LAS Pro January-February 2022 by aalasoffice

January/February 2022

Exploring the Care and Use of Nonhuman Primates in Research How NHPs Are Contributing to Anxiety Research Increasing the Quality of Macaque Housing with Custom Caging Back Onsite! Recapping 2021 National Meeting

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January/February 2022 Vol. 10 Issue 1

INSIDE THIS ISSUE...

Assisting with Anxiety

Choices and Motivation

Pandemic Impact on NHPs

18 20 24 28

First Polio, What Next?

How NHP research advances anxiety disorder research.

Cooperation through food and drink preferences of rhesus macaques.

How traditional NHP management changed in the face of COVID-19.

NHP research in biomedicine and its importance.

The Right Home

Custom caging to enhance animal welfare.

Give Me Five!

5 topics in NHP neuroscience research.

It's A Wrap!

The 2021 AALAS National Meeting wrapped up in Kansas City and virtually, setting its sights on Louisville for 2022.

Kelly Ethun, DVM, PhD, DACLAM, is a Senior Veterinary Scientist and Assistant Research Professor at the Yerkes National Primate Research Center at Emory University in Atlanta, GA. Working closely with Liz Rozanski, she chose topics and recruited authors from across the country for this NHP issue of LAS Pro. She further lent her NHP expertise by reviewing and editing all articles. We cannot thank Dr. Kelly Ethun enough for all the hard work that allowed this publication to be what you are reading today.

28 January/February 2022

2 Laboratory Animal Science Professional January 2022

On the cover: Rachelle L Stammen, DVM, DACLAM, is a Clinical Veterinarian at Yerkes National Primate Research Center at Emory University in Atlanta, GA.

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DEPARTMENTS 5 Publisher’s Note

42 DIY

6 People & Places

46 Tech Tips

The Significance of Leadership

New hires, promotions, awards, memorials

8 PROfiles

Meet Tracy Meeker

36 Inside the IACUC

Setting the stage for 2022

40 Career & Training

Thoughtful course design and workforce training

4 Laboratory Animal Science Professional January 2022

Creative enrichment ideas

Insights on techniques and tactics

67 Crossword – NHPs 68 AALAS Connection 2022 AALAS Election

72 Ad Index

PUBLISHER’S NOTE Staff

The Significance of Leadership

Publisher Ann Turner Associate Publisher Chris Lyons

I was recently joined by my colleagues for our annual holiday luncheon celebration, where we spent some much-needed quality time together. We carried on with positive conversations, fun stories, and there was laughter abounding. It did my heart good to be all together again, seeing everyone’s smiling faces, and feeling the warmth that we have cultivated as a community. We also took the opportunity to honor those of us who had spent many wonderful years here at AALAS, myself included with 20 years. Within those 20 years, I worked with some truly incredible people. Below you’ll see a photo of myself and some of AALAS past presidents who attended the National Meeting in Kansas City this past year. I have gotten to know the elected officials and board of trustees very well, and I treasure the relationships, both as individuals and leaders. We share many things in common. We’ve all attended branch meetings, completed presentations, developed strategies, and so much more. These things, among others, are crucial in AALAS. Serving in a volunteer position may enhance one’s career, but you see, leadership is more than just a resume builder it’s being willing to serve, to put the association’s wellbeing over your own desires. That requires some solid problem-solving skills and confidence to make those tough, but necessary, calls. Good leadership stems from companionship with your colleagues and it is cultivating a culture of togetherness that aids in team building. Without these components, this association would not be what it is today. Volunteering within AALAS, whether it be serving on the Board of Trustees, a national committee, in your branch or district organizations, or various ad hoc volunteer opportunities, is truly an honor and a privilege. I encourage you to research the roles to determine what will be your best fit and complete the volunteer form on the website to be placed in the volunteer pool. The “fire in the gut,” as I like to call it, is crucial to take on the mantle of leadership. Begin attending branch/district meetings, get to know people, and identify ways to use your skills and talents. You may find that your interest is serving at the national level and there are several leadership positions which will be filled in the upcoming election. Check the AALAS website for open positions and the qualifications for each position as well as the process for becoming a candidate. Soon enough, through hard work and determination, you’ll find yourself in the very spot you belong. It’s worth the time and effort.

Managing Editor John Farrar Associate Editor Morgan McCloud Ad Sales John Farrar Design/Production Zara Garza

Editorial Advisory Board Leslie Birke Louisiana State Univ Andrew Burich Benaroya Research Institute Bob Dauchy Tulane Univ School of Medicine David DeOrnellis Champions Oncology Penny Devlin Pennsylvania State Univ College of Med Sonia Doss Duke Univ Medical Center Kelly Ethun Emory University Glenn Jackson Cornell University Richard Marble Alpha Genesis Inc Elizabeth Nunamaker Charles River Laboratories Sara Oglesby Abbvie Karuna Patil Seattle Children's Research Institute Amy Pierce Tulane Univ School of Medicine Stacy Pritt UT Southwestern Medical Center Robin Tucker Georgetown Univ

Mission Statement Laboratory Animal Science Professional (LAS Pro) is the official magazine for American Association for Laboratory Animal Science members. LAS Pro provides a wide range of useful resources and knowledge to the association’s 14,000 laboratory animal science professionals who are involved in advancing responsible laboratory animal care and use to benefit people and animals. All signed articles, including, committee reports, news, and commentary, reflect the individual views of the authors and are not official views of AALAS. Authorization to photocopy portions for personal or internal use is granted by the American Association for Laboratory Animal Science. Photocopying for purposes of resale or outside distribution is prohibited unless written approval is obtained from the AALAS Director of Communications. Copyright 2022 by the American Association for Laboratory Animal Science. Laboratory Animal Science Professional (USPS 010-730) is published bimonthly by the American Association for Laboratory Animal Science, 9190 Crestwyn Hills Drive, Memphis, TN 38125. Periodicals Postage paid at Memphis, TN 38101 and additional mailing offices. POSTMASTER: Send address changes to AALAS, 9190 Crestwyn Hills Drive, Memphis, TN 38125-8538.

Ann Turner

Publisher Executive Director American Association for Laboratory Animal Science

American Association for Laboratory Animal Science 9190 Crestwyn Hills Drive Memphis, TN 38125-8538 Phone: 901-754-8620 Fax: 901-753-0046 E-mail: info@aalas.org Web: www.aalas.org

January 2022 Laboratory Animal Science Professional 5

PEOPLE & PLACES

New hires, promotions, meeting updates, and memorials.

!NHP Educational Resource

Allentown, LLC will be hosting an on-demand webinar entitled “The Challenges Facing the NHP Supply Chain: The Threat to Public Health and US Leadership in Biomedical Research.” This session dives into the reasons for the supply shortage, the impact they are having on research, and potential solutions. This session will also highlight the critical role nonhuman primates have in biomedical research, referring to their assistance in the COVID vaccine development. Presenters are Dr. Chris Abee, Professor Emeritus of Comparative Medicine at the University of Texas MD Anderson Cancer Center and Matt Bailey, President of NABR and FBR, with Cindy Buckmaster, PhD, CMAR, RLATG as the moderator. The viewing link can be accessed here: https://bit.ly/nhp-webinar

!Beware of Ratz Wins Pumpkin Decorating Contest The Animal Resource Center at Case Western Reserve University held a pumpkin decorating contest for technicians in October 2021. The contest was a big hit and showcased the staff ’s creativity. Pumpkins were submitted anonymously, and the favorite was voted on by all employees. The winner was the "Beware of Ratz" pumpkin and the technician won a gift card.

'Beware of Ratz' (above) was the winning design for the pumpkin decorating contest.

All of the submissions for the pumpkin decorating contest held at the Animal Resource Center at Case Western Reserve University.

6 Laboratory Animal Science Professional January 2022

!Donating for the Holidays

UT Southwestern held their annual canned food drive competition during the month of November. Each area collected food, which was donated to the local North Texas Food Bank. This year, the staff donated over 600 items, totaling 575lbs of food! To celebrate this year’s accomplishment a new trophy was created, which will be used for future canned food drives. Thank you to everyone who donated this holiday season!

Winning Facility Supervisors with the new Canned Food Drive trophy.

This year’s new trophy for the annual canned food drive.

IN MEMORIAM James M. Kling

Dr. James Malcolm Kling died August 26, 2021, in Perry, GA with his daughter by his side. Dr. Kling was born January 21, 1936, in Menlo, GA to George Thomas and Lula Whaley Kling. He married Anne Starr Kling in 1956 and together they had one daughter. Dr. Kling obtained his Doctor of Veterinary Medicine at the age of 23 from the University of Georgia in 1959. He went on to pursue a master’s degree from Auburn University in physiology and biochemistry in 1961 and a doctoral degree in pharmacology and toxicology at the University of Florida in 1967. He returned to the University of Georgia College of Veterinary Medicine in 1961 where he taught anatomy for two years. His professional career began in 1967 at the Medical College of Augusta where he served as the Director of Office of Research and Support Services, Attending Veterinarian, and Professor until his retirement in 2001. He taught numerous future veterinarians, physicians, physician assistants, and even medical illustrators and won Teacher of the Year on numerous occasions. He also served as Interim Vice-President for Research and interim Chair of Pharmacology. In 2009 he was awarded an Alumni of the Year distinction from the UGA CVM which coincided with the 50th anniversary of his graduation. Dr. Kling was one of the groundbreaking leaders of establishing ethical and humane treatment of laboratory animals as well as ethical research standards. In 2001 in recognition of his contributions the Malcolm Kling Lectureship in Research Ethics was established at MCG. He is preceded in death by his parents and wife. He is survived by his daughter, Melissa Kling-Newberry (Pep. In lieu of flowers contributions can be made to Heart of Georgia Hospice in Warner Robins, GA or to the charity of choice.)

Crystal Sims, BS, RVT, RLAT

Crystal Sims, Central Ohio Branch AALAS member and past president, passed away on Saturday, November 6. She was a co-worker, friend, supporter, and beloved member of our lab animal science community for many years and will be greatly missed by all who knew her. Crystal was a graduate of OSU, a RVT, and also received her LAT AALAS certification. Prior to that she served in the US Navy for 9 years. She worked at ULAR as an Animal Health Technician for 20+ years. Crystal was a long-standing member of the Ohio Association of Veterinary Technicians, National AALAS, and COB AALAS. She served on Council for COB AALAS since 2013 and was the 2020 President. Many of us who have served on Council can say they are involved in COB AALAS because of Crystal. She had many accomplishments in her career and received many awards and well-deserved recognition over the years. Most notably and recently she won the 2021 Distinguished Service Award for COB and D5 and the 2017 National AALAS Technician of the Year Award. She also volunteered her time as a docent at the Columbus Zoo and as a member of the Medical Reserve Corp of Franklin County, OH. Crystal went above and beyond in her dedication and advocacy to animals inside and outside her work environment. She was selfless, well-respected, giving, and was such a positive force that she impacted everyone who was fortunate enough to know her. Memorial donations can be made to: Columbus Humane Society (www.columbushumane.org).

January 2022 Laboratory Animal Science Professional 7

PRO-files in LAS

minutes with...

Tracy Meeker, CMAR

Facility/Employer: Yerkes National Primate Research Center Field Station at Emory University (Atlanta, GA)

Current Job Title: Colony Manager Degree: BS in Psychology and Animal Behavior interdisciplinary studies

What is the mission of your unit? The mission of the Colony Management unit at Yerkes

is to maintain the social health and diversity of the NHP colony to meet Yerkes' production and scientific needs.

How long have you worked at your current facility? I have been at the Yerkes Field Station for 26 years.

