The practical application of statistical power is becoming an increasingly important part of experimental design, data analysis, and reporting. Power is essential to estimating sample size as part of planning studies and obtaining ethical approval for them. Furthermore, power is essential for publishing and interpreting negative results. In this manuscript, we review what power is, how it can be calculated, and reporting recommendations if a null result is found. Power can be thought of as reflecting the signal to noise ratio of an experiment. The conventional wisdom that statistical power is driven by sample size (which increases the signal in the data), while true, is a misleading oversimplification. Relatively little discussion covers the use of experimental designs which control and reduce noise. Even small improvements in experimental design can achieve high power at much lower sample sizes than (for instance) a simple t test. Failure to report experimental design or the proposed statistical test on animal care and use protocols creates a dilemma for IACUCs, because it is unknown whether sample size has been correctly calculated. Traditional power calculations, which are primarily provided for animal number justifications, are only available for simple, yet low powered, experimental designs, such as paired t tests. Thus, in most controlled experimental studies, the only analyses for which power can be calculated are those that inheriently have low statistical power; these analyses should not be used because they require more animals than necessary. We provide suggestions for more powerful experimental designs (such as randomized block and factorial designs) that increase power, and we describe methods to easily calculate sample size for these designs that are suitable for IACUC number justifications. Finally we also provide recommendations for reporting negative results, so that readers and reviewers can determine whether an experiment had sufficient power. The use of more sophisticated designs in animal experiments will inevitably improve power, reproducibility, and reduce animal use.

Micronutrient deficiency is one of the most prominent public health concerns; in particular, vitamin A and iron are determinants of appropriate development, and vitamin A influences iron homeostasis and metabolism. Here we compared the effects of diets that were sufficient and insufficient in vitamin A and iron on the hematologic parameters and body weight of rats. Male Wistar rats were randomly divided into 5 dietary groups (n = 7 per group): adequate in iron and vitamin A (control); adequate in iron but low in vitamin A (FesvAi); adequate in iron but lacking vitamin A (FesvAd); low in iron but adequate in vitamin A (FeivAs); and low in both iron and vitamin A (FeivAi). After 6 wk, rats showed significant differences in serum iron relative to the control diet (control, 256 ± 44 μg/dL; FesvAi, 220± 16 μg/dL; FesvAd, 181 ± 15 μg/dL; FeivAs, 131 ± 44 μg/dL; FeivAi, 75 ± 19 μg/dL). Rats on iron-deficient diets showed reduced Hgb values relative to the control diet (control, 15.9 ± 0.7 g/dL; FeivAs, 13.2 ± 1.6 g/dL; FeivAi, 12.9 ± 1.0 g/dL), MCV (control: 57 ± 10 fL; FeivAs, 48 ± 10 fL; FeivAi, 44 ± 3 fL), and Hct (control, 53% ± 2%; FeivAs, 44% ± 5%; FeivAi, 42% ± 8%). All of the experimental dietary groups showed significant differences in reticulocyte count when compared with the control group (control, 2.7% ± 2.2%; FesvAd, 0.6% ± 0.2%; FesvAi, 0.3% ± 0.1%; FeivAs, 1.2% ± 0.2%; FeivAi, 0.6% ± 0.5%). The mean difference in body weight for the experimental groups, relative to the control group, was 30 ± 10 g. These results suggested that, in young male Wistar rats, both iron and vitamin A are essential to cause increases in body weight and various hematologic parameters.

Appropriate calculation and use of reference intervals have widespread clinical and research implications. Unfortunately, reference intervals for blood pressure in one of the most commonly used NHP species, rhesus macaques (Macaca mulatta), have never been calculated. Although anesthetic drugs and noninvasive methods of blood pressure measurement both have known effects on blood pressure values, their use provides the safest, fastest, and most widely used approach to clinical evaluation and blood pressure collection in this species. We analyzed noninvasive blood pressure measurements from 103 healthy, ketamine-sedated, adult (age, 8 to 16 y) rhesus macaques, representing both sexes, with various body condition scores by using 2 types of sphygmomanometers at 3 different anatomic locations. Reference intervals were calculated for each device, in each location, thus establishing normative data beneficial to clinical veterinarians assessing animal health and encouraging researchers to use noninvasive methods. Age, body condition score, sex, type of sphygmomanometer, and location of cuff placement were all found to influence blood pressure measurements significantly, providing important information necessary for the appropriate interpretation of noninvasive blood pressure values in rhesus macaques.

