Pheromones—chemical signals that can elicit responses in a conspecific—are important in intraspecies communication. Information conveyed by pheromones includes the location of an animal, the presence of food or a threat, sexual attraction, courtship, and dam–pup interactions. These chemical messages remain intact and volatile even when animals, such as rodents, are housed in laboratories rather than their natural environment. Laboratory protocols, such as the cage cleaning and sanitation processes, as well as general housing conditions can alter a rodent's normal production of pheromones in both amount and type and thus may affect behavior. In addition, some procedures induce the release of alarm pheromones that subsequently alter the behavior of other rodents. To prevent pheromonal interference and stress-induced pheromonal release in their research subjects, experimenters should assess current laboratory protocols regarding cage cleaning processes, housing designs, and behavioral assays. Here we discuss how the most commonly used laboratory procedures can alter pheromonal signaling and cause confounding effects.

The lowest observed adverse effect level for bisphenol A (BPA) in mice and rats is currently poorly defined due to inconsistent study designs and results in published studies. The objectives of the current study were to (1) compare the estrogenic content of rodent diets, bedding, cages, and water bottles to evaluate their impact on the estrogenic activity of BPA and (2) review the literature on BPA to determine the most frequently reported diets, beddings, cages, and water bottles used in animal studies. Our literature review indicated that low-dose BPA animal studies have inconsistent results and that factors contributing to this inconsistency are the uses of high-phytoestrogen diets and the different routes of exposure. In 44% (76 of 172) of all reports, rodents were exposed to BPA via the subcutaneous route. Our literature review further indicated that the type of diet, bedding, caging, and water bottles used in BPA studies were not always reported. Only 37% (64 of 172) of the reports described the diet used. In light of these findings, we recommend the use of a diet containing low levels of phytoestrogen (less than 20 μg/g diet) and metabolizable energy (approximately 3.1 kcal/g diet) and estrogen-free bedding, cages, and water bottles for studies evaluating the estrogenic activity of endocrine-disrupting compounds such as BPA. The oral route of BPA exposure should be used when results are to be extrapolated to humans.

Compared with earlier editions, the eighth edition of the Guide for the Care and Use of Laboratory Animals recommends more cage floor space for female rats with litters. As such, conventional rat cages often do not supply the recommended floor space to maintain 2 adult rats and a litter in the same cage. We evaluated 2 breeding schemes using traditional cages that afford 140 in.² (903 cm²) of floor space: (1) monogamous pairs housed continuously and (2) monogamous pairs cohoused intermittently with removal of the male rat after parturition. The results did not demonstrate a significant difference between breeding schemes in generation time, number of litters per breeding pair, percentage of litters weaned, number of pups born per breeding pair, and number of pups weaned per breeding pair. However, the average weaning weight of pups was significantly higher with scheme 1 compared with scheme 2. Collectively, these results indicate continuous housing of monogamous breeding pairs may be preferable to intermittent housing when conventional cages are used.

Light is potent in circadian, neuroendocrine, and neurobehavioral regulation, thereby having profound influence on the health and wellbeing of all mammals, including laboratory animals. We hypothesized that the spectral quality of light transmitted through colored compared with clear standard rodent cages alters circadian production of melatonin and temporal coordination of normal metabolic and physiologic activities. Female nude rats (Hsd:RH-Foxn1^rnu; n = 6 per group) were maintained on a 12:12-h light:dark regimen (300 lx; lights on, 0600) in standard translucent clear, amber, or blue rodent cages; intensity and duration of lighting were identical for all groups. Rats were assessed for arterial blood levels of pO₂ and pCO₂, melatonin, total fatty acid, glucose, lactic acid, insulin, leptin, and corticosterone concentrations at 6 circadian time points. Normal circadian rhythms of arterial blood pO₂ and pCO₂ were different in rats housed in cages that were blue compared with amber or clear. Plasma melatonin levels (mean ± 1 SD) were low (1.0 ± 0.2 pg/mL) during the light phase in all groups but higher at nighttime in rats in blue cages (928.2 ± 39.5 pg/mL) compared with amber (256.8 ± 6.6 pg/mL) and clear (154.8 ± 9.3 pg/mL) cages. Plasma daily rhythms of total fatty acid, glucose, lactic acid, leptin, insulin, and corticosterone were disrupted in rats housed in blue or amber compared with clear cages. Temporal coordination of circadian rhythms of physiology and metabolism can be altered markedly by changes in the spectral quality of light transmitted through colored standard rodent cages.

