Euthanasia of Mouse Fetuses and Neonates

BRENDA A. KLAUNBERG, MS, VMD,1 JAMES OMALLEY, 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 P8P14). 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 animal use for basic research in our institute. Vendors frequently include extra dams to ensure that the scientist receives the requested number of animals at the appropriate day of gestation (e.g., embryonic day 12 [E12], E14, etc.). Consequently, facility staff must euthanize pregnant animals if they are unable to locate other potential users of the extra animals. Discussions during training classes on proper euthanasia techniques revealed employee concern about fetal death. Employees asked how they would know whether the fetuses were dead. Removing the uterus and physically examining each fetus is an option to ensure that fetuses are dead, but we consider that impractical. We were unable to find documented evidence that commonly used euthanasia methods rapidly and painlessly kill fetuses. The only published references to fetuses or neonatal animals in relation to euthanasia were admonitions against the use of carbon dioxide because newborn animals are more resistant to hypoxia (1, 2). These publications further state that inhalation anesthetics will not kill fetuses and that newborn pups 10 days of age seem to react more like fetuses than adult animals (3). Complicating the problem of fetal and neonatal euthanasia is the phenomenon of neonatal hypoxia tolerance, in which adaptive mechanisms to acute oxygen loss include decreased metabolism, decreased body temperature, and reduced blood pH (4). Any mention of proper euthanasia of fetuses and neonates cautions that the animals may appear insensate but are still alive. Recommendations for fetal euthanasia include removal from the uterus followed by decapitation or concussion (3) or, if the fetus weighs < 4 g, it may be placed directly into liquid nitrogen for euthanasia (3). None of these methods were considered practical by our staff. We describe the use of ultrasound imaging to determine cardiac arrest after administration of a euthanasia method to mice. The purpose of this study was to determine whether any of the common
10 Center Drive, Bldg 10, Room B1D69, NMRF, DIR, NINDS, NIH, DHHS, Bethesda, Maryland 208921; 36 Convent Drive, Bldg 36, Room B306, AHCS, DIR, NINDS, NIH, DHHS, Bethesda, Maryland 20892-44142; 31 Center Drive, Bldg 31, Room B1C37, OACU, OIR, OD, NIH, DHHS, Bethesda, Maryland 20892-22523 * Corresponding author Work was conducted at the Mouse Imaging Facility, National Institutes of Health, Bethesda, Maryland The views expressed are those of the authors and do not reflect the official policy or position of the National Institutes of Health or the United States Government.
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methods of euthanasia for adult rodents would also result in an acceptable death for the fetus or neonate. We wanted to find 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 investigated six methods of euthanasia, but not every method was applied to each group. The study was divided into two phases, which were based upon the ages of the pups. Phase one examined fetal mice at 14 days of gestation or more ( E14). Phase two examined neonatal mice, which were divided into two groups: pups 1 to 7 days old (P1-P7) or 8 to 14 days old (P8-P14).

Materials and Methods

Mice. Virus antibody-free mice (n = 166) identified as culls of various genotypes were used. The experimental group included inbred, outbred, hybrid, and mutant mice. The mice were housed in a National Institutes of Health animal facility that is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International. The mice were maintained in a specific pathogen-free animal care holding room; sentinel Swiss-Webster mice (Taconic, Germantown, N.Y.) were on racks in the same animal holding room and were evaluated monthly to monitor the colony for pathogen exposure. In addition, colony animals identified for euthanasia were monitored for pathogen exposure. Sera from the sentinel and cull mice consistently tested negative for the following microorganisms: CAR bacillus, ectromelia virus, mouse rotavirus, mouse encephalomyelitis virus, lymphocytic choriomeningitis virus, murine cytomegalovirus, mouse hepatitis virus, mouse adenovirus, minute virus of mice, Mycoplasma pulmonis, mouse parvovirus, polyoma virus, pneumonia virus of mice, reovirus 3, and Sendai virus. Mice were free of pinworms and fur mites. The animal holding room was maintained with restricted access via electronic cipher-coded door locks. Mice were housed in polycarbonate (19.56 cm 30.91 cm 14.92 cm) ventilated cages (Thoren Caging Systems, Hazelton, Pa.) on CareFRESH Ultra bedding (Quality Lab Products, Inc., Elkridge, Md.). Cages were changed twice weekly. The animal holding room was maintained under environmental conditions of 22 1C, relative humidity of 50% 10%, 20 air changes/h, and a 12:12-h light:dark cycle. Mice received pelleted rodent diet (NIH Open Formula Rodent Diet NIH-07, Zeigler Brothers, Inc., Gardners, Pa.)
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Table 1. Experimental age groups and methods of euthanasia applied (+) Fetuses (E14-20) Neonates (P1-7) Neonates (P8-14) Carbon dioxide asphyxiation Barbiturate overdose Cervical dislocation Halothane overdose (5% by vaporizer) Potassium chloride under anesthesia Cervical dislocation under anesthesia + + + + + + + not done not done + not done not done + + not done + not done not done

