of euthanasia for adult rodents would also result in an ac- animal use for basic research in our institute. Vendors frequently ceptable death for the fetus or neonate. We wanted to find a method include extra dams to ensure that the scientist receives the requested that was rapid, free of signs of pain or distress, reliable, and mini- number of animals at the appropriate day of gestation (e.g., embry- mally distressful to the person performing the procedure and that onic day 12 [E12], E14, etc.). Consequently, facility staff must minimized the amount of handling required to perform the proce- euthanize pregnant animals if they are unable to locate other poten- dure. We investigated six methods of euthanasia, but not every method tial users of the extra animals. Discussions during training classes on was applied to each group. The study was divided into two phases, proper euthanasia techniques revealed employee concern about fetal which were based upon the ages of the pups. Phase one examined death. Employees asked how they would know whether the fetuses fetal mice at 14 days of gestation or more (≥ E14). Phase two exam- were dead. Removing the uterus and physically examining each fetus ined neonatal mice, which were divided into two groups: pups 1 to 7 is an option to ensure that fetuses are dead, but we consider that days old (P1-P7) or 8 to 14 days old (P8-P14). impractical. We were unable to find documented evidence that com- monly used euthanasia methods rapidly and painlessly kill fetuses. Materials and Methods The only published references to fetuses or neonatal animals in rela- Mice. Virus antibody-free mice (n = 166) identified as “culls” of tion to euthanasia were admonitions against the use of carbon dioxide various genotypes were used. The experimental group included in- because newborn animals are more resistant to hypoxia (1, 2). These bred, outbred, hybrid, and mutant mice. The mice were housed in a publications further state that inhalation anesthetics will not kill fe- National Institutes of Health animal facility that is accredited by the ≤ tuses and that newborn pups 10 days of age seem to react more like Association for Assessment and Accreditation of Laboratory Animal fetuses than adult animals (3). Care, International. The mice were maintained in a specific patho- Complicating the problem of fetal and neonatal euthanasia is the gen-free animal care holding room; sentinel Swiss-Webster mice phenomenon of neonatal hypoxia tolerance, in which adaptive mecha- (Taconic, Germantown, N.Y.) were on racks in the same animal nisms to acute oxygen loss include decreased metabolism, decreased holding room and were evaluated monthly to monitor the colony for body temperature, and reduced blood pH (4). Any mention of proper pathogen exposure. In addition, colony animals identified for eutha- euthanasia of fetuses and neonates cautions that the animals may nasia were monitored for pathogen exposure. Sera from the sentinel appear insensate but are still alive. Recommendations for fetal eutha- and cull mice consistently tested negative for the following microor- nasia include removal from the uterus followed by decapitation or ganisms: CAR bacillus, ectromelia virus, mouse rotavirus, mouse < concussion (3) or, if the fetus weighs 4 g, it may be placed directly encephalomyelitis virus, lymphocytic choriomeningitis virus, murine into liquid nitrogen for euthanasia (3). None of these methods were cytomegalovirus, mouse hepatitis virus, mouse adenovirus, minute considered practical by our staff. virus of mice, Mycoplasma pulmonis, mouse parvovirus, polyoma vi- We describe the use of ultrasound imaging to determine cardiac rus, pneumonia virus of mice, reovirus 3, and Sendai virus. Mice arrest after administration of a euthanasia method to mice. The pur- were free of pinworms and fur mites. The animal holding room was pose of this study was to determine whether any of the common maintained with restricted access via electronic cipher-coded door locks. Mice were housed in polycarbonate (19.56 cm × 30.91 cm × 10 Center Drive, Bldg 10, Room B1D69, NMRF, DIR, NINDS, NIH, DHHS, Bethesda, 14.92 cm) ventilated cages (Thoren Caging Systems, Hazelton, Pa.) Maryland 