Although efficient transcervical transfer of embryos in mice would provide many advantages over a surgical method, the low success rate of transcervical transfer has hampered its acceptance and use. Here, we describe a novel device and protocol for transcervical embryo transfer in mice. Blastocysts from CD1 female mice were transferred into the uteri of 2.5-d pseudopregnant CD1 mice by using this method resulted in the successful development of 66.7% to 73.5% of the transferred blastocysts into live-born fetuses. Our method is as efficient as surgical embryo transfer yet is much simpler, easier, and markedly less traumatic to the recipient. In addition, our method provides hygienic and economic advantages and conforms to the principles of humane experimental technique. More importantly, our method provides a model for studying transcervical embryo transfer in cattle, other large animals, and humans.

In the past decade, the use of genetically engineered rats has increased exponentially; therefore, the ability to perform embryo transfer (ET) in rats to rederive, reanimate, or create mutant rat lines is increasingly important. However, the successful generation of pseudopregnant female rats for ET represents a limiting factor. We here evaluated the subcutaneous administration of 40 μg luteinizing hormone releasing hormone agonist (LHRHa) for estrus synchronization during the development and implementation of a rat ET program. Our first experiment assessed endogenous estrus cycling patterns by examining vaginal cytology without administration of LHRHa in 5-wk-old peripubertal Sprague–Dawley female rats. These rats then received LHRHa at approximately 7 wk of age; 57% of the rats were synchronized in proestrus or estrus as assessed by vaginal cytology 96 h later. In a second experiment, 8-wk-old virgin, unmanipulated Sprague–Dawley female rats received LHRHa; 55% were synchronized in proestrus or estrus 96 h later. Copulatory plugs were confirmed in 28% and 82% of the rats that had been synchronized in the first and second experiments, respectively, and mated with vasectomized male rats. Embryo transfer surgery was performed, and live pups were born from both fresh and cryopreserved transgenic rat embryos. Our results indicate that subcutaneous administration of 40 μg LHRHa followed by examination of vaginal cytology 96 h later is an effective technique to generate multiple pseudopregnant recipient rats for use in an ET program.

The use of a commercial 4-drug diet has been shown to eradicate Helicobacter spp. from immunocompetent mice and those with innate immunodeficiencies. However the efficacy of this diet has not been confirmed in mice with altered adaptive immunity. We hypothesized that an 8-wk treatment with medicated diet would eradicate H. hepaticus and H. typhlonius from young naturally infected nude and Rag1 mice lacking functional T cells (Foxn1^nu) or T and B cells (B6.129S7-Rag1^tm1Mom/J), respectively. We evaluated helicobacter status, body weight, and gross and histologic changes between medicated and control diet in groups of infected and uninfected mice throughout treatment and at 8 wk after treatment completion. Initial infection status was confirmed by fecal PCR at weaning and 3 wk later, with study initiation in 7-wk-old mice. PCR testing demonstrated that independent of strain and sex, all treated mice tested negative for Helicobacter spp. after 4 wk of treatment and remained negative for the duration of the study. Irrespective of infection status, nude and Rag1 mice fed 8 wk of medicated diet gained less weight than did their untreated controls. Both strains normalized body weight while on control diet for the 8 wk after treatment. Mice fed medicated diet developed severe gastroesophageal hyperkeratosis, suggestive of reduced feed consumption, and enlarged ceca. These conditions improved or resolved after the return to control diet. This report is the first to demonstrate the efficacy and physical effects of providing medicated diet for the eradication of Helicobacter spp. from mice with adaptive immune deficiencies.

Environmental enrichment in rodents may improve animal well-being but can affect neurologic development, immune system function, and aging. We tested the hypothesis that wood block enrichment affects the interpretation of traditional and transcriptomic endpoints in an exploratory toxicology testing model using a well-characterized reference compound, cyclophosphamide. ANOVA was performed to distinguish effects of wood block enrichment separate from effects of 40 mg/kg cyclophosphamide treatment. Biologically relevant and statistically significant effects of wood block enrichment occurred only for body weight gain. ANOVA demonstrated the expected effects of cyclophosphamide on food consumption, spleen weight, and hematology. According to transcriptomic endpoints, cyclophosphamide induced fewer changes in gene expression in liver than in spleen. Splenic transcriptomic pathways affected by cyclophosphamide included: iron hemostasis; vascular tissue angiotensin system; hepatic stellate cell activation and fibrosis; complement activation; TGFβ-induced hypertrophy and fibrosis; monocytes, macrophages, and atherosclerosis; and platelet activation. Changes in these pathways due to cyclophosphamide treatment were consistent with bone marrow toxicity regardless of enrichment. In a second study, neither enrichment nor type of cage flooring altered body weight or food consumption over a 28-d period after the first week. In conclusion, wood block enrichment did not interfere with a typical exploratory toxicology study; the effects of ingested wood on drug level kinetics may require further consideration.

