Rhesus macaques (Macaca mulatta) are a common model for the study of human biology and disease. To manage coagulopathies in these animals and to study their clotting changes, the ability to measure coagulation biomarkers is necessary. Currently, few options for coagulation testing in NHP are commercially available. In this study, assays for 4 coagulation biomarkers—D-dimer, antithrombin III, protein C, and soluble P-selectin—were developed and optimized for rhesus macaques. Whole blood was collected from 28 healthy Chinese-origin rhesus macaques (11 male; 17 female) ranging in age from 5 to 20 y. Coagulation biomarkers were measured by using bead-based sandwich ELISA technology. The ranges (mean ± 90% confidence interval) for these biomarkers were: antithrombin III, 124.2 to 133.4 μg/mL; protein C, 3.2 to 3.6 μg/mL; D-dimer, 110.3 to 161.3 ng/mL; soluble P-selectin, 0.12 to 0.14 ng/10⁶ platelets. These reference values did not differ significantly according to sex or age. These new assays for coagulation biomarkers in rhesus macaques will facilitate the evaluation of in vivo hemostasis.

Mouse cage and bedding changes are potentially stressful to mice and are also labor- and resource-intensive. These changes are often performed on a calendar-based schedule to maintain a clean microenvironment and limit the concentrations of ammonia to which mice and workers are exposed. The current study sought to establish a performance-based approach to mouse cage-changing that uses urine spot characteristics as visual indicators of intracage ammonia levels. Colorimetric ammonia indicators were used to measure ammonia levels in individually-ventilated cages (IVC) housing male or female mice (n =5 per cage) of various strains at 1 to 16 d after cage change. Urine spot characteristics were correlated with ammonia levels to create a visual indicator of the cage-change criterion of 25 ppm ammonia. Results demonstrated a consistent increase in ammonia levels with days since cage change, with cages reaching the cage-change criterion at approximately 10 d for IVC containing male mice and 16 d for those with female mice. Ammonia levels were higher for male than female mice but were not correlated with mouse age. However, urine spot diameter, color, and edge characteristics were strongly correlated with ammonia levels. Husbandry practices based on using urine spot characteristics as indicators of ammonia levels led to fewer weekly cage changes and concomitant savings in labor and resources. Therefore, urine spot characteristics can be used as visual indicators of intracage ammonia levels for use of a performance (urine spot)-based approach to cage-changing frequency that maintains animal health and wellbeing.

Factors that are considered when selecting laboratory mouse bedding include animal health and comfort, cost, effects on personnel, and bioactive properties. Corncob is economical and facilitates low intracage ammonia but has undesirable influences on some endocrine studies. Rice hulls are an economical material that has not been well characterized as a bedding substrate. In this pilot study, we compared various aspects of bedding performance of rice hulls and other materials. On a per-volume basis, rice hulls were less absorbent than was corncob bedding. Rice hulls had higher odds than did corncob or reclaimed wood pulp of having moisture present at the bedding surface. The results of the absorbency tests coupled with the results of preliminary monitoring of intracage ammonia raised concern about the ability of rice hulls to control ammonia levels sufficiently in cages with high occupancy. However, ammonia was negligible when cages contained 5 young adult female mice. The relative expression of 3 cytochrome p450 genes was compared among mice housed on rice hulls, corncob, reclaimed wood pulp, or pine shavings. The expression of Cyp1a2 was 1.7 times higher in the livers of mice housed on rice hulls than on pine shavings, but other differences were not statistically significant. This study provides information on the merits of rice hulls as laboratory mouse bedding. Their relatively poor moisture control is a major disadvantage that might preclude their widespread use.

Behavior and health, including the incidence of chronic idiopathic diarrhea, can vary widely among NHP reared indoors. We hypothesized that factors during gestation account for some of the variability in chronic diarrhea risk that cannot be explained by postnatal environment, genes, or known physiologic deficits. We hypothesized that, among macaques reared indoors postnatally, outdoor housing during gestation (when the dam engaged with a large, species-typical social group) would be protective against diarrhea as compared with gestation experienced in an indoor setting. We also hypothesized that exposure to routine husbandry and veterinary care in utero would increase diarrhea rates in offspring. We built models to test the influence of specific events during pregnancy as well as their interactions with anxiety-related genotype as a way of understanding gene×environment interaction on the development of diarrhea in indoor-reared rhesus macaques. Although previous reports have suggested that rearing by the mother in an indoor environment is preferable to nursery rearing, we found that whether gestation occurred indoors (in single or pair housing) or outdoors (in a large social group) better explained the variability in diarrhea rate in our study population of indoor-reared macaques. Furthermore, the diarrhea incidence was associated with nervous temperament and serotonin transporter promoter genotype. Several significant interactions indicated that some of these effects were specific to subsets of animals. Our results demonstrate that the prenatal environment can have unexpected lasting health consequences.

