In vitro fertilization (IVF) is used to produce mouse embryos for a variety of reasons. We evaluated the effect of the method of euthanasia on the fertilization rate in 2 different IVF protocols. Oocytes collected from C57BL/6J female mice euthanized by CO₂ inhalation or cervical dislocation were used in IVF with fresh sperm from either wild-type or genetically engineered C57BL/6J. Compared with CO₂ inhalation, cervical dislocation improved the resulting rate of fertilization by 18% in an IVF method using Cook media and by 13% in an IVF method using methyl-B cyclodextrin and reduced glutathione. The lower fertilization rate due to euthanasia by CO₂ inhalation was accompanied by changes in blood pH and body temperature despite efforts to minimize temperature drops. In our hands, euthanasia by cervical dislocation improved fertilization rates and consequently reduced the number of egg-donor mice required.

The Guide for the Care and Use of Laboratory Animals recommends housing rats and mice separately to reduce the potential for environmental stress to mice. The literature presents equivocal support for this practice, and housing practices vary widely. According to the existing literature, it is unclear whether visual, olfactory, or combined stimuli are responsible for stress-related changes in mouse physiology and behavior. To determine the extent to which exposure to visual, olfactory, or combined stimuli produce stress-related changes, measures of physiologic and behavioral stress were evaluated in mice after cohousing them in a room with rats. Adult, male C57BL/6NCrl mice (n = 8 per group) were randomly assigned to control, isolator cage, visual stimuli, olfactory stimuli, or visual+olfactory stimuli groups. After 15 d of exposure, body, and adrenal weights did not differ between groups. None of the groups of mice experienced significant increases in corticosterone or stress-related behavior in the open-field test after exposure to rat stimuli. These results suggest that the stress-related effects of cohousing with rats are negligible in mice and have implications for housing rats and mice in shared rooms, thereby al- lowing efficient use of research resources.

The goal of this project was to evaluate the efficacy of a porch in decreasing feces painting in captive rhesus macaques. The porch is a small extension that is hung on the outside of a monkey's primary home cage. Porches provide many potential benefits to indoor-housed macaques, including opportunities to perch above the ground, additional space, and increased field of view. Rates of feces painting, an abnormal behavior in which the animal smears or rubs feces on a surface, were compared in 3 situations: with porch enrichment, with 'smear board' enrichment (a foraging device commonly used to decrease feces painting), and without either enrichment item. Feces painting was evaluated daily by using a 5-point scale that ranged from 0, no feces present, to 4, multiple large areas of feces. We found that subjects received significantly lower feces painting scores when given porch enrichment or smear board enrichment compared with baseline. Furthermore, subjects received significantly lower feces painting scores with porch enrichment than smear board enrichment. These results demonstrate that the porch is an effective tool to decrease feces painting in captive macaques.

Few standardized methods of cleaning and disinfecting equipment in zebrafish facilities have been published, even though the effectiveness of these procedures is vital to preventing the transmission of pathogenic organisms. Four chemical disinfectants and rinsing with municipal tap water were evaluated for their ability to disinfect nets used to capture zebrafish. The disinfectants included benzalkonium chloride+methylene blue, sodium hypochlorite, chlorine dioxide, and potassium peroxymonosulfate+sodium chloride for a soak time of 5 or 30 min. Disinfection effectiveness was evaluated by using an ATP-based system that measured the reduction in absolute number and percentage of relative light units. In addition, nets were cultured aerobically on blood and MacConkey agar plates to determine the number of bacteria remaining after disinfection procedures. Soaking nets in sodium hypochlorite for 30 min and in potassium peroxymonosulfate+sodium chloride for 5 or 30 min were effective means of disinfection, according to at least 90% reduction in the number of relative light units and no bacterial growth after cleaning. These results will aid facility managers, veterinarians and investigators in selecting net cleaning and disinfection protocols.