Hair loss is a common problem in captive macaque colonies. A potential factor is the possible influence of stressful environments in the development of hair loss. We examined the relationship between hair loss and chronic hypothalamic–pituitary–adrenal (HPA) axis activity by measuring cortisol in hair. Adult male and female rhesus macaques housed at 3 primate facilities in the United States were screened for degree of hair loss and observed for evidence of hair-plucking behavior. Hair samples and photographic data were obtained from 99 subjects, none of which were hair-pluckers. Macaques with greater than 30% hair loss (alopecia group) showed higher concentrations of hair cortisol than did those with less than 5% hair loss (control group), a finding that was unrelated to age, body weight, or the month in which the sample was collected. Hair loss scores were positively correlated with hair cortisol levels across all monkeys and within the alopecic group alone. In addition, the strong relationship between hair cortisol and alopecia was noted in 2 but not the third facility. Friction with cage surfaces appeared to contribute to hair loss in 18 monkeys. These findings suggest that stress may be one of several factors related to hair loss in some captive nonhuman primates, although whether this relationship is causal or merely correlational is unclear. Moreover, the source of the additional cortisol in the hair of alopecic monkeys (that is, from the circulation or from local synthesis in the skin) remains to be determined.

Jacketing of nonhuman primates (NHP) is a commonly used practice in the laboratory animal setting to support data collection with reduced direct human-to-animal interaction. NHP often wear jackets for several weeks, potentially leading to the formation of dermal lesions ranging from mild alopecia to severe full-thickness ulceration. We sought to evaluate the addition of a commercially available undershirt for primates as a possible refinement practice for our jacketed rhesus macaques. In this study, we compared the lesion count, location, and severity and differences in rectal body temperature between jacketed NHP with undershirts with those wearing the jackets alone. In both groups, most lesions (75%) were located at either the underarm or shoulder. The percentages of total lesions in the back and neck were lower in jacketed NHP that wore undershirts than in those that did not. In addition, the estimated odds of increased severity scores in jacketed NHP without undershirts was 1.80 times that for NHP that wore both jackets and undershirts. Both groups of NHP showed a significant decrease in dermal scores with time, indicating adaptation to the jackets with or without undershirts. However, there was no statistically significant decrease in lesion count, severity, or location in jacketed NHP that wore undershirts compared with those that did not.

Personal protective equipment (PPE) frequently is used to reduce the risk of spreading adventitial diseases in rodent colonies. The PPE worn often reflects the historic practices of the research institution rather than published performance data. Standard PPE for a rodent facility typically consists of a disposable hair bonnet, gown, face mask, shoe covers, and gloves, which are donned on facility entry and removed on exiting. This study evaluated the effect of a reduced PPE protocol on disease spread within an endemically infected mouse colony. In the reduced protocol, only the parts of the wearer that came in direct contact with the mice or their environment were covered with PPE. To test the reduced PPE protocol, proven naïve mice were housed in a facility endemically infected with murine norovirus and mouse hepatitis virus for 12 wk. During that time, routine husbandry operations were conducted by using either the standard or reduced PPE protocols. All study mice remained free of virus antibody when reduced PPE was implemented. These results indicate that reduced PPE is adequate for disease containment when correct techniques for handling microisolation caging are used. Reducing the amount of PPE used in an animal facility affords considerable cost savings yet limits the risk of disease spread.

In North America, the biomedical research community faces social and economic challenges to nonhuman primate (NHP) importation that could reduce the number of NHP available for research needs. The effect of such limitations on specific biomedical research topics is unknown. The Association of Primate Veterinarians (APV), with assistance from the Centers for Disease Control and Prevention, developed a survey regarding biomedical research involving NHP in the United States and Canada. The survey sought to determine the number and species of NHP maintained at APV members' facilities, current uses of NHP to identify the types of biomedical research that rely on imported animals, and members' perceived trends in NHP research. Of the 149 members contacted, 33 (22%) replied, representing diverse facility sizes and types. Cynomolgus and rhesus macaques were the most common species housed at responding institutions and comprised the majority of newly acquired and imported NHP. The most common uses for NHP included pharmaceutical research and development and neuroscience, neurology, or neuromuscular disease research. Preclinical safety testing and cancer research projects usually involved imported NHP, whereas research on aging or degenerative disease, reproduction or reproductive disease, and organ or tissue transplantation typically used domestic-bred NHP. The current results improve our understanding of the research uses for imported NHP in North America and may facilitate estimating the potential effect of any future changes in NHP accessibility for research purposes. Ensuring that sufficient NHP are available for critical biomedical research remains a pressing concern for the biomedical research community in North America.