208921; 36 Convent Drive, Bldg 36, Room B306, AHCS, DIR, NINDS, NIH, DHHS, Bethesda, Maryland 20892-44142; 31 Center Drive, Bldg 31, Room on CareFRESH Ultra bedding (Quality Lab Products, Inc., Elkridge, B1C37, OACU, OIR, OD, NIH, DHHS, Bethesda, Maryland 20892-22523 Md.). Cages were changed twice weekly. The animal holding room *Corresponding author was maintained under environmental conditions of 22 ± 1°C, rela- Work was conducted at the Mouse Imaging Facility, National Institutes of Health, tive humidity of 50% ± 10%, 20 air changes/h, and a 12:12-h Bethesda, Maryland The views expressed are those of the authors and do not reflect the official policy or light:dark cycle. Mice received pelleted rodent diet (NIH Open For- position of the National Institutes of Health or the United States Government. mula Rodent Diet NIH-07, Zeigler Brothers, Inc., Gardners, Pa.) Volume 43, No. 5 / September 2004 CONTEMPORARY TOPICS © 2004 by the American Association for Laboratory Animal Science 29 Table 1. Experimental age groups and methods of euthanasia applied (+) turned on. While in the CO2 chamber, mice could not be physically Fetuses (E14-20) Neonates (P1-7) Neonates (P8-14) examined, so a visual assessment was made. Once the mouse lost its righting reflex, stopped moving, or appeared to be unconscious, that Carbon dioxide ++ +time was recorded, and the animal was immediately transferred to a asphyxiation custom-made facemask for continued delivery of CO2. The heart- Barbiturate + not done + beat of the neonate, or a randomly chosen fetus, was identified as overdose quickly as possible with ultrasound imaging. The times to cardiac Cervical dislocation + not done not done arrest and, if possible, respiratory arrest were recorded. Halothane overdose ++ +For barbiturate overdose, two age groups were studied: fetuses (E14- (5% by vaporizer) 20) and P8-14 neonates. Sodium pentobarbital (800 mg/kg) was Potassium chloride + not done not done administered via intraperitoneal injection to manually restrained neo- under anesthesia nates or gravid mice. The injection time was recorded. When the injected animal stopped ambulating, we identified the heartbeat of Cervical dislocation + not done not done under anesthesia the fetus or neonate with ultrasonography as quickly as possible. The times to respiratory arrest and cardiac arrest were recorded. and water ad libitum. This study was reviewed and approved by our Cervical dislocation of conscious animals was studied in gravid Institutional Animal Care and Use Committee. females only. The time the dam was euthanized by cervical disloca- Experimental design. Transthoracic echocardiography was used tion was recorded. The heartbeat of a randomly chosen fetus was to study selected methods of euthanasia for the three age groups of identified by ultrasonography, and the time to fetal cardiac arrest mice. Death was defined by cardiac arrest; cardiac arrest was defined was recorded. by cessation of cardiac contractions. Echocardiography was termi- In order to compare our experimental methods of euthanasia with nated at 20 min if cardiac arrest did not occur, and the animals a currently recommended method of fetal euthanasia, we performed subsequently were euthanized using a physical method of euthana- cesarean section and decapitation on fetuses of dead dams (n = 19) sia. For cervical dislocation, loss of consciousness and cerebral death from other study groups. Timing began at the initiation of the cesar- were considered immediate. The first study examined fetal death in ean section and ended at the time of the last pup’s decapitation. The relation to euthanasia of the dam; gravid females at 14 to 20 days of number of fetuses for each dam was counted. The number of fetuses gestation were used. The second study examined neonatal euthana- per dam varied, so an average time of procedure per gravid dam was sia. Neonatal pups were divided into two experimental age groups: calculated. P1 to P7 days old and P8 to P14 days old. Anesthesia methods. For general anesthesia, mice were placed in The pregnant animals used in this study were in the late stages of the previously described chamber for induction, and 5% halothane pregnancy (E14-20).