Zebrafish (Danio rerio) are a popular vertebrate model in biomedical research, but information describing the effects of environmental enrichment on fertility and fecundity of zebrafish is sparse. In the current study, 18 breeding pairs were placed in divided 1.5-L breeding tanks containing 1 of 3 enrichment conditions: plastic grass (n = 6), plastic leaves (n = 6), or no enrichment (n = 6, control). The pairs were allowed to spawn for 3 h the next day, after which eggs were counted and breeding pairs were returned to holding tanks for use in subsequent sessions. Spawning sessions were repeated at 7-d intervals until the completion of 9 trials, with pairs rotating to a different condition at each interval. Total egg count (mean ± SEM) after 3 h was greater for zebrafish spawning in the grass environment (48.0 ± 7.7 eggs) than in the leaf or control environments (29.4 ± 5.3 and 20.4 ± 3.7 eggs, respectively). An interaction emerged between enrichment type and the age of the spawning pair on the number of fry at 6 d postfertilization (dpf). Initially, more fry were obtained from 110- and 160-dpf pairs with the grass enrichment, but from 173- and 180-dpf pairs there were more obtained with leaf enrichment than grass. A separate experiment showed that enrichment type did not have an effect on fry survivability. Overall, our data indicates that, under certain conditions, zebrafish fertility and fecundity are greater in a breeding tank containing environmental enrichment than in a bare tank.

Repeated injection of urethane (ethyl carbamate) is carcinogenic in susceptible strains of mice. Most recent cancer studies involving urethane-induced tumor formation use p53^+/– mice, which lack one copy of the p53 tumor suppressor gene. In contrast, the same protocol elicits at most a single tumor in wildtype C57BL/6 mice. The effect of repeatedly injecting urethane as a component of a ketamine–xylazine anesthetic mixture in the highly prevalent mouse strain C57BL/6 is unknown. Male C57BL/6J mice (n = 30; age, 3 mo) were anesthetized once monthly for 4 mo by using 560 mg/kg urethane, 28 mg/kg ketamine, and 5.6 mg/kg xylazine. The physical health of the mice was evaluated according to 2 published scoring systems. The average body condition score (scale, 1 to 5; normal, 3) was 3.3, 3.3, and 3.4 after the 2nd, 3rd, and 4th injections, respectively. The visual assessment score was 0 (that is, normal) at all time points examined. Within 1 wk after the 4th injection, the mice were euthanized, necropsied, and evaluated histopathologically. No histopathologic findings were noteworthy. We conclude that repeated monthly injection with urethane as a component of an anesthetic cocktail does not cause clinically detectable abnormalities or induce neoplasia in C57BL/6J mice. These findings are important because urethane combined with low-dose ketamine, unlike other anesthetic regimens, allows for accurate recording of neuronal activity in both the brain and retina. Longitudinal neuronal recordings minimize the number of mice needed and improve the analysis of disease progression and potential therapeutic interventions.

Postoperative analgesia in laboratory rats is complicated by the frequent handling associated with common analgesic dosing requirements. Here, we evaluated sustained-release buprenorphine (Bup-SR), sustained-release meloxicam (Melox-SR), and carprofen gel (CG) as refinements for postoperative analgesia. The aim of this study was to investigate whether postoperative administration of Bup-SR, Melox-SR, or CG effectively controls behavioral mechanical and thermal hypersensitivity in a rat model of incisional pain. Rats were randomly assigned to 1 of 5 treatment groups: saline, 1 mL/kg SC BID; buprenorphine HCl (Bup HCl), 0.05 mg/kg SC BID; Bup-SR, 1.2 mg/kg SC once; Melox-SR, 4 mg/kg SC once; and CG, 2 oz PO daily. Mechanical and thermal hypersensitivity were tested daily from day–1 through 4. Bup HCl and Bup-SR attenuated mechanical and thermal hypersensitivity on days 1 through 4. Melox-SR and CG attenuated mechanical hypersensitivity–but not thermal hypersensitivity–on days 1 through 4. Plasma concentrations, measured by using UPLC with mass spectrometry, were consistent between both buprenorphine formulations. Gross pathologic examination revealed no signs of toxicity in any group. These findings suggest that postoperative administration of Bup HCl and Bup-SR—but not Melox-SR or CG—effectively attenuates mechanical and thermal hypersensitivity in a rat model of incisional pain.

