Most biomedical facilities that use rhesus macaques (Macaca mulatta) limit the amount of blood that may be collected for experimental purposes. These limits typically are expressed as a percentage of blood volume (BV), estimated by using a fixed ratio of blood (mL) per body weight (kg). BV estimation ratios vary widely among facilities and typically do not factor in variables known to influence BV in humans: sex, age, and body condition. We used indicator dilution methodology to determine the BV of 20 adult rhesus macaques (10 male, 10 female) that varied widely in body condition. We measured body composition by using dual-energy X-ray absorptiometry, weight, crown-to-rump length, and body condition score. Two indicators, FITC-labeled hydroxyethyl starch (FITC–HES) and radioiodinated rhesus serum albumin (¹²⁵I-RhSA), were injected simultaneously, followed by serial blood collection. Plasma volume at time 0 was determined by linear regression. BV was calculated from the plasma volume and Hct. We found that BV calculated by using FITC–HES was consistently lower than BV calculated by using ¹²⁵I-RhSA. Sex and age did not significantly affect BV. Percentage body fat was significantly associated with BV. Subjects categorized as having 'optimal' body condition score had 18% body fat and 62.1 mL/kg BV (by FITC–HES; 74.5 mL/kg by ¹²⁵I-RhSA). Each 1% increase in body fat corresponded to approximately 1 mL/kg decrease in BV. Body condition score correlated with the body fat percentage (R² = 0.7469). We provide an equation for calculating BV from weight and body condition score.

An important task facing both researchers and animal core facilities is producing sufficient mice for a given project. The inherent biologic variability of mouse reproduction and litter size further challenges effective research planning. A lack of precision in project planning contributes to the high cost of animal research, overproduction (thus waste) of animals, and inappropriate allocation of facility resources. To examine the extent daily prepartum maternal weight gain predicts litter size in 2 commonly used mouse strains (BALB/cJ and C57BL/6J) and one mouse stock (Swiss Webster), we weighed ≥ 25 pregnant dams of each strain or stock daily from the morning on which a vaginal plug (day 0) was present. On the morning when dams delivered their pups, we recorded the weight of the dam, the weight of the litter itself, and the number of pups. Litter sizes ranged from 1 to 7 pups for BALB/cJ, 2 to 13 for Swiss Webster, and 5 to 11 for C57BL/6J mice. Linear regression models (based on weight change from day 0) demonstrated that maternal weight gain at day 9 (BALB/cJ), day 11 (Swiss Webster), or day 14 (C57BL/6J) was a significant predictor of litter size. When tested prospectively, the linear regression model for each strain or stock was found to be accurate. These data indicate that the number of pups that will be born can be estimated accurately by using maternal weight gain at specific or stock-specific time points.

Limited guidance is available on practical approaches for maintaining genetic diversity in large NHP colonies that support biomedical research, despite the fact that reduced diversity in these colonies is likely to compromise the application of findings in NHP to human disease. In particular, constraints related to simultaneously housing, breeding, and providing ongoing veterinary care for thousands of animals with a highly complex social structure creates unique challenges for genetic management in these colonies. Because the composition of new breeding groups is a critical component of genetic management, here we outline a 3-stage protocol for forming new breeding groups of NHP that is aimed at maximizing genetic diversity in the face of frequent restrictions on age, sex, and numbers of animals per breeding group. As an example application of this protocol, we describe optimal combinations of rhesus macaques from an analysis of candidate animals available for breeding in July 2013, selected from among the approximately 4000 macaques maintained at the Oregon National Primate Research Center. In addition, a simulation study to explore the genetic diversity in breeding groups formed by using this protocol, indicated an approximate 10-fold higher genome uniqueness, 50% lower mean kinship, and an 84-fold lower mean inbreeding coefficient among potential offspring within groups, when compared with a suboptimal group design. We conclude that this protocol provides a practical and effective approach to breeding group design for colony managers who want to prevent the loss of genetic diversity in large, semiisolated NHP colonies.

To determine how housing density and ambient temperature interact to influence the physiology and behavior of mice, we systematically varied housing density (1 to 5 mice per cage) and ambient temperature (22, 26, or 30 °C) and measured effects on body weight, food intake, diurnal patterns of locomotor activity and core temperature, fecal corticosterone, and serum cytokine and adipokine panels. Temperatures inside cages housing 5 mice were 1 to 2 °C higher than the ambient temperature. As the housing density decreased, in-cage temperatures began to fall at a density of 2 or 3 mice per cage and did not differ from ambient temperature at 1 mouse per cage. Ambient temperature, but not housing density, significantly affected food intake. Although neither ambient temperature nor housing density affected core temperature or activity, hyperthermia and behavioral activation occurred during the 12-h period after cage change. Fecal concentrations of corticosterone metabolites and serum cytokines, chemokines, insulin, and leptin were not influenced by cage density and were only sporadically influenced by ambient temperature. Our data document that the number of mice housed per cage influences the intracage environmental conditions and that ambient temperature influences food intake even when temperatures are within or near recommended or thermoneutral ranges. We conclude that investigators should be cautious when changing the number of mice housed in a cage over the course of a study, because doing so significantly alters the cage environment to which remaining mice are exposed.

