Journal of the American Association for Laboratory Animal Science最新文献

Cryopreservation of epididymal sperm collected after euthanasia is a common method to preserve and distribute valuable mouse models worldwide. However, the euthanasia method used prior to sperm collection must not adversely affect sperm quality. The most common method of euthanasia in mice is CO₂ asphyxiation, but its effect on the quality of sperm collected postmortem is largely unknown. The objective of this study was to determine the effects of CO₂ euthanasia of C57BL/6 mice on both freshly recovered sperm and sperm subjected to freezing and thawing. First, sperm concentration, progressive motility, curvilineal velocity (VCL), average path velocity (VAP), and progressive velocity (VSL) were analyzed for mice euthanized by cervical dislocation (CD), high flow CO₂ (100%), or low flow CO₂ (30%) displacement/minute, respectively. Then, in-vitro fertilization and embryonic development rates were determined using frozen-thawed sperm from each euthanasia method. Neither fresh nor frozen-thawed sperm showed significant differences in sperm concentration, progressive motility, VAP, or VCL when compared to CD and CO₂ groups. However, frozen-thawed sperm collected from CD mice had higher VCL values than did those collected from the low flow mice (P = 0.039). VCL was not different in fresh or frozen-thawed sperm collected after mouse euthanasia by CD as compared with high flow CO₂ or by high flow as compared with low flow CO₂. Frozen-thawed sperm showed no differences among the 3 euthanasia groups for fertilization (P = 0.452) or blastocyst development rates (P = 0.298). The results indicate that CO₂ euthanasia can be used as an alternative to CD to obtain optimal quality mouse sperm for cryopreservation while remaining compliant with welfare requirements.

Maximizing operational efficiency while maintaining appropriate animal housing conditions is a continuous focus of research animal care programs. Our institution's longstanding approach to cage-change management included scheduled cage changes every 2 wk, with spot changes if cages met established visual criteria during the intervening period. This 2-wk plus spot changing (2WS) practice for mice housed in IVC was problematic during the COVID-19 pandemic when the need arose to minimize workload to reduce on-site staffing out of concern for employee health and possible absenteeism. With the approval of the IACUC, a spot-change-only (SCO) process was adopted, with the requirement to evaluate microenvironmental parameters under both practices to confirm acceptable equivalence. These parameters (humidity, temperature, and ammonia) were evaluated in a controlled study that found no significant difference between the 2 groups. Ammonia levels did not exceed 10 ppm in any group throughout the study. To assess operational differences between these 2 approaches, we collected cage-change data and employee feedback from facilities operating under these schemes. The SCO method required fewer cage changes than did the 2WS method (10.3% per day with 2WS and 8.4% per day with SCO). Despite this benefit, through a Plan-Do-Check-Act process that has been regularly employed at our institution, employee feedback identified important operational challenges associated with the SCO practice. The SCO approach was thus refined into a scheduled spot change (SSC) practice that builds on the SCO model by incorporating a scheduled focused cage evaluation period. Based on subsequent feedback, the SSC was found to retain the efficiency benefits afforded by the SCO model and simultaneously alleviated staff and operational concerns. This result underscores the importance of integrating staff feedback with a performance standard-based approach when assessing cage-change management.

Laboratory animal professionals (LAP) may experience situations that contribute to compassion fatigue (CF). The goal of this research was to better understand CF in LAP in and across employment categories. Surveys were distributed through LAP organizational listservs in the European Union (EU), China, and Japan, and results were analyzed to identify CF prevalence, personal and work-related factors, coping mechanisms, and beneficial work-support programs. Independent χ²-tests compared personal and work-related factors and feelings of CF. Feelings of CF and coping mechanisms were compared with personality scores using independent sample t tests. There were 302 respondents from the EU, 39 from China, and 77 from Japan. Over half of respondents from the EU (52%) and China (56%) reported experiencing CF (52%), with fewer (32%) reporting CF in Japan. No major differences were found based on employer type. Personality scores were significantly related to feelings of CF and preferred coping mechanisms. Work-related factors that contributed to feelings of CF in over half of respondents included staffing levels, workplace relationships, and availability of programs geared to address CF. Across regions, talking to someone, physical activity, getting away from work, and self-care were effective coping mechanisms in over 50% of respondents. Fewer than 30% of respondents indicated that their place of employment had CF support programs, and even fewer (8% to 28%) indicated that these programs were helpful. The study results suggest that to be effective, employer CF programs for LAP should consider providing quiet places at work and programs for self-care, promoting physical and mental health and social support systems, and establishing opportunities to memorialize animals.