Current husbandry and care guidelines for laboratory animals recommend social housing for nonhuman primates and all other social species. However, not all individuals of a social species are compatible, which can lead to psychosocial stress on certain members. Because stress affects immune responses, we undertook the present study to determine whether psychosocial stress associated with changes in the group housing of nonhuman primates affected allergic responses in a nonhuman primate model of allergic asthma. Historic records from 35 cynomolgus macaques (Macaca fascicularis) sensitive to house dust mites (HDM) and enrolled in asthma studies from 2007 to 2011 were reviewed for variations in response to aerosolized HDM that could not be explained by clinical or experimental interventions. We then compared these variations with husbandry and clinical records to determine whether the unexplained variations in responses were associated with events known to induce psychosocial stress in this species, including restructuring of social groups, temporary isolation of group members, and changes in cage or room configurations. Adult macaques in stable social groups exhibited little variation in responses to aerosolized antigen. Changes in group membership (conspecifics), cage configurations, and temporary isolation of a group member were associated with decreased responses to HDM. This attenuation lasted 2 to 3 mo on average, although some macaques showed prolonged responses. No evidence for a stress-induced increase in allergic responses was noted. These results demonstrate that acute stress in HDM-sensitive cynomolgus macaques diminishes the physiologic response to inhaled allergen.

Spironucleus muris is a protozoan that can colonize the intestinal tract of many rodent species. Although its effects on animal health and research are debated, S. muris is often included on exclusion lists for rodent facilities. Common diagnostic tests for S. muris are insensitive and typically are performed at postmortem examination. We sought to develop a PCR-based diagnostic test with sufficient sensitivity and specificity for use on fecal samples from live rodents. We designed and optimized a PCR assay that targeted the 16S-like rRNA gene of S. muris. The assay was highly specific, given that samples from mice contaminated with S. muris were PCR positive, whereas samples from mice contaminated with other protozoa were negative. The assay also was highly sensitive, detecting as few as 5 template copies per microliter diluent. All mice positive for S. muris on postmortem exams also were positive by fecal PCR. Moreover, S. muris was detected by PCR in mice negative by postmortem examination but from colonies known to be contaminated as well as in rats and hamsters. To assess protozoal loads in mice of differing ages, the PCR assay was adapted to a quantitative format. Fecal loads of S. muris were highest in 4-wk-old mice and declined with age. The PCR assay developed promises to be a highly specific antemortem diagnostic assay with higher sensitivity than that of existing postmortem tests.

Tail-tip biopsy for genotyping of genetically modified mice older than 21 d typically is performed by using isoflurane anesthesia. Isoflurane-induced changes in behavior and metabolism can result in unexpected complications and death. We investigated whether cryoanalgesia by using ethylene chloride spray would be an effective local anesthetic for tail-tip biopsies in mice. C57BL/6J mice were allocated randomly into 4 groups (n = 10 each) to receive isoflurane anesthesia with tail biopsy, ethylene chloride spray on the tip of the tail before biopsy, ethylene chloride spray without biopsy, or no treatment. Blood glucose was measured periodically in both groups undergoing tail biopsy, and the tail-pinch assay was performed in all mice that received ethylene chloride spray. Body weight, water, and food intake were measured daily for 2 wk. In both groups undergoing tail biopsy, blood glucose levels at 15 min were significantly higher than those after 2 min. This elevation was greater and more prolonged after 30 min in mice that received isoflurane compared with ethylene chloride spray. Tail-pinch latency at 20 min was greater than that after 2 min in all mice that received ethylene chloride spray. All mice gained weight, and there was no difference in food and water intake among groups. We conclude that ethylene chloride spray is an effective local anesthetic and a valuable alternative to isoflurane.

We evaluated the effect of repeated intraperitoneal administration of tribromoethanol on various parameters in C57BL/6NHsd mice. Mice (n = 68) were randomly assigned to 1 of 7 groups to receive tribromoethanol (500 mg/kg IP) on day 0 or days 0 and 8; vehicle (tert-amyl alcohol in sterile water) only on day 0 or days 0 and 8; sterile water injection on day 0 or days 0 and 8; or no treatment. A single dose of tribromoethanol failed to produce loss of pedal reflex and had no effect on median food and water consumption but altered median body weight on days 1 through 4 when compared with that in mice that received vehicle only or no treatment. Median body weight did not differ between mice that received a single dose of tribromoethanol and those that received an injection of water. Among mice given 2 doses of tribromoethanol, induction time, anesthetic duration, and recovery time varied widely. Repeated administration of tribromoethanol had no effect on median food and water consumption or body weight compared with those in controls. Median liver weight was significantly greater in mice that received 2 doses compared with a single dose of tribromoethanol. Median liver weight did not differ between untreated mice and those that received tribromoethanol. No significant organ or tissue pathology was observed in any study animal. Although tribromoethanol did not produce morbidity, mortality, or pathologic changes in treated animals, we urge caution in use of tribromoethanol in C57BL/6NHsd mice due to its variable anesthetic effectiveness.

