Abstracts of Scientific Presentations: 2021 AALAS National Meeting Kansas City, Missouri

Animal Welfare, Training, and 3Rs Posters

P1 Refinements for Mice Housed in Metabolic and Behavior Phenotyping Systems

A Healy*¹, C Yeung^2,3, M Choe², H Sung^2,3

¹Animal Resources and Compliance, The Center for Phenogenomics, Toronto, Canada; ²Translational Medicine Program, The Hospital for Sick Children Research Institute, Toronto, Canada; ³Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada

Metabolic and behavior monitoring systems are commonly used in laboratory animal facilities. Most animal monitoring systems require mice to be singly housed, often for a period of several days, without environmental enrichment and nesting material to ensure that there is no interference with the collection of metabolic measurements. However, social isolation in barren environments negatively affects animal welfare and can introduce unintended variation in metabolic phenotypes induced by factors such as stress and impaired thermoregulation. Our study aims to determine the amount of nesting material that may be provided without interfering with data collection when using metabolic and behavior monitoring systems. It is also of interest to know if providing 6g of nesting material, the minimum required to create a sufficient nest, affects mouse metabolism. Twenty-four male C57BL/6J mice were used in this study. All mice were singly housed on corn cob bedding with a red dome and wood stick for enrichment, and provided with either 0g, 3g or 6g of shredded paper-strip nesting material. Oxygen consumption, carbon dioxide production, respiratory exchange ratio, locomotion activity, food intake and water intake were measured 24 h/d over 3 d after a 2-d acclimation period. Body weight was measured at the beginning and end of the study. Environmental temperature was maintained between 22-23°C. Metabolic phenotypes observed between groups was nearly homogenous indicating that the nesting material provided did not interfere with data collection and metabolism in mice. Interestingly, there was a slight, yet statistically significant reduction in locomotion in the 3g nesting condition on d 2 and 3 of data collection. We conclude that the addition of a red dome, wood stick, and 6g of nesting material can be used in metabolic and behavioral monitoring systems without affecting an animal’s physiological data and that metabolic parameters remain stable when 6g of nesting material is provided. Access to sufficient nesting material, in addition to enrichment consisting of a shelter and wood stick, are important refinements to maintain mouse welfare when using similar metabolic and behaviour phenotyping systems.

P2 Standardized Cooperative Group-housed Training with Olive Baboons (Papio anubis)

AD Hawkins, A Tresler*, T Stevens, S Lane Comparative Medicine, Oklahoma University Health Science Center, Norman, OK

Group-housed settings can present problems with daily health and behavioral observations of olive baboons (Papio anubis). Injury or medical care often require an animal to be separated from the group for staff to examine in detail. Olive baboons have highly established social hierarchies and removal of an individual animal from the group can create stress and disrupt said hierarchy. In order to prevent these disruptions, we created a standardized, cooperative, group-housed training program for the baboons. The goal was to reduce the stress on the baboons by having them voluntarily approach staff for training through use of positive reinforcement methods. This grants staff better visibility of the animals for veterinary care without having to remove them from the group. Multiple practical behaviors were reinforced through use of clickers and food rewards. Trained behaviors were intended for functional purposes such as shifting animals between enclosures, allowing for easy physical observation and positioning at a designated location. Staff were able to train multiple individuals of varying ranks in close proximity simultaneously. Other behaviors were taught in hopes that it could aid in the visualization of digit injuries which are common in this species. The standardized, cooperative, group-housed training program resulted in 20 of the 25 baboons successfully learning the basic shifting behavior with many advancing to additional behaviors. This training program has proven successful in that these training techniques aided in examination, shifting, and moving the baboons without the use of separation. Future training will be aimed at aiding staff in their approach to medical treatments, such as voluntary injections, ultrasound scans, and blood draws.

P3 Evaluating Effects of CO₂ Levels on Severe Combined Immunodeficiency Disease (SCID) Mouse Models

A Updike*, D Calantropio-Covington, K Mueller, B Rendon, D Seiber, M Sowick, KD Walton

Bristol Myers Squibb, Redwood City, CA

In response to several notable studies conducted over the last 5 y, the American Veterinary Medical Association (AVMA) revised their recommendations for CO₂ euthanasia of rodents in the 2020 release of the AVMA Guidelines for the Euthanasia of Animals. The newly proposed 30-70% CO₂ displacement rates represent a shift from the previous opinion that a gradual displacement of the air within the chamber is more humane, and suggests that a higher displacement rate results in more rapid loss of consciousness and reduced pain and distress. Upon review of these revisions, we took initiative to align our rodent euthanasia practices to comply with the new guidance. A 50% displacement was selected as a starting point and visual assessments were performed using up to 20 C57Bl/6 and Balb/c mice to evaluate 3 parameters: equipment function, animal welfare indicators, and total time to euthanasia. No significant concerns were identified and a 50% displacement rate was implemented for rodent euthanasia. Over time, numerous reports of aversive responses to the increased displacement rate by severe combined immunodeficient disease (SCID) mice were communicated to leadership. Follow-up trials observing up to 10 animals (group and single-housed) of this strain identified an increased incidence of behaviors such as jumping, spinning, and erratic escape attempts. In addition, lateral recumbency was delayed 10-15 s longer than observed for other strains. Subsequent evaluations performed at multiple locations confirmed that this behavior could be replicated in SCID mice with different equipment at a 50% displacement rate. These observations prompted further trials of approximately 10 animals to titrate the CO₂ displacement rate for SCID mice, and 30% was identified as the maximum rate to eliminate all aversive behavior. Historical literature does allude to the potential for strain-related differences in response to CO₂ euthanasia, however to our knowledge, this is the first detailed report of the responses of SCID mice as compared to other common wild-type models. This data will be valuable to inform future investigations into clinical, physiological, genetic, or immune-mediated mechanisms of variable CO2 responses.

P4 Design of a Modular Swine Restraint Frame for Use in a BSL-3Ag Biocontainment Facility

AD Moffitt*^1,2, MB Gold^1,2, TJ Morgan¹

¹Animal Resource Branch, Plum Island Animal Disease Center, Orient, NY; ²The McConnell Group, Inc., Landover, MD

Commercial breeds of swine, ranging in size from 15 to 150 kg, are regularly used in studies at our BSL-3Ag facility. When performing procedures on awake swine, secure physical restraint is essential for personnel safety, animal safety, and procedural efficiency. While pig slings and restraint crates have been used successfully by others, we have faced feasibility challenges with these systems due to our unique facility and biosecurity constraints. In order to increase our mechanical restraint options, we designed a custom modular swine restraint frame (SRF). The major design criteria was that all parts of the apparatus must be able to be fully decontaminated or disposed of. Additional design considerations were ease of portability, ease of storage, and flexibility of use. The SRF can be fully disassembled for ease of decontamination, portability, and storage. Its modular design allows it to be used with a manually lifted pig sling, or as a restraint crate, accommodating animals of a wide size range. The SRF enables safe, low stress restraint of swine for procedures such as blood collection or clinical evaluation, while meeting the biosecurity needs of a BSL-3Ag environment.

P5 Refinement of Jugular Vein Catheterization with Vascular Access Button in Mice

A Lapierre*¹, K Kane¹, B Lyons²

¹Surgical Services, The Jackson Laboratory, Bar Harbor, ME; ²The Jackson Laboratory, Bar Harbor, ME

Mice implanted with a jugular vein catheter with a vascular access button (VAB) are used extensively in research for studies requiring repeated infusions over time. Multiple designs of VABs for use in rodents are available but most were originally developed for rats and therefore not optimized for use in the mouse. We found, across multiple strains of mice, that the use of a VAB with a polyester felt disk elicited a strong inflammatory reaction with poor wound healing and pressure necrosis due to the composition and stiffness of the disk. To increase the biocompatibility of the VAB disk, we designed a silicone mesh disk in collaboration with the manufacturer. The disk is perforated medical-grade silicone which provides greater flexibility to mold to the body contour and enables tissue ingrowth through the perforations for optimal healing. Initial studies were conducted using the silicone disk. The silicone disk was well tolerated with normal wound healing without evidence of pressure necrosis. A further refinement was to exteriorize the VAB through a circular incision made with a skin punch rather than through a traditional linear incision. This technique was performed (n = 70) and the circular incision eliminated the need for skin sutures and maintained the VAB in position on the dorsal midline. These refinements align with the 3Rs by optimizing wound healing and improving animal welfare.

P6 Institutional Management of Minors in Academic Animal Research

A Duval*

Animal Care and Use Office, University of Michigan, Brooklyn, MI

Academic institutions are on the forefront of scientific discovery and it is the institutional responsibility to protect and educate our young animal research scientists. Some potential concerns arise however when our young researchers are also a vulnerable population of minors. At an institutional level, there is often not a clear yet comprehensive best practice that addresses minor’s participation requirements in animal research. To address this need, we gathered data from multiple major animal research institutions, internal administrative bodies, and resource entities in attempts to consolidate, streamline, and develop consistent oversight around our minors in research program. The specific data collected from other institutions included requirements for parental consent, training, and if minors are permitted to work with ABSL-2 hazards. Currently there are limited documents that define clear regulatory requirements on how to include minors in institutional programs, and the best concise steps that should be taken. As the IACUC office oversees compliance at each institution, it is their responsibility to ensure the minors are properly approved to work with animals and they remain safe at all times. In efforts to have a definitive resource, consolidation of the data, departmental feedback, and an evaluation of policy enforcement strategies has contributed to this best practice guideline. During the development phase we worked with Unit for Laboratory Animal Medicine (ULAM) department, added the requirements for approval from our Environment, Health and Safety (EHS) department, and the university’s policies on involving minors on campus. This best practice guidance now informs the PI of all the proper steps that must be taken prior to the minor’s arrival at the institution. It points them in the right direction to get started, gives clear concise steps that must be followed, and who to contact if they need clarification. Guidance documents are only as affective as their communication so involving all departments that are involved in animal research is paramount while being mindful of the extra documentation or logistical burden of the research community.

P7 Heat Supplementation during Anesthesia: Does Type and Homeothermic Control Matter for Short Procedures in Mice?

F Scorrano², BN Gaskill*¹

¹Animal Welfare & Compliance, Novartis, Cambridge, MA; ²Comparative Medicine, Novartis, Basel, Switzerland

The Guide states that physiological function, such as body temperature, should be monitored during surgery, but this is not common practice for acute anesthesia or minor procedures. While the provision of heat supplementation is common practice, systems with homeothermic feedback capabilities (H) are rarely used outside major invasive surgeries. Further, literature comparing the efficacy and benefits of different warming devices with or without H control do not exist. Therefore, we hypothesized that when H control is used, there would be no differences in mouse body temperature between different systems (electric vs far infrared) while under brief (<20 min) anesthesia. However, differences would be observed if H was not used. Mice (C57BL/6JCrl; 12 males; 12 females) were randomly assigned to one of the following treatments in a 2 x 2 factorial design: electric (E) or far infrared heating pad (F), with or without homeothermic control (H or X). Mice were anesthetized with isoflurane in an induction box, one at a time, then maintained under anesthesia for 20 min. Rectal temperature (T_R) was averaged every minute and thermal images were taken every 5 min. After 20 min, the vaporizer was turned off, the animal left on the heating pad to recover with the administration of 100% O2 and time to ambulation recorded. Data were analyzed with a General Linear Model. After 3 min in the induction box, mean T_R was 36.3°C (34.7-38.1°C), 1.2°C below our pad set point (37.5°C). Heating system and H control affected T_R over the 20min study (F_1,474=422.3; P < 0.001), but differences were only seen in animals assigned to X. The T_R for FX mice increased above 37.5°C after 10 min and was warmer than EX at 20 min (P < 0.05). The T_R of EX mice stayed below the set point the whole 20 min. No treatments differences in thermal images were observed (P > 0.05). Time to ambulation was shorter in mice assigned to F (F_1,20=11.5; P = 0.003). In mice, body temperature is lost quickly, based on T_R at time zero, thus heat during induction may be important to consider. Further, heat loss may be exacerbated due to preparation and the surgical procedure itself. If >10 min of anesthesia is required, H control is necessary to keep F pads from overheating and E from under heating mice. Finally, F pads may be beneficial to speed recovery after anesthesia.

P8 Shake-and-Bake Health Monitoring: An Alternative to Soiled Bedding Mouse Sentinels for Racks not Compatible with Exhaust Plenum Sampling

B Hibl*¹, CL Perkins², KS Henderson², D Hamilton¹

¹Department of Comparative Medicine, University of Tennessee Health Science Center, Memphis, TN; ²Charles River Laboratories, Wilmington, MA

Maintaining healthy colony animals is pertinent for obtaining valid study results, thus it is important to ensure colony animals are free from excluded pathogens. Historically, rodent health monitoring has been accomplished through the use of soiled bedding sentinel (SBS) animals. Exhaust air dust (EAD) sample analysis has become an alternative to SBS. EAD sampling, however, is not easily accommodated by rack systems without exhaust plenums or those that rely on cage-level filtration. Filters, or portions of filters, from cages containing soiled bedding are used by some institutions for pathogen detection, however to achieve pathogen transfer to the filter, cages must have some form of agitation, such as a mouse, to aerate and distribute pathogens. The purpose of this study was to determine if animal-free colony health monitoring could be used with single-isolation-unit cages. Three cages received soiled bedding from all colony animals on a rack (sentinel 1, EAD 2, EAD 3). Sentinel 1 was a standard SBS mouse while EAD 2 and EAD 3 contained no animals. EAD 2 received 1 flocked swab and 1 sticky swab in the bedding at each weekly cage change; all swabs remained in the cage for the study duration. EAD 3 contained only soiled bedding. EAD 2 and EAD 3 were shaken in a circular fashion 2 times per week (Monday/Thursday) for approximately 20 s. At the end of a standard 3-mo health monitoring period, survival samples were collected from the SBS animals (feces, fur swab, oral swab, and blood) and swabs from EAD 2 were pooled by type for PCR. Exhaust filters were collected monthly and pooled by cage or once at the end of the collection period for PCR. All samples were sent to a commercial diagnostics laboratory for pathogen detection. Based on both positive results and copy numbers of detected pathogens, animal free health monitoring methods were found to be comparable to or superior to SBS in the detection of most non-excluded bacteria, parasites, and viruses. Though excluded pathogens were not present, we suspect they would be detected with equal efficacy as the non-excluded pathogens. As such, animal free health monitoring, especially using sticky swabs within a shaken cage, is a viable alternative to SBS for racks with cage-level filtration.

P9 Use of a Modified External Inline Exhaust Collection Device for Detection of Murine Pathogens within a Double-sided Ventilated Rack

B Hibl*¹, CL Perkins², KS Henderson², D Hamilton¹

¹Department of Comparative Medicine, University of Tennessee Health Science Center, Memphis, TN; ²Charles River Laboratories, Wilmington, MA

Historically, rodent colony health has been accomplished through proper quarantine to prevent introduction of pathogens into an animal facility and screening of soiled bedding sentinels (SBS) for the presence of infectious agents. Exhaust air dust (EAD) sampling has become another common method of colony monitoring without the need for sentinel animals. This method relies on continual, unfiltered air flow towards a point of collection. Swabbing exhaust plenums has been successful in detecting certain highly shed pathogens, however other infectious agents are not as easily detected with this method. Two styles of inline filters have been designed to increase the detection of commonly shed pathogens. These inline collection devices fit within the rack corresponding to the manufacturer of the filter, thus making it challenging to apply a uniform EAD monitoring program if different rack types are present within a facility. A third inline filter device was recently created that is placed external to the rack exhaust plenum, thus potentially allowing EAD collection from racks of varying manufacturers. One rack type present at our facility operates as a flow-through system, however no inline filters have been created for use with this system to date. For this study, the external inline collection device was modified to be connected to the exhaust from a double-sided rack (maximum 140 cages). As the rack operated normally, the filters in the collection device collected dust. Pathogen screening by a diagnostic laboratory of material collected over a 3-mo sentinel period was compared to health monitoring results obtained from a standard SBS program (1 sentinel cage per rack side). Based on both positive results and copy numbers of detected pathogens, the inline filter was comparable to or superior to SBS in the detection of most non-excluded bacteria, parasites, and viruses. Though excluded pathogens were not present, we suspect that they would be detected through EAD with equal efficacy as the non-excluded pathogens. As such, EAD using the modified external collection device is a viable alternative for health monitoring on this rack system.

P10 The Review of Rodent Surgery Records: A Postapproval Monitoring Tool to Improve Animal Welfare in Large Academic Institutions

C Elkhoraibi*, N Matthias, G Alvino, J Sharp

Animal Care and Use Program, UCSF, Albany, CA

We have a large and decentralized biomedical research program (>500 active protocols, >200 survival surgery laboratories, ~1000 rodent surgeons, >10 vivaria). The Institutional Animal Care and Use Program (IACUP) currently reviews surgical records as a postapproval monitoring tool to assess compliance with protocol requirements, university policies, and to optimize animal welfare. Each year, the IACUP requests a sample of records from those laboratories that are approved for rodent survival surgeries. While this method may present caveats, such as potential for sample bias (as laboratories decide which records to submit), it still provides a reliable snapshot of compliance. In 2020, we hypothesized that at least 60% of records would be in full compliance, based on previous unpublished findings. We requested 275 records and received 62; this gap may be explained by the number of laboratories (127) not conducting surgeries due to COVID constraints and the number of nonresponsive laboratories (86). We developed the following scoring system: 1 (finding(s) with a serious impact on welfare), 2 (some impact on welfare), 3 (administrative deficiencies), or 4 (compliant). Of the 62 records reviewed, 47% were in full compliance. The top 3 items associated with scores of 1 (5%) and 2 (31%) were failure to provide NSAIDs, buprenorphine, or local anesthetics as per the approved protocol. The deficiencies associated with a score of 3 (17%), were a lack of recorded drug volumes and undocumented postoperative checks. Bottlenecks identified with our process include high staffing efforts involved in records request, low response rate from labs, and an immediate need to streamline our surgical template. Overall, this process has provided useful insight about the various deficiencies and potential noncompliances that directly impact animal welfare in at least 35% of cases (combined scores of 1 and 2). To refine our workflow, we subsequently streamlined the surgical template to remove nonessential administrative items contributing to scores <4, we developed a decision tree describing immediate action triggers for low scores and are evaluating options to improve response rates following initial records request.

P11 Using Rubrics and Scoring Systems as Refinement Tools to Determine Experimental Specific Humane Endpoints

C Bennett*¹, V Cook¹, E Jackson², EL Mitchell², M Leblanc²

¹CTSI, Wake Forest School of Medicine, Winston-Salem, NC; ²Animal Resource Program, Wake Forest School of Medicine, Winston-Salem, NC

Appropriate humane endpoints for those experimental procedures that can result in necessary pain or distress, failure to thrive, morbidity, or death must be identified. Classical humane endpoints are often too generic and fail to be accurate predictors of pain, distress, or imminent death, meaning that animals are euthanized too early or are found dead leading to loss of valuable data and animal lives. Experimental procedures are often innovative or very specific, which makes the definition of clear and quantifiable endpoints that much harder. Defining better endpoints, using the right clinical algorithm in the context of an experimental procedure to accurately predict pain, distress, failure to thrive, morbidity, and death while meeting experimental objectives, is an important refinement. Such an exercise needs concertation between the veterinary and research community and often a bit of research. We developed 6 rubrics and score sheets specific to common rodent procedures that often result in pain, distress, morbidity, and death (tumor burden, brain cancer, irradiation, sepsis, seizure, fasting). For each procedure, animal oversight staff worked with clinical veterinarians and PIs to create a procedure-specific rubric and scoring system. Including veterinarians and PIs was critical to ensure that all relevant signs of pain/distress and a realistic scoring system were identified and agreed upon. Research teams were trained by oversight and vet staff on how to use the rubric and score sheets using their own experimental animals. Since the implementation of the first rubric and score sheet, we have seen improved study consistency (within research teams), institutional consistency (between research teams) and increased communication between the research teams and vet staff regarding euthanasia decisions. Creating a standardized tooldet that provides a more objective approach for determining accurate, experimental specific humane endpoints is a refinement that alleviates pain or distress while meeting experimental objectives.

P12 Preliminary Use Of An Otoscope Camera As A Rodent Intubation Training Ai

D Bince*, L Sturrock

St. Michael’s Hospital, Toronto, Canada

Over the past few years, our facility has seen a shift in the predominant use of rodent intubation from facilitating respiratory support during surgical procedures, to a route of intra-tracheal administration of compounds to induce lung injury. With the significant increase in the number of researchers performing rodent intubation, our facility now provides a specialized course to standardize the training available to all researchers, moving away from the self-taught model. Due to the small size of the rodents, learning how to perform this technique is difficult for most. In an attempt to reduce the challenges involved with the limited visibility, we sought out an additional training aid, a commercially available otoscope camera. A preevaluation was performed on a small number of adult mice and rats to determine the feasibility of re-purposing this equipment. The preliminary observations were favorable, as the ultra slim waterproof otoscope camera and HD monitor display were able to provide a real-time enhanced visual of the larynx and entrance to the trachea during training. Further trials will be performed to evaluate whether or not this equipment will beneficial to the outcome of the training.In the meantime, the still image and video recording features of the device enabled us to generate digital training aids that are now provided to research staff to familiarize themselves with the procedure prior to training.

P13 Identification of Refinements in a Murine Model of Lipopolysaccharide Systemic Inflammation in C57BL/6 Mice

DR Goulding*¹, RA Wiltshire¹, M Shi², TL Blankenship-Paris¹

¹Comparative Medicine Branch, National Institute of Environmental Health Sciences, Durham, NC; ²Biostatistics & Computational Biology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, NC

The aim of this study was to identify an optimal monitoring schedule and a more humane experimental endpoint than moribundity for the commonly used murine model of lipopolysaccharide (LPS) used for investigating systemic inflammation. We developed a scoring sheet for behavioral and physical changes following IP injection of LPS that included hunched posture, reduced activity, and reduced response to stimuli. Body temperatures and body weights were also recorded. Male (n = 10) and female (n = 10) C57BL/6 mice were subcutaneously implanted with temperature transponders for remote measurement of body temperature prior to injection with LPS. Animals were socially housed in static caging and provided with twice the standard amount of nesting material. Mice were injected intraperitoneally with either 5 mg/kg (n = 10) or 10 mg/kg (n = 10) LPS. Mice were monitored every 2 h for 48 h following injection with LPS and scored for each parameter. Moribundity was considered the clinical endpoint. At 48 h, any remaining mice were euthanized. We observed moderate to high degrees of correlation between low body temperature and high clinical scores in the 5mg/kg male (Pearson correlation coefficient r = -0.42), 10 mg/kg male (r = -0.76), 5 mg/kg female (r = -0.67), and 10 mg/kg female (r = -0.73) groups. Body temperature proved to be an early indicator of mortality in mice dosed with LPS. Results showed that mice with temperatures less than 24°C did not recover to normothermia and reached moribundity. We found the most critical times for observations (lowest body temperature and highest clinical sign scores) were 14-20 h post-injection. Based on these findings, we now recommend a combination of refinements to our investigators that include: monitoring every 2 h beginning at 12 h post injection, removal of animals with body temperatures less than 24°C, providing supplemental wet food (mash), and group housing in static caging with extra nesting material for thermoregulation.

P14 An Extended Reality Ecosystem: Innovating to Improve Connectivity and Efficiency in an Increasingly Virtual World

E Bryant Hall*¹, E Spofford¹, C Boyle², X Shen², S Motzel²

¹LAR, Merck, Boston, MA; ²LAR, Merck, West Point, PA

In what seems like a lifetime ago, inter/intrasite and visitor travel was common and encouraged to facilitate networking, collaboration, and efficiency. A virtual platform to replace these face-to-face interactions while continuing to enable these tasks is advantageous to the Safety Assessment and Laboratory Animal Resources (SALAR) team within the company. This group is critical to on-site in vivo capabilities, but still requires innovative solutions for social distancing. Extended reality technology facilitates 2 types of virtual interactions: Remote Assist and Guided Augmented Task Execution (GATE). The former allows for 1 colleague wearing smart glasses equipment to project their real-time view to a remote colleague or vendor. This device is useful for a myriad of tasks including animal health consultations between husbandry, veterinary, and research personnel; equipment troubleshooting, calibration and set-up; safety and quality assurance audits and inspections; and procedure training and review. The latter capability, GATE, enables training to take place without the need for a designated trainer, as smart glasses or tablets can be programmed with step-by-step instructions which require user interaction to earn credit. This innovation increases efficiency, facilitates social distancing, and provides consistency for learning novel techniques. Additionally, for interactions with external vendors, we have developed a standard process for ensuring the security of data and intellectual assets while protecting our external reputation. This innovative technology has significantly improved efficiency of training and problem-solving within SALAR and will continue to facilitate efficient collaborations even as the pandemic recedes.

P15 Public Opinion on Animal Research in a Time of COVID-19

EC Maciejewski*

Communications, Foundation for Biomedical Research, Washington, DC

Mainstream news reporting on the role of animal models in the development of vaccines and treatments has increased significantly since the start of the COVID-19 pandemic. The Foundation for Biomedical Research conducted 3 polls in 2020 and 1 this year to gauge public opinion about animal research in light of COVID-19 vaccine and treatment development. While public opinion continues to be split, only 1 in 5 Americans is opposed to animal research and more than a third of Americans are undecided. We asked the same question in several ways to better understand the sentiment of undecided participants and came up with key takeaways on how to communicate effectively with the public about animal research. We will examine the poll questions and topline findings to show which messaging strategies achieved broad public resonance.

P16 A Novel Method for Training Cervical Dislocation in Mice

HN Splawn*, E Thomas, L Akin, EA Nunamaker

Animal Care Services, University of Florida, Gainesville, FL

Our IACUC mandated a hands-on rodent biomethodology training for all research personnel working with animals. Techniques taught in this class include how to perform daily health checks, restraint, SQ injections, IP injections, and euthanasia. The training team invokes the use of nonanimal models when viable to support the 3Rs. This replacement technique has helped minimize the use of live animals in our training program, reduced stress on new animal users, and decreased the labor involved with maintaining a large animal colony. Since cervical dislocation is the prevailing form of physical euthanasia, we wanted to ensure proficiency of the technique during our mandatory hands-on class. To continue our replacement efforts, our team sought an inexpensive, nonanimal model to accurately represent the technical skill of cervical dislocation. We were left perplexed; how could we accurately represent cervical dislocation while avoiding unnecessary euthanasia of animals? After failing to find a commercially available nonanimal model to accurately depict and practice this technique, we began brainstorming alternatives. Our team felt the “pop” of dislocating the skull from the cervical vertebrate could be conceptualized with inexpensive children’s jewelry beads. We purchased and assembled them into lines to mimic the spinal column. The beads feature a ball and socket joint which click together to create chains and easily disconnect with pressure from a finger pinch. These are an ideal replacement for live animals because they provide the feel and visualization of the cervical dislocation procedure. After implementing them to our classes, trainees expressed positive feedback to our new nonanimal model. Along with low cost, the beads are reusable. Our team is committed to improving this training technique for cervical dislocation by prototyping 3D printed and stuffed mouse models with the same joint and socket feel as the beads. Overall, this novel method has been a remarkable success and an easy solution to a difficult training problem.

P17 Using a Hack-a-Thon to Engage Students as Design Partners for Incorporating Diversity, Equity, and Inclusion into Laboratory Animal Science Courses

JL Booth*, TL Whitcomb

Comparative Medicine, Penn State College of Medicine, Hershey, PA

Initiatives are underway nationwide to incorporate diversity, equity, and inclusion concepts into education at all levels. Evidence from medical education suggests that incorporating students as design partners with faculty allows students to feel more connected to the curriculum. Students report that the experience is value-added and faculty report that students are more engaged. In response to an identified gap in our curriculum, we launched an initiative to include diversity, equity, and inclusion concepts into our 3-y master’s degree program in laboratory animal medicine. We designed a 2-d curriculum retreat for residents, faculty, and staff that incorporated features of a hack-a-thon to support creativity and collaboration. Hack-a-thon is a term that was coined by the technology field to describe a rapid software development event in which participants work in collaborative small groups to strategize practical solutions to a specific software challenge. The concept has been applied to health professions education and used in curriculum refinement efforts. Here we describe the details of the hack-a-thon components and provide the end product of the process, which is a detailed framework of competencies, assessments, and lesson plans. Student experiences, as well as pitfalls, will be discussed. The use of this curriculum design technique stands to benefit educators of all levels who are looking for meaningful ways to involve students as co-designers and incorporate diversity, equity, and inclusion into training programs.

P18 Moving Beyond Needs Assessment: Developing an Emotionally Engaged Culture to Build Resiliency and Compassion Satisfaction

J Murray*¹, PV Turner¹, R Beall¹, A Foster², DM Abney³, C Bauer⁴, N Vilminot², C Brunette⁵, K Dhondt⁶

¹Global Animal Welfare & Training, Charles River Laboratories, Wilmington, MA; ²Veterinary Services, Charles River Laboratories, Mattawan, MI; ³LAM Behavioral Management, Charles River Laboratories, Reno, NV; ⁴Laboratory Animal Medicine, Charles River Laboratories, Ashland, OH; ⁵InVivo Mgmt Tech Ops FT, Charles River Laboratories, Senneville, Canada; ⁶Dir Scientifique/VPS, Charles River, Lyon, France

Recent crosssectional studies related to compassion fatigue and mental well-being in laboratory animal personnel have identified areas, both operationally and through peer support and engagement, which can create an emotionally engaged culture that supports building resiliency and increases compassion satisfaction. To address the effects of compassion stress and fatigue, we developed and implemented an impactful corporate program to meet the needs of personnel across multiple sites and geographies. The program raises awareness with the management of compassion fatigue, its impact on our work, and positive supports for building resiliency. There is further training for supervisors, managers, human resources representatives, and veterinarians focused on building an emotionally engaged culture at their facilities. The program is supported by a network of Resiliency Building Ambassadors at multiple sites throughout the organization that are trained in empathetic listening for peer support. The Resiliency Building Ambassadors share tools for building resiliency and personal wellness, support adoption and rehoming efforts, strengthen communication between research and animal care personnel during studies, develop tributes to the research animals, and collaborate with other groups on-site in wellness programs and ways to recognize the import science our work supports. Based on participant enrollment levels, demand for new workshops, internal surveys, and anecdotal feedback, the program is continuing to grow and gain momentum across sites and countries in which facilities exist.

P19 Using Calibrated Photographic Images and Thermal Data to Investigate Trends in Tumor Conditions in Different Mouse Models during Oncology Studies

A Smith², K Turley*¹

¹Marketing , Fuel3D, Oxford, , United Kingdom; ²Sales & Data Science, BioVolume, Oxford, United Kingdom

In oncology studies investigating solid tumours, levels of ulceration should be incorporated into endpoints. Necrosis resulting in skin breakdown can be grounds for termination. However, there is no standardized process for capturing calibrated images or recording tumor condition. Limited investigation from visual inspection of mice on trends of when ulceration occurs, whether it is more common in specific mouse models, or which visual signs proceed ulceration have been conducted. We aim to show how a 3D and thermal imaging system can provide key information on ulceration trends to scientists when selecting specific mouse models for a given study. An extensive dataset was used, comprising 5,677 scans collected in 26 laboratories across 276 studies with 117 users, 34 mouse strains, and 120 cell lines and captured using the 3D and thermal imaging system. Scans were carried out by holding the mouse up to the system and automatically capturing red/green/blue (RGB) and thermal and 3D images of the mouse and tumour body. This process took less handling time than recording length and width with callipers. Once captured the system then automatically processes this data to segment, record, and generate volume information for each scan. Manual visual inspections were carried out on all images throughout the duration of the study. Initial ulceration date was recorded and compared to all other mice within the same model group, verifying any trends in the data. A secondary inspection of the images was carried out from the date of ulceration, to look for any visual commonalities of tumor condition during the pre-ulceration stage. Based on extensive visual analysis of ulcerated tumor condition within the captured images, it was shown that there are significant trends in tumor condition in both the days leading up to and the days following the point of ulceration, within certain mouse models. This point of ulceration can therefore be pre-empted through the visualisation of pre-ulceration symptoms displayed in the captured images, such as redness and tightness of skin. One particular mouse strain where this hypothesis was seen to be true was in 10 C57BL/6 mouse strain with an MC38 Cell line studies taken from the dataset, where on average ulceration began to present on d 15. It can be determined that monitoring tumor condition using calibrated photographic and thermal images could play an important role in determining the suitability of certain mouse models for studies and offers greater precision in observing expected and unexpected events, better supporting 3Rs and mouse welfare. Further investigations on how machine learning could be deployed to automatically track these changes in tumor condition at scale should be investigated.

P20 Transitioning to a Blended Learning Classroom to Increase Active Learning and Competency-based Assessment

K Head*, S Hunt, S Chadbourne, R Callicott

Animal Resource Center, UT Southwestern Medical Center, Dallas, TX

The blended learning classroom combines online learning with in-person learning into one cohesive experience. In order to increase the active learning time in one of our longest classes, the blended learning model was applied to Principles of Aseptic Technique in Rodent Survival Surgery, a required component of training for any researcher conducting survival surgery. Prior to transitioning to blended learning, this class was typically 3 h long, including a 1-h lecture, with 8 students. An online interactive module was created to cover the lecture portion of the class, containing discussion and video on aseptic technique, program policies, and standard operating procedures. Students are required to complete the online module prior to the in-person class. During the in-person class, students have the opportunity to ask questions from the material covered in the online module, review specific topics, and then show competency with specific tasks covering concepts in aseptic technique while simulating survival surgery. After incorporating the online module, more class time was dedicated to hands-on simulation and competency assessment. Less time was spent on lecture. The class time overall was shortened by 30–45 min. For students who do not model competency with specific tasks and techniques, the material is reviewed until students show final understanding and competency. While our online module was completed just before the COVID-19 pandemic, the blended learning approach became a critical component as training and research continued. This blended approach helped decrease unnecessary exposure time as students were able to learn a majority of basic principles of aseptic technique online.

P21 Veterinary Supportive Care Influencing Research Results: Creating a “MICU”

KE Smith*¹, WD Hardie², C Davidson², S Tummala¹

¹Division of Veterinary Services, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; ²Division of Pulmonary Medicine, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH

The Division of Pulmonary Medicine experienced several challenges in a longitudinal lung fibrosis model, including difficulty obtaining sufficient blood sample volumes and high mortality rates. Upon collaboration with Veterinary Services, an intensive supportive treatment plan and Mouse Intensive Care Unit (MICU) was established. (22) Doxycycline-inducible TGFalpha transgenic mice were used over a duration of 16 wk and were expected to become critically ill and exhibit symptoms of peak fibrosis (dyspnea, weight loss, prolonged anesthetic recovery) between 6-8 wk. Study goals included standardization of retroorbital blood collection techniques to increase sample size and minimize variables, increased frequency of monitoring, provision of supportive care, and decreased mortality rates. Blood collection was limited to 1% of the mouse’s body weight and was performed by veterinary technicians following induction with isoflurane (2.0-3.0%). During blood collection, supplemental heat was provided using a recirculating water blanket set at 38°C. Recovery occurred within a 30°C incubator with provision of 100% oxygen and additional nesting material placed in the cage. Warmed subcutaneous fluids were provided pre and post blood collection. Ongoing supportive care included monitoring of daily body weights, provision of diet gel and chow on the cage floor, 50% oral dextrose-subcutaneous fluids, and housing within a 26°C incubator with oxygen support as needed. The survival rate dramatically increased from 40% to 86% and the mean average of blood collected per mouse increased from ~0.05ml to 0.28ml. The collaborative efforts of the research team with veterinary personnel can refine animal welfare and survival rates thus improving data acquisition and reducing animal numbers needed to complete project aims.

P22 Behaviorally Pre-screening Rhesus Macaques for Task-compatible Traits Improves Data Acquisition and Animal Well-being

K Thurman*, A Sorrells

Neuralink, Pacifica, CA

The unique personality traits of rhesus macaques have been well documented by primatologists. Similar to humans, animals have preferences that their personalities guide and adherence to that is linked to well-being. In behavioral and neuroscientific research, task acquisition often requires an animal to leave the comfort of their home to perform a variety of tasks. An ideal candidate should possess traits that help meet the goals of the project while engaging the animal in a positive practice. Often, rather, animals are assigned to projects based on availability and health status. This puts many animals on projects that they may not be well suited for, increasing latency to quality data output as well as reducing animal well-being. Behavioral indicators were used to predict how animals would perform in various task acquisition challenges. A simple numerical scale that can be easily used by animal technicians was used to identify subjects with traits needed for the tasks being trained and the potential to thrive in a training environment. The animal’s hierarchical status, engagement with novel stimuli such as difficult forage devices and electronic games, willingness to take food from a stranger’s hand, and their recovery time in the presence of a common stressor are examples of criteria used to measure participation fitness for the study. Behaviors such as lip smacking, fear grimacing, threatening, lunging, cage shaking, and tooth grinding were tallied over the course of 20 min and taken into consideration when scoring the animal’s qualification level for the intended project. We found that the performance of rhesus macaques who were pre-screened met training benchmarks more quickly and with greater competency than those who had not been assessed for behavioral compatibility with the project’s goals. In evaluated animals, latency to voluntary chairing decreased and duration in chair increased, together indicating an improvement of animal well-being, but further indicators such as behavior in chair and time spent engaged in task must be studied to confirm impacts on welfare.

P23 Improvement of Animal Welfare Using Enrichment Devices for Poultry in a Research Setting

L Rose*, L Jensen

Animal Research Support, Zoetis, Kalamazoo, MI

Animal welfare is a great ethical focus within the research community. Practicing a gold standard of animal care and welfare is not only important for humane reasons but is critical for public trust and acceptance of this discipline. The Five Freedoms state that in addition to general husbandry and veterinary care, animals have the right to express normal behavior and to be free from fear and distress. One predominant and well-known method to achieving these freedoms is enrichment. In today’s market, there is a real lack of enrichment devices designed for and available for use with poultry. What is available, is inappropriate either in use within the research environment (caging) or is a risk for contamination (noncertified pathogen free) in pathogen free animals. In order to improve our enrichment program, we had to creatively discover ways to evoke the natural behaviors of foraging, pecking, dustbathing, exploratory, and roosting behaviors while using products marketed for other animal species. Various enrichment devices were chosen and placed with chickens housed in different environments. Over several months, the chickens were observed while interacting with these enrichment devices. Observations were recorded and findings were discussed to determine the effectiveness of these enrichment devices in eliciting the natural behaviors mentioned previously. This method was repeated several times and end of trial necropsies were performed for additional investigation into product safety. Once these enrichment devices were included within their environments, chickens readily expressed natural behaviors in an otherwise unnatural environment. The chickens also displayed levels of reduced fearfulness while in the presence of humans. With the incorporation of these enrichment devices in future studies, we will improve our animal welfare standards by more effectively implementing the Five Freedoms.

P24 Use of a Novel Scoring System to Assess Agreement in a Mouse Sepsis Model

LT Ferguson*¹, Z Liang², T Yumoto², J Anyalebechi², D Swift², M Sorrentino Hernandes³, CM Coopersmith², VK Lee¹

¹Division of Animal Resources, Emory University, Atlanta, GA; ²Department of Surgery, Emory University, Atlanta, GA; ³Department of Medicine, Emory University, Atlanta, GA

Sepsis is a dysregulated host inflammatory response to infection that significantly impacts mortality for hospitalized patients. Animal models are a cornerstone in preclinical research to elucidate the pathophysiology of sepsis and to pilot therapies. Currently, scoring systems for the monitoring of humane endpoints are few and are not specific for a cecal ligation and puncture (CLP) mouse model of sepsis. Thus, a novel sepsis scoring system for a mouse CLP model was created, which includes respiratory status, activity and response to stimulus, and eye quality as parameters. It is intended that this scoring system be simple, be used by diverse animal users to track disease progression, and be put into use to prompt humane euthanasia, as a refinement in animal welfare. First, the project aims to validate the reliability of the novel scoring system between different animal users. A second aim not covered here is to validate the novel scoring system to predict mortality in the CLP model. To assess for inter-observer agreement, a veterinarian and 5 laboratory investigators were paired to monitor postoperative CLP mice on various experiments. We hypothesized that inter-observer agreement for scores of the novel scoring system would improve following training. A formal training program was implemented and consisted of a presentation, videos, and interactive polls for veterinary staff and laboratory investigators. The overall agreement before training between laboratory investigators and a veterinarian was 78.2%. Following the training session, it has been observed that the overall agreement has increased. Among veterinary staff, the overall agreement post-training was acceptable at 90.0%. This novel scoring system will be further validated for the CLP model by determining the scores that are predictive of mortality. Once the scoring system is validated for the CLP model, we recommend that a training program be implemented to improve the use of the novel sepsis scoring system by diverse users involved in animal research.

P25 Ultrasonic Vocalization Analysis as a Novel Metric to Assess Home Cage Welfare in Rats

LJ Bigelow*, AJ Cohen, RH Pimm, JB Knight, PB Bernard

Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Canada

Laboratory rodent housing conditions vary significantly across laboratories and facilities. Variation in housing is implicated in animal stress leading to study variability and subsequently the ability to replicate experimental findings. Optimization and standardization of animal housing conditions are necessary to improve animal welfare and data consistency, thereby reducing the number of animals necessary to detect treatment effects. While interest in environmental enrichment is increasing, many studies do not examine the behavior of animals within the home cage, neglecting important aspects of enrichment. To determine the impact of increased vertical home cage area on animal welfare, double-decker cages (enriched) (n = 4), which allow animals to upright stand, were compared to single-level cages (standard) (n = 4), which impede the ability to upright stand. Home cage welfare was assessed by analyzing ultrasonic vocalizations, fecal corticosterone, upright standing, and agonistic behavior. Ultrasonic vocalization content was further explored by analyses of call type as defined by a 14 call-type schematic. Rats housed in enriched cages spent more time fighting t(6)= 2.13, P = 0.0775, produced less 50 kHz calls (t(6)= 2.66, p=0.0378) and had higher levels of fecal corticosterone (t(11)=3.70, P = 0.00350). Standard caged animals attempted to upright stand more (F (1, 6) = 28.8, P = 0.00170), but stood upright for a shorter amount of time due (F (1, 6) = 34.7, P = 0.00110) to the height limitation imposed by standard cages. Standard cages restrict some naturalistic behaviors such as upright standing, but reduce agonistic behavior which may be attributable to their single-tier organization. Increased home cage height is beneficial, as it permits rats to engage in normal ethological behavior; however, the inclusion of a continuous shelf appears to increase dominance/aggressive behavior among cage mates. To build on the research described herein, further studies will examine the influence of the removal of the shelf in enriched cages.

P26 Maintaining Compliance through Technology during a Pandemic: Virtual Facility Inspections and Program Reviews

J Harvey¹, M Hoopes*¹, L Jarrell¹, S Ayvazian¹, L Coiro¹, R Hockett¹, M Kahl¹, C Lilavois¹, D Brumbaugh², W Cascio¹, J Graff¹

¹Enivronmental Protection Agency, Research Triangle Park, NC; ²Public Representative, Durham, NC

Animal care and use facility inspections and program reviews are mandated by statute to occur every 6 mo. Public health actions imposed during the 2020 COVID-19 pandemic caused many organizations to suspend or limit the number of people allowed on campuses and travel for inspections of animal housing facilities. This is especially problematic for inspecting geographically dispersed facilities. Three facilities are overseen by the unified EPA ORD Eco-IACUC because of program size and the similar types of ecological research with fish and amphibians conducted at each facility. To meet requirements for our inspections and reviews, the Eco-IACUC investigated performing our inspections and reviews remotely using a variety of available technologies. Initially, cell phones were used with limited success. Subsequently, the use of laptops with cameras allowed for real-time communication through compatible wireless access points, meeting security requirements. Inspection team members were able to direct staff on-site in real time to pan, zoom, or stop during the inspections. We employed wireless access point heatmapping to identify, test, and fix areas of low connectivity in each laboratory before inspection. This method proved to be the most successful. Eco-IACUC balanced several risks: COVID infection risk for personnel required to perform the inspection(s); risk to animals if inspections were not performed adequately; and risk to the institution for noncompliance with regulatory requirements. We were able to minimize infection risk for personnel while thoroughly inspecting the facilities and maintaining regulatory compliance.

P27 Animal Care Trivia (ACT): A Socially Distant Training Program

M Nigro*

Comparative Medicine Resources, Rutgers University, Piscataway, NJ

The COVID-19 pandemic had a great impact on the world around us, personally and professionally. Necessary COVID-19 restrictions were placed to prevent the spread and protect employees, which eliminated the ability for the training team to hold in-person training sessions. Pre-pandemic, all animal related trainings were held in-person, including monthly group training sessions for the animal care staff that covered topics such as husbandry SOPs, equipment usage, animal health, cage-side observations, and safety. Yearly refresher trainings were also held in certain areas, including animal health observations, common health concerns, and sentinel husbandry and sampling. As the pandemic and social distancing restrictions persisted, our training department sought to develop a way to continue training the animal care staff while taking into consideration pandemic restrictions, accessibility, and the overall well-being of the staff. Meetings were now being held virtually, however most of the care staff did not have access to computers during the workday. The staff also began rotating schedules to limit the amount of people in an area at one time and to help with area coverage.To overcome these obstacles, we designed the Animal Care Training program, or ACT. The ACT is a socially distant trivia game used to refresh and retrain staff on common topics. Questions are provided to the animal care staff that they can answer within the work week by writing answers down and putting them into an answer box. Questions were grouped into topics that spread over several weeks, with 2-3 questions provided each week. This created a flexible training program that the animal care staff enjoyed participating in. The ACT program will continue even as pandemic restrictions begin to lift, as the program provides an effective learning experience and a way to incorporate consistent refresher trainings.

P28 Who Gets What Enrichment? Benchmarking the Status Quo for Laboratory Animals

MR LaFollette*¹, S Cloutier², C Brady³, M O’Haire³, BN Gaskill³

¹The North American 3Rs Collaborative, Arvada, CO; ²Independent Scientist, Ottawa, Canada; ³Purdue Universtiy, West Lafayette, IN

Enrichment is important for laboratory animal welfare, scientific quality, and public approval. Anecdotally, enrichment provision varies greatly between species, enrichment type, and individual institutions. However, the reasons for this are not entirely understood. Our objective was to characterize the current application of enrichment and possible associated factors. A total of 1164 laboratory animal personnel in the US and Canada were recruited via widespread online promotion to complete an online survey about enrichment frequencies for the animal type they worked with most, enrichment desire/control, animal stress/pain, and demographics. Participants worked with a variety of research topics (e.g., basic, applied), institutions (e.g., industry, academia), and animals (e.g., mice, nonhuman primates). Enrichment frequencies were summarized into 2 outcome variables: overall diversity and frequency per enrichment category (i.e., social, human-animal interaction, physical, nutritional, and sensory). Data was analyzed using general linear models. Results showed a significant interaction between enrichment category and species (P < 0.0001). For most animals, physical, nutritional, and sensory enrichment was provided less often than social enrichment. Between species, nonhuman primates received most enrichment categories more frequently than other animals. Individuals providing limited enrichment wished they could provide more (P < 0.0001). Enrichment provided to rats, mice, and other animals was half the diversity for nonhuman primates (P < 0.0001). Both enrichment frequency and diversity level were reported to be higher by individuals with more control over provision (P < 0.0001), those working in Canada (P < ), and those working for more years in the field (P < 0.03). Our findings show that enrichment implementation differs by animal type and category of enrichment. In particular, physical, nutritional, and sensory enrichment appear to be underused categories. Enrichment is also impacted by factors such as control over enrichment and country. Overall, there is potential to address efforts to increase enrichment particularly in the aforementioned categories and for specific species, such as rats and mice.

P29 Reducing Barking and Stress Behaviors for Animal Shelter Dogs through Behavioral Self-Regulation: Applications to Research Facilities

MR Shyan-Norwalt*^1,2, N Bertoia¹

¹Psychology, University of Cincinnati, Cambridge City, IN; ²Companion Animal Problem Solvers, Inc., Cambridge City, IN

Animal shelters experience chronic barking, chronic licking/self-mutilation, and stereotypic/repetitive behaviors in a number of their canine residents, which are similar issues encounteted with research dogs. Shelter dogs have the additional stress-factor of a constantly changing population, as dogs are adopted, replaced, and removed. Although other factors contribute to noise (e.g., cleaning, HVAC systems, human noise), barking accounts for as much as 80% of shelter noise. Concomitant with this, noise levels in canine animal shelters and laboratory kennels (exceeding 100 dBs) cause damage to canine and human hearing, increases stress, and decreases welfare. Both shelters and research facilities have tried mitigating techniques, including noise-deadening acoustic panels, music, separating dogs visually, olfactory manipulations, and others, with limited success and/or undesirable side effects. Research with other species show that animals’ ability to self-regulate behavior reduces abnormal and stereotypical behaviors and cortisol-measured stress. At an animal shelter in a pretest/posttest design, we evaluated the effects of attaching aluminum partial privacy panels to 1 lower quarter of each front wall of kennel runs. Thirty dogs were evaluated as a group. Dogs could self-regulate when to see/be seen by other dogs, while maintaining access by potential adopters. Pre/post comparisons included numbers/bouts of barking and behaviors (behind panel, lay down, sit, stand, locomotion). Pre-panel, 10 one-minute recordings were collected 1 mo before. Post-panel, 10 one-minute samples were recorded 24 h after installation. Additional recordings were not made as the population of dogs was constantly shifting. After introducing the panels, animals showed a 90% reduction in stress barking. Dogs showed a statistically significantly reduction in activity levels (X ²=7.5, P <.006). Qualitative analysis, from visitor and staff interviews indicated a significant reduction in tension in the facility. It is suggested that these results have application in for laboratory dogs in facilities where similar problems occur. This is a cost-effective, hygienic, and easy technique to reduce chronic barking and undesirable stress related behaviors.

P31 Different Approaches to Virtual Teaching of Laboratory Animal Medicine Courses for Veterinary Students

NL Herndon*, JJ Xu, S Allison

Division of Animal Resources, University of Illinois Urbana-Champaign, Urbana, IL

Due to the COVID-19 pandemic, instructors worldwide have been challenged to develop interactive virtual course content. Laboratory animal medicine faculty attended a campus-sponsored online teaching academy to create virtual courses using a popular video conferencing platform within the online learning management system. Professors developed content for virtual presentation of 2 didactic courses and the fourth-year clinical laboratory animal medicine rotation. Three different virtual course formats were used for these courses: 1) virtual synchronous (VSynch), 2) virtual asynchronous (VAsynch), and 3) virtual “flipped classroom” (VFC). The lecture-based courses were provided in both VSynch and VAsynch. One of these courses was taught in the VSynch manner with the students attending the course remotely at scheduled times and dates. The professor presented lectures in real time with opportunity for question-and-answer sessions, virtual polling, and student-led presentations. With VAsynch lectures, the instructor prepared presentations in advance, allowing students flexibility in time to view course content. Students took quizzes or commented on lecture material asynchronously using the online learning management system. This course culminated in a real-time presentation to the instructors and their peers at the last class session. The 2-wk rotation was delivered using the VFC method. With VFC, course content was provided to students in advance prior to daily virtual class sessions. The course was broken into daily modules focusing on a topic for the day. Course content included PDF lectures, prerecorded content, reading assignments, podcasts, and other relevant media. The instructor conducted daily video conferencing sessions for discussion of content, answering module questions, and dialogue among students. The benefits of converting traditionally in-person courses to a virtual format included students’ ability to continue to learn about the laboratory animal field with the added flexibility in scheduling, physical location, or both depending on the teaching style. Students participated in end-of-course surveys to provide valuable feedback to instructors relating to course structure and content. We will demonstrate the implementation of each of these virtual teaching styles and well as each format’s benefits and drawbacks.

P32 Using Adoption Questionnaires to Determine Best Practices for Preparing Retired Research Dogs to Forever Homes

NL Herndon*

Division of Animal Resources, University of Illinois Urbana-Champaign, Urbana, IL

An academic institution has practiced adopting retired research animals for more than 30 y. The goals of this study are to identify best practices for preparing retired research dogs for life in a forever home and to implement the determined best practices as standard procedures when preparing retired research dogs for adoption. To address the first goal, questionnaires were sent to past adoption families to determine the time families spent training their adopted dog in anticipated common activities, such as walking on a leash or house-training. The questionnaire also asked for detailed descriptions of the behavior of the dogs when exposed to other dogs, new toys, and outside foliage. Lastly, the questionnaire left an open question for families to share any insights on ways to better prepare the dogs for life outside of a research setting. Upon receiving results from the questionnaires, the division of animal resources began implementing the use of an outdoor fenced area during regular socialization periods, called the outdoor enrichment program. While in the outdoor enrichment program, dogs are walked on leashes to a grassy, fenced area and provided with a variety of toys and manipulanda not offered in the standard university research setting. The same adoption questionnaire is sent to families of dogs that go through the outdoor enrichment program in efforts to determine if enrollment in the outdoor enrichment program reduces the time spent training dogs in the common activities or causes any observable changes in behaviors when exposed to novel toys or foliage. We will share what practices we have implemented into our own enrichment program given the findings of these questionnaires.

P33 Optimizing an Experimental Method for Continuous Long-term Monitoring of Glucose in the Blood and Interstitial Fluid of Rats

P Lachcik*¹, CA Rohde-Johnson²

¹Discovery Center, Inotiv, West Lafayette, IN; ²Research Products, BASi, West Lafayette, IN

Continuous blood glucose monitoring is challenging due to blood collection limits and study design challenges. Interstitial fluid (IF) sampling is a technique for continuous sampling of compounds and can be performed without negative health impacts. This study assessed whether continuous IF samples for glucose measurements could be used as a replacement for blood collection for studies lasting 2 wk. The design also compared alternative housing and surgical methods to improve animal health, increase study longevity and reduce costs. Four rats were implanted with 2 IF sampling probes and 1 CAC blood sampling catheter. Rats were assigned to an intermittent or a continuously connected group. Body weights were collected throughout the study. All rats were connected to an automated blood sampler. Two commercially available glucometers were used to evaluate IF vs blood glucose readings and to determine differences in performance of the 2 devices for the rat model. While there was probe failure, some probes remained viable for the full 14-d study. There were differences in glucometer performance both in terms of ability to read the IF samples, and the blood glucose results, indicating that only certain glucometers are compatible with this model. In the disconnected rats, there were more failures than with the continuously connected animals, but there were no major health impacts related to housing. Probe longevity differed between the 2 implanted probes, and anchor beads helped reduce probe movement and incidence of probe damage. While there are still improvements to be made to this method, we were able to collect viable IF samples and learned valuable lessons to optimize this protocol for future studies. This will allow us to explore this paradigm with our analytical team and to refine these studies and reduce animal numbers.

P34 Breeding Dummy for Romeo; Novel Environmental Enhancement and Semen Collection Device for Rabbits

S Martin*^1,2, J Hamilton^1,2, J Ekins^1,2, MC Rodriguez^1,2

¹SoBran Bioscience, SoBran, Inc, Norfolk, VA; ²Comparative Medicine, Eastern Virginia Medical School, Norfolk, VA

An 8-y-old male NZW rabbit, Romeo, presented with a chronic history of unsuccessful semen collections (SC) using fleece sleeve, artificial vagina (AV), and teaser methods. In addition, Romeo presented chronic intermittent anorexia without weight loss and a lack of activity in the cage and playpen. Clinical work-ups included physicals, imaging, and clinical pathology analyzes and yielded no abnormalities. Having ruled out common physiological conditions to explain the anorexia, our veterinary core looked into behavioral explanations. Linking dull behavior with anorexia and unsuccessful SC, we suspected discomfort due to epididymal hypertension (EH) as a condition that could explain both research and clinical observations. Aiming to appeal to the buck’s libido, we developed a novel SC method that also served as an environmental enhancement. Imitating the use of dummies for SC in other species, we crafted a surrogate dummy from a rabbit doll coupled with an AV to attempt SC. We also placed the dummy in playpen during biweekly playtime. Romeo mounted and ejaculated on the dummy during playtime and SC. We noted complete cessation of anorexic episodes and an increased activity in his cage and playpen. Reasons for Romeo’s improvements could be the dummy provided a more natural breeding position, or that he was apprehensive to standard SC.Identified advantages of this novel SC method include no need for a teaser female, elimination of pathogen exposure between animals, and no potential trauma or accidental breeding to the teaser. Our novel SC device represents a reduction of animals and a procedural refinement. Lastly, we cannot establish a relation between Romeo’s anorexia, lack of performance, and EH. Nevertheless, this is something that merits more research. The success of this case reminded us that individual animals’ preferences should be considered when performing research procedures.

P35 Social Learning May Decrease the Time Required to Train Socially Housed Juvenile Pigtailed Macaques (Macaca nemestrina) Using Positive Reinforcement Training

B Carlson¹, S Guerrero-Martin*^1,2, A McNamara¹, K Pate^1,2, E Hutchinson¹, S Queen¹, R Adams¹, J Clements¹, J Mankowski¹, L Hopper¹

¹Johns Hopkins SOM, Dundalk, MD; ²Division of Comparative Medicine, MIT, Cambridge, MA

Social learning, the process by which animals learn behaviors by observing conspecifics, has been observed across many species, including nonhuman primates (NHPs). As we strive to improve the welfare of research animals, refinements such as positive reinforcement training (PRT) can be used to alleviate stress associated with laboratory procedures. Additionally, the increased social housing of laboratory NHPs provides a unique opportunity to study the influence of social learning on PRT in the research environment. Eight juvenile, PRT-naïve pigtailed macaques (Macaca nemestrina) were utilized to examine whether social learning during PRT occurs in a laboratory setting. Four of the macaques were trained to present their hip for injection, while the other 4 macaques had unobstructed views of the training but did not receive any PRT themselves. Once the first 4 macaques successfully presented for injection, we trained the 4 who had observed PRT. The number of PRT sessions needed for each macaque to complete training was recorded. Although not statistically significant, the macaques who had observed PRT require fewer sessions to complete hip presentation training than the animals who were naïve to training (12.25 and 18.75 sessions respectively). Our results warrant further investigation into whether social learning could reduce the time necessary to complete a PRT program for socially housed macaques, which could encourage the adoption of these practices that both encourage cooperation with necessary procedures and enrich the lives of NHPs.

P36 Positive Reinforcement Training of Rhesus Macaques Reduces Plasma Cortisol Changes following Simian Immunodeficiency Virus Infection

B Carlson¹, S Guerrero-Martin*^1,2, A McNamara¹, S Brill³, K Pate^2,1, S Queen¹, R Adams¹, J Clements¹, J Mankowski¹

¹Johns Hopkins SOM, Dundalk, MD; ²Division of Comparative Medicine, MIT, Cambridge, MA; ³College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO

It is becoming apparent to the biomedical research community that laboratory animal husbandry practices can impact the data gleaned from the associated animal models. Particularly, studies focusing on physiologic stress responses during disease processes may be confounded by typical laboratory animal handling practices. This project aimed to assess the impact of positive reinforcement training (PRT) on plasma cortisol in macaques prior to and following simian immunodeficiency virus (SIV) infection. We compared plasma cortisol levels from 11 male juvenile rhesus macaques who completed a positive reinforcement training program to that of 8 untrained, age- and sex-matched rhesus macaques prior to and immediately following SIV inoculation. Prior to SIV inoculation, there was no observed difference in the cortisol levels between the trained and untrained groups; however, at d 7 and d 10 post-inoculation during acute SIV infection, the untrained animals had significantly higher plasma cortisol levels (P = 0.0006 and P = 0.0046 respectively, Mann-Whitney). To further understand the potential benefits of PRT in this macaque model, blood samples were obtained from 8 non-sedated juvenile macaques that had been trained using PRT to facilitate awake, voluntary blood collection. Blood samples were obtained again at time-matched points from the same 8 macaques following sedation with ketamine. This enabled us to assess whether sedation to facilitate sampling may potentially impact the data obtained from the animals, prior to and following SIV inoculation. Plasma cortisol levels in the macaque samples obtained voluntarily, without sedation were significantly lower prior to and following SIV infection compared to the plasma cortisol levels obtained while the animals were sedated with ketamine (P < 0.0001, paired t-test). Further, the non-sedated data was less variable compared to the data obtained from the animals while sedated (SD=56.29 and 99.7, respectively). In sum, this study shows that the process of PRT itself can reduce the physiologic stress levels of rhesus macaques in response to SIV infection, and that training to facilitate voluntary sample collection may lead to less confounded and more reproducible data compared to samples obtained from sedated animals.

P37 Refinements to the Guinea Pig Maximization Sensitization Test Have Led to Better Results

S Fowler*, T Moose, M Dauterman, A Tillman, DW Eaker, K Crapnell

CPDT, BD, Apex, NC

Internal refinements to the guinea pig maximization sensitization (GPMax) test have resulted in more reliable data, and also enabled a reduction in the number of animals used in testing. Prior to these updates additional animals were routinely added to our GPMax test cohorts to account for animal removal due to severe lesions, bandage loss, random deaths, or other losses to study. Our refinements include changes in cage type from stainless steel wire bottom cages to larger plastic solid-bottom cages, group housing versus single housing, change in orientation of induction I injection sites to allow injections to be further apart and not cause severe lesions due to artifacts of site, change in type of wrapping materials for induction II, and addition of Timothy Hay following bandage removal in induction II and challenge. Larger cages with solid bottoms allow for guinea pigs to be socially housed with hiding huts and soft bedding. This allows these social animals to be more comfortable and enriched. Our overall refinements to induction I, induction II and challenge phases have decreased the severity of Freund’s Complete Adjuvant (FCA) induced lesions thus decreasing animal discomfort, and the change in bandage material has improved body condition of the animals. The addition of Timothy hay following bandage removal (induction II and challenge phases) has greatly reduced animal stress. In conclusion, our refinements have led to fewer veterinary treatments and optimization of animal usage, which has reduced the overall annual number of animals by approximately 6%.

P38 Incorporating Technology into Lab Animal Medicine

S Hall-Ursone*

SNPRC, Texas Biomedical Research Institute, San Antonio, TX

Technology is one of the fastest growing industries and as the capabilities expand it has the potential to increase productivity while decreasing potential human error. We recently incorporated 2 different technology applications which include drone usage and markerless motion capture technology in order to increase our efficiency with observations. Drones have been used in many industries and at our facility we incorporated the use of this device to determine the best method to obtain the most optimal observations of our 6-acre corral that houses approximately 150 male baboons. Standard observations consist of only visual observations performed by caretakers daily, technicians twice a week and a veterinarian once a week. The study used 3 different methods, physical observation, security camera observation, and drone footage twice a week. The time allotted averaged 20-30 min and was kept as close as possible for each method of observation. All 3 methods were performed on the same days one right after the other and were kept to the same day of the week only changing if weather conditions did not permit the drone to be flown. Preliminary data has shown that visual observations yield more information than the security camera observation as far as having full visualization of the corral and identifying animals who are injured. Drone video footage has not yet been evaluated by a separate individual to be compared to the visual and camera observations. The markerless motion capture technology was developed originally for use in sports medicine. The biomechanics technology was adapted for use in baboons with the overarching goal of determining if this technology could be used noninvasively to obtain information from animals that would electronically determine normal and abnormal movement. With the markerless motion analysis we hypothesized that by using predetermined data we could identify early signs of injury or disease. So far we have been able to update the neural network so that it more accurately identifies all joints in the baboon and are still evaluating the data for each animal and comparing our results with what has been identified on physical examination. This technology has the potential to give us additional insight on how diseases affect the research model and could clinically aid in allowing intervention earlier for pain management of chronic illnesses.

