Animal research facilities are noisy environments. The high air change rates required in animal housing spaces tend to create higher noise levels from the heating and cooling systems. Housing rooms are typically constructed of hard wall material that is easily cleaned but simultaneously highly reverberant, meaning that the sound cannot be controlled/attenuated so the sounds that are generated bounce around the room uncontrolled. (Soft, sound-absorbing surfaces generally cannot be used in animal facilities because they collect microbes; various wall surface features and sound control panel options are available, although rarely used.) In addition, many of our husbandry tasks such as cage changing, animal health checks, cleaning, and transporting animals produce high levels of noise. Finally, much of the equipment we have increasingly employed in animal housing spaces, such as ventilated caging motors, biosafety, or procedure cabinets, can generate high levels of background noise, including ultrasound. These and many additional factors conspire to create an acoustic environment that is neither naturalistic nor conducive to a stress-free environment. The acoustic variability both within and between institutions can serve as an enormous confounder for research studies and a threat to our ability to reproduce studies over time and between research laboratories. By gaining a better appreciation for the acoustic variables, paired with transparency in reporting the levels, we might be able to gain a better understanding of their impacts and thereby gain some level of control over such acoustic variables in the animal housing space. The result of this could improve both animal welfare and study reproducibility, helping to address our 3Rs goals of Replacement, Reduction, and Refinement in the animal biomedical research enterprise.Abstract
Intrafacility transport of mice is an essential function for both laboratory and husbandry personnel. However, transport may induce a stress response that can alter research findings and negatively impact animal welfare. To determine minimally adverse intrafacility transport methods, in-cage noise and vibration exposure during transport on a variety of transport vehicles (hand carrying, stainless steel rack, flatbed cart, metal teacart, plastic teacart, and a cart with pneumatic wheels) were measured. Under-cage and in-cage padding was tested for its ability to decrease noise and vibration on each vehicle. Behavioral (open field test and elevated plus maze) and corticosterone responses of mice were then measured following transport on the most adverse (metal teacart) and least adverse (pneumatic cart) methods of multicage transport. Behavioral measures showed no difference between transported mice and untransported mice in both single- and group-housed settings. Plasma corticosterone was significantly elevated in mice transported on the metal teacart immediately following transport and continued to have elevated trends in circadian peaks during the 48 h of sampling. The cart with pneumatic wheels was most effective at reducing noise and vibration, reflected in posttransport corticosterone readings that remained equivalent to those in untransported mice. This study demonstrates that mitigation of noise and vibration during cart transport may decrease the impact of transport on certain stress parameters in mice.Abstract
The ability to apply findings from animal studies efficiently and effectively is predicated on an understanding of biology and pathobiology, how that biology relates to the human systems being modeled, and how the studies are conducted and reported. This overview discusses various factors in research within the animal environment (referred to as extrinsic factors) that the NIH now expects to be documented to foster replicability in science and expand interpretations of study outcomes. Specifically, an important extrinsic factor in research with animals is that of individual personnel who perform handling practices, participate in research interactions, and share an overall presence in the housing facility with animals, all of which can confound reproducibility efforts in biomedical science. An improved understanding of the influences and behaviors of animal research personnel on animal responses is critical with regard to research results and the interpretation of data collected from animal models of biomedical disease.Abstract
There are limited evidence-based husbandry recommendations for laboratory zebra finches (Taeniopygia guttata), including appropriate light sources. Light-emitting diode (LED) technology has been shown to improve circadian regulation and reduce stress in some laboratory animal species, such as mice and rats, when compared with cool-white fluorescent (CWF) lighting, but the effects of LED lighting on zebra finches have not been published. We compared the effects of broad-spectrum, blue-enriched (6,500 Kelvin) CWF and flicker-free LED lighting on the behavior, stress, and reproductive outcomes of indoor-housed zebra finches. Using breeding pairs housed in cubicles illuminated with either CWF or LED lighting, we compared the reproductive output as determined by clutch size, hatching rate, and hatchling survival rate. We also compared the behavior of group-housed adult males, first housed under CWF followed by LED lighting, using video recordings and an