and water ad libitum. This study was reviewed and approved by our Institutional Animal Care and Use Committee. Experimental design. Transthoracic echocardiography was used to study selected methods of euthanasia for the three age groups of mice. Death was defined by cardiac arrest; cardiac arrest was defined by cessation of cardiac contractions. Echocardiography was terminated at 20 min if cardiac arrest did not occur, and the animals subsequently were euthanized using a physical method of euthanasia. For cervical dislocation, loss of consciousness and cerebral death were considered immediate. The first study examined fetal death in relation to euthanasia of the dam; gravid females at 14 to 20 days of gestation were used. The second study examined neonatal euthanasia. Neonatal pups were divided into two experimental age groups: P1 to P7 days old and P8 to P14 days old. The pregnant animals used in this study were in the late stages of pregnancy (E14-20). Gravid animals < 14 days gestation were not used for a variety of reasons, but the most important one is that fetal development of the heart and vascular system do not achieve definitive prenatal configuration until E15 (5). Six methods of euthanasia were studied, but not every method was applied to every group (Table 1). Three methods were used with animals under general anesthesia, and three methods were used in unanesthetized animals. Methods of euthanasia performed under general anesthesia included an overdose of inhalant anesthesia delivered by a vaporizer, intravenous potassium chloride injection (Pharmaceutical Partners, Inc., Los Angeles, Calif.), and cervical dislocation. Methods of euthanasia without general anesthesia included carbon dioxide asphyxiation, barbiturate overdose (Beuthanasia-D Special, Shering-Plough Animal Health Corporation, Union, N.J.), and cervical dislocation. Carbon dioxide euthanasia chamber. For carbon dioxide asphyxiation or inhalant anesthesia induction, a clear acrylic chamber with a positive-sealing gasket lid was used (no. 941444, VetEquip, Inc., Pleasanton, Calif.). The total chamber volume was 2000 cc (22.86 cm [width] 9.52 cm [depth] 9.52 cm [height]). A 0- to 500-cc/min flow meter (Multitube Frame 03215-56 Cole-Porter Instruments Co., Vernon Hills, Ill.) was connected between the CO2 tank and chamber. The gas flow rate was set to 400 cc/min to achieve 20% volume displacement per minute. The CO2 gas concentration of displaced gasses exiting the chamber was measured with a capnograph (CO 2 SMO Monitor, Novametrix Medical Systems, Inc., Wallingford, Conn.). The time when the chamber measured 100% CO2 was noted. Between animals, the chamber was turned upside down and emptied of residual CO2 gas, then closed and flushed with 100% oxygen until the exhaust CO2 concentration was 0%. Euthanasia methods without anesthesia. Carbon dioxide asphyxiation was studied in all age groups. The animal was placed in the chamber described above and timing began when the CO2 gas was
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turned on. While in the CO2 chamber, mice could not be physically examined, so a visual assessment was made. Once the mouse lost its righting reflex, stopped moving, or appeared to be unconscious, that time was recorded, and the animal was immediately transferred to a custom-made facemask for continued delivery of CO2. The heartbeat of the neonate, or a randomly chosen fetus, was identified as quickly as possible with ultrasound imaging. The times to cardiac arrest and, if possible, respiratory arrest were recorded. For barbiturate overdose, two age groups were studied: fetuses (E1420) and P8-14 neonates. Sodium pentobarbital (800 mg/kg) was administered via intraperitoneal injection to manually restrained neonates or gravid mice. The injection time was recorded. When the injected animal stopped ambulating, we identified the heartbeat of the fetus or neonate with ultrasonography as quickly as possible. The times to respiratory arrest and cardiac arrest were recorded. Cervical dislocation of conscious animals was studied in gravid females only. The time the dam was euthanized by cervical dislocation was recorded. The heartbeat of a randomly chosen fetus was identified by ultrasonography, and the time to fetal cardiac arrest was recorded. In order to compare our experimental methods of euthanasia with a currently recommended method of fetal euthanasia, we performed cesarean section and decapitation on fetuses of dead dams (n = 19) from other study groups. Timing began at the initiation of the cesarean section and ended at the time of the last pups decapitation. The number of fetuses for each dam was counted. The number of fetuses per dam varied, so an average time of procedure per gravid dam was calculated. Anesthesia methods. For general anesthesia, mice were placed in the previously described chamber for induction, and 5% halothane or isoflurane (Fluotec or Isotec, SurgiVet/Anesco, Waukesha, Wis.) was delivered to the chamber with 100% oxygen at a rate of 400 ml/ min. When the animal lost its righting reflex, stopped moving, or appeared to be unconscious, the animal was transferred to a custommade facemask for continued delivery of anesthetic gasses. Anesthesia was maintained with 1% to 2% halothane or isoflurane and an oxygen flow rate of 250 ml/min. Halothane was used only for inhalant anesthetic overdose, and isoflurane was used for all other methods involving general anesthesia. Every anesthetized animal was placed in dorsal recumbency on a specialized mouse board with heating elements and electrodes for measuring heart rate or the electrocardiogram (THM 100 Electronic Control Module, Indus Instruments, Houston, Tex.). This board was placed on top of a 37C heated recirculating water pad (Temperature Therapy Pad TP-21B, Gaymar Industries, Orchard Park, N.Y.). When possible, rectal body temperature and heart rate were monitored continuously with the Indus Instruments apparatus. Euthanasia methods with anesthesia. Halothane overdose was studied in all age groups. Gravid dams were anesthetized as previously described. Once the heartbeat of a randomly chosen fetus was identified by ultrasonography, the anesthetic vaporizer was increased to 5% halothane, and timing began from that point. Neonates were anesthetized in the previously described chamber, and 5% halothane was delivered to the chamber with 100% oxygen at a rate of 400 ml/min. Timing began when the halothane was turned on. When the neonate stopped moving or appeared to be unconscious, the time was recorded, and the pup was immediately transferred to a custommade mask for continued delivery of 5% halothane. For both groups, the heartbeat was identified as quickly as possible with ultrasonography, and times to cardiac arrest and, if possible, respiratory arrest were recorded. Potassium chloride euthanasia was studied in gravid females only. The dam was anesthetized as previously described. A butterfly cathVolume 43, No. 5 / September 2004