What type of nonhuman primate species do you have at your facility, and how are they housed? The monkey species at the Yerkes Field Station include rhesus macaques and sooty mangabeys. Most are living in large social groups (20-150 animals each) in seminatural environments.

Getting Personal What companion animals do you have?

I have 2 sweet 11-year-old cats, George and Stephie, and a dog, Maggie, who always keeps me on my toes.

Favorite weekend hobbies?

I resell LEGO in my spare time. I also volunteer for my friends’ nonprofit that supports families dealing with cancer.

Where is your favorite vacation spot?

I thought Hawaii was beautiful, but I especially loved the family trip we took to the UK. We spent most of the time in the London area and took a day trip to Edinburgh to see my grandmother’s birthplace. The history there is amazing.

How did you get into this field? As a child, I knew I wanted to work with animals and got

an early start as a teenager working at a small animal veterinary clinic. In college, I was employed as a zookeeper at the Baltimore Zoo, working with various species. My first role at Yerkes was in the Animal Care unit, and after a year, I transferred to a research lab working on an aging and stress project. During that time, the Specific Pathogen Free (SPF) rhesus colony was born, and my role shifted to Program Coordinator to oversee activities associated with the growth of that colony. The Colony Management unit was established in 2005, and my contributions fell in line with that unit’s mission. There have been many advancements in the Colony Management unit in the years since inception – most recently, fine-tuning methods for a data-driven approach to the management of the colony.

Who were your mentors? I’ve had several mentors over the years including, Tom Gordon and Dr. Mark Wilson. They both taught me specialized animal handling skills and the importance of having procedures in place to work towards goals. I currently report to Dr. Brianne Beisner, who is my new mentor. She is an expert in the field of NHP social networks.

What motivated you to seek CMAR certification? I obtained a CMAR certification in 2017 to increase my institutional knowledge and advance in my career. Having CMAR certification has allowed me to contribute more to the administrative aspects of my job. What is your favorite part of your job? I love any aspect that allows me to see the benefit of our efforts to the animal colony. I feel fortunate to be able to observe and record NHP behavioral data and get paid for it.

What advice do you have for others beginning their career at a nonhuman primate facility? The advice I would offer is to follow your heart. I didn’t imagine working in a

research facility when I was a child, but I’m so glad that I had this opportunity to use my passion to care for animals in this setting.

What is the most rewarding aspect of your career? Looking back at all the rewarding

aspects of my career, I have to say that the successful socialization of 12 nursery reared sooty mangabeys was the most rewarding aspect.

What is your favorite nonhuman primate species, and why? I find DeBrazza monkeys

fascinating — they are strikingly beautiful, and their vocalizations are so remarkable. At Yerkes, sooty mangabeys have found a special place in my heart. They remind me of cats in the way they walk and have the most interesting facial features.

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FEATURE

NHPs in Research

NHPs Help Advance Childhood Anxiety Disorder Research Read an interview with Margaux Kenwood, PhD who studies rhesus macaques to better understand how we can better prevent and treat childhood anxiety. By Logan France, DVM, DACLAM

f you tuned in to the Biomedical Research Awareness Day (BRAD) 2021 webinar in September, you heard Dr. Margaux Kenwood discuss her fascinating research on anxiety disorders in children. Dr. Kenwood is a post-doctoral research fellow in the Psychiatry Department at the University of Wisconsin – Madison and will soon be transitioning to Weill Cornell. She studies nonhuman primates, rhesus macaques specifically, to better understand individual differences in anxious temperament and how we can better prevent and treat childhood anxiety. She is also the ACNP/AMP BRAD Fellow for 2021-2022. Here we learn more about her research and the vital role of nonhuman primates. France: For those who may have missed the webinar, can you tell us a little bit more about your research?

Kenwood: Everyone gets anxious at some time or another – public speaking, meeting new people, so many situations can cause us to feel anxious. For the most part, that anxiety can help us to prepare for and succeed in those situations. For a subset of people, anxiety can become uncontrollable and overwhelming, in what we consider to be an anxiety disorder. Our work is centered around understanding the brain circuits and molecules that contribute to who develops anxiety disorders, focusing on the early adolescent time point when anxiety disorders tend to emerge. In order to better characterize the brain regions, cells and molecules that contribute to pathological anxiety, we rely on the use of a nonhuman primate model, which allows us to probe and manipulate the brain circuits we think are important. Rhesus monkeys have naturally occurring individual differences in anxiety-related temperaments that look a lot like the anxiety-related temperaments we can characterize in humans, and our research relies on understanding how the brain contributes to those individual differences. France: Monkeys to study childhood anxiety – for some that may seem like a big jump. Why are monkeys a good model for this type of research? Kenwood: It’s not as big a jump as you would think! Rhesus monkeys and humans diverged in evolution roughly 23 million years ago, which can seem like a long time but is actually relatively recent in the scope of billions of years of evolution. Rhesus monkeys share a lot of really important similarities with respect to understanding human anxiety: they live in interconnected social groups, have long periods

10 Laboratory Animal Science Professional January 2022

Read more about Dr. Kenwood’s research here:

of maternal care, interact with the world through a well-developed visual system, the list goes on and on! Importantly for Kenwood, MM, Kalin, NH, Barbas H. The role of the primate prefrontal our research, rhesus cortex in anxiety and anxiety disorders. monkeys display Neuropsychopharmacology Reviews, individual differencpublished online August 17, 2021 es in anxiety-related temperaments that Kenwood, MM, Kalin, NH. Nonhuman look really similar primate models to explore mechanisms to temperaments underlying early-life temperamental in children that we anxiety. Biological Psychiatry, April 1, know are strongly 2021. linked to the later emergence of anxiKenwood, MM, Roseboom, PH, Oler, JA, Kalin, NH. New Insights into the ety disorders. They Mechanisms of Ketamine's Antidepresalso have a lot of sant Effects: Understanding the Role of similarities in terms VEGF in Mediating Plasticity Processof brain structure es. American Journal of Psychiatry, and function, parMay 1, 2019. ticularly within the frontal lobe, which is the region that I study. Because we have so many tools at our disposal to characterize and manipulate the function of brain regions, cells and molecules in rhesus monkeys that are not available in humans, we can use rhesus monkey to really characterize the brain circuits and molecules that are related to extreme anxious temperament, which should give us insight into how to better treat anxiety disorders in humans. France: How do you decide which species of monkey to work with? Kenwood: We have worked primarily with rhesus monkeys. For my work specifically, the similarities between the rhesus monkey frontal lobe and human frontal lobe are very important. Rhesus monkeys have many of the same subregions that make up the frontal lobe as humans do, and understanding the interaction between those subregions, as well as the molecular makeup of those regions, is a central theme of my work. Neuronal connections among frontal regions, as well as between frontal regions and other parts of the brain are organized into big fiber bundles, which act like electrical cables and transmit signals between connected regions. You can think of them like big highway systems in the brain, along which information travels in the form of electrical signals. Rhesus monkeys have a lot of similarities in terms of the organization of these fiber bundles to humans, and part of my work has focused on the effect of disrupting these fiber bundles, which impacts anxiety and anxiety-related brain function. While other model organisms, both primate and otherwise, are very important in understanding anxiety, we have favored the rhesus monkey model

because of the conserved anxiety-related temperaments and important similarities in frontal lobe structure and function. France: Animal research is controversial despite its proven importance for human and animal medical advancements. How do you advocate for animal research, specifically nonhuman primate research? Kenwood: I’ve found that participating in outreach and education efforts is one of the most important ways to advocate for the importance of animal research. A lot of the controversy around animal research, specifically nonhuman primate research, is driven by misinformation about how and why we’re doing the work that we do. Using animals in our work is a huge privilege and comes with the responsibility to educate the public on how our research will ultimately translate into better interventions to decrease the significant suffering associated with anxiety disorders. I think that having conversations, albeit difficult ones, about the importance of what we do and encouraging transparency about research practices is essential. Most of my outreach has been focused on elementary and middle schoolers, and I’ve found that our participants are engaged and willing to have challenging conversations about how and why animal research is done. A side benefit is that this kind of outreach is really fun and reminds me how lucky I am to have a career in such an interesting and impactful field! Part of my work as a BRAD fellow will be to develop outreach kits so that others can engage in outreach events targeted to a variety of audiences, so stay tuned for that!

More About BRAD

BRAD is an international outreach program through Americans for Medical Progress that seeks to increase public awareness about animal roles in biomedical research and the resulting medical advancements that benefit both humans and animals. BRAD highlights the importance of animal studies, including those involving nonhuman primates, and demonstrates research organizations’ commitment to providing excellent animal care. It also builds a research advocate community and showcases biomedical research and laboratory animal care and medicine careers. Since its April 2016 inaugural celebration, BRAD participation has grown from 20 events in the U.S. to over 200 celebrations annually around the world. Beyond educating the public, BRAD can increase community engagement, unite an institution’s research community, boost employee morale, and allow individuals to discuss their biomedical research roles. Follow BRAD on Facebook (@BRADglobal), Instagram (@brad_global), and Twitter (@amp_brad). Visit BRADglobal.org or contact BRAD@amprogress.org to learn more and to join the celebration! Logan France, DVM, DACLAM, is Assistant Director, Clinical Services & Training in University Research Animal Resources and Clinical Veterinarian in the Research Integrity and Safety Office of Research at the University of Georgia in Athens, GA.

January 2022 Laboratory Animal Science Professional 11

FEATURE

NHPs in Research

Considering Sustenance Preference to Motivate and Enhance NHP Learning and Performance By Aaron Gregoire, BA

inding and utilizing the specific taste preferences of the rhesus macaque in the laboratory setting can be a valuable step towards promoting cooperative, positively reinforced training. It can also be a wonderful tool for enrichment by allowing the subject to have a level of control in their daily routine. Individual monkeys have unique personalities with specific preferences, and it is no exception when it comes to what they like to eat and drink. When brought into the research setting, primates lack control and tend to be quite anxious. The responsibility for their welfare and well-being falls largely on the humans in the laboratory. From the very start, the primate pays close attention to the humans. If we are patient and allow the necessary time to return that attention, there is great potential of establishing a positive connection. This connection is essential to reduce anxiety in the primate, thus building a healthy bridge for learning new research-related tasks.

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Working with non-human primates over the years, I have seen a wide variation in what they like to eat and drink, and what motivates them for training. In addition to maintaining an enriching environment, positive reinforcement training in primates is effective and highly beneficial in the laboratory setting because it allows the monkey to be a participant rather than just a subject, and gives the animal a level of control. I have observed that when primates have more control in their routine, they often engage and explore their options in unique and creative ways. I have also observed many benefits from paying close attention to individual preference, particularly around taste. Catering to preferred taste, mostly with single-housed primates, has opened the door to create a pathway towards healthy, positive interactions. It has also boosted active engagement in the primate when attending to research-oriented requests and tasks. Early in my laboratory animal career, I took a position as a research assistant in a cognitive neuroscience laboratory in the university’s NHP facility. Here, as part of an IACUC approved protocol, rhesus macaques voluntarily participated in training and cognitive testing trials at their own pace for food or drink reward. During these trials, I made the observation that the food and drink items used to reward the NHPs for the correct performance of tasks offered limited variety. Some labs within my research division used one type of juice to reward all their animals. When performing visual tasks, animals were automatically rewarded with a squirt of juice for each correct decision. Animals would often start off strong before quickly losing interest and disengaging. At this point, the recording session would be paused or aborted. In hopes of benefiting both the research and animals, I thought about what refinement may provide the NHPs increased control during their training and testing trials, which led me to consider the juice reward as a means of boosting interest. After receiving permission from the research team, I began implementing daily juice tastings in the animal colony room. I used needleless 10 ml syringes with two flavors of juice for each round of tasting. I allowed each monkey two small samples of 100% juice, which included apple, berry, fruit punch, cherry, grape, strawberry banana, orange tangerine, and tropical. I held the two choices close to their mouth to see which they would gravitate towards first. I began to document their individual preferences as well as the overall popular flavors, which initially included

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the orange tangerine and berry. I made sure I did not have the same juice in the same hand during tastings to keep the selection process random. It became clear, as the primates gravitated to their choice flavor, that each monkey had a unique preference. Although the monkeys would tend to drink all offers of any juice flavor, they were eager to receive their favorite juice flavor first. Although some choices occasionally changed over time, the observation that individual NHPs had distinct drink preference was consistently reinforced with these tastings. Positive outcomes in task performance were fully realized and appreciated when the favored juice preference and individual choice was incorporated into the testing trials. Most notably, the NHPs immediately exhibited greater enthusiasm when they were given their desired juice preference as a food reward for completing a task correctly. Additionally, there was more engagement in the task, with less distraction for longer periods of time. What may have been a minor adjustment in routine had additional benefits which improved the general connection between the researcher and the NHP. This led to increased curiosity and willingness on the part of the NHPs to cooperate with the daily routines. This breakthrough also encouraged the research team to begin looking for other ways to give the NHPs more choice during their testing trials, which promoted further interest and engagement not only in tasks but also in relationships. Although this is just one step of positive reinforcement training, it became the building block that strengthened and further enhanced animal welfare and scientific outcomes through communication and choice. Acknowledgements: Many special thanks to Stacy Toabe, MA, LMHC .