Rodent pinworms persist in many institutions, suggesting deficiencies in eradication and diagnostic processes. When pinworms are detected, treatment success is common, but false-negative test results during health surveillance or after treatment likely contribute to the continued presence of this parasite. PCR testing is not always practical, and increased information regarding the life cycle and general epidemiology of pinworm infestations could improve the sensitivity of traditional nonPCR detection methods and improve eradication efforts. We therefore investigated a pinworm (Syphacia muris) infestation in Sprague–Dawley rats (Rattus norvegicus) to develop a more accurate testing strategy. In addition, we sought to determine the duration of egg viability by using an in vitro hatching protocol to assess environmental persistence. Finally, we tested the ovicidal efficacy of a disinfectant used at our institution. Eggs were shed in higher numbers in the midafternoon as compared with other times of the day, and the sex of the host had no consistent effect on egg shedding. Egg shedding showed periodicity over time, with shedding decreasing to 0 at 2- to 3-wk intervals. Neither cecal examination nor tape tests alone reliably predicted pinworm infestation, and results of the 2 tests did not necessarily coincide. Eggs aged for as long as 7 mo remained viable, indicating a potential for recontamination from the environment. Finally, gaseous chlorine dioxide was an effective ovicidal agent, with a kill rate of 99.7%. These results suggest that strategies for S. muris eradication can be optimized to increase detection and elimination.

We used high-fidelity PCR to amplify a portion of the small ribosomal subunit (18S rRNA) of Pseudocapillaroides xenopi, a nematode that parasitizes the skin of Xenopus laevis. The 1113-bp amplicon was cloned, sequenced, and aligned with sequences from 22 other nematodes in the order Trichocephalida; Caenorhabditis elegans was used as the outgroup. Maximum-likelihood and Bayesian inference phylogenetic analyses clustered P. xenopi in a clade containing only members of the genus Capillaria. Our analyses support the following taxonomic relationships: 1) members of the family Trichuridae form a clade distinct from those in the family Trichocephalida; 2) members of the genera Trichuris and Capillaria form 2 distinct clades within the family Trichuridae; and 3) the genus Trichuris includes 2 distinct clades, one representing parasites that infect herbivores and the other representing parasites that infect omnivores and carnivores. Using 18S rRNA sequence unique to P. xenopi, we developed a Taq Man quantitative PCR assay to detect this P. xenopi sequence in total DNA isolated from aquarium sediment. The assay's lower limit of detection is 3 copies of target sequence in a reaction. The specificity of our assay was validated by using negative control DNA from 9 other pathogens of Xenopus. Our quantitative PCR assay detected P. xenopi DNA in the sediment of 2 of 12 aquaria from the source institution of the specimen used to develop the assay; these aquaria had been treated with ivermectin 6 mo previously.

Butorphanol and buprenorphine are common analgesics used in laboratory mice. Inadvertent attenuation of the antinociceptive effects of these analgesics via the administration of an anesthetic reversal agent could result in postprocedural pain and distress, with subsequent negative effects on animal welfare, study outcomes, and regulatory compliance. This study was undertaken to determine whether atipamezole reverses ketamine–dexmedetomidine anesthesia and alters the antinociceptive effects of butorphanol and buprenorphine in female C57BL/6J mice. Atipamezole reliably reversed the anesthetic effects of ketamine–dexmedetomidine, and mice were ambulatory 17.4 ± 30.6 min after administration of the α₂-adrenoreceptor antagonist. Atipamezole alone had no significant effect on tail-flick latency and did not alter the antinociceptive properties of butorphanol or low-dose (0.05 mg/kg) or high-dose (0.1 mg/kg) buprenorphine in female C57BL/6J mice. After reversal of ketamine–dexmedetomidine anesthesia, tail-flick latency at 30, 60, and 150 min after analgesic treatment differed significantly between mice treated with atipamezole alone and those given atipamezole followed by butorphanol or high-dose buprenorphine. These results suggest that the analgesic effects of butorphanol and buprenorphine are not affected by atipamezole. Buprenorphine (0.1 mg/kg) administered 30 min prior to or at the time of anesthesia resulted in a greater magnitude of antinociception after antagonism of anesthesia than when given at the time of reversal. Given these results, we recommend the use of ketamine–dexmedetomidine anesthesia with buprenorphine administered either preemptively or at the time of anesthetic induction to provide a defined period of surgical anesthesia that is effectively reversed by atipamezole.