P39 Professional Development COVID-Style

TM Thomas*¹, JR Champion², AL Murray¹, D Calantropio-Covington²

¹Insourcing Solutions , Charles River, Frederick, MD; ²Bristol Meyers Squibb, New York, NY

The COVID-19 pandemic dramatically changed everyday lives, specifically work life. From remote working to job loss, the pandemic affected colleague and customer interactions and how we see and manage roles and expectations. Professional development, training, and cross-training are concepts institutions are now converting into reality. Onsite development limitations can be overcome, and we review methods to engage development even during a pandemic. Explore professional development and the challenges of developing professionally during a pandemic. Learn why it is important to have a flexible and diverse professional development plan in place including lessons learned with staff training and professional development during the pandemic. The presentation will cover how to provide individual training if you are working remotely. Learn how to keep staff engaged while working remotely. Receive information on how to guide remote development while maintaining a motivated workforce. Plus, get tips on developing alternative development paths and prioritizing the staff’s emotional wellbeing. The presentation will appeal to both junior and senior staff with a desire to grow and improve personal and professional lives. Real solutions will be provided to assist in maximizing new workflows established since the onset of COVID-19. Hear lessons learned including specific policy changes, workflow changes, and approaches to communication.

P40 Vascular Access Button Implantation Technique in Laboratory Rabbits (Oryctolagus cuniculus)

AM Edmunson¹, N Harrison¹, D Tracey¹, TR Gleason*², D Harrison³

¹Laboratory Animal Medicine, Charles River Labs, Ashland, OH; ²Infusion Toxicology, Charles River Labs, Ashland, OH; ³Developmental and Reproductive Toxicology, Charles River Labs, Ashland, OH

Rabbits are a species commonly used in embryo/fetal developmental toxicity studies. Vascular access devices support animal welfare for dosing and blood collection, and vascular access buttons (VAB) minimize the discomfort inherent with vascular access ports and the infection risk of percutaneous catheters. Twenty-two female New Zealand White rabbits (Oryctolagus cuniculus) were placed under general anesthesia and the left jugular vein was implanted with a VAB; embryo/toxicologic data was collected. An incision was made over the left jugular vein and approximately 3 cm of the catheter was placed into the jugular vein, secured in place with nonabsorbable suture ligatures, and locked with taurolidine citrate solution. Another incision was made on the dorsum and a subcutaneous pouch was made for placement of the VAB. The port of the button was passed through a skin punch hole. The animals were given multimodal analgesia on the day of surgery and provided 2 additional days of anti-inflammatories. One animal was euthanized post-op for a reason unrelated to the procedure. Animals were maintained for 43 d. Fourteen rabbits were noted to have unidirectionally patent catheters, allowing dosing but not blood collections, and 6 were bidirectional throughout the study. All but 1 rabbit was successfully bred after surgery, 19 of 20 were gravid. A constant rate infusion of 0.9% sodium chloride at a rate of 0.5mL/kg/hr was administered via the VAB on gestational day (GD) 7 through GD 20. At necropsy (GD 29), in 9 of the 14 unidirectionally patent rabbits, the end of the catheter was near the thoracic inlet, which may have affected patency. Two rabbits had the catheter tip in a jugular anastomosis. These findings did not impact dosing. Overall, the VABs were successful and may be a refinement to consider for studies involving repeated intravenous dosing in rabbits.

P41 Assessing a Novel System for Biliary Elimination Studies in Rats

Z Tarazi*

QPex Biopharma, Inc., San Diego, CA

Biliary excretion studies involve multiple complicated catheterization surgeries and delicate system manipulations. They have historically been among the most challenging rodent study models for a variety of reasons, including maintaining bile flow and infusion of replacement bile into the duodenum. At our facility, bile study significance requires 3 animals. Previously, this necessitated 4 catheterized animals (to ensure 3 working animals), 4 researchers, and a minimum of 30 min to connect the animals to the tether system. For a recent biliary excretion study, we implemented a new catheter access button system with the goal of reducing animal use, staff, and stress, while refining our collection methods. Sprague Dawley Rats (60-d-old) had catheters and catheter access buttons surgically implanted by a vendor prior to delivery and acclimation at our facility. On the day of the study, set-up consisted of removing a specialized magnetic bile shunting cap which completes the bile circuit, and magnetically attaching a tether which allows continuous infusion and sampling. The rats were then administered the novel compound via a 1-h intravenous infusion. Blood and bile were sampled from the rats over 8 h to assess pharmacokinetics and biliary recovery. The catheter access button system required fewer animals (3 instead of 4), fewer staff (2 instead of 4 four), and cut set-up time down from 30 min to 5 min while providing a more comprehensive data set. Subsequent studies using this system confirmed improved animal comfort, had no need for anesthesia, and reduced stress for both animal and researcher. As intended, the catheter access button system significantly reduced animal use, manipulation, stress, and researcher time when compared to previous studies performed with standard externalized catheters.

P42 Use of Buccal Swab DNA Collection for Molecular Genotyping

M Osborne*, T Dehm, K Taylor, T Strout, M Greene

Colony Strategy, GenoTyping Center of America, Ellsworth, ME

The requirement of genotyping laboratory animals for alleles of interest is a constant necessity in biological research. In general, this requires the acquisition of a tissue sample from the animal for DNA extraction followed by PCR genotyping. In many cases, genotyping needs to be additionally confirmed when there is a need to select animals of a specific genotype among segregating genotypes. For example, if the animals enter a longitudinal study, if they are put into matings, or they harbor inducible alleles, where the administration of doxycycline or tamoxifen causes a germline recombination event, it may be required to repeat molecular testing of the same animal. It is the responsibility of the research professional to minimize animal stress and pain at every possible touch point. One source of such stress is the acquisition of biological samples, and the repeated sampling of individual animals-requiring both analgesia and sometimes anesthesia. We present a method of buccal swab sample collection that is less invasive and does not require the use of analgesia or anesthesia. This sampling method produces a sufficient quantity of DNA for multiple assay applications including, quantitative PCR, standard PCR, and endpoint PCR. We demonstrate the applicability of this method across multiple age points from pre-wean to adulthood. Additionally, sample collection requires no special handling at the vivarium level. Buccal swabs can be placed directly into deep well plates, kept at ambient air temperature, and are stable to withstand 48-h shipping. Over 1200 samples from buccal swab or tail were screened for the Apc^min mutation. The results of these data demonstrate nearly 100% concordance rate of genotype calls, and slightly elevated assay failure in buccal swab (1.86% buccal vs 0.12% tail). In summary, this method of sample collection is minimally invasive, produces sufficient DNA for multiple types of molecular testing and provides a means of repeat testing the same animal without the need for pain management.

Clinical Posters

P100 Emergency Care of Orphaned Unique Transgenic Rabbit Kits after Sudden Loss of All Nursing Does

AK Seidel*¹, KF McClain¹, C Chang², T Bryan², X Peng²

¹Transgenic Core Facility, Department of Comparative Medicine, Penn State University, College of Medicine, Hershey, PA; ²Comparative Medicine, Penn State College of Medicine, Hershey, PA

Animal models are indispensable to the discovery of causes, diagnoses, and novel treatments of disease. Breeding genetically modified animal models, especially larger species like rabbits which are not commercially available and can be challenging to create, is vital to future research and genetic integrity. When faced with the sudden loss of all nursing dams and 1 kit over a 2-wk period, the future of a unique and complex genetic line of Audiogenic EIII/JC-HLA-A2.1 inbred transgenic rabbits was jeopardized. It became imperative to develop an immediate emergency care plan that would allow the 5 remaining 23-d-old kits to live and thrive. The quickly formulated care plan included hand-feeding the kits infant formula, as well as giving them free access to critical care herbivore formula, moistened pellets, hay, and water. Each animal was uniquely identified daily, with an indelible mark. Measured and warmed kitten milk replacer-based formula was fed daily by a small group of designated personnel, and an accurate record was kept of the amount of formula consumed each day. To socialize the kits and allow them to bond with the people feeding them, they were allowed playtime as a group before and after each feeding. The health of the animals was closely monitored several times a day by the people feeding them, as well as veterinary and animal care staff. The kits were eventually weaned by progressively diluting the formula until the kits lost interest. The 4 females and 1 male genetically modified kits all reached normal and healthy weights and continue to thrive as adults. The emergency plan that was formulated proved successful and could easily be applied in the future to other similar situations in our facility. It is recommended that other animal facilities have a similar plan to deal with any situation that could lead to the loss of important research rabbit lines.

P101 Abdominal Distension in Ovariectomized C57bl/6j Mice Undergoing Hormonal Treatment

A Christodoulou*, N Reyes, JM Snyder

Department of Comparative Medicine, University of Washington, Seattle, WA

An 8-mo-old female C57BL/6J mouse, in a study evaluating the role of hormonal regulation on anxiety behavior, presented for a marked abdominal distension. A 1 cm fluctuant mass was palpated in the caudal abdomen, and clear fluid was expressed from the vulvar opening during palpation. Bilateral smaller, firm abdominal masses were noted cranially. Ovariectomy was performed 4 mo prior, followed by 6 wk of high dose hormonal treatment and behavioral testing. Hormonal therapy consisted of subcutaneous estradiol benzoate at 48 and 24 h prior to testing (10ug and 5ug respectively) and 500ug of progesterone subcutaneously 6 h prior to testing. Mice rested for at least 1 d before repeating this cycle. Differential diagnoses included cystic endometrial hyperplasia, pyo/hydrometra, and mechanical or functional urinary obstruction. This mouse was submitted for necropsy 1 mo following presentation. Gross exam found severe bladder distension and bilateral hydronephrosis. The uterus appeared grossly normal. Urine specific gravity was 1.020 with no other abnormal findings on urinalysis. Histopathology confirmed bilateral hydronephrosis with associated atrophy of the renal tubules. The urinary bladder was dilated with mild cystitis. Cystic endometrial hyperplasia with squamous metaplasia of the epithelium was noted in the uterus. No site of urethral obstruction secondary to inflammation, fibrosis, calculi, or masses was identified grossly or microscopically. Similar histopathologic lesions of mild unilateral hydronephrosis and hydroureter were seen in 1 asymptomatic mouse whose bladder distension spontaneously resolved prior to necropsy. In previous reports, increased urethral resistance has been hypothesized as the mechanism for urine retention in mice implanted subcutaneously with sustained-release estrogen pellets. We have found similar urinary tract lesions in these mice treated with repeated, cyclical subcutaneous injections of estrogen, which hasn’t been described previously. Urine retention should be considered a potential complication of long-term estrogen supplementation, and mice should be monitored for clinical signs associated with the development of renal lesions and urinary tract infections.

P102 Orbital Cellulitis in a Rhesus Macaque

A Garcia*¹, C Boisvert², Y Kawamura³, F Yoshida^4,5, M Leblanc^6,7, SP Shannon⁴, NJ Fabian¹, M Mahnke⁸, JJ Ballesteros⁹, J Roy⁸, JG Fox¹

¹Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA; ²Ophthalmology, Duke University School of Medicine, Durham, NC; ³Cardiovascular Research Center, Massachusetts General Hospital, Boston, MA; ⁴McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA; ⁵Saga Medical School, Saga University, Saga, , Japan; ⁶Animal Resources Program, Wake Forest Baptist Health, Winston-Salem, NC; ⁷Pathology, Wake Forest Baptist Health, Winston-Salem, NC; ⁸The Picower Institute for Learning and Memory and Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA; ⁹Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, MA

A 12-y-old male rhesus macaque with a cephalic implant but without craniotomies presented with an acute onset of severe periocular swelling and epiphora of the right eye (OD) and mild ventral eyelid swelling of the left eye. Approximately 4 w prior to presentation, the animal fell from the cage perch and experienced a seizure-like episode. On physical exam, T=102°F, the OD pupil exhibited miosis suggesting intraocular inflammation, and its conjunctiva was swollen. A CBC revealed a normal white blood count. Initial treatment included triple antibiotic ophthalmic ointment, buprenorphine, meloxicam, and famotidine. The periocular inflammation was consistent with preseptal cellulitis. The cephalic implant chamber (adjacent to the right upper eyelid), skin-implant interface, and conjunctiva of both eyes were sampled for culture and sensitivity. Empiric antibiotic treatment with ceftriaxone for 10 d led to clinical improvement, but periocular swelling recurred. Culture results revealed multiple isolates, including aerobic and anaerobic bacteria, and Bacteroides fragilis and Corynebacterium spp. were cultured from the chamber, skin-implant interface, and OD conjunctiva. Lesions observed on MRI were consistent with orbital cellulitis in the OD. Re-institution of ceftriaxone and addition of clindamycin to target anaerobic bacteria resulted in improvement. However, extension of antibiotic treatment coincided with diarrhea positive for C. difficile toxins A and B. Ceftriaxone was discontinued, and diarrheic condition subsequently resolved. Surgical explantation of the chamber revealed a minute cranial orifice associated with the right orbital space. Bacteroides fragilis and Corynebacterium sp. were cultured from the explanted screws. Clinical improvement was achieved, but a slight periocular swelling persisted. In conclusion, orbital cellulitis may be a serious complication of cephalic implantation that requires prompt and aggressive antibiotic therapy. MRI is useful for revealing the extent of the disease. Empiric treatment with a third-generation cephalosporin is warranted, but ultimately, cultures for aerobes and anaerobes, antibiotic susceptibility testing, and explantation are important for achieving clinical improvement.

P103 Doppler and Oscillometric Measurements Accurately Represent Direct Blood Pressure in Rabbits under Sevoflurane Anesthesia

A Davis*¹, K Jampachaisri², S Baker¹, C Pacharinsak¹

¹Comparative Medicine, Stanford University, Palo Alto, CA; ²Mathematics, Naresuan University, Phitsanulok, Thailand

Noninvasive blood pressure measurement is more straightforward to perform and less likely to result in complications than invasive measurement. However, information regarding the agreement between noninvasive and invasive blood pressure methods in rabbits is limited. This study investigated the agreement of noninvasive Doppler (Dop) and oscillometric [Os, systolic (Os-sys), mean (Os-mean), and diastolic (Os-dias)] blood pressure measurements with invasive blood pressure measurements in rabbits under sevoflurane anesthesia. We hypothesized that Dop and Os accurately estimate invasive blood pressure under sevoflurane anesthesia. New Zealand White rabbits, induced with ketamine (35 mg/kg)/xylazine (5 mg/kg), were maintained with 1.5-2x minimal alveolar concentration of sevoflurane in 100% O₂. Invasive blood pressure [systolic (I-sys), mean (I-mean), diastolic (I-dias)] was assessed via a femoral artery. Noninvasive measurements were obtained via blood pressure cuffs of width equal to 30-40% of limb diameter placed proximal to the carpus on either forelimb. Blood pressure readings were obtained every 5 min for up to 120 min. Estimation of invasive blood pressure using Dop and Os was assessed using the Bland-Altman method and validated using criteria published by the American College of Veterinary Internal Medicine (ACVIM): (1) bias between noninvasive and invasive blood pressure methods is ±10 mm Hg; (2) 50% of all noninvasive measurements lie within 10mmHg of the paired invasive measurements; and (3) 80% of all noninvasive measurements lie within 20mmHg of the paired invasive measurements. During normotension, 1) Dop met ACVIM criteria with 64.18% and 98.51% of readings within 10 and 20mmHg of I-sys respectively; and 2) Os-sys met ACVIM criteria with 59.70% and 88.06% of readings within 10 and 20mmHg of I-sys respectively. During hypotension, 1) Dop met ACVIM criteria with 78.57% and 96.43% of readings within 10 and 20mmHg of I-sys respectively; 2) Os-mean met ACVIM criteria with 92.86% and 100% of readings within 10 and 20mmHg of I-sys respectively; and 3) Os-dias met ACVIM criteria with 92.86% and 96.43% of readings within 10 and 20mmHg of I-sys respectively. Os-sys did not meet ACVIM criteria for estimation of systolic blood pressure during hypotension. These results indicate that Dop and Os can be used to accurately estimate invasive blood pressure under sevoflurane anesthesia in rabbits, with appropriate care taken to distinguish between normotensive and hypotensive states. Additionally, these data suggest that during normotension, Dop and Os-sys underestimate I-sys; and during hypotension, Dop, Os-mean and Os-dias overestimate I-sys, I-mean, and I-dias, respectively.

P104 Conjoining the Systems: How Parabiosis Can Assist with Studying Tumorigenesis

A Le*¹, S Holtorf², R Morris²

¹Animal Vivarium, The Hormel Institute - University of Minnesota & Mayo Clinic, Austin, MN; ²Stem Cells and Cancer, The Hormel Institute - University of Minnesota & Mayo Clinic, Austin, MN

Multi-stage chemical carcinogenesis, a 2-step process requiring an initiator and promoter, provides an effective way for scientists to study the stages of tumor development. Previous studies have shown that bone marrow derived cells are recruited into the tissue during tumorigenesis. However, these studies require X-ray irradiation followed by a bone marrow transplant. We sought to remove the need for X-ray irradiation to ensure that it was not a contributing factor in our tumorigenesis model. We sought to remove the need for a bone marrow transplant to see if bone marrow derived cells would travel from the bone marrow into the tumor. Parabiosis provided a more biologically relevant model by removing the need for X-ray irradiation and a bone marrow transplant. Prior to parabiosis, 1 mouse would receive the initiator, and the other would receive the promoter. The mice would be surgically conjoined until chimerism, a shared circulatory system, was established. Tumor formation would require both the initiator and the promoter. The presence of tumors would indicate that the carcinogen-exposed bone marrow of the promoter mouse would have traveled through the shared circulatory system and into the tissue of the initiator mouse. There was no surgeon, class, or manual about parabiosis available to guide us, making preparation challenging. IACUC, our veterinarian, and our in-house animal facility were invaluable resources in the planning of this procedure. Preparation involved writing the IACUC protocol and reading through the literature. Suturing, aseptic technique, and anesthesia classes were taken to prepare for surgery. Watching a parabiosis surgery was not available to us, so we practiced on euthanized mice to get our technique down. A pilot study was conducted to practice the technique through non-survival and survival surgeries. Through these surgeries, we found how best to recover the animals through antibiotics in the water, changing the bedding type, and giving fluids, along with additional supportive care. After performing multiple parabiosis surgeries, we were unable to continue this work due to lack of funding. We plan to revisit this again in the future and apply our knowledge to developing and using parabiosis with a multistage carcinogenesis model.

P105 Acute Mortality of Blue Tilapia in an Aquaponics System

C Si*, MJ Hoenerhoff, J Keller

University of Michigan, Ann Arbor, MI

A colony of 82 4-mo-old blue tilapia (Oreochromis aureus), sourced from 1 vendor and housed in a single 700-gal tank as part of an aquaponics system managed by the laboratory, experienced an outbreak of acute mortality. Over the course of 1 mo, several fish were found dead every 1-2 d, with colony mortality eventually reaching greater than 50% (n = 46). Rarely, moribund animals were found isolated and swimming listlessly at the water’s surface. On gross necropsy, affected fish exhibited petechiation along the ventrum, operculum, and at the base of the fins, blunting and pale discoloration of the gill filaments, and diffusely pale livers. Microscopically, there was lamellar blunting and fusion of secondary gill lamellae due to epithelial hyperplasia, and mixed inflammatory infiltrates, as well as diffuse hepatocellular vacuolation in the liver. A subset of fish showed filamentous bacteria adhered multifocally along the surface of secondary gill lamellae as well as inflammation of the coelom and associated organs, including the heart, kidneys, swim bladder, gastrointestinal tract, ovaries, and body wall. Acute morbidity and mortality in tilapia and other aquatic animals often stem from poor water quality, particularly decreased dissolved oxygen, increased total ammonia nitrogen, and suboptimal temperature and pH. The presentation and observed lesions in these fish were consistent with bacterial gill disease (BGD) and septicemia, likely secondary to poor water quality and consequent stress. In this case, poor water quality parameters included a prolonged deficiency in dissolved oxygen and fluctuating temperatures. There was also a failure to sufficiently treat new system water. Common etiologic agents involved in BGD include opportunistic bacteria such as Aeromonas hydrophila, Vibrio anguillarum, and Flavobacterium columnare. Attempts were made to correct water quality, but episodes of mortality persisted, and ultimately the remaining fish were euthanized. This case demonstrates the need for consistent and comprehensive monitoring of water quality, as well as regular communication and collaboration between researchers and veterinary/animal care staff in ensuring health of aquatic animals.

P106 Amoxicillin Water Treatment for a Corynebacterium bovis Outbreak: It Takes a Team!

C Nestel*, C Mullen-Fazekas, H Goold, M Fink, L Habenicht, DL Fong, JK Leszczynski, C Manuel

Office of Laboratory Animal Resources, University of Colorado Anschutz Medical Campus, Aurora, CO

Following 7 y of effort, Corynebacterium bovis was eliminated from our campus in 2018. Five months later, our surveillance program detected 3 racks of C. bovis positive immunodeficient mice in a housing room. This room contained 27 principal investigators using immunodeficient and immunocompetent mice. Due to shared-use molecular imaging and anesthesia equipment, all immunodeficient mice in this room were presumed to be exposed to or were infected with C. bovis. PCR testing was used to detect and remove shedding mice, and antibiotic prophylaxis given to the remaining immunodeficient mice. Immunocompetent mice housed in the same room were left untreated. Exposed spaces and equipment were decontaminated by hand. Amoxicillin water bottles (0.26 mg/mL) were chosen due to availability, quick implementation, and our ability to support weekly maintenance. Medicated water required 1 wk for preparation compared to amoxicillin diet, which required a 5-wk lead time for formulation, manufacturing, and shipping. Medicated water also had lower upfront costs. The disadvantages to using water bottles included increased time handling water bottles because automatic watering is the standard in this facility. Eight-seven immunodeficient mouse cages were present at the initial outbreak. After 5 wk of negative results by PCR-environmental monitoring, 43 experimental cages of immunodeficient mice were allowed to enter and immediately placed on antibiotics. After 11 wk of prophylactic antibiotics and no outbreaks, antibiotics were removed from all cages except the original 87 immunodeficient cages. These targeted cages remained on antibiotics until their experimental endpoint, up to 53 wk later. Initially, water bottle maintenance took 8.5 min/bottle, but this improved to a 4.5 min/bottle as efficiency increased. On average, this required 8.3 hr/wk for 2 staff members. Antibiotics had no noticeable impact on animal health. Prophylactic amoxicillin and environmental decontamination were 100% effective at preventing infection spread. The source of C. bovis was traced back to new arrival, vendor-sourced immunodeficient mice. Antibiotic water can be used in future C. bovis outbreaks for quick containment, and medicated feed should be considered based on the size of the outbreak.

P107 Do Oral Hypoglycemic Drugs Improve Laboratory Findings in Naturally Occurring Diabetes Mellitus in an Olive Baboon (Papio anubis)?

C Root*, S Lane, W Williams

Comparative Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK

A 17-y-old male pair-housed olive baboon (Papio anubis) presented with elevated fasting blood glucose on semiannual health examination. Follow-up evaluation of hemoglobin A1C was elevated, and the animal was diagnosed with type 2 diabetes mellitus. He was started on a low glycemic index forage to supplement standard monkey chow. While dietary intervention was successful at decreasing hemoglobin A1C, it was inadequate to return the levels to normal reference ranges. Due to the study restrictions of his mate, diet and exercise could not be adjusted further. Current medical literature suggests oral metformin, a biguanide drug, as a first-line pharmaceutical intervention and suggests that metformin may decrease hemoglobin A1C by up to 1% within 2 mo in newly diagnosed type 2 diabetic patients. After 78 d of receiving crushed oral metformin once daily at a dose of 26 mg/kg, hemoglobin A1C remained unchanged. Metformin dose was raised to 50 mg/kg once daily, and 0.125 mg/kg glipizide, a sulfonylurea drug, was added as an adjunct therapy once daily. After 35 d on the new regimen, hemoglobin A1C was unchanged at 10.9%, serum fructosamine was elevated at 386 umol/L, glucosuria was significant at 500 mg/dl on a urine test strip, glucose clearance rate was delayed at 2.4 mg/dl/minute, and there was a prolonged return to baseline in an intravenous glucose tolerance test. In published intravenous glucose tolerance test results, non-diabetic baboons returned to baseline glucose by 20 min post-bolus, while naturally occurring diabetic baboons did not return to baseline glucose within 60 min post-bolus. Diabetes mellitus is diagnosed as a fasting glucose of >126 mg/dl and hemoglobin A1C >6%; therefore, this animal had uncontrolled diabetes mellitus. Both oral hypoglycemic regimens were well-tolerated with no adverse effects or significant changes to baseline bloodwork. Oral metformin and glipizide did not improve hemoglobin A1C or other laboratory markers of diabetes mellitus. Despite these findings, it is possible that oral hypoglycemic drugs may confer a benefit in combination with other therapies such as calorie restriction, a low glycemic diet, increased exercise, or insulin.

P108 Managing a Poultry Research Flock with a Positive Salmonella enteritidis Test

CA Hayes*^1,2, S Pearson¹

¹AACUP, University of Illinois, Urbana, IL; ²College of Veterinary Medicine, University of Illinois, Urbana, IL

Many universities have agricultural facilities that serve as both production units as well as places of research. Therefore, they must comply with regulations pertaining to each use. Conventional solutions to common industry problems are not always feasible in a research unit. This case describes the management of such a flock after receiving a positive Salmonella enteritidis (SE) result. As a production facility that produces eggs, our unit participates in the United Egg Producers co-op. Regular testing for SE is performed since it is a zoonotic disease of high importance to human health and can be passed in raw or undercooked eggs. This unit maintains a breeding flock in addition to laying hens. During a routine screening with environmental swabs, a positive SE result was detected. This affected the flock replacement birds. In an industry situation, the solution would be to depopulate all the birds in this room. This was not an ideal solution since this room housed a unique genetic line of birds that produce our research chicks. Since SE is a reportable disease in our state, the research unit was placed under quarantine. None of the chicks showed any signs of clinical illness and there is no good individual animal test. Cloacal swabs were considered but due to several factors, this idea was discarded. Environmental drag swabs tested with a SE specific PCR is the current gold standard. Drag swabs were conducted in every building and every animal housing location. No additional areas of concern were identified. The decision was made to try and manage the problem in the breeder population. The layer chicks were depopulated. Management included moving the breeders to a clean environment, increasing probiotics in the feed, serial testing of the environment and increasing and improving biosecurity. Eventually this approach was successful and no additional SE was isolated. The quarantine was lifted after several months of testing. This demonstrates that there are other methods to managing an SE positive flock besides depopulation.

P109 Method for Surgical Closing of Muscle Biopsy Sites on Nonhuman Primates in Group Housing

DM Benedict*, E Stewart, B Megrath, C Cruzen

Scientific Services, ALTASCIENCES, Everett, WA

There are several benefits to group housing nonhuman primates in the research setting, but it also adds challenges to study procedures. Needing to collect punch biopsies from the gastrocnemius, latissimus dorsi, and vastus lateralis of Cynomolgus monkeys (Macaca fascicularis) twice over the course of a study, we updated our surgical and postoperative procedures to limit the amount of time the animals needed to be separated from their social partners as much as possible. The animals were sedated using ketamine (10mg/kg) and dexmedetomidine (0.05mg/kg) administered intramuscularly. The surgical sites on the back and leg were prepared, and meloxicam (0.2mg/kg) was administered intramuscularly. An incision was made over the muscle, using an 8mm biopsy punch, 2 samples per muscle were collected. The muscle and skin were closed using a simple interrupted pattern with 3.0 suture. Surgical staples and surgical adhesive were then applied to ensure that the incision sites would remain closed. After completion of the procedure, bupivacaine (1mg/kg) was administered subcutaneously at the incision sites, ceftiofur (20mg/kg) was administered subcutaneously, and atipamezole (0.25mg/kg) intramuscularly. Additional doses of meloxicam (0.2mg/kg) were administered intramuscularly for 2 d following the procedure. The previous collection only used sutures and surgical adhesive to close the sites, and ceftiofur was only administered to animals with indications of postoperative infections.With the addition of the surgical staples and ceftiofur, postoperative complications dropped from approximately 15% to 2%, and animals could be returned to group housing 24 h after the procedure. For the next 6 subsequent d, only a brief separation was needed to administer meloxicam and monitor the animals for postoperative complications.

P110 The Impact of Liraglutide Treatment on the Chinchilla (Chinchilla lanigera)

D Marchi*, C Root, S Lane, W Williams

Division of Comparative Medicine, The Univerisy Of Oklahoma Health Science Center, Oklahoma City, OK

Chinchillas are the preferred animal model for otologic research due in part to large tympanic bulla, which allows easy access to inner ear structures. Animals at our institution were enrolled in a study that included hearing tests under anesthesia, exposure to pneumatic pressure up to 25 psi, and injectable liraglutide. This particular project had an unusually high mortality rate compared to previous studies, which resulted in this retrospective analysis. Chinchillas, like many other rodents species, pose particularly difficult challenges when undergoing anesthetic procedures. These include high anesthetic-related mortality rates of over 5 % when compared to other rodents that have rates under 4%. Dilated cardiomyopathy, congenital septal defects, and valvular disease have all been reported in chinchillas, but published literature has not reported the frequency of heart disease. Liraglutide is an incretin mimetic that works as an agonist for acylated glucagon-like peptide-1 (GPL-1) receptors used for diabetes mellitus and obesity treatment. Adverse effects may include: weight loss, appetite suppression, delayed gastric emptying, decrease gastric acid secretions, substantial increase in heart rate, increased blood pressure, hypoglycemia, and decreased basal body temperature. Pressurized pneumatic procedures in this study use a specialized chamber with a pressurized air system to deliver serial pressurization of either 3-5 psi or 15-25 psi. Pneumatic pressure procedures over 19 psi have been documented to produce brain, lung, heart, intestine, kidney, and liver damage in mice, pigs, and humans. Necropsy findings from chinchillas that died before experimental endpoints exhibited one or more of these four primary pathological abnormalities: Aspiration pneumonia, congestive heart failure, hepatic steatosis, pancreatitis. This analysis showed an increased mortality rate in chinchilla receiving liraglutide 39% when compared with deaths of chinchilla not receiving liraglutide 11%. A chi-squared test showed significant evidence of association between liraglutide administration and death (p<0.0001). Liraglutide treatment leads to an increased mortality rate in chinchillas when compared to liraglutide-naïve animals undergoing pneumatic pressure procedures.

P111 Reproductive Aberrations of Rabbits: Intrauterine Growth Retardation (IUGR) in a New Zealand White Dystocic Doe

EF Tena-Betancourt*^1,2, MC Vázquez García², V Graullera-Rivera^2,3

¹ Animal Facility Services & Experimental Surgery and, Universidad La Salle México, Alcaldía Tlalpan, , Mexico; ²Departamento de Producción y Zootecnia de Abejas, Conejos y Organismos Acuáticos., Facultad de Medicina Veterinaria y Zootecnia, UNAM, CDMX, , Mexico; ³Departamento de Patología, Instituto Nacional de Cardiología Ignacio Chávez, Tlalpan, , Mexico

Reproductive disorders are complex, including extrauterine pregnancies (EP), hydrometrocolpos, and rarities as superfetation (SF) or disorders as intrauterine growth retardation (IUGR). This work reports the delayed maturation of an NZW kit discovered during an emergency C-section of a doe suffering maternal dystocia. The dam was found in extremis, prostrated and hypothermic, displaying an edematous vulva and severe vaginal hemorrhage. Abdominal palpation detected several full-term fetuses on the right uterine horn and a smaller number of kits on the opposite side. After prolonged nonproductive parturition, uterine atony was suspected, leading to emergent surgery. At first glance, a distended, semitransparent uterus showed that the right horn contained a larger kit, dead in caudal position blocking the birth canal. After retrieving the first 2 fetuses, the third constituted a grossly underdeveloped fetus among full-term kits. At this point, the mother was euthanized with barbiturics. Morphologically, fetuses weighed 65.8, 70.7, 61.3, 68.4, 53.7, and 65.3 g, respectively, while the underdeveloped fetus weighed 19.5 g showing abundant meconium, absent in the rest. Body lengths of normal kits (crown to rump) were 10.7, 11.2, 9.9, 10.9, 8.6, and 9.2 cm, whereas 3.9 cm for the underdeveloped fetus. Placental weights were analogous among all normal kits (5.97g), while for the blighted fetus was only 2.32 g. No malformations, infection, or mummification were observed. Based on clinical-surgical observations, the primary diagnosis was obstructive dystocia followed by secondary uterine inertia, while the fetal underdevelopment was estimated the result of partial uteroplacental insufficiency possibly related to complex factors, involving in this case, a superior number of fetuses located in the right uterus, advanced age of the dam showing multiple litters during the last 2.7 y, as factors that could influence or impaired placental growth associated with IUGR reported in several domestic and laboratory animal models.

P112 Integrative Therapies for Treatment of Radial Nerve Paralysis in a Specific Pathogen-free Kitten

E Houston*¹, T Holt², J Ayers¹, LV Kendall¹

¹Department of Laboratory Animal Resources, Colorado State University, Fort Collins, CO; ²Veterinary Teaching Hospital, Colorado State University, Fort Collins, CO

An 8.5-wk-old female intact, domestic shorthair kitten presented with acute non-weight bearing lameness on the right front leg following weaning and single housing for fecal collection. The cat was part of a closed breeding colony free from feline herpesvirus, panleukopenia virus, calicivirus, and coronavirus. On physical exam, she was non-weight bearing on the right front limb when standing but readily ambulated on the right front carpus with no evidence of discomfort. Moderate laxity of the right scapula was noted. No fractures or bony abnormalities were palpable. Deep pain was intact, though proprioception of the right front was decreased. A carpal radiograph was taken and confirmed that no skeletal abnormalities were present. The kitten was diagnosed with radial nerve paralysis and administered meloxicam. No improvement was noted after 2 wk of confinement and NSAID therapy, making spontaneous resolution of the peripheral neuropathy without additional intervention unlikely. Before pursuing surgical amputation, passive range of motion exercises were initiated 2-3 times per day. Acupuncture needles were applied to multiple points on the right front limb daily for 3 treatments. Laser therapy was administered daily from the scapula to carpus. Massage of the digits was included following treatments. Three days after initiating this regimen, the kitten began bearing weight on the paw, with medial deviation of the distal limb. On the fifth day of treatment, the medial deviation had resolved, and ambulation remained normal. The patient was able to enter her study with no abnormalities or need for chronic treatments. Integrative therapies such as acupuncture, laser, and massage are tools for treatment in peripheral neuropathies that occur in the laboratory environment. The combination of these therapies contributes to resolution without the need for surgical intervention or removal from study.

P113 Acute Hemothorax Due to a Metastatic Thoracic Amelanotic Melanoma in an Indoor-housed Aged Adult Rhesus Macaque (Macaca mulatta)

E Franklin*¹, S Muthupalani¹, D Annamalai¹, I Napier¹, NJ Fabian¹, C Atkinson¹, A Garcia¹, JG Fox¹, S Carrasco^2,1

¹Division of Comparative Medicine, Massachusetts Institute of Technology, Somerville, MA; ²Laboratory of Comparative Pathology, Memorial Sloan Kettering Cancer Center/Weill Cornell Medicine, New York, NY

A 21-y-old male rhesus macaque (Macaca mulatta) presented for clinical workup due to an acute onset of lethargy and coughing. On sedation, bronchovesicular sounds were muffled bilaterally. Thoracic radiographs revealed significantly retracted lung borders, marked loss of soft tissue detail, and gas-filled esophagus and stomach. Ultrasound-guided thoracocentesis yielded 100mL serosanguinous fluid. Euthanasia was elected due to grave prognosis and decompensating condition. Postmortem examination revealed marked atelectasis, pleural effusion, fibrous adhesions, and numerous soft, white to pale-pink, friable, dome-shaped masses (0.5-3.5 cm diameter) throughout the lung and thoracic cavity, including mediastinum, parietal pleura, diaphragm, and costal muscles. Differential diagnoses included hemangiosarcoma rhabdomyosarcoma, primary pulmonary carcinoma, and mesothelioma. Histologically, the lung and thoracic tissues were effaced by well-demarcated, nonencapsulated, densely cellular expansile masses comprised by pleomorphic neoplastic polygonal to spindloid cells arranged in cords, streams, and/or aggregates and supported by a fibrovascular stroma. Neoplastic cells have distinct cell margins and moderate to abundant amounts of pale-eosinophilic granular cytoplasm with round to oval nuclei with finely stippled to lacy chromatin; anisocytosis, anisokaryosis, and mitotic figures are common. The neoplasms were well-vascularized and dissected by fibrous connective tissue and necrotic regions. Immunohistochemistry/special stains showed the neoplastic cells were positive for mesenchymal (vimentin), melanin (Fontana-Masson), neural crest (S-100), and neural/neuroendocrine markers (neuron-specific enolase). These cells were negative for hemosiderin (Perls), epithelial (pancytokeratin), neuroendocrine (chromogranin-A, synaptophysin), neural (synaptophysin), mesothelial (Wilm’s tumor antigen-1), and smooth muscle/muscle (α-smooth muscle actin, desmin) markers. Together these findings suggest these neoplasms originated from neuroectodermal cells from the thoracic cavity with metastasis to the lungs. This case features an atypical presentation for a spontaneous, multicentric, pleomorphic amelanotic melanoma (presumptive) in an aged adult rhesus macaque.

P114 Osteosarcoma in a Juvenile Male Rat

E Pearson*¹, J Cavasin², ER Feldman¹, G Jackson¹

¹CARE, Cornell University, Ithaca, NY; ²AHDC, Cornell University, Ithaca, NY

A 1.3 y-old, male Sprague Dawley rat was reported for a 1 cm firm subcutaneous mass over the right lateral thorax on the ventral half of the rat. Differential diagnoses were benign fibroadenoma vs benign sebaceous cyst. The rat was otherwise within normal limits. This rat was on an aging study and had no prior study intervention. Diagnostics involved obtaining a fine needle aspirate and monitoring. Seven days after initial presentation the rat was found deceased. Gross external necropsy findings included a subcutaneous hard, immobile mass on the right lateral thorax adhered to the rib cage extending into the thoracic cavity, and a diffusely swollen right hindleg with a hard distal femoral mass. Internal gross necropsy findings included serosanguinous abdominal fluid, a 2 by 1.5 cm firm, nodular mass adhered to mesentery in the dorsal-caudal abdomen, a severely enlarged right kidney, a dilated left kidney, and multifocal, diffuse, tan, firm nodules comprising 90% of the lungs. Histologically, these masses (skeletal muscle, lung, abdominal lymph node, mediastinal mass) are consistent with metastatic osteosarcoma, characterized by a densely cellular proliferation of spindle cells arranged in interlacing bundles and streams within a collagenous stroma. The cells produce eosinophilic amorphous to globular extracellular material (osteoid), which is partially mineralized. The nuclei are highly pleomorphic with moderate anisocytosis and anisokaryosis, multifocal areas of necrosis, and extensive vascular invasion. Renal changes were considered incidental and included severe unilateral hydronephrosis, lymphohistiocytic interstitial nephritis, fibrosis, and glomerulosclerosis. Osteosarcoma in rats (rattus norvegicus) is limited to experimentally induced models. This account characterizes spontaneous metastatic osteosarcoma in a 1.3-mo-old Sprague Dawley rat.

P115 Arterial Pseudoaneurysm in an Intermittent Non-weight Bearing Cynomolgus Macaque (Macaca fascicularis)

G Mendoza*², AB Le Roux¹, C Cheleuitte-Nieves²

¹Diagnostic Imaging, The Animal Medical Center, New York, NY; ²Tri-Institutional Training Program in Laboratory Animal Medicine and Science, New York, NY

A 7-y-old, 4.35 kg, intact-male, pair-housed cynomolgus macaque (Macaca fascicularis) presented for intermittent right hind limb, non-weight bearing lameness. One year prior to presentation, the animal was enrolled in a study in which radionuclide-tagged antibodies were administered IV, and CT/PET imaging and blood collection were performed monthly. During a routine annual physical examination, conducted 4 mo prior to presenting with lameness, a bounding pulse and thrill were palpated in the right femoral triangle (RFT). Two additional examinations 1 mo apart revealed that the RF arterial pulse was stronger than on the left, but a thrill was no longer appreciated. Upon presentation of the intermittent right hind limb lameness during ambulation, no obvious lesions were observed, and the animal was able to stand normally using the affected limb. The patient was treated with meloxicam (0.2 mg/kg once then 0.1 mg/kg q24hr IM) for 3 d. The lameness persisted unchanged, and 3 circular red discolored areas (~0.5 - 1.5 cm in diameter) developed in the skin of the RFT. A day after the last meloxicam treatment, the animal was again sedated for examination. Thoracic auscultation and musculoskeletal and neurologic exams remained normal. On palpation, the discolored areas in the RFT were nodular, and a prominent thrill was again detected. Clinical pathology revealed hypoalbuminemia, hypocalcemia, and a mild stress leukogram. Differential diagnoses included arteriovenous fistula, aneurysm, arterial stenosis, and pseudoaneurysm. A focused ultrasound of the vascular structures of the inguinal region revealed a small (7 x 9 mm) saccular luminal dilation of the RF artery with turbulent intralesional blood flow on color flow Doppler echo. The adjacent RF vein was normal, and an RF arterial pseudoaneurysm was diagnosed. The patient’s lameness gradually resolved 1 wk later. The clinical features, research significance, and treatment of pseudoaneurysms will be presented.

P116 Septicemia in Catheterized Rats Caused by Novel Emerging Nosocomial Pathogen, Serratia marcescens

HM Wilson*, MJ Hoenerhoff, G Hish

Lab Animal Medicine Unit , University of Michigan, Ann Arbor, MI

A 6-mo-old, intact male Long Evans rat (Rattus norvegicus) singly housed in static microisolator caging presented with a 2-d history of lethargy and anorexia. The rat was on a chemical dependency protocol utilizing chronic indwelling jugular catheterization for self-administration of remifentanil and belonged to an experimental cohort that experienced acute loss of approximately 50% (n = 7) of animals. Physical examination revealed dull mentation, thin body condition (BCS 2/5), moderate dehydration, cold extremities, and tachypnea. Differential diagnoses for lethargy and anorexia included inflammatory, infectious, and neoplastic disease. Examination of complete blood count (CBC) revealed a mild neutrophilia with a left shift, moderate thrombocytopenia, and anemia. Clinical chemistry abnormalities included elevated BUN, creatinine, ALT, and AST. Due to poor prognosis, the animal was euthanized and submitted for diagnostic necropsy. Grossly, there were multifocal pale foci within the left lung lobe, which correlated histologically with suppurative embolic pneumonia with thromboembolism. In addition, there was severe suppurative valvular endocarditis of the tricuspid valve and diffuse adrenal hemorrhage, consistent with bacterial seeding to the lung and generalized septicemia. Culture of the lung and catheter tip showed growth of Serratia marcescens, a gram-negative, rod-shaped bacteria that has emerged as a serious nosocomial pathogen over the last several decades. Environmental testing was performed, and the source of the infection was determined to be the catheter flushing device, which was used on multiple animals. Aseptic technique and proper catheter maintenance were reviewed with laboratory staff. While Serratia marcescens is a frequent isolate in outbreaks of hospital infections in adult and pediatric human patients, and has recently been recognized as a growing concern in companion animals, the literature lacks reports of spontaneous infection in laboratory rats. This case emphasizes the importance of nosocomial infections in chronically catheterized rats and appropriate control measures to help prevent intravascular catheter-related infections.

P117 Assessment of Ovarian Follicular Dynamics during Natural and Gonadotropin-stimulated Cycles in the Common Marmoset (Callithrix jacchus)

M Stair¹, I Napier*¹, Q Zhang², S King², MA Burns³, RM Kramer¹

¹Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA; ²McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA; ³Novartis Institutes for Biomedical Research, Cambridge, MA

There is limited publication on ovarian follicular dynamics in the common marmoset (Callithrix jacchus). Such information is valuable for breeding colonies to meet the high demand for marmosets in various research areas. Trans-abdominal ultrasonography is a useful, noninvasive imaging modality that allows repeated monitoring of ovaries to facilitate assisted reproduction. This study documents the use of high-resolution ultrasound to follow and characterize folliculogenesis in naturally cycling and hyper-stimulated marmosets (N = 6) starting from day 1 of follicular phase. During natural cycles, adults produced 2-5 mature ovulatory follicles, consistent with the average marmoset litter size of 2-4 offspring. In unstimulated animals, ovulation occurred on day 11.5 of the cycle and preceded a rise in serum progesterone. Ovarian size increased in the luteal phase (day 21 mean = 7.85 mm) as compared to the follicular phase (day 5 mean = 6.17 mm) (P = 0.02). Notable observations included atresia of medium-sized (0.6 - 1 mm) follicles in mid-to-late follicular phase; continuous presence of small follicles (< 0.6 mm) through all stages; and the persistence of cyst-like structures in some animals through apparently normal cycles. Animals (N = 5) were then followed through an ovarian hyper-stimulation cycle for surgical oocyte collection. To induce hyper-stimulated conditions, 25 IU of human follicle-stimulating hormone were administered from day 3 to day 10, followed by 75 IU of human chorionic gonadotropin on day 11 of the cycle. Gonadotropin-stimulated animals produced a greater number of follicles than naturally cycling ovaries (P < 0.05). However, mean ovary size was similar under both conditions (P > 0.05). Surgical oocyte retrieval from four hyper-stimulated marmosets resulted in 19-94 oocytes collected per procedure, well above the number of ovulatory follicles in naturally cycling animals. A portion of collected oocytes was implanted in surrogates, resulting in multiple pregnancies and live births. In conclusion, trans-abdominal ultrasonographic imaging is a valuable tool for monitoring of ovarian follicular development and ovulation for accurate timing of assisted reproductive procedures for breeding the common marmoset.

P118 Dosing Accuracy of Sustained-release Buprenorphine Formulations for Rodent Analgesia

JL Haupt*, NJ Fabian

Massachusetts Institute of Technology, Cambridge, MA

Buprenorphine is the most commonly used opioid analgesic in laboratory animal species. Sustained-release formulations are used for long-term pain management. The viscosity of these formulations represents a challenge on 2 fronts: maintenance of controlled drug logs and accurate dosing of small volumes. The current formulations of sustained-release buprenorphine for rodent use include buprenorphine SR-LAB (BSR), a non-pharmaceutical grade formulation, and a novel extended-release (NER) formulation FDA-indexed for mice and rats. This project compares the drug loss and dosing accuracy of BSR and NER using needle and syringe combinations as per the manufacturer’s recommendations. We evaluated 50 ul of both NER and BSR (0.5 mg/ml) using serial weights. Three syringe and needle combinations (n=10) were assessed: 1 ml low dead space and standard syringe with 18 g needle to draw up and 23 g needle to dispense, and 0.5 ml insulin syringe with 28 g needle (NER only). A calibrated pipette was used to weigh formulations using a precision balance. A significant amount of drug was retained in the needle and syringe when using the low dead space and standard syringes compared to the insulin syringe. The hub loss or the amount withdrawn from the bottle compared to the amount dispensed via injection accounted for 76% NER from the standard syringe (139 ul), 74% BSR from the standard syringe (135 ul), 73% NER using the low-dead space syringe (108 ul) and 64% B SR using the low-dead space syringe (106 ul). An insulin syringe for dosing of NER resulted in minimal (8% or 6 ul) drug loss. Dosing accuracy also varied by formulation and syringe combination. Doses were significantly higher when using NER with an insulin syringe (+27% or 64 ul) and BSR with a low-dead space syringe (+17% or 59 ul). Lower doses were delivered with NER using a standard (-13% or 44 ul) and low-dead space (-20% or 40 ul) syringe. The most accurate dose was provided using BSR in a standard syringe (-4% or 48 ul). This project highlights the potential inconsistencies in controlled substance logs using small volumes of viscous substances and the difficulty of providing accurate doses for rodent analgesia using sustained-release formulations.

P119 A Comparison of 3 Sedation Protocols to Facilitate Awake Ocular Exams in New Zealand White Rabbits (Oryctolagus Cuniculus)

KL Navarro*, S Baker

Comparative Medicine, Stanford University, Stanford, CA

Rabbits are a model organism for ophthalmic research due to ease of handling and similarity in eye size to the human eye. However, certain imaging techniques may cause stress to awake animals as large instruments must be in close proximity to the eye (e.g., slit-lamp microscopy, ocular coherence tomography, and tonometry), or the eye must be manipulated (e.g., speculum placement and fluorescein staining). Herein we investigated the ability of 3 different injectable sedation protocols to facilitate ocular exams in awake animals. The aim was to assess whether any of these protocols would allow the animals to be examined with minimal stress and without the use of anesthetic gas. Sixteen adult female New Zealand White rabbits were randomly assigned to 1 of 3 treatment groups: 1) Xylazine (20 mg/mL, 2.5 mg/kg IM once); 2) dexmedetomidine (0.5 mg/mL, 40 mcg/kg IM once); 3) midazolam + butorphanol (5mg/mL & 10 mg/mL, 0.5 mg/kg & 0.3 mg/kg respectively, IM once). Rabbits underwent sedation protocol for this study once. Rabbits were sedated, placed in a restraining device, an ocular speculum placed in the right eye, followed by an eye examination (slit-lamp microscopy, ocular coherence tomography, then fluorescein staining). Respiratory rate, heart rate, and oxygen saturation were monitored beginning after restraint and throughout the procedure. Results showed moderate bradycardia after xylazine and dexmedetomidine sedation. midazolam + butorphanol sedated rabbits showed comparatively higher heart rates versus the xylazine or dexmedetomidine sedation protocols but caused comparatively decreased oxygen saturation (92-94% SpO2) in several rabbits. All sedation protocols allowed for awake restrained ocular exams to be performed. These data suggest administration of either single agent xylazine (2.5 mg/kg) or dexmedetomidine (40 mcg/kg) provides sufficient sedation to allow for awake examinations without compromising oxygen saturation among the 3 protocols investigated.

P120 Tamoxifen-associated Scrotal Hernias in Male Mice

KW Kopf*, JM Poczobutt, MD Rosenbaum, I Petrache

National Jewish Health, Denver, CO

We are reporting a case of numerous scrotal hernias in male mice that were given intraperitoneal injections of tamoxifen while concurrently undergoing smoke exposure in 2 different studies using the same strains of mice Cdh5(PAC)-CreERT2 x B6.129S6-Ugcg^tm4Rlp/Mmucd. In one study, the mice were given 1 mg IP tamoxifen in sterile corn oil with 5% ethanol for 3 consecutive days, every 3 wk for 6 mo during a concurrent 6-mo smoke exposure. For the second study, mice were given an IP injection of 1 mg tamoxifen in sterile corn oil once weekly for 5 wk during concurrent 5-wk smoke exposure. Although the exact number of males with scrotal swellings were not recorded, they were noted in the majority of male mice from both studies. In the 6-mo study, the swellings were noted at mo 4 and in the 5-wk study, the swellings were noted when the mice were sacrificed at the termination of the study. The most likely differential diagnoses for scrotal swellings in mice include hernias and testicular neoplasia. Tamoxifen has been reported in the literature to occur when male mice are treated with tamoxifen administered through tamoxifen chow and so the presumptive diagnosis of scrotal hernia was given. The mice did not show signs of distress or weight loss and so the studies were followed to completion while continually monitoring affected mice for signs of distress. The final diagnosis could not easily be made until gross necropsy at the termination of the study. Upon termination of the study, the scrotums of several male mice were opened to confirm the presumed diagnosis of scrotal hernia. Mice had varying degrees of abdominal contents contained in their scrotum, confirming the diagnosis of scrotal hernia. Some mice only had fat protrusion but most mice had fat, intestines, and occasionally bladder in the scrotum. Tamoxifen is frequently used but hernias have not been observed frequently in other strains at this institution. The exact mechanism of hernia formation is a direction of future research; however; a genetic cause is suspected. The mice have a defect in ceramide metabolism which may interact with collagen metabolism, leading to hernia formation.

P121 Endometrial Adenocarcinoma in 2 Rhesus Macaques (Macaca Mulatta)

KN Wharton*, C Zeiss, D Ekanayake-Alper, PC Smith

Comparative Medicine, Yale University School of Medicine, New Haven, CT

In humans, uterine carcinoma is the fourth leading cancer among women. Clinical signs include heavy and irregular menstruation after menopause, abdominal pain, and weight loss. This case report describes 2 clinically distinct cases of endometrial adenocarcinoma in rhesus macaques. Rhesus A (16 y of age) presented with vaginal hemorrhage, pain, a palpably firm caudal abdominal mass, and a hemorrhagic mass extruding into the vagina. She had a 3-y history of painful cycling managed with monthly depot medroxyprogesterate acetate (DMPA). Due to uncontrolled pain, euthanasia was elected. Necropsy revealed a firm necrotic and hemorrhagic mass terminated by a large blood clot that extended through a dilated cervix into the vagina. Histopathology revealed multinodular cytokeratin, CA-125 and p53 immunopositive, and estrogen receptor-negative neoplastic masses limited to the endometrium, consistent with endometrial adenocarcinoma. Rhesus B (21 y of age) presented acutely with respiratory distress after a 5-y history of painful cycling managed with DMPA. Thoracic radiographs revealed an extensive nodular pulmonary pattern, and euthanasia was elected. Gross pathology revealed multiple firm, nodular tan masses at the uterine-cervical junction extending to the right ovary. Approximately 80% of lung was occupied by numerous solid to umbilicated firm tan masses. Histopathology identified an endometrial serous adenocarcinoma with metastasis to the lungs, spleen, and pancreas.There are few reports of endometrial carcinoma in nonhuman primates. Like humans, each macaque in this report experienced prolonged, irregular, heavy menses and weight loss. These cases also presented initially as firm uterine masses palpable during abdominal and rectal exams. In both, progression was clinically silent until the sudden onset of illness accompanying advanced pathology. The cryptic nature of clinical progression in both cases stresses the importance of monitoring uterine diameter in all intact females.

P122 Evaluating Menstrual Cycle Suppression in Rhesus Macaques (Macaca mulatta) Implanted with Subcutaneous GnRH-agonist

K Carroll*, A Mackiewicz, A Ardeshir, K Christe

CNPRC, UC Davis, Woodland, CA

A commercially available GnRH agonist formulated as a 4.7mg subcutaneous implant may offer a relatively noninvasive, long-lasting, and reversible alternative to standard methods of contraception used in nonhuman primates. Based on its successful use in various species, we hypothesized that a single 4.7mg implant would suppress the production of estrogen and progesterone in 6 adult female rhesus macaques for at least 5 mo with a corresponding cessation of menses. For the duration of the study, animals were monitored cageside daily for detection of menses. In the month before implantation, blood was collected on the second day of observed mense and at 6 subsequent timepoints throughout the month. At the onset of the next cycle, animals were sedated with ketamine and implanted subcutaneously with 4.7mg GnRH agonist in the interscapular region. For the month following implantation, blood collection mirrored the pre-implant schedule and continued on a weekly basis for the remainder of the study. Samples were submitted for serum analysis of estradiol and progesterone levels. Between 1-3 d after implantation, a transient rise in hormones was seen, representing the initial stimulatory phase of the GnRH agonist. By day 6 post-implantation, serum estradiol and progesterone concentrations had decreased to basal levels and remained suppressed for variable durations. Return to hormonal cyclicity was observed in 2 animals at 82 and 99 d post-implantation, as evidenced by a rise in estradiol to >30 pg/ml, followed by a rise in progesterone to >0.2 ng/ml 14 d later. This cyclicity was corroborated by observation of menses 11-12 d after the progesterone peak. The remaining 4 animals had not exhibited a return to cyclicity by 5 mo postimplantation, although 1 animal continued to have sporadic vaginal bleeding. This study showed that GnRH agonist suppressed hormone cyclicity in female rhesus macaques for at least 82 d and up to >150 d.

P123 Diagnosis of H3N2 in Swine Using Human Rapid Diagnostic PCR Influenza Tests

LE Riddle*¹, KD Bean¹, RJ Cybulski²

¹Veterinary Support Group, US Army Institute of Surgical Research, San Antonio, TX; ²Microbiology, Brooke Army Medical Center, Fort Sam Houston, TX

Fifteen swine (13 Yorkshire Cross and 2 Durocs) were received from an approved vendor at a government military agency. After a 3-d acclimation period, blood samples were drawn for baseline bloodwork, to include CBC and chemistry. On day 7 of housing, 1 pig (Patient Zero) had bloodwork results that likely indicated an ongoing infectious process. At the same time, 3 pigs in one room (to include Patient Zero) developed bilateral nasal discharge. The vendor was contacted to ascertain if there had been any issues in the vicinity of where Patient Zero had been housed at the vendor’s facility. The vendor informed veterinary staff that Patient Zero came from a barn that had confirmed cases of H1N1 detected via oral fluid samples after shipment. Human rapid diagnostic pan-influenza tests were obtained from a military medical center to quickly test pigs in the vivarium. The 15 new pigs, as well as 9 pigs that had already been housed in the vivarium prior to receipt of the shipment, were sampled by nasal swabbing. Test results indicated that seven out of 15 new swine were positive for Influenza A. At that time, the decision was made to euthanize the 15 swine to prevent further spread within the vivarium and to protect personnel. The positive cases were retested for subtyping to further delineate the specific strain. It was found that the seven positive cases were actually H3N2, and the vendor was contacted with the updated information. The use of human rapid diagnostic influenza tests may be a convenient, practical way to quickly obtain results for suspected cases of influenza in swine with respiratory clinical signs.

P124 Project Bum Bum: Clinical Scoring System and Assessment of Medical Treatments for Rectal Prolapses in Mice

LF Chaffee*¹, G Nault¹, H Goold¹, M Fink^1,2, L Habenicht^1,2, DL Fong^1,2, JK Leszczynski^1,2, C Manuel^1,2

¹Office of Laboratory Animal Resources, University of Colorado Anschutz Medical Campus, Aurora, CO; ²Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, CO

Rectal prolapses occur frequently in mice used for biomedical research. Little information is available on standardized methods for assessment or successful treatments; thus, many cases result in euthanasia. We hypothesized that inflammation was the primary contributor to rectal prolapses and that anti-inflammatory drugs may aid in resolution. From 2019-2021, mice found with rectal prolapse on routine health checks were enrolled in a therapy study after investigator approval. Once enrolled, mice were distributed into 4 treatment groups which included: 5 d of meloxicam (2 mg/kg SC) with daily topical triple antibiotic ointment (TAB); daily TAB with dexamethasone; daily TAB alone, and daily application of sterile lube. Researchers could request exclusion from the NSAID and steroid arms if it confounded their ongoing studies and remove mice from our study at any time. All treatment groups were evaluated on 0, 7, and 28 d. A novel scoring system was used to assess tissue health (scored 1-5), and tissue protrusion and diameter were recorded. In addition, 14 demographic factors were also recorded. Humane endpoints included severe mucosal ulceration, progression of comorbidities, decrease in overall health, or an inability to defecate. In total, 48 cases were enrolled in the study. Of those, 41 (85%; n = 9-11/group) and 27 (56%; n = 5-9/group) cases reached the 7 and 28 d time points for analysis. Mean mucosal health scores were compared within each treatment group to the initial observation and between treatment groups at each time point for evidence of benefit. No treatment option consistently decreased the severity or led to resolution of rectal prolapses at 7 or 28 d. Nevertheless, the primary factor found most useful for clinical assessment was mucosal tissue health. Overall, size of the prolapsed tissue did not correlate with deterioration of the animal’s condition. No trends were identified in demographic data to help anticipate spontaneous rectal prolapse. Our results suggest that daily application of sterile lube was just as effective as other treatments to maintain tissue health and may be the most reasonable approach to maintaining rectal prolapses in mice.

P125 Spontaneous Neural Hemangiosarcoma in a Transgenic Mouse

LT Ferguson*¹, L Birdwell³, T Nagy², VK Lee¹

¹Division of Animal Resources, Emory University, Atlanta, GA; ²Department of Pathology, University of Georgia, Athens, GA; ³Division of Animal Resources, Yerkes National Primate Research Center, Emory University, Atlanta, GA

A male breeding APP/PSEN1 mouse was evaluated for hindlimb paresis. On examination the mouse was bright, alert, responsive, and kyphotic. On neurologic examination, the mouse exhibited proprioceptive deficits of the hindlimbs, right hindlimb paresis, left hindlimb paralysis, and absent nociception at the left hindlimb. Differential diagnoses included neoplasia of the nervous system such as schwannomas or neurofibroma, peripheral neuropathy, spinal injury, significant age-related orthopedic conditions such as osteoarthrosis, or a vascular event. Euthanasia was elected due to a poor prognosis, and a necropsy was pursued to elucidate the causative disease. Necropsy did not reveal significant abnormal findings. Histopathology of the region of the lumbar vertebrae showed an extensive, infiltrative, and densely cellular spindle cell neoplasm. The neoplasm was irregular and marked by multifocal islands of newly formed cancellous bone within it, as well as the presence of numerous dilated vascular channels within the neoplastic cells. The vascular channels were lined by several layers of spindle cells and effaced the center of a vertebral body. From the effacement of the neoplastic proliferation with neural tissue, the white matter, spinal nerves, and peripheral sciatic nerves were found to be undergoing extensive Wallerian degeneration. Histopathology also confirmed that there were no splenic lesions. On immunohistochemistry, neoplastic cells were positive for Factor VIII-related antigen (von Willebrand factor). With this endothelial-specific marker, the neoplasm was most consistent with hemangiosarcoma. The diagnosis of hemangiosarcoma in the spinal cord of a mouse is rare. Clinically, fatal blood loss is an urgent concern with hemangiosarcoma lesions upon rupture. Spinal cord injury from the expansion of this lumbar mass may have affected the mutated presenilin 1 gene in this transgenic mouse, additionally contributing to the clinical signs. This case presentation is striking since it highlights a rare neoplasm of the nervous system in mice. Although hemangiosarcoma in this location is rare, mesenchymal neoplasms should be considered as a differential diagnosis for mouse neural lesions.