Pancuronium is a long-duration neuromuscular blocking drug (NMBD) that has been used in anesthetized rabbits at 0.1 mg/kg. However, there are limited data regarding the time course for recovery from this dose either spontaneously or with pharmacologic reversal. Here we defined the potency, onset, and recovery characteristics for the intermediate-duration NMBD cisatracurium and CW002 (a novel cysteine-inactivated molecule) in the rabbit, and test the hypothesis that these drugs may be alternatives to 0.1 mg/kg pancuronium for survival procedures. New Zealand white rabbits anesthetized with isoflurane were studied in a cross-over design. Potencies of cisatracurium and CW002 were defined as the effective dose for 95% depression of evoked muscle twitch (ED₉₅). Responses to 3×ED₉₅ were used to define onset (time to maximal effect), recovery index (RI; time from 25% to 75% recovery of twitch), and duration (time to complete recovery). Responses to all drugs were determined with and without reversal by neostigmine–glycopyrrolate or L-cysteine. CW002 was 4-fold more potent than was cisatracurium, but their onset, RI, and duration were similar. Pancuronium had similar onset and RI but longer duration, compared with cisatracurium and CW002. Reversal shortened the recovery index and duration for all 3 drugs. At 3×ED₉₅, cisatracurium and CW002 had the same onset as did standard-dose pancuronium, but durations were shorter and more predictable. In addition, CW002 can be reversed without the potential side effects of cholinergic manipulation. We conclude that cisatracurium and CW002 are viable alternatives to pancuronium for survival studies in rabbits.

Isoflurane, ketamine, and propofol are common anesthetics in human and nonhuman primate medicine. However, scant normative data exist regarding the response of neonatal macaques to these anesthetics. We compared the effects of isoflurane, ketamine, and propofol anesthesia on physiologic parameters in neonatal rhesus macaques. Neonatal rhesus macaques (age, 5 to 7 d) were exposed to isoflurane (n = 5), ketamine (n = 4), propofol (n = 4) or no anesthesia (n = 5) for 5 h. The anesthetics were titrated to achieve a moderate anesthetic plane, and heart rate, blood pressure, respiratory rate, end tidal carbon dioxide, oxygen saturation, and temperature were measured every 15 min. Venous blood samples were collected to determine blood gases and metabolic status at baseline, 0.5, 2.5, and 4.5 h after induction and at 3 h after the end of anesthesia. Compared with ketamine, isoflurane caused more hypotensive events and necessitated the administration of increased volumes of intravenous fluids to support blood pressure throughout anesthesia; no significant differences were observed between the isoflurane and propofol groups for these parameters. In addition, isoflurane resulted in a significantly shorter average time to extubation, compared with both ketamine and propofol. Due to supportive care, other physiologic variables remained stable between anesthetic regimens and throughout the 5-h exposure. These data improve our understanding of the effects of these 3 anesthetics in neonatal rhesus macaques and will aid veterinarians and researchers as they consider the risks and benefits of and resources required during general anesthesia in these animals.

Although antibiotics frequently are added to the drinking water of mice, this practice has not been tested to confirm that antibiotics reach therapeutic concentrations in the plasma of treated mice. In the current investigation, we 1) tested the stability of enrofloxacin and doxycycline in the drinking water of adult, female C57BL/6 mice; 2) measured the mice's consumption of water treated with enrofloxacin, doxycycline, amoxicillin, or trimethoprim–sulfamethoxazole; and 3) used HPLC to measure plasma antibiotic concentrations in mice that had ingested treated water for 1 wk. Plasma concentrations of antibiotic were measured 1 h after the start of both the light and dark cycle. The main findings of the study were that both enrofloxacin and nonpharmaceutical, chemical-grade doxycycline remained relatively stable in water for 1 wk. In addition, mice consumed similar volumes of antibiotic-treated and untreated water. The highest plasma antibiotic concentrations measured were: enrofloxacin, 140.1 ± 10.4 ng/mL; doxycycline, 56.6 ± 12.5 ng/mL; amoxicillin, 299.2 ± 64.1 ng/mL; and trimethoprim–sulfamethoxazole, 5.9 ± 1.2 ng/mL. Despite the stability of the antibiotics in the water and predictable water consumption by mice, the plasma antibiotic concentrations were well below the concentrations required for efficacy against bacterial pathogens, except for those pathogens that are exquisitely sensitive to the antibiotic. The findings of this investigation prompt questions regarding the rationale of the contemporary practice of adding antibiotics to the drinking water of mice for systemic antibacterial treatments.

During the past several years, trauma resuscitation in human patients has evolved from decreased use of crystalloids to increased use of blood products. Of high interest is the role of platelets in trauma resuscitation. Because conducting prehos- pital resuscitation in human trauma patients is very difficult, swine are often the animal model of choice for such studies because their coagulation and hemodynamic systems are similar to those in humans. However, consistent production of sufficient swine platelets for such studies has not previously been achieved. We developed a method for producing swine platelets by using standard human techniques and equipment. We assessed pH, pO₂, pCO₂, lactate, thromboelastography, and platelet aggregation over 5 d of storage to determine whether the swine platelet product met the American Association of Blood Banks (AABB) standards for transfusion. Swine platelets met AABB standards at 24 h but not at later time points. In addition, we fluorescently labeled nonautologous platelets and then measured their percentage recovery over 5 h (the time used in subsequent experimental studies) when transfused into a recipient pig. We showed that 80% of the platelets stored for 24 h remained in the circulation and increased the recipient pigs' thromboelastographic responses, indicating that the platelets were viable and active. Therefore, swine platelets stored for 24 h by using standard human products met the AABB criteria and were functional.