Buprenorphine HCl (BUP) is a μ-opioid agonist used in laboratory rodents. New formulations of buprenorphine (for example, sustained-released buprenorphine [BUP SR], extended-release buprenorphine [BUP ER]) have been developed to extend the analgesic duration. In a crossover design, 8 adult rats were injected subcutaneously with either BUP, BUP SR, BUP ER, or saline, after which voluntary running-wheel activity, arterial blood gases, and thermal withdrawal latency were assessed. Wheel running was decreased at 24 h compared with baseline in all treatment groups but returned to baseline by 48 h. Arterial pH, HCO₃^–, and CO₂ were not changed between groups or over time. However, arterial oxygen was lower than baseline in the BUP (–8 ± 2 mm Hg), BUP SR (–7 ± 1 mm Hg), and BUP ER (–17 ± 2 mm Hg) groups compared with saline controls (3 ± 2 mm Hg); the BUP ER group showed the greatest decrease when all time points were combined. BUP increased the withdrawal latency at 1 h (15% ± 3%), whereas BUP ER increased latencies at 4, 8, 12, and 48 h (35% ± 11%, 21% ± 7%, 26% ± 7%, and 22% ± 9%, respectively) and BUP SR prolonged latencies at 24, 48, and 72 h (15% ± 6%, 18% ± 5%, and 20% ± 8%, respectively). The duration of thermal analgesia varied between buprenorphine formulations, but all 3 formulations reduced voluntary-running activity at 24 h after injection and might cause hypoxemia in normal adult rats.

The objective of this study was to compare isoflurane with a combination of dexmedetomidine and ketamine, administered intramuscularly, for anesthesia in chinchillas (Chinchilla lanigera). In a prospective, complete crossover study, adult chinchillas (n = 8; age, 2 to 5 y) were anesthetized with a combination of dexmedetomidine (0.015 mg/kg IM) and ketamine (4 mg/kg IM). Atipamezole (0.15 mg/kg) was injected subcutaneously 45 min after dexmedetomidine–ketamine administration. For comparison, anesthesia also was induced and maintained with isoflurane in 100% oxygen, delivered by facemask. Anesthetic and physiologic parameters were recorded during each anesthesia, including various reflexes, heart rate, respiratory rate, body temperature, and SpO₂. Food intake, fecal output, and body weight were recorded daily for 6 d after each anesthetic trial. Induction time, heart rate, respiratory rate, and body temperature did not differ significantly between the 2 anesthetic protocols. Recovery times were shorter and SpO₂ was higher in animals that received isoflurane delivered in 100% oxygen. Food intake and fecal output were reduced in the dexmedetomidine–ketamine group for as long as 3 d after anesthesia, whereas isoflurane had no signifcant effect on food intake or fecal output. Both anesthetic protocols provided effective anesthesia in chinchillas. However, when anesthetized with dexmedetomidine–ketamine, chinchillas received room air and became hypoxemic. Future studies are needed to evaluate the effect of oxygen supplementation on anesthetic recovery and on the recovery of food intake and fecal output in chinchillas.

Providing lidocaine, ketamine, and an opioid greatly decreases the minimum alveolar concentration (MAC) of volatile anesthetics in dogs. However, the efficacy of this combination shows marked interspecies variation, and opioids are likely to be less effective in pigs than in other species. The aim of the study was to determine the effects of constant-rate infusion of lidocaine and ketamine combined with either morphine or fentanyl on the MAC of sevoflurane in pigs. In a prospective, randomized, crossover design, 8 healthy crossbred pigs were premedicated with ketamine and midazolam, and anesthesia was induced and maintained with sevoflurane. Pigs then received ketamine (0.6 mg/kg/h) and lidocaine (3 mg/kg/h) combined with either morphine (0.24 mg/kg/h; MLK) or fentanyl (0.0045 mg/kg/h; FLK) after a loading dose; the control group received Ringers lactate solution. The anesthetic-sparing action of the 2 infusion protocols was calculated according to the MAC, by using dewclaw clamping as the standard noxious stimulus. The sevoflurane MAC (mean ± 1 SD) was 2.0% ± 0.2%, 1.9% ± 0.4%, and 1.8% ± 0.2% in the control, MLK, and FLK groups, respectively. No differences among groups or treatments were found. In conclusion, the administration of MLK or FLK at the studied doses did not reduce the MAC of sevoflurane in pigs.

Neonatal mice (that is, pups younger than 6 d) must be exposed to CO₂ for as long as 50 min to achieve euthanasia. Alternatively, other inhalant anesthetic agents have been used to euthanize laboratory rodent species. We investigated the efficacy of isoflurane at saturated vapor pressure to euthanize neonatal mice. Neonatal mice (n = 76; age, 1 or 2 d) were exposed to isoflurane in a sealed, quart-size (0.95-L) plastic bag at room temperature. Righting and withdrawal reflexes were absent in less than 2 min. After 30 min of exposure to isoflurane, pups were removed and monitored for recovery. All pups were cyanotic and showed no detectable signs of life when they were removed from the bag. However, after 30 to 120 min after removal from the bag, 24% of isoflurane-overexposed pups began gasping and then resumed normal respiration and regained a normal pink coloration. These results demonstrate that isoflurane overexposure at saturated vapor pressure for 30 min is insufficient to euthanize neonatal mice and that isoflurane overexposure must be followed by a secondary means of euthanasia.