Aaron Gregoire, BA, is a Behaviorist and Primate Lab Manager at Brown University in Providence, RI. REFERENCES: 1. Bayne KA, Dexter SL, Strange GM. 1993. The effects of food treat provisioning and human interaction on the behavioral wel1-being of rhesus primates (Macaca mulatta). Contemp Top Lab Anim Sci 33(2): 6-9. PMID: 16471479. 2. Laule GE, Bloomsmith MA, Schapiro SJ. 2003. The use of positive reinforcement training techniques to enhance the care, management, and welfare of primates in the laboratory. J Appl Anim Welf Sci 6(3): 163-173, DOI: 10.1207/ S15327604JAWS0603_02. PMID: 14612265. 3. Perlman JE, Bloomsmith MA, Whittaker MA, McMillan JL, Minier DE, McCowan B. 2012. Implementing positive reinforcement animal training programs at primate laboratories. App Anim Behav Sci 137(3-4): 114-126.

14 Laboratory Animal Science Professional January 2022

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FEATURE

NHPs in Research

Management of a National Primate Research Center Animal Colony during the COVID-19 Pandemic By Joyce Cohen, VMD, DACLAM

he National Primate Research Centers (NPRCs) are NIH-funded translational research centers that focus on nonhuman primate (NHP) research. They are spread across the country in seven locations (Figure 1) and collectively house more than 22,000 NHPs.3

When the COVID-19 pandemic emerged in the United States in early 2020, it had an immediate and tremendous impact on the NPRCs, beginning with the need for extra measures to protect the animals. NHPs are widely known to be susceptible to many of the same diseases as humans, which makes them an important research model for human diseases. NHPs are especially susceptible to zoonotic diseases transmitted by an aerosol route, such as tuberculosis, measles, and many other respiratory viruses. 2,4 Personnel working with NHPs routinely use personal protective equipment (PPE) to prevent transmission of zoonotic disease. However, there have been documented cases of respiratory viruses transmitted from people to animals in research settings, zoos, and in the wild. 7,8 Because of this, managers of the captive NHPs colonies at the NPRCs were immediately concerned about human-to-animal transmission of SARS-CoV-2 within their facilities. An outbreak of SARS-CoV-2 within these NHP colonies would have detrimental effects on animal health and their availability for use as animal models for COVID-19 vaccine and therapeutic research. In addition to these animal concerns, NPRC managers also navigated challenges facing the entire research community, including PPE shortages and personnel health and well-being. Here, I will highlight the unique challenges and responses of managers maintaining captive colonies of NHPs at NPRCs throughout the COVID-19 pandemic.

Prevention of Human-to-NHP Transmission

Because SARS-CoV-2 was a newly emerging disease and limited data on viral transmission was available at the beginning of the pandemic, we had to make decisions based on previous coronavirus transmission data. From the start of the pandemic, basic principles of disease transmission prevention were reinforced with animal care staff, such as the importance of wearing PPE appropriately, wearing a mask and gloves during NHP food preparation, and staying home at any sign of clinical illness. Supply chain shortages impacted PPE availability, and many centers initiated PPE conservation efforts, including extended use of face shields and masks as well as the sterilization of some PPE such as Tyvek suits and faceshields to allow reuse. Another simple measure adopted quickly was minimizing the number of people in animal areas. This included restricting access to animal areas to essential personnel

16 Laboratory Animal Science Professional January 2022

only and the cessation of center tours. Veterinarians at some NPRC facilities also established quarantine procedures for NHP clinical cases with respiratory symptoms. To ensure the availability of COVID-19 free animals for vaccine research if an outbreak was to occur, one NPRC established a COVID-19 barrier facility with a high-efficiency particular air (HEPA) system which provided housed animals with additional respiratory protection. Many facilities considered using N95 respirators rather than surgical masks to prevent disease transmission to animals, but this was not possible due to supply chain shortages of N95s. The NPRCs also worked diligently to set up a SARS-CoV-2 screening program for colony animals. A previously established NPRC consortium, the Pathogen Detection Working Group, collaborated to rapidly develop assays and protocols for monitoring NHP colonies for SARS-CoV-2. By March 2020, some NPRCs were screening their NHP colonies by using PCR in serum samples, while the remainder of the NPRCs used both serology and real-time PCR detection methods. By mid-summer 2020, the NPRCs had collectively screened more than 10,000 NHPs for the SARS-CoV-2 virus by using PCR or antibody testing but identified no positive cases. Fecal PCR assays were also established for noninvasive screening of large animal groups. Most NPRCs have integrated SARS-CoV-2 screening into routine colony testing. To date, the NPRCs have performed more than 25,000 tests, but no positive cases have been identified. The SARS-CoV-2 virus, however, has been detected in NHP species at other facilities, including one case report of a SARS-CoV-2 positive African green monkey at a research facility 5 and multiple reports of positive great apes at zoos across the country.1,6 Additional information about the NPRC COVID-19 animal screening program is available on the Pathogen Detections Working Group website (https://nprcresearch. org/primate/pathogen-detection/pathogen-detection-whats-new.php).

Personnel Management

Another challenge faced by all NHP research centers throughout the COVID-19 pandemic was the management and protection of personnel. Given the value of work with NHPs, the NPRC’s were unable to significantly reduce ongoing research and even ramped up activities with COVID-19 projects, thus requiring the majority of personnel to remain on site. Some facilities

Given the close relatedness of NHPs to humans, the threat of emerging zoonotic diseases will always be a concern for facilities managing large groups of NHPs. The lessons we learned from the COVID-19 pandemic will be invaluable preparation for any future outbreaks. Joyce Cohen, VMD, DACLAM, is the Associate Director of Animal Resources and an Associate Professor of Psychiatry and Behavioral Sciences at Yerkes National Primate Research Center at Emory University in Atlanta, GA.

REFERENCES

Figure 1. The seven NPRCs include: California NPRC, Oregon NPRC, Southwest NPRC, Tulane NPRC, Washington NPRC, Wisconsin NPRC and Yerkes NPRC. (from https://nprc.org/about-nprcs/) developed teams of staff that worked on alternating days or staggered shifts to minimize overlap between personnel. Centers identified creative solutions to space staff out for lunches and in locker rooms. Dedicated personnel continued to provide care and research support throughout the lockdown periods and the entire pandemic despite any personal challenges. Several of the NPRCs were able to set up employee COVID-19 testing centers for weekly screening early on when public testing was not widely available. In addition, many employees at the centers had early opportunities for COVID-19 vaccines as essential personnel.

Development of Research Models

In conjunction with working to develop methods to protect and screen the NHP colonies from SARS-CoV-2, the NPRCs worked to rapidly develop NHP models of COVID-19 for evaluation of vaccines and therapeutics. Institutions with ABSL3 capabilities for NHPs quickly pivoted to SARS-CoV-2 research. The NIH helped facilitate NPRC collaboration with other academic and contract research organizations to share data on NHP model development in real time. This collaboration included an NPRC coronavirus working group and a Coronavirus Vaccine and Therapeutic Evaluation Network (COVTEN) whose members met weekly to share data, discuss plans to ensure rapid development of disease models, and avoid potential duplication of research efforts. These collaborations facilitated pre-publication information sharing between groups doing COVID-19 research and helped researchers develop better animal models based on real- time data. The COVTEN group also created a series of harmonized standard operating procedures (SOPs) for NHP COVID-19 research that allowed data collected across the NPRC program to be comparable. This level of collaboration facilitated more efficient and rapid model development. These groups continue to meet regularly to discuss ongoing and future projects using NHPs for COVID-19 research. Throughout the pandemic, the NPRCs have faced many challenges in the management and protection of their large NHP colonies. Collaboration among the NPRCs, along with support from the NIH, helped guide the centers to rapidly respond to the pressures of COVID-19 and ensure the well-being of our animal colonies.

1. AP News. [Internet]. 2021. COVID-19 infections spread through gorillas at Atlanta zoo. [Cited 29 November 2021]. Available at: https:// www.science.org/content/article/captive-gorillas-test-positive-coronavirus 2. Lowenstine LJ, Osborn Kent G. 2012. Respiratory system diseases of nonhuman primates. nonhuman primates in biomedical research. Nonhuman Primates in Biomedical Research. 413-481. DOI: 10.1016/ B978-0-12-381366-4.00009-2 3. National Primate Research Centers. [Internet]. 2021. About NPRCs. [Cited 29 November 2021]. Available at: https://nprc.org/ 4. Qin C, Wang J, Wei Q, She M, Marasco WA, Jiang H, Tu X, Zhu H, Ren L, Gao H, Guo L, Huang L, Yang R, Cong Z, Guo L, Wang Y, Liu Y, Sun Y, Duan S, Qu J, Chen L, Tong W, Ruan L, Liu P, Zhang H, Zhang J, Zhang H, Liu D, Liu Q, Hong T, He W. 2005. An animal model of SARS produced by infection of Macaca mulatta with SARS coronavirus. J Pathol. 206(3):251–9. doi: 10.1002/path.1769. PMID: 15892035; PMCID: PMC7167940 5. Ricks KM, Herbert AS, Koehler JW, Kuehnert PA, Tamara L. Clements, Charles J. Shoemaker, Ana I. Kuehne, Cecilia M. O’Brien, Susan R. Coyne, Korey L. Delp, Kristen S. Akers, John M. Dye, Jay W. Hooper, Jeffrey M. Smith, Jeffrey R. Kugelman, Brett F. Beitzel, Kathleen M. Gibson, Sara C. Johnston, Timothy D. Minogue. [Internet]. 2020. Animal model prescreening: pre-exposure to SARS-CoV-2 impacts responses in the NHP model. [Cited 29 November 2021]. Available at: https://www.biorxiv.org/content/10.1101/2020.07.06.189803v2 doi: https://doi.org/10.1101/2020.07.06.189803 6. Science Insider. [Internet]. 2021. Captive gorillas test positive for coronavirus first case of COVID-19 in endangered apes detected at San Diego wildlife park. [Cited 29 November 2021]. Available at: https:// www.science.org/content/article/captive-gorillas-test-positive-coronavirus 7. Science Insider. [Internet] 2020. Primatologists work to keep great apes safe from coronavirus. [Cited 29 November 2021]. Available at: https://www.science.org/content/article/primatologists-work-keep-great-a pes-safe-coronavirus 8. Willy ME, Woodward RA, Thornton VB, Wolff AV, Flynn BM, Heath JL, Villamarzo YS, Smith S, Bellini WJ, Rota PA. 1999. Management of a measles outbreak among Old World nonhuman primates. Lab Anim Sci 49(1):42-8. PMID: 10090093