Table 2. Time (min; mean 1 standard deviation) to cardiac arrest (CA) and loss of righting reflex (RR) for each method of euthanasia Dams Carbon dioxide asphyxiation CA RR Barbiturate overdose CA RR Cervical dislocation CA RR Halothane overdose (5% by vaporizer) CA RR 2:50 0:14 n = 5a 1:54 0:17 n=7 11:02 7:35 n = 6b 1:51 0:04 n=7 2:07 0:25 n=7 not determined 18:56 2:37 n = 6c not determined 0:11 0:03 n = 6d not determined 3:17 2:09 n=7 not determined Fetuses (E14-20) 20:00 0 n=7 not determined 20:00 0 n=7 not determined 20:00 0 n=7 not determined 20:00 0 n=7 not determined 20:00 0 n=7 not determined 20:00 0 n=7 not determined Neonates (P1-7) 4:41 0:37 n = 11 3:44 0:43 n = 11 not determined not determined not determined not determined 20:00 0 n=7 3:38 1:16 n = 6c not determined not determined not determined not determined Neonates (P8-14) 3:52 1:02 n = 15 1:56 0:32 n = 15 6:03 1:19 n = 12 0:30 0:05 n = 12 not determined not determined 19:36 1:06 n=7 2:01 0:26 n=7 not determined not determined not determined not determined

Potassium chloride under anesthesia

CA RR

Cervical dislocation under anesthesia

a

CA RR

did not measure time to CA in the first two dams. missed CA time point in one animal. c discarded one value greater than 2 standard deviations from the mean. d missed one dams time to CA.
b