ethogram. Fecal samples were collected from these males at the end of each recording period, and basal fecal corticosterone metabolite (FCM) levels were compared. A FCM assay for adult male zebra finches was validated for efficacy and accuracy using a capture-restraint acute stress response and parallelism analysis, respectively. The breeding pairs had no significant difference in the clutch size or percent hatching rate, but percent hatchling survival improved under LED with an increased proportion achieving 100% survival. There was no significant difference in FCM between the lighting treatments. However, the activity budgets of the birds were altered, with a reduction in flighted movement and an increase in enrichment manipulation under LED. Overall, these results support the use of blue-enriched, broad-spectrum flicker-free LED as a safe alternative to CWF lighting for breeding and nonbreeding indoor-housed zebra finches.Abstract
Due to a lack of evidence-based standards for cage-change intervals for antelope ground squirrels (AGS, Ammospermophilus leucurus), we evaluated cage ammonia accumulation in our colony of adult, wild-caught AGS and identified factors that influenced ammonia levels. Intracage ammonia was measured daily in singly housed AGS in static caging that contained a running wheel and 1/2, 3/4, 1, or 2 quart (qt) of corncob bedding. Cages were changed when ammonia levels reached greater than 50 ppm, our upper acceptable limit for ammonia based on mouse studies of ammonia aversion and toxicity. We also measured average daily water consumption over 2 wk to examine any correlation between water use and ammonia accumulation. We hypothesized that the desert-dwelling AGS would not reach intracage ammonia levels of greater than 50 ppm in a 2-wk interval at any bedding volume. Our data showed that intracage ammonia was highly variable among individuals and was significantly associated with water consumption and bedding volumes. Seventeen percent of AGS on 1/2 qt of bedding and 18% on 3/4 qt of bedding reached greater than 50 ppm ammonia before 7 d. All AGS on 1 and 2 qt of bedding remained below 50 ppm ammonia for 1 wk. Even when maintained on 2 qt of bedding, not all AGS remained below 50 ppm ammonia for 2 wk. Therefore, we concluded that the most appropriate option was weekly cage change for singly housed AGS on 1 qt of bedding in static caging.Abstract
Despite the long-term contributions of the spiny mouse (Acomys cahirinus) to research, basic knowledge of appropriate nutrition is lacking for this species. In the wild, spiny mice eat a high-fiber, high-protein food source. In the research setting, spiny mice are prone to obesity that can lead to diabetes mellitus. Common dietary modifications for weight control in humans with diabetes mellitus consist of increased fiber and protein. We hypothesized that increasing the dietary protein or fiber of spiny mice would reduce weight gain and improve their glycemic control, whereas the combination of protein and fiber in the diet would achieve optimal weight management and glycemic control without diet-related pathologic changes. We randomly assigned cages of young adult spiny mice (n = 34) to one of 4 diets: high protein (HP), high fiber (HF), a combination of both high protein and high fiber (HPF), or the base (control) diet (BD). Over the 8-wk study, spiny mice given HF diets maintained baseline weights despite the elevated dietary protein. None of the diets altered blood glucose levels; all diet groups maintained mean blood glucose levels within normal ranges. Spiny mice seem particularly sensitive to changes within their environment, as seen by increased food waste and transient elevated blood glucose levels when the spiny mice were transitioned to novel diets. The short-term elevations in protein and fiber that we tested were well tolerated by spiny mice. Although HF was effective in controlling weight, the ideal percentage of fiber still needs to be determined. The combination diet (HPF) maintained weight and body condition scores and showed a nonsignificant elevation of blood glucose that warrants a longer diet trial before our recommending this specific combination.Abstract
Nonhuman primates used in biomedical research may experience clinically significant weight loss for a variety of reasons. Episodes of anorexia (complete loss of appetite) or hyporexia (decreased appetite) can result in significant weight loss, potentially altering animal welfare and scientific studies. The FDA has approved several appetite stimulants for use in domestic species, but currently none are approved for use in NHP. Treatment of inappetence and weight loss in NHP often relies on the extralabel use of these compounds. Capromorelin is a ghrelin receptor agonist. As a growth hormone secretagogue, capromorelin increases appetite, leading to weight gain. Studies in several species have shown a positive correlation between capromorelin administration and weight gain; in 2017, an oral solution of capromorelin received FDA approval for use in dogs. We tested this solution in healthy adult rhesus macaques (n = 3 males and 3 females) for its effects on body weight and insulin like growth factor-1 (IGF-1). A control group (n = 2 males and 2 females) was used for comparison. Treated macaques received a 3 mg/kg oral dose daily for 7 d. Clinical signs were observed daily. Weights were