The optimal choice of euthanasia method for laboratory rodents depends on a number of factors, including the scientific goals of the study, the need to minimize animal pain and/or distress, applicable guidelines and laws, the training and proficiency of personnel, and the safety and emotional needs of the personnel performing the euthanasia. This manuscript aims to provide guidance to researchers so they may select the method of euthanasia that results in minimal experimental confounds, such as the creation of artifact and alteration of tissues and analytes. Specific situations addressed include euthanasia of large numbers of rodents and euthanasia of neonates. Recent literature supports the notion of significant strain-dependent differences in response to euthanasia methods such as CO₂ inhalation. To assist researchers in selecting a strain-appropriate method of euthanasia, the authors present a summary of methodologies for assessing the effectiveness of euthanasia techniques, including elements and parameters for a scoring rubric to assess them.

Intraperitoneal (IP) injection of sodium pentobarbital (PB) is an accepted method of euthanasia for mice. However, this method has important drawbacks, including the potential for pain or misinjection. The objective of this prospective, randomized, blinded study was to determine whether intrahepatic (IH) injection of PB is more effective than IP delivery for mouse euthanasia. Secondary objectives were to: 1) determine whether IP ethanol (ET) is a suitable alternative to PB and 2) study the effect of isoflurane anesthesia on euthanasia with either PB or ET. Eighty adult CD1 mice were randomly assigned to 6 different treatment groups, were euthanized by using IP or IH injections of either PB or ET, and were either anesthetized or conscious before injection. Variables of interest were: 1) misinjection rates (based on necropsy evaluation), 2) time from injection to apnea and 3) time to cessation of heartbeat (CHB). The misinjection rate for IH injections was 93% (28/30). Two successful IH injections resulted in death within 4 s, but this method cannot be recommended due to the possibility for intrathoracic injection ( n = 4). In nonanesthetized mice, time to apnea and CHB was significantly shorter with IP ET (apnea: 72.5 s [median], CHB: 115 s) than with IP PB (apnea: 136 s, CHB: 176 s). Anesthesia at time of injection was associated with a shorter CHB time for IP PB. These data show the difficulty in achieving successful IH injections in mice, but confirm that IP ET is a viable and potentially superior alternative to IP PB. Lastly, anesthesia can shorten time to death after IP injection of PB.

Larval, or tadpole-stage Xenopus laevis frogs are a popular research model for developmental biology and disease studies. Existing euthanasia guidance documents offer recommendations for both eggs and adult stages, yet do not specifically address the larval stage. Data evaluating effective euthanasia methods for groups of X. laevis tadpoles would therefore be useful. The goal of the current study was to evaluate the efficacy of various immersion euthanasia procedures on tadpoles: tricaine methanesulfonate (MS222) at 6 g/L, eugenol at 800 μL/L and rapid chilling (2 to 4 °C). We also evaluated tadpoles at various developmental stages (NF stages 46, 47 and 49). Tadpoles (n = 70) were exposed to euthanasia solution for 15 min, and controls (n = 40) were placed in housing tank water for 15 min. All animals were then placed in recovery tanks containing housing tank water for 4 h to confirm irreversibility of each agent. Cessation of the heartbeat was assessed at the end of euthanasia solution exposure and at each hour thereafter. We found that immersion in a 6 g/L solution of MS222 resulted in 100% euthanasia of all larval stages tested. Conversely, eugenol produced variable euthanasia rates that were affected by both age group and batches of stock solutions. Rapid chilling was completely ineffective as a euthanasia method in our study. Based on our findings, we recommend MS222 as an effective and practical means of euthanizing large numbers of X. laevis tadpoles.

Jamaican fruit bats (Artibeus jamaicensis) are used as an animal model for several viruses, including Middle East respiratory syndrome virus, dengue virus, Zika virus, and Tacaribe virus. However, despite ongoing studies regarding these pathogens, little is known regarding the bats' normal physiology. In this study, phlebotomy of the propetagial (cephalic) vein was performed to establish baseline hematologic parameters in an apparently healthy, captive population of Jamaican fruit bats. Furthermore, we compared results from physically restrained and isoflurane-anesthetized bats. Our findings indicate significant increases in WBC count, lymphocytes, and monocytes in the anesthetized bats. However, RBC and platelet parameters were not different between the 2 groups. This information on the normal hematologic parameters of Jamaican fruit bats, adds to our overall understanding of the normal physiology of this species, and expands our knowledge on bat species in general.