eter (27 gauge 1/2", Surflo winged infusion set, Terumo Corporation, Tokyo, Japan) was inserted into the tail vein of the anesthetized gravid dam. Once the heartbeat of a randomly chosen fetus was identified by ultrasonography, a lethal dose of KCl (1 to 2 mmol/kg) was administered. The time of injection was recorded. The times of respiratory arrest of the dam and cardiac arrest for both the dam and fetus were recorded. Euthanasia by cervical dislocation under anesthesia was studied in gravid dams only. The dam was anesthetized and maintained under anesthesia as previously described. Once the heartbeat of a randomly chosen fetus was identified by ultrasonography, an assistant performed cervical dislocation of the dam. The times of respiratory arrest for the dam and cardiac arrest for both the dam and fetus were recorded. Ultrasound image acquisition. Transthoracic echocardiography was performed with an Acuson Sequoia Echocardiography system (model C256, Siemens Medical Solutions, Mountain View, Calif.). The variable range transducer offered ultrasound frequencies of 8, 12, and 14 MHz. These frequencies generated images with microscopic resolution of approximately 200 m. Two-dimensional B-mode images were obtained from parasternal long and short axis views by using the 14-MHz linear transducer with optimized gain and depth settings. Some data were stored in DICOM file format or video images. B-mode images were obtained for all pups, and when possible, Doppler blood flow images were obtained also. Images were viewed and analyzed with the Acuson software (DIMAQ workstation). If necessary, the hair was removed from the mouses ventral thorax prior to imaging. Time points were measured with a clock timer and recorded in minutes. The data points were converted to seconds for calculations and then converted back to minutes for reporting. For statistical analysis, the value of 1200 sec (20 min) was used for data points that exceeded the 20-min limit. Calculations and Student t tests were performed using Excel (Microsoft, Redmond, Wash.).
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Results
Study 1: fetal death relative to the dams. According to our definition of death by cardiac arrest, no euthanasia method used in this study successfully caused fetal death (Table 2). With dams breathing 5% halothane anesthesia delivered by a vaporizer (n = 7), fetal heartbeats continued beyond the 20-min limit. Most dams (n = 6; 1 data point more than 2 standard deviations [SD] from mean) also failed to undergo cardiac arrest within 20 min of breathing 5% halothane. Intraperitoneal injections of sodium pentobarbital (800 mg/kg) caused cardiac arrest in the dams at approximately 6 min (n = 6, one data point not recorded). Fetal heart rates continued beyond the cut-off of 20 min, but there was considerable variation in the rates (24 to 148 beats per min [bpm]) under pentobarbital. In addition, fetal cardiac arrest did not occur within 20 min after the dam received intravenous KCl, carbon dioxide asphyxiation, or cervical dislocation performed alone or under anesthesia. Conversely, these were excellent methods of euthanasia for the dam. After KCl injection, the mean time until cardiac arrest of the dams was 11 sec (n = 6, one value not recorded). Dams developed cardiac arrest under carbon dioxide inhalation in an average of 2:50 min (n = 5, 2 data points not recorded). Pregnant animals that were first anesthetized with isoflurane then euthanized by cervical dislocation experienced cardiac arrest in an average of 3:17 min (n = 7); however, the fetal hearts continued to beat after 20 min, with an average of 31 bpm. Performing cervical dislocation on conscious pregnant females (n = 7) yielded similar results. The average time of death of the dam was 2:07 min, but 20 min later, fetal hearts were still beating at an average rate of 22 bpm. The time required for cesarean section and decapitation of fetuses will vary with the number of fetuses present in the uterus. In our study, dams selected for the mock cesarean section and decapitation of fetuses experiment had an average of 10 fetuses per dam. The entire procedure averaged 2:13 min per dam. Study 2: neonatal mice. For neonatal mice, we found that the
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most rapid method of euthanasia was carbon dioxide asphyxiation (Table 2). We examined two age groups of neonates, pups 1 to 7 days old and those 8 to 14 days old. The average time for cardiac arrest after carbon dioxide inhalation was 4:41 min in P1-7 pups (n = 11) and 3:52 min in P8-14 pups (n = 15). The higher dose (800 mg/kg) of intraperitoneal sodium pentobarbital produced acceptable euthanasia results for P8-14 neonates, in which cardiac arrest occurred at an average time of 6:03 min (n = 12). Sodium pentobarbital was not evaluated in the younger age group. The neonates confirmed our earlier findings that 5% halothane inhalation delivered by a vaporizer is not an effective means of euthanasia for mice. After 20 min of breathing 5% halothane, neonatal heartbeats remained strong and regular, with an average rate of 404 bpm for P1-7 (P8-14, data not recorded). Both methods of euthanasia studied in the P1-7 group involved inhalation techniques. Although the carbon dioxide asphyxiation produced acceptable times to cardiac arrest in P1-7 pups, there was no significant difference in the time to loss of righting reflex when CO2 was compared to 5% halothane. P1-7 pups lost their righting reflexes after breathing carbon dioxide for an average of 3:44 min (n = 11) and 3:38 min (n = 6, 1 data point > 2 SD from mean) after breathing 5% halothane. Similarly, there was no significant difference in the time to loss of righting reflex of the P8-14 pups when CO2 (1:56 min, n = 15) was compared with 5% halothane (2:01 min, n = 7). It is noteworthy, however, that the time to loss of righting reflex for the P1-7 pups is nearly twice that of the older pups for both CO2 (P = 0.18E-5) and halothane (P = 0.0237). It is well known that the neonatal mouse is resistant to the effects of hypoxia (4), and this phenomenon may account for the delayed loss of righting reflexes in the younger pups.