P126 Idiopathic Megacolon in a Recently Shipped, Purpose-bred Feline (Felis domestica)

MM Walker*, CL Freed

Office of Research, The Ohio State University, Columbus, OH

A 7-mo-old, purpose-bred, intact male domestic shorthair presented with firm colonic distension, low body condition, and lack of anal tone upon arrival to the facility. A complete blood count and chemistry were performed to rule out underlying hypothyroidism and metabolic (e.g., hypokalemia, hypercalcemia) causes for megacolon; all parameters were within normal limits. Surgical consultation determined a poor prognosis due to the presumed congenital origin and chance of recurrence even with colectomy. It was further discussed that an enema is not an appropriate option for severe cases due to extent of fecoliths, inability to address colonic distension, and potential for traumatic tears. The feline was bright, alert, and responsive on arrival but progressed to quiet, alert, responsive, and weak with moderate dehydration, hindlimb weakness, and respiratory effort by 48 h. Euthanasia was performed. Necropsy confirmed the morphologic diagnosis of megacolon with a 7 cm colonic diameter at its widest portion and total distension length of 32 cm. There was no evidence of a pelvic fracture, foreign body, tumor, or any other outlet obstruction. Histopathologic examination did not support a congenital cause, revealing normal nerve ganglia in both the small and large intestines. There were no microscopic abnormalities to suggest underlying or comorbid disease in any tissues submitted. Megacolon in the feline pet population is relatively common, the incidence of which is 62% idiopathic, 70% in male cats, and 46% in domestic shorthair breeds. Physical abnormalities were not noted prior to shipment; this case highlights an unusual presentation of idiopathic megacolon in a young cat as a possible outcome of shipping stress and demonstrates the extent of pathologic changes that can occur in the interval from pre-shipment examination, which can occur as early as 10 d prior to shipping, to arrival examination. Lastly, this case underscores the importance of abdominal palpation, to which many felines may be resistant, as a critical component of the arrival veterinary exam.

P127 Idiopathic Megacolon in a Female Cynomolgus Macaque (Macaca fascicularis)

M McKinney*, K Shuster, C Pinelli

Vanderbilt University Medical Center, Nashville, TN

A 15-y-old female cynomolgus macaque presented with a 1-wk history of progressive hyporexia and constipation following recovery from a routine semi-annual exam. The animal was individually housed with no husbandry or dietary changes and no recent experimental history other than a cranial implant of 4.5 y duration. Physical exam revealed severe abdominal distension with diffuse percussive tympany on auscultation. Additionally, rectal examination revealed a mild degree of luminal narrowing of the rectum. Blood was collected for routine CBC and chemistry panels. With suspicion of gas in the abdomen, 3-view abdominal radiographs were obtained to ascertain etiology. Radiographs revealed marked, diffuse colonic dilatation secondary to intraluminal gas. Following recovery, treatment options included aggressive medical management, surgical exploration, or humane euthanasia. Given the anesthetic risks, rapid progression of clinical signs, and poor prognosis, humane euthanasia was elected. Remarkable gross necropsy findings included severe colonic dilatation and left lateral liver lobe torsion believed to be secondary to an increase in intraabdominal pressure. Histology revealed colonic serosal edema with hemorrhage as well as a mild to moderate typhlocolitis. Due to the absence of a specific etiology, idiopathic megacolon was suspected. Idiopathic megacolon, or acute colonic pseudo-obstruction, is pathologic dilatation of the colon without any underlying mechanical cause. Many risk factors have been identified and well documented in numerous veterinary species. There are a few publications in cynomolgus macaques, and although sample size is small, females appear to be overrepresented. Based on our findings, idiopathic megacolon warrants consideration as a differential for progressive abdominal distension and inappetence in cynomolgus macaques.

P128 Challenges Associated with Diagnosis of Clinical Hypervitaminosis D in a Cohort of Guinea Pigs

ML Granzow*¹, P Fishnick¹, D Ferguson^1,2, FC Hankenson^1,2

¹Campus Animal Resources, Michigan State University, Lansing, MI; ²Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI

Over a period of 4 wk, several guinea pigs (N=4; 5 months old, strain Mdh:(SR[A])) presented with reduced body condition, lethargy, and discolored extremities to the veterinary service. Animals in this colony serve as healthy controls for an interinstitutional collaboration. Guinea pigs arrive at ~4-wk-old, are housed socially in sex-specific groups within stainless-steel cages with bottled RO water and ad libitum guinea pig chow. Affected guinea pigs were euthanized and necropsies (n=3) performed by an internal diagnostic laboratory. Gross lesions were noted in liver, kidneys, cecum, bladder, and lungs and histopathology identified metastatic mineralization in multiple organs. Our differential diagnosis included underlying endocrine imbalance, musculoskeletal, genetic, or nutritional disease. We had a concern of hypervitaminosis D from the feed source or another environmental source; however, feed analysis from three distinct lots confirmed that Vit D levels were consistent with the expected nutrients, and no extrinsic variables could be identified. The cluster of clinical cases did not reflect the typical health status of the colony, yet we elected to assess further animals to determine if there were subclinical issues. Clinically normal animals (n=16) were randomly selected at the time of planned endpoint. All animals underwent necropsy (n=16), and a subset (n=8) had complete blood counts and serum chemistries performed. As expected, necropsy and histopathology were unremarkable, with minimal tubular mineralization of kidneys (n=8) and hepatocellular necrosis (n=1) documented. Serum concentrations of 25-hydroxyvitamin D from these healthy guinea pigs ranged between 310-896 nmol/L (anecdotal normal levels are estimated as <350nmol/L), which were unexpectedly high given the lack of clinical signs and nutritionally sound diet. To validate the results from the internal lab, additional animals (n=4) had blood sampled, serum divided, and sent simultaneously to the internal and an external diagnostic lab for Vit D levels. Results between labs were significantly different, and the external lab results were all within normal Vit D ranges; clarification in differences in the lab results will be crucial. This experience reinforced the need to define Vit D reference ranges for guinea pigs in determining clinically acceptable blood values.

P129 Micro-Computerized Tomography as a Diagnostic Imaging Aid Investigating Nodular Lesions on the Distal Phalanges of the Xenopus tropicalis Hindlimb

M Zhang*¹, S Wilson³, KM Casey¹, P Thomson², S Green¹

¹Stanford University, Cupertino, CA; ²Institut national de la recherche scientifique, Quebec City, Canada; ³Diagnostic Imaging Service, University of California-Davis, School of Veterinary Medicine, Davis, CA

A 7-y-old female, laboratory Xenopus tropicalis (SVL 5.4 cm), recently retired from the researcher’s colony, presented with a firm, pale, 3-4 mm nodular lesion surrounding the claw on the distal aspect of digit III of left pelvic limb. The frog was otherwise healthy. Differential diagnoses included metabolic bone disease (MBD), gout, pseudogout, infectious disease, or neoplasia. The frog was humanely euthanized in buffered tricaine sulfate, and the whole carcass was analyzed with micro-computed tomography (microCT) at 40kV and 200μA with 21μm in-plane pixel size, and 21 μm slice thickness. Image reconstruction and evaluation were performed with multiplanar reconstruction and 3D voluming rendering. MicroCT scans revealed alteration of shape of the distal phalanx, osteoarthritis on both hindlimbs, and showed a large peripherally mineralized, bulbous expansile lesion on digit III of the left hindlimb with fat-attenuating material between the remnant phalanx and the peripheral mineral. Histologically, the nodule was composed of a thick rim of cortical bone surrounding adipose and hematopoietic tissue. The presence of osteoarthritis was confirmed histologically. There was no evidence of infection, gout, neoplasia, or metabolic bone disease. In this report, MicroCT aided in the diagnostics of the affected frog by producing high-resolution images, allowing the study of bone microarchitecture and enabling 3D reconstruction of the bone structure. Although the etiopathogenesis of this condition is unknown, we hypothesize that this frog’s geriatric age, the unique growth pattern of the Xenopus claw, and biomechanical forces on the appendicular skeleton all played a role. In laboratory animal medicine, microCT is uniquely suited as a diagnostic for conditions in small animal species such as frogs, mice, fish, and others.

P130 Identification of Stenotrophomonas Maltophilia with Unique Morphology Caused by Antibiotic-induced Filamentation in a NZW Rabbit Model of Invasive Bacterial Pneumonia

MN Campbell*, I Miranda, RJ Ricart Arbona

Tri-Institutional Training Program in Laboratory Animal Medicine and Science, Memorial Sloan Kettering Cancer Center, Weill Cornell Medicine, The Rockefeller University, New York, NY

A 6-mo-old female cannulated (jugular vein) New Zealand White rabbit (Oryctolagus cuniculus) was immunosuppressed with cytosine arabinoside and methylprednisolone intravenously for 2d prior to sedation for intratracheal inoculation of Stenotrophomonas maltophilia. The model of invasive bacterial pneumonia in a neutropenic patient was used to study the efficacy of a new-generation cephalosporin antibiotic, which was administered intravenously once daily. Eight days post-inoculation, the rabbit was found dead without prior clinical signs. A complete necropsy was performed. On gross necropsy, the lung contained multifocal to coalescing, flat, dark red areas extending throughout the parenchyma, with similar lesions present on the epicardium, myocardium, endocardium, serosa of the small and large intestines, and in the renal cortex and medulla. Histopathologic evaluation revealed myocarditis, glomerular capillary thrombosis, and tubular necrosis, interstitial pneumonia, and centrilobular and midzonal microvesicular hepatic lipidosis all associated with bacterial emboli. Gram stain revealed intravascular, Gram-negative long filamentous bacteria inconsistent with S. maltophilia’s morphology. Pulmonary aerobic bacteria culture was negative, and several untreated control rabbits from the same experimental cohort inoculated with S. maltophilia presented with similar clinical and pathologic findings, except the bacterial morphology and cultures were consistent with S. maltophilia. Antibiotic-induced filamentation (AIF) was suspected based on the experimental history, histopathology, and culture results. AIF, as well as the potential implications on therapeutic animal models of bacterial diseases, will be discussed.

P131 Shigella-induced Arthritis in a Rhesus Macaque

R Donocoff*, C Kissel, P Latourette, C Dyer

Gene Therapy Program, University of Pennsylvania, Philadelphia, PA

A 5-y-old captive-bred, naïve male rhesus macaque (Macaca mulatta) with an unremarkable clinical history presented with pelvic limb weakness and tremors on 12/19/20. The animal was quiet, alert, and responsive with normal mentation. Treatment with meloxicam was initiated for presumed soft tissue injury. Clinical signs did not resolve after 2 d, and a cage-side neurologic exam revealed mild tremors and ataxia in the pelvic limbs. A sedated physical exam, CSF tap, nerve conduction velocity (NCV) studies, fluoroscopic images with myelography, and CBC and chemistry panels were performed. The animal was administered sustained-release buprenorphine and meloxicam. On physical exam, there was diffuse bilateral atrophy of the pelvic limbs. Although serology for cytomegalovirus (CMV) antibody titers was positive, PCR was negative, ruling out CMV. Fluoroscopic images of the caudal spinal canal revealed a small area of ventral deviation of the dorsal contrast line at the L6-L7 vertebrae, possibly due to intervertebral disc disease. A mild osteomyelitis lesion in the left hip joint was noted. Other diagnostics did not reveal significant findings. The animal’s clinical signs progressed to moderate, predominantly left-sided pelvic limb paresis. An animal housed in the same room had recently been diagnosed with Shigella. Fecal culture and PCR were submitted, and PCR results were positive for Shigella on 01/05/21. After treatment with Enrofloxacin and TMS was initiated, the paresis improved but was still present. A tapering dose of dexamethasone was started on 01/14/21 for presumptive intra-articular inflammation due to Shigella-induced arthritis. The animal improved significantly after steroid administration, and all treatments were discontinued on 01/22/21. Due to the mild nature of abnormalities noted on fluoroscopy, images were not repeated. Clinical signs were caused by reactive arthritis, a systemic inflammatory disorder characterized by aseptic arthritis. The process is triggered by an infection at an extra-articular site, and clinical signs occur up to 4 wk after enteric infection. Treatment for Shigella-induced arthritis includes antibiotics, analgesics, and steroids. The animal recovered completely, regained muscle mass, and full use of the pelvic limb with no additional musculoskeletal or neurologic abnormalities.

P132 Molar Apical Elongation in a Colony of Prairie Voles (Microtus ochrogaster)

SD Alaniz*, T Gluckman, KE Saunders

Department of Comparative Medicine, Oregon Health and Science University, Portland, OR

Prairie Vole (Microtus ochrogaster) dentition consists of entirely elodont teeth that require friction through mastication of fibrous food and gnawing enrichment to maintain appropriate attrition and occlusion. In the fall of 2019, our institution obtained a colony of prairie voles which we maintained on their previous diet; a 2:1:1 mixture of high fiber rabbit food cracked corn, and crimpled oats, respectively, as well as aspen blocks and manzanita sticks for gnawing enrichment. Within the first few months of managing this colony, multiple voles were found dead with their incisors caught on the wire bar lids or with an undetermined cause of death. The high mortality was eventually attributed to a dental pathology sometimes referred to as molar apical elongation (MAE), which has been reported in various elodont species such as degus, chinchillas, guinea pigs, rabbits, as well as other vole species. This condition consists of retrograde displacement of molar apices with or without secondary incisor malocclusions. In severe cases, this retrograde displacement can invade the cranial floor and/or ventral mandibular wall leading to clinical signs such as abnormal mentation, ataxia, dyspnea, anorexia, or death. We then screened the entire colony by mandibular palpation and assessed a 40% prevalence with or without secondary incisor malocclusions. The condition was managed by culling severe cases and adjusting the diet to prevent selective eating of low fiber components. The dietary grain was discontinued, and timothy hay was added for additional fiber and dietary variety. New breeding stock was added to the colony, and after 6 mo, the prevalence of MAE had been reduced to 14%, in which nearly all were mild cases. As prairie voles become a more widely used model for understanding social affiliations and their neural mechanisms, it is prudent that this species is also managed with their dental health in mind. Further investigation to determine genetic versus nutritional effects on MAE development is needed to help improve animal welfare and minimize research complications.

P133 Topical Oclacitinib as a Treatment for Murine Ulcerative Dermatitis in Laboratory Mice

SE Davison*¹, KM Emmer¹, B Ugiliweneza^2,3, LC Sherwood¹

¹Comparative Medicine Research Unit, University of Louisville, Louisville, KY; ²Department of Neurosurgery, School of Medicine, University of Louisville, Louisville, KY; ³Department of Health Management and Systems Science, School of Public Health and Information Science, University of Louisville, Louisville, KY

Murine ulcerative dermatitis (UD) is a common, multifactorial skin disease of C57BL/6 and C57BL/6-background strains of mice. Many treatment options have been previously reported but have been variably successful and may interfere with specific research studies. Janus kinase (JAK) inhibitors, such as oclacitinib, have been used to treat allergic dermatitis in humans, dogs, and other species. Additionally, topical oclacitinib was shown to improve an induced model of dermatitis in mice. We hypothesized that topical application of oclacitinib in conjunction with hind limb nail trimming would improve UD lesion scores more than our institutional standard treatment regime using meloxicam, topical antibiotic ointment, and nail trimming or nail trimming alone. To test this, mice with naturally-occurring UD were recruited to the study and assigned to 1 of 3 treatment groups (n = 14/group): nail trim only; nail trim plus meloxicam and topical triple antibiotic ointment; or nail trim plus topical oclacitinib. UD was assessed on days 1, 7, and 14 for all treatment groups and scored based on a previously published scoring system that quantitatively scored UD lesions based on pruritus, character of the lesion, size of lesion, and location of lesion. Here we found that mean UD scores decreased from day 1 to day 7 and from day 1 to day 14 for all treatment groups. However, there was no significant difference in mean UD score between the treatment groups at any timepoint. These data show that topical oclacitinib improved UD lesion scores comparably to our institutional standard treatment and nail trimming alone. However, further studies may be warranted to investigate other potential applications of oclacitinib to treat UD.

P134 Pregnancy Associated Thrombocytopenia and Bone disease in a Breeding Colony of Common Marmosets

S Maddur Ganesan*, S Muthupalani, RM Kramer, JG Fox, JL Haupt

Division of Comparative Medicine, Massachusetts Instiute of Technology, Cambridge, MA

Marmosets are increasingly in demand for use as animal models in various areas of biomedical research but are limited in supply. Breeding is essential for maintaining and expanding marmoset colonies. Marmosets are efficient breeders with a 5-mo gestation and generally produce 2 litters/year. Pregnancy monitoring includes serial ultrasound examinations and routine bloodwork. During routine health screening, we identified thrombocytopenia (< 250k/ul) in seven pregnant marmosets in the last month of gestation out of the 16 marmosets that we evaluated. Among these cases, 57% were clinically asymptomatic (4/7), but 43% (3/7) presented with lameness. Further evaluation revealed that all of marmosets presenting with lameness had radiographic changes consisting of varying degrees of lytic bone lesions and osteopenia. Radiographic lesions were generally unilateral, primarily affecting the long bones of fore and hindlimbs. Animals presenting with lameness and thrombocytopenia had increased alkaline phosphatase (ALP) in comparison to late gestation pregnant animals without thrombocytopenia (n=9). One animal with severe thrombocytopenia (76 k/ul) presented with pinpoint hemorrhages and left forelimb lameness. The animal was euthanized due to worsening thrombocytopenia(64k/ul) despite 72 h of supportive care and severe osteolytic lesions of the humerus, radius, and ulna. Histopathology for this case showed severe bone disruption, osteoclastic resorption and bone remodeling, hemorrhage, stromal proliferation consistent with fibrous osteodystrophy, and reactive proliferative bone lesions. Close monitoring of the other two animals with bone lesions showed improvement in lameness about 3-4 wk after parturition, with simultaneous return of normal platelet counts (>250k/ul) and significant improvement in lytic bone lesions. Outcome of pregnancy and infant survival was not affected by thrombocytopenia. The results of this study highlight the need for careful observation of marmosets in late gestation, including hematological evaluation and radiography for any animal presenting with lameness.

P135 Streptococcus suis Valvular Endocarditis in a Swine Model of Atrial Fibrillation

T Jones*, DM Molk, A Slate, DM Jarrell

Center for Comparative Medicine, Massachusetts General Hospital, Charlestown, MA

A 53kg, 1-y-old castrated male Yucatan minipig (Sus scrofa domesticus) on a study of pacemaker-induced atrial fibrillation experienced acute onset of ataxia, inability to stand, and emesis. The animal had undergone pacemaker implantation and atrioventricular node ablation several weeks prior. Aseptic technique and intraoperative antibiotics (cefazolin 17 mg/kg) followed by a 1 wk course of cephalexin (10 mg/kg PO q12 hrs.) were used. Experimental design entailed placement of a dual-lead pacemaker with 1 lead rapidly pacing the left atrium and 1 providing normal physiological contractions of the left ventricle. On presentation, the heart rhythm was irregularly irregular, and oxygen saturation could not be measured due to limitations of pulse-oximetry with patients experiencing arrhythmias. Differential diagnoses included heart failure, infectious disease, and pacemaker malfunction. Pacemaker performance had been consistently unsatisfactory, and the investigative team recommended performing voltage adjustments in order to improve signal transduction and function. Following these alterations, the animal improved and remained stable over the next several days. Experimentally, however, pacemaker performance was poor in that atrial fibrillation was unable to be induced consistently. The animal was deemed sufficiently healthy to undergo surgical revision of the pacemaker per the approved protocol. Echocardiography prior to surgery revealed a suspect vegetative lesion associated with the left atrioventricular valve. Given the poor experimental and clinical prognosis, euthanasia was elected. Post-mortem examination confirmed a left atrioventricular vegetative lesion. Culture and histopathology identified Streptococcus suis endocarditis with broad antimicrobial susceptibility, including first-generation cephalosporins. S. suis is a common cause of endocarditis in post-weanling swine and is often carried by adult swine. Although susceptible to the antibiotics used peri- and postoperatively, it is noteworthy that S. suis forms biofilms. The role of the pacemaker, in this case, remains unclear, but this finding nonetheless underscores the importance of echocardiogram in cardiac studies of swine.

P136 A Combination of Tiletamine/Zolazepam and Dexmedetomidine with Tramadol Provide Effective General Anesthesia in Rats

V Limprasutr*^1,2, PE Sharp³, K Jampachaisri⁴, C Pacharinsak⁵, S Durongphongtorn⁶

¹Department of Pharmacology and Physiology, Chulalongkorn University, Faculty of Pharmaceutical Sciences, Bangkok, , Thailand; ²Research Clusters: Preclinical Toxicity and Efficacy Assessment of Medicines and Chemicals, Chulalongkorn University, Bangkok, Thailand; ³Animal Resources Centre, Murdoch, , Australia; ⁴Department of Mathematics, Naresuan University, Faculty of Sciences, Phitsanulok, Thailand; ⁵Department of Comparative Medicine, Stanford University, School of Medicine, Stanford, CA; ⁶Department of Veterinary Surgery, Chulalongkorn University, Faculty of Veterinary Science, Bangkok, Thailand

When gas anesthesia is unavailable or contraindicated, injectable anesthesia can be used. Here, we evaluated the effectiveness of a tiletamine/zolazepam and dexmedetomidine combination with and without the opioid tramadol in rats. We hypothesized that tiletamine/zolazepam and dexmedetomidine with tramadol would achieve a surgical plane of anesthesia better than tiletamine/zolazepam and dexmedetomidine without tramadol. Adult male and female Sprague-Dawley rats (n=18) were randomly divided into 2 groups: Group 1) tiletamine/zolazepam (10 mg/kg) and dexmedetomidine (0.25 mg/kg) with an analgesic, tramadol (12.5 mg/kg) - TDT; and Group 2) tiletamine/zolazepam (10 mg/kg) and dexmedetomidine (0.25 mg/kg) - TD. Following subcutaneous administration of the drugs (T0) and paw withdrawal reflex loss, the thigh was aseptically prepped, incised (0.5 cm), and sutured close. Rats were anesthetized for 45 min (T45). After T45, atipamezole (1 mg/kg) was subcutaneously administered as a dexmedetomidine reversal agent. During anesthesia, anesthetic parameters (heart rate, respiratory rate, oxygen saturation, and body temperature) were monitored every 5 min for 45 min. Duration parameters monitored following T0: 1) righting reflex loss; 2) paw withdrawal reflex loss; 3) movement; 4) standing; 5) walking. Respiratory rate, oxygen saturation, and body temperature had no group or time point differences. Compared to T0 heart rate values, the TDT and TD groups were significantly lower at 30 and 20 (and 25) min, respectively. There were no differences for duration parameters [righting reflex loss (3 min); paw withdrawal reflex loss (4-5 min); movement (50-51 min); standing (51-52 min); walking (52-53 min). Data indicate that TDT achieves a surgical plane of anesthesia similar to TD for 45 min in a rat minor procedure.

P137 Pharmacokinetics of Sustained-release Buprenorphine in Adult Baboons (Papio anubis)

W Williams*¹, A Norris¹, X Ding²

¹Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK; ²Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK

Baboons (Papio anubis) are the largest Old World primate and are used in approximately 6% of biomedical research models involving nonhuman primates. As a key component of research refinement, a multimodal analgesic regimen for potentially painful procedures is standard practice among our veterinary care team. However, there exists a paucity of literature addressing effective dosing strategies for this species. Buprenorphine is a key component of veterinary multimodal analgesic strategies due in part to its relatively long half-life and minimal cardiorespiratory effects. Sustained-release buprenorphine is a long-lasting formulation with claims of at least 72 h of efficacious drug levels (at least 0.1ng/mL) after 1 subcutaneous injection in several laboratory species, but there is no such description for baboons. Therefore, the purpose of our project was to evaluate the pharmacokinetic profile of sustained-release buprenorphine in adult male and female baboons. After administration of 1 subcutaneous injection of sustained-release buprenorphine (0.2 mg/kg) to 5 male and 5 female baboons, we collected serial plasma samples from 5 min to 120 h post-administration. We then analyzed the samples by liquid chromatography-tandem mass spectrometry. We found the plasma concentrations were above 01.ng/ml within 30 min of drug administration and remained here for at least 5 d in all animals. The time to maximal concentration was 24 h, and the terminal half-life was 465 h in males compared to 194 h in females while total plasma clearance was 2088 L/hr in males and 1663 L/hr in females. Side effects were mild and included injection site redness in 4/10 animals and generalized mild pruritis in 4/10 animals. Our data suggest that 1 dose of sustained-release buprenorphine administered at 0.2mg/kg subcutaneously provides at least 5 d of therapeutic plasma concentration. These data provide the first information to guide accurate dosing in adult male and female baboons.

P139 Spontaneous Plasma Cell Myeloma in an Audiogenc Inbred Rabbit (Oryctologus cuniculus)

X Peng*, MC White, HM Atkins

Department of Comparative Medicine, Penn State University College of Medicine, Hershey, PA

A 2.5-year-old female Audiogenic inbred (EIII/JC) New Zealand White rabbit born in our breeding colony presented for acute paralysis of both hind limbs. Radiographs showed a subluxation of L7 from S1. The rabbit was euthanized due to poor prognosis. At necropsy, there was a region of hemorrhage within the fascia overlying the left aspects of the epaxial muscles in the lumbar region. The joint between the sacrum and caudal lumbar vertebra was easily manipulated. No additional gross lesions were noted, and the vertebral column fixed in toto and decalcified for 1 wk in Formical-4 (StatLab, Columbia, MD). Tissues were paraffin-embedded and then sectioned at 5 um before being stained with routine hematoxylin and eosin (H&E) stain. Microscopic examination of the affected lumbar vertebrae identified that the normal bone marrow architecture was completely obscured by a monomorphic, non-encapsulated, highly cellular neoplasm, consisting of round cells arranged in sheets supported by a fine stroma. The neoplastic cells had scant eosinophilic cytoplasm, perinuclear clearing, and moderately basophilic nuclei with a finely stippled chromatin pattern. There were 4-5 mitotic figures per 40X high-powered field. The neoplasm extended through the bone (pathologic fracture) and into the surrounding adipose tissue and joint space. A few isolated regions of densely eosinophilic bone lacking cytoplasmic and nuclear detail remained (necrosis). Viable bone adjacent to the neoplasm has increased osteoclast activity (osteolysis). The microscopic appearance was highly suggestive of a plasma cell myeloma with multifocal, moderate vertebral osteolysis. To our knowledge, this is the second case of plasma cell myeloma reported in a rabbit, but the first in an inbred New Zealand White rabbit with bone invasion of vertebrae and paralysis of hind limbs.

P140 Methodology Improvements for Colonoscopy Preparation in Cynomolgus Macaques

T Rinaldo*, M Wood, E Voss

Comparative Medicine, Abbvie, North Chicago, IL

An ongoing study required serial distal colonoscopies of cynomolgus macaques. Initial colonoscopies were hampered by residual fecal material, which made visibility difficult. Original colonoscopy preparation included prolonged fasting (24-36 h) and the administration of laxative solutions, softened/liquid nutrition, subcutaneous fluid therapy, and saline enemas on chair restrained animals. As this method did not provide the optimal visualization of the distal colon mucosa, refinements needed to be made. The first refinement made was the removal of laxatives from the protocol, followed by subcutaneous fluid therapy. Other changes included the reduction of fasting period by offering softened biscuits (offered 48 h prior to event), followed by soft foods, such as applesauce or yogurt (~30 h prior to event), finally providing only liquids (24 h prior to event). The next refinement was to discontinue saline enema administration on awake animals. Instead, the enema would be performed after the animal was anesthetized for the colonoscopy. This allowed for complete body relaxation, making a smoother insertion of the red rubber catheter. Body positioning was also changed. Raising the upper body during expression allowed gravity to further aid in the enema process. The adjustments were made over a period of 1-2 y and made a marked improvement on the removal of solid fecal material for the visualization of the colon tissue, thereby improving study results. The refinements also resulted in improved animal welfare by decreasing or eliminating potentially stressful preparation procedures and reducing the procedure time for each colonoscopy.

Husbandry/Management Posters

P200 Creative Enrichments for Nonhuman Primates

A Medina*

MICV, Carnegie Mellon University, Pittsburgh, PA

Providing novel and creative enrichments for nonhuman primates is an important aspect of all husbandry programs for mental wellbeing. Preparing engaging food enrichment can be challenging, as familiar, repetitive foods may create a quickly fading excitement. Even when toys and feeders are periodically cycled out, the novelty is lost when those objects are not as entertaining. If primates were encouraged to engage with their enclosure and enrichment devices in differing ways, this may prevent the onset of boredom with these items. The implementation of creative techniques to reimagine these same enrichment foods encouraged further interest. These changes were implemented during afternoon enrichment times. Destructible enrichment was created using origami shapes, such as a crane or balloon, which allowed for an interactive foraging experience. Seeds scattered after these novel shapes were broken open created more opportunity for foraging. Paper rolls or hollowed-out cucumbers were filled with shredded lettuces and peanuts, and openings covered to promote exploration of food enrichment. Likewise, forage feeders may be made more challenging to use by blocking holes with hay or shredded lettuce. By implementing these smaller changes to enrichment for the nonhuman primate, a greater interest and shown excitement for otherwise familiar foods can be observed. Longer enrichment interaction times and slower eating have also been observed. The use of these creative enrichments have continued to be implemented as part of the husbandry program.

P201 Time to Play: Incorporating Exercise, Human Interaction, Conspecific Socialization, and Environmental Stimulation into a Chinchilla (Chinchilla lanigera) Husbandry Enrichment Plan

D Marchi*, T Stevens

Division of Comparative Medicine, The Univerisy Of Oklahoma Health Science Center, Oklahoma City, OK

Chinchilla are a rodent species originating from the Andes Mountains in South America. In the wild, chinchilla are a rambunctious, agile rodent that spend a large portion of their waking hours bounding across rocky landscape. Husbandry practices for chinchillas play an important role in their overall welfare. Deficient husbandry (limited enclosure size, lack of enrichment, overcrowding, poor ventilation, and minimal exercise) can lead to abnormal behavior and acute physiological dsyhomeostasis contributing to chronic systemic illness. Stereotypic behaviors have been frequently documented in the fur farming industry and have shown association with high animal stress. These behaviors include bar chewing, fur chewing, scratching at the cage and back-flipping. The chinchilla at our institute are single housed. Caging provided consists of IVC caging or a metal rabbit bank caging system. The caging, although spacious, did not allow for the chinchilla to run, jump, or climb. Our institution wanted to add increased exercise to their enrichment. The updated enrichment plan for the chinchilla consists of cage-level enrichment items, food enrichment, weekly dust baths, positive human interaction, and the newly instituted conspecific playtime in large playpen structures. For playtime, we bring the animals out of their home cages in same-sex pairs then place them into 2 separate but conjoining playpens for 1 hr once a week. The playtime enclosures are enclosed on all 4 sides and top to prevent animals from escaping. Animals were able to socialize with their playpen neighbor and were provided additional cage enrichment including chew sticks and 1-2 elevated platforms. During playtime the animals exhibited natural curiosity, exploration, socialization, and great feats of boldness and athleticism, lovingly referred to as chinchilla parkour. Since incorporating our playtime program into the chinchilla’s enrichment plan we have seen improvement in eating habits as well as improvement in mood and temperament during handling.

P202 Validation of a Sanitation Process for the Automated Watering System in Racks

D Hajosi*, CC Castagna, VV Ifill, MG Morris, AJ McLuckie, CL Winnicker

Institute of Comparative Medicine, Columbia University, New York, NY

Facilities are facing staffing shortages due to the current pandemic, which makes it necessary to reflect on husbandry operations. We assessed a sanitation protocol of racks with an automated watering system in order to streamline the sanitation process. We evaluated a 2-step sanitation process, flushing rack water manifolds with 20 ppm chlorinated water following sanitation in rack washer at 82^◦ C for 15 min. Swabs were collected for ATP detection and bacterial culture (n = 146) at 3 time points: prior to rack washer sanitation, immediately after rack washer sanitation, and immediately after chlorinated water flush. Sterile cotton-tipped swabs were placed ~1cm directly into the opening of randomly selected lixit valves and rotated to have contact with the entire inner surface of the opening. Swabs were analyzed using an automated ATP detection device and colony forming units (CFU) were counted on agar plates at 24 h and 48 h incubation. Relative light units (RLU) were significantly lower following rack wash and chlorinated water flush when compared to baseline. However, RLU did not decrease significantly following chlorinated water flush compared to rack washer sanitation alone. CFU decreased significantly at both 24- and 48-h timepoints between soiled lixits and after the rack wash and the chlorinated water flush, respectively. Additionally, the number of CFU on lixits decreased significantly between rack washer sanitation and chlorinated water flush. The detection of ATP as RLU indicates the presence of organic matter and can provide a total hygiene status, however, the formation of CFU is a more reliable method in detecting live contaminant microorganisms. While studies have shown that residual microbial contamination of racks occurred after rack washer sanitation with manifold flushing with RO water, we could not find any publications using a chlorinated flushing. The results of this study suggest that the chlorinated water flush reliably eliminates live microorganisms from the automated watering system, and should be routinely performed in addition to rack washer sanitation for sanitation of racks with automated water.

P203 False Positive Result with Exhaust Dust PCR Testing due to Potential Backflow of Contaminated Dust

ET Carbone*¹, E Roswall²

¹Animal Resources, Genentech, Inc., South San Francisco, CA; ²Facilities Operation & Maintenance, Genentech, Inc., South San Francisco, CA

Positive exhaust dust PCR results for an occupied rack, for an agent thought to be eradicated, raised the question of whether contaminated dust can linger and even backflow to an upstream sampling site. To investigate the possibility of backflow, we connected permanent ceiling HVAC fixtures to an autoclaved, unoccupied rack, and collected dust samples at 7 locations along the inner walls of the rack exhaust plena, flexible exhaust hose, and permanent ceiling duct. Samples were collected both at the time of rack placement, and 4-5 mo later, using a sterile flocked swab. At the latter time point, multiple samples tested positive for Staphylococcus xylosus, as far upstream as the rack’s horizontal exhaust plenum. Test results demonstrate that false positives may occur due to backflow of PCR-positive dust from permanent downstream components of the exhaust system. Additional research is indicated to quantify this risk and to investigate mitigation measures, such as exhaust pre-filtration downstream of the sampling site.

P204 Hypovitaminosis A in Wild-caught Birds and Dietary Correction

E Wegner*¹, SM Hale², AJ McLuckie¹

¹Institute of Comparative Medicine, Columbia University, New York, NY; ²Aronov Lab, Department of Neuroscience, Zukerman Mind Brain Behavior Institute, Columbia University, New York, NY

An appropriate diet is fundamental for the use of wild-caught birds in research. Our wild-caught black-capped chickadees (Poecile atricapillus) and tufted titmice (Baeolophus bicolor) were fed a mixed diet of pellets, sunflower seeds, and dried-mealworms. Approximately 70% of the colony presented with suboptimal health and respiratory disorders. A diagnosis of hypovitaminosis A was made. Providing a mixed diet allowed selective consumption of seeds and mealworms, inadequate in nutrition. The diet was corrected by eliminating the seeds and mealworms to provide only pellets, excepting weekly enrichment of kale and egg. This was done in cohorts of 20-40 birds over a transition period of 6-8 wk by gradually decreasing the portions of seeds and mealworms per part per week. Black-capped chickadees and tufted titmice are known to be neophobic, with high preference for seeds and mealworms, so an estimated 10-15% loss was expected presuming anorexia and related illness. A total of 264 birds transitioned per cohort from September to May. The percent success varied, ranging from 0% to 15% failure per cohort, averaging about 6% failure per cohort. A total of 31 birds failed to transition, yielding the 10-15% loss expected. Birds that failed to transition more commonly had symptoms of poor body condition, hypothermia, lethargy, with infrequent cases of respiratory issues and death. Overall, the majority did acclimate, thus better suited for use in research. In conclusion, providing a wholesome pelleted diet exclusively is ideal for wild-caught birds. Transitioning from the improper diet can elicit malnutrition, therefore the process should be gradual with meticulous monitoring of body condition. Once acclimated, wild-caught birds can thrive in the research setting.

P205 A New Feeder Device to Provide Cleaner and Accessible Diet Gel to Laboratory Rodents

F Scorrano*¹, S Tavani²

¹Comparative Medicine, Novartis International AG, Basel, Switzerland; ²Engineering Center, Novartis International AG, Basel, Switzerland

A common issue in using diet gel for laboratory rodents is that it becomes soiled quickly due to cage density, type of strain, and amount of bedding. The rodents can also flip upside down the cup or bury it under the bedding. Besides, pups and weak mice may have difficulties accessing the product. This raise some concerns for the welfare of our animals that might not have continuous access to the diet or to the medicament administered. On the market only few gel holder solutions are available but still they show limited efficacy with low scalability. Using 3D printing technology, our team decided to create a new feeder device to keep the gel clean under different experimental conditions, accessible to pups or ill rodents and that could be used with both mice and rats. The device was printed with polycarbonate material and had 3 main components: a bracket to secure the gel cup, a vertical structure with a screw to regulate the height and angle of the holder, and 2 magnets to fix it to the wall of the cage. These prototypes were tested for more than 6 mo with both mice and rats, with different strains and experimental models. Cages using this feeder showed less gel contamination and evaporation compared to the ones with the cup left directly on the bedding. Clean access to the gel could last up to 6 d even in high-density cages with adult mice. The angle for gel presentation was regulated depending on the age and health condition of the animals and the position of the feeder adjusted from the outside without opening the cage. The material used was sanitizable and safe for the animal to ingest. By using this feeder, we were able to consistently provide clean gel to both mice and rats and to get different solutions in only one product. The blueprint of this device is available, free of charge, on a public hosting platform and can be printed, remixed, and used by other facilities according to their needs and budget.

P206 Evaluation of Outcomes of a Common Marmoset (Callithrix jacchus) Foster Program on Infant Survival

KA Thompson, M Jamiel, JL Haupt*

Massachusetts Institute of Technology, Cambridge, MA

Despite the growing use of the common marmoset (Callithrix jacchus) in biomedical research, there is a lack of information regarding infant survival following fostering. Currently, only one publication describes a foster success rate of 33% with a small number of attempts (n = 6). A greater understanding of this practice and factors associated with success can significantly impact marmoset infant survival. This project aimed to assess our program’s success in fostering marmoset infants. Factors evaluated included the reason for foster (triplet litter, poor weight gain, dropped, C-section); infant demographics (weight, age, birth method); foster family demographics (size of family, parity of female/male in cage, date since birth, number of infants, previous litters/size, weight/BCS dam); methods of introduction to the family (bathing, incubator with other infants); and success/failure rates (survival to weaning, notes for the reason of failure). Chi-square analysis and Fisher Exact Test were used to evaluate differences. Twenty-one foster attempts involving 19 infants were evaluated over a 4-y period. Overall infant survival following fostering was 63% (12/19) and foster attempt success was 57% (12/21). All infants with successful foster attempts survived to weaning. The reason for the foster attempt was significantly different between successful and failed foster attempts, with all failures occurring in infants born via cesarean section (P = 0.05). Evaluation of factors associated with success revealed a significant association of infant age at the time of foster with successful fosters being older (2.2 days) than failed fosters (0 days of age, fostered the day they were born) (P = 0.02). There was no effect of infant weight, infant feeding before foster, foster dam experience, number of helpers in the foster cage, number or age of nursing infants with foster dam, and introduction method on infant survival or foster success. Based on these findings, we suggest that infants born via cesarean section may benefit from being nursery-reared for at least 24 h before the foster attempt, given that both delivery via cesarean section and fostering on the day of birth were associated with failed attempts of fostering.

P207 Use of an Alternative Water Bottle Design to Reduce Off-cycle Water Refills in a Meadow Vole (M.pennsylvanicus) Colony

J Abazaris*, C Paulson, M Stoltz, D Franklin, A Duffy, S Hashway

University of Colorado Boulder, Boulder, CO

Voles (Microtus spp) are an excellent model for examining the neural and molecular basis of complex social behaviors. At our institution, meadow voles (M.pennsylvanicus) are used to investigate the neural circuits involved in pair bonding. General recommendations for vole husbandry in the laboratory have been published, but specific recommendations for water delivery systems have not. In over 6 y of caring for voles at our institution, we have found that use of water bottles with non-ball bearing sipper tubes results in excess water loss via mechanical interference by the voles, psychogenic polydipsia (as determined by lack of glucosuria), or excess loss due to dripping during routine cage movement. This has often resulted in over 50% of water bottles needing refills between weekly cage changes, with subsequent increased frequency of cage disturbances and technician time. Attempts at replacement with hydropacs, sipper sacks, and ball-bearing sipper tubes have failed due to either continued excessive manipulation by the voles or failure of the voles to adapt to the delivery system. This study examined use of a shoulder hole bottle design in which animals access water via a small hole inserted directly into the body of the bottle, with no sipper tube or lixit system. We hypothesized that this system would significantly reduce the frequency of off-cycle water refills while maintaining animal welfare in our meadow vole colony. Two groups of 10 cages were each randomly assigned either a shoulder hole or sipper tube bottle. Off-cycle refills (required when volume dropped below 1/3 filled volume) were counted over a 4-wk period. One cage in the shoulder hole group was removed from study after signs of dehydration developed in one of the voles on d 2. No other adverse effects were noted in any animals during the study period. Statistical analysis using 1-way ANOVA revealed a significant decrease in the number of off-cycle water refills in the shoulder hole group, compared to the sipper tube group. This study demonstrates the efficacy of shoulder hole water bottle design in providing adequate hydration while eliminating excess water loss and reducing the number of off-cycle water refills required in a meadow vole colony.

P208 Use of Cageside Body Condition Scoring in Weight Monitoring of New Zealand White Rabbits (Oryctolagus cuniculus)

J Cordingley*, LA Burlingame, PA Lester

ULAM, University of Michigan, Ann Arbor, MI

Maintaining a healthy weight in rabbits is essential to overall animal health and longevity, minimizing surgical risk, and optimizing research output and fecundity. At our facility, feed optimization of overweight rabbits is being implemented as a method to achieve a healthy body weight and body condition while promoting research benefits. Monitoring the weight loss of rabbits on feed reduction is imperative to preventing adverse effects of overly rapid weight loss, but increased monitoring can also have adverse effects due to the increased handling, including animal stress and increased workforce requirements and allergen exposure of staff. Thus, we sought to explore the use of cageside body condition scoring via visual assessment and palpation as an adjunct to weight monitoring. Ten New Zealand white rabbits (Oryctolagus cuniculus) determined to be overweight by having a body weight of at least 4 kg and a body condition score of at least 4/5 were reduced from ¾ to ½ cup of commercial rabbit chow daily. The cohort consisted of 5 males and 5 females ages 2-5 y of a variety of transgenic and wild type genotypes. All rabbits continued to receive free choice hay and regularly scheduled food enrichment. The rabbits were weighed and body condition scored out of 5 by a single rater at the initiation of feed restriction and then weekly for 3 wk. Linear regression data indicated a 66% correlation between body condition score and measured body weight. Additionally, the majority of rabbits exhibited statistically significant weight loss from baseline that was reflected in reduced body condition scores without surpassing 5% loss of total body weight during the observed time period. This initial data suggests body condition scoring via visual assessment and palpation appears to have a strong correlation with actual body weight, though some variability exists even with a single rater. Additionally, chow reduction alone is an effective means of inducing gradual weight reduction in overweight New Zealand white rabbits. Cageside body condition scoring (with palpation) is an effective, efficient, and potentially low-stress means of estimating the weight status of New Zealand white rabbits, though it should be used as an adjunct to periodic measurement of actual body weights for the greatest assessment accuracy.

P209 Development of Testing Method for Leaking Mouse Cage Water Bottles

JA Gustafson*, M Foskett, E Rossow

The Jackson Laboratory, Bar Harbor, ME

Water leakage into a mouse cage can lead to discomfort and even death. As water bottles age, they become more prone to leaking. Pinpointing the cause of a leaking bottle can be challenging as there are 3 primary components to a water bottle: the bottle itself, the cap, and the O-ring that provides a seal between the bottle and the cap. A defect in any of these components can lead to a leaking bottle. Our challenge is to determine when to discard a leaking bottle and when it can be returned to circulation without posing risk to our mice. We developed the following procedure to assess leaking water bottles. When a leaking water bottle is submitted for evaluation, our first step is to identify any defects that could cause leaking such as cracks or chew marks. These defective bottles are immediately removed from circulation. The second step is to take measurements of the water bottle opening. We measure both at the seam and at a 90 degree rotation to assure that the opening is round and meets our specifications. We have seen that a difference of 1/1000 of an inch can cause a bottle to leak. If the bottle is within specifications, then the O-ring is replaced and a new cap is put on the bottle. The final step is to perform a leak test. The bottle is filled with water and placed on a mouse cage. The cage is then put onto an air rack similar to those used in our mouse rooms to simulate actual conditions as much as possible. The bottle and cage are then checked 24 h later. If the cage is dry and the bottle is still full, then the bottle can safely be returned into circulation. If the cage is wet and/or the bottle is not still full, then the bottle is discarded. This program has allowed us to more readily identify and remove water bottle mill dates that are prone to leaking. We have also used this data to establish guidelines for shelf life of our water bottles.

P210 Pig Enrichment Toy Durability and Its Effects on Pig Behavior

K Hall*¹, J Bousema², C Bousema²

¹Premier BioSource, Rensselaer, IN; ²Premier BioSource, Ramona, CA

Pigs benefit from environmental enrichment in laboratory and commercial settings. Due to their curious and playful nature, finding enrichment toys or devices that are entertaining and durable can be difficult. Two popular industry enrichment toys were selected for 2 separate 8-wk observational studies. The first toy design was blue and yellow, natural rubber, 12-pointed star toys. The second toy design, hanging, elastic plastic chew sticks. Durability and length of enrichment interaction were the criteria used to measure toy enrichment. These toys were chosen with the intent of finding a toy that would encourage play at a young age without needing to be sized up or replaced as the pigs grew. Four groups of approximately 20 domestic farm pigs and 20 Yucatan minipigs were studied concurrently at weaning. For 8 wk the pigs were observed for 10 min per pen Monday through Friday after feeding. The length of time in which pigs interacted with the enrichment within the 10 min was recorded, and images were taken to assess enrichment toy durability. There were obvious differences in toy durability by the study’s conclusion. Both toys placed with the Yucatans deteriorated faster than those with farm pigs. This is believed to be due to physiological differences between species and relative sexes as well as differences in the materials in which the toys where crafted. Overall, the star toys were better able to withstand both species’ interactions, while maintaining interest longer. This being said both toys proved beneficial but varied in long- vs short-term price, cleanliness, and time before replacement. Enrichment toys divert negative behaviors such as mounting, and fighting with pen mates but must also be durable and economical for continued use in a facility. Based on these results we will continue to provide our pigs with star enrichment toys to alleviate negative behaviors.

P211 Development of a Real-time Database to Efficiently Manage Overlapping Animal Studies in a High-containment Facility during the COVID-19 Pandemic

KL Gully*¹, KH Dinnon², RS Baric^3,4, VK Baxter^5,1

¹Comparative Medicine, University of North Carolina at Chapel Hill, Raleigh, NC; ²Microbiology & Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC; ³Epidemology, University North Carolina at Chapel Hill, Chapel Hill, NC; ⁵Pathology & Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC

Many challenges are associated with maintaining a biosafety level-3 laboratory, including reserving adequate time and space for experimental procedures. Our lab primarily focuses on coronavirus research, and the COVID-19 pandemic has resulted in an increased demand in productivity and surge in SARS-CoV-2-related mouse studies. This increased demand brought on new challenges with a greater complexity in scheduling supplies, reserving biosafety cabinet time, and maintaining schedule integrity all while having limited animal rack space for different experiments. Without effective communication methods, coinciding studies across multiple users in the lab became increasingly difficult to manage, resulting in the inability to maximize experimental output. With limited animal study space and a critical need to sustain the surge in capacity, facilitation of a more collaborative method of communication between researchers was crucial to the lab’s ability to schedule concurrent studies and maintain adequate supplies. In order to enhance experimental output while maintaining lab safety and biosecurity, a real-time electronic inventory database that researchers could access across the entire lab was created and implemented. The database shows active or upcoming studies and their cage space requirements, and each study’s timeline, featuring infection and harvest dates, is shown to optimize inventory turnover for additional studies. As researchers plan experimental schedules and edit cage inventory when studies lengthen or conclude, a real-time inventory count changes to ensure adequate space before exceeding capacity. Researchers are able to edit the database themselves, further augmenting efficiency, but the key to success lies within them in maintaining an updated and current inventory. By implementing this database, we have been able to accommodate the higher volume of overlapping animal studies all while operating continuously at full capacity, thereby promoting maximum experimental output.

P212 Solutions to Minimize Urine Spraying in Single-housed Male New Zealand White Rabbits (Oryctolagus cuniculus)

LA Burlingame*^1,2, A Morgan¹, SE Thurston^1,2, T Martin^1,2

¹Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI; ²Refinement and Enrichment Advancements Laboratory, Ann Arbor, MI

Wild rabbits are strongly territorial and often react aggressively to strangers invading their home range. Scent marking is one way rabbits mark territory. Within our colony of New Zealand White rabbits (Oryctolagus cuniculus), we observed urine spraying in singly housed males. Urine spraying can be associated with health conditions such as fur matting, dermatitis, and urine scalding, but little information is available on prevention of this behavior. We hypothesized that rabbits engaged in urine spraying as a territorial marking response related to the nearby presence of other dominant males and that alternating neighboring rabbits would change the social hierarchy. We identified 20 single-housed intact male rabbits with a history of varying degrees of urine spraying due to nonclinical issues and used them to develop a Urine Spraying Scale. Interventions occurred in 3 phases. Phase 1: Increased enrichment when initial urine spraying was noted. Phase 2: Relocated the rabbits to a new cage, directly next to a rabbit without a history of urine spraying. Phase 3: Moved the rabbit to a single freestanding rack, which eliminated any direct neighbor. Results showed that of 17 affected rabbits (3 lost to attrition), 13 (76%) showed elimination and 2 showed reduction of spraying following movement away from initial neighboring rabbits. This data supports our hypothesis that urine spraying in male rabbits is a territorial marking behavior, perhaps meant to reinforce dominance hierarchies. When rabbits were placed next to a new neighbor, the majority (88%) stopped or reduced urine spraying. For rabbits that continued to spray, we trialed novel scented enrichment to assess whether this could reduce spraying. Preliminary data showed transient reduction, but was not consistent or statistically significant. Additional studies are warranted to examine the underlying cause of and mitigation methods for urine spraying in the minority of animals that continue to spray.

P213 Ultrasonic Vocalization Detection in a Social Housing Paradigm Comparison

M Deselem*², C Maples¹, J Manro³, ID Bolton¹

¹Veterinary Resources, Eli Lilly and Company, Greenfield, IN; ²Medicine Innovation Hub, Eli Lilly and Company, Indianapolis, IN; ³Discovery/Development Statistics, Eli Lilly and Company, Indianapolis, IN

Ultrasonic vocalizations (USVs) have been determined to be effective in measuring contextual positive and negative situations in rats. Additionally, these vocalizations and alarm cries are attributed to pro-social behavior. This work compared responses of single and pair-housed rats subjected to standard laboratory procedures by observing 22 kHz USVs, a frequency associated with negative contexts, using a heterodyne bat detector and behaviors including human-audible (audible) vocalization, kicking, and tongue flicks. USVs and behavioral indicators from 14 single-housed and 14 pair-housed male Sprague Dawley rats were collected over an 11-d period during restraint and oral gavage procedures. Week 1 consisted of restraint procedures only and wk 2 included both restraint and oral gavage procedures. Restraint procedures consisted of hand restraint for a 5-s duration. Oral gavage was performed using the same hand restraint method but incorporated a 200ul tap water gavage using an 18 gauge 3” gavage needle with a 2 mm ball-tip was inserted into the esophagus of the animal. Week 1 results revealed that restraint procedures elicited more USVs from the pair-housed group than the single-housed group. Week 2 results demonstrated similar patterns to those of wk 1 with pair-housed animals vocalizing ultrasonically more than the single-housed group when subjected to restraint and oral gavage procedures. Additionally, single-housed rats vocalized in the audible range more often than the pair-housed group during both weeks. Statistical analysis showed that USV emission, audible vocalizations, and tongue flicks were correlated with housing condition. Contrastingly, kicking behavior was found to be correlated with handling as opposed to housing. Thus, pair-housed rats demonstrate pro-social behavior in the form of USVs more often than single-housed rats confirming that housing conditions can alter certain pro-social behaviors in rats.

P214 Converting an Indoor Rodent Room into an Indoor Free-range Chicken Room

M Saenz*, D Saldana, T Armenta, NN Lee, R Ermel

Center for Comparative Medicine, City of Hope, Duarte, CA

To ensure optimal animal health and well-being, species-specific differences in physical housing conditions need to be established. With no established guides, here we describe the creation of a free-range chicken environment in an indoor setting. Macroenvironment components that were considered include air quality, ventilation, lighting levels, ability for social interactions, and the acclimation of the hens to an indoor free-range environment. However, our facilities were designed to house rodents; thus, we implemented the following changes to convert a rodent room into a free-range chicken room. The ventilation system was adjusted to handle the increased dust in the environment. The floor was prepared using surface protection tape with artificial grass and bed shavings. To prevent inadvertent transfer of bedding and waste materials from the room entrance to the outside door area, a baby gate with 4-inch-wide strips at the bottom were installed creating a buffer zone at the room entrance. The light cycle was switched from 12:12 (light:dark) to 16:8 and the light intensity was adjusted to 5-10 lux. For husbandry components, elevated feed and water sources, and crushed oyster shells were provided. A 2-tier galvanized metal nesting hutch (10-spots) containing nesting pads and artificial eggs were provided to encourage egg-laying in the hutch. For enrichments, dust bath (80% sand, 20% diatomaceous earth), toys (plastic toy balls, xylophones, and bells), wall mirrors, and two perches made using cage wash catching racks with clear plastic carpet running on the bottom shelves and 1 ¼” dowels placed at three different levels were provided. Approximately 1 mo after being housed in the converted indoor free-range chicken room, the hens began using all 3 levels of the perches, laying eggs in the nest boxes and appeared to have acclimatized. Converting a rodent-housing room into a free-ranging chicken room can be accomplished using tools and supplies from a local hardware store, online poultry vendor and equipment that is available to a rodent research facility.

P215 Comparison of Corn Cob and Refined Cellulose Product as Bedding Material for Mice

M Archer*¹, T Green¹, K Carter², J Plunkard¹, JS Villano¹

¹Research Animal Resources, Johns Hopkins University, Havre de Grace, MD; ²Johns Hopkins University, Baltimore, MD

The laboratory animal microenvironment is integral to ensure integrity and reproducibility of research studies and animal welfare. Bedding materials for rodents come in various forms, with corn cob being the most commonly used because it is readily available and economical. However, its disadvantages include poor absorbency and hardness. Here, we compared a new bedding material, refined virgin diced cellulose product (P) to corn cob (C). BALB/cAnNCrl mice (n = 2 cages of males and 3 cages of females/bedding type; 5 mice/cage) were housed in cages with ¼” deep of either bedding. At d 1, 3, 7, 9, and 14 of a 14-dc age change cycle, cage gas levels (ammonia, oxygen, and carbon dioxide) were measured. Additionally, 2 husbandry personnel assessed cage dirtiness using a novel scoring system of a scale of 0 (clean) to 3 (severely soiled with fecal pellets and food crumbs and with > ¼ floor space with urine spots). Three trials were conducted, alternating bedding type used for each cage per trial. Results revealed that at every time point for both bedding type, O₂ and CO₂ had an inverse correlation, and cages were consistently hypoxic with mean O₂ levels over time of (C) 20.21% ± 0.28 and (P) 20.31% ± 0.14, which were below normal room oxygen concentration of 21%. At all time points, CO₂ levels (mean over time of 0.53 ± 0.24) for corn cob bedding exceeded the OSHA recommendations of 0.5%. In contrast, mean CO₂ levels for paper bedding was 0.45 ± 0.14, with only the d14 levels exceeding the 0.5%. Interestingly, the mean NH₃ levels (P: 3.4 ± 12.23; C: 4.57 ± 12.9 ppm) remained below the NIOSH recommended exposure limit for humans (25 ppm) at all time points. All gas values were not statistically significant between bedding type. Not surprisingly, cage dirtiness mean scores were not significantly different between bedding types and started at 0 on d1 and ended at (C) 2.1 ± 0.67 and (P) 1.97 ± 0.56 on d14. Inter-rater reliability was strong, except for d3 and d14 for both bedding type. In conclusion, the cellulose bedding provides a viable alternative to corn cob. Additional studies are warranted to investigate other variables such as effects on animal health, reproduction, and research.

P216 Monitoring Air Flow in Mouse Cage Pressurized Individually Ventilated Racks

JA Gustafson, M Foskett*, E Rossow

The Jackson Laboratory, Bar Harbor, ME

It is easy to adopt a “set it and forget it” approach to air flow monitoring in pressurized individually ventilated (PIV) mouse cage racks. However, this may not account for inconsistencies due to drift in air flow over time. So, we implemented a program of monitoring air flow in the PIV racks as part of our annual equipment certification process for our mouse rooms. For the annual monitoring program, we took air flow readings using 2 different air flow meters. One of the air flow meters monitors air flow velocity in liters per minute (LPM) and the other monitors air pressure in inches of water. We also developed a standardized approach to measure air flow in consistent, representative sections of the rack, to better understand variability within a rack and adjusted air flow at a rack or room level, to assure optimal conditions were achieved. After completing at least 2 y of monitoring for 40 different standard, high, and maximum-barrier mouse rooms containing PIV racks, we have learned that there can be significant drift in air flow rates in mouse cage racks from year to year which cannot be detected at the level of the building automation system. Results of the monitoring program indicate that as many as 30% of rooms require readjustment of air flow at either a room or rack level on an annual basis. Age of the HVAC system, ability to manually adjust air flow at the rack level, and total number of cage racks in a room were variables found to affect stability of air flow within a room. Based on our study we recommend annual verification of air flow rates. We recommend using calibrated equipment and found that measurements using one of the air flow meters to be the most informative for making adjustments to flow rates as well as being easiest to use. Additionally, we recommend re-zeroing the magnahelic gauges for supply and exhaust at least annually since these gauges can “freeze” and give inaccurate readings as the instrument ages.

P217 Implementing and Evaluating Enrichment for Short-term Housed Farm Animals

P Gordnier*¹, R Anderson¹, C May¹, S McMillan¹, MT Young Lipschutz²

¹Preclinical, Kelly Professional & Industrial for Ethicon Endo Surgery, Cincinnati, OH; ²Preclinical, Ethicon Endo Surgery, Cincinnati, OH

Enrichment is an essential element of superior animal care and welfare. Limited information is available for building a robust enrichment program for short-term (typically between 7-10 d) housed farm animals. Each laboratory is unique; therefore, each enrichment program must be customized. It is prudent that enrichment methods focus on triggering natural, species-specific behaviors and promoting positive human interaction. The enrichment program should allow for efficient evaluation and flexibility. At our facility, animals are raised in a significantly different environment than most laboratory settings, so development of an enrichment program that supports this transition is critical. An evaluation form, which included sections on desired behaviors, observed behaviors, length of interaction, and novelty of the item, was created to allow technicians to assess how animals interacted with novel enrichment. Novel enrichment items included fresh fruits and vegetables, a water tub, foraging boards, manipulable items attached to the pen walls, and grooming tools. Varying enrichment items were offered multiple times a day, encouraging activity of the animals and allowing for direct observation of their behaviors. Based on multiple evaluations, technicians were able to determine if previous enrichment methods were successful or if new methods were required. Technicians worked within standard operating procedures and research protocols to customize enrichment to each study and species with the aid of the evaluation forms. The existing enrichment program was expanded, with the addition of new toys and edible enrichment, resulting in an observable increase in natural behavior expression. Technicians were more engaged with the animals throughout the day and noticed a smoother acclimation to the laboratory environment. Catering an enrichment program for farm animals used in acute research is engaging and rewarding to both animals and their caregivers. Animal welfare is placed further in the foreground of the staff’s mind in an ever-changing environment, empowering caregivers and promoting creativity. The new program resulted in an increase in variety of enrichment methods, frequency of presentation, and evaluation of the animals’ behavior.

P218 An Updated Digital Approach to Regulatory Recordkeeping

S Lane*

Comparative Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK

The management and upkeep of an animal facility can be a tiresome process. When taking into consideration the regulatory oversight needed for an animal facility and the potential consequences of not providing the required level of care to an animal, that task becomes more burdensome. With these requirements comes ample recordkeeping. Many facilities use paper records for tracking the duty assignments of animal care staff. This often leads to a tremendous amount of paper accumulation and space required to store it for the years it is required. In order to streamline this task and reduce the paper accumulation, we have implemented a digital recordkeeping tool to ease the burden on the managers and staff. This software can be structured to suit the facility it is used in and can be modified to suit the needs of the staff using it. This cloud-based software has allowed us to remove 90% of the paper records that we have used for decades and has reinvigorated staff to complete all tasks as assigned. It has provided a ‘buy-in’ for the staff as they are actively involved in the fine-tuning of the program and are able to see in real-time the completion of their daily work. This has noticeable benefits to those people who perform repetitive tasks. The platform has been incorporated into 2 facilities at our institution and we are actively engaged in getting it in place at all on-site facilities with hopes of completion by 2022. After just 6 mo of full use, it has increased efficiency of staff, improved the overall quality of work, and ensured a stable and consistent level of care for the animals they supervise. This has not only improved the lives of the users, but also those responsible for recordkeeping, review of duties, and regulatory oversight.

P219 Positive Behavioral Impact of Environmental Enrichment on Red-eared Slider Turtles Trachemys elegans

M Mayo^2,1, S Lane*¹, W Williams¹

¹Comparative Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK; ²Oklahoma School for Science and Math, Oklahoma CIty, OK

There is little research in the topic of reptile well-being, specifically in relation to animal husbandry. In those studies that have occurred, providing enrichment that replicates the reptile’s habitat is shown to increase well-being, measured through the amount of positive species-specific behaviors. Environmental enrichment has become a staple in the husbandry and management of the more common laboratory species including mice, rats, dogs, cats, and nonhuman primates. Well-defined enrichment plans can be found outlined at research institutions for these species. No such enrichment design was set in place for the housing of turtles and the implementation was largely left to comparative medicine staff and researchers. With that in mind, this project aimed to outline the impact that a defined environmental enrichment plan would have on the behaviors and well-being of red-eared slider turtles (Trachemys elegans). In order to implement a well-designed strategy, proof of positive impact was required. In order to test the impact of environmental enrichment, we compared defined positive and negative behaviors of a control group over a set period to those of the same group following addition of environmental enrichment. During the control and experimental phase of this experiment, three, 1-h recordings of the turtles were taken over a period of 10 d, and their behaviors were recorded and analyzed. The experimental group showed significant increases in positive behaviors as well as significant decreases in negative behaviors. This study supports the use of environmental enrichment for red-eared sliders to improve their well-being, which is shown through the significant changes in species-specific behaviors.