Nest building and burrowing are highly motivated natural behaviors in rodents, and changes in these behaviors can serve as welfare assessment tools. In this study, we investigated: 1) the limits of agreement between 2 observers for a refined scoring method for nest-building behavior; 2) the effect of repeated exposure to 15 min of isoflurane on nest-building behavior; 3) the effect of 24 h of grid-floor housing, repeated exposure to 15 min isoflurane, and daily intraperitoneal injection of 0.2 mL 0.9% isotonic saline for 3 d on burrowing behavior; and 4) the effect of exposure to grid-floor housing, isoflurane, and intraperitoneal injections on fecal corticosterone metabolites, body weight, fur status, and sucrose preference in mice. SPF C57BL/6NTac female mice (n = 27) were included in the study and were assessed first for burrowing behavior, followed by 2 wk of rest and then for nesting behavior. The refined scoring method for nest-building activity had good inter observer agreement. According to this method, a single exposure to anesthesia with isoflurane led to a decrease in nest-building activity and sucrose preference; a second exposure to anesthesia with isoflurane had no effect on nest building. Neither grid-floor housing nor repeated exposure to isoflurane anesthesia had any effect on burrowing behavior in mice. In contrast, intraperitoneal injections increased burrowing behavior. In conclusion, a refined scoring method for nest-building activity test that we developed for this study proved to be objective and sensitive to the effect of an initial exposure to anesthesia with isoflurane.

Identifying early indicators of distress in mice is difficult using either periodic monitoring or current technology. Likewise, poor pain identification remains a barrier to providing appropriate pain relief in many mouse models. The Time to Incorporate to Nest Test (TINT), a binary measure of the presence or absence of nesting behavior, was developed as a species-specific method of identifying moderate to severe distress and pain in mice. The current study was designed to evaluate alterations in nesting behavior after routine surgery and to validate the TINT's ability to measure pain-related behavioral changes. CD1 mice undergoing carotid artery catheterization as part of a commercial surgical cohort were randomly assigned various nesting, surgery, and analgesia conditions. To provide context for the TINT outcomes, we measured other variables affected by pain, such as weight loss, food consumption, and scores derived from the Mouse Grimace Scale (MGS). Mice that had surgery were more likely to have a negative TINT score as compared with controls. All mice were more likely to fail the TINT after receiving postoperative buprenorphine, suggesting that buprenorphine may have contributed to the failures. The TINT, MGS live scoring, and scoring MGS images all loaded strongly on a single component in a principal component analysis, indicating strong convergent validity between these measures. These data indicate that the TINT can provide a quick, objective indicator of altered welfare in mice, with the potential for a wide range of uses.

Some captive breeding colonies of rhesus macaques live in large outdoor multimale, multifemale social groups. These groups are composed of several matrilineal families, governed by a clear female dominance hierarchy. Aggression within the same or between different matrilineal families due to social instability can result in trauma and mortality. Therefore, a primary management goal is to detect emerging social unrest before the onset of significant fighting and wounding. Accordingly, groups are monitored routinely for changes in dominance and alliance relations as well as for increases in trauma frequency and severity. Decreased food intake is a normal physiologic response to acute stress; therefore, inappetence in key animals or groups of monkeys might be used as an indicator of increased social stress and emerging instability. An incident of intrafamily aggression occurred recently in a breeding group at our facility and resulted in considerable fighting. Because this compound was equipped with an automated feeding system that tracks the caloric intake of individual animals, we retrospectively analyzed feeding data to determine whether significant reduction in caloric consumption occurred prior to the onset of aggression, compared with baseline values. Neither the entire group nor individual families showed any significant differences in total caloric intake between baseline and previous 24 h values; however, the affected family exhibited a 20% reduction in total caloric during the 24 h prior to the aggression. Most notably, the deposed subfamily showed a marked 58% reduction in caloric intake during the prior 24 h, whereas remaining subfamilies showed no significant changes in intake. High-ranking animals of the group, including the α female, β female, and α male, similarly exhibited marked decreases in caloric intake during that period. These findings indicate that automated feeders can assist management staff with monitoring social stability in breeding colonies of rhesus macaque.