Few studies have evaluated the long-term effects of providing environmental resources to mice. This consideration is important given that mice are often maintained in vivaria for months. We evaluated the effects of providing simple cage resources (wood wool, cotton nesting material, a plastic tunnel, and oat cereal) compared with standard housing (solid-bottom cage with hardwood chips) to group-housed adult male and female C57BL/6 and BALB/c mice (n = 20/sex/strain/group) over 6 mo to determine whether these resources had a lasting effect on animal physiology, anatomy, and behavior. Body weights increased in all groups over time but were proportionately higher in male and female BALB/c mice housed in resource-supplemented environments. Throughout the study, adding environmental resources had no effect on hematology and lymphocyte subsets, fecal corticoid metabolite levels, response to LPS injection, or dendritic spine length or density. Strain- or sex×environmentspecific changes occurred in dark–light activity and thermal nociceptive responses. Dominant agonistic behaviors, abnormal conspecific sexual behaviors, and social nonagonistic behaviors demonstrated sex and strain×environment interactions such that fewer maladaptive social behaviors were noted in mice that were provided with environmental resources. This association was particularly evident in male mice of both strains in resource-supplemented environments. A small but significant increase in brain weight:body weight ratios occurred in mice in resource-supplemented environments. Under the conditions evaluated here, consistent use of simple environmental resources had a positive long-term effect on the behavioral wellbeing of male and female BALB/c and C57BL/6 mice yet minimally affected other aspects of murine physiology and neuroanatomy.

Agonistic behavior in group-housed male mice is a recurring problem in many animal research facilities. Common management procedures, such as the removal of aggressors, are moderately successful but often fail, owing to recurrence of aggressive behavior among cagemates. Studies have incorporated enrichment devices to attenuate aggression, but such devices have had mixed results. However, these studies did not include research manipulations when assessing the benefits of various enrichment devices. We obtained 100 male athymic nude mice and studied the efficacy of various enrichment devices, including cotton squares, paper rolls, shredded paper, nylon bones, and a mouse house and wheel combination in the reduction of fighting during an ongoing study that involved randomization followed by prostate and intratibial injections. Groups were evaluated according to a numerical grading system for wound assessment. Examination of the data revealed that the enrichment devices had no effect on the presence of wounds, thus none of the devices tested affected fighting in nude mice. However, when mice began experimental use, fight wounds increased significantly at cage change and after randomization, reflecting a disruption of existing social hierarchies. Therefore, in the context of an actual research study that involves common manipulations, the specific enrichment device had less effect on aggression in male nude mice than did the destruction and reconstruction of social structures within each group.

Excessive environmental vibrations can have deleterious effects on animal health and experimental results, but they remain poorly understood in the animal laboratory setting. The aims of this study were to characterize train-associated vibration in a rodent vivarium and to assess the effects of this vibration on the reproductive success and fecal corticosterone metabolite levels of mice. An instrumented cage, featuring a high-sensitivity microphone and accelerometer, was used to characterize the vibrations and sound in a vivarium that is near an active railroad. The vibrations caused by the passing trains are 3 times larger in amplitude than are the ambient facility vibrations, whereas most of the associated sound was below the audible range for mice. Mice housed in the room closest to the railroad tracks had pregnancy rates that were 50% to 60% lower than those of mice of the same strains but bred in other parts of the facility. To verify the effect of the train vibrations, we used a custom-built electromagnetic shaker to simulate the train-induced vibrations in a controlled environment. Fecal pellets were collected from male and female mice that were exposed to the simulated vibrations and from unexposed control animals. Analysis of the fecal samples revealed that vibrations similar to those produced by a passing train can increase the levels of fecal corticosterone metabolites in female mice. These increases warrant attention to the effects of vibration on mice and, consequently, on reproduction and experimental outcomes.