Vaginal cytology is the most common method of monitoring the estrous cycle in rats; however, this test requires specific technical training and can be subject to interpretation. Vaginal impedance offers a quicker and less technically challenging alternative and has been used successfully to identify estrus in normally cycling breeder rats. We hypothesize that vaginal impedance can also be used to stage the estrous cycle in rats that have been given luteinizing hormone releasing hormone (LHRH) for timed mating. Vaginal impedance measurements and vaginal cytology were performed in LHRH-primed female rats (n = 36) at the expected peak of proestrus and paired with proven stud males. Breeding success was determined by gross necropsy to detect embryo implantation sites in the female rats. We found that the predictive rates of vaginal cytology and impedance measurement for proestrus were similar; however, both methods resulted in high proportions of false positive and false negative determinations (28% and 31%, respectively). We further hypothesized that females respond to LHRH at variable rates, resulting in variable times of peak proestrus. To test this, vaginal impedance measurements were performed multiple times throughout the expected day of proestrus in LHRH-primed female rats (n = 36). Females were either paired with a male 24 h after reaching the proestrus threshold (n = 18) or paired according to our standard protocol at 1300 h on the day after the expected proestrus (n = 18). Sequential measurements reduced false positive and negative rates (14% and 8%, respectively). Pregnancy rates did not differ based on the time of pairing during expected estrus. Overall, we determined vaginal impedance can be more successful than vaginal cytology at identifying proestrus in the rat, but only if multiple measurements are taken.

According to the Guide, cage change frequencies must be considered when cage density requirements are exceeded. We monitored ammonia, carbon dioxide, cage wetness, health status, and breeding parameters of trio and pair breeding cages containing CD1 mice in ventilated and static microisolation caging (4 cages per condition) daily for approximately 6 wk. Minimum cage change frequencies for each condition were determined on the basis of performance data. At 3 d after cage change, static trio and pair cages had average ammonia levels of 74 and 38 ppm. Ventilated cages remained below the 25ppm threshold reported to be potentially deleterious for mice until at least day 7 after cage change. By 7 d after cage change, ammonia levels had risen to an average of 100 ppm and 64 ppm in static trio and pair cages and to 34 ppm and 20 ppm in ventilated trio and pair cages, respectively. Ammonia levels in ventilated cages continued to rise slowly through day 14 after cage change. CO₂ levels exceeded 5000 ppm in all groups at 2 d after cage change. Pair breeders in ventilated cages took the longest—10 to 14 d—to reach cage wetness threshold scores. On day 7, pups in trio static cages were noted to have decreased and squinted eyes, whereas in ventilated cages containing trios and pairs, these clinical signs were rare to absent. Histologically, there was an increasing incidence and severity of nasal lesions in weanlings with increasing housing density and decreasing ventilation, consistent with nasal epithelial toxicity. Given these parameters, we concluded that under the current husbandry conditions, it may be necessary to change breeders in static cages more frequently than every 7 d. Additional studies are necessary to evaluate the effects of more frequent cage changes on reproductive parameters, given that cage changing is stressful for mice and affects breeding results.

Both standard and sustained-release injectable formulations of buprenorphine (Bup and BupSR, respectively) are used as preemptive analgesics, potentially affecting gas anesthetic requirements. This study tested the effects of Bup and BupSR on isoflurane requirements and confirmed that buprenorphine could reduce isoflurane requirements during a laparotomy in mice. We hypothesized that both Bup and BupSR would significantly decrease the required minimum alveolar concentration (MAC) of isoflurane. C57BL/6 mice received either isotonic crystalloid fluid (control), Bup (0.1 mg/kg), or BupSR (1.2 mg/kg) subcutaneously 10 min prior to the induction of anesthesia. Each anesthetized mouse was tested at 2 isoflurane concentrations. A 300-g noxious stimulus was applied at each isoflurane concentration, alternating between hindfeet. In addition, a subset of mice underwent terminal laparotomy or 60 min of anesthesia after injection with Bup, BupSR, or saline to ensure an appropriate surgical plane of anesthesia. Mice were maintained at the lowest isoflurane concentration that resulted in 100% of mice at a surgical plane from the aforementioned MAC experiments (control, 2.0%; Bup and BupSR, 1.7%). Analysis showed that both Bup and BupSR significantly decreased isoflurane requirements by 25.5% and 14.4%, respectively. The isoflurane MAC for the control injection was 1.80% ± 0.09%; whereas Bup and BupSR decreased MAC to 1.34% ± 0.08% and 1.54% ± 0.09%, respectively. Sex was not a significantly different between the injection groups during MAC determination. All of the mice that underwent surgery achieved a surgical plane of anesthesia on the prescribed regimen and recovered normally after discontinuation of isoflurane. Lastly, heart and respiratory rates did not differ between mice that underwent surgery and those that were anesthetized only. Bup and BupSR are MAC-sparing in male and female C57BL/6 mice and can be used for effective multimodal anesthesia.