January 2022 Laboratory Animal Science Professional 17

FEATURE

NHPs in Research

Why Primates? A Broad Review of the Current Uses of NHPs in Biomedical Research By Diana G. Scorpio, DMV, MPH, DACLAM

on-human primates (NHPs) of various species are known to be excellent models of disease in humans and other animals. NHPs have historically been associated with the eradication of polio, the discovery of life-saving medications, and the numerous complex functions of the mind/ brain interface. Their impact on the scientific community is quite significant and widespread even though they represent a small percentage of animals used in biomedical research. Approximately 0.5% of animals used in the USA and globally are NHPs, and the remaining are a mix of small and large animal species, mostly rats and mice. Among NHP species in NIH funded research, rhesus macaques (Macaca mulatta) comprise 65% of all planned use, followed by cynomolgus macaques (Macaca fascicularis) at 15%, baboons (Papio spp.) at 5.5%, and common marmosets (Callithrix jacchus) at 3.1%. 2 Many NHPs are used in basic research investigations; however, they are also used extensively in translational research, drug development, and medical device testing. Regarding planned use by research area, studies in simian immunodeficiency virus (SIV) as a model for HIV/AIDS comprise a very large percentage of

Pictured above: A cynomolgus macaque (Macaca fascicularis)

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Indian-origin rhesus macaque use. Indian-origin rhesus share similarities with humans in MHC alleles associated with HIV infection and development of AIDS. Rhesus macaques are also used extensively in the fields of neuroscience and other infectious diseases (non-AIDS). 2 Table 1 lists all federal and non-federal institutions that breed NHPs for biomedical research. Many of the facilities are funded in part or entirely by the NIH. Eight primate research centers breed and provide the largest and most diverse species of NHPs. All primate centers together have a combined estimated NHP holding capacity of 31,500 animals.2 The animals are either used for intramural studies or sold to outside institutions. Serving outside institutions is a critically important requirement for primate centers. However, it’s important to note that non-federal sources also provide many NHPs for federally funded research, whether by breeding or importation, which is less common. There are some unique features of NHPs used in biomedical research compared to rodents. NHPs are regulated by the USDA/APHIS, unlike rats and mice.1 They have a lawful requirement for environmental enrichment, social housing, and processes to promote psychological wellbeing unless exemptions are put in place.1,4 NHPs require a dedicated space specifically for surgical procedures, 1,4 and NHPs can be held in one single enclosure with a large number of their conspecifics, even exceeding 100 individuals, unlike the way in which rodents, dogs, cats, and many other mammalian species are housed. Recently, there has been a significant and growing interest in using the smaller common marmoset (Callithrix jacchus) in biomedical research. They have become increasingly essential in the fields of neuroscience, aging, and the gut/brain axis. Smaller common marmosets have some unique traits that set them apart from the other NHP species listed here. Smaller common marmosets can be commonly handled without sedation, have a shorter lifespan, which is attractive for aging studies, and usually give birth to twins and sometimes triplets, allowing for greater reproductive capacity and colony growth. Species such as macaques and baboons typically only give birth to 1 offspring per year. As listed in the table below, there are only 3 institutions currently breeding marmosets in moderately sized colonies (up to only 400 individuals), making them an exceptionally difficult resource to procure and use for studies. This shortage is a common theme that began in 2019. Many NHPs can no longer be imported into the U.S., so reliance on adequate animal supply is predominantly based on breeding and production in the USA and U.S. Territories.

Species

NIH-Sponsored Centers

Other Sources

African Green (Vervet) Monkey

• Wake Forest University

• New Iberia Research Center

Baboon

• Southwest NPRC • The Mannheimer • University of Foundation Texas (Keeling Center)

Common Marmoset

• Southwest NPRC • The Johns Hopkins • Wisconsin NPRC University

Cynomolgus Macaque

• New Iberia Research Center • The Mannheimer Foundation • Primate Products Inc.

Dusky Titi Monkey

• California NPRC

Japanese Macaque

• Oregon NPRC

Owl Monkey

• MD Anderson (Keeling Center)*

Pigtail Macaque

• The Johns Hopkins University • Washington NPRC

Rhesus Macaque

• California NPRC • Caribbean Primate Research Center • Oregon NPRC • Southwest NPRC • Tulane NPRC • Wisconsin NPRC • Yerkes NPRC

Sooty Mangabey

• Yerkes NPRC

Squirrel Monkey

• University of Texas (Keeling Center)

• Covance Laboratories • The Johns Hopkins University • New Iberia Research Center • The Mannheimer Foundation • Primate Products Inc. • University of Texas (Keeling Center)*

Table 1. U.S. NHP Breeders *Baboon and squirrel monkey colonies at the University of Texas are sponsored by the NIH; their owl and rhesus macaque colonies are not.

Regarding chimpanzees, apes once used for studies in HIV, Hepatitis B, and Hepatitis C are no longer allowed to be utilized in research. Those animals supported by past federal funding are protected from unnecessary euthanasia and

have mostly been transferred to a federal retirement system (CHIMP Act, signed into law by President Clinton in 2000). However, their historical contributions must not be forgotten, as they have contributed to advances in HIV and Hepatitis C. In fact, the successful cure for Hepatitis C was studied and developed following years of research in chimpanzees.3 Many institutions with chimpanzees may still study them ethologically and behaviorally so the scientific field can derive valuable information from them. Scientists have also used clinical samples from veterinary cases or routine physical exams to compile data on aging chimpanzees and diseases that mimic aging human populations.

Conclusion

To conclude, NHPs represent a small percentage of animals used in biomedical research, but they have made a significant contribution to many of the world’s infectious and non-infectious diseases. NHPs have contributed to the production of vaccines, therapeutics, and life-saving measures currently in use by the medical and veterinary fields. As with all animals in biomedical research, they must be used when other alternatives do not exist for scientific advancement. They are a species to be used judiciously and in conjunction with robust in vitro techniques and preliminary in vivo studies conducted in rodent models. It is most important to recognize how valuable NHPs have been throughout our current SARS CoV-2 pandemic. Studies using both rodents and NHPs as animal models for COVID accelerated the development of vaccines and therapeutics in record time. These contributions by scientists with the use of animals protect human populations from diseases of global impact. Diana G. Scorpio, DVM, MPH, DACLAM, is the Associate Director of Veterinary Resources, Attending Veterinarian at Southwest National Primate Research Institute, and Professor of the Host-Pathogen Interactions Program at the Texas Biomedical Research Institute in San Antonio, TX. REFERENCES 1. Animal and Plant Health Inspection Service. 2020. Animal Welfare Act and Animal Welfare Regulations. USDA Animal Care. APHIS 41-35-076. [Cited 3 December 2021]. Available at: https://www.aphis.usda.gov/aphis/home/ 2. Feister AJ, DiPietrantonio A, Yuenger J, Ireland K, Rao A. Nonhuman primate evaluation and analysis. part 1: analysis of future demand and supply. 2018. National Institutes of Health, Office or Research Infrastructure Programs. 3. Lanford RE, Walker CM, Lemon SM. The Chimpanzee Model of Viral Hepatitis: Advances in Understanding the Immune Response and Treatment of Viral Hepatitis. 2017. ILAR Dec 1;58(2):172-189. 4. National Research Council. 2011. Guide for the care and use of laboratory animals, 8th ed. Washington (DC): National Academies Press.

January 2022 Laboratory Animal Science Professional 19

FEATURE

NHPs in Research

Enhancing Animal Welfare by Providing Custom Caging Space for Nonhuman Primates By Rachelle L. Stammen, DVM, DACLAM, Joyce K. Cohen, VMD, DACLAM, Fawn R. Connor-Stroud, DVM, DACLAM, Kelly F. Ethun, DVM, DACLAM

erkes National Primate Research Center (YNPRC) is one of seven National Primate Research Centers in the United States. Similar to other nonhuman primate (NHP) facilities supporting NIH-funded biomedical research, the YNPRC is committed to continuously enhancing animal welfare and refining the socialization, enrichment, and housing options for their NHPs. The YNPRC recently renovated two NHP housing areas with a novel facility design and equipped with a customized caging system that offers additional outdoor access, socialization, and enrichment opportunities for NHPs assigned to infectious disease studies, awaiting biomedical research assignment, or in quarantine. Conventional NHP caging has limited housing square footage and only allows for socialization of pairs indoors. This state-of-the-art customized caging system provides opportunities for trio or group housing, larger floor space, complex caging configurations, and increased climbing and perching opportunities. Cage Customizations

The Division of Animal Resources at the YNPRC collaborated with a caging company to customize a novel caging system to enhance the welfare of the NHPs by providing opportunities for group housing, trio housing, as well as pair-housing, with both indoor and outdoor access. The custom caging consists of a top and bottom cage integral rack unit that has the capability to attach directly to other cage rack units or outdoor runs (Figure 1). Two indoor-only buildings were custom-designed and renovated into indoor-outdoor facilities with this special caging in mind. Each animal room in these buildings

Figure 2: A group of juvenile rhesus macaques utilize the access doors that open to an outdoor run equipped with a variety of enrichment devices including perches, spin poles, and toys. Figure 1: The custom caging consists of a top and bottom cage integral rack unit that can attach directly to other cage rack units or to individual outdoor runs. Operating windows provide staff visual access to the outdoor runs. All metal components of the custom caging are #304 stainless steel and autoclavable.

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contain square access doors that provide animals access to 51 sq ft outdoor run, outfitted with various enrichment devices, perches, and toys (Figure 2). On the inside portion of the animal housing area, stainless steel door plates attach to the square openings adjacent to each cage unit. Once the top and

Figure 3: Operating windows are used for training and shifting animals between inside and outside. The windows also provide additional visual enrichment for the NHPs.

bottom cage units are securely attached to the square openings, the animals have access to the run. Each animal room in the renovated buildings is equipped with two operating windows that can be used for training and shifting animals inside and outside (Figure 3). In addition to horizontal socialization, the custom caging has a removable grid floor and ceiling panels that allow opportunities for vertical socialization, maximizing passageways and space for the NHPs to exhibit species-specific behaviors (climbing and perching). When the cages are connected vertically, a wire floor panel is placed to create the vertical opening while still providing a half-floor of space for NHPs to perch upon while in the upper cage unit. These wire floor panels also provide flexible caging configuration options to accommodate different size social groups as needed. To expand socialization space further, custom tunnel connectors were designed to connect directly to the end of each caging system (Figure 4). These vertical tunnels provide an enriched perch-type environment for the NHPs, offering them increased visual access of the inside portion of the animal housing area. The vertical tunnels are also used for cage change procedures as part of routine husbandry. Additionally, the vertical tunnels help facilitate the shifting of group-housed animals when individual animals are needed for veterinary or experimental procedures. Also, conventional caging restricts the size of the animals that can be housed in them. The new custom cages have a larger square footage of 6.25 sq ft versus 4.3 sq ft and can be configured to even larger sizes (8.0 sq ft), thus accommodating larger animals.

Figure 4: The custom cage racks can be connected horizontally. Additionally, half-floor wire panels can be inserted into the top cage pan area providing vertical access to the top and bottom portions of the cage rack. A custom designed vertical tunnel also connects directly to end of each caging system, providing a supplemental perching opportunity and increased visual access of the animals to the inside portion of the animal room.