Balanced anesthesia-the use of a combination of drugs to achieve a desired anesthetic plane-offers many benefits, including smoother induction and recovery and fewer adverse effects than occur with individual drugs. Although premedication prior to inhalant anesthesia is routine in other species, mice are commonly induced with gas anesthesia alone. The hypothesis of this study was that premedication with ketamine or xylazine would safely reduce the stress of isoflurane induction and lower the minimum alveolar concentration (MAC) of isoflurane. Young adult male and female C57BL/6J mice were premedicated with ketamine (100 mg/kg), xylazine (4 mg/kg), or isotonic crystalloid (0.1 mL) and were used in 4 experiments. First, isoflurane induction was video recorded under all test conditions, and the videos were scored according to a behavioral ethogram to identify signs of distress. Mice in the ketamine group experienced tremors and ataxia before and dur- ing induction. Therefore, ketamine was given after induction with isoflurane in subsequent experiments. Second, the MAC value for each anesthetic protocol was determined by using quantal and bracketing analysis. Third, mice were anesthetized according to the 3 protocols, and vital parameters were monitored for 60 min. Finally, anesthetized mice were challenged with hypoxia and hypovolemia, and vital parameters were monitored. Premedication with xylazine significantly reduced the stress scores for isoflurane induction (control, 7.3 ± 1.5; ketamine, 6.0 ± 3.0; xylazine, 3.1 ± 1.0). Ketamine and xylazine both reduced the MAC of isoflurane (control, 1.89%; ketamine, 0.96%; xylazine, 1.20%). All mice survived 60 min of anesthesia and the hypoxia-hypovolemia challenge. Premedication with xylazine reduced the stress of induction and lowered the necessary dose of isoflurane in C57BL/6J mice to maintain a surgical plane of anesthesia. We recommend administering xylazine before isoflurane induction and anesthesia of healthy mice that are undergoing procedures in which 100% oxygen is provided and anticipated blood loss is less than 10% to 15% of the total blood volume.

Maintenance of an appropriate microenvironment for rodents used in research is of paramount importance because changes in environmental parameters such as O₂ and humidity can influence animal health and welfare and potentially alter research results. Here we evaluated the microenvironment of mouse and rat disposable cages after removal from mechanical ventilation in order to guide recommendations for their use. Cages with sealed IVC lids, unsealed lids (partially ajar), and lids without the exhaust filter (for rats) or static lids (for mice) were removed from the ventilated rack and were thereafter monitored CO₂, O₂, and NH₃ levels. For mice, effects were investigated under both standard (set point of 72°F/22°C) and thermoneutral (set point of 82°F/28°C) temperatures. When IVC with sealed lids and group-housed C57BL/6J male mice were removed from ventilation under standard temperatures, CO₂ started at 6,600 ± 265 ppm at 0 h and rose to 42,500 ± 7,263 ppm at 1 h, with mice showing a visibly elevated respiratory rate in 1 of the 3 cages; CO₂ stabilized at 26,150 ± 3,323 ppm at 8 h. In contrast, CO₂ levels in cages with single mice were stable after 1 h (1,350 ± 409 ppm at 0 h, 9,367 ± 802 ppm at 1 h, and 8,333 ± 1,115 ppm at 8 h). Findings were similar at thermoneutral temperatures: sealed group-housed mice cages started at 3,617 ± 475 ppm at 0 h and rose to 39,333 ± at 5,058 ppm at 1 h, whereas sealed cages with 1 mouse started at 1,117 ± 247 ppm at 0 h and were 7,500 ± 1,997 ppm at 8 h. IVC with sealed lids and pair-housed Crl:CD(SD) female rats rose to 48,000 ± 2,828 ppm CO₂ and over 70% humidity within 1 h. By 3 h, IVC with sealed lids and singly housed rats had 40,167 ± 5,132 ppm CO₂, and rats were displaying a visually elevated respiratory rate. O₂ levels had an inverse relationship with CO₂ levels. Removing the rat lid exhaust filter was not helpful. However, leaving the IVC lid ajar ameliorated the rise in CO₂ and fall in O₂ for both species. Therefore, IVC with sealed lids and group-housed mice should not be removed from ventilation more than 1 to 2 h; IVC containing pair- or singly-housed rats IVC should not be removed for more than 1 or 3 h, respectively. Whenever possible, such cages should be fitted with static lids, left partially ajar and monitored, or replaced on ventilation.