Researchers often consult with laboratory animal veterinarians for suggestions on how to improve their protocols. We assisted a researcher in performing serial liver biopsies in rats (Rattus norvegicus) to assess the transport of iron on a cellular level. We developed a novel collection approach that used laparotomy through a midline abdominal incision and disposable biopsy punches to obtain liver samples at 3 different times at various intervals. We hypothesized the survival of the subjects undergoing the multiple survival procedures would be independent of the weight loss or gain sustained throughout the study. Although 2 rats died during the study, the results were statistically significant with regard to survival when comparing the Belgrade rats to the Sprague Dawley rats and Swiss Webster mice and were independent of the weight loss or gain incurred during the study. We also performed a pilot study in mice (Mus musculus), using the same method as in the rats, with equivalent results. Our study showed the survival of rodents that underwent multiple laparotomies and liver biopsies was independent of the weight gain or loss throughout the study.

Body temperature is a common physiologic parameter measured in both clinical and research settings, with rectal thermometry being implied as the 'gold standard.' However, rectal thermometry usually requires physical or chemical restraint, potentially causing falsely elevated readings due to animal stress. A less stressful method may eliminate this confounding variable. The current study compared 2 types of digital rectal thermometers—a calibrated digital thermometer and a common digital thermometer—with an implantable subcutaneous transponder microchip. Microchips were implanted subcutaneously between the shoulder blades of 16 ferrets (8 male, 8 female), and temperatures were measured twice from the microchip reader and once from each of the rectal thermometers. Results demonstrated the microchip temperature readings had very good to good correlation and agreement to those from both of the rectal thermometers. This study indicates that implantable temperature-sensing microchips are a reliable alternative to rectal thermometry for monitoring body temperature in ferrets.

The incidence of human spinal column disease remains high, and animal models still play important roles in prophylactic, diagnostic, and therapeutic research. Because of their similar size to humans, pigs remain an important spine model. For pigs to serve as a model for the human spine, basic similarities and differences must be understood. In this study, morphometric data of the lumbar spine of Munich miniature pigs (Troll) were recorded radiologically, evaluated, and compared with recorded human data. Whereas humans have a constant number of 5 lumbar vertebrae, Munich minipigs had 5 or 6 lumbar vertebrae. Compared with their human counterparts, the lumbar vertebral bodies of the minipigs were remarkably larger in the craniocaudal (superior–inferior) direction and considerably smaller in the dorsoventral and laterolateral directions. The porcine vertebral canal was smaller than the human vertebral canal. The spinal cord extended into the caudal part of the porcine lumbar vertebral canal and thus did not terminate as cranial, as seen in humans. The lumbar intervertebral spaces of the pig were narrower in craniocaudal direction than human intervertebral spaces. These differences need to be considered when planning surgical actions, not only to avoid pain and irreversible damage to the minipigs but also to achieve accurate scientific results.

Handheld, point-of-care glucometers are commonly used in NHP for clinical and research purposes, but whether these devices are appropriate for use in NHP is unknown. Other animal studies indicate that glucometers should be species-specific, given differences in glucose distribution between RBC and plasma; in addition, Hct and sampling site (venous compared with capillary) influence glucometer readings. Therefore, we compared the accuracy of 2 human and 2 veterinary glucometers at various Hct ranges in rhesus macaques (Macaca mulatta), sooty mangabeys (Cercocebus atys), and chimpanzees (Pan troglodytes) with that of standard laboratory glucose analysis. Subsequent analyses assessed the effect of hypoglycemia, hyperglycemia, and sampling site on glucometer accuracy. The veterinary glucometers overestimated blood glucose (BG) values in all species by 26 to 75 mg/dL. The mean difference between the human glucometers and the laboratory analyzer was 7 mg/dL or less in all species. The human glucometers overestimated BG in hypoglycemic mangabeys by 4 mg/dL and underestimated BG in hyperglycemic mangabeys by 11 mg/dL; similar patterns occurred in rhesus macaques. Hct did not affect glucometer accuracy, but all samples were within the range at which glucometers generally are accurate in humans. BG values were significantly lower in venous than capillary samples. The current findings show that veterinary glucometers intended for companion-animal species are inappropriate for use in the studied NHP species, whereas the human glucometers showed clinically acceptable accuracy in all 3 species. Finally, potential differences between venous and capillary BG values should be considered when comparing and evaluating results.