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Benefits

Figure 5: (a) Cages are attached to outside door plates and panels can be removed to allow access to an outdoor run. (b) Full-swing removable cage-front door used to remove anesthetized animals from the cage. The double handles are squeezed and pulled up to open the front of the cage for easier access to anesthetized NHPs. (c) Venipuncture hole used for conscious blood collections and training. (d) Built-in rotatable forage board with turf material to provide forage items to the NHPs. (e) Built-in socialization panels including solid panels, clear panels, small holes for grooming contact, and grooming bars.

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The complex behavioral and socialization requirements of nonhuman primates were also considered when designing these cages (Figure 5). The new caging has built-in socialization panels in every unit to facilitate the introduction process of housing partners, including solid panels, clear panels, small holes for grooming contact, and grooming bars. Another convenient built-in feature, and a favorite element among YNPRC behavior management specialists, is the non-removable forage boards. Each cage is equipped with its own forage board with a turf mat for forage materials. The forage boards can be rotated downward for easy cleaning, which is advantageous for the animal care technicians. These built-in features also eliminate the need for panel and forage board storage. To accommodate training or conscious blood collection, each cage has a small square “venipuncture hole” in which the animals can easily present a limb to collect blood consciously from the saphenous vein. Lastly, in addition to the traditional vertical sliding door, the entire front of the cage can be opened as an access door to remove anesthetized animals more easily and safely from the cage. Both veterinary and research technicians who frequently access anesthetized animals have expressed how much they appreciate this feature. Because these animals can go from outdoor housing directly into caging, this increases socialization opportunities for animals assigned to infectious disease studies and in quarantine. YNPRC and other NHP research institutions have worked closely with researchers to identify ways to socially house animals on infectious disease research studies. Usually, this is a difficult, and sometimes impossible, task when animals are housed in larger social groups with outside access. However, in these new facilities and new caging systems, animals can be shifted indoors,

separated into individual cages, accessed for experimental procedures and treatments, and then easily reunited. In addition, YNPRC is currently performing research with their Behavioral Management Unit to evaluate the benefits of pair housing animals during quarantine by monitoring stress indicators. Animals that are brought in from external facilities and go through quarantine may come from large social groups, and the custom caging system allows Yerkes to maintain these animals in their social groups while in quarantine, thus reducing stress and enhancing welfare.

Lessons Learned

Innovations and novel projects, as beneficial they may be, are regularly accompanied by challenges, considerations, and lessons learned. Some challenges with the custom cages were understood upfront during the design process, such as the heavier weight and increased height of the caging. The large size makes transport to cagewash more difficult, especially if the closest cagewash is in a different building. Therefore, it is important to train staff on safely moving the heavy cages with equipment designed for moving wheeled loads on uneven surfaces, such as tug machines or forklifts. Additionally, the weight of the wire floor panels is very heavy and can require more than one individual to remove or insert them. One of the most challenging aspects of the custom caging is ensuring the cages are safely secured. Due to the ability of the animals to have horizontal and vertical access to adjacent cages, there are several locks on the custom cages compared to traditional NHP cages (Figure 5). Thus, it is extremely important to properly train staff on the use of the cages and their unique intricacies, such as tethering adjacent cages, affixing cage units to runs, and verifying the numerous locks are secure. It is also important to consider the total time animals will have access to the outdoor run (i.e., 24-hour access versus daytime-only access). YNPRC animal care staff has learned that the longer the animals have access to the outdoor runs, the longer it takes to perform husbandry tasks. Therefore, more animal care technicians may be necessary to support these facilities to ensure

husbandry is finished in a timely manner. In addition, training animals on how to shift indoors and outdoors can take extra time and effort. Personnel who routinely observe and interact with the animals in the new custom caging system have subjectively noticed a positive impact on the animals. For example, YNPRC animal behavior management specialists have noted that the ability to go outside has been positive for the animals. Animals have more opportunities to forage, exercise, play, have larger social groups, and access different enrichment devices such as swings, spin poles, water enrichment, different styles and levels of perching, and large toys such as rocking horses or balls. Staff love to watch them play, interact with each other, and sunbathe. Likewise, veterinary technicians agree that the animals appear happier, enjoy having the option to go outside, and spend considerably more time outdoors compared to indoors. YNPRC Behavior Management Unit is currently researching the effects of this new caging system, with outdoor access, on animal welfare with more objective methods and measurements. All photos credited to Rachelle Stammen, DVM, DACLAM for the Yerkes National Primate Research Center, Emory University. Rachelle L Stammen, DVM, DACLAM, is a Clinical Veterinarian at Yerkes National Primate Research Center at Emory University in Atlanta, GA. Fawn R. Connor-Stroud DVM, DACLAM is a Senior Veterinarian and Assistant Division Chief of the Division of Animal Resources at Yerkes National Primate Research Center at Emory University in Atlanta, GA. Kelly F. Ethun DVM, DACLAM is a Senior Veterinarian and Co-Director of the Biomarkers Core at Yerkes National Primate Research Center in Atlanta, GA.

Joyce K. Cohen VMD, DACLAM is the Attending Veterinarian and Associate Director of the Division of Animal Resources at Yerkes National Primate Research Center in Atlanta, GA.

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January 2022 Laboratory Animal Science Professional 23

FEATURE

NHPs in Research

Five Hot Topics in Refinement of Nonhuman Primate Neuroscience Research

Christine L. Buckmaster, BS; Denyse Levesque, DVM; Megan LaFollette, MS, PhD; Meaghan Loy, MS; Jennifer L. McMillan, BS; Mark J. Prescott, PhD; Sally Thompson-Iritani, DVM, PhD

onhuman primates (NHPs) play an important role in neuroscience research and in advancing our understanding of normal neurological function, behavior, and diseases such as Parkinson’s, Alzheimer’s, and spinal cord injury. This important biomedical research is supported by continual evaluation of opportunities to improve the lives of research animals in line with the refinement principle of the 3Rs.24 The purpose of this article is to highlight and provide resources for five recent evidence-based NHP refinement advances in neuroscience. It is important to always remember that modifications need to be reviewed and implemented in partnership with the researchers, veterinary and animal care staff as well as oversight bodies (e.g., IACUC (Institutional Animal Care and Use Committee), AWERB (Animal Welfare and Ethical Review Board), NIH (National Institutes of Health) to ensure they are incorporated in a thoughtful and measured approach that delivers both improved animal welfare and optimal data. Members of the North American 3Rs Collaborative (NA3RsC) refinement initiative have chosen to summarize developments in five hot topics of interest: 1. 2. 3. 4. 5.

Tips for welfare-friendly transport, chairing, and restraint Guidance on refining food and fluid control Protective caps for cranial implants MRI Neuroimaging Retirement: an optional experimental endpoint

1. Tips for Welfare-friendly Transport, Chairing, and Restraint

Traditionally, NHPs are often restrained to facilitate safe handling and obtain critical research outcomes. However, restraint may cause stress for some animals, potentially confounding study results.25,27 Therefore, it is important to refine these methods whenever possible. It is now common in many neuroscience labs to train animals to voluntarily exit their home enclosure, enter a primate chair, accept restraint, and sit in place for an extended period. In fact, in only 20 days, rhesus macaques can be trained without pole-and-collar to voluntary transfer from the home enclosure into a primate chair, and accept neck-plating.11 Overall, the chairing procedures can be improved – made faster and less stressful - through a greater understanding of operant conditioning and how to best incorporate positive reinforcement training techniques.13 Choosing an appropriate chair design can also support improved handling and animal welfare. Two main types of chairs exist: box and open. In a box chair, animals are trained to enter and extend their heads through an aperture on the top. In an open chair, animals are transferred manually or through the pole-and-collar process. In a

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recent survey, two-thirds of labs using restraint indicated using a ‘box chair’ compared to one-third of labs which used an ‘open chair.’12 Literature also suggests the ‘box chair’ can improve training success, decrease training time, and reduce stress compared to the ‘open chair.’8 Alternatives to physical restraint should also be considered as recommend by the Guide for the Care and Use of Laboratory Animals.9 For example, attaching a touch screen computer tablet directly to the cage allows NHPs to work continuously and stay free-moving in their home enclosure during initial training on cognitive tasks, before moving to the laboratory for electrophysiology. The ‘Mymou’ wireless, tablet-based system incorporates real-time facial recognition, allowing the animals to remain socially housed.4 Beyond tablets, training programs can also eliminate the need for restraint. For example, researchers at the University of Minnesota were able to eliminate chair restraint after implementing a training program focusing on the handler-animal relationship including hand feeding and drinking, shifting and limb presentation.5 All of the above refinements may improve the overall well-being of animals involved in neuroscience research, benefiting the animals, handlers, and science.

2. Guidance on Refining Food and Fluid Control Control of food and fluid have long been used to motivate NHPs to perform certain activities for food and fluid rewards during neuroscience and behavioral studies. However, international research institutions have recognized that if food and water regulation are not conducted appropriately, they can have animal welfare implications.20 Therefore, food and fluid management require careful consideration, ethical review, and approval as well as close work with oversight and clinical staff. Fluid restriction may be particularly distressing as water is a physiological requirement. Any restriction needs to be carefully monitored for individual animals to ensure adequate food consumption and body weight/growth are maintained.1 Extensive guidance on refining these protocols is available from the NC3Rs (National Centre for the Replacement, Refinement and Reduction of Animals in Research) and APV (Association of Primate Veterinarians).1,20 For some studies, food and water restriction can be avoided or relaxed by optimizing positive reinforcement training. For example, identifying individually preferred rewards can improve performance in cognitive tasks, which can be advantageous because the NHPs may work reliably on a more relaxed fluid restriction regimen.6 In large contract research organizations, highly preferred items such as yogurt, sports drinks, raisins, and nuts have been used to train sexually mature male macaques for procedures (such as blood sample collection). The increased time needed for training and minimal expense of treats may be outweighed by reduced animal distress and increased positive interactions between animals and technicians. Ultimately, positive reinforcement can be successful in NHPs of both sexes and all ages and elicit a variety of behaviors.19

insertions and compound injections), and monitor and diagnose health issues.17, 21 Finally, MRI results can be combined with behavioral assessment to gauge psychological well-being to better understand the impact of neuroscience procedures on the welfare of NHPs.2 Across the entire field, NHP welfare and the 3Rs can be advanced through collaboration, open resources, resource sharing, and international harmonization. Some key resources include the International Neuroimaging Data-sharing Initiative (INDI), the PRIMatE Data Exchange (PRIME-DE) which is an open resource for NHP MRI neuroimaging data15 and the PRIMatE Resource Exchange.14 In a recently held PRIME-DE meeting entitled, Accelerating the Evolution of Nonhuman Primate Neuroimaging,23 key program priorities were decided and it was agreed additional transparency on NHP welfare standards and regulations is needed when sharing data sets. International harmonization of animal welfare standards requires mutual recognition and implementation of the 3Rs3 and this would apply to the neuroimaging community's effort towards formulating its common set of NHP welfare regulations.

5. Retirement: An Optional Experimental Endpoint

Photo provided courtesy of the Yerkes National Primate Research Center.

3. Protective Caps for Cranial Implants

Neuroscience studies often require NHPs to be fitted with cranial implants to stabilize the head or gain access to the brain for scientific purposes (e.g., electrophysiological recordings), but wound management after surgery can be difficult. Technical and research staff from the University of Oxford and Newcastle University collaborated to develop an easily modifiable head cap to protect the sutured skin margins after surgery, restricting the monkey’s fingers from accessing the wound while allowing air to circulate to promote healing.18 Made from plastic sheeting, the cap is affixed to the implant post-surgery and can accommodate a variety of different implant types and head posts. Once secured, and while the animal is still anesthetized, the sheeting is molded around the implant. The protective cap, which is now being used routinely in several laboratories in the UK, EU, and USA, has reduced wound dehiscence (opening), the need for re-suturing, and the number of days antibiotics and analgesia are required. Further advances in cranial implant design, implantation and maintenance are given in the NC3Rs chronic implants wiki (https:/www.ciwiki.net).