Discussion
Selection of an appropriate euthanasia method for pregnant rodents is a difficult task, and one must consider several factors. If euthanasia is to be performed in the laboratory, the use of fetal or neonatal tissue will influence the method of euthanasia chosen. If the animal facility staff performs euthanasia, one must consider the human emotions involved and choose a method that is not offensive. In this situation, efficiency and human sensitivities must be balanced with the knowledge that the selected method will humanely and rapidly kill both the dam and the fetuses or neonates. In our study, the time-to-death (cardiac arrest) of the dams was much longer than anticipated for most euthanasia methods, although the expected variability between the animals time-to-death was not as large as anticipated (Table 2). As there was almost no variation in fetal experimental time points, we reduced the experimental group number from 10 to 7 animals. We terminated echocardiography after 20 min if the fetal or neonatal hearts continued to contract. The dams and fetuses were then euthanized using a physical method of euthanasia. In a pilot study, we found that none of the adult animals experienced cardiac arrest within 20 min of breathing 5% isoflurane delivered through a vaporizer. Further investigation of this method of euthanasia was abandoned, and halothane was used for inhalant anesthesia overdose instead of isoflurane. We were surprised that no method of euthanasia studied rapidly killed fetuses aged E14 or older. We found that fetal cardiac contraction occurred long after the dam was clinically dead. Although carbon dioxide asphyxiation appears to be an effective method of euthanasia for adult, gravid, and neonatal mice, it did not cause fetal death by our criteria. Within 1.5 to 3 min after initial exposure to 100% CO2, adult mice lost their righting reflex, exhibited muscle flaccidity, and (in some cases) were unresponsive to deep pressure applied to the hind paw. Under 100% CO2, animals displayed short-term apnea and brief muscle twitching. This agonal
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respiratory pattern continued for 1.5 to 2.5 min prior to cessation of all respiratory efforts. It is important to note that we observed asystolic neonatal pups take deep agonal breaths that could revive them. As the pup took a deep agonal breath, its body would flex convulsively like a person doing a sit-up exercise. On two occasions, a cyanotic and asystolic pup exposed to room air turned pink and a regular cardiac rhythm returned after this convulsive gasping procedure. Indeed, the pups began crawling! It was as if the agonal gasps and body contractions of the pup caused auto-cardiopulmonary resuscitation (CPR). This CPR phenomenon only occurred in pinky pups (P1-7) and was not observed in older pups (P8-14). Duffy et al. (6) found that the length of terminal gasping correlated well with the length of anoxic survival, whereas Fazekas et al. (7) observed that respiratory gasps in many neonatal animals could be evoked by peripheral stimulation. Viemari et al. (8) also described this phenomenon; their study documented the relationship of hypoxia to gasping and its role in establishing a respiratory rhythm in E18 and P0-2 pups. We recommend that when mouse pups (P1-7) are euthanized in a CO2 chamber, they should be kept in the chamber for several minutes after they are cyanotic and all signs of life have ceased. To ensure death with no chance of revival, pups should be carefully removed from the chamber with minimal stimulation and placed directly into the freezer. Perhaps a secondary physical method of euthanasia should be performed when euthanizing neonates with CO2. Our results support those in the current literature and demonstrate the fetal and neonatal mouses tolerance to hypoxia. In the CO2 chamber, the P1-7 pups took nearly twice the amount of time to lose their righting reflex (3:44 min, n = 11) when compared to pups aged 8 to 14 days (1:56 min, n = 15). A similar phenomenon occurred with pups breathing 5% halothane. The P1-7 pups averaged 3:38 min (n = 6, one value 2 SD from mean) to lose their righting reflex whereas the P8-14 pups lost their righting reflex in 2:01 min (n = 7). A number of physiological mechanisms enable young animals to tolerate hypoxia. In contrast to that in adults, any impairment to the oxygen supply of a fetal or neonatal animal triggers bradycardia, which is accompanied by shunting of blood from the periphery to central organs (4). Singer (4) describes the left shift of the fetal oxyhemoglobin dissociation curve, in which the aerobic environment at the cellular level is prolonged. Furthermore, fetal and neonatal animals have lower cerebral metabolic needs compared with those of adults (7). Body temperature is another important factor in metabolism and tolerance to hypoxia. As the fetus or neonate begins to lose body temperature, its metabolic rate slows to exert a protective effect against hypoxia. In fact, a reduction in ambient temperature is most effective in prolonging the survival time of asphyxiated mammalian neonates (7). During the study, all pups were placed on a recirculating warm water blanket when placed in a facemask for delivery of CO2, but despite our efforts, pups lost body heat and became palpably cool. We suspect that hypothermia may have prolonged the time to cardiac arrest because of the slowing of metabolic rate; therefore, we suggest that maintaining a warm environment may reduce the tolerance to hypoxia when using CO2 as the method of euthanasia for neonatal rodents. The use of 5% halothane for euthanasia did not produce acceptable results in our study. The time to cardiac arrest for all groups was either or close to 20 min or not achieved (Table 2). When comparing the time to cardiac arrest with 5% halothane with that of the other inhalational method (CO2), statistical significance was found between all groups (Students t test; dams, P = 0.205E-5; pups P1-7, P = 1.820E-15; pups P814, P = 2.199E-12) This statistical significance was not surprising because CO2 inhalation produced acceptable times to cardiac arrest, whereas 5% halothane delivered by vaporizer