collected before, during and at the end of treatment. Blood was drawn before, during and after treatment for measurement of IGF-1 levels and standard hematology and biochemistry parameters. Baseline-adjusted mean body weights and IGF-1 levels were significantly higher in treated as compared with control monkeys after 7 d of beginning treatment (body weight of 10.5 ± 0.1 kg (mean ± SEM) and 10.1 ± 0.1 kg, respectively; IGF-1 of 758 ± 43 ng/mL and 639 ± 22 ng/mL, respectively). Capromorelin administration was not associated with appreciable changes in hematologic and biochemical values in treated macaques. These findings suggest that capromorelin may be useful for treating inappetence and weight loss in NHP, and based on blood analysis, a 7-d course of treatment does not appear to cause acute toxicity.Abstract
The use of soiled-bedded sentinels (SBSs) has historically been the standard for colony health surveillance monitoring at our institution. With the advent of newer technologies in which dust collected from filters is tested by PCR, we compared traditional SBS with PCR testing of both exhaust air dust collected from a filter in the downstream vertical plenum (exhaust dust test [EDT]) and the SBS cage-level exhaust filter (SCEF). Our hypothesis was that both methods of filter testing would identify more pathogens than SBS testing. Twenty-five individually ventilated mouse racks that used disposable caging were sanitized and placed into rotation. Rack plenums were tested by PCR to verify negative results before the study start. Exhaust dust collection media were placed in the exhaust plenum (n = 25). SBS cages were placed on each side of the rack with 2 mice per cage (n = 42 mice), with the remaining cage slots occupied by research animals. At each triweekly cage change, the exhaust air filters were carefully removed from the cage top, placed in sterile 50-mL conical tubes, and pooled for submission. After 3 mo, the SBS mice were tested via serology for bacterial and viral agents and by PCR for Helicobacter species, pinworms, and ectoparasites. In addition, the EDT filter and SCEF were collected for PCR to evaluate for the same agents. Our results indicate that the SCEF consistently detected agents more frequently than the EDT filter placed in the plenum and that the EDT filter media detected agents more frequently than did the SBS mice. Our data suggest that both PCR methods of detection are superior to SBS for individually ventilated disposable rodent cages and that the SCEF is superior to EDT. These data supported our movement of institution toward environmental monitoring as a method of rodent colony health surveillance.Abstract
The search for alternatives to live animal sentinels in rodent health monitoring programs is fundamental to the 3Rs (Reduction, Replacement, and Refinement) of animal research. We evaluated the efficacy of a novel battery-operated tumbler device that rotates soiled bedding in direct contact with sample media against the use of exhaust sample media and soiled bedding sentinel (SBS) mice. Four rodent racks were used, each with 3 test cages: a cage with a tumbler device that rotated for 10 min twice a week (TUM10), a cage with a tumbler device that rotated for 60 min twice a week (TUM60), and a cage housing 2 female Crl:CD1(ICR) mice. Every 2 wk, each test cage received soiled bedding collected from all cages on each respective rack. In addition to soiled bedding, the tumbler device contained various sample collection media: a contact Reemay filter (3 mo-cRF) that remained in the tumbler for the duration of the study, a contact Reemay filter (1 mo-cRF) that was replaced monthly, adhesive swabs (AS) that were added at every biweekly cage change, and an exhaust Reemay filter located at the exhaust outlet of the cage. All analyses were performed by direct PCR for both sample media in the animal-free methods, and fecal pellet, body swab, and oral swabs were collected from sentinel mice. Out of 16 total pathogens detected, assessment of 1 mo-cRF from both TUM10 and TUM60 cages detected 84% and 79% of pathogens, respectively, while SBS samples detected only 47% of pathogens. AS in TUM60 and TUM10 cages detected the fewest pathogens (24% and 13%, respectively). These results indicate that the novel tumbler device is an effective and reliable tool for rodent health monitoring programs and a suitable replacement for live animal sentinels. In this study, 1 mo-cRF in TUM10 cages detected the highest number of pathogens.Abstract
Heat supplementation during surgery is a common practice; however, thermal support is not commonly used during anesthesia induction. Mice lose body temperature quickly, and air movement can exacerbate this, potentially putting mice at a thermal deficit before surgery. Whether the method of warming during induction affects overall heat loss during anesthesia is unknown. We hypothesized that the method of heating would affect body temperature (Tb) during anesthesia induction, maintenance, recovery, and once placed back on the rack. Mice (C57BL/6NHsd-6M/6F [C57BL/6]; Hsd:Athymic Nude-Foxn1nu [Nude]; N = 24;12M/12F) were assigned to a treatment in a factorial design: thermal chamber (TC; ambient temperature [Ta] = 28.8 °C); heating pad (HP; induction chamber placed on an electric heating pad;Ta = 28.4 °C); and control (Ctrl; Ta = 21.6 °C). During induction, one mouse at a time was