Pathogen monitoring and colony health management are critical components of any rodent research program. From an operational perspective, rodent facilities are protected from unwanted infectious agents by facility-specific bioexclusion criteria, sanitation of the physical environment, and personal protective equipment. Another important preventative measure is the use of room health levels to provide traffic patterns for animal care and research staff as they move between rooms of differing health status. For mice, our institution uses a tiered room level system with 6 defined categories, ranging from level 1 (strictest entry criteria) to 6 (least stringent entry criteria). Level 6 is defined as rooms with mice that have tested positive for mouse parvovirus (MPV) or mouse rotavirus (MRV) or both on sentinel serology at any point in time in the past and no decontamination. Because many of our mouse rooms had historically been positive for MPV and/or MRV and because of the high financial and logistic challenges of using repeated test-and-cull for elimination, we had tolerated the potential presence of MPV and MRV and had developed management practices that would promote 'burnout' (that is, elimination of infectious agents due to absence of susceptible hosts) of these pathogens. Analysis of sentinel data showed that we had 28 rooms in 4 facilities for which excluded pathogens had not been identified in 3 y or more. We therefore developed a hybrid testing strategy involving both PCR analysis and serology and implemented it in sentinels and in select colony mice to determine whether the rooms had undergone successful burnout and were free of MPV and MRV. All test results obtained during the assessment were negative for both viruses, and the rooms were subsequently upgraded to level 5 (free from excluded pathogens and allowing two-way movement in and out of housing room). All upgraded rooms have remained negative on subsequent quarterly routine sentinel serology for over 3 y. Our testing strategy for confirming pathogen burnout may be a useful and cost-efficient model for other academic rodent research programs that face a similar situation.

PCR testing is increasingly important for microbial control in SPF facilities. However, most current PCR methods are timeconsuming and require compromise between high sensitivity and high multiplexing. We developed a one-tube multiplex nested PCR strategy (MN-PCR) for simultaneous direct (that is, without culturing) detection of multiple pathogens. We first aligned sequences for the 16S rDNA genes of selected target bacteria and a panel of closely related organisms. From these data, we designed a pair of universal primers and multiple sets of species-specific PCR primers to amplify the target sequences; the universal primers were modified to include various degenerate bases and locked nucleic acids. In a single tube, 16S rDNA sequences were amplified by using the nested PCR primers under high temperature (that is, above 65°C) during the first stage of the MN-PCR procedure, when the target-species-specific PCR primers do not support amplification due to their short length. In addition, the concentration of the nested PCR primers during the first stage was adjusted to ensure that they were consumed and did not yield visible bands themselves. During the second stage, the enriched 16S rDNA sequences then served as templates for amplification of the species-specific fragments by using the multiple PCR primers at low annealing temperatures (that is, below 60°C). The results showed that our MN-PCR method detected as little as 1 fg of target bacterial DNA in a 20-μL reaction volume, whereas conventional multiplex PCR detected a minimum of 1 pg only. Compared with traditional multiplex PCR assays, our MN-PCR system is an effective and efficient culture-free process.

Inappetence is a welfare concern in rabbits (Oryctolagus cuniculus), as it can lead to potentially fatal gastrointestinal stasis. In other species, inappetence is commonly treated with appetite stimulants; however, few published studies have evaluated the efficacy of appetite stimulants in rabbits. We performed 2 studies to evaluate the effects of capromorelin and mirtazapine on appetite in New Zealand White (NZW) rabbits. In the first study, healthy rabbits ( n = 9) were evaluated using a randomized crossover design and 9 treatments: capromorelin 4 mg/kg oral (PO) once a day (SID), capromorelin 8 mg/kg PO SID, saline control PO SID, capromorelin 4 mg/kg PO twice a day (BID), capromorelin 8 mg/kg PO BID, saline control PO BID, mirtazapine 0.5 mg/kg transdermal (TD) SID, mirtazapine 1 mg/kg TD SID, and saline control TD SID for 3 d with a 1-wk washout period between treatments. Treatment efficacy was assessed by measuring daily feed intake and fecal output and by weighing rabbits twice a week. Overall, feed intake and fecal output were higher for all treatments as compared with controls, except for fecal output in the capromorelin 4 mg/kg and 8 mg/kg PO SID groups. Feed intake and fecal output were significantly higher with mirtazapine as compared with capromorelin. Body weight and erythema/petechia of the pinnae were greater in the mirtazapine 1 mg/kg TD SID group than in the control group. A second study evaluated rabbits that had undergone surgery (castration, n = 7) and then received one of 3 treatments: capromorelin 8 mg/kg PO BID, mirtazapine 1 mg/kg TD SID, or saline PO BID for 3 d postoperatively. Feed intake and fecal output in the postoperative mirtazapine group were not significantly different from those of the capromorelin and control groups. Due to its superior efficacy as compared with capromorelin in healthy NZW rabbits, we recommend considering mirtazapine as a treatment for inappetence in NZW rabbits.