Efficient, effective cage decontamination and the detection of infection are important to sustainable biosecurity within animal facilities. This study compared the efficacy of cage washing at 110 and 180 °F on preventing pathogen transmission. Soiled cages from mice infected with mouse parvovirus (MPV) and mouse hepatitis virus (MHV) were washed at 110 or 180 °F or were not washed. Sentinels from washed cages did not seroconvert to either virus, whereas sentinels in unwashed cages seroconverted to both agents. Soiled cages from mice harboring MPV, Helicobacter spp., Mycoplasma pulmonis, Syphacia obvelata, and Myocoptes musculinus were washed at 110 or 180 °F or were not washed. Sentinels from washed cages remained pathogen-free, whereas most sentinels in unwashed cages became infected with MPV and S. obvelata. Therefore washing at 110 or 180 °F is sufficient to decontaminate caging and prevent pathogen transmission. We then assessed whether PCR analysis of debris from the bedding disposal cabinet detected pathogens at the facility level. Samples were collected from the prefilter before and after the disposal of bedding from cages housing mice infected with both MPV and MHV. All samples collected before bedding disposal were negative for parvovirus and MHV, and all samples collected afterward were positive for these agents. Furthermore, all samples obtained from the prefilter before the disposal of bedding from multiply infected mice were pathogen-negative, and all those collected afterward were positive for parvovirus, M. pulmonis, S. obvelata, and Myocoptes musculinus. Therefore the debris on the prefilter of bedding-disposal cabinets is useful for pathogen screening.

Recent efforts have focused on mitigating anesthetic gas emissions during laboratory animal experiments. A recently developed, digitally controlled, integrated digital vaporizer (IDV) using a syringe pump has been designed to use and administer anesthetic gas to mice and rats more efficiently. The entire IDV system can be placed on a laboratory bench, requires fewer charcoal filters to act as passive scavengers when used at a low gas flow rate, and does not need compressed gas to operate, a requirement for traditional passive systems. The objective of this study was to compare isoflurane usage between a traditional vaporizer (TdV) and an IDV system at both the same settings and those recommended by the manufacturer. We used 10 C57BL/6 male mice and administered isoflurane through either nose cones or tracheal tubes connected to a pulsatile ventilator. The results showed that isoflurane usage is highly dependent on the flow rate of the carrier gas, but the IDV system was more precise and handled low flow rates (150 mL/min) better than did the TdV system. We observed only slight differences in heart rate, respiration rate, core body temperature, time to loss of the righting reflex, and recovery time between group averages for both systems when set to manufacturer-recommended settings. Although observed decreased levels of waste anesthetic gas at low flow rates are expected from the IDV system, further work is needed to assess environmental anesthetic gas levels and exposure to laboratory personnel.

Buprenorphine is a potent analgesic commonly administered to alleviate pain in sheep used in research. Sustained-release buprenorphine (SRB) is an alternative to conventional buprenorphine hydrochloride (which must be injected repeatedly). To compare SRB with a typical conventional buprenorphine regimen (0.03 mg/kg every 8 h for 72 h), we used a simple 1:1 conversion to calculate a total SRB dose of 0.27 mg/kg per injection. The pharmacokinetics and thermal nociceptive effects of SRB were analyzed in 4 healthy adult sheep after a single intramuscular injection plus a washout period then a single subcutaneous injection. For both routes in all 4 sheep, plasma buprenorphine concentrations exceeded 0.1 ng/mL, considered the minimal threshold for therapeutic benefit, after 12 h and maintained a steady state for at least 72 h Likewise, for both routes in all sheep, thermal thresholds increased significantly between baseline and 12 h; lack of response persisted for at least 72 h. The average maximal plasma buprenorphine concentrations and bioavailability were similar for both routes. No clinical adverse effects occurred. Using a dose equivalent to the total course of conventional buprenorphine, this pilot study suggests that SRB is a well-tolerated, effective, and long-acting analgesic that can be administered as a single intramuscular or subcutaneous injection. SRB confers steady plasma concentrations and continuous analgesia in thermal nociception for at least 72 h. When compared with conventional buprenorphine, SRB has considerable advantages in improving wellbeing by minimizing handling-associated stress of repeated injection and limiting the likelihood of end-of-dose breakthrough pain.

Compassion, professional ethics, and public sensitivity require that animals are euthanized humanely and appropriately under both planned and emergent situations. According to the 2013 AVMA Guidelines for the Euthanasia of Animals, intraperitoneal injection of ethanol is “acceptable with conditions” for use in mice. Because only limited information regarding this technique is available, we sought to evaluate ethanol by using ECG and high-definition video recording. Mice (n = 85) and rats (n = 16) were treated with intraperitoneal ethanol (70% or 100%), a positive-control agent (pentobarbital–phenytoin combination [Pe/Ph]), or a negative-control agent (saline solution). After injection, animals were assessed for behavioral and physiologic responses. Pain-assessment techniques in mice demonstrated that intraperitoneal injection of ethanol was not more painful than was intraperitoneal Pe/Ph. Median time to loss of consciousness for all mice that received ethanol or Pe/Ph was 45 s. Median time to respiratory arrest was 2.75, 2.25, and 2.63 min, and time (mean ± SE) to cardiac arrest was 6.04 ± 1.3, 2.96 ± 0.6, and 4.03 ± 0.5 min for 70% ethanol, 100% ethanol, and Pe/Ph, respectively. No mouse that received ethanol or Pe/Ph regained consciousness. Although successful in mice, intraperitoneal ethanol at the doses tested (9.2 to 20.1 g/kg) was unsuitable for euthanasia of rats (age, 7 to 8 wk) because of the volume needed and prolonged time to respiratory effects. For mice, intraperitoneal injection of 70% or 100% ethanol induced rapid and irreversible loss of consciousness, followed by death, and should be considered as “acceptable with conditions.”