To monitor rodent colony health in research facilities, soiled-bedding sentinel (SBS) animals have traditionally been used. SBS can be tested by various methods, which may include serology, PCR analysis, and necropsy. Several pathogens are unreliably detected by using SBS or transmitted poorly through soiled bedding, and collection and evaluation of SBS samples can be time-intensive. Recently, exhaust air dust (EAD) testing through PCR analysis has emerged as an adjunct or replacement method for rodent colony health monitoring. EAD monitoring may provide a more efficient, sensitive, and humane method for monitoring health status. Using both EAD and SBS health monitoring, we evaluated colony health over the course of 1 y in 3 research barrier rooms in which mice were housed exclusively on IVC racks. Three pathogens—Helicobacter spp., Rodentibacter spp. (previously Pasteurella pneumotropica), and murine norovirus (MNV)—were not excluded in 2 of the rooms, and we expected that these mice would test positive with some regularity. EAD monitoring was significantly more sensitive than SBS for detection of the bacterial agents. SBS failed to detect Helicobacter spp. at time points when EAD had 100% detection in the rooms that did not exclude the bacteria. The detection of MNV did not differ between health monitoring systems at any time point. The findings suggest that EAD is especially valuable in detecting bacteria poorly transmitted through soiled bedding. In addition, the corresponding results with MNV detection suggest that EAD surveillance can reliably be implemented as an alternative to SBS monitoring in a facility in which mice are housed exclusively on IVC racks.

Appropriate aseptic technique is a crucial component of rodent survival surgery. Ease of technique, surgical space constraint, batch surgery, and cost are factors that may affect researcher compliance with appropriate aseptic technique. The first part of this study compared 3 antiseptic preparation agents with the standard triplicate application of povidone-iodine and alcohol. Euthanized mice (n = 40) were shaved on the dorsum, and culture swabs were taken for RODAC plating and bacterial identification. Shaved sites were prepared by using one of the 4 antiseptic preparation agents. Culture samples were obtained immediately and at 20 min after antiseptic preparation. In the 2nd part of the study, 8 mice (n = 2 per group) were prepared for a survival surgical procedure by using one of the 4 antiseptic preparation agents to evaluate whether the antiseptic preparation agents caused skin irritation or impaired healing. Results from this study indicated that all 3 of the antiseptic agents evaluated were equally effective at reducing bacterial populations immediately and at 20 min after preparation. Histopathologic examination of the incision sites revealed signs of normal healing without lesions adjacent to the incision site. We conclude that all 3 of the products evaluated are comparable to traditional povidone–iodine and alcohol as agents for aseptic preparation of surgical sites.

Laboratory animals are widely used in imaging studies, including infection, heart, and brain research. Compared with rodents, pigs are especially useful because of their large organ sizes, ability to tolerate long-term anesthesia, and substantial blood volume, which allows repeated blood sampling. These factors are particularly important in positron emission tomography studies of potential new radioactive tracers, because the scans often are prolonged; in addition, kinetic studies involving repeated blood sampling may be performed to establish the optimal scan time. However, protracted studies may affect the cardiovascular system, brain, and other organs. This raises the question of how to monitor and counteract the effects of longterm anesthesia in pigs in a typical experimental setting yet prevent introducing bias into the experiment. To address this question, we investigated the effects of long-term anesthesia (maximum, 18 h), repeated blood sampling (maximum of 20 mL blood per kilogram body weight), and road transportation (as long as 1.5 h between 2 imaging centers) on key variables of lung, heart, and brain function in the context of a well-established pig model of Staphylococcus aureus infection. Pulse rate, oxygen saturation, body temperature, arterial pressure of CO₂, and urine production were stable during anesthesia for at least 16 h, whereas blood glucose slowly decreased. Hct and leukocyte count decreased due to repeated blood sampling. During road transportation, blood lactate levels increased 5 fold and arterial pressure of O₂ decreased by 50%. Repeated CT scans, necropsy results, and histopathology findings documented progressive lung changes and acute cardiac necrosis. No lesions indicative of hypoxia were found in brain. The study data show that the typical monitoring parameters do not fully depict the cardiovascular state of pigs during prolonged anesthesia. We recommend streamlining experimental protocols for imaging studies in pigs to avoid organ pathology.