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appears to be inadequate for euthanasia. Interestingly, there was no statistical significance between the effects of 5% halothane and CO2 on the righting reflex of neonates within the age groups. We believe that the data support the theory of neonatal tolerance to hypoxia. In our hands, the use of intraperitoneal pentobarbital at the published dosage of 200 mg/kg did not produce euthanasia within an acceptable period of time. In a pilot study, adult non-gravid mice dosed with 200 mg/kg intraperitoneally did not undergo cardiac arrest until approximately 15:16 7:07 min (n = 11); increasing the dose to 400 m/kg reduced the time to cardiac arrest to 5:18 2:18 min (n = 10). Time to loss of righting reflex also decreased with the increased dose, requiring an average of 2:20 (n = 10, one point not recorded) or 1:36 min (n = 10) for the 200 or 400 mg/kg dose, respectively. We were not satisfied with the time of death until we increased the pentobarbital dose to 800 mg/kg, when cardiac arrest of adult non-gravid animals occurred in 2:40 0:30 min (n = 10). Acceptable results also were achieved when 800 mg/kg was administered intraperitoneally to neonatal pups (P8-14). The P8-14 pups lost their righting reflexes in an average of 0:30 min (n = 12) and appeared to be unconscious; however, cardiac arrest did not occur until an average of 6:03 min (n = 12) from the time of administration of pentobarbital. In contrast to the non-gravid mice in the pilot study, echocardiography of pregnant females given 800 mg/kg of intraperitoneal pentobarbital was stopped at 20 min because none of the fetuses had achieved cardiac arrest. Pentobarbital-injected pregnant females took longer to lose their righting reflexes (1:51 min, n = 7) than did non-gravid adults (1:04 min, n = 10). Pregnant animals consequently took longer to develop cardiac arrest (11:02 min, n = 6, one value not recorded) than did non-gravid adults (2:40 min, n = 10). The increased length of time needed for pentobarbital to have the anticipated effect in gravid females was unexpected. One explanation for the difference in results between gravid and non-gravid mice is accidental in utero injection rather than injection into the peritoneal space. When performing the mock C-sections, we noticed that the uteruses completely filled the abdomen; thus, unintended in utero injection would be an easy mistake. Perhaps gravid females (> E14) should be given pentobarbital intravenously if it is used for euthanasia. According to echocardiography, fetuses of gravid females appeared to be unaffected by intraperitoneal pentobarbital. A plausible explanation for the lack of fetal effects of pentobarbital could be the following: although cardiac arrest in the dam did not occur until 11:02 min after injection, effective blood circulation may have stopped soon after administration. The European Union Working Partys (1) recommendations for euthanasia of experimental animals suggest that fetuses often are not anesthetized by inhalation anesthetics unless the dam is maintained under anesthesia for a prolonged period of time. The report further suggests that fetuses frequently are not anesthetized (any type of anesthesia) for potentially painful procedures. The implication is that even if drugs cross the placenta, they rarely achieve the desired (or presumed) effect. We do not recommend intraperitoneal pentobarbital as a sole method of euthanasia for fetuses > E14. Potassium chloride achieved rapid death in both gravid and nongravid mice; however, similar to pentobarbital, KCl did not appear to affect the fetuses. All echocardiography exams of gravid mice were stopped at 20 min because fetal heartbeats were still present. We speculate that sufficient KCl did not cross the maternalplacental barrier due to the rapid cardiac death of the dam. Cervical dislocation (CD), with or without anesthesia, did not result in fetal death by our criteria. Dams achieved cardiac arrest within acceptable times, but it appears that isoflurane anesthesia may have an effect on the time to cardiac arrest after cervical dislocation is applied. Dams that had CD alone achieved cardiac arrest in 2:07
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0:25 min (n = 7). Interestingly, dams that had CD under isoflurane anesthesia took longer to achieve CA (3:17 min), but the standard deviation was 2:09 min (n = 7). We have no explanation for this difference; perhaps there was variability in the CD method. When the time to CA for CD alone versus CD under anesthesia is compared using Students t test, there is no statistical significance. We concluded that cardiac arrest was not a good measure of death. Our definition of cardiac arrest was cessation of all cardiac contractions. We did not anticipate that cardiac arrest would be so difficult to assess. Although the hearts continued to beat, rates were often dramatically lower than normal, and we suspect that blood was not moving effectively in either pups or dams. When we described our observations to a pediatric cardiac specialist (9), she suggested that our observations were similar to those in human infants. In terminally ill premature human neonates for whom life support is discontinued, the heart activity may persist for an hour or more. In human medicine, a similar clinical condition called pulseless electrical activity (PEA) is a well-recognized phenomenon. Physicians characterize PEA as the presence of coordinated electrical activity of the heart muscle without peripheral pulses. In humans, PEA is associated with major cardiac