4. MRI Neuroimaging

Magnetic resonance imaging (MRI) is playing a significant role in applying the 3Rs to neuroscience studies. For individual research facilities MRI can contribute to refinement in many ways. MRIs can be used to enhance the selection and assignment of NHPs to studies, guide the manufacture of custom-fitted recording and head fixation devices, target brain regions more accurately (e.g., for electrode

For most NHPs, euthanasia is a common endpoint due to experimental requirements and necessary collection of post-mortem data. However, when euthanasia is not a necessary endpoint, there is an opportunity to rehome NHPs to sanctuaries.10,22 NHP rehoming is clearly of interest to the research community as evidenced by recent platform sessions during annual meetings of AALAS, the APV, and the ASP (American Society of Primatologists). Incorporating retirement and rehoming into research programs may be an opportunity to strengthen the well-being of personnel working in research animal facilities and decrease euthanasia associated stress.16,26 Some institutions in the USA are working toward novel and sustainable options for rehoming New World and Old World NHPs when research studies are completed and are or have set up financial partnerships with specific sanctuaries or are building on-site sanctuaries.7,28 In summary, this is a high-level overview of current refinements to consider when working with NHPs to support neuroscience research. It is important to recognize that each refinement must be considered in context with the scientific objective and that personnel must be properly trained to implement each technique. Finally, all modifications need to be reviewed and implemented in partnership with the researchers, veterinary, animal care staff, and oversight bodies (e.g. IACUC, AWERB, NIH) to ensure they are incorporated in a thoughtful and measured approach that delivers both improved animal welfare and optimal data. For additional ideas of evidence-based refinements for non-human primates visit NC3Rs or NA3RsC non-human primate research hubs. Christine L. Buckmaster is Senior Research Staff in the Department of Comparative Medicine, Stanford University, Palo Alto, CA. Megan LaFollette, MS, PhD, is Program Manager at the North American 3Rs Collaborative, Denver, CO. Denyse Levesque, DVM, is a Senior Veterinarian at the Yerkes National Primate Research Center and IACUC Vice-Chair at Emory University, Atlanta, GA. Meaghan Loy, MS, is the Category Director of In Vivo Services at Scientist.com, Solana Beach, CA. Jennifer L. McMillan is Training Manager for the Division of the Animal Resources at the Yerkes National Primate Research Center, Atlanta, GA. January 2022 Laboratory Animal Science Professional 25

Photo provided courtesy of Christine Buckmaster, BS.

Mark J. Prescott, PhD, is Director of Policy and Outreach at the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), in London, UK. Sally Thompson-Iritani, DVM, PhD, is the Assistant Vice Provost, Animal Care, Outreach &3Rs at the University of Washington in Seattle, WA. REFERENCES

1. Association of Primate Veterinarians. [Internet]. 2014. Guidelines for use of fluid regulation for nonhuman primates in biomedical research. [Cited 29 November 2021]. Available at: https://www.primatevets.org/guidance-documents 2. Basso MA, Frey S, Guerriero KA, Jarraya B, Kastner S, Koyano KW, Leopold DA, Murphy K, Poirier C, Pope W, Silva AC, Tansey G, Uhrig L. 2021.Using non-invasive neuroimaging to enhance the care, well-being and experimental outcomes of laboratory non-human primates (monkeys). Neuroimage. Mar;228:117667. doi: 10.1016/j.neuroimage.2020.117667. Epub 2020 Dec 24. PMID: 33359353; PMCID: PMC8005297. 3. Bayne K, Turner PV. 2019. Animal welfare standards and international collaborations. ILAR J. 60 (1): 86–94. doi: 10.1093/ilar/ily024. PMID: 30624646. 4. Butler JL, Kennerley SW. 2019. Mymou: A low-cost, wireless touchscreen system for automated training of nonhuman primates. Behav Res Methods. 51:2559–2572. 5. Graham ML, Rieke EF, Mutch LA, Zolondek EK, Faig AW, Dufour TA, Munson JW, Kittredge JA, Schuurman HJ. 2012. Successful implementation of cooperative handling eliminates the need for restraint in a complex non-human primate disease model. J Med Primatol. 41(2):89106. doi: 10.1111/j.1600-0684.2011.00525.x. Epub 2011 Dec 12. PMID: 22150842; PMCID: PMC3309152. 6. Gray H, Thiele A, Rowe C. 2019. Using preferred fluids and different reward schedules to motivate rhesus macaques (Macaca mulatta) in cognitive tasks. Lab Anim. 53(4):372-382. doi: 10.1177/0023677218801390. Epub 2018 Oct 3. PMID: 30282500. 7. Grimm, D. Should aging lab monkeys be retired to sanctuaries? [Internet]. 2019. Science. [Cited 29 November 2021.] Available at: https://www.science.org/content/article/should-aging-lab-monkeys-be-retired-sanctuaries

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8. Houser LA, Ramsey C, de Carvalho FM, Kolwitz B, Naito C, Coleman K, Hanna CB. 2021. Improved training and semen collection outcomes using the closed box chair for macaques. Animals (Basel). 11(8):2384. 9. Institute for Laboratory Animal Research. 2011. Guide for the care and use of laboratory animals, 8th ed. Washington (DC): National Academies Press. 10. Lankau EW, Turner PV, Mullan RJ, Galland GG. 2014. Use of nonhuman primates in research in North America. J Am Assoc Lab Anim Sci. 53:278-282. 11. Mason S, Premereur E, Pelekanos V, Emberton A, Honess P, Mitchell AS. 2019. Effective chair training methods for neuroscience research involving rhesus macaques (Macaca mulatta). J Neurosci Methods. 317:82-93. 12. McMillan J, Bloomsmith MA, Prescott MJ. 2017. An international survey of approaches to chair restraint of nonhuman primates. Comparative Medicine 67(5):1-10. 13. McMillan JL, Perlman JE, Galvan A, Wichmann T, Bloomsmith MA. 2014. Refining the pole-and-collar method of restraint: emphasizing the use of positive training techniques with rhesus macaques (Macaca mulatta). Journal of the American Association of Laboratory Animal Science 53:61-68. 14. Messinger A, Sirmpilatze N, Heuer K, et al. 2021. A collaborative resource platform for non-human primate neuroimaging. NeuroImage 226. 117519. 15. Milham MP, Ai L, Koo B, et al. 2018. An open resource for non-human primate imaging. Neuron 100: 61–74. 16. Murray J, Bauer C, Vilminot N, Turner PV. 2020. Strengthening workplace well-being in research animal facilities. Front Vet Sci 7:1-10. 17. Ortiz-Rios M, Haag M, Balezeau F, Frey S, Thiele A, et al. 2018. Improved methods for MRI -compatible implants in nonhuman primates. Journal of Neuroscience Methods 308: 377-389 18. Perry BAL, Mason S, Nacef J, Waddle A, Hynes B, Bergmann C, Schmid MC, Petkov CI, Thiele A, Mitchell AS. 2021. Protective cranial implant caps for macaques. J Neurosci Methods 348:108992. 19. Prescott MJ, Bowell VA, Buchanan-Smith HM. 2005. Training laboratory-housed non-human primates, part 2: resources for developing and implementing training programmes. Animal Technology and Welfare 4:133-148. 20. Prescott MJ, Brown VJ, Flecknell PA, Gaffan D, Garrod K, Lemon RN, Parker AJ, Ryder K, Schultz W, Scott L, Watson J, Whitfield L. 2010. Refinement of the use of food and fluid control as motivational tools for macaques used in behavioural neuroscience research: Report of a working group of the NC3Rs. J Neurosci Methods 193:167-188. 21. Prescott MJ, Poirier C. 2020. The role of MRI in applying the 3Rs to non-human primate neuroscience. NeuroImage 225:117521. 22. Prescott MJ. 2006. Finding new homes for ex-laboratory and surplus zoo primates. Laboratory Primate Newsletter 45:5-8. 23. PRIMatE Data Exchange (PRIME-DE) 2020. Global collaboration workshop and consortium. Neuron 105: 601-603. 24. Russell WMS, Burch RL. 1959. The principles of humane experimental technique. Wheathamptead (UK): Universities Federation for Animal Welfare Publishing. 25. Ruys JD, Mendoza SP, Capitanio JP, Mason WA. 2004. Behavioral and physiological adaptation to repeated chair restraint in rhesus macaques. Physiol Behav 82:205-213. 26. Schlanser TV, Rabinowitz PM, Thompson-Iritani, S. 2021. Compassion fatigue and satisfaction in US army laboratory animal medicine personnel. J Am Assoc Lab Anim Sci. 60(4): 422-430. 27. Shirasaki Y, Yoshioka N, Kanazawa K, Maekawa T, Horikawa T, Hayashi T. 2013. Effect of physical restraint on glucose tolerance in cynomolgus monkeys. J Med Primatol. 42:165-168. 28. Zardonella C. [Internet]. 2019. Princeton establishes long-term care program for retiring research monkeys. [Cited 29 November 2021]. Available at: https://research.princeton.edu/news/princeton-establishes-long-term-care-program-retiring-research-monkeys

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2021

AALAS AWARD WINNERS William J. White, VMD, MS, DACLAM, DECLAM Consultant

Nathan R. Brewer Lifetime Achievement Award

Dr. White is currently a consultant in lab animal medicine and biosecurity and was the Corporate Vice President at Charles River where he headed global biosecurity and professional services activities. He serves on the International Air Transport Association’s (IATA) Live Animals and Perishables Board’s animal care team. Prior to joining Charles River, he was on the Comparative Medicine faculty at the Milton S. Hershey Medical Center where he conducted basic research in numerous areas including the effects of environment on laboratory animals. He was a member of the 1996 Guide committee and was president of ACLAM and IACLAM.

Betty R. Theriault, DVM, DACLAM

Director, Gnotobiotic Research Animal Facility Professor, Department of Surgery Clinical Veterinarian, Animal Resources Center The University of Chicago

Pravin N. Bhatt Scientific Investigator Award

Dr. Theriault earned her veterinary degree from the University of California Davis, completed a small animal medicine and surgery internship at UPenn, and is an ACLAM Diplomate. Scholarly and clinical pursuits include transplantation immunology research early career, animal models of human disease mid-career, and focus on germ free/gnotobiotic mouse models spanning the past 15 years. She is past president of Chicago Branch AALAS, past president and presidentelect of the Association for Gnotobiotics, and current president for the International Association for Gnotobiology. AALAS activities include sharing her gnotobiotic expertise through poster presentations, platform sessions, seminars, workshops, publications, and affiliate organization engagement.

Neil S. Lipman, VMD

Executive Director and Professor Memorial Sloan Kettering Cancer Center Weill Cornell Medicine

Charles River Prize

Neil Lipman is the Executive Director, Center of Comparative Medicine and Pathology, Memorial Sloan Kettering Cancer Center (MSK) and Weill Cornell Medicine (WCM); Professor of Veterinary Medicine in Pathology and Laboratory Medicine, WCM; and, Laboratory Member, Sloan-Kettering Institute, MSK. A graduate of the University of Pennsylvania’s School of Veterinary Medicine, Dr. Lipman completed postdoctoral training at MIT. An ACLAM Diplomate, he has held professional and faculty appointments at MIT, Brown University, Tufts University, and the University of Chicago, served as an AAALAC ad hoc Consultant and on the committee to update the Guide (8th Edition), and has been actively engaged in research, vivarium design and operations, and training of laboratory animal specialists throughout his career.