P220 Improving Nonhuman Primate Food Enrichment Management to Reduce Waste and Costs

T Xiong*², LC Impelluso¹

¹Research Animal Resources, University of Minnesota, Minneapolis, MN; ²Research Animal Resources, University of Minnesota, Minneapolis, MN

Nonhuman primates (NHP) in research institutions require detailed food husbandry procedures to maintain their welfare. Efficient organization and management of the program is important to ensure there is limited wastage. Over the last several years, we observed an increase in NHP biscuit wastage believed to be associated with a lack of proper management of food enrichment (FE). In order to improve the FE management so we can reduce wastage and costs, we created some new approaches to our food husbandry procedures. One approach was to designate a single employee to perform all FE ordering and tracking versus the previous decentralized process. FE ordering, spending, and biscuit wastage was recorded and analyzed on a regular basis to compare. Another approach was to emphasize the purchase of seasonal produce with lower costs and higher quality. More frequent, smaller FE purchases were instituted to decrease spoilage instead of 2-wk intervals. We also changed our feeding procedures by feeding biscuits twice a day vs once a day. Lastly, we created new ideas and guides on how to conserve FE that would spoil more quickly. For example, overripe bananas were frozen to turn into novel enrichment like smoothies. During a 5-mo trial period with the updated management processes, we saw approximately a 70% decrease in NHP biscuit waste. We also decreased our FE expenditures by approximately 52%. Researchers who use NHPs for behavioral studies anecdotally reported improved performance on behavioral tasks. In conclusion, review and reassessment of NHP FE practices and procedures has led to dramatic improvements in the institution’s enrichment program.

P221 Ergonomic Rodent Survival Surgical Suite

WS Johnson*, A deCastro, M Donnelly

Veterinary Sciences, Bristol Myers Squibb, Lawrenceville, NJ

The purpose of the renovation was to design the suite so it could handle the significant increase of rodent surgeons. Instead of sticking with basic industry standards in design and equipment, the team saw this as an opportunity to become innovative and develop a state-of-the-art surgical suite. The team did a significant amount of research for this project by identifying best practices from colleagues at other institutions, attending rodent surgical wet labs, and working with vendors to learn about the latest and greatest equipment on the market. This research led us to focus on ergonomics, efficiency for high throughput, and animal welfare. An ergonomic rodent survival surgical suite is imperative for a surgeon’s comfort and the ability to perform high-quality surgical procedures. Surgeons can experience certain challenges during rodent surgery, such as performing microsurgery on multiple animals for multiple days in a row. This can cause musculoskeletal fatigue and injuries. We used an ergonomic approach for our renovated rodent survival surgery suite to maximize the efficiency and safety for our surgeons and the surgical procedures they are performing. In addition to ergonomics, we focused on identifying industry best practices and equipment to allow for efficient surgical procedures and the best of care for the surgical patient throughout the process. We report the renovations, specialized equipment, and how these changes have created an ergonomic environment resulting in increased efficiency and productivity.

P222 The Chinchilla Consortium and Its Role in Improvement of Standard Practices for the Laboratory Chinchilla

M Williams¹³, R Beiler², DJ Coble³, A Darbyshire⁴, GL Dobek⁵, R Esquivel⁶, M Fink⁷, DL Fong⁷, L Habenicht⁷, G Jackson⁸, S Lewis⁹, RA Malbrue³, EL Mitchell¹⁰, M Ortega¹¹, A Osborne¹², C Thurman², W Williams*¹, C Manuel⁷

¹Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK; ²Boston University, Boston, MA; ³Nationwide Childrens Hospital, Columbus, OH; ⁴Purdue University, West Lafayette, IN; ⁵Tulane University, New Orleans, LA; ⁶Boston Children’s Hospital, Boston, MA; ⁷University of Colorado AMC, Aurora, CO; ⁸Cornell University, Ithaca, NY; ⁹University of Texas SMC, Dallas, TX; ¹⁰Wake Forest University, Winston-Salem, NC; ¹¹Massachusettes Eye and Ear, Boston, MA; ¹²University of Alabama, Birmingham, AL; ¹³CBSET, Boston, MA

Sourcing laboratory animals, particularly less commonly used species, can be a challenge. High-quality lab animal research requires that animals are in excellent health; ideally free of pathogens, or at least of known status for pathogens that could impact animal health and study outcomes. Chinchillas (Chinchilla lanigera, C. chinchilla) are a niche species used almost exclusively for otologic research (otitis media/interna, deafness, tinnitus, toxicologic testing of novel compounds, etc.). A combination of relatively low demand for laboratory chinchillas, long reproductive cycles, and low fecundity compared to other lab animals has resulted in difficulty obtaining appropriate animals for research needs. Among available chinchilla vendors, problems include lack of USDA licensure, inability to satisfy cohort numbers and timelines, and levels of health monitoring, veterinary care, and husbandry conditions that do not meet standards expected by the laboratory animal science community. Recently, several institutions have formed a chinchilla consortium to assess options for sourcing research-appropriate chinchillas. We have met virtually to discuss purchasing, husbandry, and health monitoring practices. In doing so, we discovered many of our import and husbandry practices varied considerably. To promote best practices in laboratory chinchilla management, we compiled information on import testing, prophylactic care and quarantine, as well as common health and zoonosis concerns in laboratory chinchillas. Our goal is to provide the lab animal community with practical information regarding responsible acquisition and high standards of care and welfare for laboratory chinchillas.

P223 Sentinel Swabs: An Alternative to Sentinel Animals in Rodent Colony Health Monitoring

A Leon*, G Miner, J Lankasky, R Walters, C Zao, R Berger, D Pitts

VRL, Gaithersburg, MD

To date, the number of pathogens tested using sentinel swabs as an alternative to sentinel animals has been limited to a couple of pathogens. We have expanded the number of pathogens to include an exhaustive list of the most prevalent agents that infect mice and have shown that the swab methodology is a feasible alternative. Reduction of sentinel animals in rodent colony health monitoring has been one of the driving forces in the 3Rs and animal welfare initiatives. Different approaches for the reduction of sentinel animals are in practice or have been proposed, among them rack plenum filters, direct sampling from animals, and increases to the intervals between tests. In addition to these alternatives, incubating other types of capture media with the soiled bedding has been proposed. To determine if the use of swabs could be used as an alternative to sentinel animals and capture media, we tested swabs exposed to soiled bedding. The soiled bedding was collected from 4 cages containing animals positive for several infectious agents, including prevalent viruses, bacteria, and parasites. Swabs were incubated for up to 3 mo with the soiled bedding and tested by PCR for the presence of several agents. For comparison, fecal samples from the infected animals were tested by PCR. The group of pathogens tested consisted of 6 viruses, 4 bacteria, and 3 parasites. Notable in this group included MPV, MNV, Helicobacter, Rodentibacter pneumotropica, and pinworms. This data supports the use of soiled exposed swabs for the monitoring of rodent colony health. In conclusion, swabs detect the presence of infectious agents effectively and are a feasible alternative to sentinel animals and fecal pellet pools.

Laboratory Investigations Posters

P300 Plasma and Joint Tissue Pharmacokinetics of Oral Cannabidiol in a Guinea Pig Model of Osteoarthritis

AP Spittler*¹, J Helbling¹, S McGrath², D Gustafson², KS Santangelo¹, M Sadar²

¹Microbiology, Immunology, and Pathology, Colorado State University, Erie, CO; ²Clinical Sciences, Colorado State University, Fort Collins, CO

Cannabidiol (CBD) has gained widespread attention due to its anti-inflammatory effects as a treatment for osteoarthritis (OA). However, there is minimal scientific evidence regarding dosing for OA and whether CBD can be detected in joint tissues. The aim of this study was to determine plasma and joint tissue pharmacokinetics after oral CBD administration in Dunkin Hartley guinea pigs, which spontaneously develop OA at 3 mo of age. Ten 5-mo-old male guinea pigs were randomly assigned to receive 25 (n = 5) or 50 mg/kg (n = 5) CBD oil once orally. Blood samples were collected at 0, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h timepoints. To assess potential side effects on activity levels from CBD administration, open-field enclosure monitoring was performed before and after treatment. After euthanasia, knee cartilage and infrapatellar fat pads were collected to quantitate CBD. CBD concentrations were determined using a validated liquid chromatography-mass spectrometry method, and pharmacokinetic parameters were calculated using noncompartmental analysis. The area under the plasma concentration-versus-time curve was 379.5 and 873.7 hr*ng/mL, maximum plasma concentration was 42 and 96.8 ng/mL, time to maximum plasma concentration was 1.6 and 4.8 h, and terminal half-life was 8.1 and 10.8 h for the 25 and 50 mg/kg doses, respectively. CBD was detected in joint tissues of all animals, suggesting its potential to directly exert effects within the joint. Mobility parameters did not differ between baseline and post-CBD timepoints. These findings may be utilized for future studies to determine the efficacy of CBD for OA.

P301 Assessing Microchip Locations for Mouse Temperature Data Collection

A Hernandez*, T Sutterfield, L Long, B Lyons, M Campagna

Jackson Laboratory, Sacramento, CA

RFID microchips are a convenient and reliable mechanism for collecting experimental data, while minimizing the amount of stress experienced by research animals. Temperature programmable microchips are capable of providing both identification and noninvasive temperature monitoring. This is particularly valuable in studies of inflammatory responses or infectious disease. This study was designed to determine if the location of implanted microchips has a significant effect on temperature readings in mice. We hypothesized that temperature readings would vary depending on location. C57BL/6J mice (5 male and female per method) were placed under isoflurane anesthesia and were implanted percutaneously in the following locations: intrascapular, inguinal, flank, intraperitoneal, or dual intrascapular and intraperitoneal. Body weights were obtained at baseline and at 2 wk, 4 wk, and 4 mo post-implantation. Mice were assessed for microchip retention and temperature values daily for 1 wk post-implantation, weekly for the following 4 wk, and again at 4 mo post-implantation. Temperature assessments were made either intermittently, using a handheld microchip scanner, or continuously, using a plate reader placed directly under the cage. At the end of the study, mice were euthanized and necropsy was performed to confirm microchip placement and observe for gross pathology. Temperature data analysis was performed using ANOVA and Tukey’s post-hoc test. No adverse effects were noted from any of the groups and weights did not vary significantly between members of the same sex. Average temperature per group ranged from 35.1 to 38.7 °C. Inguinal and flank temperatures trended lower than the other locations in both sexes, with a significant (P < 0.05) difference noted in females. In males, intrascapular temperatures were significantly higher (P < 0.05) than other locations. In conclusion, all locations assessed were considered reliable for collecting temperature data, although consistency of location is likely to be important for ensuring comparable results between studies.

P302 Assessing the Preference for Thermoneutral Temperature or Reduced Light in Laboratory Mice, Mus musculus

HJ Davis, AJ Barabas*, BN Gaskill

Animal Science, Purdue University, West Lafayette, IN

Aspects of the laboratory are aversive to mice, such as being housed under bright lights and at temperatures below their thermal comfort, causing stress and poor welfare. However, it is unknown which of these is more aversive. Since 30-32° C is the preferred temperature range for mice, we hypothesized that mice would prefer a dark (red) environment when the thermal options available are below 30° C, but the opposite will be observed when temperatures exceed 30° C. Seven pairs of 41-wk-old female CD-1 mice were randomly allocated to 2 connected cages (a red-tinted cage and a clear heated cage) for 8d, each containing 8g of nesting material. Every 48 h, the heated cage temperature was changed to 20, 24, 28, or 32° C, in a random order. After an initial 24 h of exposure, nest complexity and the amount of nesting material was weighed in each cage, and inactive behavior was observed using scan sampling. All measures were analyzed with general linear mixed models with temperature as a fixed effect and cage ID as a random effect. Temperature did not significantly alter nest weights (F_1,19.43 = 0.71; P = 0.409). However, it did impact nest scores: more complex nests were built in the red cage when 20-28°C were available (F_1,19.37 = 5.38; P = 0.032). Further, mice spent more time in the red cage when they had access to 20 and 24°C (F_1,20=8.99; P=0.007). These temperatures do not outweigh the preference for lighting conditions; thus, aversion to typical laboratory lighting may be more important than previously assumed.

P303 Comparison of Catheter Tip Style, Placement, Locking Solutions and their Influence on Catheter Patency in Sprague Dawley Rats

AJ Evans*¹, S Gledhill¹, P Sparks¹, TR Gleason¹, V Karicheti²

¹Infusion Toxicology, Charles River, Ashland, OH; ²Surgery, Charles River, Raleigh, NC

Transcutaneous buttons are a proven refinement for intravenous (IV) infusion and blood sampling compared to traditional vascular access ports by reducing pain and distress from repeat needle punctures; however, there are still issues with patency. To further explore patency issues, an 8-wk study on patency was conducted in Sprague Dawley (SD) rats comparing catheter tip styles, placement, and locking solutions with and without anticoagulant. Surgeries were conducted at the animal vendor, and animals were transported to the study site. Twenty animals/sex were surgically implanted with a femoral vein catheter (FVC) with a round-tip placed at the approximate level of the renal veins (FVC-RV); 20 animals/sex received a FVC with the tip placed in the vena cava (FVC-VC) (10/sex with round-tip (RT), 10/sex with blunt-tip (BT)); 20 animals/sex received a jugular vein catheter (JVC) with tip placed close to the atrium (10/sex RT, 10/sex BT). Patency was checked weekly, and catheters were locked with 20 IU/mL heparinized saline except for 10 animals/sex (FVC-RV), which were locked with 0.9% sterile saline. Animals were socially housed; body weights and clinical observations were collected weekly. When comparing catheter tip styles regardless of placement, 79% of animals with RT style catheters maintained bidirectional patency for the duration of the study compared to 35% with BT. Seventy percent of FVC-RV and FVC-VC animals combined remained bidirectionally patent, while JVC animals had a 53% patency rate. Animals with FVC-RV placement were 87% bidirectionally patent compared to 70% of FVC-VC animals. Eighty-five percent of FVC-RV animals locked with saline remained bidirectionally patent compared to 89% of FVC-RV animals locked with heparinized saline. All animals were patent for infusion. FVC-RV with RT style catheters provided the best outcome for blood sampling and IV infusion, indicating tip style and position are important for long-term patency.

P304 Sterility of Commonly Used Cements and Adhesives Used in Rodent Implants

Ä Sipos*¹, MJ Crim², D Eckhoff², JA Scholz¹

¹Department of Comparative Medicin, Mayo Clinic, Rochester, MN; ²Microbiology and Aquatic Diagnostics, IDEXX, Columbia, MO

An important component of aseptic technique in rodent surgery is ensuring that all materials and instruments that enter the sterile field, including implants, are sterile. Surgical placement of cranial implants is commonly performed in rodents, and often a cement or adhesive material is used as a component of the implant. These products may or may not be medical grade and are rarely labeled as sterile. There is a lack of data on these products’ sterility and contamination risk, and most manufacturers do not perform sterility testing. The aim of this project was to determine the sterility of 5 commonly used medical-grade and non-medical grade cement or adhesive products and whether these products are inhibitory to bacterial growth when contaminated. The sterility of the compounds was tested by direct culture and method suitability tests. Several of the adhesives require 2 or more components to be mixed priorbeforeication, and individual components were evaluated separately for these adhesives. For direct culture, samples of previously unopened components were cultured for bacterial and fungal growth in triplicate. For the method suitability test, each component was inoculated with 5 bacterial or fungal species, including Aspergillus brasilensis (formerly Aspergillus niger), Bacillus subtilis, Candida albicans, Pseudomoas aeruginosa, and Staphylococcus aureus, and organism recovery was evaluated. No bacterial or fungal growth was detected by direct inoculation from samples of any cement or adhesive components. The method suitability testing revealed no inhibitory properties of any product on bacterial and fungal organisms’ growth. In conclusion, all cement and adhesive components tested in this study were sterile upon opening but did not inhibit bacterial or fungal growth when contaminated. Therefore, these products can be considered sterile when opening, and appropriate handling is essential to maintain sterility if used for sterile surgery.

P305 Mid-Tibiofibular Amputation as an Alternate Method of Terminal Blood Collection in Xenopus laevis.

BC Dixon*¹, M Culbreth¹, D Kumsher², C Carbaugh², D Fetterer¹, C Reiter¹

¹Veterinary Medicine Division, US Army Medical Research Institute of Infectious Diseases (USAMRIID), Myersville, MD; ²Experimental therapeutics, Walter Reed Army Institute of Research, Bethesda, MD

The African clawed frog, Xenopus laevis, is a widely used model for biomedical research. X. laevis could be more useful as a model with a better method for collection and analysis of its blood and serum. However, blood collection in X. laevis can be challenging due to their small size, lack of peripheral vascular access, and species-specific hematology variables. This study aimed to compare cardiocentesis, the current gold standard terminal blood collection method, with a mid-tibiofibular leg amputation technique using a standard single-edged razor blade. Blood samples were collected from 24 laboratory-reared X. laevis, randomized to either the cardiocentesis or leg amputation method, with 6 males and 6 females in each group. All frogs were deeply anesthetized by immersion in buffered MS222 before blood collection and subsequent euthanasia. Hematology and serum biochemistry were also conducted to identify any lymph contamination in the samples. On average, the leg amputation method produced over 2.7 times more blood by volume and as a percentage of body weight in less than half the time compared to cardiocentesis. No significant differences in clinical pathology parameters were found between blood collection methods. These results indicate that terminal blood collection by leg amputation may be a valuable approach for increasing the utility of an already valuable biomedical research model.

P306 Major Survival Surgery Combined with an Immunosuppressive Agent Induces Body Weight Loss in a Xenogeneic Human Chondrocyte Transplantation in a New Zealand White Rabbit Model

C Wangdee*¹, S Wongin², K Viravaidya-Pasuwat³, K Jampachaisri⁴, PE Sharp^5,6, C Pacharinsak⁷

¹Department of Veterinary Surgery, Chulalongkorn University, Bangkok, Thailand; ²Biological Engineering Program, Faculty of Engineering, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand; ³Department of Chemical Engineering and Biological Engineering Program, Faculty of Engineering, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand; ⁴Department of Mathematics, Naresuan University, Phitsanulok, Thailand; ⁵Murdoch University, School of Veterinary and Life Sciences, Murdoch, Australia; ⁶Office of Research and Economic Development, University of California Merced, Merced, CA; ⁷Department of Comparative Medicine, Stanford University School of Medicine, Stanford, CA

Weight loss after a major survival surgery impacts animal health, welfare, and research outcomes. Although rodent weight loss after a major survival surgery is a common occurrence, limited rabbit information is available. The rabbit has been used as an animal model in human tissue-engineered grafts. Here, we investigated the effects of a major survival surgery combined with cyclosporin A (CyA), an immunosuppressive agent, on rabbit body weight loss after xenogeneic human chondrocyte transplantation. We hypothesized that a bilateral stifle arthrotomy combined with CyA would induce significant weight loss in rabbits. Following anesthesia [ketamine (35 mg/kg)/xylazine (5 mg/kg) with isoflurane in 100% O₂] and aseptic stifle preparation, adult (13-wk-old) female New Zealand White rabbits (n=29) underwent a bilateral stifle arthrotomy (D0) creating an osteochondral defect filled with transplanted human chondrocyte cells at the femoral condyles. Morphine (2 mg/kg, SC, once) and carprofen (2.2 mg/kg, SC, 5 days) were provided postoperatively. To suppress xenogenic rejection after human chondrocyte implantation, CyA (15 mg/kg, SC) was administered to all rabbits daily on D-3 to D7 and every other day thereafter until D28. Body weights (BW) were recorded on D-3 (baseline weights), D0 (arthrotomy), D7, D14, D21, D28, D35, D42, D48, and D56. Mean BW differences (compared to D-3) were calculated. Compared to D-3 values, 1) Mean BW differences on D0 (0.12±0.07 kg) were not significantly different; 2) mean BW differences on D7 (-0.28±0.07 kg), D14 (-0.35±0.07 Kg), D21 (-0.35±0.07 Kg), and D28 (-0.34±0.07 Kg) were significantly lower; 3) mean BW differences thereafter (D35 to D56) were not significantly different. Results indicate CyA alone (for 3 d) does not induce significant body weight loss while a combination of major surgery, bilateral stifle arthrotomy, with CyA induces significant body weight loss in rabbits.

P307 Biodegradation of Different Collagen Membranes in Guided Bone Regeneration: Histomorphometric Study in Rat Calvarium

S Srisuwatanasagul³, S Pisamai¹, K Mai-ngam², O Jaturakan¹, S Charoenla², C Wangdee*¹

¹Department of Veterinary Surgery, Chulalongkorn University, Bangkok, Thailand; ²Assistive Technology and Medical Devices Research Center, National Science and Technology Development Agency, Pathumthani, Thailand; ³Department of Anatomy, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand

Collagen membranes were introduced for guided bone regeneration, especially in dentistry, since they could serve as an occlusive barrier for periodontal tissue regeneration. The ideal collagen membranes should provide the occlusive properties which last until the bone has healed; therefore, the biodegradation rate of collagen membranes is a crucial factor for guided bone regeneration success. In our present study, the newly developed cross-link collagen type I membrane (NC) was compared with the non-crosslink commercial collagen type I and III membrane (CC). Sixteen male Wistar rats aged 12 wk were used, and the calvaria defects with the diameter of 5 mm were made on each side of parietal bone. The defects were replaced with either NC and CC on each side of the rat calvaria, and the histomorphometric data were compared between 2 types of collagen membranes. The evaluation of histomorphometric data, which were the height of residual collagen membrane and the area of collagen fiber by Masson’s Trichrome staining, was done at 4 and 8 wk. The area of collagen fiber, which evaluated by an image analysis program, revealed that the percentage of collagen fiber per 1 mm² was 12.04 + 5.54 and 11.38 + 3.11 at 4 wk and were 17.36 + 4.16 and 15.72 +3.44 at 8 wk for NC and CC respectively. The results showed no significant difference on any parameters between these 2 kinds of collagen membranes. In conclusion, the newly developed collagen membrane (NC) could be comparable to the commercial collagen membrane (CC) regarding the biodegradation rate on the rat calvaria defects.

P308 The Effects of Brucella Infection on Bovine B-cell Function and Antigen Presentation

CR Moley*

Veterinary Pathobiology, University of Missouri, Columbia, MO

Brucella spp. are a diverse group of gram negative, facultative, intracellular bacteria that cause a severe disease process in humans and ruminants known as Brucellosis. Despite infecting an estimated 12 million individuals per year and causing widespread economic losses in livestock, relatively little is known about the pathogenesis of these bacterial species and their unique ability to evade the immune system, causing prolonged infections. Previous studies in our lab have found B cells to be a target of Brucella infection in murine models, and that mice lacking B cells display significantly reduced Brucella loads. Few studies have been published examining the pathogenesis of B. abortus infections in cattle, and while previous studies have demonstrated the pathogen’s ability to bind to bovine B cells, it is still unknown whether or not the pathogen can enter and reproduce within these cells. Here, by extracting blood from various Holstein cows and isolating and infecting the B cells, we demonstrate that B. abortus strains can enter bovine B cells following infection via live imaging by automated microscopy. Furthermore, we have isolated peripheral blood mononuclear cells (PMBC’s) from Holstein blood samples, and infected them with strains of B. abortus for surface antigen analysis via flow cytometry. By comparing various molecules involved in antigen presentation (CD14, CD79, CD80, MHCII), we demonstrated significant differences in the expression of these molecules on infected B cells when compared to uninfected B cells. Namely, a significant (P < 0.05) upregulation in the costimulatory molecule, CD80, was seen on infected cells when compared to the uninfected control. Collectively, these findings indicate that B. abortus can enter and alter the cellular characteristics of bovine B cells.

P309 Assessing the Impact of Soiled Bedding Sentinel Age in Pathogen Transmission

CL Perkins*, G Mulder, KS Henderson

RADS, Charles River Laboratories, Wilmington, MA

To reduce the number of animals used in research, many institutions are considering maintaining soiled bedding sentinels (SBS) for longer than 3 mo. While some reports suggest pathogen detection may not be compromised by this practice, others suggest that there may be some impact of age on susceptibility. We investigated the transmission of pathogens in aged mice versus young mice to determine effectiveness as SBS. Eight female CD-1 (SOPF/VAF-Elite) mice at 3-6 wk (young) and 8 mice at 17-19 wk (aged) were placed as SBS. Baseline testing of mice by PCR using ante mortem sampling confirmed the absence of rodent pathogens. Six cages of conventionally-reared pet shop mice infected/infested with multiple agents, including viruses, bacteria, protozoa, and parasites, were used to provide 15% soiled bedding at biweekly cage changes. One 3-4 wk contact sentinel (CS) was placed in each of 2 cages housing 6-10 wk old pet shop mice to detect active shedding and increase pathogen load to SBS cages. After 3-mo exposure, SBS mice from each cage were submitted for testing by PCR and traditional methods. MFIA interpretation: negative <2.0, 2-3 equivocal, >3 positive. Comparable MNV MFIA scores were obtained for young (20.5) vs aged (20.6) SBS; and PCR copy numbers were 12.2K vs 11.6K, respectively. TMEV MFIA scores were 11.8 vs 13.5, while PCR copy numbers were 3.3K vs 1.5K, respectively. MVM was detected in one of the young SBS by PCR (2 copies) and in 4 of the young SBS by serology (mean MFIA = 25). MVM was not detected in the aged SBS by either PCR or serology. MHV was detected in 1 of the young SBS by serology (MFIA =14) but was not detected by PCR. Adenovirus, K virus, MCMV, and rotavirus were detected by PCR in the pet shop mice upon arrival but were not transmitted to SBS for either age group. Helicobacter was detected by PCR in the aged SBS only (13.3K copies). Pinworms and entamoeba were detected in both SBS age groups by PCR and parasitology. PVM, BHS group B, M. pulmonis, Rodentibacter, Cryptosporidium, Tritrichomonas, and fur mites detected in the CS were not detected in SBS. While showing some slight variation in transmission by agent, there is some suggestive data that supports that aged mice are appropriate for use as soiled bedding sentinels.

P310 Vaginal Inoculation of Uropathogenic E. coli during Estrus Leads to Renal and Genital Tract Colonization in Mice

CK Robinson*, P Saenkham-Huntsinger, B Hanson, S Subashchandrabose

Veterinary Pathobiology, Texas A&M College of Veterinary Medicine, College Station, TX

Urinary tract infection (UTI) is one of the most prevalent bacterial infections, particularly in women, children, and the elderly. Uropathogenic E. coli (UPEC) is the predominant etiological agent of UTI. Current models utilize transurethral route with bacterial instillation in the urinary bladder by a catheter. We assessed whether vaginal inoculation of UPEC led to UTI and how stages of estrous would impact bacterial colonization in 6-8-wk-old female CBA/J mice. Mice in proestrus (N=9), estrus (N=14), metestrus (N=11), and diestrus (N=8) were identified by vaginal cytology. UPEC in PBS (10⁹ CFU/ml) was instilled by lavage of the vaginal tract. Mice were euthanized 1-d post-infection, and bacterial loads in the urinary tract, genital tract, liver, and spleen were enumerated. This endpoint was selected based on previous successful transurethral inoculation. Mice in estrus exhibited the highest and consistent UPEC burden in all organs except the bladder. The vaginal inoculation route did not result in bladder colonization. In contrast, transurethral inoculation of UPEC led to colonization of the bladder and other tissues (N=10). Importantly, inoculation by both routes led to vaginal and uterine colonization and concomitant systemic dissemination to spleen and liver. We then evaluated the kinetics of bacterial colonization over time by vaginal inoculation (N=10/group). 50% of animals exhibited positive UPEC bladder colonization by 7 d but were undetectable by 14 d. Colonization in other organs was comparable over time. We are currently conducting ELISAs, and histopathological evaluation to determine the extent of inflammation triggered by vaginal instillation of UPEC. Our findings suggest that estrogen creates a conducive milieu for UPEC colonization since animals in estrus were heavily colonized. In summary, vaginal inoculation of UPEC in mice during estrus represents a novel model to investigate infection of the upper urinary and genital tracts, as well as systemic dissemination from the urogenital tract.

P311 Efficacy of Alfaxalone versus MS-222 Immersion Anesthesia for Rainbow Trout (Oncorhynchus mykiss)

D Savson*¹, EK Daugherity¹, B Singh¹, S Zenilman², C Smith², R Getchell²

¹Center for Animal Resources and Education, Cornell University, Ithaca, NY; ²Cornell University, Ithaca, NY

Options for anesthesia of sport fish used for research purposes are limited and often require lengthy withdrawal times before release, precluding the release of fish back into the field. Alfaxalone, a synthetic neuroactive steroid, has recently been explored in ornamental fish as an immersion anesthetic alternative, but the safety and efficacy of alfaxalone in sport fish have not been investigated. This study presents the first exploration of alfaxalone as an immersion anesthetic in sport fish. The objective of this study was to compare the physiologic and behavioral effects of alfaxalone versus tricaine methanesulfonate (MS-222) delivered by water immersion for the anesthesia of rainbow trout (Oncorhynchus mykiss). Fish (n=48) were anesthetized via immersion, held for 10 min in a recirculating system with low-dose alfaxalone (A_low; 5mg/L induction, 1mg/L maintenance), high-dose alfaxalone (A_high; 5mg/L induction, 2mg/L maintenance), or MS-222 (MS; 150mg/L induction, 100mg/L maintenance), and opercular rate, response to caudal fin pinch, time-to-induction (TTI), and time-to-recovery (TTR) were recorded. Fish received each of the 3 treatments, separated by a minimum 18-d washout period, in a complete crossover design. We hypothesized 1) that A_low and A_high immersion would provide a stable plane of anesthesia in rainbow trout, with a dose-dependent TTI and TTR; and 2) that the anesthetic profile would be equivalent to that of MS-222. Alfaxalone yielded a significantly longer TTR (p<0.0001) but shorter TTI (p=0.001) than for MS-222, and TTR was significantly shorter for A_low than for A_high (p<0.0001), confirming dose-dependence. All treatments produced reductions in opercular rate and response to stimuli. Our results support hypothesis #1 and produced clinically insignificant differences in induction and recovery lengths between anesthetic agents. We conclude that alfaxalone is a viable alternative to MS-222 for the anesthesia of rainbow trout.

P312 Effect of a Gelatinous Protein Mixture on Growth Kinetics of Patient-derived Xenograft (PDX) Tumors in NSG Mice

D VanBuskirk*, K Pineda, EL Jocoy, M Cheng, J Yang

Product Development, The Jackson Laboratory, Citrus Heights, CA

Patient-derived xenograft (PDX) tumor models have emerged as useful tools for oncology research as the characteristics observed in patients’ tumors are maintained. Moreover, these models accurately predict drug efficacy responses. However, long latency periods, poor tumor take rates, and the need for overage mice to meet the proper number of mice for drug studies limit the ability of researchers to use PDX models. A commercially available gelatinous protein (GP) mixture is commonly used to provide a basement membrane structure to support engrafted tumors. In this study, growth-factor reduced GP was used to determine if growth kinetics are altered for two PDX models (J000106532 and TM01510) in 7-wk-old female NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG^TM) mice. Extracted tumors were processed as fragments (a chunk of 5-10 mm³ tumor) or minced (tumor physically minced with a razor blade) for engraftment. Our laboratory uses fragment tumors to establish PDX models, while minced tumors are preferred for drug efficacy studies to allow a large cohort to enroll in a study within a narrow timeframe. The prepared tumors were subcutaneously engrafted with or without GP (n = 9-10/group), and tumor volumes were measured weekly once tumors became measurable (~50mm³). Tumor fragments were coated with GP (25µl), while minced tumors were mixed with GP (20% or 50% total volume). For both PDX models with minced tumors, the first measurable tumor developed earlier with GP (14d for J000106532; 7d for TM001510). Time for at least 50% of mice to reach enrollment criteria (tumor volume 75-150mm³) was also shorter with GP; J000106532 minced took 74 d while minced+50% GP took 46 d, and TM001510 minced took 57 d while minced 50% GP took 29 d. Minced+GP had a larger average tumor volume compared to minced when the first mouse reached tumor volume endpoint criterion (>2000mm³) for both PDX models; J000106532: 109.7mm³, 696.1mm³, and 321.6mm³; and TM001510: 534.7mm³, 959.1mm³, and 1341.5mm³, for minced, 20% GP, and 50% GP, respectively. These data suggest that studies using minced tumor+GP will be more efficient by increasing the number of mice reaching enrollment criteria sooner and decreasing the total number of enrollments required per study. However, the effect of GP on tumor fragments varied depending on the PDX model; GP enhanced tumor growth kinetics for J000106532 but not for TM01510. Further research with additional PDX models is warranted to determine the effect of GP on tumor fragments. In summary, GP facilitates minced tumor growth, and this technique may allow researchers to initiate PDX studies faster and use fewer mice.

P313 Effect of Tail RFID Microchips on Growth Kinetics and Immune Response in Preclinical Oncology Mouse Models

D Germain*², S Barnes², D Draper², S Urs², H Nielsen², S Wise², SE Clark^1,2

¹Animal Welfare and Veterinary Services, Covance, Ann Arbor, MI; ²Covance, Ann Arbor, MI

Mice are the most common laboratory animal for preclinical oncology studies, and because they are socially housed, a simple and efficient method for identifying individuals is required. Various types of radiofrequency identification (RFID) microchips are available for animal identification. There was a concern that implant of foreign objects, such as microchips, could affect an animal’s immune response to immune-modulating compounds or immuno-oncology tumor models. Many microchips also require a large gauge needle for insertion in the subcutaneous space, which could interfere locally with a subcutaneous tumor model. For this project, 67 female BALB/c mice were implanted with RFID tail microchips, and 66 were not. Microchip implant occurred 2-4 d before subcutaneous implant of CT26.WT murine colon carcinoma tumors. Eight days post-implant, mice were sorted into groups for efficacy analysis (increase in time on study, incidence of regression, tumor doubling time) or sample collection based on tumor volume. Pro-inflammatory cytokine levels were measured through blood collections 1 d prior and 1, 7, and 14 d post microchip implant. Levels of several cytokines were minimally elevated in naïve mice with RFID microchips compared to those without the microchips 24 h post microchip implant; however, differences were no longer seen by 7 d post-implant. Immunohistochemistry of mouse tail sections (base of tail at site of RFID chip implant) also revealed no meaningful immune infiltration at the site of RFID microchip implantation. RFID microchips were shown to have minimal impact on baseline tumor growth kinetics and response to anti-mPD-1 in study mice. Flow cytometry analysis of tumors, spleen, and blood also revealed little to no difference in myeloid or lymphoid cell populations between microchipped and control mice. The use of the tail RFID microchips for identification does not appear to have a biological impact on the immune system of mice used for preclinical oncology research studies and is a technique refinement in RFID microchip systems.

P314 Extended-Release Buprenorphine, an FDA-indexed Analgesic, Attenuates Mechanical Hypersensitivity in Rats

ED Alamaw*¹, B Franco¹, K Jampachaisri², MK Huss¹, C Pacharinsak¹

¹Comparative Medicine, Stanford University, Stanford, CA; ²Department of Mathematics, Naresuan University, Phitsanulok, Thailand

A new extended-release buprenorphine (XR), an FDA-indexed analgesic, was recently introduced to the laboratory animal community. The efficacy and dosing of XR for rats have not been extensively evaluated. This study aimed to investigate XR’s efficacy in attenuating postoperative hypersensitivity in rat incisional pain models. We hypothesized that postoperative hypersensitivity attenuation achieved with XR (a low or high dose) would be similar to that of sustained-release buprenorphine in this model. Male adult Sprague-Dawley rats (n = 32) were randomly assigned to 1 of the 4 treatment groups: 1) Saline (Saline, 0.9% NaCl, 5 mL/kg, SC; once); 2) sustained-release buprenorphine (Bup-SR; 1.2 mg/kg, SC, once); 3) low dose extended-release buprenorphine (XR-Lo; 0.65 mg/kg, SC, once); 4) high dose extended-release buprenorphine (XR-Hi; 1.3 mg/kg, SC, once). A 1 cm longitudinal skin incision was made on the plantar hind paw 5 min after drug administration. Mechanical and thermal hypersensitivity was evaluated 1 d before surgery (D-1), 4 h after surgery (D0), and 3 d after surgery (D1, D2, and D3). The plasma buprenorphine concentration (n = 39) was measured from D0-D3. Clinical observations were recorded daily, and a gross necropsy was performed on D3. Mechanical (D0-D2) and thermal (D0-D3) hypersensitivity were observed in the Saline group. Bup-SR, XR-Lo, and XR-Hi effectively attenuated mechanical hypersensitivity for D0-D2. Plasma buprenorphine concentrations remained above the efficacious concentration of 1 ng/ml on D0 and D1 in all treatment groups. There were no abnormal clinical signs; however, injection site reactions were evident on D3 in the Bup-SR (71%), XR-Lo (75%), and XR-Hi (87%) groups. This study indicates that postoperative hypersensitivity attenuation with a low or high dose of XR is similar to Bup-SR. XR 0.65 mg/kg is recommended to attenuate postoperative mechanical hypersensitivity for up to 48 h in rats in an incisional pain model.

P315 Long-lasting, Highly Concentrated Buprenorphine Solution Provides Prolonged Therapeutic Blood Levels and Prolonged Mechanical Pain Responses In Rats

E Houston*¹, SM Tan¹, S Thomas¹, M Burton¹, H Knych², LV Kendall¹

¹Department of Laboratory Animal Resources, Colorado State University, Fort Collins, CO; ²Department of Veterinary Molecular Biosciences and K.L. Maddy Analytical Chemistry Laboratory, School or Veterinary Medicine, University of California, Davis, CA

A long-lasting, highly concentrated veterinary formulation of buprenorphine has been used to provide prolonged analgesia in cats, nonhuman primates, and mice. The use of a long-lasting formulation could prove beneficial in the management of pain in rats, which typically require dosing every 4-6 h to maintain adequate analgesia. LHC-Bup was evaluated in this study to determine if this formulation would provide similar prolonged analgesia in Sprague-Dawley rats. The pharmacokinetics were assessed after subcutaneous injection of 0.5 mg/kg LHC-Bup in male and female rats. Blood was collected via jugular venipuncture into a heparinized tube, and plasma levels were evaluated at 0.25, 0.5, 1, 2, 4, 8, 12, 24, 36, 48, and 72 h. Both male and female rats had a peak plasma level of Bup at 15 min after administration with a steady decrease by 24 h to 0.72 ± 0.3 ng/ml and 1.35 ± 0.51 ng/ml, in males and females, respectively. Administration of LHC-Bup maintained plasma levels of Bup above the purported therapeutic level of 1 ng/mL for over 12 h. The efficacy was assessed using a Randall-Selitto analgesiometer to measure mechanical pain tolerance after LHC-Bup administration. Male rats had an increased paw withdraw at 1 (p < 0.01), 3 (p < 0.01), 6 (p=0.02), and 12 (p=0.09) hours post-administration, tolerating a maximum pressure of 817.81 ± 215.48 g during the testing period, compared to the saline control that never tolerated more than 228.46 ± 114.96 g. Females had an increased paw withdraw at 1 (p < 0.01), 3 (p < 0.01), 6 (p < 0.01), 12 (p=0.08) and 24 (p= 0.04) hours post-administration, reaching a maximum threshold of 898.15 ± 204.50 g compared to 255.53 ± 116.40 g in the saline control. Based on these findings, LHC-Bup is a suitable formulation for the treatment of pain in rats at a dose of 0.5 mg/kg subcutaneously every 12 h in males and every 24 h in females.

P316 Ability of the Colony Stimulating Factor 1 Receptor (csf1r)-inhibitor, Plx5622 to Ablate Resident Retinal Microglia in Sprague Dawley Rats

E Kaalberg*, L Wiley, M Lang, R Mullins, I Han, B Tucker

Ophthalmology , University of Iowa Institute for Vision Research, Iowa City, IA

Retinal microglia participate in the pruning and maintenance of retinal neurons during development but are also active participants in phagocytosis of photoreceptors in retinal degenerative diseases. However, their exact contribution to photoreceptor degeneration remains unclear. We developed 2 novel rat models of inherited retinal disease, Pde6b^-/- and Mak^-/-, via CRISPR-Cas9-meditated genome editing to characterize the role of retinal microglia and test whether depletion of these resident immune cells is feasible in the rat retina. Photoreceptor loss in Pde6b^-/- and Mak^-/- rat lines were characterized using standard immunohistochemistry against common retinal proteins and activated microglia. To deplete microglia, we treated 2-mo-old wildtype Sprague-Dawley rats (3 males and 3 females) with rat chow supplemented with the compound, PLX5622, a specific inhibitor of colony stimulating factor 1 receptor (CSF1R). Rats were either fed normal chow, chow containing 1200 parts per million (ppm) PLX5622, or 2400 ppm PLX5622 for a duration of 2 wk. Microglial depletion was assessed using anti-Iba1. Each rat model displays significant photoreceptor loss and thinning of the outer nuclear layer (ONL). Pde6b^-/- and Mak^-/- rat lines each also displays translocation of Iba1- and CD68-positive activated microglia to the ONL at the site of photoreceptor degeneration. Compared to wildtype rats (1 male and 1 female) maintained on a normal, PLX5622-free diet, wildtype rats fed PLX5622-supplemented chow have a drastic decrease in resident retinal microglia. Moreover, 2400ppm PLX5622-chow results in more complete depletion of retinal microglia compared to the standard 1200ppm dose. Importantly, PLX5622-mediated depletion of retinal microglia was observed in male and female rats and neither dose resulted in any obvious thinning of the outer nuclear later (i.e., photoreceptor loss) or retinal damage. Although previously reported in mice and in rat brain, to our knowledge this is the first study demonstrating depletion of microglia in rat retina. Future aims will include using PLX5622 to deplete microglia in Pde6b ^-/- and Mak ^-/- models to test the extent to which loss of retinal microglia alters the cadence of photoreceptor degeneration.

P317 Bacterial Stress Management: Evaluation of Diverse Pathogen Stress Responses

E Knebel*¹, P Brown^2,1

¹Veterinary Pathobiology, University of Missouri, Columbia, MO; ²Biological Sciences, University of Misssouri, Columbia, MO

All bacterial pathogens encounter environmental stressors when invading their host. How these pathogens sense, respond, and ultimately overcome such stressors can yield better understanding of host-pathogen interactions and identify new therapeutic targets. In this study, 2 closely related bacteria that occupy vastly different lifestyles were evaluated for their responses to stressors. Genomic comparisons suggest both pathogens use evolutionarily conserved pathways to promote survival in challenging conditions. Here, we explored how Agrobacterium tumefaciens, a plant pathogen, and Brucella abortus, a mammalian pathogen, respond to stressors encountered during host invasion, including acidic conditions, oxidative stress, antimicrobial peptides, and cell-surface targeting antibiotics.In addition to the conserved stress response pathways, we hypothesize that there will also be niche-specific stress responses with unique phenotypes.There are distinct, observable responses to several of these stressors in vitro. For example, A. tumefaciens secretes an exopolysaccaharide, expresses a Type 6 secretion system (T6SS), and reduces cell growth in response to acidity, whereas B. abortus reduces expression of a T4SS and decreases its metabolism and growth. Exposure to sublethal concentrations of faropenem, a cell wall-targeting antibiotic, induced growth pole swelling in A. tumefaciens but results in spheroplasting in B. abortus. In addition, we find stress tolerance also depends on the culture media, highlighting the importance of identifying conditions translatable to the pathogens in vivo environment. The pathways promoting survival in stressful conditions will be further characterized by high-throughput sequencing of transposon mutagenized libraries in order to identify the genes essential to survive these stressful conditions. This work will help identify both conserved and niche-specific mechanisms of cell stress detection and responses.

P318 A Multi-facility Examination of Novel Technology for Gnotobiotic Decontamination: Efficacy within Semi-rigid and Soft-sided Isolators

FM Grinstead*

CURIS System, Oviedo, FL

Relying on steam, chemicals, or radiation, existing sterilization products, and processes for isolators, transfer ports, and supplies have remained largely unchanged for many years. Despite wide use, these methods entail common limitations: they are often labor-intensive, subject to human error, and time-consuming. Full sterilization often requires days to achieve at the expense of stalled research and higher labor costs. High operating vapor concentrations may also pose exposure risks. This study assesses the feasibility of hybrid hydrogen peroxide (HHP) via pulse technology in overcoming flaws inherent to other methods while maintaining the required efficacy. To determine success, 3 facilities tested an automated modular HHP system’s ability to decontaminate soft-sided and semi-rigid isolators using a 7% hydrogen peroxide solution and validated efficacy using 323 Geobacillus stearothermophilus (1 x 10^6) biological indicators (BIs) placed throughout the chambers, including gloves and HEPA filters. In contrast to current methods, isolator inlet and exhaust filters remained connected to the closed-loop system for the duration of the decontamination process. For each test, the HHP system delivered primary and secondary injection, extraction, and neutralization, returning the isolator to operational conditions. After each complete cycle, BIs were collected and incubated. All 323 BIs were successfully inactivated, indicating simultaneous sterilization of the chambers, filters, and contents, posing the potential for reuse, rather than replacement, of critical and expensive components. The ability to achieve decontamination of a chamber, its filters, and its contents in 1 treatment while condensing turnaround time from multiple days to as little as two hours, combined with system automation and lower operating concentration of 150 ppm versus >1,000 ppm, demonstrates the HHP system is an effective alternative for isolator decontamination with the potential to save money, decrease reprocessing time, and lower risks to staff and equipment.

P319 Evaluation of Media Conductivity and a Combination of Iodine and Sodium Hypochlorite Surface Disinfection on Zebrafish (Danio rerio) Embryo Viability and Morphology

G Mendoza*, AA Afolalu, K Lertpiriyapong, C Lieggi, NS Lipman

Tri-Institutional Training Program in Laboratory Animal Medicine and Science, New York, NY

The health status of zebrafish (Danio rerio) imported into research facilities is frequently unknown. It is recommended that imported adults are housed on an independent system, and embryos generated from imported fish are surface disinfected by immersion in either an iodine or sodium hypochlorite solution prior to introduction of the larvae from those embryos into an existing colony. Iodine (12.5 - 25 ppm) is effective at eliminating several Mycobacterium spp., whereas sodium hypochlorite (50-100 ppm) reduces Pseudoloma neurophilia in a dose-dependent manner. Unfortunately, exposing embryos to higher concentrations of either solution results in significant mortality. No studies have evaluated whether the conductivity of the embryo culture media and disinfecting solution impacts embryo survival. In addition, there are no published safety evaluations of the consecutive use of iodine and sodium hypochlorite solutions for embryo disinfection. We evaluated survival, hatching success, and morphological defects daily for 5 d post fertilization (dpf) of embryos reared in media of differing conductivity (0-10 µS, 100-200 µS, 750-950 µS, and 1500-2000 µS) with and without exposure to sodium hypochlorite solution (100 ppm) at 6-8 h post fertilization (hpf). Subsequently, we evaluated the consecutive use of iodine (12.5 ppm for 2 min) followed by sodium hypochlorite (75 or 100 ppm for 10 min) and application of the disinfectants in reverse order with pronase and sodium thiosulfate. The goal was to identify a combination disinfection protocol resulting in an acceptable level of embryo loss and defects that would be expected to maximize pathogen elimination. We determined that the conductivity of embryo rearing media does not have an impact on embryo viability; however, embryo viability is significantly reduced after exposure to sodium hypochlorite solution in media with conductivity >750-950 µS and when sodium hypochlorite is used before iodine.

P320 Assessment of Biologic Safety Risk for Murine Xenograft Models Containing Human Pathogens

J Duncan, E Cromwell, A Santo, G Roble*

Comparative Medicine, Fred Hutchinson Cancer Research Center, Seattle, WA

Patient-derived xenograft (PDX) and cell-derived xenograft (CDX) models are critical tools in preclinical cancer research. Researchers derive these models through the implantation of tumor tissue directly into immunocompromised mice. Many animal facilities house these implanted mice at ABSL-2 conditions unless confirmed negative for human blood-borne pathogens. Given the extra costs for testing and housing at ABSL-2, we aimed to determine whether a select subset of blood-borne pathogens would remain in and/or be shed from tumor lines passaged through immunocompromised mice (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG)). We hypothesized that human viral materials would likely survive passage in the tumor but not lead to viremia and viral shedding, thus reducing the risk to handlers and altering the research outcome. We established a PDX model using tissue positive for both human papillomavirus (HPV) and Epstein-Barr virus (EBV) (n=10) and a CDX model using a cell line positive for Hepatitis B (n=10) in NSG mice. We collected samples at multiple time points (d=0, 7, initial tumor growth, tumor diameter 1 cm, and endpoint (2 cm)) for the presence of human virus using PCR. Samples included blood, feces, soiled bedding, and a cage swab. We collected tumor tissue and salivary glands at endpoint for testing. Testing found HPV in the blood of 2/10 mice; one mouse at 2 consecutive time points (1 cm and endpoint) and one mouse at day 7 post-implantation. HPV was also detected in 2 other samples: fecal sample day 7 (n=1/10) and one cage swab at endpoint (representing 5/10 mice). Hepatitis B was found in the blood of a mouse (n=1/10) and from a cage swab (representing 5/10 mice), both at endpoint. Explanted tumors tested positive for either HPV or Hep B, respectively. EBV was not detected at any point post-implantation. Although not consistent, HPV and Hep B positive tumor-bearing mice may shed virus into their environment. Further work is needed to establish whether the detected viral DNA is associated with infective virus. However, our recommendation is to continue to house immunocompromised mice implanted with tissue with either an unknown pathogen status or known to be positive for human virus in ABSL-2 facilities.

P321 LPS Dose Dependently Induces Body Temperature Change in Rabbit

HB Culp*¹, I Petrescu¹, P Szczerba¹, S Chia³, X Ping¹, L Yang¹, C Nunes¹, J Destefano¹, T Schnaufer², M Washington², M Ault¹, L Gichuru¹, L Handt¹, X Li¹, S Motzel¹, X Shen¹

¹SALAR, Merck, West Point, PA; ²Clinical Pathology, Merck, Lansdale, PA; ³Pacific Translational Biomarkers, Merck, San Francisco, CA

Lipopolysaccharide (LPS), a component of the gram-negative bacteria outer membrane, has been generally used for experimental modeling of inflammation in different species. In rabbits, the LPS causes a reversible inflammatory status which clinically appears as lethargy, ruffled fur, shivering and body temperature (BT), and hematologic changes. In this poster, the effects of LPS (Escherichia coli O55:B5, 3 x 10⁴ endotoxin units [EU]/µg) on body temperature were investigated in New Zealand rabbits. BT telemetric transponders were implanted subcutaneously at the base of the animal ear. LPS was injected intravenously at doses of 1 and 3 µg/kg (3 x 10⁴ and 9 x 10⁴ EU/kg accordingly), and animal BTs were monitored via telemetric transponders in intervals as short as 5 min. As a result, hyperthermia (+1.5^oC ± 0.3^o C at 2 h) was observed in the 1 µg/kg group for up to 6 h. Meanwhile, hypothermia (-1.9^o C ± 0.31^o C at 2 h) was observed in the 3 µg/kg group for up to 8 h and was back to normal in 24 h; the hypothermic rabbits were provided with external temperature support for 6-7 h post LPS inoculation. Recorded data suggests that the endotoxin appeared to have a dose-dependent effect on the body temperature of the rabbits. Blood chemistry and hematology were also conducted in the 3 µg/kg group. Decreasing white blood cell, platelet, neutrophil, and lymphocyte counts were confirmed. In conclusion, this poster indicates that the different concentrations of endotoxin could be a critical factor causing either hyperthermia or hypothermia in rabbits.

P322 Evaluation of Active Warming and Surgical Draping Practices for Perioperative Thermal Support in Laboratory Mice

KT Bailey², SR Jantre³, FR Lawrence³, FC Hankenson^1,2, JM Del Valle*¹

¹Campus Animal Resources, Michigan State University, Lansing, MI; ²College of Veterinary Medicine, Michigan State University, East Lansing, MI; ³Center for Statistical Training and Consulting, Michigan State University, East Lansing, MI

Surgical procedures are commonly performed using mice but can have major effects on their core body temperature, including development of hypothermia. The aim of this study was to evaluate active perioperative warming with and without surgical cling wrap draping for rodent surgery to refine practices and improve animal welfare. Mice (n=48, 24F + 24M; age, 3-6 mo; CD1) were randomized into treatment groups (n=6; 8 mice (4F + 4M) per group). Treatments included placement in a small animal forced air incubator set to 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 as well as surgical draping (Both/Drape), and another draping only without incubator warming (Control/Drape). The final group received neither warming nor draping (Control). Mice had subcutaneous temperature transponders placed, and ~5 d later, were anesthetized with 100 mg/kg ketamine/10 mg/kg xylazine IP and a laparotomy was performed. 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 or in the incubator. Mice prewarmed in the incubator for 30 min (pre, both, both/drape) demonstrated significantly increased subcutaneous body temperatures (average, 39.97 °C) compared to mice that were not prewarmed (average, 38.0 °C; p<0.05 for all pairwise comparisons). Mice placed in the incubator (pre, post, both, both/drape) had significantly longer anesthetic recovery times (average, 45.16 min) compared to mice that did not receive incubator warming (average, 21.39 min; p<0.05 for all pairwise comparisons) Mean intraoperative rectal temperatures of both/drape mice tended to be greater than mice in the both group (mean temperature at end of surgery: Both, 34.71 °C, Both/Drape, 36.80 °C), suggesting a warming benefit of surgical draping, although this difference was not statistically significant (p>0.05). Use of a forced air incubator and cling wrap draping mitigated body temperature loss in mice during surgery and throughout postoperative recovery.

P323 Testing Optimization and Time of Day Effects for Overhead Enclosure Monitoring in the Dunkin Hartley Guinea Pig (Cavia porcellus)

J Helbling*¹, AP Spittler¹, M Sadar², KS Santangelo¹

¹Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO; ²Department of Clinical Sciences, Colorado State University, Fort Collins, CO

The Dunkin Hartley guinea pig (Cavia porcellus) is utilized in orthopedic research as a naturally occurring animal model of nontraumatic osteoarthritis. In addition to structural joint deterioration, these animals demonstrate decreased movement with disease progression. Overhead enclosure monitoring can provide objective mobility measurements; however, protocols for trial optimization have not been established. Specifically, it is not known whether (i) testing duration or (ii) time of day effects influence movement patterns. First, it was theorized that a total testing period of 10 min would be adequate. Second, given their crepuscular nature, it was hypothesized that these guinea pigs would display periods of heightened activity during early and late testing periods. To assess these variables, 10 5-mo-old male Dunkin Hartley guinea pigs were used. Animals were housed in filter top caging on corncob bedding with a 12:12 h light:dark cycle. No treatments were administered. Voluntary movement in a 4 ft diameter, walled enclosure containing a red hut shelter was allowed for 14 min. Monitoring software was calibrated to divide the total testing duration into 2-min intervals, allowing incremental but uninterrupted detection of activity patterns. Overhead monitoring was performed at 4 different times of day (0530-0700, 0930-1100, 1115-1330, 1515-1700). Key quantitative mobility measures included total distance traveled, total time mobile, average speed while mobile, and total time spent in the hut shelter. Statistical analyses (repeated measures one-way ANOVA) demonstrated: (i) no significant differences within 2-min testing intervals across the 14-min protocol; and (ii) when compared to other time points, animals in the 0530-0700 testing interval spent significantly more time mobile (142 s vs 40 s, P < 0.03) and traveled a significantly farther distance (15.5 m vs 4.5 m, P < 0.05); mobility measures for other time points did not differ from one another. Given our findings, a trial period greater than 10 min is likely unnecessary. Notably, the impact of time of day on mobility measures should be accounted and controlled for when possible.

P324 Effects of Different Grades of Carbon Dioxide (CO₂) on Euthanasia of Mice

JE Stuckey*, S Makhija, S Putta, L Bright, D Reimer

Comparative Medicine Resources, Rutgers University, Piscataway, NJ

Many animal research studies involve euthanasia to relieve pain and/or as an experimental endpoint for the study. Euthanasia literally means “good death” and should include ways to minimize or eliminate any pain or distress due to the process of euthanasia. Guidelines for the euthanasia of animals have been established by the American Veterinary Medical Association (AVMA), and include the use of carbon dioxide (CO₂) as an acceptable means for euthanasia of many laboratory species including mice. Euthanasia research to date has focused on the flow rate of CO₂ into the euthanasia chamber or mixtures of CO₂ with other gases. However, possible effects of different grades of CO₂ on euthanasia are not clear or published. In a 2012 Office of Laboratory Animal Welfare (OLAW) webinar, OLAW, United States Department of Agriculture (USDA), and AAALAC International asserted that different grades of CO₂ including non-pharmaceutical industrial grade were acceptable as they all provide a minimum purity of CO₂ of 99.0%. There are currently 3 grades of CO₂ being used commonly: USP medical (>99.2% CO₂), Bone Dry (>99.9% CO₂) and industrial (>99.0% CO₂). Animal care programs source and use all 3 grades of CO₂ depending on availability. For this study, the inclusion criteria consisted of: mice already slated for euthanasia and age >10 d old. Age, strain, and gender were blocked, and then mice were randomized to each of the 3 grades for euthanasia. Based on the pilot data, time to recumbency and time to cessation of respiration were on average 35-38 s and 132-149 s, respectively. Bone Dry was the quickest and medical grade the longest to effect, although no significant differences were detected. Tissues from a subset of animals were submitted for histopathology to assess the effects on the respiratory tract including the nasal epithelium and lungs. This data confirmed that the grade does not compromise animal welfare. Therefore, standardizing the use of industrial grade CO₂ would provide approximately a $300 per month per average facility cost savings to the program, and all grades may serve as alternative options in disaster management.

P325 Buprenorphine Does Not Attenuate Postoperative Hypersensitivity in NSG Mice

J Arthur*¹, ED Alamaw¹, K Jampachaisri², C Nagamine¹, MK Huss¹, C Pacharinsak¹

¹Department of Comparative Medicine, Stanford University, Stanford, CA; ²Faculty of Science, Naresuan University, Phitsanulok, Thailand

Buprenorphine is one of the most commonly prescribed analgesics for management of postoperative pain in mice. Although various forms of buprenorphine have proven to be effective for commonly used immunocompetent mouse strains, very little is known about the effectiveness of buprenorphine in immunodeficient mice. This study aimed to evaluate the efficacy of 3 different buprenorphine formulations for attenuating postoperative hypersensitivity in the immunocompromised NSG mouse using a plantar incisional pain model. We hypothesized that regular, sustained-release, and extended-release buprenorphine would attenuate postoperative mechanical and thermal hypersensitivity for NSG mice using this model. Male and female NSG mice (n=40) were randomly allocated to 1 of 4 treatment groups: 1) Saline (0.9% NaCl, 1 ml/kg, SC; once); 2) bup-HCl (buprenorphine HCl, 0.1 mg/kg, SC, BID for 48h); 3) SR (sustained-release buprenorphine, 1 mg/kg, SC, once); 4) XR (extended-release buprenorphine, 3.25mg/kg, SC, once). Mechanical and thermal hypersensitivity assessments were conducted at 24 h presurgery and then at 4, 8, 24, 48, and 72 h postsurgery before euthanasia and necropsy. For mechanical hypersensitivity: 1) Saline, SR, and XR groups did not exhibit mechanical hypersensitivity at any time point; 2) Bup-HCl group exhibited mechanical hypersensitivity at all time points. For thermal hypersensitivity: 1) Saline group exhibited thermal hypersensitivity at 4, 8, 24, and 48 h; 2) Bup-HCl, SR, and XR groups exhibited thermal hypersensitivity at 4, 8, 24, 48, and 72 h. No abnormal clinical observations or gross pathologic findings were seen in any groups. Results indicate that regular, sustained released, and extended-release buprenorphine do not attenuate postoperative hypersensitivity for NSG mice in this model.

P326 An Investigation of Bupivacaine for Euthanasia in African Clawed Frogs (Xenopus laevis)

KL Navarro*¹, K Jampachaisri², D Chu¹, C Pacharinsak¹

¹Comparative Medicine, Stanford University, Palo Alto, CA; ²Mathematics, Naresuan University, Phitsanulok, Thailand

The 2020 AVMA Euthanasia Guidelines accept the use of MS-222 immersion (5 g/L; minimum 1 h) for euthanasia of African clawed frogs (Xenopus laevis). Pilot studies using bupivacaine at 1.5 g/L indicated a similar effect on heart rate as MS-222 revealing a potential use as a euthanasia agent for Xenopus laevis. The aim of this project was to investigate whether bupivacaine overdose of African Clawed frogs via immersion ceases heart function similar to MS-222. In the first part of the study, frogs (n = 10/group) were randomly immersed for 1 h in 1 of 3 treatment groups: 1) MS-5 (MS-222, 5g/L); 2) MS-10 (MS-222, 10 g/L); or 3) Bupi-1.5 (0.5% Bupivacaine, 1.5 g/L). Frogs were then removed from solutions, rinsed with system water, and placed into a recovery cage. Heart rate was evaluated audibly via doppler ultrasound over 1 min at immediate removal (T1) and at 2 (T2), and 3 (T3) h in the recovery cage. In the second part of the study, frogs (n = 10/group) were randomly immersed for 5 h in either MS-5 or Bupi-1 (0.5% Bupivacaine, 1 g/L). Heart rate was assessed twice at 2.5 (T2.5) and 5 (T5) h. Righting reflex and withdrawal reflex of the hindlimb were tested during both experiments at 1 h of immersion. For part 1, neither MS-5, MS-10, or Bupi-1.5 ceased heart rate at T1. Heart rate in Bupi-1.5 was significantly lower than that in MS-5 at T2 and T3. In the part 2 results, neither MS-5 nor Bupi-1 ceased heart rate at T2.5 or T5. Bupi-1 animal heart rates were significantly lower at T5 than at T2.5. No groups exhibited righting reflex or withdrawal reflex. These data suggest that immersion of African clawed frogs in an overdose of bupivacaine does not cease heart function, thus is not an adequate euthanasia agent. These data also further reinforce the use of secondary methods for euthanasia in African clawed frogs.

P327 A Comparison of Tumor Volume Accuracy Taken with Calipers and a Tumor Measurement System When Compared to Excised Tumor Weights at the Point of Termination

K Turley*

Marketing , Fuel3D, Oxford, , United Kingdom

In animal studies, tumor size is used to assess responses to anti-cancer therapy. The current standard for volumetric measurement of subcutaneous tumors is by external caliper by taking a length and width measurement, a method often affected by error. It has been historically shown that excising a tumor and taking a weight in grams (g) has a 1:1 relationship with the tumor’s true volume (mm3). We wanted to investigate if taking a length, width, and height measurement automatically a novel 3D and thermal imaging system more closely aligned with the excised weight and, therefore, the true tumor volume. Three tumor growth studies were selected across NSG, Nude, and C56BL/6 animal strains, at varying termination points for each animal. A final measurement of length and width were taken with calipers, and a volume calculated using a standard length, width, width equation. A final scan was also taken, which automatically calculated the length, width, and height to generate the tumor volume. The tumors were excised from the animals and weighed. This weight was then compared to the tumor volumes calculated for both calipers and the 3D and thermal imaging system, plotting each method’s volume variation and relative percentage variation from the excised weight. Results show that the 3D and thermal imaging system has a mean difference across the 3 studies of just – 77mm3 to the mean excised weight while caliper’s mean difference is +210mm3. In the 3 studies, the 3D and thermal imaging system performed consistently and always offered a volume more closely correlated to excised weight; however, the same was not true for calipers. In one particular study, calipers had a mean difference of +1000mm3 and 132% relative difference, while the scan remained consistent and was only –188mm3 away from the mean excised weight and 15.4% relatively. From this study, it can be concluded that including a true height measurement of a subcutaneous tumor is essential to get an accurate tumor volume. Further results continue to be collected across varying operators, laboratories, and animal strains to strengthen the conclusions drawn and further understand its impact on animal welfare and observation of therapeutic efficacy.