insult, frequently secondary to respiratory arrest or hypoxia (10). Progressive acidosis exacerbates PEA. In humans, the heartbeat is often the last parameter to cease in death, usually because of overwhelming metabolic acidosis from major organ system failure (10). Furthermore, PEA is not considered to be a conscious state. In our pilot study, we observed electrical activity by electrocardiogram monitoring that did not always correspond to cardiac contractions in dying adult mice. After learning about PEA, we suspect that these mice may have been in PEA when their heart rate fell below the normal range. Mice in this condition (PEA) may also be considered unconscious and not suffering. Normal fetal development is a complex process. Production of normal, viable offspring is dependent on the coordination of many systems all developing at the chronologically appropriate time, developing within the correct anatomical location, and ultimately functioning properly. Until approximately E15, the developing fetus is completely dependent on a viable interface between fetal and maternal tissues. This dependence is initially accomplished through placentation. The placental vascular bed of both rodents and humans is classified as hemochorial. In this configuration, maternal blood perfuses a space lined by fetal trophoblast cells (11) where gas exchange occurs. Prior to E10, fetal mouse oxygen exchange is dependent completely on the trophoblast placental system. With minimal circulatory development, embryonic tissues are bathed in fetal fluids, and gas exchange is direct. As the fetus develops, it must contribute various components to its own survival, despite its dependence on the dam for oxygen, nutrients, and waste removal. The developing embryonic mouse heart achieves strong regular heartbeats at E9, yet blood components remain primitive, and the heart is not fully developed until E15 (5). Bifurcation of the common carotid arteries is not detectable until late pregnancy (E17) (5). After E10, embryonic red blood cells are needed to carry oxygen to sustain fetal mouse life. The importance of embryonic red blood cells to embryonic viability is demonstrated by EKLF mice, which are deficient in erythropoietic transcription factor. The EKLF fetal mice have normal embryonic hematopoiesis but fail to make definitive red blood cells. The lack of normal red blood cells causes these mice to die in utero between E13.5 and E14 (12, 13). Continued fetal viability is dependent on not only an effective placenta and maternal blood supply but also appropriate fetal contributions. Failure of any component of this complex matrix will result in fetal death. Thus, until E15, the fetus is dependent on the maternal-placental system for survival.
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In addition to other developing systems, the central nervous system is not complete in the mouse until after birth. Pain perception is considered unlikely in fetal mice less than E15 because neural development is incomplete. The mouse cerebral cortex is still differentiating at E17 (5). Fetuses cannot survive ex utero until late E18-E19 (8, 14); however, there may be strain variation (11, 15, 16). By our criteria of death, we found no method of euthanasia for fetuses that satisfied all aspects of the definition of humane euthanasia; however, for several of the physiological reasons discussed above, we believe that fetal (E15 or younger) death should be considered equivalent to death of the dam. Although the fetal hearts appeared to be beating, we suspect that the contractions were not effectively moving blood, and the fetuses may have been exhibiting PEA. With the removal of a maternal blood supply (death of dam), the fetus presumably experiences increasing hypoxia and progressive metabolic acidosis. We feel that these factors are incompatible with fetal life. Because of late brain development, we also suggest that these fetal mice are most likely not experiencing any pain or discomfort. Our assessment of fetal death was a difficult task, and the results were not as objective as we had hoped. Perhaps a future study of effective fetal hemodynamics (blood flow) or blood gas analysis may yield a more definitive parameter for determining the viability of a fetus. In summary, we do not recommend inhalation anesthesia overdose (delivered by a vaporizer) for euthanasia of laboratory rodents of any age. If CO2 asphyxiation is used for neonatal pups (P1-7), a second method of euthanasia is strongly recommended to ensure death. We also suggest that during CO2 euthanasia, keeping pups warm may decrease the time it takes for neonatal death. In our hands, the commonly used euthanasia dose of intraperitoneal pentobarbital (100 to 200 mg/kg) is not effective for euthanasia; the higher dose of 800 mg/kg intraperitoneally produces acceptable euthanasia results in non-gravid animals. If pentobarbital is used for euthanasia of gravid mice, intravenous administration may be a better route of administration to achieve the desired effect. For euthanasia of adult mice, intravenous potassium chloride, carbon dioxide asphyxiation, and cervical dislocation alone or under anesthesia are effective and acceptable methods. These findings are in agreement with Cartner et al. (17). Finally, measurement of cardiac arrest is not a good indicator of fetal death. Fetuses E14 likely die at the time of maternal death. Because cortical structures in the embryonic mouse brain are poorly differentiated during late stages of gestation (5), we feel that these fetuses are not conscious or capable of perceiving pain. Because we were unable to find a method of euthanasia that caused fetal (E1420) death by our definition (asystole), we recommend using an additional, physical method of euthanasia (cervical dislocation or decapitation) to ensure death for the fetuses of gravid mice in late gestation ( E14).

