The IACUC comprises the key component of animal research oversight at any institution or facility and thus has the responsibility to review and approve proposed animal activities. As the primary oversight unit that ensures the quality of animal welfare and therefore contributes to overall research quality, the IACUC can support reproducibility in research by ensuring rigorous experimental design, standardization of care and management, and assessment of the validity of research. An IACUC that is constituted as required by the Animal Welfare Act and the PHS Policy incorporates a wide range of expertise. Here we explore the contributions of the various IACUC members and discuss how each can help to enhance rigor and mitigate issues regarding irreproducibility in biomedical research involving animals.

Embryo transfer (ET) is a frequent procedure in contemporary animal and transgenic facilities. We compared the reproductive performance of mice after unilateral and bilateral ET of 15 to 18 two-cell embryos per recipient. The genetic backgrounds of the donors were C57BL/6J (B6J), C57BL/6N (B6N), or fewer than 5 generations of backcrossing to B6 (unknown substrain, <5G B6). The pregnancy rate was significantly higher for bilateral than for unilateral ET for B6J lines (85.4% compared with 79%) but similar between modes for B6N (73.7% compared with 77.9%) and <5G B6 (77% compared with 74.5%) lines. The birth rates after unilateral and bilateral ET were 30.8% and 33.0% for B6J lines, 24.5% and 26.9% for B6N lines, and 31.0% compared with 28.2% for <5G B6 lines, with no significant difference between the modes of ET. Birth rate was significantly higher for B6J lines than B6N lines after both unilateral and bilateral ET. For B6J and B6N lines, the number of pups born per litter was significantly higher for bilateral than unilateral ET. Unilateral ET yielded 0.24 to 0.31 pup per embryo transferred compared with 0.27 to 0.33 pups after bilateral ET. Over all genetic backgrounds, 3.03 to 4.09 embryos were required to produce a single pup. The present study provides data to aid in decision-making as to whether unilateral or bilateral ET should be performed. Bilateral ET results in a larger litter but increases pain and discomfort in recipients. However, unilateral ET saves time and contributes to refinement because surgical trauma is reduced.

Obesity is a public health problem in both developed and developing countries, and the negative effects of obesity on reproductive physiology have been highlighted recently. We evaluated the effects of porcine obesity index, sex hormones, and peptide hormones on litter size in various breeds of minipigs. Blood samples were collected from sedated 8-,10-, and 12-mo-old minipigs to measure preovulatory levels of sex hormones (follicle-stimulating hormone, luteinizing hormone, estradiol, progesterone, testosterone, and prolactin) and peptide hormones (insulin-like growth factor, glucagon, cortisol, growth hormone, free thyroxine, free triiodothyronine, insulin, and leptin). We also measured weight, abdominal circumference, neck circumference, and body length and then calculated the porcine obesity index. Data were analyzed by one-way ANOVA, and means were compared by least significance difference testing. Pearson correlation between parameters and litter size was analyzed. Prepregnancy porcine obesity index and litter size were negatively correlated in primiparous minipigs. Litter size was influenced by luteinizing hormone, estradiol, progesterone, testosterone, prolactin, follicle-stimulating hormone, cortisol, insulin-like growth factor 1, growth hormone, free thyroxine, insulin, and leptin. In conclusion, prepregnancy obesity reduces litter size in primiparous minipigs.

Sleep disruption in humans, caused by shift work, can be detrimental to physical and behavioral health. Nocturnal laboratory mice may experience a similar disruption caused by human daytime activities, but whether this disruption affects their welfare is unknown. We used 48 mice (CD1, C57BL/6, and BALB/c of both sexes) in a factorial design to test a sleep disruption treatment, in which mice were disturbed by providing routine husbandry at either 1000 or 2200 during a 12:12-h light:dark cycle, with lights on at 0700. All mice were exposed for 1 wk to each disruption treatment, and we used a noninvasive sleep monitoring apparatus to monitor and record sleep. To determine whether providing nesting material ameliorated effects of sleep disruption, we tested 4 amounts of nesting material (3, 6, 9, or 12 g) and continuously recorded sleep in the home cage for 2 wk. C57BL/6 mice, regardless of sex or disruption timing, slept the least overall. There was a strong interaction of sex and type of mouse on sleep across 24 h. Mice slept less during the first day of the daytime disturbance than on day 6. These results suggest that disturbance timing affects sleep patterns in mice but not their overall amount of sleep and that the changes in sleep patterns vary between mouse type and sex. In addition, mice appear to both anticipate and acclimate to human activity during the day. Our welfare checks were possibly too predictable and inconsequential to induce true sleep disruption.