P328 Evaluating the Application of Microchips to Monitor Temperature Change after Immunotherapy

KE Potts*¹, A Hernandez², B Matran¹, G Yang¹, M Campagna², J Jiao¹, M Cheng¹

¹Innovation and Product Development, The Jackson Laboratory, Sacramento, CA; ²The Jackson Laboratory, Sacramento, CA

Immunotherapy provides a powerful strategy to eradicate cancer; however, it can trigger potentially severe side effects, including Cytokine Release Syndrome (CRS). The excessive release of cytokines can cause a change in temperature and potentially severe organ failure. Therapeutic induced CRS can be monitored using humanized mouse models. A rectal thermometer is commonly used to determine the change in temperature in mice. Due to the limited usage of the rectal thermometer, it is difficult to capture the exact time point when mice experience the change in temperature after immunotherapy. Temperature monitoring microchips, paired with under-cage matrix readers, allow for continuous monitoring of temperatures in mice. To more closely and noninvasively monitor the temperature change due to CRS, 32 female MHCI/II KO NSG mice were humanized by injection with human peripheral blood mononuclear cells (PBMC). To ensure microchip implantation itself did not affect CRS, 20 of those mice were implanted with microchips in the intrascapular area while under anesthesia, and 12 were not. Mice were IV injected with either PBS or OKT3 (0.25 mg/kg) to induce CRS. The microchipped mice were then placed on matrix readers for continuous temperature monitoring. All mice received daily clinical observations and body weight measurements. At 6 h post-treatment, tetracaine, a local anesthetic, was applied to the right eye, and all mice were retro-orbital bled for serum cytokine analysis. Mice had significant cytokine release at 6 hours post OKT3 treatment, and significant body weight loss was observed between day 1 and day 4 post-treatment, compared to the PBS group. Cytokine analysis and bodyweight comparison determined there was no statistically significant difference between the microchipped and non-microchipped groups. Data obtained from continuous temperature monitoring showed OKT3 treated mice had a significant decrease in temperature between 7-9 h and 35-40 h post treatment, which otherwise could not be determined due to daily rectal temperature measurement restrictions. In summary, temperature microchips are an effective approach to determine the changes in temperature due to CRS.

P329 Stable Transfer of Gut Microbiota in Flexible-film Isolator Reared Immunodeficient Mice Following Transfer of Isolators between Facilities

KY Jen*, KP Nickerson

Charles River Laboratories, Wilmington, MA

The mouse microbiome (MB) is an important variable underlying research outcomes, study reproducibility, and mouse health. It has been established that many factors influence the structure of the gut MB (GMB), including animal transfers between facilities. GMB studies have investigated conventional shipping methods such as placing animals into shipping containers and transported via a third-party transportation vehicle and analyzing GMB via next-generation sequencing before and after transport. In this study, whole mouse isolator movement between 2 rodent product facilities was monitored through the evaluation of the GMB pre- and post-transport to assess GMB stability. For 16S rRNA GMB assessment and community composition determination, freshly expelled feces were collected from 5 males and 5 females 4-6-mo-old breeders per isolator at multiple time points, before and after a scheduled transfer due to closing of a facility. Nine isolators of BALB/c nude (CAnN.Cg-Foxn1^nu/Crl) and 23 isolators of athymic nude (Crl:NU(NCr)-Foxn1^nu) mice were assessed. GMB sequence data were classified using One Codex. Relative stability of the commensal community was seen in both strains. It was also observed that the same total taxonomy of microbes in the athymic nude commensal community present before transport remained consistent through shipment and 1 wk out, with gradual increases in species alpha diversity over the next few months. The relative abundance of some members of the microbial community did change, likely in response to the stress of moving, but most were restored within 1 wk of acclimation. Throughout the entirety of the study, only 1 species remained reduced after shipment, Acetatifactor muris, while others (Bacteroides thetaiotaomicon and Muribaculum intestinale) saw a decrease in the relative abundance after the initial transport but stabilized over the duration of the study. In conclusion, transport of animals housed within an isolator environment was successful in retaining the microbial community composition present in the animals at their original facility. By controlling for outside exposures through stringency of intact transport environment, we successfully moved animals without significant disruption to their GMB.

P330 Analysis of Novel Tumorigenic Factors in Colorectal Cancer

K Hiura*¹, Y Takahashi¹, M Watanabe¹, T Maruyama¹, K Nakano², T Okamura², H Sasaki¹, N Sasaki¹

¹Kitasato University, Towada, Japan; ²National Center for Global Health and Medicine, Shinjuku, Japan

Colorectal cancer is the third cause of death worldwide. Chronic inflammation plays a key role in colorectal carcinogenesis. In inflammatory bowel disease, fibrosis of the intestinal tissue resulting from chronic inflammation is thought to be involved in carcinogenesis. We have found NSAS1 to activate inflammation or fibrosis in renal tubular epithelial cells in a nephropathy mouse model. Overexpression of NSAS1 was observed in colorectal cancer patients compared to healthy non-cancer patients in the Cancer Genome Atlas database, but it is unclear whether it contributes to proliferation and malignancy. In the present study, we examined the relationship between NSAS1 and the malignant transformation of intestinal tumors using NSAS1 KO mice and human colorectal cancer cell lines (SW620 and HCT116). NSAS1 KO mice were generated using CRISPR/Cas in the FVB/NJ background and appeared to be normal in appearance, viability, and breeding. Next, both KO and WT male mice (8-10 wk of age) were injected intraperitoneally with azoxymethane (AOM, 10 mg/kg) and maintained on a regular chow and water. After 5 d, the mice were fed with 2.5% dextran sodium sulfate (DSS) in the drinking water for 5 d, followed by 14 d of regular drinking water for recovery. This cycle was repeated 3 times; after that, mice were fed with regular chow and water till the end. The number and size of AOM/DSS-induced colorectal tumors were significantly reduced in KO mice (n=13) compared with wild-type controls (n=7) (P<0.01). Furthermore, in the colony formation assay using human colorectal cancer cell lines, SW620 and HCT116, NSAS1 knockdown significantly reduced the proliferation of both cell lines (SW620: P<0.05, HCT116:P<0.01). Thus, these data suggest that NSAS1 is a potent driver of intestinal tumorigenesis. Targeting NSAS1 may be useful for the development of novel therapeutic agents for colorectal cancer.

P331 Clinicopathologic Characteristics of Pancreatic Islet Amyloidosis in the Rhesus Macaque (Macaca mulatta) and Sooty Mangabey (Cercocebus atys)

L Birdwell*, D Levesque, F Connor-Stroud, D Kodandera, S Gumber

Yerkes National Primate Research Center, Emory University, Atlanta, GA

Diabetes mellitus type 2 has been linked to pancreatic islet amyloid deposition in humans and nonhuman primates, including rhesus macaques (RM) and sooty mangabey (SM). In this study, we characterized the amyloid deposits by performing histological and immunohistochemistry evaluation of collected pancreatic tissue samples from RMs and SMs. A retrospective study was conducted comparing 3 groups of animals that had pancreatic amyloid deposits: 15 diabetic RMs (2 male, 13 female), 14 non-diabetic RMs (2 male, 12 female), and 15 diabetic SMs (12 male, 3 female). Pancreas from diabetic RMs showed higher histopathology scores for islet amyloid distribution (average score = 3.0), amyloid severity (average score = 4.0), and calcification deposits (average score =2.0) than non-diabetic RM (averages of 2.0, 3.0, and 0.2, respectively). These results suggest that clinical diabetes in RMs may be associated with a larger percentage of amyloid-affected islets with a more diffuse distribution and calcification deposits. The diabetic rhesus macaques also had elevated scores for the percentage of islet area immunopositive for islet amyloid polypeptide (IAPP) with an average score of 5.0 and serum amyloid P (SAP) with an average score of 4.9. Non-diabetic RM had average scores of 4.3 for IAPP and 4.0 for SAP. This data suggests that IAPP and SAP amyloid components may be in greater quantities for diabetic RM as compared to non-diabetic RM. As expected, tissues from the diabetic RM (average score = 2.1) had lower scores for the percentage of islet area immunopositive for insulin as compared to non-diabetic RM (average score= 4.8). The diabetic SMs (average score = 3.2) showed significantly lower scores for the percentage of islet area immunopositive for glucagon than diabetic RMs (average score= 4.4), suggesting a potentially lower amount of glucagon present in the islet amyloid affected pancreatic tissue of the diabetic SMs. An additional group of non-diabetic SMs pancreatic amyloid deposits is currently under evaluation. Future research may examine the genetic basis of mangabey diabetes and evaluate potential biomarkers and therapeutics based on characterization results.

P332 Assessment of Transfer Tubes Compared with Tail-lift Handling in a Breeding Colony of C57BL/6J Mice

MA Hull*¹, PS Reynolds², EA Nunamaker¹

¹Animal Care Services, University of Florida, Gainesville, FL; ²Anesthesiology, University of Florida, Gainesville, FL

Current methods of handling mice during cage change procedures induce stress to the point of potentially compromising welfare. Recent behavioral work with lab mice showed that use of transfer tunnels elicits less stress and provides a simple and efficient tool for staff during cage change. To date, there is no published data concerning the effect of transfer tunnels on breeding mice. We hypothesized that transfer tunnels would decrease handling stress for pregnant females and improve reproductive performance outcomes compared to animals handled with traditional transfer forceps (tail lift). The C57BL/6J mouse strain is commonly used for general research. Sixty 6-8-wk-old B6 breeding pairs were randomized into 2 groups: the control group (industry standard) handled by tail-lift with transfer forceps, and the experimental group handled with transfer tunnels. Pairs were followed for 6 mo. Number of pups born and weaned per litter, average weaned litter weight, breeding productivity, and pup survival data were collected and analyzed. Compared to tail-lift animals, tunnel handling reduced complete litter attrition by 43% (p=0.024). Tunnel handling resulted in a 32% increase of all litters born surviving to weaning compared to tail-lift (p=0.006). These results translate to approximately 1 extra pup per pair, 1 extra litter weaned per 3 pairs, and up to 10% more breeding pairs with no litter losses for tunnel-handled compared with tail-lift pairs. Transfer tunnel handling improves breeding outcomes when compared to tail-lift handling with transfer forceps in breeding C57BL/6J mice.

P333 Using Genetically Modified Mouse Models for Faster Research Results in Liver Disease

M Seays*

Sales, GemPharmatech, Wilmington, DE

Nonalcoholic steatohepatitis (NASH) is inflammation and damage of the liver caused by excessive fat accumulation. NASH is the most prevalent chronic liver condition in the US and the most severe form of nonalcoholic fatty liver disease (NAFLD). When NASH exacerbates, it can cause scarring of the liver, which leads to cirrhosis and even hepatocellular carcinoma. Mice fed on a Western diet (WD) are valuable in vivo tools for NASH studies and therapeutic development. Typically, it takes ~20 wk for the phenotype to develop before the mice can be used in research. LepR and Alms1 genes assist with metabolic regulation. The loss of function of Lepr and Alms1 genes in humans, leads to metabolic syndrome. Using CRISPR-Cas9 technology, we constructed Lepr and Alms1 knockout mice (KO) on BKS and B6 genetic backgrounds, respectively. Both BKS-db and B6-Alms1 KO mice developed early onset metabolic disorders, including obesity, type 2 diabetes, and dyslipidemia with a WD treatment. WD treatment rapidly worsens the pathomorphological features of NASH in the liver of these mice, including steatosis, inflammation, and ballooning. The genetically modified mice required a much shorter WD treatment (~4 to 6 wk vs ~20 to 30 wk in other traditional mouse models). NAFLD activity scores and fibrosis scores were both significantly increased upon 4-7 wk of WD treatment. The BKS-db and the B6-Alms1 KO mice had significantly increased body weight, cholesterol level, LDL level, ALT and AST levels within 4 to 6 wk of WD treatments. BKS-db and B6-Alms1 KO mice were tested for efficacy evaluation of candidate drugs, resulting in promising phase II data with significantly improved steatosis, inflammation, and ballooning. This data demonstrates that BKS-db and B6-Alms1 KO mouse models accelerate study start times, reduce costs associated with animal care resources, and improve overall result timelines for liver disease research and drug discovery.

P334 Optimization of Biomimetic Nanofiber Scaffolds to Promote Periosteal Expansion

MM Walker*¹, H Powell^2,3

¹Office of Research, The Ohio State University, Columbus, OH; ²Department of Materials Science & Engineering, The Ohio State University, Columbus, OH; ³Department of Biomedical Engineering, The Ohio State University, Columbus, OH

Traumatic injuries, neoplasia, and osteolysis can result in large segmental bone defects that present particular clinical challenges. Autogenous bone grafts, allografts, and synthetic bone implants have been used to manage large defects; due to poor incorporation with the patient’s bone; however, complications such as nonunion, malunion, and infection are common. The current project harnesses the osteogenic properties of periosteum to investigate its potential for segmental defect healing when used in conjunction with bioengineered nanofiber scaffolds. The periosteum provides vasculature, cytokines, proteins, and mesenchymal stem cells that are central to new bone formation. Biomimetic scaffolds act as a foundation on which periosteal cells can expand to incorporate with host tissue. This investigation specifically aims to demonstrate isolation strategies for porcine periosteal cells based on human periosteal cell isolation protocols. Tissue from human femoral and porcine tibial specimens was minced, cultured, and quantified using both migration assay and enzymatic digestion with collagenase. To the authors’ knowledge, this is the first instance in which porcine periosteal cells, the tissue for which is readily available in the laboratory animal setting, were propagated in this manner. The central objective of isolating these cells is to demonstrate that nanofiber scaffolds can promote the expansion of periosteal cells. Bovine-extracted collagen nanofiber scaffolds were created in either an aligned or random orientation. Half of the scaffolds were doped with hydroxyapatite (HA). Immunohistochemical staining was used to determine the orientation (random vs aligned) and architecture (collagen vs HA-doped collagen) that resulted in maximum cellularity. Pilot results support the hypothesis that periosteal cell migration is successfully promoted on biomimetic scaffolds; in vitro optimization of this project will inform future trials in small and large animal models. This work will translate to the ultimate goal of clinical implementation of the tissue and bioengineered construct in human patients.

P335 Development of New Adriamycin Nephropathy Murine Model in C57BL/6 Genetic Background

M Watanabe*¹, Y Takahashi¹, K Hiura¹, K Nakano², T Okamura², H Sasaki¹, N Sasaki¹

¹veterinary medicine, Kitasato University, Towada, Japan; ²National Center for Global Health and Medicine, Shinjuku, Japan

The number of people with chronic kidney diseases (CKD) is increasing worldwide, and the increase in the numbers of dialysis patients has been putting pressure on the health budgets of each country. In developed countries, over an estimated 10% of adults will develop CKD. Many patients with CKD have podocytopathies caused by their primary diseases, such as diabetes and hypertension. In nephrology research, several rodent experimental models of podocytopathies are available. Adriamycin (ADR)-induced nephropathy is one of the most frequently used models for understanding the pathogenic mechanisms of podocytopathies and CKD. However, the application of this model in mice is limited to a few strains, such as BALB/c. The most commonly used mouse strain, C57BL/6 (B6), is resistant to ADR-induced nephropathy, as are all mouse strains with a B6 genetic background. Reportedly, the R2140C variant of the Prkdc gene is the cause of susceptibility to ADR-induced nephropathy in mice. To verify this hypothesis, we produced Prkdc mutant B6 mice, termed B6-Prkdc^R2140C, that possess the R2140C mutation. After male B6- Prkdc^R2140C (8 wk of age, n=7) and male BALB/c mice (8 wk of age, n=7) were administered ADR (10 mg/kg, single dose), large amounts of proteinuria were both observed. In addition, glomerular diseases and renal tubular disorders were histologically examined, and no significant distinction was indicated between B6-Prkdc ^R2140C and BALB/c. These findings demonstrated that B6-Prkdc^R2140C show ADR-induced nephropathy susceptibility at a similar level to BALB/c and that the PRKDC R2140C variant causes susceptibility to ADR-induced nephropathy. In the future, ADR-induced nephropathy may become possible in various kinds of genetically modified mice with a B6 background by mating with B6-Prkdc^R2140C.

P336 Evaluation of Tarsal Lesions in Immunocompromised Mouse Strains

M Campagna*¹, A Hernandez¹, K Ramsay², M Maglaty², D Imai-Leonard², J Serrano¹, A Schile¹, R Doty¹

¹The Jackson Laboratory, Sacramento, CA; ²Comparative Pathology Laboratory, UC Davis, Davis, CA

The NSG (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ) mouse and its variants are highly immunocompromised animals with specific husbandry needs, including strict biosecurity practices. The major clinical manifestations in these strains result from their immune status, but as with any other inbred strain, there exists a potential for unexpected phenotypes. One such phenotype that has been intermittently detected in NSG mice in our research facility is a progressive tarsal lesion, which was recently reported at a higher frequency in the MHC double knock-out strain [DKO; NSG-(K^bD^b)^null(IA)^null]. This lesion starts as an abnormal gait and swelling of 1 or both heels, advancing to skin ulceration. Our veterinary team assessed the prevalence of, risk factors for, and pathology associated with these lesions in both NSG and DKO strains. Initial investigations were performed to determine the rate of occurrence in production colonies and develop a clinical scoring system to better define the progression of disease in these mice. Then, a prospective study was conducted involving 10 groups ( n= 10 each) of NSG and DKO mice maintained under different husbandry conditions, to determine the influence of strain, health status, cage type, scruff restraint frequency, and handler on rate and severity of these lesions. Mice were monitored for clinical signs for 6 consecutive wk. A subset of these mice was evaluated for gross and histological pathology. The prevalence in our production colonies at the time of evaluation was 0%. Comparison of experimental groups indicated that restraint frequency and handler were the most important disease determinants, resulting in differential pathology of the calcaneal tendon between groups. Regardless of housing conditions, gross or histological lesions did not occur in unrestrained groups. Lesions were only grossly visible after handling by one particular individual, with rates reaching up to 30%. The major conclusion is that the extent and manner of handling is a vital consideration when working with strains predisposed to this condition. Lastly, these clinical and histologic findings may be potentially valuable as a model of Achilles tendinopathy.

P337 Effects of Depilatory Cream on Murine Skin

M Reichert*, N Koewler

Research Animal Resources, University of Minnesota, Minneapolis, MN

Depilatory creams have widespread use in research to remove hair in preparation for surgery, imaging, and other procedures. To date, there are few studies on the physiologic and pathologic effects of these creams on murine skin and how they may impact animal welfare and research outcomes. We aim to determine the effects of depilatory cream application duration on murine skin using a popular commercially available brand. A pilot study was performed in C57BL/6 mice (n = 8, ~3-wk-old females) to refine the study by determining how many days post-application skin tissue should be collected to see peak inflammation. Nair was applied to 1 flank of each animal for 2 min, and skin tissue was collected at 3, 5, 7, and 10 d post-application, with the contralateral flank acting as a control. Photos of the skin were taken daily and blind-scored using a modified Draize skin scoring rubric. There were no discernable differences in skin erythema, ulceration, or edema across groups. Total histopathology scores were generated by a dermatopathologist and were based on dermal inflammation, follicular changes, fibroplasia, acanthosis, and changes to the stratum corneum. Histopathology scores of the treated skin were higher at 3 and 5 d post-application, with histopathology scores in the former group being higher with less variability. Based on these results, 3 d post-application of the product was determined to be the peak inflammatory time point for future studies. Further research into the use of depilatory creams on murine skin will help determine best practices for its use in research to promote better animal welfare and improve research outcomes.

P338 Extended Release Buprenorphine Effectively Attenuates Laser-Induced Thermal Hypersensitivity in an Incisional Model in Neonatal Rats

M Zhang*¹, ED Alamaw¹, K Jampachaisri², MK Huss¹, C Pacharinsak¹

¹Stanford University, Cupertino, CA; ²Department of Mathematics, Naresuan University, Phitsanulok, Thailand

Research on the effectiveness of neonatal rat analgesic techniques is limited. In this study, we investigate if a newly released, lipid bound, extended-release buprenorphine effectively attenuates thermal hypersensitivity in a neonatal rat incisional pain model. We hypothesized that lipid bound, extended-release buprenorphine would attenuate laser-induced thermal hypersensitivity during postoperative period for rat pups in this model. Male and female postnatal day 5 Sprague Dawley rat pups (n=38) were randomly assigned to 1 of 4 treatment groups: 1) 0.9%NaCl (Saline), 0.1 mL, once SC; 2) sustained release buprenorphine (Bup-SR), 1mg/kg, SC once; 3) low dose extended-release buprenorphine (XLo), 0.65 mg/kg, SC once; 4) high dose extended-release buprenorphine (XHi), 1.3 mg/kg, SC once. Pups were anesthetized with sevoflurane, a 5 mm long skin incision was made over the left lateral thigh, the underlying muscle was dissected, and the skin was closed with surgical glue. Thermal hypersensitivity and clinical observations were evaluated at 1 h prior to surgery/analgesic administration and then subsequently at 1, 4, 8, 24, 48, 72 h. Plasma buprenorphine concentration levels were evaluated at 1, 4, 8, 24, 48, 72 h. Thermal hypersensitivity in the Saline group was significantly enhanced at 1, 4, and 8h. The enhanced thermal hypersensitivity was effectively attenuated in the Bup-SR and XLo groups at all 3 time points. The XHi group showed thermal hypersensitivity attenuation at 1 and 4 h. Plasma buprenorphine concentration for all treatment groups remained near the clinically effective concentration of 1 ng/mL at least 8 h and no abnormal clinical observations were evident. Results indicate that extended-release buprenorphine attenuates laser-induced thermal hypersensitivity during postoperative period for rat pups in this model. Specifically, XLo attenuated postoperative laser-induced thermal hypersensitivity for at least 8h while XHi attenuated post-operative hypersensitivity up to 4 h.

P339 Comparison of Microenvironmental Parameters in Single- and Pair-housed Static Rat Cages

MG Stover*, JS Villano

Johns Hopkins University, Baltimore, MD

Social housing and microenvironmental parameters are important considerations when housing rats for research purposes. The objective of this study was to determine if microenvironmental parameters (cage temperature, humidity, NH₃, CO₂, O₂) vary significantly between single- and pair-housed static rat cages housing large (>500g) adult (>6 mo) Crl:CD®(SD)IGS rats. Microenvironmental parameters were measured at 3-timepoints (days 0, 2, 4) over the first week following cage change and were measured within 4 cages each of single- (n=4) and pair-housed (n=8) rats. We hypothesized that pair-housed cages would have higher temperatures, humidity, NH₃, CO₂, and lower O₂ when compared to single-housed cages. There were no significant differences when comparing 1-wk mean cage temperatures (means±1 SD, singles = 76.2±1.6°F, pairs = 76.8°F±1.4°F), humidity (singles = 59.2±15.5%, pairs = 54.7±6.4%), NH₃ (singles = 82.5±110.7 ppm, pairs = 16.8±20.7 ppm), CO₂ (singles = 0.24±0.17%, pairs = 0.17±0.05%), and O₂ (singles = 20.7±0.2%, pairs = 20.7±0.1%) (P values > 0.05). Although 1-wk means were not significantly different, at the final timepoint a paradoxical effect was noted as single-housed cages had significantly higher NH₃ (208.3±39.2ppm) and CO₂ (0.4±0.2%) and lower O₂ (20.5±0%) when compared to pair-housed cages (NH₃ = 40.0±10.5ppm, CO₂ = 0.2±0.2%, O₂ = 20.6±0.1%) (P values < 0.05). These data reject our hypothesis that pair-housed cages would have higher temperatures, humidity, NH₃, CO₂, and lower O₂ compared to single-housed cages. This study further supports the recommendation for social housing of rats whenever possible unless contraindicated for research or medical purposes.

P340 Refinement of a Traditional Hormone Treatment to Improve Ovulation Rates in Wild Type C57BL/6 Donors for Pronuclear Injection (PNI)

OL Franco Mahecha*, P Qi, G Du, AN Viana, E Umaña, TE Chavarria, JG Fox, SE Erdman

Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA

Pronuclear injection (PNI) is the most widely used procedure to transfer genetic material in the mouse. PNI requires large numbers of high-quality zygotes routinely collected from juvenile 3-4-wk-old donors superovulated with 5IU pregnant mare serum gonadotropin (PMSG) + 5IU human chorionic gonadotropin (hCG). Building on the concept of the 3Rs to reduce, refine, and replace, we hypothesized that using larger amounts of hormones in older pubertal females may improve zygote number and quality while using fewer embryo donors. Two hormone treatments were evaluated: 7.5IU-PMSG/5IU-hCG (7.5-P/5-CG) and 7.5IU-PMSG/7.5IU-hCG (7.5-P/7.5-CG) in 4-, 5-, and 6-wk-old C57BL/6NTac females (n = 5/group). The standard 5IU-PMSG + 5IU-hCG (5-P/5-CG) dose was used as control treatment. We first examined number of oocytes and found that 7.5-P/5-CG improved the number of oocytes/female in 4-, 5-, and 6-wk-olds (43.2±14.6, 46.2±14.9, and 27.0±3.7) versus the control (36.8±9.5, 27±5.2, and 21±7), respectively. The 7.5-P/7.5-CG treatment increased the number of oocytes produced in 4-wk-old females (51.6±10.5), but not in the other 2 age groups. Additionally, the number of injectable zygotes produced in each treatment group was evaluated. Females treated with 7.5-P/5-CG produced more injectable zygotes/mouse (42±14.3, 35±15.2, and 17.2±5.7) than the control (34.8±8.6, 19.8±6.3, and 14.4±5) in each age group, respectively. The 7.5-P/7.5-CG treatment did not improve the number of injectable zygotes compared to the 7.5P/5CG group, suggesting that increasing hCG does not provide any benefit to the superovulation protocol. Yield of live offspring/female improved significantly in 5- and 6-wk-olds treated with the 7.5-P/5-CG versus the control (5.5 and 4 vs 0 and 0), respectively. Four-wk-olds treated with 7.5-P/5-CG, or the control, yielded 8.5 pups per female. This study suggests that increasing the amount of PMSG to 7.5IU in older pubertal 5-wk-old donors improves reproductive parameters of total oocytes, injectable zygotes, and live births. According to these results, and in support of the 3Rs principles, refinement of the hormone priming protocol for superovulation helped to reduce the number of donors required for PNI and optimize scientific productivity in our transgenic facility.

P342 Acidified Water and Total Body Irradiation Best Support Human T Cell Engraftment in NOD.Cg-Prkdc^scidIl2rg^tm1Sug/JicTac Mice

PS Allen*, E Lin, A Hinkle, W Zhang

Boehringer Ingelheim Pharmaceuticals Inc, Ridgefield, CT

An investigation was conducted into the best practices for management of NOD.Cg-Prkdc^scidIl2rg^tm1Sug/JicTac (NOG) mice in an experimental design involving total body irradiation (day 0), subcutaneous flank NCI-H716 carcinoma cell implantation (day 1), and subsequent intravenous purified human T cells transfer (day 15). Initial work with the model resulted in 18-40% mortality, with consistent loss on days 6-8 post-irradiation, and tissue histology confirming lesions consistent with septicemia. It was hypothesized the provision of sterile acidified water would be superior to antibiotic water in mitigating the septicemia, and non-irradiated mice would have similar human T cell engraftment to irradiated mice. Fifty-four NOG mice, group-housed under sterile barrier conditions, were randomly assigned to 3 groups (n=18): acidified water/no irradiation, acidified water/200cGy irradiation, or trimethoprim sulfamethoxazole antibiotic water/200cGy irradiation. Treated water was provided at least 7 d before day 0. All mice then underwent the standard experimental design above, and human CD3+ T cell populations in peripheral blood of mice were evaluated by flow cytometry on day 18 to assess engraftment success. An 11% mortality rate was observed in mice receiving antibiotic water, with tissue histology consistent with septicemia, versus 0% mortality in acidified water groups. The average 72-h human T cell engraftment rate was 5.8% in non-irradiated mice but 16.2% and 15.1% in irradiated mice receiving acidified and antibiotic water respectively. It was concluded that the provision of acidified water mitigated the previously observed NOG mortality in a manner superior to antibiotic water, and total body irradiation with 200cGy was preferred in this experimental paradigm based on the improved human T cell engraftment rate.

P344 Evaluation of Ketamine, Xylazine, Midazolam, and Alfaxalone-containing Sedation Protocols in the Syrian Hamster (Mesocricetus auratus)

RE Cooper*, A Werts

Department of Comparative Medicine, Lovelace Biomedical Research Institute, Albuquerque, NM

The Syrian or golden hamster (Mesocricetus auratus) is a valuable model for SARS-CoV-2 research, as it demonstrates respiratory tract viral replication, pulmonary pathogenesis, aerosol transmission, and clinical disease similar to that in humans. When sedation is needed but inhalant isoflurane is not feasible, e.g. for respiratory-focused dosing or sampling procedures, intraperitoneal (IP) ketamine (K)-xylazine (X) is broadly used. However, at a standard sedative dose (K 80 mg/kg, X 5 mg/kg; K80X5), K-X produces a wide effect range, from failure to achieve loss of righting reflex (LORR) to a LORR of up to 65 m (n = 86). In the context of complex, high-throughput studies in an ABSL-3 environment, a predictable window of sedation is vital. To identify an injectable protocol producing more consistent sedation, we dosed 20 male hamsters with 4 anesthetic cocktails in a crossover design: K80X5, K 80 mg/kg-midazolam 1 mg/kg (K80M1), alfaxalone 15 or 25 mg/kg-midazolam 1 mg/kg (A25M1 or A15M1), and alfaxalone 25 mg/kg (A25). We assessed probability of, time to, and duration of LORR; vital signs at 2-min intervals; and quality of induction and recovery. Probability of LORR was highest for K80M1 (95%) and lowest for A15M1 (40%) (P = 0.06). Mean time to LORR was shortest for A25M1 (3.1±0.9 m) and longest for K80X5 (4.7±1.4) (P = 0.06). Duration of LORR was shortest for A15M1 (17±4.4 m) and longest for K80X5 (23±9.9 m) (P = 0.09); the cocktail with the least % variability was A25M1 (22±5.0 m). K80M1 was associated with the highest heart rate (HR) (345±47.5), and K80X5 resulted in the lowest (167±24.7) (P = 0.00). Assessed by a blinded observer, both induction and recovery were smoothest in K80M1 and roughest in A25; K80X5 and K80M1 outperformed all alfaxalone-containing protocols. In sum, K80M1 and A25M1 both matched or outperformed K80X5 in proportion of animals to achieve LORR, time to LORR, and variability of duration of LORR; both K80M1 and A25M1 were associated with higher HR than K80X5. Considering sedation variability and duration, vital parameters, and induction/recovery quality, all of which have implications for animal welfare and work efficiency in high-throughput contexts, K80M1 is recommended as a refinement over K80X5 for IP sedation of hamsters.

P345 Use of Ultrasound Imaging to Monitor Pathologic Changes on a Mouse Model of Endometrail Cancer

R Vistein*¹, X Guo¹, S Ishiyama³, T Wang², I Shih², K Gabrielson¹

¹Molecular and Comparative Pathobiology , Johns Hopkins University, Baltimore, MD; ²Department of Gynecology/Obstetrics, Johns Hopkins University, Baltimore, MD; ³School of Medicine, Johns Hopkins University, Baltimore, MD

Ultrasonography is used to monitor soft tissue changes in both clinical and research settings. In this study, we assessed the feasibility of detecting morphologic changes in the iPAD mouse model of endometrial cancer (Pax8-Cre Arid1a-Pten conditional knockout) via serial ultrasound. We developed a simple protocol to capture micrometer resolution images of the mouse uterus over an 8-wk treatment with doxycycline in feed to induce neoplastic change. For 10 mice, in weekly sedated ultrasound exams, we were able to differentiate normal (week 0/ week of transition to medicated feed) tissue from two distinct patterns of structural change; fluid-filled (serous) and coalescent soft tissue uterine masses as they developed in individuals. These changes were confirmed by gross pathology and histology at week 7 or earlier if clinical conditions arose (3/10 mice). We conclude that ultrasound can contribute both to experimental studies of uterine disease in mice and diagnosis of clinical reproductive disease in research mice.

P346 Comparison of Egg Shedding Patterns in Nude and NSG Mice Inoculated with Nippostrongylus brasiliensis

RJ Floyd*¹, RJ Ricart Arbona^1,2, NS Lipman^1,2

¹Tri-Institutional Training Program in Laboratory Animal Medicine and Science, New York, NY; ²Center of Comparative Medicine and Pathology, Memorial Sloan Kettering and Weill Cornell Medicine, New York, NY

Nippostrongylus brasiliensis (Nb), known as the rat hookworm, is a nematode used to study Th2 type immune responses. While the rat is the natural host, mice are used as a model due to the availability of a large number of inbred and genetically engineered strains. The life cycle of Nb is direct and is approximately 2 wk in length. In immunocompetent strains, maximum egg production occurs 8 to 9 d post-inoculation (DPI) followed by a gradual decline and worm expulsion at 10-15 d. Once animals have cleared the infection, lifelong immunity persists. In contrast, athymic nude mice are reported to become chronically infected and shed eggs for extended periods. As part of a study examining the potential for horizontal transmission of Nb in mice, female J:NU (nude; n = 6) and NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG; n = 12) mice were inoculated SQ with 750 infective L₃ larvae and egg shedding was monitored via fecal floatation daily for 30 consecutive d. Egg shedding in NSG mice has not been previously examined. Patency was achieved in all NSG mice. NSG mice were chronically infected, shedding on average of 17,422, 16,688, and 6,736 eggs per gram of feces (EPG) at 8, 16, and 28 DPI, respectively. In contrast and surprisingly, patency was detected in only 2 of 6 nude mice which shed on average < 25 EPG at only 8, DPI. To determine if the inability to infect nude mice was stock dependent, Hsd:Athymic Nude-Foxn1^nu mice (n = 6) were similarly infected with L3 larvae. Only 2 of 6 mice shed eggs at 8 DPI shed < 25 EPG. Eggs were not detected at days 16 or 28 DPI. While NSG mice became chronically infected, nude mice did not. The results of this study, possible explanations, and their implications on research are reported.

P347 Improved Detection and Transmission of Proteus mirabilis in Immunodeficient Mice

R Tierce*¹, A Winn², T Albers³, W Poueymirou¹, E Levee¹, B Reddyjarugu¹, S Woods¹

¹VelociGene, Regeneron Pharmaceuticals inc., Tarrytown, NY; ²Small Animal Division, Bioqual inc, Rockville, MD; ³Research Animal Diagnostic Services, Charles River Laboratories, Wilmington, MA

Opportunistic pathogen exclusion is important for protecting animal health and ensuring desired research outcomes in immunodeficient mice. Proteus mirabilis has been associated with gastrointestinal tract lesions, septicemia, pyelonephritis, splenomegaly, and hepatitis and can influence select mouse models. To inform colony health surveillance practices following difficulty excluding P. mirabilis, we aimed to determine the likelihood of detecting P. mirabilis positive immunocompromised (SRG; n=32), immunovague (Fbn1^+/-; n=16), and immunocompetent (CD-1; n=16) colony mice by culture and PCR; to evaluate transmission by 2 sentinel approaches (direct contact, n=16 cages; indirect dirty bedding transfer, n=16 cages); and to further characterize associated pathology (n=24 mice). We hypothesized immunocompromised mice would be better detectors and transmitters of P. mirabilis. Multiple logistic regression models were used for analysis and included PCR copy number, repeat testing, age (various), sex (both), and diet (Trimethoprim and Sulfamethoxazole) as covariates. After 10 wk of repeat testing, P. mirabilis colonized immunocompromised mice were 95 times more likely than immunocompetent mice to test positive by culture (P=0.006) and 30 times more likely by PCR (P=0.022). Sentinel mice were 15 times more likely to test positive by PCR for P. mirabilis if exposed by direct contact compared to dirty bedding (P=0.006) and 18 times more likely to test positive if exposed to positive immunocompromised colony mice instead of immunocompetent (P=0.001). After 10 wk of exposure, 3.8% of bedding sentinel PCR tests were positive, as opposed to 30.7% of contact sentinels (P<0.0001). Only immunocompromised mice on antibiotic diet developed lesions of the urogenital tract and abdominal cavity consistent with known pathology of P. mirabilis (37.5%). Our findings suggest that PCR testing of dirty bedding sentinels alone is not sufficient for detecting P. mirabilis in mouse colonies and direct contact sentinels, testing colony mice, especially immunocompromised, with adjunct culture indicated for successful bioexclusion.

P348 Feasibility of Using a Non-contact Infrared Thermometer to Monitor Disease Progression and Make Endpoint Decisions in SARS-CoV-2-infected hACE2 Hamsters

RA Byrum*, D Ragland, M St. Claire

Comparative Medicine, NIAID Integrated Research Facility, Frederick, MD

Several animal models for SARS-CoV-2 have been established to investigate therapeutics, test vaccines, and understand the pathogenesis of COVID-19. Our laboratory uses hamsters to evaluate therapeutic efficacy and elucidate the pathogenesis of COVID-19. Because fever is a common clinical sign in human patients with COVID-19, measuring temperature for evidence of fever in the hamster model is desired. By contrast, SARSCoV2infected hACE2 hamsters demonstrate a precipitous drop in the body temperature, which has been effectively used as part of the endpoint criteria for experiments using this model. Typically, transponders are implanted in hamsters to avail temperature monitoring; however, when studies involve computed tomography or magnetic resonance imaging, the transponders produce local artifacts that significantly compromise image evaluation. Developing a reliable alternative method to monitor the temperature of hamsters has received great interest. Since these studies are performed in a biosafety level 4 laboratory and body temperature is obtained at least twice a day, a safe, minimally invasive technique was needed. We explored the use of a non-contact infrared thermometer, which we have used successfully in mice. To determine the feasibility of using infrared temperature, we compared temperatures obtained by infrared thermometer and programmable temperature transponders implanted in 1 female and 4 male adult hamsters. Temperatures were recorded on 10 daytime occasions over a 30-d period. Infrared temperatures were obtained immediately after opening the cage lids. Each hamster was quickly removed from the cage, and a previously shaved ventral area was scanned with an infrared thermometer. Not surprisingly, implantable transponder temperatures were usually higher than skin surface temperatures; however, the alignment between these 2 methods was evident, as demonstrated by correlation coefficients ranging from 0.67 to 0.94, obtained with a correlation function calculation within spreadsheet software. These results demonstrated the feasibility of using a non-contact infrared thermometer to monitor temperature in SARS-CoV-2-infected hamsters and make endpoint decisions without compromising imaging performed during studies.

P349 Immunomodulators as Therapy for Canine Inflammatory Triple-negative Mammary Cancer

S Caceres*¹, B Crespo¹, M ILLERA¹, G SILVAN¹, AJ Alonso-Diez¹, PC Carlos², I DIEZ-PRIETO², JC Illera¹

¹Animal Fisiology. Fac Veterinaria, University Complutense of Madrid, Madrid, Spain; ²Departamento de Medicina, Cirugía y Anatomía Veterinaria, UNIVERSIDAD DE LEON, Leon, Spain

Immunotherapy constitutes a novel, successful, and very active research area in breast cancer. However, little is known about its efficacy in inflammatory breast cancer and even less in the canine species. The aim of this study was to determine the efficacy of an immunomodulator in mice xenotransplanted with the canine inflammatory mammary cancer cell line in terms of tumor growth and levels of estrogens, androgens, and glucorticoids. 24 SCID mice: 12 male and 12 females Fox Chase SCID® Beige CB17.Cg-PrkdcscidLystbg-J / Crl mice, 6-8-wk-old were used. A suspension of 106 IPC-366 cells was implanted orthotopically into the mammary fat pad. When tumors developed, mice were administered orally with a solution of 1 mg/kg/d of Immunocin solution for 4 wk; both experiments were done simultaneously. Mice were inspected twice/wk for the development of tumors. If tumors were detected, they were monitored weekly by palpation and measured by calipers. When tumors reached 1500 mm3 of volume, mice were euthanized, and tumors and organs were used. A portion of tumor was used for hormonal analysis. Steroid hormones determination in serum and tumor homogenates (testosterone (T), 17β-estradiol (E2), cortisol (C), and corticosterone (CT) were assayed by EIA previously validated. In our results, a decrease of 56% in females and 36% in males is observed in tumor progression. The intratumoral E2 levels of mice treated with the immunomodulator are higher in males than in females; however, the levels of testosterone in treated females are significantly higher than in the case of males. Serum and intratumoral cortisol levels are higher in both treated males and females. No differences were found between control and treated mice in serum and intratumoral CT concentrations. In conclusion, the administration of the immunomadulator (Immunocin) produces a significant reduction in tumor progression mediated by the action of steroids. Giving evidence that immunomodulatory drugs could be an effective treatment for canine triple-negative mammary cancer.

P350 Efficacy and Duration of Various Buprenorphine Preparations in a Mouse Hind Paw Incision Model of Pain

SJ Mangosing*¹, HR Martin^1,2, C Lieggi^1,2, DE Willis^3,4, NS Lipman^1,2

¹Tri-Institutional Training Program in Laboratory Animal Medicine and Science, New York, NY; ²Center of Comparative Medicine and Pathology, Memorial Sloan Kettering Cancer Center & Weill Cornell Medicine, New York, NY; ³Burke Neurological Institute, White Plains, NY; ⁴Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY

The ideal surgical analgesic regimen has adequate efficacy, sufficient duration, and minimal side effects. Buprenorphine, a synthetic opioid (partial μ agonist and k antagonist), is a common analgesic used to manage postoperative pain in mice, but its duration of action is short, ranging from 4-12 h, minimally requiring administration twice daily. We chose to evaluate 2 buprenorphine products reported to have an extended duration of action: an FDA-approved highly concentrated buprenorphine (up to 24 h in cats) and an FDA-indexed extended-release buprenorphine (up to 72 h in mice), utilizing a validated mouse hind paw incision model. We aimed to determine a dose for these formulations resulting in analgesia for at least 72 h with minimal adverse effects, evaluating doses higher than have been assessed previously. Male C57BL/6J mice (6-8-wk-old; n=6 per group) were administered highly concentrated buprenorphine (10, 30, 60, or 100 mg/kg), extended-release buprenorphine (3.25, 9.75, or16.25 mg/kg), or saline, 1-2 min before hind paw incision. Mechanical allodynia and thermal hypersensitivity of the incised and non-incised paw (using von Frey and Hargreaves tests, respectively) and clinical parameters were assessed 4 and 1 d before, then 4, 24, 36, 48, 60, 72, and 96 h after surgery. Open-field tests quantified activity levels 4 d and 1 d before surgery, then 30 min, 1, 2, 4, 12, 24, and 48 h after treatment. Buprenorphine plasma concentrations were also measured by liquid chromatography-mass spectrometry at 1, 4, 24, 36, 48, 60, 72, and 96 h after treatment. Plasma concentrations were maintained above the minimum therapeutic levels of buprenorphine for up to 48 h for highly concentrated buprenorphine at 30 mg/kg and up to and 36 h for extended-release buprenorphine at 3.25 mg/kg; therefore, these were determined to be the lowest doses to attenuate pain in the incised paw for 72 h with no adverse effects or significant changes in weight or activity.

P351 Evaluation of Compassion Fatigue in Laboratory Animal Personnel During the COVID-19 Pandemic

SE Thurston*^1,2, G Chan¹, LA Burlingame¹, J Jones¹, PA Lester¹, T Martin^1,2

¹Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI; ²Refinement and Enrichment Advancements Laboratory, Ann Arbor, MI

Compassion fatigue (CF) is commonly observed in care-associated professions, including both human and animal care. The COVID-19 pandemic challenged laboratory animal research institutions to implement new guidelines while maintaining essential animal care operations. These ranged from shift changes to last-resort measures such as culling animal colonies to comply with reduced staffing regulations. As a result, laboratory animal personnel experienced increased stress, isolation, and helplessness, all of which could increase CF risk. To gauge whether CF had risen among laboratory animal personnel due to the pandemic, 200 individuals involved with animal research were recruited for an online survey. The survey consisted of questions examining professional quality of life, self-assessed levels of CF, institutional changes, perceived changes in animal welfare, and institutional measures enacted to alleviate CF. 86.4% of participants experienced CF at some point in their career, with 41.2% experiencing a CF event (new or worsening symptoms of CF) during the pandemic. 89.7% of participants that reported a CF event also reported an impact on their personal or professional lives as a result. Health, employment, and animal-related stressors that arose due to the pandemic significantly impacted CF scores. 95.7% of respondents were considered essential workers, but 67% did not feel as valued for their work as other essential personnel. 88% of personnel responsible for the euthanasia of healthy animals who experienced a CF event also reported that CF impacted their personal life, professional life, or both. 78.2% responded that interventions from internal CF programs or leadership did not help alleviate CF symptoms. The COVID-19 pandemic and resultant institutional changes will likely have lasting impacts on individuals and organizations. By determining and subsequently mitigating sources of CF, we can better assist the laboratory animal community through future crises.

P352 Optimizing a Rat Surgical Model for Recurrent Laryngeal Nerve Regeneration Strategies

SK Fudge*, JL Schmid, R Akin, C Seiller, K Osman, S Kington, R Thompson, C Smith, N Nichols, T lever

University of Missouri, Columbia, MO

During anterior neck operative procedures in human and animal patients, the recurrent laryngeal nerve (RLN) is at risk for surgical sacrifice and iatrogenic injury, leaving a gap in the nerve that requires nerve grafting. However, recovery of the RLN may take several months to years and full return of normal physiologic function rarely occurs, resulting in lifelong dyspnea, dysphagia, and dysphonia. Therefore, we sought to establish a rat surgical model of RLN transection with nerve grafting repair to optimize our novel intraoperative vagal nerve stimulation (iVNS) treatment strategy, with the goal of restoring normal laryngeal function. In our pilot study, we performed ventral neck surgery on 10 Sprague Dawley male rats, which all underwent right-sided RLN transection and conduit repair with hydrogel. All 10 rats were food restricted for 4-6 h prior to being anesthetized intraperitoneally with Ketamine + Xylazine for the microsurgery and laryngoscopies. During the surgical procedure, 5 of the 10 rats had iVNS for 30 min immediately after nerve repairment, while the remaining 5 rats underwent sham-treatment for 30 min without electrical stimulation. Behavioral assays were performed to determine upper airway function prior to and following surgery; laryngoscopy, video-fluoroscopy, and whole-body plethysmography were used to assess vocal fold (VF) motion, swallow function, and respiratory function, respectively. The immediate post-surgical results showed that RLN transection created ipsilateral VF paralysis and laryngeal adductor reflex (LAR) impairment. Thus far, our preliminary data demonstrates evidence of bilateral VF movement and improved LAR/airway protection in both groups of rats, but to varying degrees. Monthly testing will continue until 4 mo post-surgery. Additionally, our plan is to create another cohort to increase group sample sizes for statistical analysis. This study is a work in progress for surgical protocol refinement and method development to promote long-term survival and analyze in-depth characterization of neuromuscular recovery and treatment effects. We envision this rat surgical model of RLN transection with nerve grafting repair will serve as a translational platform to investigate a variety of therapeutic interventions that may ultimately accelerate and improve restoration of laryngeal functions in human and veterinary patients.

P353 Sex Differences in the Role of Vascular Endothelin-1 in Diabetic Kidney Disease

SD Greene*^2,1, SN Biswal², CD Miguel²

¹Animal Resources Department , University of Alabama at Birmingham, Birmingham, AL; ²Section of Cardio-Renal Physiology and Medicine, Division of Nephrology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL

Renal diseases progress faster in males than females; however, diabetic kidney disease advances at a similar rate in both sexes. In type 1 diabetes, little or no insulin is produced, resulting in high blood glucose. Endothelin-1 (ET-1) is a vasoactive peptide critical in kidney injury. In the kidney, endothelial and tubular cells produce ET-1. ET-1 is important in diabetic kidney disease; however, whether there are sex differences in the role of vascular ET-1 in the development of diabetic nephropathy remains unclear. We hypothesized that lack of vascular ET-1 attenuates kidney damage in males and worsens it in females. Diabetes was induced in male and female vascular endothelial cell ET-1 knockouts (VEET KO) and floxed ET-1 mice with streptozotocin (STZ, 50 mg/kg i.p., 5 consecutive days). Hyperglycemia (glucose > 250 mg/dl) was confirmed 10 d after the last injection, and no insulin pellet was implanted. 24-hour urine production and water consumption were measured at the end of the study to assess kidney injury. During the 10-wk study, the mice were weighed, and health status was documented twice/week. At the end of the study, levels of glycemia were similar between the sexes. After 10 wk of diabetes, male VEET KO mice displayed similar urinary protein excretion (floxed ET-1 vs VEET KO: 1.606±0.346 vs. 1.926±0.429 mg/ml n = 4-6/group, P = 0.8536), increased nephrin excretion (6.252±2.320 vs 40.118±10.757 mg/ml n = 3-5/group, P = 0.0196) and increased KIM-1 excretion (608.810±183.298 vs 2740.415±938.095 mg/ml n = 6/group, P = 0.0084) compared to the floxed ET-1 control mice, indicating that they developed diabetic kidney damage. On the other hand, female VEET KO mice presented decreased values for urinary excretion of protein (3.186±0.907 vs. 0.8631±0.183 mg/ml n = 10/group, P= 0.0218), nephrin (17.25±4.813 vs. 5.830±2.244 mg/ml n = 8/group, P = 0.0495) and KIM-1 (1277±250.767 vs. 555.9±118.742 mg/ml n = 8/group, P = 0.0210). In conclusion, these results demonstrate that vascular ET-1 has a different role in males and females during diabetes, where it seems to be protective in males and detrimental in females.

P354 Cell Injection Technique Impacts the Shape and Rate of Tumor Growth in a Syngeneic Tumor Mouse Model

T Gonzales, C Pyles, E Dunkin, K Mayer, L Loos-Austin, E Slosarek, C Atwood, S Vadakkadath Meethal*

Heartland Vivarium, Fitchburg, WI

Syngeneic mice are a widely used model of carcinogenesis for drug testing. We examined how cell injection and tumor measurement techniques impact the growth curves and tumor ulceration of a breast cancer cell line in a syngeneic model. Female C57/Bl6 mice (n = 40) were injected with EMT6 breast cancer cells (1 million cells/mouse) into the sub-dermis of the right flank by four animal technicians (n = 10 each), and tumor size and ulceration determined on post-injection d 2, 4, 7, 9, 11, 14, 16, 18, 21, and 23 using the same caliper. Tumors formed in all mice. Spherical tumors with the lowest standard deviations (as measured by all 4 technicians) were obtained from injectors that were medium and highly skilled but not from the first-time injector suggesting that injection technique greatly impacted the resulting tumors. Mice injected by the first-time injector revealed no ulceration (curve of best fit: y = 660 - 383x + 79x² - 7x³ + 0.29x⁴ - 0.0044x⁵; R² = 0.99; n = 15) likely due to the spreading of cells into different sub-dermal areas. In medium and highly skilled injectors where tumors were localized to one area, 80% and 100% of tumors, respectively, became ulcerated by d 23 (curve of best fit: y = 15.40e^0.17x; R² = 0.99; n = 25). A simple correlation analysis indicated that enhanced growth rate between days 16-23 predicted increased ulceration rate (85%). These results suggest that injection technique greatly impacts the shape of the tumor, its exponential growth, and its ease of measurement. Furthermore, growth rate analysis can be predictive of ulceration in this syngeneic tumor model, and that fast-growing tumors are more likely to ulcerate. Future studies to assess the role of cell number on the rate of tumor growth and the timing of ulceration are warranted.

P355 Assessment of Blood Pressure in Total Body Irradiated Rhesus Macaques (Macaca mulatta)

S Achilles*¹, JD Olson², M Cline¹

¹Animal Resources Program, Wake Forest University College of Medicine, Winston-Salem, NC; ²Pathology/Section on Comparative Medicine, Wake Forest University College of Medicine, Winston-Salem, NC

Elevated blood pressure (BP) affects over 1 billion people globally and is one of the major risk factors for cardiovascular events such as stroke. In humans, BP increases with obesity and age. There is also evidence that circulatory disease morbidity and mortality is associated with radiation exposure. Nuclear accidents and acts of terrorism have the potential to expose thousands of people to high-dose total body irradiation (TBI). Those who survive the acute radiation syndrome are at risk of developing chronic, degenerative radiation-induced diseases. Determining whether changes in BP levels occur as a result of radiation exposure could improve our understanding of humans that have experienced radiation exposure and have experienced these outcomes. We hypothesized that radiation would increase BP in a dose-dependent way. In this work, we utilized a cohort of nonhuman primates [(NHP); rhesus macaques, Macaca mulatta] long-term survivors of a single high dose TBI (1.1-8.5 Gy, n = 132) and controls (n = 38) to evaluate the effects of TBI on BP and obesity. NHP were males between 3 and 22 y of age (median 9 y). BP was measured 1-14 y post-irradiation (median 4 y). NHPs were sedated with a combination of 15mg/kg ketamine and 0.1mg/ kg midazolam, and systolic, diastolic, and mean arterial pressures were measured using a high definition oscillometer (HDO). Obesity was defined by dual-energy X-ray absorptiometry (DEXA) body fat percentage >35%. Statistical analysis indicated significant increases in BP with increasing age, with a magnitude effect of ~15mmHg difference of systolic BP in adult vs adolescent animals. Obese animal BP was 10-13mmHg higher than non-obese animals. There was no significant effect of TBI either alone or as a modifier of other factors. We conclude that TBI does not increase BP, but our findings suggest the potential of the rhesus macaque as a model for obesity and age effects on BP.

P356 A Small Animal Model for the Study of HIV Cardiovascular Comorbidities: The HIV-1 Transgenic Rat

S Williams*^1,2, MD Worthington², CC Krauss², M Awolowo², F Benedetti¹, H Davis¹, D Zella¹, J Bryant¹, F Denaro^2,1

¹Institute of Human Virology, University of Maryland, Baltimore, MD; ²Department of Biology, Morgan State University, Baltimore, MD

Great strides have been made in the treatment of HIV infection, but patients on antivirals can still display noninfectious HIV comorbidities. Presently, there are few animal models to examine the multi-system-comorbidities found in HIV infection, including cardiovascular comorbidity. It is believed that these comorbidities occur because of incomplete viral eradication, with low levels of virus or HIV proteins (GP120) that can still bind to cellular receptors (CXCR4). This then initiates a series of events that leads to cell dysfunction and cell death. A model in which the comorbidities can be studied is the HIV-1 Transgenic rat (HIV-1TGR). It has been demonstrated that the rat possesses a CXCR4 receptor capable of binding to GP120. As a result, host/viral protein interactions can be examined at the level of GP120 and cell receptor CXCR4. This then provides a hypothesized mechanism for this observed cardiovascular pathology. Hearts were examined for signs of pathology in correlation with CXCR4 expression. Histological examination with H&E (5 animals approximately 1 y in age) revealed inflammatory foci, myocyte dropout, and fibrosis. Immunohistochemistry to nitrotyrosine revealed positive cells in areas of inflammation. Immunocytochemistry to GP120 revealed positive lymphocytes but no positive myocytes. Immunocytochemistry to CXCR4 displayed a number of positive cells with a dendritic morphology. These cells were found in areas of inflammation as well as in areas without inflammation. The morphology of CXCR4 positive cells is suggestive of either macrophages or the resident cardiac dendritic cells. Additional characterization of this cell type is critical for understanding the observed pathology. CXCR4 positive cells found in the HIV-1TGR heart suggest a possible mechanism for the production of pathology. GP120 binding to these cells could initiate a series of events leading to free radical stress and bystander effect of myocytes and other cells.

P357 Investigating the Role of Scavenger Receptor CD36 in the Pathogenesis of Lyme Borreliosis

S Maddur Ganesan*¹, AL Armijo¹, AL Aguilar¹, YL Feng¹, JG Fox¹, S Carrasco^1,2

¹Division of Comparative Medicine, Massachusetts Instiute of Technology, Cambridge, MA; ²Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY

Lyme disease is the most common vector-borne disease in the United States. It is caused by the spirochete Borrelia burgdorferi (Bb) and transmitted via infected Ixodes ticks. The disease causes chronic inflammation of joints, the heart, and nervous system. The mouse model of Lyme borreliosis (LB) has been instrumental to understand the pathogenesis of Lyme arthritis and carditis. Macrophages play a central role in host defense and modulating inflammation at different stages of LB. Scavenger receptors class-A and CD36 are receptors involved in phagocytosis of bacteria. CD36 is involved in oxidative stress, modulates inflammation and host defense against bacterial infections. Little is known about the function of CD36 in the pathogenesis of LB. This study aims to understand the role of CD36 in the pathogenesis of Lyme carditis and arthritis using the mouse model of LB. We confirmed CD36 expression in peritoneal macrophages (PMs), cardiomyocytes, synovial cells, and resident macrophages in tibiotarsal joints of mice. Further, fluorometric-based phagocytosis assay showed significantly decreased phagocytosis of spirochetes in CD36-deficient (CD36^-/-) primary PMs ex vivo. Blocking assays using anti-CD36 antibody markedly reduced phagocytosis of spirochetes by PMs. To test the role of CD36 in vivo, C57BL/6J and CD36^-/- mice, 8 to 11 in each group, were subcutaneously infected with Bb and necropsied at 14 and 28 d post-inoculation (dpi). During the early phase of infection (14dpi), we found that CD36^-/-mice exhibited similar bacterial burden and histopathology in heart and joints. In contrast, arthritis and aortitis/periaortitis were significantly increased in CD36^-/- mice during late LB (28 dpi). Bacterial burden was significantly elevated in the heart but similar in the joints of infected CD36^-/- mice at late LB. Cytokine and chemokine analysis from 28 dpi revealed a significant increase in several proinflammatory cytokines and macrophage and lymphocyte-derived chemokines in CD36^-/- mice. In summary, our results suggest that CD36 mediates phagocytosis of Bb and plays a role in downregulating inflammation during the late phase of LB.

P358 Transepithelial Nasal Potential Difference Measurement in a Ferret Model of Cystic Fibrosis

TR Yellowhair*¹, S Raju², M Smith³, A Werts¹

¹Lovelace Biomedical Research Institute, Albuquerque, NM; ²University of Alabama at Birmingham, Birmingham, AL; ³Spirovant Sciences, Philadelphia, PA

Determining the translational potential of current assays for cystic fibrosis (CF) severity in novel preclinical models is challenging. Over 2,000 mutations in the human cystic fibrosis transmembrane conductance regulator (CFTR) gene cause this disease, with different mutations resulting in a range of decreases in CFTR function. A common in vivo diagnostic tool for CF is a transepithelial nasal potential difference (NPD) measurement, which can detect underlying dysfunction in CFTR chloride channel activity. The CFTR-deficient ferret has emerged as a valuable preclinical model for CF research as it shares similar lung physiology and comparable CF phenotypes to humans. In an attempt to assess the translational utility of NPD in this ferret model, we measured total CFTR activity in wild-type (WT, n = 3 female) and CFTR-deficient (n = 4; 1 male, 3 female) ferrets using a modified human NPD protocol. Changes in voltage across the nasal mucosa were recorded following infusion of 1) Ringer’s solution, 2) Ringer’s solution with sodium channel inhibitor amiloride (100 µM), 3) chloride-free ringers, 4) CFTR-agonist forskolin (20 µM), and CFTR-specific inhibitor GlyH101 (10 µM). We determined that CFTR-deficient ferrets present with electrophysiologic tracings similar to human CF patients. Compared to WT ferret recordings, CFTR-deficient ferrets exhibited a more negative baseline potential difference, a depolarizing response to amiloride, and an absence of hyperpolarization following perfusion with chloride-free Ringer’s and forskolin. To our knowledge, these preliminary data are the first to report NPD measurements in the CFTR-deficient ferret model, supporting the utility of NPD as an important preclinical assay. Future studies will utilize NPDs to detect improvements in CFTR function secondary to novel test articles such as small molecules and gene therapies and help predict outcomes in CF patients.

P359 Nutrient Stability in NASA Spaceflight Experiment Rodent Food Bars

T Marsh*¹, V VERMA²

¹Space Biosciences, KBR/NASA, Moffett Field, CA; ²Space Biosciences, ASRC Federal/NASA, Moffett Field, CA

Nutrient-upgraded rodent food bars are the standard diet for mice involved in spaceflight experiments aboard the International Space Station as part of the National Aeronautics and Space Administration’s Rodent Research Project. These food bars are a twin-screw extruded American Institute of Nutrition 1993 Growth phase diet-based product with nutrient additives to preserve a healthy and complete diet for the animals, and the bar form factor is necessary for use in the microgravity environment. Due to the nature of spaceflight projects and the lengthy production process of the food bars, a shelf-life assessment was undertaken to determine nutrient stability over an extended storage time. To assess palatability and stability over an extended duration, a panel of lipid oxidation markers and both fat- and water-soluble vitamins were assessed under various time and temperature conditions via Association of Official Analytical Collaboration International standardized methodology. Thiamine was the least stable vitamin at room temperature. Bars that had been continually kept at 4 °C (t=6, 12, 27 mo) showed significantly higher levels of thiamine (via student’s t-test) compared to all other bars that underwent some degree of ambient temperature storage. This suggests that refrigeration is a key component in maintaining adequate nutrient levels in these relatively high moisture (average 29.3%) food bars. Also of note was the level of vitamin A in the form of retinol. There was no correlation to storage temperature, but rather the only factor having an effect on retinol levels was time. A slow yet consistent decrease in retinol was observed over the course of the experiment, though at no time did the retinol levels drop below the rodent guidelines from the National Research Council’s Nutrient Requirements for Laboratory Animals. In conclusion, over the 27-mo time course, all analyzed vitamins and lipid oxidation factors remained relatively stable and at levels that provide fully sufficient nutrition to rodents during spaceflight. Some groups, however, displayed higher-than-expected aerobic and anaerobic bacteria colony-forming units, which is believed to stem from a since-revised handling technique.