Small mammals have difficulty maintaining body temperature under anesthesia. This hypothermia is a potential detriment not only to the health and comfort of the animal but also to the integrity of any treatment given or data gathered during the anesthetic period. Using an external warming device to assist with temperature regulation can mitigate these effects. In this study, we investigated the ability of an advanced warming device that uses far-infrared (FIR) heating and responds to real-time core temperature monitoring to maintain a normothermic core temperature in guinea pigs. Body temperatures were measured during 30 min of ketamine–xylazine general anesthesia with and without application of the heating device. The loss of core body heat from anesthetized guinea pigs under typical (unwarmed) conditions was significant, and this loss was almost completely mitigated by application of the FIR heating pad. The significant difference between the temperatures of the actively warmed guinea pigs as compared with the control group began as early as 14 min after anesthetic administration, leading to a 2.6 °C difference at 30 min. Loss of core body temperature was not correlated with animals' body weight; however, weight influences the efficiency of FIR warming slightly. These study results show that the FIR heating device accurately controls core body temperature in guinea pigs, therefore potentially alleviating the effects of body heat loss on animal physiology.

Rabbits are a common animal model in eye research and in safety testing of novel chemical agents. In addition, ocular disease is a routine presentation in clinical practice. However, few studies have quantitatively examined lacrimation kinetics in this species. This study used a noninvasive method of tear measurement (the Schirmer tear test, STT) to quantify values for basal and reflex tearing and to determine the kinetic nature of tear production in 76 New Zealand white rabbits. We obtained a value of 7.58 ± 2.3 mm/min for the standard 1-min STT. Calculated values for mean residual tear volume and reflex tear flow were 1.95 μL and 0.035 μL/s, respectively. In addition, this study provides preliminary evidence for an interaction effect between eyes given that higher STT values were obtained from the second eye tested.

Despite the routine collection of oocytes from African clawed frogs (Xenopus laevis) for use in research, few studies have evaluated methods for preparing their skin for surgery. We evaluated 3 skin preparatory agents by examining their antibacterial efficacy and the gross and microscopic appearance of Xenopus skin after exposure. Frogs (n = 14) were sedated and treated (contact time, 10 min) with 0.9% sterile NaCl on one-half of the ventrum and with 0.5% povidone–iodine or 0.75% chlorhexidine on the other half. Bacterial cultures were obtained before and after skin treatment; bacteria were identified by mass spectrometry. To assess inflammation and degenerative changes, the incision sites were photographed and biopsied at 0, 1, and 7 d after surgery. We isolated at least 22 genera of bacteria from the skin of our frog population (mean ± SE, 5.21 ± 0.82 genera per frog). Iodine (2.00 ± 0.44 genera) and chlorhexidine (0.29 ± 0.76 genera) both had greater antimicrobial activity than did saline. Skin erythema did not correlate with treatment group. Histologic evidence of epidermal degeneration and necrosis was greater on days 1 and 7 after chlorhexidine treatment than after iodine or saline. In addition, frogs treated with chlorhexidine had a higher incidence of clinical illness associated with the exposure site. In summary, although chlorhexidine has adequate antimicrobial activity against organisms on X. laevis skin, it leads to skin damage and subsequent clinical complications. We therefore do not recommend chlorhexidine as a preoperative preparation agent in Xenopus.

During the acclimation phase of a preclinical safety study involving Syrian golden hamsters, some of the cages of treatment-naïve animals were noted to contain blue-tinged bedding; the urine of these hamsters was not discolored. We sought to understand the underlying cause of this unusual finding to ensure that the study animals were healthy and free from factors that might confound the interpretation of the study. Analysis of extracts from the blue bedding by using HPLC with inline UV detection and high-resolution mass spectrometry indicated that the color was due to the presence of indigo blue. Furthermore, the indigo blue likely was formed through a series of biochemical events initiated by the intestinal metabolism of tryptophan to an indoxyl metabolite. We offer 2 hypotheses regarding the fate of the indoxyl metabolite: indigo blue formation through oxidative coupling in the liver or through urinary bacterial metabolism.