Effective environmental enrichment is used by animals, promotes species-typical behavior, and decreases abnormal behavior. Porches attached to the front of an animal's cage provide additional space, perching opportunities, and a better view of the surroundings. Here we assessed the effectiveness of porches as a form of enrichment and identified characteristics of the animals most likely to use the porches. We videorecorded and scored the behavior of 18 (9 male, 9 female) singly housed cynomolgus macaques (Macaca fascicularis) during 3 observation intervals (15 min each) the week before, during, and after exposure to the porches. Changes in abnormal and tension-related behaviors (pacing, yawning, scratching) and speciestypical behaviors were compared across the 3 wk of observation. Novel object temperament tests were performed before and after the study. During observation periods, subjects spent an average of 75% of time in the porch. No changes in pacing or tension-related behaviors occurred, but activity decreased during and after porch exposure, rest increased during the porch exposure, and consumption decreased afterward. Eight subjects were categorized as having a bold temperament, and the remaining 10 subjects had an intermediate temperament. Sex and a temperament×cage location interaction were predictors of porch usage. Males used the porches more than did females, and those with an intermediate temperament were less likely to use the porches when they were located in the lower cages. Porches are beneficial in that they are used for extensive periods of time, but the benefits can vary according to the individual animal.

Sanitation frequency of mouse cage components can be determined through verification of microenvironment, including microbiologic load and air quality within the cage. Here we demonstrate a consistent microbiologic load on wire IVC lids that were used for as long as 8 continuous weeks to house 4 or 5 mice and significant decreases in the microbial load on filter tops at 4, 6, and 8 wk compared with 2 wk. In addition, air quality, represented by intracage ammonia concentration at the time of bedding change, did not differ between 2-, 4-, and 6-wk time points in cages containing same-sex groups of 4 or 5 male or female adult mice. We propose that the lack of significant differences represents justification for an extended sanitation frequency of as long as 6 wk for cage top components in mouse IVC housing and represents a performance standard that might be reproduced by similar facilities to determine appropriate sanitation frequencies for mouse caging components.

Selection of an appropriate method of euthanasia involves balancing the wellbeing of the animal during the procedure with the intended use of the animal after death and the physical and psychologic safety of the observer or operator. The recommended practices for anesthesia as compared with euthanasia are very disparate, despite the fact that all chemical methods of euthanasia are anesthetic overdoses. To explain this disparity, this study sought to determine whether perception bias is inherent in the discussion of euthanasia compared with anesthesia. In this study, participants viewed videorecordings of the anesthesia of either 4 rats or 4 mice, from induction to loss of consciousness. Half of the participants were told that they were observing anesthesia; the other half understood that they were observing euthanasia. Participants were asked to rate the distress of the animals by scoring escape behaviors, fear behaviors, respiratory distress, and other distress markers. For mice, the participants generally rated the distress as high when they were told that the mouse was being euthanized, as compared with the participants who were told that the mouse was being anesthetized. For rats, the effect was not as strong, and the distress was generally rated higher when participants were told they were watching anesthesia. Because the interpretation of distress showed bias in both species—even though the bias differed regarding the procedure that interpreted as distressing—this study demonstrates that laboratory animal professionals must consider the influence of potential perception bias when developing policies for euthanasia and anesthesia.

Laboratory animal programs and core laboratories often set service rates based on cost estimates. However, actual costs may be unknown, and service rates may not reflect the actual cost of services. Accurately evaluating the actual costs of services can be challenging and time-consuming. We used a time-driven activity-based costing (ABC) model to determine the cost of services provided by a resource laboratory at our institution. The time-driven approach is a more efficient approach to calculating costs than using a traditional ABC model. We calculated only 2 parameters: the time required to perform an activity and the unit cost of the activity based on employee cost. This method allowed us to rapidly and accurately calculate the actual cost of services provided, including microinjection of a DNA construct, microinjection of embryonic stem cells, embryo transfer, and in vitro fertilization. We successfully implemented a time-driven ABC model to evaluate the cost of these services and the capacity of labor used to deliver them. We determined how actual costs compared with current service rates. In addition, we determined that the labor supplied to conduct all services (10,645 min/wk) exceeded the practical labor capacity (8400 min/wk), indicating that the laboratory team was highly efficient and that additional labor capacity was needed to prevent overloading of the current team. Importantly, this time-driven ABC approach allowed us to establish a baseline model that can easily be updated to reflect operational changes or changes in labor costs. We demonstrated that a time-driven ABC model is a powerful management tool that can be applied to other core facilities as well as to entire animal programs, providing valuable information that can be used to set rates based on the actual cost of services and to improve operating efficiency.

Biodecontamination is important for eliminating pathogens at research animal facilities, thereby preventing contamination within barrier systems. We enhanced our facility's standard biodecontamination method to replace the traditional foggers, and the new system was used effectively after creating bypass ducts in HVAC units so that individual rooms could be isolated. The entire system was controlled by inhouse-developed supervisory control and data-acquisition software that supported multiple cycles of decontamination by equipment, which had different decontamination capacities, operated in parallel, and used different agents, including H₂O₂ vapor and ClO₂ gas. The process was validated according to facility mapping, and effectiveness was assessed by using biologic (Geobacillus stearothermophilus) and chemical indicator strips, which were positioned before decontamination, and by sampling contact plates after the completion of each cycle. The results of biologic indicators showed 6-log reduction in microbial counts after successful decontamination cycles for both agents and found to be compatible with clean-room panels including commonly used materials in vivarium such as racks, cages, trolleys, cage changing stations, biosafety cabinets, refrigerators and other equipment in both procedure and animal rooms. In conclusion, the automated process enabled users to perform effective decontamination through multiple cycles with realtime documentation and provided additional capability to deal with potential outbreaks. Enabling software integration of automation improved quality-control systems in our vivarium.