P360 Use of Groβ with AMD3100 to Mobilize Hematopoietic Stem Cells in the Nonhuman Primate Model

U Stasula*^1,2

¹College of Veterinary Medicine, University of Illinois at Urbana Champaign, Chicago, IL; ²Cellular and Molecular Therapeutics Branch, NHLBI, National Institutes of Health, Bethesda, MD

Hematopoietic stem cell (HSC) transplantation using a mobilized, apheresis-harvested graft is associated with increased cell collection efficiency, improved cell quality, and better patient outcomes compared with marrow-derived grafts. Granulocyte colony-stimulating factor (G-CSF) and plerixafor (a CXCR4 antagonist) are standard agents used alone and in combination to mobilize stem cells from marrow into the circulation, but poor mobilization responses can occur, necessitating multiple apheresis procedures. Importantly, G-CSF administration can also precipitate pain crises in sickle cell patients. Groβ, a CXCR2 agonist, has recently been evaluated in animal models to mobilize HSCs, and no adverse effects were identified. We sought to determine if co-administration of Groβ and plerixafor could effectively mobilize CD34+ HSCs in nonhuman primates. Nine rhesus macaques (6m/3f) received 0.5 mg/kg Groβ SQ alone (n = 9), 1 mg/kg plerixafor SQ alone (n = 9), or Groβ plus plerixafor with plerixafor administered 30 min prior to Groβ (n = 9). Serial blood samples were collected for flow cytometric analysis. Mean(SD) peak CD34+ counts were 33(16)/uL (Groβ alone), 231(99)/uL (plerixafor alone), and 365(353)/uL (combination),representing an increase of 1.1(0.8)-fold (Groβ), 8.9(3.0)-fold (plerixafor), and 29(44)-fold (combination) in circulating CD34+ cells, compared to baseline unstimulated counts. Peak mobilization occurred at 0.9(0.4) h (Groβ), 4.7(1.0) h (plerixafor), and 3.2(1.6) h (combination). The CD34+CD45RA+ subset mobilized by 45 min, reaching a plateau at 4-6 h. The CD34+CD45RA+CD117+CD90-subset, representing an immature CD34+ progenitor cell population, had the fastest and highest relative mobilization peak. Whereas G-CSF mobilization is associated with an early, paradoxical neutropenia, this effect was not observed for either agent. Our results demonstrate that Groβ plus plerixafor is a rapid, effective HSC mobilization regimen, resulting in a synergistic 29-fold increase in CD34+ cell numbers in nonhuman primates. Groβ plus plerixafor may prove to be an optimal mobilization regimen in humans. Studies to determine Groβ plus plerixafor safety and efficacy are ongoing in human subjects.

P361 Molecular Hydrogen Protects Mouse Sperm Mitochondria from Oxidative Stress and Improves Sperm Motility

Y Noda*¹, T Nemoto¹, T Endo¹, I Ohsawa²

¹Animal Facility, Tokyo Metoropolitan Institute of Gerontology, Itabashi-ku, , Japan; ²Biological process of aging, Tokyo metropolitan institute of gerontology, Itabashi, , Japan

Oxidative stress caused by the imbalance between reactive oxygen species (ROS) and biologic antioxidant system leads to increased damaged human sperm and subsequent male infertility. Because molecular hydrogen (H₂) acts as therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals, we hypothesized that H₂-treatment is beneficial for the prevention of oxidative stress-induced impairment in mice sperm. To investigate the effect of H₂ on injured sperm, they were collected from the epididymis tail of B6D2F1/Crlj mice (12-15-wk-old, 3 males) and suspended in TYH medium. One hour after preincubation, it was made into a sperm suspension. Hydrogen peroxide diluted with water was added to the sperm suspension to a concentration of 0.3 mM, and the sperm suspension was treated with. In addition, they were incubated for an additional 20 min with or without H2 saturated culture. To assess sperm damage, the number of untreated motile sperm was compared to the number of motile sperm in the damaged sperm. The H2 saturated culture solution was added, and the H2 functional recovery effect was observed. To further investigate the H2 effect on ROS-dependent mitochondrial damage, we stained sperm with Nonyl Acridine Orange (NAO), mitochondrial cardiolipin binding dye, and MitoTracker Red, mitochondrial membrane potential-dependent dye, and observed them with STED (Stimulated emission depletion microscopy) super-resolution microscope. The structure of sperm was observed by SEM (Scanning Electron Microscope). Fresh sperm (motility rate: 82.4%) were treated with hydrogen peroxide, resulting in damaged sperm with low motility rate (14.6%). H₂-treatment significantly restored their motility rate (63.9%), accompanied by improvement of intrasperm ATP content. Damaged sperm fertilization rate was markedly improved from 37.6% to 59.2% by H₂-treatment. It was defined as the percentage of fertilized eggs cleavage 24 h after insemination. Transfer of 2-cell stage embryos obtained from H₂-treated damaged sperm showed normal ontogeny (94.6%). SEM imaging and STED imaging of NAO-stained sperm showed mitochondrial appearance and intra-mitochondrial cristae network, respectively. Treatment of sperm with hydrogen peroxide-induced a decrease in mitochondrial membrane potential. Because of the rapid diffusion and high membrane permeability, H₂ can reach and react with intrasperm ROS, including hydroxyl radical, possibly in mitochondria and improve low sperm motility. Our results strongly suggest that H₂ is a new promising tool for male infertility treatment.

P362 Development of a Minimally Invasive Skin Swab Protocol to Quantify Corticosterone Levels in Skin Secretions of African Clawed Frog (Xenopus laevis)

Z Bault*, DL Hickman

Laboratory Animal Resource Center, Indiana University School of Medicine, Indianapolis, IN

African clawed frogs (Xenopus laevis) are an important research model used in studies investigating genetics, neurobiology, ophthalmology, cardiology, embryology, and tissue regeneration. Over the past years, a large stride has been taken to improve animal welfare of vertebrates used in biomedical research through behavioral testing; however, Xenopus has been one vertebrate model that has had little investigation. Xenopus live in wetlands, ponds, and lakes in the wild, and to replicate or provide enrichment similar to these in a lab setting is challenging. A factor that makes it difficult to gauge if proposed housing and enrichment is beneficial to Xenopus is the lack of an assay to consistently determine if the frog is experiencing stress. Quantifying stress metabolites can be challenging in Xenopus due to poor venous access to collect blood antemortem and water contamination of any feces or urine produced from the frog. This project aims to develop an assay that can consistently quantify corticosterone in skin secretions of Xenopus using a minimally invasive swab technique that has been successfully used in other Anuran species. Samples were collected by swabbing a 2.54 cm (1 in) along the mid dorsum using a sterile cotton-tipped swab, and corticosterone levels were measured using a competitive enzyme-linked immunosorbent assay (ELISA). We measured if the assay could detect corticosterone after swabbing six acclimatized frogs at 0, 6, 24, and 60 h during a trial period. The data collected does show that corticosterone can be quantified using this swab technique with an average range of 6.6 to 26.12 pg/ml. Short-term goals are to refine this assay for optimal and consistent results and evaluate corticosterone levels’ response when the frogs are exposed to ACTH hormone as a positive control. Long-term goals include using this assay along with other parameters of stress to optimize Xenopus housing conditions.

P363 PDX and CDX Tumor Growth Comparisons in the Ultra-immunodeficient NOD.CB17-Prkdc^scid IL2rg^tm1/Bcgen (B-NDG) Mouse Model

SJ Wildt, M Melton*, T Rothrock

Veterinary Services, Envigo, Indianapolis, IN

We sought to characterize a new immunodeficient model to better support cancer research and the development of new therapies. The B-NDG (NOD.CB17-Prkdc^scid IL2rg^tm1/Bcgen) mouse model was designed and generated by a company located in Beijing, China. The entire coding region of the IL2rg (common gamma) gene was deleted (exons 1-8) using CRISPR technology. The model was generated on an inbred NOD-scid genetic background and no backcrossing was required. The B-NDG mouse model lacks mature B cells, T cells, NK cells, and has a deficiency in cytokine signaling. This triple immunodeficiency enhances tumor cell acceptance and results in decreased leakiness as compared to other SCID models. There is also a high humanization capability for human immune cell engraftment with minimal rejection. Five female B-NDG, NSG, and Athymic nude mice were implant subcutaneously on the flank with tumor cells. Tumors were measured and mice weighed twice per week. We describe results for several tumor model comparison studies between the B-NDG mouse model and other commonly used immunodeficient models, including NSG^TM and Athymic nude mice. The tumor models studied, include but are not limited to, the PDX breast cancer model, WHIM5, the PDX melanoma model, WM4071-2, the PDX acute myeloid leukemia model Kasumi-1, and the CDX non-small cell lung cancer model NSCLC. Results: In all studies described, the B-NDG mouse model performed better than Athymic nude mice and the same as or better than the NSG^TM mouse model.

Platform Sessions

PS1 How Lean Management Helped One Institution Remain Agile during the COVID-19 Pandemic

LA Ingram¹, DM Molk¹, C Warren², GM Cronin*¹

¹Center for Comparative Medicine, Mass General Hospital, Charlestown, MA; ²CCM, Massachusetts General Hospital, Charlestown, MA

In March 2020, COVID-19 forced our institution, like so many others in our industry, to make major changes quickly to the way we operated our facilities and provided the care and services to our research community. Contributing factors included but were not limited to 1) a limit of onsite personnel to essential employees, 2) a limit of research efforts while supporting new research associated with COVID-19, 3) new 6-ft minimum physical distancing practices, 4) continuous mask wearing, and 5) accommodations associated with greater than 6 feet distancing when eating without a mask. These mandated safety measures required a quick and drastic change to staffing numbers and scheduling. We follow a lean management operations business philosophy which proved to be a key contributor to successfully demonstrate the required agility necessary to meet the emerging and rapidly changing mandates. These critical management principles and tools facilitated our ability to make streamlined changes from a 5- to 7-d workweek; established mechanisms for collecting and sharing employee feedback; supported evaluation of performance standards for changes to rodent cage change practices; and allowed for constant metric collection.

PS2 How to Find a Project Management and Scheduling Tool that Works for your Team

A Holk*, RE Cooper

Physiology, Lovelace Biomedical, Albuquerque, NM

Research using animal models is complex and requires the coordination of many uniquely skilled individuals, including technicians, investigators, administrators, and clinicians. Organizing such a large group of people toward a common goal can be a challenging and at times overwhelming process. At a midsized CRO scheduling 10-20 new studies a week with 200 personnel across 10 departments, miscommunications between teams, ineffective scheduling programs, and rapidly evolving study schedules led to a search for more inclusive and adaptive scheduling tools that both address traditional project management needs and meet the more unique challenges common to the animal science industry. We used 7 major criteria to compare some of the most popular project management tools on the market, including the ability to incorporate into current workflows, automation, flexibility to meet a wide range of needs, ease of use, processing power to handle large studies including interdepartmental coordination, customer service, and affordability. There are limited tools that are focused on the specific complexities of organizing and scheduling people around animal work. However, based on the criteria we set forth, a small handful of tools struck a successful balance between affordability and functionality, and were translatable from the standard corporate model to animal science teams. While many tools available offered beautiful graphics and user-friendly technology, the tradeoff was oftentimes failure to accommodate a wide variety of study designs or to handle large, interdepartmental studies. Customized solutions to these issues were often available but cost-prohibitive. We identify some of the unique challenges encountered when applying project management tools to animal science teams, discuss the features of some of the most popular programs available today, and characterize the common pitfalls to avoid when looking for a program that best fits your team’s needs.

PS3 Husbandry and Veterinary Considerations when Facilitating Gut Microbiota Research

P Arreola*, A Buehler, J Finlay, NN Lee, R Ermel

Center for Comparative Medicine, City of Hope, Duarte, CA

Conducting a study involving gut microbiota (GM) requires meticulous coordination between the researcher, husbandry, and veterinary technicians in order to be successful. Scientific curiosity in the GM has been increasing as it has shown to modulate immunologic and physiologic responses that determine underlying pathogenesis of several systemic diseases such as diabetes and colorectal cancer. The GM can shift with changes in experimental manipulations, husbandry practices, and veterinary interventions; thus, it is important to reduce variabilities introduced by often well-intended inadvertent changes. This abstract was created to better communicate husbandry and veterinary considerations when facilitating GM studies. Our study required several weekly food and/or water alterations to see GM changes, so it was important to have exceptional communication from all involved staff. Problems arose at the start of the study because husbandry staff was uncertain of what to do if emergencies like floods and low water levels were to be seen. To avoid miscommunication and introducing unintentional variabilities, details of the study and personnel involvement were relayed to all parties with several different forms of communication. We communicated with chow type stickers on the cage cards, marking water levels on bottles, cage cards indicating cage and water changes, and emails between management, husbandry, and veterinary staff. Lastly, the PI provided a summary of the study timeline which was shared with the research support staff. The active verbal and written communication between all parties allowed for a successful completion of the GM study while minimizing unintended experimental variabilities with positive feedback from the research support staff. It is important to keep husbandry factors consistent and this further highlights the importance of clear communications between all parties involved when conducting GM studies.

PS4 Caught in the Middle: What Veterinary Residents Learn About Relationship-centered Care

M Nowland*¹, P Haidet³, TL Whitcomb²

¹Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI; ²Comparative Medicine, Penn State Hershey College of Medicine, Hershey, PA; ³Department of Medicine, Division of Internal Medicine, Penn State College of Medicine, Hershey, PA

The practice of relationship-centered care (RCC), which emphasizes development of authentic partnerships between clients and veterinarians, is crucial both for the mental wellbeing of veterinarians and for client satisfaction. Mixed messages around this concept are common in undergraduate veterinary education, but the post-graduate environment has not been assessed. The purpose our qualitative study was to explore what messages about RCC residents are internalizing in laboratory animal medicine (LAM) training programs. We interviewed 24 residents from seven LAM training programs in the Eastern United States. Four themes and 3 sets of subthemes emerged from qualitative analysis of the de-identified interview transcripts. Themes included: 1) responsibility for maintaining relationships with researchers (building rapport, practicing clinical empathy, overcoming language barriers, communicating in the “authorized” way, and navigating e-mail limitations), 2) challenges with professional identity (acting as the middleman between stakeholders, overcoming the stigma of the policing role, experiencing a lack of power to impact change), 3) operating within a culture of conditional value of veterinary knowledge, and 4) outlets for frustration (reliance on residency mates, limitations of venting). Our findings suggest that residents find themselves caught in the middle of an imperative to practice relationship-centered care, while at the same time balancing the conflicting demands of stakeholders and experiencing a lack of power to influence change, all within a culture in which their knowledge is frequently undervalued. These results have implications for the learning of RCC and provide an important background for refining curricula and creating effective support systems for residents.

PS5 Replacing Analog Husbandry Room Logs with a Flexible Tablet-based Digital Solution

TR Meier*, N Rindels, J Willhite

Comparative Medicine, Mayo Clinic, Rochester, MN

Animal research facilities are required to document a variety of animal husbandry activities. These activities are often documented on weekly or monthly room logs that are affiliated with a room or area. Room logs serve as a guidance document for staff as they outline what tasks need to be completed on a given day. Management uses room logs to document adherence to standard operating procedures and manage personnel performance. Our institution has multiple animal facilities in multiple buildings; every animal holding room has at least 1 or more room log. Manual review of paper-based logs is a time-consuming and inefficient way to monitor operations. At our institution, we leveraged our existing Filemaker application to create a completely self-supported and customizable digital method of tracking husbandry activities. The staff is assigned to rooms to support, species-specific tasks are then assigned to the rooms, and a dashboard outlines the tasks that are due, overdue, and completed for each room in the facility. Within the application, the animal care staff role is limited to task completion. The management role can complete tasks as well as create tasks and add, edit, or delete them from a given digital room log. The application was designed to operate without the ongoing need for a developer. Staff were issued tablets and provided 1 h of training on the system prior to converting to digital documentation. Feedback from animal care staff has been positive. They report feeling more confident that they have completed all of their work. In addition, management can review whether there are any overdue tasks in their facilities in less than a minute. Room Tasks also tracks cage changing volumes making it an important tool for monitoring weekly/daily changing volumes as a component of our staffing-to-workload and employee safety model. Overall, Room Tasks has simplified the documentation of activities for front-line staff, simplified monitoring staff performance for management, and provides a mineable database for metrics of interest.

PS6 Comparison between Artificial Sentinels and Soiled Bedding Sentinel Mice to Refine the Health Surveillance Process

AJ Osborne*¹, CL Perkins²

¹Animal Resources Program, University of Alabama at Birmingham, Birmingham, AL; ²Laboratory Services, Charles River, Wilmington, MA

The 3Rs are guidelines for research that encourage the most humane and most scientifically relevant information simultaneously. Health surveillance of research colonies is necessary to ensure an appropriate health status and to prevent a potential impact on research. However, traditional health surveillance with soiled bedding sentinel (SBS) mice often does not coincide with the 3Rs; at the end of a predetermined period, the mice are euthanized for sample collection. Furthermore, the literature documents that many infectious agents do not successfully transfer to SBS mice. To reduce animal use and improve the efficacy of health monitoring programs, many institutions are seeking to replace live mice with environmental testing or other artificial sentinel means. A pilot project was initiated to compare the effectiveness of artificial sentinel samples (flocked swabs or filter media) to a traditional SBS mouse. Our hypothesis is that the artificial sentinel sampling will be just as effective if not better than SBS samples for monitoring organisms of interest (specifically Helicobacter spp. and MNV). Soiled bedding from 2 rooms historically positive for MNV and Helicobacter spp. was split between a SBS mouse cage and an artificial sentinel cage at each weekly cage change. Once weekly a flocked swab was added to each artificial sentinel cage, and twice weekly these cages were manually agitated for 15 s. Media was exposed for the duration of the study. At the conclusion of the 12-wk experimental period, samples were collected including the flocked swabs, media filter, filter top cage lids, and fur swabs, oral swabs, and Opti-spot blood from the SBS mice. Artificial sentinel samples were more sensitive at detecting Klebsiella oxytoca, Rodentibacter spp. (R. heylii and R. pneumotropicus), Demodex genus, Helicobacter spp., and Tritrichomonas genus as compared to the SBS mouse samples. Artificial and SBS sentinel samples were equivalent at detecting MNV, Astrovirus, and Entamoeba genus. Overall, this pilot study shows that usingartificial sentinels for health surveillance is just as sensitive and even surpasses SBS mice, allowing opportunity for refinement in techniques and reduction of live animal use.

PS7 Clopyralid Herbicide in Laboratory Animal Bedding and Feed Waste

DM Kurtz*¹, P Johnson², W Steinmetz², G Caviness¹, P Poliachik³, D Gaffney¹, RA Wiltshire¹, K Laber¹

¹Comparative Medicine Branch, National Institute of Environmental Health Science, Research Triangle Park, NC; ²Health & Safety Branch, NIEHS, RTP, NC; ³Facilities Operations Team, NIEHS, RTP, NC

A rodent-only facility housing approximately 19,000 cages was generating approximately 24,000 lbs. of non-hazardous, bedding and feed waste per month which was transported to the municipal landfill. As part of our effort to reduce landfill waste, we started to compost this waste. Within a short period of time, we received notification from the composting company that our waste contained unacceptable concentrations (> 5.0 ppb) of the herbicide Clopyralid. Clopyralid (3,6-dichloro-2-pyridinecarboxylic acid) is a selective herbicide for control of broadleaf weeds used extensively on food-producing crops, tree plantations, and residential lawns. In the early 2000s, many states started reporting problems from gardeners using compost on a variety of numerous plant species. Analysis found the compost contained Clopyralid, and certain plants were extremely sensitive. Since then, many states have issued pesticide advisories, and Clopyralid has since been banned for use on residential lawns. Clopyralid is relatively non-toxic to mammals with an acute, oral LD₅₀ of > 5000 ppm. Previous testing of our standard, natural ingredient rodent feed by a commercial laboratory was negative for Clopyralid, and initial testing of our clean, unused bedding by the commercial lab (#2) recommended by the composting company was also negative. However, Clopyralid was detected by lab #2 in our bedding/feed waste (71–75.5 mg/kg) and subsequently in our standard rodent feed (73–172 mg/kg), as well as, another natural ingredient rodent diet (141 mg/kg). Removal of feed pellets from dirty cages prior to dumping the bedding waste did not significantly reduce the Clopyralid concentrations. Clopyralid likely has no physiological effects on our laboratory rodents at the concentrations measured. However, these findings demonstrate another example of a chemical contaminant to which laboratory animals may be exposed and underscores the importance of monitoring for external sources of experimental variability.

PS8 Implications of Lab Mouse Trap Preference on Your Pest Control Program

J Schoenberger*¹, BJ Prendergast², KR Luchins¹, B Theriault¹, G Langan¹

¹Animal Resource Center, University of Chicago, Chicago, IL; ²Department of Psychology, University of Chicago, Chicago, IL

Insects are potential vectors of disease for laboratory animals. Therefore, implementing a pest control program is a requirement for animal research facilities. The Guide for the Care and Use of Laboratory Animals emphasizes the humane use of traps, however, insect traps commonly use glues that can inadvertently ensnare escaped laboratory mice, leading to distress and injury. This presents a challenge for research facilities attempting to identify insect populations within animal housing barriers. In an effort to improve use of pest traps in animal facilities, we sought to characterize behavioral interactions of lab mice with common insect and mouse traps. Three experiments using different combinations of traps (glue trap, live trap with a clear viewing window, and live trap with a red-tinted viewing window) and arenas addressed these questions. Mice were under direct video surveillance for all experiments and were immediately euthanized if captured via glue trap. Experiments 1 and 2 were performed within a smaller arena, whereas experiment 3 was performed in an animal housing room within an SPF barrier facility. Measures included exploration of the test environment, grooming behavior, time spent near each trap, and the latency to capture by traps of each type. Data to be presented indicate that lab mice were captured significantly more quickly by live traps than by glue traps, and were far more likely to enter a live trap as compared to a glue trap. In addition, mice did not appear to differentiate between clear or red-tinted window live traps. Taken together the results support the conclusion that a live trap combined with a glue trap provides the best approach to capturing escaped mice and insects within a single environment.

PS9 Muzzle Swelling in a Nude Rat

AL Carlson*, S Monette

Memorial Sloan Kettering Cancer Center, New York, NY

An 8-mo-old, female, intact athymic nude rat (Hsd:RH-Foxn1^rnu) presented with an acute, right-sided, maxillary swelling of the muzzle. Four mo prior to presentation, the rat used as a model of Parkinson’s disease received a unilateral intracranial midbrain injection of 6-hydroxydopamine (6-OHDA) into the striatum. The rat was injected with D-amphetamine intraperitoneally 4 wk later to confirm depletion of dopamine and successful induction of the model. On physical examination, there was a poorly demarcated, firm, round, approximately 1 cm diameter swelling of the right maxillary region of the muzzle. The animal was bright, alert, responsive, able to eat and drink normally, and had a BCS of 3/5. Differential diagnoses included infectious, e.g., Staphylococcus aureus, and Rodentibacter spp., and neoplastic, e.g., squamous cell carcinoma and osteosarcoma processes. Treatment with enrofloxacin 10 mg/kg PO q 24 h was initiated. After 1 mo of treatment, the swelling had increased in size, measuring 2.5 x 1.7 x 1.5 cm and encompassing the right lateral and dorsal aspect of the muzzle, necessitating euthanasia. The rat was submitted for a complete necropsy. At necropsy, the mass was noted to infiltrate soft tissue as well as bone. Histologically, the lesion consisted of nests of squamous epithelial cells with abundant keratinization, consistent with a squamous cell carcinoma, which invaded and effaced the nasal and maxillary bones and the nasal and oral mucosae. The neoplasm appeared to have originated from the oral mucosa due to the greater involvement of the oral tissues compared to the nasal mucosa, but a nasal origin could not be definitely ruled out. Naturally occurring oral squamous cell carcinomas have rarely been reported in rats and have not been previously reported in the athymic nude stock. Differential diagnoses for muzzle lesions in rodents, with a focus on infectious and neoplastic lesions, will be discussed.

PS10 Severe Constipation in a Rhesus Macaque (Macaca mulatta)

D Celdran*¹, KM Gothard², I Ghaderi³, D Besselsen¹, CJ Doane¹

¹University Animal Care , The University of Arizona, Tucson, AZ; ²Physiology, The University of Arizona, Tucson, AZ; ³Surgery, The University of Arizona, Tucson, AZ

During routine semiannual examination, a 10-y-old naive female rhesus macaque (Macaca mulatta) presented with 700 gm weight loss and an intestinal thickening in the right cranial abdomen detected by palpation. Differential diagnosis included gastrointestinal obstruction, neoplasia, abdominal granuloma, and inflammatory enteropathies. Radiographs revealed intestinal gas distention between L3-L5. Blood chemistry revealed elevated ALT and moderate hypoproteinemia. Hematology showed no abnormalities and fecal occult blood and TST were negative. No abnormalities of appetite or fecal production were noted prior to or following the exam. One wk later an additional loss of 600 gm was noted. The enlarged intestine palpated considerably longer (~15 cm). Radiographs confirmed intestinal enlargement, fecal stasis, gas accumulation, and no evidence of metastatic nodules in the thorax. Abdominal ultrasound confirmed previous findings and revealed an enlarged gall bladder. Follow-up ALT was normal but continued hypoproteinemia with significant hypoglobulinemia. Presumptive diagnosis (adenocarcinoma or obstruction) required surgical resolution, so a laparoscopic procedure was scheduled. Laparoscopy revealed a redundant colon with associated constipation involving the entire colon and distal ileum. Treatment involving laxatives (Senna glycoside) and fiber supplements was initiated, and oral metoclopramide was added later. After 7 d of treatment, fecal output increased considerably and treatment was discontinued. Commercial and natural fiber supplements (prunes) were continued to help normal GI motility. After 6 wk of treatment body weight and blood work returned to baseline levels, with no evidence of constipation noted. Redundant colon, or dolichocolon, is an anatomic abnormality involving the presence of colon redundancies affecting 1 or all 3 segments of the colon and flexures. The extra length forms additional loops, tortuosities, and kinks that predispose to constipation. The incidence in human population is unknown, and to the knowledge of the authors, this is the first case described in rhesus macaques.

PS11 Acute Onset Hind Limb Paralysis in NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) Mice

P Yang*¹, HM Wilson¹, MJ Hoenerhoff^1,2, IL Bergin^1,2, RC Dysko¹, ZT Freeman¹

¹Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI; ²In-Vivo Animal Core, University of Michigan, Ann Arbor, MI

Nineteen 3-4 month old female NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice presented with acute unilateral or bilateral hind limb paresis or paralysis. Upon examination, mice demonstrated a range of maintained deep pain sensation with limited withdrawal reflex to a complete absence of both. These mice also had mild dehydration and poor to thin body condition with a range of BCS from 1-2.5. The mice were from a cohort (n = 30) implanted with a human patient derived breast cancer xenograft (PDX) that was in its fourth passage. The tumor was implanted in the mammary fat pad 1 mo prior to the clinically observed onset of paresis/paralysis. The mice were housed in a SPF facility designed specifically for immunodeficient strains. Based on clinical signs and history, differential diagnoses included mouse hepatitis virus (MHV), Theiler’s murine encephalomyelitis (TMEV), lactate dehydrogenase elevating virus (LDEV), and polyomavirus. Necropsies performed on affected mice (n = 10) were unremarkable with no significant gross lesions. On histopathologic examination, bilaterally within the ventral funiculi and nerve roots of the lumbar spinal cord, there was variable axonal swelling and vacuolation of myelin sheaths. Similar lesions were observed in the corresponding sciatic nerves of affected mice. Fresh PDX samples (n = 6) from affected mice obtained at the time of necropsy were submitted for a broad rodent infectious PCR panel which included the agents in the differential diagnosis list. All samples were PCR positive for LDEV, as were pooled plenum swabs from the rack on which these animals were housed. LDEV is known to cause serum elevation of the LDH enzyme, but also causes degenerative myelopathy and neuropathy in certain strains of mice, especially immunodeficient strains. LDEV may be transmitted within colonies by infected mice, or experimentally secondary to contamination of cell lines or xenografts, which represents an important source of spread.

PS12 Abdominal Distension in a Baboon (Papio anubis)

DD Chen*, D Flanagan, DM Molk, J Morris

Center for Comparative Medicine, Massachusetts General Hospital, Charlestown, MA

A 5-y-old, 17.6 kg intact male olive baboon (Papio anubis) housed singly indoors underwent a heterotopic heart xenograft, upon which the animal recovered smoothly and unremarkably. Two d post-op, the animal presented acutely for lethargy, vomiting, and lying on the cage floor. The NHP was sedated for physical examination and diagnostics. On physical exam, the abdomen was distended, mucous membranes were a pale pink, and he had an sPO2 of 89-91%. All other vital parameters were unremarkable. Serum chemistry showed 2.5 times elevation of ALP; remaining bloodwork was unremarkable. Two-view radiographs revealed a moderate amount of free gas, as well as a stomach dilated with food content and air. A gastric tube was immediately introduced, and the stomach was decompressed. A large amount of ingesta and material was removed, however post-decompression radiographs showed pockets of free air remained in the abdomen. Differential diagnoses included ileus, GI partial or full obstruction, foreign body impaction, volvulus, or pyloric stenosis. With the unknown etiology of the distended abdomen and concern for reoccurrence, the decision to perform exploratory laparotomy was made. During assessment of the bowel, a small serosal injury to a focal area of the small intestine was discovered that narrowed the intestinal lumen, causing a partial obstruction. A small portion of the small intestine that was adjacent to the heart xenograft within the abdomen was likely injured during defibrillation of the heterotopic heart xenograft. As for treatment, the swollen focal area of the bowel was overstitched with a Lembert suture to improve lumen diameter. The rest of the GI was assessed, and no other abnormalities were noted. The animal recovered smoothly with the following post-operative management plan: maintenance LRS fluids, Ketorolac 2 mg/kg IV SID for 3 d, Cefepime 50 mg/kg IV SID for 7 d, buprenorphine 0.01 mg/kg IV for BID for 3 d, NPO for 2 dand slow return to a liquid diet and then solid food. After 1 wk, the baboon made a full recovery.

PS13 Weight Loss, Intermittent Vaginal Bleeding and Pelvic Mass in a Hysterectomized Rhesus Macaque (Macaca mulatta)

RA Lang¹, S Achilles*¹, ND Kock², D Caudell², M Cline², M Leblanc¹

¹ARP, Wake Forest University, Winston-Salem, NC; ²Pathology, Wake Forest School of Medicine, Winston-Salem, NC

A 13-y-old, 7.7 kg, female Rhesus macaque experimentally irradiated with 5 Gray total body irradiation 10 y prior was evaluated for weight loss, intermittent vaginal bleeding, and palpable pelvic mass. The animal had undergone supracervical total hysterectomy (TH) 5 y earlier for a suspicion of endometriosis and a prolonged history of dysmenorrhea, menorrhagia, and anemia during menses. Clinical signs resolved with TH but the origin of menorrhagia could not be determined based on uterine pathology. Two y later, a 6.5 x 4.8 x 4.3 cm intraabdominal mass was discovered on routine CT examination with no overt clinical signs. Cytology of a needle aspirate yielded lipids and brownish fluid and prompted a diagnosis of lipoma and persistent endometriosis. Clinical evolution of both conditions was closely monitored subsequently during regular physical examinations. Three y later, the animal presented a 20% body weight loss over a 3-mo period and started to show intermittent vaginal bleeding. CBC revealed normocytic anemia and abdominal ultrasound confirmed that the mass had not evolved. An exploratory laparotomy was performed to evaluate the mass. A firm, pink, 7 x 6 x 5 cm mass at the pelvic inlet stemming from the TH stump was found. A dark colored cyst containing brown fluid was positioned cranial to the mass. A biopsy specimen was taken for histopathology and the animal recovered but was euthanized a few wk later for experimental reasons. A diagnosis of uterine leiomyoma stemming from the TH stump as well as abdominal and lung endometriosis were made based on gross necropsy, histopathology, and immunohistochemistry stains for estrogen and progesterone receptors on the lung lesions. Uterine leiomyoma and endometriosis are common estrogen-dependent reproductive pathologies in aging macaques and humans that may be hasten by aberrant estrogen signaling and local aromatase activity. TH with or without ovariectomy has been advocated for the management of severe cases in both conditions but several reports in the human and veterinary literature support that recurrence of leiomyoma from remnant uterine tissue as well as persistence of endometriosis may occur in both estrogen rich or poor environments even after surgery.

PS14 Dyspneic Mature Ewe from an Animal Science Research Herd

LA Taylor*, TH Vemulapalli

Veterinary Pathobiology, Texas A&M Univeristy, College Station, TX

A 4-y-old, 46 kg, Hampshire cross ewe presented for dyspnea. Three mo prior to presentation, the ewe had been acquired from the local sale barn for this research study. She had been naturally inseminated and was diagnosed open on rectal ultrasound. On initial examination the ewe was bright, alert, and in good body condition with an elevated respiratory rate and temperature. Auscultation revealed increased bronchial-alveolar sounds and an inspiratory stridor. She was initially treated symptomatically with florfenicol and banamine. One wk later the ewe’s condition had deteriorated. She was euthanized and submitted for a full necropsy. On necropsy the mucous membranes were diffusely pale. The abomasum contained a moderate load of Haemonchus contortus. Multiple abscesses in the right kidney were noted, with 90% of the parenchyma occupied. Approximately 40% of the diaphragm was adhered to the liver and multiple 0.7-6 cm, pale tan, round, and firm nodules were noted. The space surrounding the liver had several abscesses, up to 5 cm, that were displacing the duodenum. A 10 x 6 x 5 cm abscess was adhered to the liver, ventral ruminal wall, and sternum. The thoracic cavity revealed multiple enlarged, abscessed lymph nodes with multifocal mineralization. Multiple raised, firm, multifocal nodules (2.5-4 cm), were noted within the pulmonary parenchyma, the lungs failed to collapse, and the pleura was firmly adhered to the ribs. A 4 x 2.5 x 2 cm friable mass originating in the ethmoid mucosa and bilaterally effacing the ethmoid turbinates was found in the skull. Histopathology on the nasal tumor was consistent with Enzootic Nasal Tumor Virus-1, the lung histopathology was suggestive of Maedi-Visna virus, and bacterial culture confirmed Corynebacterium pseudotuberculosis, the causative agent of caseous lymphadenitis. Source of research animals should be carefully considered prior to purchase.

PS15 Acute Onset of Seizures in a Rhesus Macaque (Macaca mulatta) with a Cranial Implant

AL Armijo*¹, JJ Ballesteros², Y Kawamura³, F Yoshida^4,5, L Palley⁶, J Morris⁶, JL Haupt¹, I Napier¹, JG Fox¹, K Pate¹, A Garcia¹

¹Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA; ²Picower Institute for Learning & Memory, Massachusetts Institute of Technology, Cambridge, MA; ³Cardiovascular Research Center, Massachusetts General Hospital, Boston, MA; ⁴McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA; ⁵Saga University Medical School, Saga, Japan; ⁶Center for Comparative Medicine, Massachusetts General Hospital, Boston, MA

An 11-y-old, 9.0-kg, male, rhesus macaque (Macaca mulatta), in an indoor facility with a history of inappetence and weight loss (1.7 kg over 5 wk) presented with an acute onset of generalized seizures. This macaque was used in neuroscience research and had 1 right cephalic recording chamber in place. The macaque was sedated with ketamine (5 mg/kg), dexmedetomidine (250 mg/m²), and midazolam (0.1 mg/kg). Physical exam revealed thin body condition (2/5). Complete blood count revealed a leukocytosis (19.7 K/mL, reference range (RR) 4.5-19.5 K/mL) characterized by neutrophilia (18 K/mL, RR 0.7-12.7 K/mL). Serum chemistry was unremarkable. Differentials included meningitis, encephalitis, sepsis, and neoplasia. Fluids (200 mL of LRS) were administered subcutaneously. A loading dose of ceftriaxone (100 mg/kg, IM) was administered and continued once daily at 50 mg/kg. The following day, the macaque was chaired for chamber cleaning and a culture acquired from the chamber revealed ceftriaxone susceptible Corynebacterium  sp. Corynebacterium spp. are frequent contaminants of cephalic implants in nonhuman primates. Six d following the initial presentation the macaque was sedated for an MRI. Brain MR imaging revealed a ring-enhancing focal lesion adjacent to, and possibly connecting with, the head chamber and calvarium defects surrounding the chamber, suggesting a brain abscess. Follow-up bloodwork revealed resolution of the leukocytosis (8.1 K/mL) and neutrophilia (5.4 K/mL). The macaque underwent an explant surgery 3 wk after initial presentation. An MRI conducted 1 mo following explantation revealed a 69% reduction in volume. The reduction in volume of 10.6% per week assessed by MRI is similar to that observed in humans following antibiotic therapy. Brain abscessation is a potential sequela of chronic implants utilized in neuroscience. In addition to appropriate antibiotic therapy, explantation of the chamber should be considered to hasten the resolution and advanced imaging techniques, such as MRI, are instrumental in assessing therapeutic efficacy.

PS16 Necrosis of Distal Extremities in an Owl Monkey (Aotus nancymaae)

AM Braxton*, J Izzi

Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, MD

A 12-y-old, female owl monkey (Aotus nancymaae) presented for nonweight bearing lameness of the right leg 2 d after undergoing a protocol-driven splenectomy. On exam, severe soft tissue swelling and erythema affected the leg distal to the knee and ulceration was noted on the plantar surface of the metatarsus and phalanges. The animal was unable to grasp with the affected foot and lacked a withdrawal reflex. Differential diagnoses included traumatic bone fracture, soft tissue injury, and ischemia-reperfusion injury. Radiographs were performed and fracture was ruled out. Following debridement of necrotic tissue, the wound was managed with daily iodine baths, passive range of motion therapy, bandage changes and analgesics. Seven d post-presentation, granulation tissue began to form on the plantar surface of the tarsus but the animal remained nonweight bearing. During this treatment course, an ulcerated lesion developed on the dorsal aspect of the tail that ultimately became necrotic and required amputation of the distal tail. Despite aggressive management, tissue necrosis progressed and function was never regained. Approximately 1 mo post-presentation, the animal was anesthetized for further surgical debridement. Examination of the tissue revealed bone exposure of the lateral and plantar surface of the fourth metatarsal bone with minimal viable surrounding tissue. Euthanasia was elected at this time due to poor prognosis. Necropsy of the limb revealed epidermal ulceration, inflammation, and necrosis. Histopathology of the tail tip revealed diffuse coagulative necrosis and intravascular thrombi. No thrombi were identified within the limb or major vessels, however, the history of splenectomy and clinical presentation combined with clot formation in the tail led us to suspect a hypercoagulable state was present. Hypercoagulability is well reported in humans following splenectomy. Though owl monkeys are commonly splenectomized for malaria research, there are no published reports of hypercoagulability following splenectomy in this or other nonhuman primate species. Based on the clinical and histopathology findings, a diagnosis of microthrombosis leading to distal tail necrosis secondary to a hypercoagulable state following splenectomy was reached.

PS17 Auricular Swelling and Discharge in a Male Mouse

LF Mosca*, J Klug, JM Snyder

Department of Comparative Medicine, University of Washington, Seattle, WA

An experimentally naïve 28-mo-old male mouse presented for dermatitis and alopecia on the right shoulder, extending up through the lateral cervical region. Two mo prior, the mouse had experienced mild dermatitis on the intrascapular and dorsal cervical area. The previously observed dermatitis was treated with weekly toe nail trims, and resolved within 3 wk. On physical examination the newly developed dermatitis was an approximately 2-centimeter long lesion spanning the lateral right shoulder and right lateral cervical region. Another discrete, alopecic 0.5 cm x 0.5 cm lesion was also present at the base of the right ear. The mouse was given a toe nail trim, however, within 5 d the lesions had not improved. Moderate thickening and swelling of the right ear base was noted, and the ear canal was producing a serous, clear to pink-tinged discharge. Cytology of the discharged showed scattered diplococci, proteinaceous material, and red blood cells. The differential diagnosis list included otitis media and/or externa, trauma, tooth root abscess, or neoplasia. The mouse did not exhibit signs of difficulty eating or weight loss. The mouse was initiated on topical antibiotic therapy to the ear canal once daily. Within one week the swelling at the ear base was undetectable, however, serous,watery discharge was still noted in the ear canal. Cytology of the discharge showed occasional plasma cells. Four d later, a 1 millimeter portion of exposed cartilage was noted on the right ear base, surrounded by mildly erythemic skin. Due to this development euthanasia was elected. Necropsy confirmed moderate otitis of the right ear with dermatitis and ulceration. On histologic examination a densely cellular neoplasm with local invasion into the middle ear and destruction of surrounding skeletal muscle and bone was encountered, with metastasis to the lungs. The neoplasm arose from the Zymbal’s gland, a multilobulated sebaceous holocrine gland located anterior-ventral to the ear canal in rats and mice. Neoplasms reported from the Zymbal’s gland are rare, but more commonly reported in rats and include sebaceous cell adenoma, squamous cell papilloma, and carcinoma of sebaceous and/or ductal epithelial origin.

PS18 Emphysematous Facial Lesion in a Musk Shrew (Suncus murinus)

N Fogarty*, R Duran-Struuck, A Brice, T Manzi

University Laboratory Animal Resources (ULAR), University of Pennsylvania, Philadelphia, PA

An adult male musk shrew (Suncus murinus) originating from a breeding colony and used experimentally for emesis studies was reported for a lesion on the right side of the face during routine health examinations. Ventrolateral to the right orbit, a soft, round swelling approximately 1.0 cm in diameter was observed. The animal was otherwise active with appropriate body condition and hydration. Examination under general anesthesia did not identify an oral lesion, and surgical lancing of the swelling resulted in its deflation without hemorrhage or purulent discharge. A short course of Meloxicam (5 mg/kg PO SID for 3 d) was administered following the lancing. Over the next 2 mo, this emphysematous mass increased and decreased in size in correspondence with respiration, however the animal continued to thrive. The animal was removed from the experiment following the onset of the lesion, but continued to be used for breeding purposes. Computed tomography and magnetic resonance images of the skull did not reveal any abnormalities that might contribute to the development of emphysema, such as an oral or nasal fistula. As the lesion failed to spontaneously resolve, the animal was euthanized and submitted for postmortem examination. The cause of the lesion was not apparent on gross examination. Microscopically, the subcutaneous tissue was markedly distended by clear space lined by abundant epithelioid macrophages and multinucleated giant cells with fewer lymphocytes. The surrounding subcutaneous connective tissue and muscle fibers were separated by abundant clear space. Based on these findings, the lesion was diagnosed as focal subcutaneous facial emphysema (SFE). There are several documented causes of SFE in humans, such as orofacial trauma, iatrogenic injury (i.e. from dental or surgical procedures), infections (i.e. retropharyngeal abscessation), and pneumomediastinum. This case highlights a unique lesion in an increasingly popular species in the laboratory animal field.

PS19 Abdominal Mass in an Adult Common Marmoset (Callithrix jacchus)

I Napier*¹, S Muthupalani¹, RM Kramer¹, AL Armijo¹, C Atkinson¹, JG Fox¹, S Carrasco^1,2

¹Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA; ²Laboratory of Comparative Pathology, Memorial Sloan Kettering Cancer Center / Weill Cornell Medicine, New York, NY

A 10-y-old, pair-housed female common marmoset (Callithrix jacchus) presented with a firm, moveable, abdominal mass and hepatomegaly on routine semiannual physical examination. Significant clinical pathology findings included anemia; azotemia; and elevated levels of gamma-glutamyl transferase, lipase, and amylase. An abdominal ultrasound revealed a cranioventral mass (3.0-4.0 cm in diameter) and chronic changes to both kidneys. The differential list included neoplasia, pancreatitis, and hepatobiliary disease. One mo after initial evaluation, the marmoset was euthanized due to increased abdominal mass size, along with progressive weight loss and lethargy. On necropsy, all liver lobes contained multiple pale-tan to white, semi-firm masses (0.1-2.0 cm in diameter) with central necrotic areas and hemorrhage. There was an additional large, oval, mesenteric mass (5.0 x 4.0 x 3.5 cm) replacing the pancreas adjacent to the superior duodenum. On histopathology, hepatic lobules were multifocally effaced and compressed by numerous well-demarcated, non-encapsulated, densely cellular masses that often infiltrated portal tracts. The hepatic and mesenteric masses were composed of deeply basophilic neoplastic polygonal and pyramidal cells forming lobules and trabeculae, supported by a fibrovascular stroma. Neoplastic cells often formed palisades around primitive ducts containing a brightly eosinophilic matrix. Neoplastic cells were closely packed with poorly defined borders and small amounts of basophilic granular cytoplasm. Anisocytosis and anisokaryosis were moderate with high mitotic activity. Neoplastic cells exhibited cytoplasmic and nuclear immunoreactivity to beta-catenin (a marker involved in cellular development), and were negative for epithelial (pancytokeratin), mesenchymal (vimentin), neuroendocrine (chromogranin A, synapthophysin), and neuronal markers (synapthophysin). Based on histomorphology and immunohistochemical features, the hepatic masses were diagnosed as poorly differentiated embryonal tumor with multicentric involvement and pancreatic lymph node metastasis. Histological findings, differential diagnoses, and the prevalence of hepatic neoplasias in nonhuman primates will be discussed.

PS20 Establishment of a Nonhuman Primate Model of Right Ventricular Pressure Overload with Successful Engraftment of Human Induced Pluripotent Stem Cell-Derived Cardiac Lineage Cells (hiPSC-CL)

JA Scholz*¹, F Secreto², J Wobig², C Reece², B Cannon², L Wierenga³, M Berg³, K Lafferty³, T Roehling³, J Hayes³, RA Johnson⁴, H Simmons³, A Mejia³, J Kurian³, D Padley⁵, M Emborg³, T Nelson²

¹Comparative Medicine, Mayo Clinic, Rochester, MN; ²Mayo Clinic, Rochester, MN; ³Wisconsin National Primate Research Center, Madison, WI; ⁴University of Wisconsin School of Veterinary Medicine, Madison, WI; ⁵Regen Theranostics, Rochester, MN

Hypoplastic left heart syndrome (HLHS) is a complex congenital heart disease that requires multiple surgeries for any chance at survival. These surgeries result in a right heart that pumps blood to the systemic circulation with passive flow through the pulmonary circulation. Right ventricular dysfunction is a common morbidity in these patients, and cell-based therapy is a promising strategy to support ventricular function and delay or prevent the need for cardiac transplantation. The aims of this pilot study were to 1) develop a nonhuman primate model of right ventricular pressure overload, and 2) demonstrate engraftment of hiPSC-CL cells in the compromised heart. Nine rhesus macaques (Macaca mulatta) underwent pulmonary artery banding (PAB) surgery and cardiac event recorder implantation. Approximately 2 wk later, hiPSC-CL cells were injected into the right ventricular myocardium of four immunosuppressed animals. Animals were followed for 4 or 8 wk with periodic analyses of hematology and serum chemistry markers, along with performing echocardiograms prior to and subsequent of PAB. Histological analysis of all tissues was performed, including the entire heart (4 mm slices), and the cardiac tissue was stained against human cardiac troponin (hCTnI). Three animals did not complete the study due to intraoperative (n = 1) or post-operative (n = 2; first 2 animals) complications. Adjustments were made to the surgical procedure after the first 2 animals, and subsequent animals tolerated the PAB procedure well. All animals developed varying degrees of pathology evident on echocardiogram and histology consistent with right ventricular pressure overload. No significant arrhythmia events occurred in any of the 6 animals. All 4 animals administered hiPSC-CL cells exhibited significant engraftment at 4 and 8 wk. This study is the first to describe a PAB model of right ventricular pressure overload in the nonhuman primate, and is also the first to report engraftment of human iPSC-derived cardiac lineage cells in non-infarcted primate myocardium.

PS21 Reducing Inter-operator Variability Using a Novel 3D and Thermal Measurement System when Measuring Subcutaneous Tumours in Mice

K Turley*

Marketing , Fuel3D, Oxford, , United Kingdom

The key to successfully assessing therapeutic efficacy and accurately calculating tumour growth inhibition is ensuring that each group within a study has the volumes consistently measured across the life of the experiment by any operator. A previously published investigation showed a statistically significant reduction in inter-operator variability observed across 3 in vivo efficacy studies conducted by multiple users when using a novel 3D and thermal imaging system. We now explore how this reduction in inter-operator variability can be seen across a much larger dataset.Our dataset includes 5,677 scans and 4,743 corresponding calliper measures collected in 26 laboratories across 276 studies with 117 users, 34 mouse strains, and 120 cell lines. Scans were conducted by holding the mouse up to the system and automatically capturing red/green/blue (RGB), thermal, and 3D images of the mouse and tumor body. This process took less handling time than recording length and width with callipers. Once captured, the system then automatically processes this data to segment, record, and generate volume information for each scan. After analysing the variability of the 2 methods using coefficient of variation (CoV), intra class correlation (ICC) subset analysis, and significance testing, we can see that the 3D and thermal imaging system produced a median CoV of 0.176 compared to a median calliper CoV of 0.197. Significance testing produced a P value of 0.01794 indicating that this is a statistically significant result, further backed up by the ICC subset analysis. We conclude that introducing a novel 3D and thermal imaging system to a subcutaneous tumour volume collection methodology can offer a significant improvement in inter-operator variability and help to improve study reproducibility across a wide range of studies, mouse strains, and cell lines.

PS22 MSNASH, A Novel Mouse Model of Heart Failure with Preserved Ejection Fraction

Y Shi², X Chen², JP Morin², K Tam², A Puca¹, S Vadvalkar¹, A Schile¹, RA Miller², DK Hirenallur-Shanthappa*²

¹The Jackson Laboratory, Farmington, CT; ²Pfizer, Cambridge, MA

Metabolic syndrome has been associated with development of diabetes and heart failure. Although the pathogenesis of heart failure with preserved ejection fraction (HFpEF) is not clearly understood, many reports suggest that metabolic dysfunction and hypertension are comorbidities, which are the major underlying mechanisms for HFpEF. Thus, animal models that show robust metabolic dysfunction in the absence of high-fat diet feeding or genetic mutation are invaluable tools for understanding the pathogenesis of HFpEF as well as advancing therapeutic drug development. Recently developed MSNASH model, resulting from a cross between the AKR/J and C57BL/6J (B6/J) mouse strains, spontaneously develops robust metabolic syndromes in the absence of high-fat feeding, with intact leptin pathway. However, the cardiac systolic and diastolic phenotype of the MSNASH mouse has not been evaluated. We hypothesized that metabolic dysfunction in the MSNASH model induces progressive development of HFpEF. Systolic and diastolic functional phenotype of male MSNASH and B6/J (control) mice were evaluated using echocardiography at 11, 19, 24 and 33 wk of age, and the metabolic phenotype was evaluated at 12 and 25 wk of age. Echocardiography and contrast enhanced ultrasound (CEUS) imaging was performed under isoflurane (1 to 3%) anesthesia. MSNASH mice showed obesity (32% higher body weight), hyperinsulinemia (1321% higher), hyperglycemia (41.7% higher), glucose intolerance and insulin resistance (ipGTT AUC ~55 % higher) compared to control B6/J as early as 12 wk old. Further, MSNASH mice displayed preserved systolic function (ejection fraction 69.1 ± 2.7% in MSNASH vs 73.7 ± 1.6 % in B6 control at 33 wk old) with progressive increase in left ventricular (LV) mass ( 146.9 ± 4.8 mg at 11 wk old vs 234.6 ± 13.1 mg at 33 wk) and LV posterior wall thickness at diastole (LVPWD) (0.90 ± 0.04 at 11 wk vs 1.16 ± 0.08 at 33 wk) and decrease in radial diastolic strain rate (-14.05 Pk(1/s) in MSNASH vs -19.83 Pk(1/s) in control B6 at 33 wk old), a measure of diastolic dysfunction from 11 to 33 wk of age compared to age matched B6/J mice (LV mass 102.4 ± 3.2 mg at 11 wk vs 140.7 ± 16.3 mg at 33 wk; LVPWD 0.92 ± 0.04 at 11 wk vs .87 ± 0.04 at 33 wk). Notably, oxygen consumption at peak exercise capacity was significantly reduced in MSNASH mice (35.1 % less peak VO₂ consumption) compared to control mice at 30 wk of age. Furthermore, poor skeletal muscle vascular perfusion (CEUS time to peak readout 44.82 ± 3.09 s) was observed in MSNASH mice compared to control B6/J (CEUS time to peak 18.95 ± 4.45 s) mice at 34 wk of age. Results indicate that MSNASH mice show development of a progressive HFpEF phenotype in conjunction with metabolic syndrome.

PS23 The Influence of Animal Facility Lighting on Growth of Human Breast Cancer Xenografts in Nude Mice

RT Dauchy*, DE Blask, S Xiang, M Anbalagan, SM Hill

Laboratory of Chrono-Neuroendocrine Oncology, Tulane University, New Orleans, LA

Animal facility light exposure influences temporal coordination of circadian rhythms of normal and neoplastic tissues in laboratory animals in an intensity-, wavelength-, and duration-dependent manner. Recent evidence indicates that rotating night shift workers have an increased risk of developing breast and prostate cancers, which has been directly associated with light at night (LAN) as the principal risk factor. Little is known, however, regarding exposure to blue-enriched (460-480 nm) light-emitting diode (LED) light at daytime (bLAD), the most rapidly emerging lighting technology globally. Our previous studies demonstrated that exposure of human prostate tumor-bearing nude rats to bLAD amplified production of the nighttime circadian neurohormone melatonin resulting in decreased tumor growth and metabolism. In this GLAS-supported study we tested whether dim-LAN (dLAN) stimulates, while the bLAD-enhanced nocturnal melatonin signal suppresses, human breast tumor growth. Athymic female nude mice (Crl:NU(NCr)-Foxn1^nu; 4-5 wk-old) under an IACUC-approved protocol were maintained in an AAALAC-accredited facility for 8 wk on 1 of 3 lighting regimens: Group I, 12L cool white fluorescent (CWF, control):12D (0 lux); Group II, 12L(CWF):12dLAN (0.02 lux); and Group III, 12bLAD:12D (0 lux). All groups (n = 10/group) were maintained in a common light phase (lights on 0600h) lighting photoperiod (83.36 ± 2.64 lux, within cage). Following two weeks light exposure all mice were inoculated subcutaneously with MCF-7 human breast cancer cells (3 x 10⁶ cells) and measured for tumor growth. Latency-to-onset of tumor growth (first palpable mass) and tumor growth rates were significantly faster in II (dLAN), and slower in III (bLAD), compared to I (control) mice (P > 0.001). Peak nighttime (2400 h) plasma melatonin levels (mean ± 1 SD) for groups I – III were significantly different at 140.2 ± 7.9 pg/mL, 16.5 ± 3.1 pg/mL, and 934.6 ± 27.5 pg/mL, respectively. Final harvest tumor weights for groups I-III were, 0.84 ± 0.08 g (d 38), 1.04 ± 0.02 g (d 27), and 0.36 ± 0.10 g (d 50), respectively. These findings show that animal facility dLAN stimulates, while bLAD-amplified nocturnal melatonin levels inhibit, growth activity in MCF-7 human breast cancer in vivo.

PS24 Inhibition of Human Cancer Growth in Nude Rats by Dietary Melatonin and Omega-3 Fatty Acids

RT Dauchy*¹, EM Dauchy², DE Blask¹

¹Laboratory of Chron-Neuroendocrine Oncology, Tulane University, New Orleans, LA; ²Medicine, Louisiana State Health Sciences Center, New Orleans, LA

Melatonin (MLT), produced by the pineal gland in mammals primarily at night, and omega-3 fatty acids (FAs) regulate linoleic acid (LA)-dependent metabolism and growth in rodent and human tumors in vivo. These agents inhibit LA-uptake and conversion to the mitogenic agent 13-hydroxyoctadecadienoic acid (13-HODE). Previous studies showed that proliferative, signal transduction and LA metabolic activity were abrogated in tissue-isolated human PC3 prostate and FaDu hypopharyngeal squamous cell xenografts perfused in situ in nude rats with donor blood replete with MLT (1nM) or eicosapentaenoic acid (0.4 mM). In this IACUC-approved study, human PC3 and FaDu xenografts were grown in male nude rats (Hsd:RH-Foxn1^rnu) maintained on a 5% corn oil (CO) diet for a period of 4 wk. PC3 and FaDu tumor latency-to-onset (first palpable mass) and growth rates were, respectively, 18 and 26 d, and 0.60 ± 0.04 and 0.48 ± 0.06 g/day (n = 8/group). When estimated tumor weights were 3.5 g, tumor-bearing animals were divided into 2 groups (n = 4). Control tumor-bearing rats were maintained on the 5% CO diet, while experimental groups were placed on a combination test diet consisting of MLT (50 mg/d)/5% fish oil (FO). Control group tumors continued to grow as before, while those in the test diet groups regressed at a rate of 0.27 ± 0.03 (PC3) and 0.19 ± 0.01 g/day (FaDu), respectively. Final mean tumor weights of controls were 6.65 ± 0.56 g (PC3) and 5.90 ± 0.28 g (FaDu), while those of test diet rats were 2.38 ± 0.18 g (PC3) and 2.25 ± 0.16 g (FaDu). Prior to complete tumor regression in test groups, tumors were prepared for arteriovenous blood measurements. LA uptake and 13-HODE release by PC3 and FaDu tumors in control animals were, respectively, 3.1 ± 0.9 and 3.6 ± 0.7 µg/min/g (LA), and 21.3 ± 1.9 and 18.8 ± 0.9 ng/min/g (13-HODE), and was completely suppressed in tumors of test diet rats. PC3 and FaDu tumors in test diet animals exhibited a reduction of over 65% in tumor cAMP levels, compared to controls. Results indicate that a combination of decreased dietary LA and increased dietary MLT and omega-3 FAs elicit a strong growth inhibitory effect on these two human tumors, and may encourage efforts to reduce LA and increase omega-3 and MLT consumption.

PS25 Fully Automated Mouse Echocardiography Analysis Using Deep Convolutional Neural Networks

C Duan*, M Montgomery, DK Hirenallur-Shanthappa

Pfizer INC, Cambridge, MA

Echocardiography (echo) is a translationally relevant ultrasound imaging modality widely used to assess cardiac structure and function in preclinical models of heart failure during drug development. Though echo is a valuable tool for quantifying endpoints, the image analysis is a time consuming, resource demanding process, and susceptible to inter-reader variability. Recent advancements in machine/deep learning have enabled researchers to automate image processing and analysis and reduce analysis time and inter-reader variability in the field of medical imaging as well as in preclinical research. We propose to utilize deep learning to develop and validate an automation tool for the analysis of parasternal long axis brightness (B)-mode and (M)-mode echo images of mouse. Fully convolution neural networks were used to segment the left ventricle (LV) in B-mode, and to segment the LV anterior wall and posterior wall in M-mode images. The neural networks were trained and tested using manually segmented B-mode and M-mode images. The segmented images were then further processed to automatically identify the diastole and systole phases of the heart cycle, and to compute cardiac structural and functional metrics. Bland-Altman analysis, Pearson’s correlation, and Dice Similarity Coefficient (DSC) were used to evaluate the performance of the proposed deep learning-based echo analysis pipeline. The neural networks achieved high DSC (0.92 to 0.96) in the segmentations, and excellent correlations (r = 0.85 to 0.99) between automated analysis and manual analysis outputs. Bland-Altman analysis also showed excellent agreements between the automated analysis and the manual analysis. The use of this automated analysis tool reduced manual analysis time by 90%. In conclusion, we developed an automated echocardiography image analysis pipeline based on deep learning. This tool allows for fast and accurate analysis of B-mode and M-mode mouse echo data and mitigates the issue of inter-reader variability in manual analysis.

PS26 Fat Imaging: A Refined Micro-CT Approach

S Ullas*¹, M Montgomery¹, B Faherty², T Ross², DK Hirenallur-Shanthappa¹

¹Comparative Medicine, Pfizer INC, Cambridge, MA; ²Pfizer, Cambridge, MA

Assessment of fat distribution is essential to understanding the pathophysiology of obesity and obesity-associated disorders and the development of therapies against these disorders. Magnetic resonance imaging (MRI) and computed tomography (CT) have the ability to provide quantitative information on fat distribution in different compartments of the body. However, both these tools have low throughput. We evaluated a refined, high throughput micro-CT imaging technique in conjunction with an automated image analysis tool to assess fat distribution in diet-induced obesity (DIO) mouse model. In addition to control and DIO groups (18 to 20 wk old), a DIO plus Liraglutide treatment (0.3 mg/kg, subcutaneous, once daily for 10 d), was included to validate a refined micro-CT imaging technique. Liraglutide is a glucagon like peptide‐1 (GLP‐1) receptor shown to induce weight loss clinically, when administered subcutaneously. Each group consisted of 10 mice, for a total of 30. Animals were anesthetized using isoflurane (1 to 3%), and underwent micro-CT imaging of whole body and abdominal region between lumber vertebra 1 to 4, and whole body dual X-ray absorptiometry (DEXA) imaging. Data from micro-CT was then compared with outputs from Echo-MR (echo-magnetic resonance), DEXA and excised visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) using 1-way ANOVA with post hoc Turkey test and Pearson’s correlation coefficient analysis. Results of total fat from micro-CT (5704 ± 278 mm³ in DIO vs 1776 ± 232 mm³ in control), DEXA (19.74 ± 0.9 g in DIO vs 7.7 ± 0.66 g in control) and ECHO-MR (18.47 ± 1.23 g in DIO vs 4.24 ± 0.70 g in control) showed significantly higher fat content in DIO mice compared to control group, and the liraglutide-treated group showed significantly lower total body fat in DIO mice (micro-CT total fat: 3862 ± 425.7 mm³; DEXA 14.69 ± 0.92 g) compared to untreated DIO (micro-CT total fat: 5704 ± 278 mm³ ; DEXA 19.74 ± 0.9 g). Total whole body and abdominal fat, and SAT values from the micro-CT imaging correlated well (Pearson’s ‘r’ coefficient values > 0.80) with excised total body fat and SAT values. However, the VAT values from micro-CT didn’t correlate well (Pearson’s ‘r’ coefficient values < 0.50) with excised VAT. The micro-CT, DEXA imaging and echo MR readouts of total fat, VAT and SAT values correlated well with one another with Pearson coefficient ‘r’ value > 0.94. Importantly, refinement to micro-CT imaging technique and use of automated image analysis reduced image acquisition and analysis time by 50 %. Our results suggest that a refined micro-CT imaging technique with automated analysis can be used to assess total and subcutaneous adipose tissue in preclinical models of obesity in a high throughput manner.

PS27 Reproductive Factor Influences Spontaneous Tumors in FVB/N Mice

Q Zhang¹, C Leung^1,2, L Gerweck¹, P Huang*¹

¹Radiation Oncology, Massachusetts General Hospital & Harvard Medical School, Boston, MA; ²Division of Urology, Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, , China

The FVB/N strain of mice is widely used in cancer research, including the development of transgenic mouse models. Previous studies have reported the development of spontaneous tumors as a function of mouse age, gender, and the presence or absence of specific genes. However, little is known about the influence of reproductive factors on the spectrum and incidence of spontaneous tumors. Our aim was to record the spontaneous tumors in our retired breeders and naïve nonbreeding FVB/N mice, and analyze the relationship between tumor incidence, reproduction, and histopathologic tumor type. A total of 843 mice (507 retired breeders and 336 nonbreeding mice) were observed during their natural lifespans (average ages of 724±139 and 731±229 d, respectively). A complete necropsy was performed on all mice that died during the study, on those euthanized when they were sick or developed tumors. All grossly observed lesions were collected for histopathologic study. The overall incidence of spontaneous tumors was 56.0% (284/507) in breeders and 50.0% (168/336) in nonbreeding mice (P = 0.086). The tumor incidence was 57.3% (102/178) and 55.3% (182/329) in male and female breeders; and 45.7% (43/94) and 51.7% (125/242) in male and female nonbreeding (all P > 0.05). Histopathologic findings showed that the most common neoplasm in both male and female breeders was lung alveolar-bronchiolar tumor (43.3% and 22.8%), as well as in nonbreeding FVB/N mice (38.3% and 21.5%). In female retired breeders, the second most common was mammary gland tumor (14.6% incidence); but only 0.4% incidence was detected in female nonbreeding mice (P < 0.0001). There was a higher tumor incidence of histiocytic sarcoma (10.7%) in female nonbreeding mice compared to a 3.6% incidence in retired female breeders (P = 0.001). The reproductive factor did not affect the overall incidence of spontaneous tumors in our FVB/N mice but significantly increased the risk of mammary tumor in female breeders and protected them from developing histiocytic sarcoma. The tumorigenesis profile shift with age, gender, and reproduction should be carefully considered when using FVB/N and FVB background transgenic mice in cancer research.