manuscript and offering many helpful suggestions. We thank Dr. Linda Leatherbury, a pediatric cardiologist, for stimulating conversations and insight into the difficulty of determining pediatric death. Finally, we would like to thank our pets, as conversations about them provided comfort to us while we performed the technical aspects of this investigation. The views, opinions, and findings contained in this report are those of the authors and do not reflect official policy or positions of the Department of Human Health Services, the National Institutes of Health, or the United States Government.

References
1. Close, B., K. Banister, V. Baumans, et al. 1996. Recommendations for euthanasia of experimental animals: part 1, compiled by the EU Working Party. Lab. Anim. 30:293-316. 2. Bertens, A. P. M. G., L. H. D. J. Booij, P. A. Flecknell, et al. 1993. Anesthesia, analgesia, and euthanasia, p. 267-298. In L.F.M. van Zutphen, V. Bauman, and A. C. Beynen (ed.), Principles of laboratory animal science. Elsevier Publishing Co., Amsterdam. 3. Close, B., K. Banister, V. Baumans, et al. 1997. Recommendations for euthanasia of experimental animals: part 2, compiled by the EU Working Party. Lab. Anim. 31:1-32. 4. Singer, D. 1999. Neonatal tolerance to hypoxia: a comparative-physiological approach. Comp. Biochem. Physiol. Part A. 123:221-234. 5. Kaufman, M. H. 1995. The atlas of mouse development, p. 183, 212, 215, 295, and 431. Academic Press, San Diego, Calif. 6. Duffy, T. E., S. J. Kohle, and R. C. Vannucci. 1975. Carbohydrate and energy metabolism in perinatal rat brain: relation to survival in anoxia. J. Neurochem 24:271-276. 7. Fazekas, J. F., F. A. D. Alexander, and H. E. Himwich. 1941. Tolerance of the newborn to anoxia. Am. J. Physiol. 134:281-287. 8. Viermari, J-C., H. Burnet, M. Bevengut, et al. 2003. Perinatal maturation of the mouse respiratory rhythm-generator: in vivo and in vitro studies. Eur. J. Neurosci. 17:1233-1244. 9. Leatherbury, L. 2003. Personal communication. 10. Martin, S. K., C. H. Shatney, J. P. Sherck, et al. 2002. Blunt trauma patients with prehospital pulseless electrical activity (PEA): poor ending assured. J. Trauma 53:876-881. 11. Adamson, S. L., Y. Lu, K. J. Whiteley, et al. 2002. Interactions between trophoblast cells and the maternal and fetal circulation in the mouse placenta. Dev. Biol. 250:358-373. 12. Nuez, B., D. Michalovich, A. Bygrave, et al. 1995. Defective haematopoiesis in fetal liver resulting from inactivation of the ELKF gene. Nature 375:316-318. 13. Perkins, A. C., A. H. Sharpe, and S. H. Orkin. 1995. Lethal bthalassaemia in mice lacking the erythroid CACCC-transcription factor EKLF. Nature 375:318-322. 14. Hatta, T., K. Moriyama, K. Nakashima, et al. 2002. The role of gp130 in cerebral cortical development: in vivo functional analysis in a mouse exo utero system. J. Neurosci. 22(13):5516-5524. 15. Bronson, F. H., C. P. Dagg, and G. D. Snell. 1968. Reproduction, p. 187-204. In E. L. Green (ed.), Biology of the laboratory mouse, 2nd ed. Dover Publications, Inc., New York. 16. Theiler, K. 1989. The house mouse. Atlas of embryonic development, 2nd ed. Springer-Verlag, Inc., New York. 17. Cartner, S. C., S. C. Barlow, and T. J. Ness. 2003. Loss of cortical function in mice following decapitation, cervical dislocation, potassium chloride injection, and CO2 inhalation. Poster Session, ACLAM Forum, Induced animal models of human diseases, Sanibel Harbour Resort, Ft. Myers, Fla., 14 to 17 May 2003.

Acknowledgments
We would like to thank Ms. Kathryn Sharer who provided pregnant female mice for this study and Mr. Daryl DesPres and Mr. Dan Schimel who shared their technical expertise. We thank Ms. Joan Timberlake (University of Maryland and Johns Hopkins University) for her critical reading of the

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CONTEMPORARY TOPICS 2004 by the American Association for Laboratory Animal Science

Volume 43, No. 5 / September 2004