anesthetized with isoflurane over a 3 min period and then maintained under anesthesia for 10 min on a hot water heating pad (33 °C). Then isoflurane was stopped and time to ambulation was recorded. Tb and activity were tracked in the home cage on the rack before and after anesthesia. During induction, Ctrl mice lost significantly more heat (−2.8 °C) than did TC (+0.2 °C) and HP mice (+0.1 °C) but TC and HP were not different. During anesthesia maintenance, Ctrl mice regained 1 °C, but their Tb was still lower than that of the treated groups. Nude mice consistently had a lower Tb than C57BL/6 mice, regardless of treatment or anesthesia phase. C57BL/6 Ctrl mice took longer to ambulate than either HP or TC mice, but the method of heating did not differentially affect Nude mice. In general, C57BL/6 as compared with Nude and females as compared with males were comparatively more active and had higher Tb during certain times of day, regardless of the heating methods. Overall, our findings support the provision of heat during anesthesia induction, regardless of method, to reduce overall Tb loss during a short anesthesia event.Abstract
The New Zealand white rabbit (Oryctolagus cuniculus) is a frequently used surgical model. Pain management after surgery is a critical aspect of animal welfare. Recently, a long-acting buprenorphine formulation (Ethiqa XR; EXR) was approved for use in rats and mice but has not yet been investigated in rabbits. The current study aimed to determine whether a single subcutaneous dose of 0.15 mg/kg of EXR could achieve and maintain therapeutic buprenorphine plasma concentrations (0.1 ng/mL) for 72 h in male and female rabbits. We also evaluated the safety profiles of EXR and the fentanyl patch (FP) by assessing fecal output after surgery, because opioids are known to decrease intestinal motility. Behavior and pain scores were compared for rabbits that received either EXR or the FP after undergoing an annulus puncture procedure to induce osteoarthritis. EXR at 0.15 mg/kg SC provided a shorter time to onset and sustained analgesia for 72 h in male and female rabbits, whereas the FP provided suboptimal analgesia after 48 h. Both EXR and FP reduced fecal output after surgery. Output returned to baseline levels within 72 h for the EXR group and remained slightly below baseline at 96 h after surgery for the fentanyl group. Grimace pain scores revealed no significant difference between treatment groups. These results suggest that EXR is a safe and effective option for postoperative pain management in rabbits.Abstract
Overdose of carbon dioxide gas (CO2) is a common euthanasia method for rodents; however, CO2 exposure activates nociceptors in rats at concentrations equal to or greater than 37% and is reported to be painful in humans at concentrations equal to or greater than 32.5%. Exposure of rats to CO2 could cause pain before loss of consciousness. We used 2 standardized loss of righting reflex (LORR) methods to identify CO2 concentrations associated with unconsciousness in Wistar, Long–Evans, and Sprague–Dawley rats (n = 28 animals per strain). A rotating, motorized cylinder was used to test LORR while the rat was being exposed to increasing concentrations of CO2. LORR was defined based on a 15-second observation period. The 2 methods were 1) a 1-Paw assessment (the righting reflex was considered to be present if one or more paws contacted the cylinder after the rat was positioned in dorsal recumbency), and 2) a 4-Paw assessment (the righting reflex was considered to be present if all 4 paws contacted the cylinder after the rat was positioned in dorsal recumbency). Data were analyzed with Probit regression, and dose-response curves were plotted. 1-Paw EC95 values (CO2 concentration at which LORR occurred for 95% of the population) were Wistar, 27.2%; Long–Evans, 29.2%; and Sprague–Dawley, 35.0%. 4-Paw EC95 values were Wistar, 26.2%; Long–Evans, 25.9%, and Sprague–Dawley, 31.1%. Sprague–Dawley EC95 values were significantly higher in both 1- and 4-Paw tests as compared with Wistar and Long–Evans rats. No differences were detected between sexes for any strain. The 1-Paw EC95 was significantly higher than the 4-Paw EC95 only for Sprague-Dawley rats. These results suggest that a low number of individual rats from the strains studied may experience pain during CO2 euthanasia.Abstract
Vehicular whole-body vibration (WBV) can have long-term adverse effects on human quality of life. Animal models can be used to study pathophysiologic effects of vibration. The goal of this study was to assess animal cooperation and well-being to determine the feasibility of a novel seated rat model for investigating the effects of WBV on biologic systems. Twenty-four male Sprague–Dawley rats were used. The experiment consisted of an acclimation phase, 2 training phases (TrP1 and TrP2), and a testing phase (TeP), including weekly radiographic imaging. During acclimation, rats were housed in pairs in standard cages without vibration. First, experimental (EG; n = 18) and control group 1 (C1; n = 3) rats were placed in a vibration apparatus without vibration, with increasing duration over 5 d during TrP1. EG rats were exposed to vertical random WBV that was increased in magnitude over 5 d during TrP2 until reaching the vibration signal used during TeP (15 min, 0.7 m·s−2 root mean square, unweighted). C1 rats were placed in the vibration apparatus but received no vibration during any phase. Control group 2 (C2; n = 3) rats remained in the home cages. Cooperation was evaluated with regard to rat-apparatus interactions and position compliance. Behavior, weight, and fecal glucocorticoid metabolite concentrations (fGCM) were used to evaluate animal well-being. We observed good cooperation and no behavioral patterns or weight loss between phases, indicating little or no animal stress. The differences in fGCM concentration between groups indicated that the EG rats had lower stress levels than the control rats in all phases except TrP1. Thus, this model elicited little or no stress in the conscious, unrestrained, seated rats.Abstract