Rabbits are widely used as an animal model for urologic research studies in which urinary bladder catheterization is required. However, standard manual retrograde urinary catheterization proved to be difficult to perform on anesthetized male rabbits in a research study, with frequent misplacement of the catheter into the vesicular gland. Attempts to reposition the catheter into the bladder after initial entry into the vesicular gland frequently failed and resulted in exclusion of the animal from the study. We assessed the normal anatomy of the lower urinary tract of male rabbits to determine the cause of catheterization misdirection into the vesicular gland and to develop a more reliable technique for urinary bladder catheterization. A modified 'digital (finger) pressure' catheterization technique was developed for successful urinary catheterization of male rabbits. Retrospective statistical analysis of 45 rabbits used for urinary catheterization studies showed improvement in the success rate of catheterization by using the digital pressure technique over the standard method of retrograde urinary catheter insertion. In addition, we here review the relevant gross and histologic anatomy of the urogenital tract of male rabbits.

As part of an enrofloxacin pharmacokinetic study, concentrations of enrofloxacin and ciprofloxacin (metabolite) were measured in various tissues (brain, heart, kidney, liver, lung, and spleen) collected from treated (subcutaneous delivery, n = 3; intramuscular delivery, n = 3; untreated controls, n = 2) adult female Xenopus laevis by using HPLC. Enrofloxacin was rapidly absorbed after administration by either route and readily diffused into all sampled tissues. Enrofloxacin and ciprofloxacin were present in the tissue samples collected at 8 h. The highest average tissue concentrations for enrofloxacin were found in kidney, with the lowest concentrations in liver. Ciprofloxacin tissue concentrations paralleled but were always lower than those of enrofloxacin for all time points and tissues except brain and kidney. These results, together with previously published pharmacokinetic data and known minimal inhibitory concentrations of common pathogenic bacteria, provide a strong evidence-based rationale for choosing enrofloxacin to treat infectious diseases in X. laevis.

Cortisol measurements of hair are becoming a valuable tool in monitoring chronic stress. To further validate this approach in domestic dogs, we compared the variability of cortisol immunoreactivity in hair with that in saliva and feces of dogs housed under constant social and physical conditions. Fecal (n = 268), and hair (n = 21) samples were collected over 3 mo from 7 dogs housed in a kennel and kept for training veterinary students in minimally invasive procedures. Salivary samples (n = 181) were collected 3 times daily twice weekly during the last month of the study. Hair and salivary samples were analyzed by enzyme immunoassay and feces by radioimmunoassay. HPLC coupled with tandem mass spectrometry was used to confirm the presence of cortisol in 3 hair samples. Variability of cortisol was compared across sample types by using repeated-measures ANOVA followed by paired t tests. Within dogs, cortisol immunoreactivity was less variable in hair than in saliva or feces. Averaged over time, the variability of fecal samples approached that of hair when feces were collected at least 4 times monthly. As predicted, the stable social and environmental condition of the dogs maintained repeatability over time and supported the hypothesis that data from hair samples reflect baseline cortisol levels. These findings indicate that determining cortisol immunoreactivity in hair is a more practical approach than is using samples of saliva or feces in monitoring the effects of long-term stressors such as social or physical environments and disease progression.

During environmental monitoring of our institution's rodent watering systems, one vivarium was found to have high bacterial loads in the reverse-osmosis (RO) automatic water system. These findings prompted evaluation of the entire RO water production and distribution system. Investigation revealed insufficient rack and RO system sanitization, leading to heavy biofilm accumulation within the system. Approximately 2 wk after discovery in the water system, one of the bacterial organisms isolated in the water supply, Sphingomonas paucimobilis, was isolated from a peritoneal abscess of a severely immunodeficient B6. Cg-Slc11a1^r Rag1^tm1Mom/Cwi mouse housed in the same vivarium, suggesting that rodents drinking from this system were being exposed randomly to fragments of biofilm. Plans were developed to sanitize the entire system. Hypercholorination was used first, followed by treatment with a combination of peracetic acid and hydrogen peroxide. Between system sanitizations, a low-level chlorine infusion was added to the system as a biocide. Heterotrophic plate counts and bacterial isolation were performed on water samples obtained before and after sanitization procedures. We here discuss the process of identifying and correcting this important water-quality issue.