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Dondrae Coble, DVM, DACLAM, CertAqV Director and Attending Veterinarian Abigail Wexner Research Institute at Nationwide Children’s Hospital

George R. Collins Education and Training Award

Dr. Coble graduated from Tuskegee University School of Veterinary Medicine prior to completing a rotating small animal medicine and surgery internship. He worked as an emergency veterinarian before pursuing residency training at Emory University and Yerkes National Primate Research Center. He became a Diplomate in the American College of Laboratory Animal Medicine and began employment at The Ohio State University with a dual appointment in University Laboratory Animal Resources and the Department of Veterinary Preventive Medicine. He is currently the Director of the Animal Resources Core and Attending Veterinarian of the Abigail Wexner Research Institute at Nationwide Children’s Hospital.

Thomas Meier, DVM, MBA, DACLAM Chair, Department of Comparative Medicine Mayo Clinic

Joseph J. Garvey Management Award

Dr. Meier is the Chair of the Department of Comparative Medicine and the Attending Veterinarian for Mayo Clinic. He completed his veterinary training at the University of Minnesota. He is a diplomat of the American College of Laboratory Animal Medicine and an Assistant Professor of Comparative Medicine in the Mayo Clinic College of Medicine. His professional responsibilities include membership on the Institutional Animal Care and Use Committee for Mayo Clinic as well as leading Mayo’s animal husbandry/veterinary care department. Dr. Meier’s passions centers around leveraging technology to improve processes, outcomes, and safety in the animal care program.

Robin M. Miller, AAS, RLATG Senior Laboratory Technician WuXi AppTec

Technician of the Year Award

Robin Miller is a Senior Laboratory Technician at WuXi AppTec. Robin has 30 years of experience in the research industry, including former employment at Medtronic. She has provided service in both the animal husbandry and research areas. She has been heavily involved in the local branch of Minnesota AALAS, including positions as president and secretary. She has served on various committees at the local and national levels. Robin’s passion for research and unending devotion to animal welfare and care can be observed in her drive to educate and promote learning opportunities at WuXi AppTec and her involvement with AALAS.

FEATURE

National Meeting - Recap

72nd AALAS National Meeting:

BY THE NUMBERS

he 2021 AALAS National Meeting was a hybrid event with an onsite and online presence. Total registration for the meeting was 2,747 with 1,569 attending onsite and 1,178 attending virtual platform. One hundred and eighty exhibitors participated this year. The event app was downloaded 1,330 times. And the virtual meeting attendee website had 2,115 unique logins.

Clinical

Door Prize and Facilitator Winners

Second Place: P134 Pregnancy Associated Thrombocytopenia and Bone Disease in a Breeding Colony of Common Marmosets (Callithrix jacchus): S Ganesan; S Muthupalani; R Kramer; J Fox; J Haupt, Division of Comparative Medicine, Massachusetts Institute of Technology-

First Place: P117 Ultrasonographic Assessment of Ovarian Follicular Dynamics during Natural and Gonadotropin-stimulated Cycles in the Common Marmoset (Callithrix jacchus): M Stair,1 ID Napier,1 Q Zhang,2 S King,2 M Burns,1 G Feng,2 R Kramer,1 1 Division of Comparative Medicine and 2McGovern Institute for Brain Research, Massachusetts Institute of Technology

Door Prize winners from the technical trade presentations on Sunday, October 17 were NAMES Each winner was awarded a $50 gift certificate. The winners of the facilitator drawings were NAME, a free 2022 National Meeting registration, and NAMES, $50 AALAS Bookstore gift cards.

Third Place: P140 Methodology Improvements for Colonoscopy Preparation in Cynomolgus Macaques: T Rinaldo; E Voss; M Wood, Abbvie

Poster Winners

This year, 167 posters were exhibited in Kansas City. Congratulations to the 2021 poster winners.

Husbandry/Management

Animal Welfare, Training, and 3Rs

First Place: P205 A New Feeder Device to Provide Cleaner and Accessible Diet Gel to Laboratory Rodents: F Scorrano,1 SB Tavani,2 1Training and Innovation, Comparative Medicine, Novartis International AG, 2Engineering Center, Novartis International AG

First Place: P5 Refinements of Jugular Vein Catheterization with Vascular Access Button in Mice: A Lapierre, R LaFleur, K Kane, B Lyons, The Jackson Laboratory Second Place: P3 Evaluating Effects of CO2 Levels on Severe Combined Immunodeficiency Disease (SCID) Mouse Models: A Updike,¹ D Calantropio-Covington,¹ D Seiber,¹ B Rendon,¹ M Sowick,¹ L Bates,¹ K Mueller,¹ D Garcia,² R Guzman,³ K Walton,¹ Bristol Myers Squibb,¹Veterinary Sciences, ²Pharmacology, ³NCD-Exploratory Toxicology Third Place: P9 Use of a Modified External In-line Exhaust Collection Device for Detection of Murine Pathogens within a Double-Sided Ventilated Rack: BM Hibl,1 CL Perkins,2 KS Henderson,2 DJ Hamilton,1 1Department of Comparative Medicine, University of Tennessee Health Science Center, 2Charles River Laboratories

Second Place: P222 The Chinchilla Consortium: Improvement of Standard Practices for the Laboratory Chinchilla: M Williams-Fritze,2 R Beiler,3 D Coble,4 A Darbyshire,5 G Dobek,6 R Esquivel,7 M Fink,8 D Fong,8 L Habenicht,8 G Jackson,9 S Lewis,10 R Malbrue,4 E Mitchell,11 M Ortega,12 A Osborne,13 C Thurman,3 J Villano,14 W Williams,1 C Manuel,8 1The Univ of Oklahoma Health Sciences Center; 2CBSET, 3Boston University, 4Nationwide Children’s, 5Purdue University, 6Tulane University, 7 Boston Children’s, 8University of Colorado Denver Anschutz Medical Campus, 9Cornell Research, 10 UT Southwestern Medical Center,11Wake Forest Baptist Health, 12Massachusetts Eye and Ear, 13 The University of Alabama at Birmingham,14Johns Hopkins University Third Place: P212 Solutions to Minimizing Urine Spraying in Single-housed New Zealand White Rabbits: Lisa Burlingame; University of Michigan

January 2022 Laboratory Animal Science Professional 29

AALAS Foundation National Meeting News

Laboratory Investigations

First Place: P311 Relative Efficacy of Alfaxalone vs MS-222 Immersion Anesthesia for Rainbow Trout (Oncorhynchus mykiss): DJ Savson,1 SS Zenilman,1 CR Smith,1 EK Daugherity,1 B Singh,1 RG Getchell,2 1Center for Animal Resources and Education (CARE) at Cornell University, 2Department of Microbiology and Immunology, Cornell University Second Place: P352 Optimizing a Rat Surgical Model of Recurrent Laryngeal Nerve Regeneration Strategies; S Fudge,1 J Schmid,2 R Akin,2 C Seiller,2 K Osman,2 S Kington,2 R Thompson,2 C Smith,3 N Nichols,3 Teresa Lever,2,3 1Comparative Medicine Program, University of Missouri College of Veterinary Medicine, 2Otolaryngology–Head and Neck Surgery, University of Missouri School of Medicine, 3Biomedical Sciences, University of Missouri College of Veterinary Medicine Third Place: P324 Effects of Different Grades of Carbon Dioxide (CO2) on Euthanasia of Mice: JE Stuckey, S Makhija, S Putta, LA Bright, D Reimer, Rutgers University

Thanks to everyone who made this year's AALAS Foundation Appreciation Reception & Live Auction a huge success, especially all our generous Live Auction donors and bidders and our Appreciation Reception sponsor, Charles River! Congrats to Texas Branch for winning the Branch POE (Public Outreach & Education) Award! Congrats to Diana Baumann for being the lucky winner of Charles River's door prize drawing—a free registration to the 2022 AALAS National Meeting! Congrats to Central Illinois Branch, winners of the AALAS Foundation Branch Auction Item Challenge drawing! Congrats to all the "Swing Into Science" contest winners. There were 87 entries into the contest. A total of $5,780 was raised in “votes” and a total of $3,045 in entry fees for a grand total of $8,825 to support the AALAS Foundation! • Individual Category Winner - Terri Swanson • Branch Category Winner - Louisiana Branch • Corporate Category Winner - Bristol-Myers-Squibb - Redwood City Institution Category Winner - Univ. of Maryland/College Park • Fan Favorite Winner - UT Southwestern Medical Center • Best of Show Winner - Abveris

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2022 Laboratory Animal Science Professional 35 thinkanesthesia.education

INSIDE the IACUC In February of 2021, Texas and parts of several other states including Oklahoma, New Mexico, and Louisiana, experienced what has been called a “once in a century” severe weather event. While different authorities and agencies vary on the exact dates, the below 0°F weather, snow, and freezing precipitation lasted for about a week, starting on February 13 (at least where I live outside of Dallas). The news reports accurately reported how under-prepared the state was for the freezing weather. This included under-estimating the severity of the weather, under-estimating the amount of energy needed by businesses and residences during the storm, and under-preparing energy-producing equipment and facilities for winter weather. For those interested in an academic report reviewing the storm’s timeline and examining what happened and why, please see The Timeline and Events of the February 2021 Texas Electric Grid Blackouts, published in July 2021 by the University of Texas at Austin Energy Institute. Within the Texas Society for Biomedical Research (TSBR), several institutional representatives felt that we should share the stories of our successes and challenges experienced during the storm. Several of us got together, with TSBR’s sponsorship, and participated in a seminar at the 2021 AALAS National Meeting entitled “Once in a Century Event: Lessons Learned from the 2021 Texas Snowstorm.” Representatives from Baylor College of Medicine, the University of Houston, the University of Texas at Austin, and the University of Texas Southwestern Medical Center discussed what went well and what opportunities remain. Over the years, I have noticed that sessions about weather events and natural disasters have typically neglected to discuss how the IACUC can support compliance and recovery operations. So, after our seminar, I asked Dr. Thomas (Thom) Todd, IACUC Veterinarian at the University of Houston, to provide some reflections about the storm and how it was managed from an IACUC perspective. I hope you enjoy reading about Dr. Todd’s experiences and thoughts about IACUCs and natural disasters. It is something you do not hear about every day. Stacy Pritt, DVM, MS, MBA, CPIA, CHRC, DACAW

IACUC Roles and Opportunities during Natural Disasters By Thomas Todd, DVM, CPIA

n February 2021, the Houston Metroplex was hit with another natural disaster. Sadly, this was nothing new for area residents, most of whom experienced Hurricane Harvey three years earlier, and all of whom were still being impacted by the COVID-19 pandemic. While these and other emergencies provided valuable practice and familiarity with natural disasters that can be applied when preparing for future disasters, the week of "Snowvid," as some have since called it, presented some unique institutional challenges at the University of Houston (UH). Most campus personnel, including IACUC Office staff, were still working remotely and had been since the beginning of the pandemic. Remote work proved to be convenient in some ways, but the damage caused by sub-freezing temperatures and ruptured pipes resulted in water and power shortages, putting animal (and human) welfare at serious risk. Indeed, this damage was not just physical. The experience took a mental, emotional, and spiritual toll on those who went through it, especially as it brought back traumatic memories for those who lived through Harvey's catastrophic flooding and damage. Many who Snowvid did not greatly touch saw their neighbors impacted and experienced bouts of survivor's guilt (i.e., why was one person so negatively affected while another, perhaps living right next door, was not?). For animal care and IACUC operations, the safety and welfare of animals and humans is our number one concern during disasters such as this one. To that end, relevant considerations, mitigation measures, and lessons learned have been discussed at length elsewhere. But, with all that has been discussed, reviewed, and evaluated on the

36 Laboratory Animal Science Professional January 2022

confluence of natural disasters and laboratory animal facilities, one overlooked aspect is the role played by IACUCs and other local oversight and compliance bodies.