Extending a surgical plane of anesthesia in mice by using injectable anesthetics typically is accomplished by repeat-bolus dosing. We compared the safety and efficacy of redosing protocols administered either during an anesthetic surgical plane (maintaining a continuous surgical plane, CSP), or immediately after leaving this plane (interrupted surgical plane, ISP) in C57BL/6J mice. Anesthesia was induced with ketamine, xylazine, and acepromazine (80, 8, and 1 mg/kg IP, respectively), and redosing protocols included 25% (0.25K), 50% (0.5K), or 100% (1.0K) of the initial ketamine dose or 25% (0.25KX) or 50% (0.5KX) of the initial ketamine–xylazine dose. In the ISP group, the surgical plane was extended by 13.8 ± 2.1 min (mean ± SEM) after redosing for the 0.25K redose with 50% returning to a surgical plane, 42.7 ± 4.5 min for the 0.5K redose with 88% returning to a surgical plane, and 44.3 ± 15.4 min for the 1.0K redose, 52.8 ± 7.2 min for the 0.25KX redose, and 45.9 ± 2.9 min for the 0.5KX redose, with 100% of mice returning to a surgical plane of anesthesia in these 3 groups. Mortality rates for ISP groups were 0%, 12%, 33%, 12%, and 18%, respectively. Mice in CSP groups had 50% mortality, independent of the repeat-dosing protocol. We recommend redosing mice with either 50% of the initial ketamine dose or 25% of the initial ketamine–xylazine dose immediately upon return of the pedal withdrawal reflex to extend the surgical plane of anesthesia in mice, optimize the extension of the surgical plane, and minimize mortality.

The opioid buprenorphine has been shown to provide adequate postoperative analgesia in both companion and laboratory animals. However, its use is still hindered by the need for multiple parenteral injections to achieve continuous analgesia. The purpose of the current study was to conduct a pharmacokinetic analysis of 2 new long-acting formulations of buprenorphine—an injectable sustained-release buprenorphine (SRB) and a transdermal buprenorphine (TDB) patch—in healthy Göttingen minipigs by using liquid chromatography–electrospray ionization–tandem mass spectrometry. Administration of 0.18 mg/kg SC SRB and 30 μ g/h TDB achieved AUC_0-Tlast of 221.6 ± 26.8 and 25.2 ± 3.9 ng × h/mL, respectively, compared with 9.7 ± 1.4 ng*h/mL for 0.02 mg/kg IV buprenorphine. By using a hypothesized therapeutic plasma buprenorphine concentration threshold of 0.1 ng/mL, therapeutic concentrations were achieved at the first study time point (5 to 30 min) and lasted an average of 8.0 ± 1.3 h for intravenous buprenorphine and 264.0 ± 32.2 h for SRB. TDB achieved therapeutic concentrations in 12 to 24 h after patch application, which lasted until the patch was removed at 72 h. The results of this study suggest that SRB and TDB are long-acting alternatives for pain management, and their use could decrease animal handling and stress, thereby simplifying pain management and improving welfare in laboratory swine.