Demodex mites are microscopic, cigar-shaped, follicular mites often regarded as commensal microfauna in mammals. Although Demodex spp. can cause dermatologic disease in any immunocompromised mammal, they are rarely reported in laboratory mice. Recent identification of Demodex musculi in a colony of immunodeficient mice with dermatitis afforded us the opportunity to investigate the comparative sensitivity of 4 antemortem diagnostic techniques to detect D. musculi—superficial skin scrape (SSS), tape impression (TI), fur pluck (FP), and deep skin scrape (DSS)—which we performed on 4 anatomic sites (face, interscapular region [IS], caudal ventrum [CV], and caudal dorsum [CD]) in 46 mice. DSS had an overall detection rate of 91.1% (n = 112 tests), with the highest detection rates in IS (93.5%), CV (89.1%), and CD (90.0%). The detection rates for SSS (62.5%; n = 112 tests), TI (57.5%; n = 138 tests), and FP (62.7%; n = 158 tests) were all lower than for DSS. IS was the most reliable site. Results from combined FP and DSS samples collected from IS and CV yielded 100% detection, whereas the face was not a desirable sampling site due to inadequate sample quality and low detection rate. Demodex eggs and larvae were observed from FP more often than DSS (19.0% of 158 tests compared with 14.3% of 112 tests). In a subset of samples, an 18S rRNA PCR assay was equivalent to DSS for detection of mites (both 100%, n = 8). We recommend collecting samples from both IS and CV by both FP and DSS to assess for the presence of D. musculi and performing further studies to assess whether PCR analysis can be used as a diagnostic tool for the detection of Demodex mites in laboratory mice.

Recent studies have revealed some of the most frequently used analgesics in mice are not effectively treating postoperative pain. Our laboratory sought to compare and assess the validity and reliability of 2 cageside pain assessments that we recently developed for use in mice—nesting consolidation and grooming transfer tests. We then applied these tests to compare the efficacy of commonly used analgesics—buprenorphine (0.1 mg/kg SC every 12 h for 48 h) and carprofen (30 mg/kg in drinking water for 72 h)—alone and in multimodal combination as a refinement for treating postoperative pain in mice. Briefly, C57BL/6 and CD1 male and female mice underwent assessment under conditions of baseline, anesthesia–analgesia, and laparotomy. Results showed that multimodal analgesia displayed the greatest analgesic coverage over the postoperative period, whereas buprenorphine showed slightly less coverage, and carprofen and saline groups displayed signs of pain at most postoperative time points. After anesthesia–analgesia, buprenorphine and multimodal mice lost significant body weight in the absence of a painful stimulus and displayed other significant drug-related changes. Animals treated with carprofen showed few drug-related changes after anesthesia–analgesia but also demonstrated minimal benefit from postsurgical analgesia. Overall, multimodal analgesia was more effective for treating postsurgical pain in mice than the single-analgesic protocols we tested; however, effects on weight loss need to be considered during analgesic selection. Nesting consolidation and grooming transfer tests were valid and highly reliable over time, in inbred and outbred mice, in male and female mice, under different housing conditions. In addition, the nesting consolidation test had excellent reliability between observers. These findings can be used in refining the detection and treatment of postoperative pain in mice.

Often few alternative anesthetics for exotic species are available, due to the small numbers of these animals used in research. In this study, we evaluated the depth and duration of anesthesia in Xenopus laevis after their immersion in 3 doses of etomidate (15, 22.5, and 30 mg/L) and in 3 doses of benzocaine (0.1%, 0.5%, and 1%) compared with the 'gold standard,' tricaine methanesulfonate (MS222; 2 g/L). We then chose an optimal dose for each alternative anesthetic according to induction time, duration of surgical plane, and time to complete recovery. The optimal etomidate and benzocaine doses (22.5 mg/L and 0.1%, respectively) as well as the MS222 dose were then used to achieve a surgical plane of anesthesia, with the addition of flunixin meglumine (25 or 50 mg/kg) administered in the dorsal lymph sac at the completion of mock oocyte harvest. Efficacy of the analgesic was assessed at 1, 3, 6, and 24 h postoperatively by using acetic acid testing (AAT). Histology of the liver, kidney, and tissues surrounding the dorsal lymph sac was performed at day 3, 14, and 28 in each group of animals. Mild to moderate myocyte degeneration and necrosis were present in tissues surrounding the dorsal lymph sac at both flunixin meglumine doses after etomidate and benzocaine anesthesia. In addition, the 50-mg/kg dose of flunixin meglumine resulted in the death of 5 of the 12 frogs within 24 h, despite an otherwise uneventful anesthetic recovery. In conclusion, benzocaine and etomidate offer alternative anesthetic regimens, according to typical requirements for an anesthetic event. Flunixin meglumine at the 25-mg/kg dose provided analgesic relief at the latest time point during etomidate dosage and at all time points during benzocaine dosage, but further characterization is warranted regarding long-term or repeated analgesic administration.