PS28 Mouse-adapted SARS-CoV-2 Infection in Mice

J Plunkard*¹, M Lowman², CF Brayton¹, K Carter², JS Villano¹

¹Department of Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, MD; ²Johns Hopkins University, Baltimore, MD

Small animal models are vital for investigating SARS-CoV-2 (SCV2) pathogenesis and potential COVID-19 treatment options. While hamsters remain the leading small animal model of SCV2 infection, laboratory mice present certain advantages such as genetic manipulation. Here, we investigated the use of a mouse-adapted SCV2 (maSCV2) capable of infecting mice that do not carry the human angiotensin 1 converting enzyme 2 (ACE2) receptor used by SCV2 for cellular entry. We hypothesized that infected mice would develop clinical disease (ex. weight loss) and would have histopathologic findings similar to those observed in human patients and other SCV2-infection animal models. Three groups of mice (6 C57BL/6J males, 6 C57BL/6J females, and 4 As:129S1 females) were inoculated with maSCV2 intranasally (50 ul/nare of infectious dose) under ketamine/xylazine sedation. These mice were then weighed and monitored daily until euthanasia on d 5 post infection. Two mice were euthanized at d 3-4 post infection because of severe clinical signs, including absence of escape response, hunched posture, squinting, increased respiratory rate, and rough hair coat. Percent body weight loss was examined by t-test and ANOVA at P < 0.05 and revealed that mice lost a significant percent of body weight. Male C57BL/6J mice lost significantly more weight (5.8 ± 2.5%) than the female C57BL/6J mice (1.2 ± 2.6%), and female As:129S1 mice lost significantly more weight (12.2 ± 3.5%) than female C57BL/6J mice. Histology revealed mild to moderate inflammatory changes, with the As:129S1 mice exhibiting more prominent peribronchiolar mononuclear inflammation. In conclusion, we demonstrated that maSCV2 infection produced clinical signs and pathology in wild-type C57BL/6J and As:129S1 mice. The development of this animal model presents a wide array of possibilities for COVID-19 researchers, especially to those wanting to use genetically modified mice. Future studies include evaluation of maSCV2 infection in outbred and inbred mice, including transgenic mice.

PS29 Effects of Buprenorphine Treatment on Influenza Pathogenesis in Ferret (Mustela putorius) Models

V Mrotz*¹, J Belser²

¹Comparative Medicine Branch, Centers for Disease Control and Prevention, Jackson, GA; ²National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA

Ferrets represent the gold standard model for influenza A virus (IAV) research due to their natural susceptibility to human and zoonotic IAV, comparable respiratory anatomy and physiology to humans, and development of clinical symptoms similar to those seen in infected humans. As presence and progression of clinical symptoms can be useful in infectious disease research, uncertainty of how analgesics may perturb research outcomes and comparable characteristics of disease progression have outweighed the concern of discomfort related to these symptoms. Nonetheless, 3Rs drive researchers to consider how this invaluable model for IAV research can be refined. Opioids offer a possible refinement option that would not directly impact the inflammatory cascade involved in IAV infection. Mirroring pathogenicity studies that utilize ferrets, we intranasally inoculated 12 ferrets (castrated male, 9-mo-old) with the H3N2 IAV A/Panama/2007/1999 and divided them into 3 treatment groups (n = 4) where 2 groups received buprenorphine (Bup) treatments on 2 different schedules based on historical peaks of clinical signs (0.04 mg/kg Bup SC BID, Group 1 d 0-4 post inoculation (p.i.) and Group 2 d 5-9 p.i.) and the third group received saline treatments corresponding to those schedules. Duration and location of viral replication, lymphohematopoietic changes, and clinical symptoms were comparable across all groups at all timepoints. High quantities of infectious virus in nasal wash specimens were detected in ferrets from all groups through d 5 p.i., indicating that Bup treatments on either schedule did not substantially alter replication kinetics in the upper respiratory tract. Ferrets receiving Bup treatments exhibited transient increases in weight loss and fever compared to the saline group, but all groups ultimately achieved similar peaks in both measurements. Collectively, these findings support the continued evaluation of Bup as a refinement for IAV challenged ferrets.

PS30 Refinement of Primary and Secondary Methods of Euthanasia in African Clawed Frogs (Xenopus laevis)

MR Nichols*, T Martin, SE Thurston, J Keller

Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI

Recommended euthanasia methods for amphibians often include a primary pharmaceutical method that deeply anesthetizes the animal followed by a secondary physical method that causes irreversible death. Reliable secondary euthanasia methods are especially important in amphibians, where death can be difficult to differentiate from deep anesthesia. However, application of these methods can be difficult, prone to error, and may have negative emotional impacts on operators and observers. We compared 5 primary methods of euthanasia and hypothermia as a secondary method in a group of 54 adult female African clawed frogs (Xenopus laevis). Six frogs per group were euthanized via the following 5 primary methods: immersion in 5 g/L tricaine methanesulfonate (MS-222), immersion in 10 g/L MS-222, intracoelomic injection with sodium pentobarbital (1100 mg/kg), ventral cutaneous application of 20% benzocaine hydrochloride gel (182 mg/kg), or dorsal cutaneous application of isoflurane (60 mL/kg). For MS-222, frogs were immersed to a depth not covering the nares until a time that euthanasia was assessed to be complete (median = 3 m, IQR = 0.5 m). Euthanasia was assessed through loss of spontaneous movement, loss of righting reflex, loss of withdrawal reflex, and expiration of buccal respiration and heartbeat. Signs of distress and ease of application were also assessed. 5 g/L MS-222 and benzocaine elicited the fewest signs of distress, with 5 g/L MS-222 causing death faster than benzocaine (P < 0.05). Isoflurane, 10g/L MS-222, and pentobarbital are not recommended due to aversive behaviors (i.e. increased activity, increased secretions or sloughing of skin, gulping of air, blinking) or difficulty in use. To assess hypothermia as a secondary method, 24 frogs were deeply anesthetized in 1.5 g/L MS-222, followed by 1 of these methods: removal of heart, placement in a refrigerator until cloacal temperature reached 5C followed by a freezer, placement directly into a freezer, or placement into a cooler containing dry ice. Frogs were monitored for cessation of buccal respiration, cessation of heartbeat, and for a cloacal temperature curve indicating complete freezing of the coelom. All secondary methods led to irreversible death. We conclude that freezing of adult Xenopus laevis frogs that have been deeply anesthetized or euthanized via cutaneous application of benzocaine gel or immersion in 5 g/L MS-222 is an easy to perform, reliable, irreversible method of euthanasia that does not result in signs of distress.

PS31 The Effect of Premedication with Ketamine or Xylazine on Isoflurane Induction and Anesthesia in C57BL/6J Mice

EM David*^2,1, JO Marx^2,1

¹Pathobiology, University of Pennsylvania, Philadelphia, PA; ²University Laboratory Animal Resources, University of Pennsylvania, Philadelphia, PA

Balanced anesthesia, the practice of using a combination of drugs to achieve a desired anesthetic plane, has many benefits including smoother induction and recovery and dose-reduction and decrease of adverse effects of individual drugs. While premedication with an injectable agent before inhalant anesthesia is routine in other species, mice are commonly induced with gas anesthesia alone. We hypothesized that premedication with ketamine or xylazine would safely reduce the stress of induction and lower the minimum alveolar concentration (MAC) of isoflurane. We premedicated mice with intraperitoneal injection of ketamine (100 mg/kg), xylazine (4 mg/kg), or isotonic crystalloid (0.1 ml) and examined the effects on isoflurane induction and MAC. Young adult (8-20-wk-old) male and female C57BL/6J mice (n = 40) were used. First, we video-recorded isoflurane induction under all test conditions and scored the videos according to a behavioral ethogram to identify signs of stress. Next, we anesthetized mice to determine the MAC value for each anesthetic protocol using quantile analysis. Lastly, using isoflurane concentrations determined to keep at mice at a surgical plane of anesthesia during the MAC testing experiment (ketamine 1.3; xylazine 1.6; control 2.2), we anesthetized mice and monitored their vital parameters under anesthesia for 60 min.. Ketamine and xylazine had a significant (P = 0.02) effect on induction scores (control 8.5±0.7, ketamine 5.3±4.0, xylazine 3.3±1.5), where a lower score represents a less stressful induction. Ketamine treated mice displayed ataxia and tremors before induction. Both drugs had a MAC-reducing effect (ketamine 0.96; xylazine 1.20; control, 1.89). All mice survived 60 min of anesthesia. The average heart rates of mice receiving xylazine (327±27) and ketamine (406±45 bpm) were significantly lower (P < .001) than those of control mice (572±72). The average respiratory rates for control mice (54±17) were significantly lower (P <.001) than those of mice given xylazine (102±14) or ketamine (115±20). The higher heart rates and lower respiratory rates in the control group were likely largely the result of respiratory suppression and hypotension due to a higher isoflurane concentration. Balanced anesthesia, specifically premedication with ketamine or xylazine, has potential benefits in mice, including reducing the stress of isoflurane induction and lowering isoflurane requirements.

PS32 Propofol as an Effective Means of Euthanasia for Adult Zebrafish (Danio rerio)

A Davis*, D Chu, JP Garner

Comparative Medicine, Stanford University, Palo Alto, CA

Despite the popularity of the zebrafish (Danio rerio) as a model organism in biomedical research, the most frequently used methods of euthanasia for these small fish (MS222 and ice bath) have multiple shortcomings with regard to animal welfare and human safety. Propofol has been known to cause rapid induction of anesthesia for many species including zebrafish, but its efficacy as a euthanasia agent for zebrafish has not been tested. Fifty-eight adult zebrafish were used in this project, with 8 to 9 fish per treatment group. Each fish was individually placed in a small tank containing water from their home tank plus ice, 250ppm MS222, 600ppm lidocaine hydrochloride, 100ppm propofol, or 150ppm propofol for a period of 20 or 30 m. Display of aversive behaviors (erratic swimming, piping at the surface), time to loss of righting reflex, time to cessation of opercular movement, time to loss of tank tap response, and time to recovery after removal to clean tank water were recorded. Propofol at both concentrations induced loss of righting reflex and opercular movement more quickly than MS222 or lidocaine, without display of aversive behaviors as seen with ice bath exposure. However, for fish euthanized with propofol, exposure duration was a significant predictor of recovery from anesthesia whereas dose was not. Of the animals exposed to propofol for 20 m, 3 animals (1 of 8 at 100ppm, 2 of 8 at 150ppm) experienced deep anesthesia with loss of all measured responses but recovered within 30 m after being returned to a clean tank. A 30-m exposure to propfol at either concentration was sufficient for euthanasia of all animals tested. These findings suggest that exposure to propofol for a duration of at least 30 m may be an effective and humane means of euthanasia for adult zebrafish.

PS33 Comparing the Effect of Capromorelin and Mirtazapine on Appetite in New Zealand White Rabbits

J Draper*, EK Daugherity, M Martin-Flores, D Savson, E Lavin

Cornell University, Ithaca, NY

Inappetence is a welfare concern in rabbits, Oryctolagus cuniculus, that can lead to gastrointestinal (GI) stasis. Causes of inappetence and GI stasis include stress, pain, disease, poor diet, and opioid use. Treatments includes fluid therapy, enteral nutrition, GI motility stimulants, and appetite stimulants. There are no published studies evaluating efficacy of appetite stimulants in rabbits. We evaluated the effect of capromorelin, a ghrelin receptor agonist, and mirtazapine, antagonist of 5HT_2c and H1 receptors, on appetite in subsequential studies in New Zealand White (NZW) rabbits. Nine male healthy NZW rabbits received 9 treatments: capromorelin 4 mg/kg PO SID, capromorelin 8 mg/kg PO SID, capromorelin 4 mg/kg PO BID, capromorelin 8 mg/kg PO BID, mirtazapine 0.5 mg/kg transdermal (TD) SID, and mirtazapine 1 mg/kg TD SID with saline PO SID, saline PO BID, and saline TD SID acting as controls for 3 d with at least a week washout period in a randomized crossover study. Feed intake and fecal output were monitored daily with body weight monitored biweekly. Statistical analysis showed significant increase in feed intake and fecal output for all groups of capromorelin and mirtazapine compared to control except for capromorelin 4 mg/kg and 8 mg/kg SID on fecal output. Mirtazapine significantly increased feed intake and fecal output when compared to capromorelin. Body weight significantly increased for mirtazapine 1 mg/kg compared to control. Next, 9 male post-operative (neuter) NZW rabbits received 1 of 3 treatments: capromorelin 8 mg/kg PO BID, and mirtazapine 1 mg/kg TD SID with saline PO BID acting as a control for 3 d. Statistical analysis did not show a significant difference in feed intake, fecal output, or body weight for any of the groups. There was a magnitude of difference regarding increased feed intake and fecal output for mirtazapine that is clinically meaningful. In summary, capromorelin and mirtazapine increased appetite in healthy NZW rabbits evidenced by increases in feed intake and fecal output with mirtazapine increasing appetite in post-operative NZW rabbits as well. We recommend using mirtazapine in rabbits due to its superior efficiency compared to capromorelin.

PS34 Impact of Supplier-derived Gut Microbiomes on Anxiety-related Behavior

K Gustafson*¹, ML Hart², J Kwan³, L Lanoue⁴, L Bower⁴, R Araiza^4,5, K Lloyd^4,5, CL Franklin^6,8, A Ericsson^7,6

¹Comparative Medicine Program, University of Missouri, Columbia, MO; ²IDEXX BioAnalytics, Columbia, MO; ³School of Veterinary Medicine, University of California (UC), Davis, CA; ⁴Mouse Metabolic Phenotyping Center (MMPC) at UC Davis, Davis, CA; ⁵Mutant Mouse Resource and Research Center at UC Davis, Davis, CA; ⁶MU Mutant Mouse Resource and Research Center (MU MMRRC), Columbia, MO; ⁷University of Missouri Metagenomics Center (MUMC), Columbia, MO; ⁸University of Missouri Department of Veterinary Pathobiology, Columbia, MO

Anxiety disorders and anxiety-related behaviors are becoming increasingly prevalent in the general population, and can lead to learning deficits in children and a burden on the healthcare industry. The gut microbiome (GM) has been shown to play a role in central nervous system function, and differences in the taxonomic composition, and richness of the GM have been associated with anxiety-related behavior. The GM of specific pathogen-free (SPF) mice varies in composition and richness across suppliers of SPF mice, and substrains of mice from different suppliers are reported to display differences in anxiety-related behavior, although it is unclear whether those differences are due to the supplier-dependent differences in the GM or substrain divergence. We sought to determine whether the difference in diversity of these GMs affects anxiety-like-behavior phenotypes. Two colonies of CD1 mice were rederived in surrogate dams harboring either a high microbial diversity (CD1GM4) or low microbial diversity GM (CD1GM1), and underwent multiple behavior tests commonly used to test for anxiety-related behavior in mice. The 3 behavior tests that were performed were open-field exploration (OFE), light/dark transition (LDT), and elevated plus maze (EPM). Our results with the OFE test showed that CD1GM4 mice with high GM diversity travelled a significantly greater distance in the center zone of the open-field test than CD1GM1 mice with low GM diversity (P = 0.003), suggesting decreased anxiety-related behavior in CD1GM4 mice. LDT test showed that CD1GM4 mice also spent significantly more time in the light zone than mice with a lower diversity GM (P < 0.001), and results from the EPM test showed CD1GM4 mice travelled a further distance within the open arms than the CD1GM1 mice (P = 0.002), again indicating decreased anxiety-like behavior in CD1GM4 mice compared with CD1GM1 mice. These results demonstrate that supplier-origin GMs modulate behavior phenotypes. The data from this study will provide the scientific community with a novel system in which to study how the GM influences anxiety disorders.

PS35 Evaluation of Microenvironmental Parameters in Trio Breeding Cages of Mice

A Maxwell*, MG Stover, J Plunkard, JS Villano

Johns Hopkins University, Baltimore, MD

Trio breeding (a male and 2 dams) is a commonly used method of breeding mice in laboratory animal research. The Guide recommends 51 in² of space for a dam with a litter, thus indicating that standard cages (~75 in²) may not be sufficient for trio breeding housing. However, the Guide also emphasizes that performance standards are important considerations for space requirements. The objective of this study was to evaluate the microenvironment of breeding trios to create performance standards for our institutional guidelines on mouse cage densities. BALB/c mice (n = 10 cages; each housing 1 male and 2 dams, each with a litter of ~ 7 pups within 7 d apart) were housed in IVCs changed every 2 wk. Microenvironmental variables (temperature, relative humidity, CO₂, O₂ and NH₃ were measured at d 0, 1, 3, 5, 10, 14 of the cage change cycle). Results revealed that at every time point, cage temperatures (78.9°F + 0.56) but not relative humidity (40.1+ 1.9%) were significantly higher (than the acceptable ranges for the mouse housing room (67-77°F; 30-70%). O₂ and CO₂ had an inverse correlation, and cages were consistently hypoxic (20 + 0.19%; below normal room oxygen concentration, 21%) at every time point, and CO₂ levels exceeded the OSHA recommendations of 0.5% starting at day 3 (0.6 + 0.19%). Interestingly, NH₃ levels (5.27 + 9.7 ppm) remained below the NIOSH recommended exposure limit for humans (25 ppm) at all time points until d 14. Of note, animals remained clinically healthy during the entire experiment and litters were weaned without issues. In conclusion, the trio breeding microenvironment cage was suboptimal due to the increased temperature, increased CO₂ and decreased O₂ levels. However, further studies are warranted to determine potential effects of these findings to the overall animal health and wellbeing. In addition, studies are lacking on the ideal rodent environment as literature often cites animal macroenvironmental or human standards.

PS36 Pharmacokinetics of Gabapentin after Single, Oral Administration in Domestic Rabbits (Oryctolagus cuniculus)

M Burton*¹, R Conway³, N Mishkin³, LV Kendall¹, K Mama⁴, H Knych², M Sadar⁴

¹Laboratory Animal Resources, Colorado State University, Fort Collins, CO; ²Department of Molecular Biosciences and the K.L. Maddy Equine Analytical Pharmacology Laboratory, University of California-Davis, Davis, CA; ³James L. Voss Veterinary Teaching Hospital, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO; ⁴Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO

Gabapentin is used to treat epilepsy and neuropathic pain and, more recently, it has been prescribed as an anxiolytic in both human and veterinary medicine. Gabapentin has gained favor for its use as an anxiolytic as it is not classified as a controlled substance and has a wide margin of safety. As rabbits are easily stressed by handling, transport, and medical procedures, we propose gabapentin could be used as an anxiolytic. Current doses recommended for rabbits are extrapolated from other species or from limited studies. In this study, 3 male and 3 female 7-mo-old New Zealand white rabbits received a single dose of oral gabapentin at 25 or 50 mg/kg in a randomized crossover design. Plasma samples were obtained at 0, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 8, 12, 24, and 48 h post administration and concentrations determined using liquid chromatography-tandem mass spectrometry. Sedative effects were also scored at all time points. Pharmacokinetic analysis revealed mean peak plasma concentrations of 17,695 and 16,830 ng/mL and mean time to maximum plasma concentrations at 1.75 and 1.5 h for the 25 and 50 mg/kg doses, respectively. These results suggest nonlinear absorption may occur, as doubling the dose did not result in increased plasma concentrations. Rabbits were mildly to moderately sedated at 2-3 h with 25 mg/kg and 1.5-3 h with 50 mg/kg with none experiencing profound sedation. Based on these results, further evaluation of oral gabapentin at a dose of 25mg/kg as an anxiolytic for rabbits is recommended.

PS37 Assessment of Extended-release Buprenorphine in Comparison to Immediate-release Buprenorphine in a Murine Laparotomy Model

G Chan*, MR Nichols, C Si, L Kennedy

Unit for Laboratory Animal Medicine , University of Michigan, Ann Arbor, MI

Buprenorphine is commonly used by researchers for postoperative pain relief in rodents. Currently, immediate-release formulations of buprenorphine (Bup-HCl) require frequent handling and restraint of animals for appropriate dosing, which can subsequently increase stress and impair healing in surgical models. Frequent dosing intervals also require increased labor and compliance from research personnel, consequently increasing the potential for falling below therapeutic concentrations. A recently approved FDA-indexed extended-release buprenorphine formulation may be an appropriate alternative to Bup-HCl in mice and rats. The purpose of this study was to investigate efficacy of the new extended-release buprenorphine (Bup-ER) in a murine laparotomy model and compare its analgesic effects to Bup-HCl in the first 72 h after surgery. Six female and 12 male C57Bl/6J mice were randomly and evenly allocated to receive either Bup-ER (3.25 mg/kg) once presurgically or Bup-HCl (0.1 mg/kg) presurgically and then every 8 h for 72 h postsurgically. Mice underwent a sham laparotomy and were weighed daily and assessed for pain based on a cageside ethogram (appearance of hair coat, coordination and posture, and overall condition), behavioral assays (nesting consolidation test, rearing frequency), and nociception via von Frey testing at 6, 12, 24, 48, and 72 h postsurgically. Response variables were analyzed with a repeated measures 2-way ANOVA and posthoc comparisons were performed. Weight was significantly decreased from baseline at 48 h in the Bup-ER group and at 72 h in both groups. There were significant changes in cageside ethogram, nest consolidation scores, and rearing frequency at early time points as compared to presurgical baseline in both treatment groups, but no significant differences were noted between treatment groups. There were no significant differences in nociception between treatment groups or from baseline. These results indicate that extended-release buprenorphine has comparable efficacy to the immediate-release formulation and may be a viable alternative for postoperative analgesia in mice.

PS38 Effects of Analgesic Regimens on Appetite in New Zealand White rabbits (Oryctolagus cuniculus)

Z Hsi*^1,2, J Theil¹, R Oates^1,2, BW Ma¹

¹Campus Veterinary Services, University of California, Davis, Davis, CA; ²School of Veterinary Medicine, University of California, Davis, Davis, CA

Opioids and NSAIDs are frequent analgesics used in rabbits, whether in a clinical veterinary or laboratory setting. Rabbits are especially sensitive to adverse effects related to these analgesic regimens and may develop poor appetite and resulting gastrointestinal stasis. However, it is unclear whether these adverse effects are solely related to the analgesics or are a response to the restraint and injections required for administration. We compared the gastrointestinal side effects of buprenorphine and carprofen to their saline controls in New Zealand White rabbits following a nonsurgical anesthetic event. Nine rabbits (3 females and 6 males, aged 8 to 20 mo) randomly rotated through 5 treatment groups with a 7-d washout period between treatment periods: anesthesia control, buprenorphine (0.05 mg/kg SC every 12 h for 72 h), carprofen (5 mg/kg SC every 24 h for 72 h), twice daily saline control (equivalent volume to buprenorphine dosage SC every 12 h for 72 h), and once daily saline control (equivalent volume to carprofen dosage SC every 24 h for 72 h). Generalized linear mixed models were used to assess food intake, water intake, and fecal output over a 7-d period following anesthesia. Post hoc analysis showed that buprenorphine-treated animals had a significant 4-d decrease in food intake and 3-d decrease in fecal output compared to baseline. No other treatment groups showed any changes in food intake or fecal output compared to baseline. Of note was that animals that received twice daily saline had significantly greater appetites than all other treatment groups on d 7. These findings demonstrate that buprenorphine significantly depresses appetite in rabbits and that increased restraint and injections have minimal effect on appetite despite the possibility of increased stress.

PS39 Identification of Ornithonyssus bacoti Infestation via Molecular Diagnostic Techniques

B Clancy*^1,2, B Theriault^1,2, J Schoenberger^1,2, C Bowers¹, CM Mitchell^1,2, G Langan^1,2, A Ostdiek^1,2, KR Luchins^1,2

¹Animal Resources Center, The University of Chicago, Chicago, IL; ²Department of Surgery, The University of Chicago, Chicago, IL

Ornithonyssus bacoti, the tropical rat mite, is a zoonotic bloodsucking mite which may infest mouse colonies via wild rodent vectors. Heavy infestations in mouse colonies have been documented to cause anemia, alopecia, and decreased reproductive performance. In mid 2020, our institution experienced increased levels of wild mice, which were found to be infested with O. bacoti diagnosed by microscopic exam and confirmed by fur swab PCR. O. bacoti is often categorized as a rat pathogen, and is not included as a common wild rodent add-on test for direct animal PCR sampling, so it is likely overlooked during surveillance of wild rodents. We elected to add O. bacoti to our quarterly health monitoring exhaust air dust (EAD) testing, increase wild mouse control measures, and treat the environment with a permethrin spray in an attempt to prevent colony animal infestation. Initial quarterly EAD health monitoring results in September of 2020 were negative for O. bacoti. However, in early 2021, multiple IVC racks tested positive for O. bacoti via quarterly testing. Historically in the literature, O. bacoti outbreaks of research mice were not identified until mite burden was high enough to cause dermatitis on animal care workers. Due to modern molecular diagnostics and proactive EAD surveillance, our institution was able to identify and initiate eradication measures in a timely manner. To the best of our knowledge, this is the first report to successfully identify O. bacoti using environmental PCR techniques. This outbreak demonstrates the importance of screening for O. bacoti in facilities with wild rodent issues, and highlights unique considerations when managing O. bacoti infestations.

PS40 The Prevalence and Contrasting Outcomes of 2 Common Types of Ulcerative Dermatitis

DL Fong*, M Fink, L Habenicht, J Taylor, CA Manuel, JK Leszczynski

OLAR, University of Colorado Denver, Aurora, CO

Ulcerative dermatitis (UD) remains a common disease in laboratory mice. At our institution, we have informally classified UD into 2 main types based on clinical presentation and response to intervention: typical UD and atypical UD. Typical UD frequently involves superficial abrasions and/or scabs on the dorsal neck or ears. As previously reported, this type of UD generally heals well following a nail trim due to attenuation of self-trauma associated with repeated scratching. Atypical UD is not currently well-characterized and involves full thickness skin lesions that are primarily located on the lateral thorax and abdomen or limbs. We colloquially call atypical UD lesions “lick-chews” as we believe that excessive grooming behavior is the primary mechanism of injury. Anecdotally, these lesions are refractory to nail trims, and other topical and systemic treatments that have been described for the management of typical UD. To provide objective data, we retrospectively examined our electronic veterinary medical records for the prevalence and outcomes of these types of UD cases over a 3-mo timeframe. The average mouse daily census was approximately 22,300 cages and there were 2,456 completed mouse health reports during this period. Forty percent of health reports were classified by veterinary staff as UD. Atypical UD lesions accounted for 16% (160/975) of all UD cases. More importantly, mice with typical UD were reported healed in 74% (603/815) of cases, whereas animals with atypical UD lesions were reported healed in 12.5% (20/160) of cases. This data demonstrates the prevalence and importance of atypical UD lesions. While many papers have evaluated treatments for UD, we do not believe that studies have specifically assessed treatment efficacy by specific UD type. Atypical UD lesions should be further characterized to help develop more efficacious treatments for this challenging clinical presentation.

PS41 Cobalamin Supplementation in the Treatment of Chronic Gastrointestinal Disease in a Cynomolgus Macaque (Macaca fascicularis)

LM Lamoureux*, LC Halliday

Biologic Resources Laboratory, University of Illinois at Chicago, Chicago, IL

An 8-y-old, 2.8 kg, female, pair-housed cynomolgus macaque presented for chronic diarrhea and poor hair coat. On initial cage side exam, mentation and appetite were appropriate. The feces in the cage were voluminous, soft, and pale. The hair coat was greasy and unkempt. There were no remarkable findings on physical exam or abdominal ultrasound. Complete blood count and serum chemistry revealed moderate hypoproteinemia and hypoalbuminemia. Fecal occult blood, floatation, and wet mount were unremarkable. Fecal PCR and cultures were negative for common enteric pathogens, but indicated heavy growth of commensal organisms. Diet modification was attempted with high fiber and high protein (25%) biscuits with no improvement. Serum cobalamin and folate analysis revealed a marked decrease in cobalamin with normal folate compared to age and sex matched conspecifics. Cobalamin supplementation was initiated resulting in clinical improvements in fecal consistency, hair coat, protein, albumin, and cobalamin. Cobalamin supplementation was continued for 10 mo and was terminated based on clinical resolution and enrollment into a neuroscience study. After 6 mo on study, the study endpoint was reached and a necropsy was performed. Grossly, the gastric and colonic mucosa were thickened, and the pancreas was small at 6 cm in length. Histopathology results revealed severe atrophic lymphoplasmacytic gastritis and diffuse acinar cell atrophy of the exocrine pancreas. Immunohistochemistry of the stomach revealed a mixed inflammatory infiltrate of T and B cells. There are reports of Helicobacter spp. causing severe gastritis in cynomolgus macaques; however, Steiner (silver) stain did not reveal the presence of argyrophilic organisms. Notably, atrophy of the exocrine pancreas has been regarded as a rare incidental finding. Although considered to have low sensitivity and specificity, evaluation of serum cobalamin and folate levels may provide useful insight into underlying pathologies and course of treatment in nonhuman primates with chronic diarrhea.

PS42 Acute Cholangiohepatitis in a Colony of Common Marmosets (Callithrix jacchus)

N Castell*, S Powers, K Gabrielson, J Izzi

Johns Hopkins School of Medicine, Baltimore, MD

Seven experimentally naïve common marmosets (Callithrix jacchus) presented individually over the course of 9 mo with acute onset of severe lethargy and dull mentation. Perimortem serum chemistries revealed marked elevations in GGT, ALP, AST, and ALT, indicative of hepatobiliary disease. Complete blood counts were mostly unremarkable, with mild neutrophilia in some cases. Abdominal ultrasounds showed increased echogenicity in liver parenchyma and within the gallbladder lumen, thickening of the gallbladder wall, abdominal effusion, or were unremarkable. Despite extensive supportive care and antibiotic therapy, all animals experienced a rapid decline in clinical condition leading to death or euthanasia within 48 h of presentation. Necropsy revealed severe necrosuppurative cholangiohepatitis with gallbladder obstruction. Postmortem cultures from multiple organs isolated predominantly Pseudomonas and E. coli spp. Based on clinical and necropsy findings, the likely cause of bacterial cholangiohepatitis was due to ascending infection from the gastrointestinal tract into the biliary tree, ultimately leading to septicemia. PCR testing for multiple viral pathogens was negative. Investigation into sources of the bacterial infection led to the discovery of contamination of water supply equipment with Pseudomonas aeruginosa. The entire water supply system was replaced immediately, and no additional cases have since occurred. Interestingly, though all animals in the colony received similar exposure through the common water supply, only a small subset of marmosets showed any indication of clinical disease. The incidence of mortality due to cholangiohepatitis in the colony was 3.4%, with predominantly adult males over 7 y of age being affected. Genetic predisposition may play a role in susceptibility, as most cases (6/7) had a common origin outside the facility. These cases demonstrate the predisposition of marmosets to Pseudomonas infection through chronic oral exposure, as well as the importance of using environmental testing to monitor and validate routine sanitization practices.

PS43 Successful Treatment of Acute Buoyancy Disorder in a Common Goldfish (Carassius auratus) Using an Improvised Hyperbaric Chamber

CF Janssen*

CLAMC, UTHealth, Pearland, TX

A common goldfish with normal anatomy presented acutely with classic signs of buoyancy disease, specifically positive buoyancy, after being overfed. The fish was actively struggling to swim downward, resulting in an abnormal head down posture. Opercular movements were rapid and pronounced. Subjectively the fish appeared to be distressed. Any cessation in effort resulting in rapid floating towards the surface. As the fish became fatigued and rested at the surface, it was noted that 1-2 mm of the body was above the water confirming significant positive buoyancy. The abdomen was not obviously distended, however, the peri-anal region did appear to bulge, suggesting intraabdominal pressure in a non-distensible abdomen. Based on the history of overfeeding, it was determined that the excess gas was as likely to be within the GI tract, ie colic, as within the swim bladder. Needle aspiration of air from the swim bladder is often recommended as treatment for positive buoyancy, however, in this instance, without available imaging or anesthesia, with suspicion that the swim bladder was not the issue, and with high risk of GI perforation, this option was rejected. It was decided to reduce the volume of the air bubble by compressing the fish in an improvised hyperbaric chamber. This immediately alleviated the tympany, potentially relieving any discomfort and potentially improving blood flow to affected tissues, and eliminated the struggle to maintain buoyancy. Subjectively the fish appeared to be less distressed, with less frantic swimming motion, when compressed. The pressure was maintained for a short period, then slowly released, allowing the bubble opportunity to be redistributed before re-expanding to the original volume. This was repeated several times in a first session, and again several times 4 h later. The fish eventually defecated a large air bubble, trapped in mucus, which remained attached to the anus long enough to be photographed. One year later the fish is well and has had no further issues. Here we suggest that hyperbaric therapy be considered as a treatment option for acute positive buoyancy disease of fish, and suggest that a better quality, less hastily improvised hyperbaric chamber might be a useful addition to the fish veterinarian’s toolbox.

PS44 Evaluation of Proparacaine for Use during Tuberculin Skin Tests in the Common Marmoset (Callithrix jacchus)

JL Haupt*, RM Kramer, M Jamiel, A Johnson

Massachusetts Institute of Technology, Cambridge, MA

Tuberculin skin test (TST) is the primary screening tool for M. tuberculosis surveillance in common marmosets (Callithrix jacchus). In our colony, all animals are tested during semiannual health examinations under alfaxalone sedation (7-10 mg/kg IM). Mammalian old tuberculin (MOT) is injected intradermally into the superior palpebral at the lateral canthus. The palpebral skin of the common marmoset is thick and many marmosets react to the injection of MOT with the movement of the head, eyelids, or scratching of the eye immediately after injection. Failed injections, multiple needle sticks, and scratching can lead to palpebral erythema, bruising, and inflammation, leading to indeterminate or false-positive test results. This project aimed to evaluate the use of proparacaine, a rapid-acting local anesthetic for ophthalmic use, to diminish adverse reactions from TST using MOT in the common marmoset. During semiannual health exams, animals (n = 66) were assigned in a blinded manner and either given 1 drop of artificial tears (control, n = 35) or proparacaine (treatment, n = 31) in the eye assigned for TST at least 1 min before intradermal placement of the MOT. The dose of alfaxalone, animal weight, body condition score, and age was recorded along with reaction to the injection. TST reaction grade was recorded at 24, 48, and 72 h post-injection. There was no difference with the use of proparacaine on needle reaction, injection reaction, movement during injection, scratching, bleeding, or bruising post-injection, and 24, 48, and 72 h TST grade reaction scores. However, higher alfaxalone doses were associated with decreased needle reaction, injection reaction, movement during injection, scratching post-injection, and 24 and 72 h TST grade reaction scores. Inverse prediction was used to identify decreased reaction to injection at an alfaxalone dose of 10 mg/kg. The results of this project show that proparacaine had no impact on TST reaction in the common marmoset. However, higher doses of alfaxalone diminished adverse reactions to injection and lead to less erythema, bruising, and indeterminate TST reaction grades in the common marmoset. Based on the findings of this project, sedation with 10 mg/kg of alfaxalone is advocated for TST in the common marmoset.

PS45 Management of Encephalitozoon cuniculi Infection in a Breeding Colony of African Spiny Mice (Acomys spp.)

DM LeMoine*, D Moorman-White, K Nolan

Comparative Medicine, University of Rochester, Rochester, NY

A 5-wk-old male African spiny mouse (Acomys spp.) born in-house was euthanized due to acute onset ataxia, lethargy, weakness, and dyspnea. Histopathology revealed meningoencephalitis and interstitial nephritis with Gram-positive microsporidia consistent with Encephalitozoon cuniculi present in both tissues. We performed follow up testing to determine the prevalence of the pathogen within the colony. PCR of urine and feces detected active shedding of E. cuniculi in 1 of 4 cages, and 55% (n = 11) of the colony was seropositive for E. cuniculi antibodies. Because E. cuniculi is a zoonotic pathogen that can infect a wide range of mammalian hosts, a multipronged control strategy was implemented to control the agent while preserving valuable breeding animals for future research needs. We quarantined the colony in place and informed personnel of additional PPE requirements. Two cages of offspring born in-house were culled for experimental use. For the remaining animals, we separated breeding pairs to prevent vertical transmission and initiated treatment with two, 4-wk courses of fenbendazole-medicated feed. Shortly after starting treatment, one additional juvenile was found dead and, although seronegative, confirmed to be infected based on PCR of brain tissue. PCR testing of urine and feces was negative by the midpoint of treatment and remained negative after completing the full 8-wk treatment course. No additional clinical signs or mortality were seen. None of the animals that were previously seronegative (n = 4) or that were born during the treatment period (n = 5) seroconverted. We believe that at least 1 of the founder animals was carrying E. cuniculi, which is not included in our routine quarantine testing, upon import. Fenbendazole treatment effectively prevented further spread of E. cuniculi within the colony. The use of multiple testing strategies was critical for fully characterizing the infection, as tissue PCR detected the pathogen in animals that were seronegative and not actively shedding spores in urine or feces. It is possible that organisms persist in the tissues of previously infected animals. Therefore, we plan to continue regularly screening for E. cuniculi by exhaust air dust PCR and tissue PCR when available.

PS46 Spontaneous Acute T-cell Leukemia in a Specific-Pathogen Free Rhesus Macaque (Macaca mulatta)

G Campellone*, M Stovall

Veterinary Medicine, Yerkes National Primate Research Center, Stone Mountain, GA

A 5-y-old, female rhesus macaque (Macaca mulatta) housed in a specific-pathogen free (SPF), outdoor breeding colony, presented to the hospital with lameness. Physical examination and radiographs revealed mild effusion in the right stifle. A soft tissue injury was suspected, and analgesics and cage rest were initiated. Bloodwork revealed anemia and a marked leukocytosis, 27.02k/ul. The animal was re-examined the following day due to reports of abnormal respiration, stertor, and bilateral epistaxis. Respiratory signs improved once the nares were cleared and the animal was returned to the cage for close monitoring. Three d later the animal was re-examined and noted with bilateral epistaxis and crackles in all lung fields. Thoracic radiographs were consistent with pneumonia, and broad-spectrum antibiotics were initiated. Repeat bloodwork revealed continued anemia and a progressive leukocytosis (40.85k/ul). Over the next 9 d, multiple examinations and radiographs were performed. While the pneumonia resolved, the hindlimb lameness progressed bilaterally and the leukocytosis consistently progressed, increasing to 47.26k/ul and then to 52.32k/ul. Acute leukemia was suspected at this time. The patient was examined the following day to obtain a bone marrow aspirate, however, severe generalized lymphadenopathy, cardiovascular decompensation, and progressive weight loss were noted on examination. Due to the animal’s decline, and the presumptive diagnosis of acute myeloid leukemia, the animal was euthanized. Gross pathology findings and histology were consistent with T-cell leukemia. No abnormalities were noted in the stifles; however, joint pain is associated with leukemia. In nonhuman primates, T-cell leukemia is typically associated with STLV-1; however, both serology submitted 8 mo prior to initial clinical presentation and PCR submitted at necropsy were negative. To the authors’ knowledge, this is the first reported case of spontaneous acute T-cell leukemia in an SPF rhesus macaque. It’s important to recognize this as a potential neoplasia, even in SPF nonhuman primate colonies.

PS47 Prevalence of Infectious Agents in Zebrafish Colonies Based on Real-time PCR (2015–2019)

MJ Crim*¹, ML Hart², CL Besch-Williford³, RS Livingston²

¹Molecular Diagnostics and Microbiology, IDEXX BioAnalytics, Columbia, MO; ²R&D and Scientific Affairs, IDEXX BioAnalytics, Columbia, MO; ³Pathology, IDEXX BioAnalytics, Columbia, MO

Naturally occurring infections can adversely affect research, including invalid or misinterpreted experimental data, zoonotic infections, or elevated morbidity and mortality. Prevalence of infectious agents is an important consideration in risk assessments for import procedures, quarantine, and colony health monitoring. We report the positivity rate of real-time PCR results for 18 infectious agents in pooled zebrafish and environmental samples submitted by 230 biomedical research institutions over a 5-y period to a major commercial diagnostic laboratory. Prevalence varied widely by agent, and for some agents varied widely among submitting institutions. The overall positivity rate also varied by sample type for each agent, with a higher percentage of positive test results in pooled zebrafish samples for Zebrafish picornavirus and all common parasites except for Myxidium streisingeri. A higher percentage of environmental samples tested positive for Mycobacterium spp., facultative pathogens that participate in system biofilms, compared to pooled zebrafish samples. The most frequently detected infectious agents in pooled zebrafish samples were Zebrafish picornavirus (26.5 %), Pseudoloma neurophilia (19.9 %), M. streisingeri (16.0 %), M. chelonae (9.7 %), Pseudocapillaria tomentosa (7.6 %), and M. haemophilum (2.6 %). In contrast, M. chelonae (44.8 %), M. fortuitum (35.1 %), M. streisingeri (23.5 %), M. gordonae (21.8 %), M. abscessus (18.7 %), and Zebrafish picornavirus (16.7 %) were the most frequently detected agents in environmental samples. Some pathogens may remain undetected by exporting institutions relying on detection via less sensitive sample types. Institutional prevalence from the exporting institution is more useful than overall prevalence for assessment of importation biosecurity risk since the institutional prevalence for several agents can differ substantially from overall prevalence. Thus, obtaining detailed zebrafish colony health information from exporting institutions, including sampling methodology, is critical to assessing the biosecurity risks associated with the importation of zebrafish lines.

PS48 Prostaglandin F2α and Dexamethasone in the Treatment of Closed-Cervix Pyometra to Preserve Breeding Function in a Bitch

L Hagan*¹, V Stora², JO Marx¹, C Vite²

¹ULAR, University of Pennsylvania, Philadelphia, PA; ²RCAM, University of Pennsylvania, Philadelphia, PA

A 5-y-old 21.0kg mixed-breed bitch heterozygous for the gene causing globoid cell leukodystrophy (Krabbe disease) was reported by animal care staff for lethargy and one episode of vomiting. On physical examination she was quiet, alert, and responsive. Her mucus membranes were pink, tacky, with a capillary refill time of < 2 seconds, and her abdomen was mildly distended and firm on palpation. Ultrasonography of her abdomen revealed uterine horns filled with hyper to hypoechoic fluid without vaginal discharge. Complete blood count and chemistry revealed dehydration (total protein 8.9 g/dL, BUN 91 mg/dL, creatinine 2.0 mg/dL), hyperphosphatemia (13.6 mg/dL), and hypercholesterolemia (538 mg/dL). Serum progesterone measured 23.3 ng/mL (normal less than 1.0 ng/mL). The diagnosis of closed-cervix pyometra was made. Following the diagnosis, a veterinary theriogenologist prescribed and initiated treatment of dexamethasone-SP 0.1 mg/kg subcutaneously once daily for 7 d, and prostaglandin F2α for luteolysis and smooth muscle contraction for the expulsion of uterine contents, (0.05 mg/kg SC BID for one day, then 0.1 mg/kg SC BID until normal serum progesterone), buprenorphine (0.02 mg/kg SC BID), amoxicillin/clavulanic acid (25 mg/kg PO BID), enrofloxacin (10 mg/kg PO SID), maropitant (1 mg/kg SC SID), ondansetron (0.5 mg/kg SC BID), and subcutaneous fluids (10-15 ml/kg SC BID). The patient improved within 3 d, with increased appetite and activity following tolerance to prostaglandin. Bloodwork abnormalities resolved after 6 d of treatment and serum progesterone reduced to 0.5 ng/mL. Prostaglandin therapy was discontinued after 8 d. Although ovariohysterectomy is commonly the treatment of choice for closed-cervix pyometra, this case demonstrates successful medical management of a closed-cervix pyometra within a valuable, middle-aged breeding bitch.

PS49 C57BL/6 Mouse Sperm Quality Analysis, Fertilization Rates, and Embryonic Development Competency following Preservation at 4°C for 18, 48, and 72 Hours

S Gerb*, K Gustafson, C Agca, Y Agca

Veterinary Pathology , University of Missouri, Columbia, NJ

Around the world, investigators are creating mouse models of human disease. These models are time consuming and expensive to create. It is not uncommon for genetically modified animals to die unexpectedly, potentially causing extinction of a line. The collection of epididymal sperm for assisted reproductive techniques, such as in vitro fertilization is a viable method to reanimate lines. However, not all institutions are equipped to perform such procedures. Therefore, there may be a need to ship animals to institutions that have such capabilities. To determine the ideal shipping conditions and timeline, 6-12-mo-old male C57BL/6 mice were randomly placed in 1 of 3 time point groups: 18 h (n = 12), 48 h (n = 14) and 72 h (n = 16). Animal were then then euthanized via CO₂, and sperm was collected from 1 cauda epididymis for fresh sperm analysis. Mice with the remaining cauda epididymis were then placed in a refrigerator at 4°C. After the appropriate time had passed (18 h, 48 h, or 72 h), sperm was collected from the remaining cauda epididymis and analyzed. Sperm was significantly less motile following 18, 48 or 72 h at 4°C (P < 0.001) however, there was no significant differences in progressive motility between groups for both fresh and aged sperm (P = 0.465). Fresh sperm and sperm from the 48 h and 72 h group was then used to perform in vitro fertilization using fresh oocytes from C57BL/6 mice super ovulated with PMSG and hCG. The percent of fresh oocytes fertilized was significantly higher in the fresh group in comparison to the 72 h group (P > 0.001) and sperm from the 48 h group fertilized significantly more oocytes than the 72 h group (P > 0.001). The percentage of fertilized oocytes that went on to develop into morula and blastocyst were assessed. There were no significant differences in morula (P = 0.324), or blastocyst development rates (P = 0.147) between groups. The data collected from this study suggests that sperm collected from animals that have been preserved at 4°C for 48 h or less would provide the best outcome for in vitro fertilization and reanimation of a line. Practically, our study suggests that mice should be shipped via 2-d shipping on ice (about 4°C) for the best outcome.

PS50 The Extracellular Antioxidant Enzyme Glutathione Peroxidase 3 Drives Tumor Progression in a Syngeneic Mouse Ovarian Cancer Model

C Chang*¹, N Hempel², R Phaeton³

¹Comparative Medicine, Penn State University College of Medicine, Hershey, PA; ²Pharmacology, Penn State University College of Medicine, Hershey, PA; ³Gynecologic Oncology, Penn State University College of Medicine, Hershey, PA

Ovarian cancer is the most deadly gynecological malignancy and fifth leading cause of cancer-related deaths in women. Most patients are diagnosed at late stages of the disease, which is marked by chemoresistance, high rates of recurrence, and significant abdominal tumor burden evidenced by ascites accumulation. We previously demonstrated that glutathione peroxidase 3 (GPX3), an extracellular antioxidant enzyme, has heterogenous expression in ovarian cancer, where high GPX3 expression is associated with decreased patient survival and increased tumor stage. The main function of GPX3 is to reduce hydrogen peroxide (H₂O₂) to water with the help of glutathione. Therefore, a decrease in GPX3 expression may leave tumor cells vulnerable to increased reactive oxygen species in the extracellular tumor microenvironment. We hypothesized that ovarian cancer with decreased GPX3 expression that has metastasized to the peritoneal cavity will be less aggressive than ovarian cancer with normal GPX3 expression. We developed 2 stable mouse ovarian cancer (ID8) GPX3 knock-down cell lines using lentiviral shRNA constructs (shGPX3 #1 and #2). Female C57BL/6 mice were injected intraperitoneally with either 1 x 10⁶ control ID8 (n = 7) or 1 x 10⁶ GPX3 knockdown ID8 cells (n = 9) and were serially imaged weekly using in vivo bioluminescence imaging for at least 8 wk when they began to develop ascites. At necropsy, the amount of ascites was quantified and tumor burden documented. Mice injected with GPX3 knockdown ovarian cancer cells either never developed tumors or showed increased survivability compared to control ID8 mice. The mice that did develop tumors showed delayed tumor progression but similar tumor burden compared to controls. These results support evidence that decreased GPX3 expression is antitumorigenic, although more research is needed to determine if this conveys an increased susceptibility to certain treatments.

PS51 Influence of Daytime LED Light Exposure on Circadian Regulation of Neuroendocrine Hormones in Adolescent Sprague Dawley Rats

AA Allen*¹, AT Pierce¹, RT Dauchy², GB Voros¹, GL Dobek¹

¹Comparative Medicine, Tulane University, New Orleans, LA; ²Structural & Cellular Biology, Tulane University, New Orleans, LA

Light can significantly influence regulation of circadian rhythms of physiology and metabolism in laboratory animals. Light-emitting diode (LED) light is a rapidly emerging technology, yet little is understood regarding its impact on animal circadian and neuroendocrine responses. Previous studies from our laboratory demonstrated that exposure to LED light enriched in the blue-appearing portion (465-480 nm) of the visible spectrum at daytime (bLAD) enhances these responses in mice compared to those housed under broad spectrum (300-740 nm) cool white fluorescent (CWF) light. Here, we hypothesize that daytime exposure of adolescent Sprague Dawley rats to bLAD, compared to CWF light, positively influences circadian regulation of select neuroendocrine hormones. Control animals (n = 36m, 36f) were maintained on a standard IVC system under CWF light, whereas experimental animals (n = 36m, 36f) were housed inside a novel light-containment IVC system emitting bLAD light; both groups were housed for 30 d under a common lighting regimen 12L (68.8 ± 5.2 lux; 168.6 ± 12.8 µW/cm²):12D (0 lux). On d 30, rats were anesthetized with ketamine/xylazine and terminal collection of arterial blood performed to quantify serum melatonin, corticosterone, insulin, and glucose across 6 circadian time points (0400, 0800, 1200, 1600, 2000, 2400). Compared to CWF males and females, rats in bLAD lighting evinced a 6-fold higher peak in nighttime serum melatonin (P < 0.05). Differences in serum corticosterone were sex dependent, as CWF and bLAD females produced more corticosterone overall than CWF and bLAD males (P < 0.05), respectively. While CWF and bLAD males exhibited significantly higher serum glucose compared to females, there was no difference in serum insulin among sex (m or f) nor lighting conditions (CWF or bLAD). This data show that housing SD rats under bLAD lighting conditions has a positive impact on circadian melatonin rhythm without increasing overall serum corticosterone production, nor altering glucose metabolism and production of serum insulin. Future studies should examine effects of long-term (>3 mo) housing of both adolescent and adult animals under bLAD lighting on circadian regulation of these neuroendocrine and metabolic parameters.

PS52 Feline Immunodeficiency Virus Treatment with Combination Antiretroviral Therapy Assessed Using Digital Droplet PCR

J Kim*¹, E Behzadi², M Nehring², CA Miller³, S Carver⁴, S VandeWoude²

¹Laboratory Animal Resources, Colorado State University, Fort Collins, CO; ²Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO; ³Veterinary Pathobiology, Oklahoma State University, Stillwater, OK; ⁴Biological Sciences, University of Tasmania, Hobart, , Australia

Feline Immunodeficiency Virus (FIV) is a naturally occurring retrovirus that infects domestic cats and causes progressive immune dysfunction. Although highly effective treatments, including a combination antiretroviral therapy (cART), have been developed for human immunodeficiency virus, there is no effective therapy for FIV. This study aimed to assess a cART, composed of 2.5 mg/kg Doulutegravir, 20 mg/kg Tenofovir, and 40 mg/kg Emtricitabine, as a treatment for FIV in domestic shorthaired cats. Eighteen 6 to 10-mo-old cats in total were used with female intact and male neutered cats equally distributed among cART, placebo, and control groups. We inoculated 12 cats with FIV in addition to 6 naïve cats serving as controls, and we collected whole blood to quantify viral and proviral loads via digital droplet PCR pre-FIV inoculation, weekly postinoculation at wk 1-6, and every other wk at wk 8-24. Beginning wk 4, we injected cART (n = 6) or a placebo (n = 6) containing 1 mL of 15% kleptose, the cART vehicle, subcutaneously once daily to the FIV-infected cats. Infection peaked at wk 2 with mean viral loads noted at 6.43e⁶ and 12.4e⁷ copies/mL among cART and placebo cats, respectively; mean proviral loads were noted at 5.43e⁴ and 4.94e⁴ copies/10⁶ cells among cART and placebo cats, respectively. Viral and proviral loads steadily declined after wk 2 and were not statistically significant. Hematologic analysis on wk 4, 6, 10, and 16 demonstrated marked neutropenia in placebo cats compared to cART (P = 0.05), lowest at week 6 with mean absolute values/uL of 651 and 1340, respectively. Mean neutrophil values in cART cats normalized by week 16, but placebo cats remained neutropenic. No clinical signs associated with neutropenia were observed. These results suggest that this cART protocol does not reduce FIV viral and proviral loads but may impact hematologic impacts of FIV infection.

PS53 Examining the Transmission of Nippostrongylus brasiliensis in Mice to Assess Biosecurity Risks when Used as an Animal Model

RJ Floyd*¹, RJ Ricart Arbona^1,2, NS Lipman^1,2

¹Tri-Institutional Training Program in Laboratory Animal Medicine and Science, New York, NY; ²Center of Comparative Medicine and Pathology, Memorial Sloan Kettering and Weill Cornell Medicine, New York, NY

Nippostrongylus brasiliensis (Nb) infected mice are commonly used to study systemic and mucosal immune responses due to the parasite’s ability to induce a potent Th2 type response. There is no clear consensus regarding what biosecurity measures should be employed when housing mice and rats infected with Nb. Transmission is anecdotally reported to not occur when infected mice are co-housed with naive mice, if the cage is changed twice weekly, as the infective L₃ larvae take approximately 5 to 6 d to develop under optimal temperature and humidity. We sought to determine what conditions, if any, favor horizontal transmission to inform biosecurity decisions when using the model. Female NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/Sz (NSG; n = 12] and C57BL/6J (B6; n = 12) inoculated with 750 Nb L₃ larvae were cohoused with naïve mice (NSG; n = 24 and B6; n = 24) at a ratio of 1 infected:2 naïve mice per cage for up to 28 d with cages changed on a 14-d interval. We also assessed whether larval development can occur to the L₃ stage when Nb ova-containing fecal pellets are maintained in 3 different environmental conditions (dry, moist, and soiled bedding) and whether infection results when naïve mice (NSG; n = 9) are housed in cages with infective L₃ larvae-spiked (10,000/cage) soiled bedding. NSG mice (n = 3) were also gavaged with embryonated and non-embryonated Nb eggs to model the potential for infection to occur following coprophagy. Both naïve NSG (8/12) and B6 (8/12) cohoused with an infected cage mate passed Nb eggs in feces within 4 d of cohousing for short intermittent periods, presumably a result of coprophagy as adult worms were not detected at euthanasia on d 28. While eggs were able to develop into L₃ larvae in 2 environmental conditions, none of the NSG mice housed in cages with L₃-spiked bedding or gavaged with eggs became infected with Nb. These findings demonstrate that horizontal transmission does not occur even when mice are housed in static caging with 14-d cage changing intervals. Results from this study can be used to make recommendations regarding biosecurity practices to employ when working with the Nb-infected mouse model.

PS54 SARS-CoV-2 Variant Pathogenesis and Effects of Preexisting Immunity in Syrian Hamsters

J Plunkard*¹, M Lowman², K Mulka¹, K Carter², JS Villano¹

¹Department of Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, MD; ²Johns Hopkins University, Baltimore, MD

SARS-CoV-2 (SCV2) has had a historical impact on the globe, with over 3.5 million COVID-19 related deaths since its discovery. As new variants emerge, it is crucial to understand differences in their pathogenesis and the influence prior infections may have. This study investigates the pathogenesis of 5 variants and the impact of preexisting immunity on variant pathogenesis using the Syrian hamster model. We hypothesize that variants will cause significant clinical disease and lung pathology, and that preexisting immunity will result in significantly less severe clinical signs. To examine variant pathogenesis, 6-8-wk-old Syrian hamsters (n = 4/sex/time point/variant) were inoculated intranasally (50 ul/nare of infectious dose) under ketamine/xylazine sedation and euthanized at 2, 4, or 7 d post-infection (dpi). Body weights (BW) and clinical scores (rough hair coat, squinting, etc.) were recorded daily until euthanasia. To examine preexisting immunity, 5 groups (n = 4/sex) were infected intranasally (as described above) with the SCV2 Washington strain. At 28 dpi, each group was then infected with another variant (as described above) and euthanized at 35 dpi (post-original infection). BW and clinical scores as above were collected on 1-10, 14, 21, and 28-35 dpi. Results were analyzed by ANOVA and t-test; P < 0.05. Male and female hamsters lost significant amounts of BW, with males losing more weight than females. The BW loss over time differed significantly between most variant groups, with variants having up to a 13.9% difference in average weight loss on d 7. Hamsters with preexisting immunity lost significantly less BW than those without, with up to a 16.9% difference seen on 7 dpi. Unkempt hair, pinned ears, and squinting were the 3 primary clinical signs recorded, and no respiratory clinical signs were observed. Histologic findings revealed mild to severe lung consolidation, atypical type II pneumocyte hyperplasia, and macrophage infiltration, with severity dependent on the timepoint. In conclusion, prior SCV2 infection provided some protection against another variant. Future studies are warranted to investigate the pathogenesis of other variants and the molecular and immunological mechanisms of preexisting immunity.

PS55 Physical Performance Is Enhanced in Old Mice Fed a Short-term Diet Medicated with Rapamycin, Acarbose, and Phenylbutyrate

M Ellis*, W Ladiges, Z Jiang

Comparative Medicine, University of Washington, Seattle, WA

Loss of physical performance, as seen in humans by decreased grip strength and overall physical fitness, is generally accepted to be a consequence of aging. Treatments to delay or reduce these changes or increase resilience to them are generally not available. We are suggesting the use of a novel diet combination to increase resilience to age-related physical decline in mice. In this preliminary study, 20-mo-old male and female C57BL/6 mice were given either a standard mouse diet or a formulated mouse diet containing rapamycin (14 ppm), acarbose (1000 ppm), and phenylbutyrate (1000 ppm), or a diet containing one half dose of each drug, for 3 mo. Ten to 12 mice were included in each group. The rationale for the drug cocktail was based on validated anti-aging effects of the individual drugs, each targeting different but overlapping processes of aging. Rapamycin downregulates mTOR and acarbose increases glucoregulatory control, both working on different aging pathways to increasing lifespan in mice. Phenylbutyrate, an ammonia scavenger, enhances physical and cognitive performance in aged mice. At the end of the study, performance on a rotarod and grip strength test was compared. In general, mice fed the full dose drug cocktail diet performed better on these assays, with significant improvements in rotarod performance in females fed the full dose cocktail and in grip strength in males fed the full dose cocktail, and females fed the low dose cocktail. These observations provide support for the concept that short term treatment with a cocktail of drugs that targets multiple aging pathways can increase resilience to aging and suggests that this prototype cocktail could be part of a clinical therapeutic strategy for delaying age-related loss of physical performance in people.

PS56 Antibody Production and Clinical Assessment of Rabbits Administered Freund’s Adjuvant With and Without Carprofen

E Perminov*¹, SC Adams²

¹ARD, Salk Institute of Biological Sciences, San Diego, CA; ²Janssen Pharmaceutical Companies of Johnson & Johnson, San Diego, CA

Freund’s adjuvants are commonly used to boost polyclonal antibody production in rabbits, however, the immunostimulatory effects, in particular with complete Freund’s adjuvant (CFA), result in inflammation and can have the potential to cause pain and/or distress. Recommended injection routes for Freund’s adjuvants include subcutaneous and intradermal but there is still debate on which is the best in terms of animal welfare. It has been previously shown that subcutaneous administration of Freund’s adjuvant in conjunction with carprofen did not alter polyclonal antibody production in rabbits. However, no one has specifically looked at antibody titers after intradermal injection of Freund’s adjuvant along with nonsteroidal antiinflammatory administration. In this study, 9 female New Zealand White rabbits were assigned to 1 of 3 groups: intradermal inoculation with carprofen (4 mg/kg SC SID for 3 d; n = 3), intradermal inoculation with carprofen (4 mg/kg SC SID for 3 d; n = 3), or subcutaneous inoculation without carprofen (n = 3). They were then immunized with antigen in CFA twice, 3 wk apart, followed by injection with antigen in IFA (incomplete Freund’s adjuvant) 3 wk later. One wk after immunization with antigen in IFA, blood was collected for circulating antibody titers. Three wk later rabbits were given a booster immunization of antigen in IFA and additional blood samples were collected 1 wk later. Titers were determined using radiolabeled ligands in solution with dilution series of the serum from each rabbit after each bleed. Clinical assessment of the rabbits was performed using an ethogram score for 20 d post both CFA injections. We found that carprofen administration did not adversely affect antibody production in response to the antigen used in this study. Ethogram scores for rabbits with intradermal injections without carprofen trended higher than for rabbits with intradermal injections with carprofen. These results indicate that carprofen may reduce the associated pain and distress of rabbits being used for polyclonal antibody production while not adversely affecting titer levels.

PS57 Stretch-induced Atrial Arrhythmia in Isolated Hearts of Aged C57BL/6 mice

JA Cayton*^1,2, Z Nourian^2,1, M Lambert^2,1, T Domeier^1,2

¹Department of Pathobiology, University of Missouri, Columbia, MO; ²Medical Pharmacology and Physiology, University of Missouri, Columbia, MO

Atrial fibrillation (AF) is the most common sustained arrhythmia, and more than 2.7 million people are living with AF in the United States. The elderly are at greatest risk for AF with prevalence estimated at ~10% in individuals over the age of 80. The aged myocardium is subjected to chronic stretch, and atrial muscle stretch is an established risk factor for atrial arrhythmogenesis. We hypothesized that increases in left-atrial pressure lead to atrial arrhythmogenesis in a mouse model of advancing age. To test this hypothesis, we used isolated hearts (excised hearts in a modified Langendorff perfusion technique) of aged (19-28 mo) C57BL/6 male (n = 6) and female (n = 2) mice subjected to atrial preloads of 0 cm and 12 cmH20 (i.e., increasing stretch). Atrial arrhythmias were monitored using left-atrial placement of an intracardiac electrocardiogram, and burst pacing stimuli were used to induce arrhythmias (bursts 2 s in duration with 20-40 ms varying cycle length, followed by a 3-min stabilization/observation period, repeated 4 times). Arrhythmias were defined as transient (premature atrial contractions and salvos of <3 premature atrial contractions) or sustained (salvos of >3 premature atrial contractions, atrial tachycardia, or AF). At 0 cmH20 transient atrial arrhythmias were observed in 6/8 hearts, and sustained atrial arrhythmias were observed in 2/8 hearts. Following elevation in left-atrial preload to 12 cmH20 pressure, transient atrial arrhythmias were observed in 7/8 hearts and sustained atrial arrhythmias observed in 6/8 hearts (P < 0.05 versus 0 cmH20, z-test of proportions). Stretch-induced sustained atrial arrhythmias were observed in both male (4/6) and female (2/2) aged mice. In conclusion, the aged mouse atrium exhibits sustained arrhythmia in response to increases in left-atrial preload pressure. The aged C57BL/6 mouse model may therefore be useful for preclinical studies of age- and stretch-induced atrial arrhythmias.