Epidural puncture in swine is technically challenging. Several combinations of limb and body positions have been suggested to increase lumbosacral interlaminar space (LSS) and lumbosacral angle (LSA). This study investigated whether cranial hyperflexion of pelvic limbs increased LSS and LSA in laterally and sternally recumbent juvenile Duroc and adult Yucatan pigs and assessed which position produced the largest LSS. Juvenile Duroc (n = 7) and adult Yucatan (n = 7) pigs were euthanized and randomly placed in 4 positions: sternal with neutral limbs, sternal with cranially hyperflexed limbs, lateral with neutral limbs, and lateral with hyperflexed limbs. LSS and LSA were measured on transverse axial CT images of the spine and compared by using multivariate ANOVA and the Student t test. In both age groups, LSS was greater in lateral flexed (juvenile, 7.0 ± 0.7 mm; adult, 15.9 ± 1.1 mm) and sternal flexed (juvenile, 7.5 ± 1 mm; adult, 17.1 ± 1.1 mm) positions than in lateral neutral (juvenile, 5.4 ± 0.9 mm; adult, 9.6 ± 1.6 mm) position. In addition, in both age groups, LSS and LSA in lateral neutral position were smaller than lateral flexed, sternal neutral, and sternal flexed positions. In adults, LSS was greater in lateral flexed and sternal flexed than in sternal neutral position. Hyperflexion of pelvic limbs increases LSS and LSA in sternally recumbent adult Yucatan pigs and laterally recumbent adult Yucatan and juvenile Duroc swine. Increased LSS from positioning pigs with pelvic limbs flexed in sternal or lateral recumbence may facilitate epidural puncture compared with neutral limb positioning.

Measuring vital signs is central to medical practice, but they are difficult to monitor in awake laboratory animals. We examined the feasibility of a noninvasive device for telemetric assessment of respiration rate, heart rate, temperature and movement in pigs. Awake piglets were monitored continuously for 31 h (interquartile range, 7) before (n = 4) and after (n = 3) surgery. Data quality was sufficient for determination of all parameters. We conclude that continuous, noninvasive monitor- ing of pigs is possible by using the evaluated device.

Maintaining effective analgesia during invasive procedures performed under general anesthesia is important for minimizing postoperative complications and ensuring satisfactory patient wellbeing and recovery. While patients under deep sedation may demonstrate an apparent lack of response to noxious stimulation, areas of the brain related to pain perception may still be activated. Thus, these patients may still experience pain during invasive procedures. The current study used anesthetized or sedated cynomolgus macaques and functional magnetic resonance imaging (fMRI) to assess the activation of the parts of the brain involved in pain perception during the application of peripheral noxious stimuli. Noxious pressure applied to the foot resulted in the bilateral activation of secondary somatosensory cortex (SII) and insular cortex (Ins), which are both involved in pain perception, in macaques under either propofol or pentobarbital sedation. No activation of SII/Ins was observed in macaques treated with either isoflurane or a combination of medetomidine, midazolam, and butorphanol. No movement or other reflexes were observed in response to noxious pressure during stimulation under anesthesia or sedation. The current findings show that despite the lack of visible behavioral symptoms of pain during anesthesia or sedation, brain activation suggests the presence of pain depending on the anesthetic agent used. These data suggest that fMRI could be used to noninvasively assess pain and to confirm the analgesic efficacy of currently used anesthetics. By assessing analgesic efficacy, researchers may refine their experiments, and design protocols that improve analgesia under anesthesia.