Surgical procedures are commonly performed using mice but can have major effects on their core body temperature, including development of hypothermia. In this study, we evaluated active perioperative warming with and without surgical draping with adherent plastic wrap to refine practices, improve animal welfare, and optimize research experiments. Mice were randomized into treatment groups (n = 6; 8 CD1 mice per group). Treatments included placement within a small-animal forced-air incubator at 38 ° C for 30 min before surgery (Pre), after surgery (Post), or before and after surgery (Both). To explore the effect of surgical draping, one group received incubator warming before and after surgery in addition to surgical draping (Both/ Drape), whereas another group received surgical draping only without incubator warming (Control/Drape). The final group of mice received neither warming nor draping (Control). Subcutaneous temperature transponders were placed in all mice. Approximately 5 d after transponder placement, mice were anesthetized with ketamine-xylazine and underwent laparotomy. Subcutaneous body temperatures were collected perioperatively from transponders, and rectal temperatures were taken every minute during surgery. For recovery from anesthesia, mice were placed either in a standard cage on a warm water blanket set to 38 °C (100.4 °F) or in the incubator. Subcutaneous body temperatures were significantly higher in mice prewarmed for 30 min (Pre, Both, Both/Drape) as compared with mice that were not prewarmed. Anesthetic recovery times were significantly longer for mice placed in the incubator (Pre, Post, Both, Both/Drape) than for those that did not receive incubator warming (Control, Control/Drape). Mean intraoperative rectal temperatures of Both/Drape mice tended to be greater than those of mice in the Both group, suggesting a warming benefit of surgical draping. Using a forced air incubator and adherent plastic draping mitigated body temperature loss in mice during both surgery and postoperative recovery.

The Guide for the Care and Use of Laboratory Animals strongly encourages the use of pharmaceutical-grade chemicals and analgesics. Sustained-release buprenorphine (SRB) is administered extralabel to rodents to mitigate moderate to severe pain. An FDA-indexed buprenorphine formulation-extended-release buprenorphine (XRB)-has recently become available and is currently the only pharmaceutical-grade slow-release buprenorphine formulation approved for use in mice and rats. However, no studies have directly compared the pharmacokinetic parameters of SRB and XRB in surgically catheterized mice. To this end, we compared the plasma buprenorphine concentrations and pharmacokinetic parameters of SRB and XRB in mice after surgical catheterization. We hypothesized that mice treated before surgery with SRB or XRB would have circulating buprenorphine concentrations that exceeded the therapeutic threshold for as long as 72 h after surgery. Male and female C57Bl/6J mice were anesthetized, treated with a single dose of either SRB (1 mg/kg SC) or XRB (3.25 mg/kg SC), and underwent surgical catheterization. Arterial blood samples were collected at 6, 24, 48, and 72 h after administration. Weight loss after surgery (mean ± SEM) was similar between groups (SRB: males, 12% ± 2%; females, 8% ± 2%; XRB: males, 12% ± 1%; females, 8% ± 1%). Both SRB and XRB maintained circulating buprenorphine concentrations above the therapeutic level of 1.0 ng/mL for 72 h after administration. Plasma buprenorphine concentrations at 6, 24, and 48 h were significantly greater (3- to 4-fold) with XRB than SRB, commensurate with XRB's higher dose. These results support the use of either SRB or XRB for the alleviation of postoperative pain in mice. The availability of FDA-indexed XRB increases options for safe and effective pharmaceutical-grade analgesia in rodents.