PS58 Hemodynamic Changes in Response to Hyperacute Spinal Trauma in an in Vivo Porcine Model of Spinal Reconstruction

ED Barras*¹, CD Hampton², C Takawira¹, T Taguchi¹, A Nourbakhsh³, M Lopez¹

¹Veterinary Clinical Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, LA; ²Large Animal Clinical Sciences, The University of Tennessee College of Veterinary Medicine, Knoxville, TN; ³Spine Surgery Division Orthopedics Department, WellStar Atlanta Medical Center, Atlanta, GA

Acute spinal cord injury (ASCI) is a devastating event that can lead to severe hemodynamic consequences that vary with injury location and severity. The hemodynamic and metabolic changes that occur post- ASCI at the cervical and high thoracic region have been reported, but there is comparably little information about the hyperacute changes that follow low-thoracic ASCI. We sought to quantify the hyper-acute hemodynamic changes observed following ASCI using subjects as their own control. Six Yucatan gilts (44.2 ± 7.5 kg and 287 ± 4-d-old) were anesthetized, and a dorsal laminectomy performed at T10-T12. Standardized blunt trauma was applied for 5 consecutive m, and hemodynamic variables and arterial blood gas samples were collected before and after ASCI at regular intervals up to 240-m post-ASCI. Data was analyzed with a mixed effects model, and significance set at P < 0.05. Diastolic, mean, and systolic arterial pressures were significantly decreased 2 m post-ASCI by 18.2% (P = 0.0468), 16.1% (P = 0.0215), and 14.7% (P = 0.0085), respectively. Arterial pressures were diminished compared to baseline at 80 and 120 m post-ASCI, although they were within the physiologic range. A compensatory increase in heart rate was seen post-ASCI, but the change did not reach statistical significance. Blood glucose progressively increased post-ASCI, reaching a 50.3% (P = 0.0025) increment from baseline between 120 and 240 m post-ASCI. Together the results indicate that low-thoracic ASCI caused a consistent but clinically minor hyper-acute decrease in arterial pressures which did not produce hypotension or metabolic changes in Yucatan gilts. When employing this T10-T12 porcine ASCI model, pre-ASCI mean arterial pressures should be maintained above 75 mm Hg to avoid hypotensive states.

PS59 Low-stress Handling Techniques for Mice And Rats: Attitudes and Perceived Barriers of Implementation Among Animal Research Professionals

C O’Malley*¹, R Hubley¹, C Moody², PV Turner^1,3

¹Global Animal Welfare and Training, Charles River Laboratories, Wilmington, MA; ²UC Davis, Davis, CA; ³University of Guelph, Guelph, Canada

Promoting positive human-animal interactions by using low-stress handling (LSH) techniques can reduce potential animal fear and stress and improve study data, but uptake of these techniques has been slow for mice and rats in North America. We sought to understand attitudes towards and impediments of using LSH with mice and rats. Interviews were conducted with 40 participants from the US and Canada, including veterinary and animal science professionals (group 1; n = 17) and in-life technicians (group 2; n = 23). Participants were asked a series of open-ended questions as well as reviewing videos of techniques being discussed such as gentle handling, habituation, and positive reinforcement. The interviews were transcribed and qualitative software, NVIVO, was used to identify themes in responses. Reduced stress (52.5%), better data (42.5%), and improved welfare (40.0%) were identified as benefits of using LSH techniques. While 50% of participants had no concerns with using LSH, US participants were more likely to mention concerns, including researcher attitudes, training personnel, and time, while Canadian participants were most concerned with training of personnel. Generally, Canadian participants were more supportive of using LSH, and some indicated that they were already using these techniques. When asked how using LSH techniques would impact their job, most participants, regardless of demographics, believed they would have improved job satisfaction, decreased stress and/or an easier job, but also increased time would be needed for training personnel. Respondents from all demographics (90%) indicated that implementing LSH would be worthwhile and would improve rodent welfare. Researcher attitudes were a perceived barrier for implementing LSH (22.5% indicating researchers would resist, and 55.0% indicating a variable researcher response). In general, research animal professionals in both Canada and the US recognized the welfare benefits that could result from more widespread adoption of LSH techniques for rodents at their institution. Addressing concerns about researcher attitudes through surveys or educational programs could be an important step for implementing LSH techniques with mice and rats.

PS60 Self-assessment Tools to Support Research Groups and Institutions to Track, Evaluate, and Benchmark 3Rs Activities

J Eddy*, V Robinson, MJ Prescott

National Centre for the Replacement, Refinement and Reduction of Animals in Research, London, United Kingdom

Obtaining a realistic evaluation of 3Rs activities is essential for researchers and organizations seeking to assess or improve their 3Rs culture as part of their animal care and use program. We have recently launched 2 3Rs self-assessment tools, 1 for research groups and 1 for research institutions. These are free-to-use, secure, interactive, online resources that are designed to allow the user to track, evaluate, and benchmark their 3Rs activities over time. Both consist of a series of questions on the 3Rs, developed in consultation with the scientific community and divided into thematic categories representative of the breadth of 3Rs-related activities that can be undertaken by a research group or institution. Once a question set is completed online, the system scores the responses and provides bespoke feedback on how improvements can be made to further support 3Rs implementation. There are a number of benefits to using the tools. Scores can be used internally to allocate resource and effort, and to focus discussions at research group meetings or in the IACUC towards the most important areas and topics. The feedback gives useful advice and examples, helping to ensure that subsequent 3Rs efforts are successful. The 3Rs self-assessment can be repeated periodically and the results compared longitudinally, allowing progress to be tracked. Finally, scores can be used externally (e.g. in grant proposals and animal study protocols; or as part of discussions with regulatory bodies or the general public). Overall, use of the self-assessment tools will encourage a more active 3Rs culture and assist in delivering on commitments to the 3Rs. This talk will introduce the tools, outline the benefits to using them and guide people through the process of submitting a self-assessment.

PS61 Creating a Digital Escape Room to Engage Technicians in Pursuit of Certifications

BA Skiles*¹, J DenHerder²

¹Laboratory Animal Resource Center, Indiana University School of Medicine, Indianapolis, IN; ²Office of the Campus Veterinarian, Washington State University, Pullman, WA

Preparing for a certification exam can be a daunting task for many adult learners. To encourage animal care staff to pursue AALAS certification, we have implemented prep courses at our respective institutions. The students come from a variety of backgrounds and have different study skillsets, test experiences, and learning styles. As teachers, we aim to address individual learning needs by using a variety of different methods to deliver the information, check for understanding, and assess the learning process. We recently discovered digital escape rooms and thought they could be an engaging way to reinforce learning and review training session information for technician certification courses. Digital escape rooms are increasingly being used by educators to add excitement to the learning process, which makes the content more memorable. Escape rooms present a scenario in which participants are “locked in” and cannot escape until they obtain the key. They do this by searching for clues and solving puzzles. We created completely digital escape rooms using an online file storage and document creation platform. Puzzles were created or adapted to review and reinforce content from the technician certification manual and workbook, and then “hidden” in the virtual escape room with clues. Participants used the information learned in class or from their reading to solve the puzzles and escape the room. We discuss our successes and challenges using this tool as a review activity for technician certification courses.

PS62 Transparent Communication and Failure Management In Animal Research

SJ Bischoff*, A Enkelmann

Animal Welfare, University Hospital Jena, Jena, , Germany

The strong need for a culture of care has now extended into animal research worldwide. Many of these care programs confirm animal research institutions’ commitment to high standards of animal care and to obtaining reproducible, excellent scientific data. However, reports about negative results gained from theseexperiments, which is essential for the progress of biomedical science, are rarely published. Consequently, animal research often lacks transparency and reflection of failure or stakeholders are burdened with feelings of guilt and fear of allegations. In order to follow the openness concept in animal research, procedural failures can be used as learning and animal welfare improvement opportunities. Sharing negative results gained from animal experiments can be seen as an evolutionary process requiring years to fully develop. The web-based platform CIRS-LAS (Critical Incident Reporting System in Laboratory Animal Science) helps scientists to reflect on negative results originating from well-designed preclinical studies. CIRS-LAS platform is gaining worldwide recognition from researchers for enabling constructive dialogue about possible causes of animal experiment failures as well as possible solutions. Incident reports mainly refer to complex laboratory animal models such as orthopedics (24%), anesthesiology (20%), and cardiovascular research (13%). The positive publication landscape trend towards a culture of care leads recognizing the high scientific value of negative results publication and critical incident reporting on CIRS-LAS.de - both increase the transparent communication in animal research. Subsequently, this effort may save animal lives and prevent unnecessary repetition of unsuccessful experiments, wastage of funds, and delay in scientific progress.

PS63 Planning a Successful Employee Engagement Program for a Remote Work Setting

KL Cavanaugh*, SL Pritt

IACUC, University of Texas Southwestern Medical Center, Dallas, TX

With the dawn of COVID-19, many IACUC offices transitioned to working remotely and even in the wake of the pandemic, many are electing to stay either partially or fully remote. Without a shared workspace, how do you keep staff engaged and foster office morale? To address this question, we developed several unique engagement strategies. First, office leadership designated one of its managers to spearhead the engagement efforts. By centralizing the responsibility, it facilitated an organized approach to planning the engagement initiative. Next, the staff were surveyed on 2 key areas: what type of virtual events they want to participate in and how often they want these events to occur. Using the results, the engagement manager planned consistently scheduled and diversified events to appeal to the expressed interests of the staff. These events ranged from social in nature to professional development driven. For instance, one event was a tour of a local llama farm, while the next was a department wide training on emotional intelligence. Soliciting the participation of staff in the planning phase allows them to feel engaged throughout the process, from design to execution. Some staff expressed a preference for more passive engagement resources (rather than events), particularly an office newsletter. Now, one year after its debut, the newsletter is prepared by rotating staff members to feature department updates, articles of interest, and personal anecdotes. Another engagement strategy deployed was to introduce conversational icebreakers at the start of each staff meeting. This was a creative way to utilize an existing group meeting and leverage it to boost engagement and build comradery. The meeting is also used to dole out employee recognition by publically highlighting staff accomplishments and exceptional work. At the end of the year, the engagement manager composes a highlight reel of these accomplishments and the video is posted on the department’s webpage. Planning a successful engagement program starts with listening to the desires of the staff, delivering diversified events and resources customized to their interests, and showing ongoing recognition of their achievements.

PS64 Extending the Use of Exposed Vascular Access Ports in Miniature Swine

E Callahan*, A Wakefield, M Haney

Veterinary Services, Sinclair Research Center, Columbia, MO

Jugular vein vascular access ports (VAPs) are commonly implanted to allow for repeated intravenous access in miniature swine. A potential complication is erosion of the port chamber through the skin, resulting in port exposure. This requires removal of the VAP to prevent further complications, such as infection. Port removal often results in the animal’s premature removal from study. The purpose of this study was to determine the efficacy of relocating exposed VAPs to a new subcutaneous (SC) position in castrated male, miniature swine to allow continuation on study. Miniswine (n = 30) with exposed VAPs underwent surgical relocation. The animals were anesthetized, intubated, and maintained on isoflurane throughout the procedure. Ketoprofen and enrofloxacin were administered intramuscularly prior to the procedure for analgesia and antibacterial prophylaxis, respectively. The site was aseptically prepared and an incision was made ~2cm cranial or caudal from the exposed VAP. A new SC pocket was created, and extended toward the exposed port. The VAP was removed through the incision, and a new sterile VAP head was placed on the intact catheter, and secured in the new pocket. The incision site was flushed with povidone solution prior to closing and closed with absorbable suture. The previous site was left open to heal by second intention. Relocation was considered a success if the VAP was functional, not noticeably infected, and not re-exposed at least 90 d after relocation. Of the 30 VAPs that were relocated, 18 met the success criteria (60% success rate). Four VAPs were noted to still be functional over 300 d after relocation. Complications contributing to failed VAP relocations included surgical site infection and repeated exposure. With a 60% success rate, relocation is a viable treatment option for exposed VAPs. This leads to longer animal retention on study, allowing more information to be obtained from individual animals. VAP relocation requires considerably less time, materials, and effort than implanting a new VAP in additional animals. Thus, VAP relocation has the potential to decrease costs and reduce animal numbers required for long-term studies, and improves data quality by enabling the continuity of individual animals on their respective studies.

PS65 Effects of Housing and Research Conditions on Ethogram and Indices of Behavioral Welfare in Rhesus Macaques (Macaca mulatta)

KR Lambert*, EL Mitchell, M Leblanc

ARP, Wake Forest University, Winston-Salem, NC

Understanding the influence of housing and research on behavior of Rhesus macaques is essential to improve their welfare. We sought to characterize the prevalence of abnormal behaviors in adult Rhesus (n = 293) of Indian (IND) or Chinese (CHI) descent enrolled in neuroscience (NEU) and non-neuroscience (NNEU) research. Animals were housed singly (36%) or socially (64%) in cages (49%) or pens (51%). Overall, 66% of animals had behavioral consultations (BC) for abnormal behaviors. Common reasons for BC were alopecia (AL, 53%), stereotypia (STY, 26%) and self-directed behavior (SDB, 7.5%). Special consideration plans (SCP) to address abnormal behaviors were developed for 45% of the population. The probabilities of requiring BC or SCP were not influenced by sex, housing (pen/rack), or descent (IND/CHI) but BC and AL animals were older. Macaques housed socially had a higher probability of needing BC or displaying AL as compared to single housing. When considering research, single housing had a negative behavioral effect in monkeys enrolled in NEU (increased BC, AL, STY) while social housing correlated with increased BC and AL in NNEU animals. These findings suggest that the impact of social housing on behavior vary based on research conditions. We also assessed the effects of sex, age, descent, housing, and research on an ethogram of 16 behaviors. In time spent, the most common behaviors were categorized as exploratory (EXP, 29%), inactivity (IA, 29%), stereotypia (STY, 12%), foraging (FOR, 11%) and self-manipulation (SMP, 8.0%). Females displayed more EXP while males showed more IA, FOR, and SMP. CHI macaques showed more EXP and less FOR than IND. Pen-housed animals spent more time in STY than those in cages. Socially housed animals spent more time in EXP behaviors and less time showing IA and SMP as compared to single housing. Macaques enrolled in NEU research spent less time EXP and more time SMP than those enrolled in NNEU. Singly housed animals enrolled in NEU foraged less as compared to those group housed, confirming that single housing in NEU is correlated with negative indices of welfare. In conclusion, age, sex, descent and housing conditions affect normal and abnormal behaviors but these effects vary based on the research program in which animals are enrolled.

PS66 Cross-facility Collaboration to Develop a Novel Welfare Assessment Tool for Research Primates

E Paterson*¹, PV Turner^1,2

¹Pathobiology, University of Guelph, Guelph, Canada; ²Global Animal Welfare and Training, Charles River, Wilmington, MA

Formally assessing the welfare of large numbers of animals can be challenging but is needed to ensure evidence-based decision-making for improving animal care and use programs. In other fields of captive animal management, such as agriculture and zoos, animal welfare frameworks and welfare assessment grids have been developed for this purpose, but no such tool exists for different species of research animals. When developing robust animal behavioral management programs, facilities should conduct periodic animal welfare assessments to support continuous program improvements and build institutional awareness of areas requiring more attention. This multifacility, collaborative project aimed to develop a novel primate welfare assessment tool (PWAT). The PWAT development involved (1) establishing an internal working group of primate behavior experts; (2) identifying main animal welfare categories and descriptors based on a literature review; (3) development of a preliminary tool; (4) beta testing the tool across facilities (6 facilities located in Canada, USA, and the EU) to ensure practicality and final consensus; (5) development of a finalized tool with automated data analysis; and (6) roll-out to 13 sites for benchmarking welfare scores for future use. The tool assesses different outcome and input-based measures of the behavioural management program as well as overall institutional culture of care using 6 different categories: physical, behavioural, training, environmental, procedural, and culture of care, and containing 167 descriptors. Measures were weighted based on assessed welfare impact and response types included nominal (yes/no) and numerical (0,1,2) values. Following initial benchmarking, the tool will be used to assess changes in the behavioral management program over time. The development process of the PWAT demonstrates that evidence-based assessment tools can be developed in practices through collaboration and consensus. The process is important for uptake across sites as well as for development of other welfare assessment tools for other research species.

PS67 Investigation of Cynomolgus Macaque Nocturnal Behavior Before and Following Surgery: Considerations for Postoperative Care and Management

E Paterson*¹, PV Turner^1,2

¹Pathobiology, University of Guelph, Guelph, Canada; ²Global Animal Welfare and Training, Charles River, Wilmington, MA

Sleep ensures good physical and mental health through interconnected immunologic, physiologic, and neurologic signaling pathways. In human medicine, a bidirectional correlation is seen between sleep quality and pain. Further, even when robust pain management protocols are provided postoperatively, human patients often report experiencing breakthrough discomfort/pain in the night. This suggests that efforts should be made to better understand and create a comfortable environment for research animals undergoing surgery to minimize discomfort and improve their rest. Research primates spend 50% of each day in a dark phase and there is limited research on their behavior during this time. This study examined dark cycle behavior of cynomolgus macaques before and following surgery to provide insight into their needs to ensure optimal recovery conditions. We hypothesized that nocturnal social interactions between familiar conspecifics, such as huddling, would be used to provide comfort and optimize thermoregulation and that increased restlessness in the postoperative nocturnal period might be seen with breakthrough pain or discomfort despite the use of multimodal analgesia. Cynomolgus macaques (n = 43) underwent routine telemetry instrumentation surgery and were treated perioperatively with buprenorphine (0.3mg/mL/animal) or buprenorphine slow release (0.2 mg/kg), injectable meloxicam (0.2 mg/kg) and oral meloxicam (0.1 mg/kg) and bupivacaine (0.25%, 0.5mL) or 50:50 bupivacaine and lidocaine (0.25%, 20mg/mL) and were video recorded 12 h before and after surgery during the dark phase. Sampling intervals of 15 min/h were used and 123 h of video was scored by an observer blinded to time, interval, and animal using a detailed ethogram. Results demonstrated a 21% increase in animal activity in the postoperative period. For social interactions, 92% and 76% of the dark phase was spent huddling pre vs postoperatively, respectively. These results suggest that breakthrough pain/discomfort may occur in the dark phase in research primates despite robust analgesic use, recognized by increased restlessness, and also emphasize the importance of maintaining social housing of familiar research primates for increasing postoperative comfort.

PS68 Will Rhesus Macaques (Macaca mulatta) Meet Standard Awake-behaving Performance Levels in a Neuro Study without Food or Water Restriction? A Refinement Reviewed

A Sorrells*, K Thurman, M Vogel

Neuro Behavior, Neuralink, Daly City, CA

While the field of behavioral neuroscience continues to advance prolifically, the methods for obtaining data from research animals remain consistent with practices developed in previous decades. Standard practices may include water or food restriction to motivate desired behavior in neuroscientific studies. These strategies require strict oversight by lab staff, veterinarians, and IACUC to mitigate the potential risks posed to the animal’s well-being. Less restrictive approaches are being examined for ability to meet similar quality and quantity of dataoutput. Our objective is to replace a standard water restriction protocol with a novel food rescheduling program that uses a variety of foods and meets the Maintenance Energetic Requirement of each animal to motivate desired behavior. To confirm behavioral performance, we collected data on the following metrics in 6 of our lab’s male macaque monkeys: trial count, bitrate, and duration of attention to research task, as well as latencies to enter chair, head immobilization, and participation. This data was compared to results of a survey distributedamongst 20 anonymous behavioral neuroscience labs. We hypothesized that macaques under less restrictive protocols would take significantly less time to enter chairs, allow head mmobilization, and begin participating in the research task than those of labs utilizing water restriction alone. We further hypothesized that macaques would accomplish similar trial counts per hour and bitrates regardless of training protocol. Both approaches require similar labor efforts and documentation for success. Anecdotal results suggest that this less restrictive approach may be successfully used to obtain high quality data without the challenges posed to animal welfare associated with restriction protocols. By ensuring their basic needs are met, the animals’ opportunity to voluntarily participate better mimics comparable human trials. This allows the data to meet standards of translational science.

PS69 Efficacy of Oral Cannabidiol in a Guinea Pig Model of Primary Osteoarthritis

AP Spittler*¹, M Sadar², J Helbling¹, K Patton¹, K Lee¹, J Becker¹, M Sykes¹, J Grubb¹, S McGrath², D Gustafson², KS Santangelo¹

¹Microbiology, Immunology, and Pathology, Colorado State University, Erie, CO; ²Clinical Sciences, Colorado State University, Fort Collins, CO

Osteoarthritis (OA) is a degenerative joint disease that commonly causes decreased and painful mobility. Due to a lack of safe and effective treatments, many individuals are seeking alternative therapeutics for discomfort. Cannabidiol (CBD), the non-psychotropic component of the cannabis plant, has gained popularity as an OA treatment due to its anti-inflammatory effects. However, there is minimal evidence regarding its efficacy for OA. The aims of this study were to assess the ability of oral CBD to prevent the structural onset and/or progression of OA and determine its efficacy for symptom modification. The Hartley guinea pig spontaneously develops OA at 3 mo of age, serving as a model for nontraumatic OA in humans. Twenty-four, 2-mo-old male guinea pigs were randomly assigned to receive 50 mg/kg CBD (n = 8), 100 mg/kg CBD (n = 8), or vehicle control oil (n = 8) twice daily for 3 mo. Treadmill-based gait analysis, overhead enclosure monitoring, and pressure sensor analysis were performed bi-weekly. At 5 mo of age, guinea pigs were euthanized to collect blood for hematology and biochemistry and knee joint tissues for histopathology. There were no clinically significant differences in weight, femur length, hematology and biochemistry, or gait/mobility between groups. Grossly, the integrity and thickness of knee joint articular cartilage was maintained in the 100 mg/kg CBD group compared to 50 mg/kg CBD and vehicle control groups. Higher concentrations of CBD and/or longer treatment duration may be needed to improve OA-related symptoms in this animal model.

PS70 Site-specific Effects of Anterior Cruciate Ligament Transection on Post-Traumatic Osteoarthritis Development in a Rabbit Model

Y Carcamo*¹, R Terracciano^1,2, A Royal¹, JD Harris³, BK Weiner³, JS Labis⁴, N Gupta⁴, CS Filgueira^1,5

¹Nanomedicine, Houston Methodist Research Institute, Houston, TX; ²Electronics and Telecommunications, Politecnico di Torino, Torino, Italy; ³Orthopedic Surgery, Houston Methodist Research Institute, Houston, TX; ⁴Clinical Radiology, Houston Methodist Research Institute, Houston, TX; ⁵Cardiovascular Surgery, Houston Methodist Research Institute, Houston, TX

A tear of the anterior cruciate ligament (ACL), a common injury in physically active individuals, is the most well-known risk factor for developing post-traumatic osteoarthritis (PTOA). Unfortunately, there is no single measure for PTOA diagnosis and no medical therapies have been shown to halt or slow its progression. Identifying early changes in articular cartilage due to ACL injury in a translationally relevant animal model is beneficial for establishing when clinical intervention should occur. To determine a treatment window, we transected the ACL in the right stifle joint of 5 female skeletally mature New Zealand White rabbits (Oryctolagus cuniculus, age 7.7 mo, weight 3.56±0.11 kg), where the contralateral knee provided an experimental control. Up to 10 wk post-surgery, we imaged the rabbits biweekly using 7T magnetic resonance imaging (MRI) to noninvasively develop a timeline of PTOA progression. For each imaging session, they were sedated via IM injection of ketamine (50 mg/kg) and midazolam (1 mg/kg) into the epaxials, intubated, and maintained with isoflurane via mechanical ventilation and monitored. After sacrifice, the femur, tibia, and patella from both knees were harvested and imaged using micro-computed tomography (microCT) to check for osteophytes, a characteristic of arthritis. The femoral cartilage was sectioned, stained with Safranin-O and Fast Green to evaluate proteoglycan content and cartilage thickness. MR imaging indicated a significant decrease in T1 values over time compared to the control, suggesting lower glycosaminoglycan content resulting from progressive cartilage degradation. MicroCT imaging showed osteophytes in the transected joints, which were not present in the controls. Safranin-O/Fast Green histology showed a decrease proteoglycan content and cartilage thickness. We found that the effects of ACL transection in rabbits 10 wk post-surgery are site-specific as evident by MRI, microCT, and histology. Significantly, we were able to demonstrate use of 7T MRI and obtain quantitative T1 maps in less than 30 min to assess PTOA progression in rabbits, which are highly sensitive to anesthesia and extended sedations. This study forms the basis of future work to develop a therapeutic strategy to halt PTOA progression.

PS71 Antibiotic Management of Corynebacterium bovis Associated Clinical Disease for NSG Mice

CA Manuel*^1,3, U Pugazhenthi², M Fink^1,3, L Habenicht^1,3, DL Fong^1,3, JK Leszczynski^1,3, MJ Schurr⁴

¹Office of Laboratory Animal Resources, University of Colorado Denver, Aurora, CO; ²Division of Endocrinology, Metabolism and Diabetes, University of Colorado Anschutz Medical Campus, Aurora, CO; ³Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, CO; ⁴Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO

Due to the difficulty in eliminating Corynebacterium bovis from immunodeficient mouse colonies, infected mice must sometimes be tolerated during remediation efforts with the ultimate goal of agent eradiation. Efficacy of therapeutic antibiotics on C. bovis clinical signs in NSG (NOD.Cg-Prkdc^scidIl2^rgtm1Wjl/SzJ) mice has never been investigated. We hypothesized that oral-amoxicillin can decrease C. bovis clinical signs in infected NSG mice. Unlike athymic nude mice, C. bovis clinical signs in NSG mice does not spontaneously resolve. Naïve, 7-wk-old, female NSG mice (n = 14) were exposed to both soiled bedding and NSG mice with C. bovis clinical signs and then confirmed positive by PCR. Five wk post inoculation (PI), at the clear presence of clinical signs, mice were either maintained as untreated controls (n = 6), or treated with amoxicillin in the water (0.26 mg/mL) for 3 wk (n = 8). Antibiotics were then either discontinued (n = 4) to assess return of clinical signs or continued (n = 4) to determine the point of complete clinical resolution. Mice were weighed and scored weekly, using 6 clinical parameters where the sum of the scores ranged from 4-18 were 4 is normal and 18 is considered clinically severe. At 5 wk PI, the mean clinical score (MCS) for the untreated controls and mice to-be-treated were 11.8 ± 0.7 and 12.4 ± 1.5, respectively. At 8 wk PI, the MCS was 17.3 ± 1.5 for untreated controls and 5.5 ± 1.0 following 3 wk of amoxicillin (P ≤ 0.001). Use of amoxicillin demonstrated a clear clinical improvement, including body weight gain after 1 wk of treatment. For the mice where antibiotics were discontinued after 3 wk, clinical signs returned were the MCS was similar to untreated controls 6 wk (P = 0.9) after treatment discontinuation. Continuous amoxicillin for 6 wk was required to achieve complete clinical resolution. For NSG mice, amoxicillin can be used to reduce or eliminate clinical signs while being administered. However, once discontinued, clinical signs reoccur, confirming a failure to eliminate the infection. Antibiotic stewardship should be considered with prolonged use for the control of C. bovis infections and must not be considered a method of C. bovis eradication.

PS72 Assessing Ambient Ultrasound and Vibration across Vivaria

AJ Barabas*¹, A Darbyshire², S Schlegel², BN Gaskill¹

¹Animal Science, Purdue University, West Lafayette, IN; ²Laboratory Animal Program, Purdue University, West Lafayette, IN

Excessive noise and vibration are detrimental to rodent welfare, yet these parameters are not often recorded in vivaria. It is unknown if suggested thresholds are exceeded in most housing environments and which specific factors may alter these parameters. Therefore, this study aimed to identify how environmental factors may alter noise and vibration at the room and cage level. Measurements were taken with an ultrasonic microphone and accelerometer. Recordings in the room (n = 64, across 9 buildings) were taken in open air at a central location while recordings taken on an animal transfer station (n = 51) or ventilated rack (n = 94) were taken inside an empty mouse or rat cage containing chow, water, and bedding. For each transfer station and rack, the manufacture year was recorded along with the species and number of cages on the rack. At each location, a baseline measurement was taken, followed by one with the transfer station on. For racks, measurements were taken at the top, middle, and bottom row. Data were analyzed with general linear mixed models. Average noise intensity was higher in rat rooms while the transfer station was functioning, in both the cage placed on the transfer station (P < 0.001) and at the wider room level (P < 0.001). Noise was lower in newer ventilated racks, but only when the transfer station was off in the room (P < 0.001). Within a cage on a rack, noise was also higher at the top when the station was on (P = 0.024), and was lower when the rack had more cages (P < 0.001). Average vibration was not impacted at the room level. On the transfer station, vibration was higher when it was on (P < 0.001). On the rack, however, vibration was lower in newer models (P < 0.001), decreased with row height (P < 0.001) and number of cages on the rack (P < 0.001). Although differences were found, the suggested vibration threshold was never met. However, the noise threshold was exceeded in rat rooms with the transfer station on. This data reveals the ambient noise and vibration that laboratory rodents experience during normal facility operations.

PS73 Development of Humanized ACE2 Mouse and Rat Models for COVID-19 Research

H Jiang¹, EM Hyddmark¹, S Gordon¹, A Bartels¹, Y Wu¹, J Warren¹, A Brown¹, M Garrett², H Ehall², G Zhao*¹

¹GEMS, Envigo RMS, St Louis, MO; ²Envigo RMS, Indianapolis, IN

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 170 million individuals and claimed 3.5 million lives as of June 1, 2021. SARS-CoV-2 infects host cells by the binding of its spike protein to the cellular surface protein angiotensin-converting enzyme 2 (ACE2). Unlike human ACE2, mouse and rat Ace2 proteins cannot efficiently mediate the SARS-CoV-2 infection. Therefore, wildtype mice and rats cannot be used for modeling the disease. Currently, the widely used mouse models for COVID-19 research are random transgenic models that artificially express human ACE2 under the control of cytokeratin 18 promoter or a constitutive promoter. While most of these mice show some COVID-19 phenotypes, none of those transgenic models fully recapitulate the disease course observed in COVID-19 patients. This is because the transgene promoter, integration site, and copy number in those models all differ from those of mouse Ace2 gene, and furthermore, the mouse endogenous Ace2 is still expressed. To overcome these limitations, we have developed humanized ACE2 mouse and rat models using CRISPR-Cas9. Specifically, we inserted a ~3kb human ACE2 cDNA cassette into the mouse and rat Ace2 gene loci to ensure that human ACE2 expression is under the control of rodent Ace2 promoter and regularity elements, while simultaneously disabling the rodent Ace2 gene. To do this, we first screened CRISPR gRNAs targeting close to the translation initiation site of Ace2 in cultured mouse and rat cells, then microinjected CRISPR/Cas9 complex and donor DNA into 1-cell stage embryos. Founder animals were screened by junction PCR and insert PCR, and the PCR products were Sanger sequenced. Tissue samples were harvested from hemizygous or homozygous animals for expression analysis. RT-qPCR and Western blot analysis data show that, in our model, human ACE2 is expressed in tissues expressing endogenous Ace2 (such as lung, kidney, and GI tract), while rodent endogenous Ace2 is absent from these tissues. Further breeding data show both hemizygous and homozygous humanized ACE2 animals appear to be normal and fertile. In summary, these data suggest that our humanized ACE2 models can be valuable for COVID-19 research.

PS74 PCR Prevalence of Human and Rodent Pathogens Detected in Human Research Biologics

CL Perkins*, J Cosentino, KS Henderson

RADS, Charles River Laboratories, Wilmington, MA

PCR is the current gold standard for non-GLP screening of cell lines, tumors, and other research biologics for the presence of infectious agents which could potentially impact research outcomes, animal welfare, and staff health. Materials derived from humans may be contaminated with human viruses. Cell lines and tumors that have been passaged in mice may have been previously exposed to rodent pathogens, therefore screening for these agents in human-derived samples is also relevant. To determine field prevalence, we compiled data for samples that were identified as human in origin between September 2011 and December 2020. Only research biologic samples submitted for panel screening were included; samples tested for individual agents were excluded to prevent the introduction of inadvertent bias. Real-time PCR was performed using proprietary assays and positive results were demonstrated to be reproducible. PCR was performed using panels testing up to 20 human viruses. The prevalence of each agent was calculated based on the total number of tests for that agent. For human viruses, Epstein-Barr virus was the most prevalent at 3.18%, followed by Herpesvirus type 7 at 2.36%, Human Immunodeficiency-virus type 1 at 0.98%, Herpesvirus type 6 at 0.72%, Human papilloma virus type 18 at 0.43%, AAV-2 at 0.28%, Human papilloma virus type 16 at 0.23%, and Hepatitis B virus at 0.19%. Eight additional human viruses were detected with prevalence <0.1%. Rodent panels contained PCR assays for up to 28 individual agents, including both viruses and bacteria. Mycoplasma was most prevalent at 5.59%, followed by C. bovis at 2.81%, and LDV at 1.60%, respectively. Murine norovirus, mouse parvovirus, polyoma virus, and Sendai virus were all detected at a prevalence of <0.1%. Although not yet detected in research biologics by our laboratory, murine chapparvovirus, a newly identified and prevalent agent in many animal facilities, should be a consideration for screening tumor cell lines. The impact and/or prevalence of many of these human and rodent infectious agents highlights the need to perform quarantine testing of any research biologic prior to its release for use.

PS75 MHV-1 and MHV-A59 Results in COVID-like Disease in A/J Mice

JD Macy*^1,2, SR Compton¹, CJ Booth¹

¹Comparative Medicine , Yale University, New Haven, CT; ²Yale Animal Resources Center, Yale University, New Haven, CT

Mouse hepatitis virus (MHV) is a natural betacoronavirus of mice with enterotropic and polytropic variants. To better understand the similarities of polytropic MHV pathology with COVID-19, the objective is to characterize the range of pathological lesions and host responses caused by 2 MHV strains with different virulence in the respiratory tract. Groups of 4 to 6 male and female A/J mice, were infected intranasally with 3 x 10⁶ or 1 x 10⁷ of MHV-A59, or 1.5 x 10⁴ or 1.5 x 10⁵ MHV-1 or media (controls; n = 2). Mice were euthanized, necropsied, and tissues were collected at 2-5 d post infection (DPI). All mice infected with MHV-A59 or -1 had significant weight loss with clinical signs of severe dehydration. Microscopically, mice had widespread pneumonia and fibrosis at 3-4 DPI. Viral titers correlated with lung pathology, with MHV-1 having consistent viral titers from 2-7 x 10⁶ pfu/g of lung whereas viral titers were highly variable in mice infected with MHV-A59, ranging from undetectable to 6 x 10⁷ pfu/g of lung. Lymphoid and hematopoietic tissues showed necrosis of lymphocytes in thymus, spleen, and lymph nodes and a 25-75% loss of bone marrow hematopoietic cells and 3-fold reduction in circulating lymphocytes in all infected mice. Gastritis and intestinal villus blunting were also present. Significant olfactory blub lesions included marked neuron pyknosis and variably edema in some mice infected with MHV-A59. Female mice infected with A59 developed severe acute hepatocellular necrosis on 3 DPI. Some mice had hypereosinophilic myocardiocytes (myocardial degeneration). MHV infection in A/J mice results in pulmonary, cardiac, brain, hepatic, hematopoietic, and gastrointestinal system disease mirroring the tissues affected by COVID-19.

PS76 MHV-1 Causes More Respiratory Disease than MHV-A59 in C57BL/6 and 129S1 Mice

JD Macy*^1,2, CJ Booth¹, SR Compton¹

¹Comparative Medicine , Yale University, New Haven, CT; ²Yale Animal Resources Center, Yale University, New Haven, CT

Mouse hepatitis virus (MHV) is a natural betacoronavirus of mice with enterotropic and polytropic variants. To better understand the similarities of polytropic MHV pathology with COVID-19, the objective is to characterize the range of pathological lesions and host responses caused by 2 MHV strains with different virulence in the respiratory tract. Groups of 4-12 male and female C57BL/6J (B6), 129S1/SvImJ (129) mice were infected intranasally with 3 x 10⁶ or 1 x 10⁷ of MHV-A59 , 1.5 x 10⁴ or 1.5 x 10⁵ MHV-1 or media (controls; n = 2). Mice were euthanized, necropsied, and tissues were collected at 4, 8, and 12 d post infection (DPI). B6 and 129 male and female mice infected with MHV-A59 or -1 had hepatic necrosis, hepatitis, olfactory bulb lesions, and lung lesions to varying degrees. Mice infected with MHV-A59 had more significant hepatic lesions compared to mice infected with MHV-1. Brain lesions resulting from MHV-A59 consisted of 1 or more of the following: shrunken neurons, dissolution of the neuropil, encephalitis, vasculitis, and/or meningitis. In contrast, significant brain lesions with MHV-1 occurred only at 8 DPI in B6 mice. Severe olfactory bulb lesions in 129 mice did not resolve by 12 DPI suggesting the potential for a long-term negative impact on olfaction. Mice infected with MHV-1 have more severe and widespread lung pathology compared to MHV-A59 infected mice and was characterized by alveolitis/pneumonia, fibrin deposition and alveolar protein persist through 12 DPI where up to 40% of the lung lobes were still affected. Viral titers in lungs of B6 mice infected with MHV-1 or -A59 on DPI 4 were variable ranging from undetectable to 2 x 10⁶ pfu/g of lung. 129 mice infected with MHV-A59 had consistent viral titers between 3 x 10⁴ and 2 x 10⁵ pfu/g of lung on DPI 4. MHV was not detected in B6 and 129 mice infected with MHV-1 on DPI 8 whereas 25% B6 and all 129 mice infected with MHV-A59 had low viral titers (6 x 10² to 4 x 10⁴ pfu/g of lung) on DPI 8. These results suggest that lung lesions persist beyond when MHV can be detected in the lung. MHV-1 is a better respiratory disease model in B6 and 129 mice than MHV-A59 because of its higher tropism for respiratory tissues and it has less impact on the brain and liver.

PS77 Assessing Mouse Kidney Parvovirus Elimination from Cages by Mechanical Washing

AL Carlson*¹, RJ Ricart Arbona¹, CL Perkins², KS Henderson², NS Lipman¹

¹Memorial Sloan Kettering Cancer Center, New York, NY; ²Research Animal Diagnostic Services, Charles River Laboratories, Wilmington, MA

Mouse Kidney Parvovirus (MKPV), a novel parvovirus of the new genus Chaphamaparvovirus, causes inclusion body nephropathy in severely immunocompromised mice and is prevalent in research mouse colonies. As non-enveloped viruses, well characterized parvoviruses of veterinary importance including canine parvovirus, feline panleukopenia virus, and murine parvovirus (MPV), which are members of the Protoparvovirus genus, are stable and resist inactivation by elevated temperatures and disinfectants. As a novel and highly divergent parvovirus, the resistance of MKPV to thermal inactivation is unknown. The goal of this study was to examine the ability of a common cage sanitization method, washing in a mechanical washer, to eliminate MKPV. Cages which had previously housed MKPV-infected mice were assigned to 1 of 3 groups: sanitization in a tunnel washer without chemical detergent (82.2°C (180^oF) final rinse for 20 s; n = 10); sanitization in a tunnel washer followed by autoclave sterilization (121°C for 20 m; n = 10); or, control (bedding change only; n = 10). The presence of MKPV on the cage was assessed by PCR performed on swabs collected from the cage interior prior to group assignments and again after treatment. After treatment, 4 MKPV-negative CD-1 female mice were housed in each cage. The processes above were repeated so that each group of CD1 mice was housed in a cage from the same treatment group at the initiation of the study and again 14 dlater. Twelve, 17, and 20 wk after the first exposure, 1 mouse per cage was sacrificed and renal tissue submitted for MKPV PCR. MKPV was detected by PCR in 63% of pre-treatment soiled cages. All cages sanitized in a tunnel washer +/- sterilization were PCR negative after treatment. Three of 10 (30%) mice in the bedding dump group were renal MKPV PCR positive at 12 wk post-exposure. The number of PCR positive mice housed in these cages doubled (6 of 10) at >17 wk post-housing. No mice (n = 40) housed in cages sanitized in a tunnel washer +/- sterilization tested positive for MKPV at any time point. This study indicates that MKPV contaminated caging can result in MKPV infection and use of a mechanical cage washer is sufficient to remove MKPV nucleic acid and prevent transmission to naïve mice.

PS78 Nasal Swabs for Virus Quantification in a Syrian Hamster SARS-CoV-2 Model: A Novel Technique

JJ Dearing*, S Caicedo, T Yellowhair, MS Vermillion, A Werts

Physiology, Lovelace Biomedical Research Institute, Albuquerque, NM

The Syrian Golden Hamster has emerged as a reliable small animal model of SARS-CoV-2 infection and disease. Intranasal inoculation of hamsters consistently results in high viral load in lung tissues, both aerosol and fecal shedding, and measurable clinical disease with successful recovery. Although this model has shown great utility for evaluating candidate vaccines and therapies, it can be optimized with focus on the 3Rs. The majority of published SARS-CoV-2 studies in hamsters include staggered necropsy dates for quantification of viral load from terminally collected respiratory tissues. Here we describe methods for in-life nasal swab collections that can be performed serially within the same animals to characterize upper respiratory tract (URT) viral replication across the study, allowing for the collection of more data with fewer animals. In this study, 12 male hamsters were inoculated intranasally with 10¹-10⁴ TCID₅₀ SARS-CoV-2 (3/dose). Nasal swabs were performed 1, 3, and 5 d post-infection (DPI) using 0.5mm fiber tip applicators. SARS-CoV-2 genomic and subgenomic RNA was quantified by reverse transcriptase quantitative PCR (RT-qPCR). Clinical disease and body weights were measured daily. Animals were euthanized 8 DPI for lung weights and quantification of tissue viral load by RT-qPCR. All titers resulted in productive viral replication, mild weight loss, and pneumonia. Peak weight loss (6 DPI) was directly correlated with inoculation titer, indicating disease severity was dependent on inoculation dose. In nasal swabs, peak viral replication occurred 1 DPI in animals inoculated with 10²-10⁴ TCID₅₀, and 3 DPI in animals inoculated with 10¹ TCID₅₀. By 5 DPI, viral load in nasal swabs was similar in all groups. Lung:body weight ratios measured 8 DPI were significantly higher in animals dosed with 10⁴ TCID₅₀ as compared to other groups despite no difference in viral load in lung tissue. In summary, these data support collection of nasal swabs as a surrogate for terminal tissue collections for quantification of viral load in the URT of SARS-CoV-2-infected hamsters. This technique promotes the 3Rs by eliminating the need for staggered necropsies and allowing direct comparison of viral replication kinetics within each animal.

PS79 Minimally Invasive Transthoracic Approach for Intrapleural Injection Improves Clinical and Research Outcomes Compared to Invasive Transabdominal Approach in a Mouse Model

JJ Xu*¹, NL Herndon¹, M Lucero², JL Ludwig¹, J Chan²

¹Division of Animal Resources, University of Illinois at Urbana-Champaign, Urbana, IL; ²Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL

Intrapleural injection of tumor cells in mice can be used to model malignant pleural disease in humans, deliver therapeutic agents, or trace neuronal pathways. Methods of intrapleural injection include a minimally invasive transthoracic injection (without surgical exposure), or a surgical exposure of either the diaphragm (via abdominal exposure) or thoracic wall (via extensive tissue dissection) to visualize the site prior to injection. While a minimally invasive approach without surgical exposure can be considered a refinement to a surgical approach, one potential drawback is the increased likelihood of an inexperienced surgeon creating an off-target effect during injection. In this pilot study, we hypothesized that a minimally invasive transthoracic approach (MI-TT) would have similar accuracy of injection into the pleural space as a surgical transabdominal approach (SX-TA), and result in improved clinical outcomes for the mice (decreased surgical time and complications). Prior to procedures, surgeons were trained in MI-TT and SX-TA approaches. Nude mice (female, 4–6-w-old, n = 10) were injected with 50 µl A549-Luc2 tumor cells either using the MI-TT (n = 8) or SX-TA (n = 2) methods under carprofen analgesia and isoflurane anesthesia. All mice recovered from anesthesia uneventfully. Tumors were monitored with bioluminescent imaging for 5 wk. Surgical time was decreased in the MI-TT group (less than 5 min) compared to the SX-TA group (15–20 min). In contrast to initial concerns that the MI-TT approach would be less reliable, all 8 MI-TT mice expressed bioluminescence signal in the appropriate location while the 2 SX-TA mice were more variable in their signal. We demonstrate that with proper training, a minimally invasive transthoracic approach for intrapleural injection both improves clinical and research outcomes compared to a surgical transabdominal approach.

PS80 Chemogenetic Studies of the Neural Basis of Anxious Behavior in Nonhuman Primates

VR Elam*¹, PH Roseboom¹, SA Mueller¹, N Kalin¹, J Oler¹, M Riedel¹, J Gomez², M Michaelides², B Christian^1,3, A Difilippo³, M Boehm², A Fox^4,5, M Olsen¹

¹Psychiatry, UW-Madison, Madison, WI; ²NIH, Baltimore, MD; ³Medical Physics, University of Wisconsin, Madison, WI; ⁴Department of Psychology, UC Davis, Davis, CA; ⁵CNRPC, UC Davis, Davis, CA

We report using chemogenetics (designer receptors exclusively activated by designer drugs, DREADDs) starting from subject enrollment through necropsy, looking for changes of any anxiety-related behavior in nonhuman primates (NHPs) post-surgical manipulation. Prior to surgical manipulation, the no-eye-contact (NEC) condition of the human intruder paradigm (HIP) was used for the initial selection of animals. Ten young rhesus monkeys (5 experimental subjects and 5 unoperated controls; 6 males and 4 females, mean age at time of surgery = 2.19 ± 0.23 years) were selected for this study. Intraoperative MRI (iMRI) surgical methods, along with anesthesia protocols, were used for the delivery of a viral vector containing the DREADDs construct (AAV5-hSYn-HA-HM4Di) mixed with gadolinium for the visualization of the infusion in real-time with MRI into bilateral amygdala. Following recovery, the animals were again tested in the HIP using a counterbalanced crossover design (DREADDs vs control; drug vs vehicle) to assess the effects of DREADDs-mediated amygdala inhibition on anxiety-like behavior. The results demonstrated that the experimental animals had less anxiety-related freezing behavior in the HIP paradigm, while coo vocalization and locomotion were unaffected. This effect was demonstrated using low dose clozapine injections; (0.03mg/kg - 0.1mg/kg I.M.) to activate the DREADDs receptors (or injections of vehicle) before testing the HIP behavior and comparing the experimental animals to the control animals. In vivo confirmation of DREADDs expression in the amygdala was achieved with [¹¹C] deschloroclozopine µPET imaging, which involved I.V. radiotracer injections along with anesthetic protocols. This talk will lean more towards the behavior paradigms, anesthesia protocols, surgical manipulation, µPet results and physiological samples collected prior to necropsy.

PS81 Increased Anesthetic Success in Rats after Handling

C Yancey*, W Williams

Comparative Medicine, University of Oklahoma, Oklahoma City, OK

Rats (Rattus norvegicus) are commonly used in research for a variety of models, including behavioral, physiological, hearing, and surgical projects. In the rat model of tinnitus and noise-induced hearing loss, Sprague-Dawley rats are anesthetized by intraperitoneal injection of ketamine and xylazine, using 80mg/kg and 10mg/kg of body weight, respectively. This anesthetic cocktail is required for specific hearing tests, as they do not influence the readings and results by suppressing certain brain functions. Anesthetic success is essential to obtain accurate results at specific time points. During experimentation with a cohort of male Sprague-Dawley rats ranging from 300 grams to 350 grams of body weight, it was noted that the rate for successful anesthetic induction was 85%. It was later observed that rats in a separate cohort with routine handling prior to anesthetic events had a successful anesthesia induction 97% of the time. These rats were handled approximately 5 min each once daily for 4 consecutive d. Given this observation of successful anesthetic inductions in these 27 rats, a control group was created with the same parameters, but routine handling was not performed. The control group was administered the same dose of ketamine and xylazine. The rats in each group were assigned either fail or pass, dependent upon if they were at an acceptable plain of anesthesia (normal and rhythmic respiratory rate, no toe pinch response, pink in color) within 10 min of injection. Fischer’s Exact Test was used to compare the handled and non-handled groups, with a P value of <0.05. This result highly suggests that handling rats prior to anesthetic procedures will provide a more successful rate of anesthetic success. Given the use of rats in a range of different research models, and the common usage of ketamine and xylazine for anesthesia, this is widely applicable across the research community. Furthermore, these results have important implications for studies that rely on consistency and reproducibility. Future studies that evaluate rat handling and anesthesia should include a larger cohort of animals, multiple strains, both sexes, and should examine the physiological responses and biomarkers of stress before and after handling to further characterize this phenomenon.

PS82 Differential Effects of 4 Intramuscular Sedatives on Cardiorespiratory Stability in Juvenile Guinea Pigs

RP Sixtus*^1,2, C Pacharinsak³, C Gray^1,2, M Berry^1,2, R Dyson^1,2

¹Paediatrics and Child Health, University of Otago, Wellington, New Zealand; ²Center for Translational Research, University of Otago, Wellington, New Zealand; ³Department of Comparative Medicine, Stanford University, Stanford, CA

Noninvasive physiological monitoring can induce stress in laboratory animals. Sedation reduces the level of restraint required, thereby improving the validity of physiological signals measured. However, sedatives may alter physiological equilibrium introducing unintended bias and/or, masking the experimental outcomes of interest. We aimed to investigate the cardiorespiratory effects of 4 short-acting sedatives in juvenile guinea pigs. Twelve healthy, 38±3 d-old Dunkin Hartley guinea pigs (6 male, 6 female) underwent 5 treatments in this blinded, randomised, crossover design study: 1 unmanipulated control (no injection), and 4 sedation treatments. Sedation was achieved by intramuscular injection using preestablished minimum effective doses of either alfaxalone (5 mg/kg), diazepam (5 mg/kg), ketamine (30 mg/kg), or midazolam (2 mg/kg) administered in random order with a minimum washout period of 48 h between agents. Sedative depth, a composite score comprised of 5 assessment criteria, was observed every 5 min from dosing until arousal. Physiological monitoring of cardiorespiratory status including measures of heart rate, blood pressure, respiratory rate, and peripheral microvascular perfusion. Ketamine and alfaxalone were most effective in inducing stable sedation suitable for physiological monitoring, and diazepam less so. Midazolam was unsuitable due to excessive hypersensitivity. All sedatives significantly increased heart rate above nonsedated control rates (P < 0.0001), without altering blood pressure or microvascular perfusion. Alfaxalone and ketamine reduced respiratory rate relative to their control condition (P < 0.0001, P = 0.05, respectively), but within normative ranges. Ketamine and alfaxalone are the most effective sedatives for inducing short duration, stable sedation with minimal cardiorespiratory depression in guinea pigs. However, alfaxalone is the most appropriate sedative for longitudinal studies requiring multiple physiological timepoints.

PS83 Postmortem Blood and Vitreous Glucose Measurement in the Mouse

A Rex*¹, J Albrecht², W Van Alstine³, E Pierpont⁴, M Rassette¹

¹Department of Veterans Affairs, Minneapolis, MN; ²Gastroenterology, University of Minnesota, Minneapolis, MN; ³Pathology, Pathway Preclinical Services, Minneapolis, MN; ⁴University of Minnesota, Minneapolis, MN

Measurement of blood glucose levels in mice is critical for investigations involving obesity, diabetic, and hepatic disease models. Variation in murine glucose levels can occur not only with experimental manipulation, but also depending on the body site sampled. The question we sought to address was whether it was possible to reliably ascertain a postmortem glucose measurement in the mouse that would approximate the level at the time of death. If possible, this could allow for additional data to be collected in the event an animal was found deceased and the time of death was able to be approximated. Using a glucometer, we measured glucose levels in blood from the tail and retroorbital venous sinus from mice euthanized from a local breeding colony (16 m and F B6.Cg-Speer6-ps1^{Tg(Alb-cre)21Mgn}/J, age range 13-14mo) at time of death, 1 h-, 2 h-, 4 h-, and 24 h postmortem. We also sampled vitreous at time of death and 2 h and 4 h postmortem. Our results demonstrated a considerable drop in measurable glucose levels occurring within 1 h after time of death, reaching very low and stable levels by 2 h after death, across all 3 sampling sites. Variability in glucose levels between collection sites, noted in other studies, was confirmed here. The average blood glucose reading from the tail at time of death was 173.5mg/dL, but by 2 h postmortem was 30.8 mg/dL. The average blood glucose reading from retroorbital venous sinus at time of death was 115.4 mg/dL, but by 2 h postmortem was 36.8 mg/dL. The average blood glucose reading from the vitreous at time of death was 201.3 mg/dL. Readings post-mortem from the vitreous could not reliably be taken; the few measures that were able to be taken showed glucose measurements in line with the other sampling sites post 2 h. Our findings do not support the use of postmortem murine glucose measurement to approximate time-of-death glucose levels. While technically possible, vitreous sampling is not a reliable method of postmortem glucose measurement, possibly due to the relatively large lens-to-vitreous ratio in the mouse globe. In addition, the variability observed between blood collection sites reinforces the importance of using a consistent sampling site for antemortem murine glucose measurements to ensure appropriate comparisons.

PS84 Comparison of 2 Methods to Train Guinea Fowl to Perform a Behavioral Task

L Brossia-Root*¹, H Heffner², R Heffner²

¹DLAR, University of Toledo, Toledo, OH; ²Psychology, University of Toledo, Toledo, OH

To study the evolution of hearing, we must determine hearing of varied species. We discovered that some birds (chickens) can hear infrasound (low-frequency sounds inaudible to humans), whereas most birds cannot (ducks). To determine why some birds can hear infrasound, additional species must be studied. Guineafowl, terrestrial birds similar to chickens, were chosen for hearing testing. To establish their hearing range, birds are trained to continuously peck a response key when there is no sound, and to stop pecking the key when a sound is presented. Training animals to perform behavioral tasks is challenging and time consuming. Per the Guide for the Care and Use of Laboratory Animals, providing a highly preferred food or fluid as positive reinforcement is recommended instead of food restriction. The goal of this study was to determine if guineafowl could be trained to peck the response key for a highly preferred food reward while fed a standard diet ad libitum, or if food restriction was required. Four guineafowl were free-fed chow and earned sunflower seeds, their highly preferred reward, for pecking the response key during training. After 30 d, the birds rarely pecked the response key for the sunflower seed reward. Therefore, this method of training was unsuccessful. Next, the guineafowl were trained with food access restricted for 23 h while earning chow as their reward for pecking the response key during their 1-h training sessions. Three out of the 4 birds were consistently pecking the response key hence were successfully trained by the end of the 30-d training period, during which body weights gradually increased. These data are consistent with previous studies of both mammals and birds demonstrating they are reliable task performers when food restriction is used during behavioral training.

PS85 Percutaneous Splenic Injection: A Minimally Invasive Route of Administration in Mice

LA Stewart*¹, KH Darrell², T Geng², J Hulse², N Poy²

¹In Vivo Surgery, Ambys Medicines, South San Francisco, CA; ²Ambys Medicines, South San Francisco, CA

Surgical procedures should be replaced by less invasive techniques whenever possible to improve animal welfare. Splenic injection, performed via laparotomy approach, is commonly used to deliver cells to the liver in rodent models of liver failure. We investigated a nonsurgical, percutaneous route of splenic injection (PS) in FRGN mice. To assess the feasibility of this procedure, a 3-point scoring system (PSS, 0-Intraperitoneal, 1-Partial, 2-Full) was devised to determine the accuracy of injection into the spleen and deposition of cells in the liver. Animals were anesthetized using isoflurane, and fur was clipped and removed with a depilatory cream. A 29 gauge needle was inserted through the skin and muscle layers to inject the test article into the spleen. In the pilot phase, green food coloring was used to verify the completeness of splenic injection. Technicians (n = 3) reported their scores immediately after injection based on gross observations such as coloring of limbs and snout due to the green dye. Gross necropsy was performed to confirm the location and extent of dye in the tissues. To further assess PS injection, 24 FRGN mice (m/f, aged ~7-wk-old) were percutaneously injected with either LEW-Tg(Gt(ROSA)26Sor-luc)11Jmsk luciferase-expressing Lewis rat hepatocytes (LELH, 1 million cells/rat) formulated in dye or LELH alone. Mice were scored immediately following injection and imaged at 1 h (n = 24) and 24 h (n = 4) post injection. Images were taken using an in vivo imaging system spectrum instrument to confirm delivery of cells to the liver. IVIS images were scored using the same 3-point system. It was observed that 91% of PS injections successfully delivered LELH to the liver: 61% with a strong concentrated dose (PSS 2) and 30% as a partial dose (PSS 1). It was also seen that 80% of the injector-reported scores were a direct match with the IVIS image score. Dye-guided LELH injections did not result in greater accuracy of injection than LELH alone. The scoring system is an effective immediate gross measure to determine the accuracy of the injection. Percutaneous splenic injection is a successful, minimally invasive method of cell administration to the liver and may be considered as an alternative to surgery in mouse models.

PS86 Development of Long-term Cecal Catheterization for Repeated Infusions in Miniature Swine

E Callahan*, M Haney

Veterinary Services, Sinclair Research Center, Columbia, MO

Implantation of infusion catheters in the small intestines has been described in multiple species. Common sites include the duodenum, jejunum, and ileum, but there is limited literature on catheter placement in the cecum or large intestines of swine. The purpose of this study was to develop a surgical method to allow liquid drug delivery directly to the cecum and bypass the stomach and small intestines, while allowing repeated, sedation-free dosing. Male, Yucatan swine (n = 6) underwent surgical implantation of an intestinal burp valve catheter into the cecum. Animals were sedated with tiletamine/zolazepam, xylazine, and atropine, intubated, and maintained on isoflurane. Ketoprofen, buprenorphine SR, and cefazolin were administered prior to the procedure. Through a left paramedian incision, the burp valve catheter was inserted into the cecum and secured. The catheter was tunneled to a subcutaneous (SC) pocket created on the lateral neck. A port head was attached to the catheter and secured to the underlying muscle. All incisions were closed with absorbable suture. Following surgery, ports were aseptically accessed and flushed with sterile saline at least monthly. Cecal catheters were successfully placed in all animals and allowed for repeated cecal infusions. Duration of infusions was up to 6 h and as often as every 4 d. Complications included port infections (n = 2), resulting in euthanasia at 65 and 125 d post-surgery. Infections were localized to the SC space along the port and catheter. Other complications involved port failure (n = 1) due to catheter separation from the port. Catheters remained functional at least 125 d post-surgery in the surviving 3 pigs. In conclusion, cecal catheter implantation is a viable option for repeated infusion dosing into the cecum. This model presents a refinement to more invasive methods by delivering consistent and targeted dosing without sedation. Though alternative methods are needed if sample collection or capsule dosing are required by study design, cecal catheters provide a sustainable route for long-term fluid administration. Thus, cecal catheters facilitate a reduction in animal numbers and improvement in data quality and reproducibility by enabling repeated dosing without the systemic effects of sedation.

PS87 Efficacy of a Heated Anesthesia Breathing Circuit for Prevention of Perianesthetic Hypothermia in Rhesus Macaques (Macaca mulatta)

PA Bowling*, M Bencivenga, C Reiter

Veterinary Medicine, USAMRIID, Frederick, MD

This study evaluated the efficacy of using a heated anesthesia breathing circuit in addition to forced-air warming in the prevention of perianesthetic hypothermia in rhesus macaques when compared with forced-air warming alone. Hypothermia is a common perianesthetic and intraoperative complication that can increase the risk of negative outcomes. Body heat is lost through 4 mechanisms during anesthesia: radiation, conduction, convection, and evaporation. Typical warming methods such as forced-air warming devices, conductive heating pads, and heated surgical tables only address radiative and conductive mechanisms of heat loss. A commercially available heated breathing circuit that delivers gas warmed to 104°F can easily be integrated into an anesthesia machine. We hypothesized that heating the inspired anesthetic gas to address the evaporative mechanism of heat loss would result in increased body temperature during anesthesia in rhesus macaques. Body temperatures were measured by esophageal and rectal thermometer at 5-m increments in a group of ten 5-7 year-old adult male rhesus macaques during two 2-h anesthetic events: one with a heated anesthesia breathing circuit in addition to forced-air warming, and one with forced-air warming alone. Animals were anesthetized with tiletamine/zolazepam to facilitate intubation and maintained under isoflurane inhalant anesthesia. The addition of a heated breathing circuit had a significant positive effect on perianesthetic body temperature, with a faster return to baseline temperature, earlier nadir of initial drop in body temperature, and higher body temperatures during a 2-h anesthetic procedure. Return to baseline was 40.5 m faster (P < 0.05) measured by esophageal temperature and 29.0 m faster (P < 0.05) measured by rectal temperature. As measured by rectal temperature at 5-m intervals, macaques on the heated breathing circuit had higher body temperatures from 70 to 120 m of anesthesia time (P < 0.05). Use of a heated anesthesia breathing circuit should be considered as a significant refinement to thermal support during macaque anesthesia, especially for procedures lasting longer than 1 h.

PS88 Continuous Monitoring of C57BL6/J Reveals a Complex Pattern of Rhythmicities of Home-cage Behaviors

B Ulfhake*, K Pernold

Karolinska Institute, Stockholm, Sweden

The spontaneous behavior of laboratory rodents is a key read-out of animal welfare, experimental interventions, and genetic manipulations. Recent technological development has made automated collection of cumulative recordings of spontaneous in-cage behaviour both feasible and affordable. We sought to examine rhythmicities in the spontaneous in-cage behaviors of C57BL/6J mice and by decomposition analysis uncover pattern of rest and activity bouts, and rhythmicities in the composition of different bouts over time. In-cage activity was recorded 24/7 using a DVC platform across several months of group-housed mice of both sexes. The data records were subjected to functional and spectral analysis to reveal any rhythmicity in activity and rest. We used decomposition analysis to probe the data for bouts of rest and activity, and the duration of bouts. Analyses of the data revealed a complex pattern of recurrent rhythmicities in behaviors such as the circadian triggered by lights-ON/OFF, rhythms generated by recurrent husbandry routines (external triggers), and an endogenous circannual rhythmicity with a period of ~100 days of unknown origin. Using decomposition analysis we classified home-cage activities in bouts of rest, (rest/very low activity), low-to-medium intense and intensive activity and show that the frequency and duration of different bouts vary with sex, number of animals in the cage, and the age of the animals.