Many experiments require the collection of serial blood samples from mice. However, the size of mice limits the volume of blood that can be safely collected as a survival procedure. In IACUC protocols, investigators may report the amount of blood they collect from mice as a number of drops. Many institutions, including ours, use an anecdotal conversion factor (1 drop of mouse blood = 25 µL) to ensure that blood-collection volumes are compliant with institutional guidelines. To our knowledge, previous work has not experimentally determined the volume of a drop of mouse blood. In this 10-wk crossover experiment, 2 phlebotomists bled 30 C57BL/6J mice from 3 sites (facial, saphenous, and tail) using one or 2 different needle gauge sizes per site. Male and female mice were weighed weekly and divided among 5 groups (n = 6): left and right tail vein, left and right saphenous vein, and facial vein. A single blood drop from each site was weighed, and the volume of each drop was calculated using the average blood density determined from 8 mice terminally bled at the end of the study. Venipuncture site and side significantly influenced blood-drop weight and thus calculated volume. Facial vein puncture produced the largest drop volume (mean: 21.7 µL), followed by the saphenous vein (mean: 9.97 µL) and tail vein (mean: 4.96 µL). Collection from the facial vein was associated with more hemorrhage and morbidity. Left-sided venipuncture was associated with slightly larger-volume blood drops, though the effect size of side was small. The results of this study may be useful in more accurately estimating blood loss via conversion of drops to volume. Our data indicate that blood collection from saphenous and tail veins minimizes blood loss relative to facial vein puncture and may optimize both serial collection of small-volume blood samples and animal welfare.Abstract
Blood collection is frequently used for neonatal and juvenile mice in toxicology, developmental, and immunology studies and is often a terminal procedure. However, the use of nonterminal blood collection techniques, including the submandibular and the submental collection techniques described for adult mice, may offer opportunities to reduce animal numbers and refine current methods. The use of the submental technique has not been described for neonatal or juvenile mice. In this study, we compared the submental and submandibular blood collection techniques to determine their suitability for use in neonatal and juvenile mice. Male and female CD1 mice, ages 7, 14, 21, and 28 d, were randomized by sex into submental (n = 16), submandibular (n = 16), or control (n = 8) groups. Each mouse was weighed, bled per its assigned group (or only restrained in the case of control mice), and then decapitated without anesthesia for terminal blood collection. Blood collection volume and corticosterone concentrations were measured. The 2 methods showed significant differences in the volume of blood collected at ages 14 and 28, with the submandibular technique yielding significantly higher volumes. No significant differences were detected in corticosterone levels between the 2 techniques based on age or sex. A subset of mice (n = 8, 2 per age group) were bled via submental or submandibular technique and were evaluated 48 h later for gross and histopathologic evidence of trauma. Seven of the 8 mice showed expected inflammation and healing at the collection sites, with 4 mice having embedded strands of fur in the tissue. These data indicate that the submental blood collection is a viable method for nonterminal blood collection method in neonatal and juvenile mice, especially when smaller amounts of blood are needed.Abstract
This corrects the article DOI: In the original article titled “Review of Intraperitoneal Injection of Sodium Pentobarbital as a Method of Euthanasia in Laboratory Rodents,” published in Vol 59, Issue 3 (May 2020), there is an error in the “Histopathologic and physiologic changes” section of Results. Specifically, the last sentence of the first paragraph incorrectly cites Arioli and Rossi (1970) as providing a review of histologic changes. The correct citation is Artwohl et al. (2006), which is included in the References.
The American Association for Laboratory Animal Science (AALAS) currently publishes two journals containing data-driven, peer-reviewed articles. The mission of Comparative Medicine (CM) is to disseminate high-quality, peer-reviewed information that expands biomedical knowledge and promotes human and animal health through the study of laboratory animal disease, animal models of disease, and basic biologic mechanisms related to disease in people and animals.General