Obtaining sufficient quantities of milk from NHP is necessary for pharmacologic and immunologic studies required for the development and safety assessment of drugs and vaccines to be used in the maternal–infant setting. We previously induced lactation in nonpregnant female rhesus macaques (RM, Macaca mulatta) and African green monkeys (AGM, Chlorocebus sabaeus) for studies of immune responses in milk, but the volume collected was variable. To improve lactation induction protocols for nonbreeding nonhuman primates, we investigated serum hormone levels and collection protocols in AGM and RM. Here, we correlated milk volume with serum levels of endogenous and administered hormones: estradiol, prolactin, progesterone, and medroxyprogesterone in RM and AGM. We also investigated whether age, parity or the timing of milk collections were associated with the volume of milk collected from the AGM and RM in which lactation was induced by using exogenous hormones. We found an inverse correlation with serum estradiol and milk volume in the RM but no significant correlation between milk volumes and the remaining serum hormone levels in the induced RM or AGM. In addition, HIL AGM had higher peak estradiol levels than did naturally lactating AGM. A revised estradiol-sparing protocol increased milk volumes in the AGM. In addition, milk volume in RM was greater in the morning than the afternoon. In conclusion, we have refined a lactation induction protocol in nonpregnant primates, which is a needed alternative to using nursing primates for the assessment of drug levels and immune responses in milk.

Fluoxetine is used to treat a number of psychiatric conditions in humans and behavioral problems in animals. Its use in pregnancy must balance maternal benefit with potential risk to the fetus. Knowledge of adult and fetal drug disposition can assist clinicians in selecting therapy that minimizes adverse effects to the fetus. Nonhuman primate models are used frequently in drug dose-translation studies, and pregnancy in baboons has many similarities to human pregnancy. Accordingly, pharmacokinetic analysis of a series of fluoxetine and norfluoxetine administrations to pregnant baboons was performed. The mean maternal baboon steady-state clearance of fluoxetine (42 mL/min/kg) was considerably higher than that in humans. Norfluoxetine, the major active metabolite, had a higher metabolite-to-drug ratio (8.7) than that found in humans, particularly with oral dosing. These results are consistent with more extensive metabolism in baboons than in humans and leads to a higher clearance than would be expected from allometric scaling. Fetal-to-maternal fluoxetine and norfluoxetine ratios under steady-state conditions were similar to those in humans, with fetal concentrations of fluoxetine 42% and norfluoxetine 47% of maternal concentrations. The fetal clearance of fluoxetine (303 ± 176 mL/min) and norfluoxetine (450 mL/min) exceeded reported placental blood flow. Understanding these species-associated differences in metabolism is a prerequisite to extrapolating data between species. Nonetheless, nonhuman primates are likely to remain valuable models for pharmacokinetic studies during pregnancy, particularly those directed toward fetal neurodevelopmental effects. Our results also are applicable to determining appropriate dosing of nonhuman primates in clinical settings.

Modern rodent colonies are housed in individually ventilated cages to protect the animals from contamination with adventitious pathogens. Standard health monitoring through soiled-bedding sentinels does not always detect infections, especially in the context of low pathogen prevalence. Recently proposed alternatives include analyzing environmental samples from the cages or rack exhaust by PCR to improve the detection of rodent pathogens but optimal sampling strategies have not yet been established for different microorganisms. Although generally very sensitive and specific, these molecular assays are not foolproof and subject to false-positive and –negative results and should always be interpreted cautiously with an overall understanding of the intrinsic controls and all the variables that may affect the results. Here, we report a limited Aspiculuris tetraptera outbreak in a mouse barrier facility that was detected by fecal PCR in sentinels and confirmed by fecal flotation and direct cecal examination of both sentinels and colony animals. The outbreak led to a widespread survey of all facilities for pinworms by using environmental PCR from ventilated rack exhaust plenums. Environmental PCR suggested an unexpected widespread contamination of all ventilated racks holding nonautoclaved cages, but results could not be confirmed in sentinel or colony animals by fecal flotation, cecal and colonic examination, or cage PCR testing. After additional investigation, the unexpected environmental PCR results were confirmed as false-positive findings due to the nonspecificity of the assay, leading to the amplification of rhabditid nematodes, which are not infectious in rodents but which contaminated the corncob bedding.