In veterinary and human medicine, gabapentin (a chemical analog of γ-aminobutyric acid) is commonly prescribed to treat postoperative and chronic neuropathic pain. This study explored the pharmacokinetics of oral and subcutaneous administration of gabapentin at high (80 mg/kg) and low (30 mg/kg) doses as a potential analgesic in black-tailed prairie dogs (Cynomys ludovicianus; n = 24). The doses (30 and 80 mg/kg) and half maximal effective concentration (1.4 to 16.7 ng/mL) for this study were extrapolated from pharmacokinetic efficacy studies in rats, rabbits, and cats. Gabapentin in plasma was measured by using an immunoassay, and data were evaluated using noncompartmental analysis. The peak plasma concentrations (mean ±1 SD) were 42.6 ±14.8 and 115.5 ±15.2 ng/mL, respectively, after 30 and 80 mg/kg SC and 14.5 ±3.5 and 20.7 ±6.1 ng/mL after the low and high oral dosages, respectively. All peak plasma concentrations of gabapentin occurred within 5 h of administration. Disappearance half-lives for the low and high oral doses were 7.4 ± 6.0 h and 5.0 ± 0.8 h, respectively. The results of this study demonstrate that oral administration of gabapentin at low (30 mg/kg) doses likely would achieve and maintain plasma concentrations at half maximum effective concentration for 12 h, making it a viable option for an every 12-h treatment.

The goal of this study is to provide quantitative data on the ideal volume for intramuscular (IM) injections into the semimembranosus muscle of guinea pigs weighing between 320 to 410 grams. This evaluation comprised 2 experiments. The first was to assess dispersion leakage of intramuscularly injected iohexol, a radiocontrast agent commonly used in Computed Tomography (CT), based on analysis of in vivo imaging. The second used varying volumes of intramuscularly injected sodium chloride (0.9% NaCl) to assess pain and pathology associated with IM injection. Hartley guinea pigs were injected IM with varying volumes of either iohexol or sodium chloride (150, 300, 500, 1000 and 1500 μL). In the iohexol experiment, results suggest IM volumes of 150 and 300 μL remain within the target muscle. In the experiment using sodium chloride, pain and pathology did not increase as IM volume increased. The pathology noted was related to needle tract through the musculature rather than the volume size of the injectate. The results did not reveal a correlation between volume of IM 0.9% NaCl and pain levels. We conclude that volume size correlates more with precision and accuracy of delivery into the intended muscle tissue. Regarding tissue distribution, our findings also suggest that the optimal capacity for IM injection in the semimembranosus muscle should be less than 500 μL.

Accurate assessment of coagulation in porcine studies is essential. We sought to establish normal values for porcine rotational thromboelastometry (ROTEM) according to the American Society for Veterinary Clinical Pathology guidelines and to assess the effects of various preanalytical parameters on those measurements. Healthy Yorkshire-cross pigs (n = 81; 46 males and 35 females) were anesthetized. By using a 18-gauge needle attached to a vacuum phlebotomy tube, blood was acquired from the cranial vena cava. Tubes were filled in the following order: evacuation clot tube, EDTA tube, heparin tube, and 2 citrate tubes. The citrate tubes were randomly assigned to 30 min with or without constant agitation on a rocker. The following parameters were reported according to the manufacturer's recommendations: clotting time, clot formation time, α, (tangent to the clot formation curve when the clot firmness is 20 mm), clot firmness after 10 and 20 min, maximal clot firmness, maximum lysis, and lysis indexes at 30 and 45 min. Reference intervals were reported as mean ± 2 SD (parametric distribution) or 2.5th and 97.5th percentile of the population's results (nonparametric distribution). The effects of sex, sampling order, and agitation on ROTEM results were analyzed through linear regression. Neither sex nor sample agitation influenced any of the ROTEM parameters. Combined reference intervals were established for each ROTEM parameter by pooling data from the nonagitated tubes for both male and female pigs. This study is the first to establish ROTEM reference intervals from a large number of male and female adult Yorkshire-cross pigs and to provide a detailed description of preanalytical sample processing.

Lactate dehydrogenase elevating virus (LDV) continues to be one of the most common contaminants of cells and cell byproducts. As such, many institutions require that tumor cell lines, blood products, and products derived or passaged in rodent tissues are free of LDV as well as other pathogens that are on institutional exclusion lists prior to their use in rodents. LDV is difficult to detect by using a live-animal sentinel health monitoring program because the virus does not reliably pass to sentinel animals. After switching to an exhaust air dust health monitoring system, our animal resources center was able to detect a presumably long-standing LDV infection in a mouse colony. This health monitoring system uses IVC rack exhaust air dust collection media in conjunction with PCR analysis. Ultimately, the source of the contamination was identified as multiple LDV-positive patient-derived xenografts and multiple LDV-positive breeding animals. This case study is the first to demonstrate the use of environmental PCR testing as a method for detecting LDV infection in a mouse vivarium.