Snowvid Lands in Houston

The official start dates for "Snowvid" vary depending on the news source and city location. In the case of UH, campus closed on February 15. This happened to be the day of a scheduled IACUC meeting. It was obvious early that morning that something was seriously wrong and that we would need to postpone the meeting, as many individuals across the institution, including those in leadership, had lost power and/ or internet access. Most phones still had service, so the IACUC Office got to work contacting the Chair, Committee members, and other stakeholders to keep everyone apprised of the continually evolving situation. Campus classes and activities were canceled for that day. The closing would be extended two more times during the week, as campus facilities and residential neighborhoods struggled to keep pace with environmental and mechanical challenges with which they had little or no prior experience. These included power outages and water shortages during dangerously cold temperatures and flooding. Once campus re-opened, after being shut down for a week, the monthly IACUC meeting was held virtually, and semiannual activities were planned. Our OLAW

Assurance required that we conduct a semiannual program review and facility inspections at specific times during the year. The inspections were challenging, as many of the labs to be visited had suffered damage. In addition, rolling power outages had affected certain equipment, including refrigerators and freezers. All of this had to be considered during the onsite inspections and in the post-visit deliberations. As already stated, this event was a challenge even for those who were not on campus. Many homes and businesses throughout the area suffered structural and/or HVAC damages. All of this contributed to increasing strains on the time and focus available to devote to IACUC matters. People affected by this event were looking for someone to lead and act as a calming force amid the post-event storm. The IACUC and IACUC Office can provide that; indeed, it may be their most important function in times like these.

Three Lessons Learned

This is not an advice column, and I do not have earth-shattering insights or new ideas to share with you due to this experience. What I do have is a handful of lessons learned and some deceptively simple but important advice you can share with your institution. It consists of three main takeaways that I present here, not in sequential order but, perhaps, as three inter-related legs of a new kind of disaster management stool. First and foremost, be prepared. Benjamin Franklin famously said that "an ounce of prevention is worth a pound of cure." Again, many manuscripts have been written and presentations given on disaster preparedness from an animal care/ welfare perspective, and this is of the utmost importance. The IACUC itself should also prepare for unforeseen circumstances. In short, this involves putting your own house in order: make sure you are up on your protocol expiration dates, and know what your semiannual program review, OLAW Assurance, and (if applicable) AAALAC Program Description say. Don't make yourself a prisoner of your local policies; be aware of the flexibilities allowed (and encouraged!) by various oversight groups. The second item is one that we have all heard before: communicate well. The most important part of this is to make everyone feel like they are necessary. In other words, involve all possible stakeholders, keeping in touch as effectively and efficiently as possible, whether that be through email, voicemail, text, Zoom, or in-person discussions. The IACUC

members (inspectors, etc.) must feel supported during these trying times, and it is perhaps an IACUC administrator's most important job to make this happen. Indeed, quality communication is probably the most impactful navigational tool amid an emergency or disaster. The third and final aspect is perhaps the hardest to quantify, and that is empathy. It should be easy to see how even a small dose of empathy will go a long way to improving both preparation and communication. We can better know how to prepare and what to prepare for if we know the situation better. Approaching others in an empathetic way will lead to better sharing of meaningful information. This, in turn, will lead to better, more fruitful communication between parties, but it is neither quick nor easy. Building solid working relationships takes time and effort but investing in people is never time wasted. I am convinced it is the best way to come out stronger on the other side of a disaster, which should be the goal.

Conclusion

While I certainly do not wish misfortune on anyone, disasters and emergencies do and will continue to happen. I will close by saying good luck with your inevitable next incident. Hopefully, the above has helped, if only as a reminder that you are not alone. We are all in this together. Thomas E. Todd, DMV CPIA, is an IACUC Veterinarian in the Research Integrity and Oversight Office in the Division of Research at the University of Houston in Houston, TX.

January 2022 Laboratory Animal Science Professional 37

Opioid Analgesic For subcutaneous use in mice and rats only. CAUTION: Federal law restricts this drug to use by or on the order of a licensed veterinarian. LEGAL STATUS--In order to be legally marketed, a new animal drug intended for a minor species must be Approved, Conditionally Approved, or Indexed by the Food and Drug Administration. THIS PRODUCT IS INDEXED--MIF # 900-014. Extra-label use is prohibited. This product is not to be used in animals intended for use as food for humans or food-producing animals. WARNING: ABUSE POTENTIAL, LIFE-THREATENING RESPIRATORY DEPRESSION, and ACCIDENTAL EXPOSURE Abuse Potential Ethiqa XR contains buprenorphine, a high concentration (1.3 mg/mL) opioid agonist and Schedule III controlled substance with an abuse potential similar to other Schedule III opioids. The high concentration of Ethiqa XR may be a particular target for human abuse. Buprenorphine has opioid properties that in humans may lead to dependence of the morphine type. Abuse of buprenorphine may lead to low or moderate physical dependence or high psychological dependence. The risk of abuse by humans should be considered when storing, administering, and disposing of Ethiqa XR. Persons at increased risk for opioid abuse include those with a personal or family history of substance abuse (including drug or alcohol abuse or addiction) or mental illness (suicidal depression). Because of human safety risks, this drug should be used only with veterinary supervision. Do not dispense Ethiqa XR. Life-Threatening Respiratory Depression The concentration of buprenorphine in Ethiqa XR is 1.3 mg/mL. Respiratory depression, including fatal cases, may occur with abuse of Ethiqa XR. Ethiqa XR has additive CNS depressant effects when used with alcohol, other opioids, or illicit drugs that cause central nervous system depression. Because of the potential for adverse reactions associated with accidental injection, Ethiqa XR should only be administered by a veterinarian or laboratory staff trained in the handling of potent opioids. DESCRIPTION Ethiqa XR is an injectable suspension of extended-release buprenorphine. Buprenorphine hydrochloride, an opioid analgesic, is the active ingredient in Ethiqa XR. Lipid-bound buprenorphine hydrochloride is suspended in medium chain fatty acid triglyceride (MCT) oil. Lipids encapsulate the buprenorphine limiting diffusion which provides for larger doses and prolonged action.1,2 Ethiqa XR has a slightly yellow to white opaque appearance. Each mL contains approximately 1.3 mg buprenorphine hydrochloride. The sterile product contains cholesterol, glyceryl tristearate, and buprenorphine hydrochloride suspended in MCT oil. Buprenorphine Formula C29H41NO4

INDICATIONS Ethiqa XR is indicated for the control of post-procedural pain in mice and rats. MOUSE DOSAGE AND ADMINISTRATION Wear protective clothing when administering Ethiqa XR (see Human Safety Warnings). Shake the vial briefly before each use to ensure uniform suspension. If stored refrigerated, bring to room temperature before use. Use aseptic techniques to withdraw the dose into a disposable 0.5 or 1 mL syringe. A 20 to 23 gauge needle should be used for injections due to the viscosity of the drug suspension. The dosage of Ethiqa XR is a single subcutaneous injection of 0.05 mL per 20 gram mouse (3.25 mg/kg body weight). Therapeutic drug concentrations are maintained for 72 hours after the initial dose. If needed, a single repeat dose may be administered 72 hours after the initial dose. Secure the mouse in a scruff-of-the-neck hold. Insert the needle into the dorsal subcutaneous space created by the scruff hold. Inject the entire dose into the dorsal subcutaneous space. An oily sheen may be observed in the dorsal fur of the mouse after injection due to leakage of the oil-based drug suspension from the injection site. The oily sheen may last for 4 to 5 days postinjection. Leakage from the injection site can be minimized by slowly injecting Ethiqa XR into the subcutaneous space. The mouse can be returned to its cage immediately after receiving Ethiqa XR. Do not return any unused drug suspension from the syringe back into the vial. Once the vial is broached, Ethiqa XR can be stored at 15° to 25°C (59° – 77°F) or refrigerated for 28 days. DO NOT FREEZE. RAT DOSAGE AND ADMINISTRATION Wear protective clothing when administering Ethiqa XR (see Human Safety Warnings). Shake the vial briefly before each use to ensure uniform suspension. If stored refrigerated, bring to room temperature before use. Use aseptic techniques to withdraw the dose into a disposable 0.5 or 1 mL syringe. A 20 to 23 gauge needle should be used for injections due to the viscosity of the drug suspension. The dosage of Ethiqa XR is a single subcutaneous injection of 0.1 mL per 200 gram rat (0.65 mg/kg body weight). Therapeutic drug concentrations are maintained for 72 hours after the initial dose. If needed, a single repeat dose may be administered 72 hours after the initial dose. Secure the rat in a passive restraint tube or by holding with a heavy glove with one person to secure the rat and a second person to administer the drug. Insert the needle in the dorsal subcutaneous space. Inject the entire dose into the dorsal subcutaneous space. An oily sheen may be observed in the dorsal fur after injection due to leakage of the oil-based drug suspension from the injection site. The oily sheen may last for 4 to 5 days post-injection. Leakage from the injection site can be minimized by slowly injecting Ethiqa XR into the subcutaneous space. The rat can be returned to its cage immediately after receiving Ethiqa XR. See CONTRAINDICATIONS and Rat PRECAUTIONS for additional information on bedding. Do not return any unused drug suspension from the syringe back into the vial. Once the vial is broached, Ethiqa XR can be stored at 15° to 25°C (59° – 77°F) or refrigerated for 28 days. DO NOT FREEZE. CONTRAINDICATIONS Only administer Ethiqa XR by subcutaneous injection. Ethiqa XR is not intended for intravenous, intra-arterial, intrathecal, intramuscular, or intra-peritoneal injection. Do not use on mice or rats with pre-existing respiratory deficiencies. Do not keep rats on wood chip-type bedding after administration of Ethiqa XR. HUMAN SAFETY WARNINGS Not for use in humans. Keep out of the reach of children. Human User Safety while handling Ethiqa XR: Two trained staff for administration: Ethiqa XR should only be handled and administered by a veterinarian, veterinary technician, or laboratory staff trained in the handling of potent opioids. To prevent human adverse reactions or abuse, at least 2 trained administrators should be present during injection of Ethiqa XR. Protective covering: To prevent direct contact of Ethiqa XR with human skin or mucous membranes when handling the suspension, protective clothing is recommended. Mucous membrane or eye contact during administration: Direct contact of Ethiqa XR with the eyes, oral or other mucous membranes of humans could result in absorption of buprenorphine and the potential for adverse reactions. If accidental eye, oral or other mucous membrane contact is made during administration, flush the area with water and contact a physician. Skin contact during administration: If human skin is accidentally exposed to Ethiqa XR, wash the exposed area with soap and water and contact a physician. Accidental exposure could result in absorption of buprenorphine and the potential for adverse reactions. Drug Abuse, Addiction, and Diversion of Opioids: Controlled Substance: Ethiqa XR contains buprenorphine, a mu opioid partial agonist and Schedule III controlled substance with an abuse potential similar to other Schedule III opioids. Ethiqa XR can be abused and is subject to misuse, abuse, addiction,

and criminal diversion. Ethiqa XR should be handled appropriately to minimize the risk of diversion, including restriction of access, the use of accounting procedures, and proper disposal methods, as appropriate to the laboratory setting and as required by law. Abuse: Abuse of Ethiqa XR poses a hazard of overdose and death. This risk is increased with concurrent abuse of alcohol and other substances including other opioids and benzodiazepines. Buprenorphine has been diverted for non-medical use into illicit channels of distribution. All people handling opioids require careful monitoring for signs of abuse. Drug abuse is the intentional non-therapeutic use of a prescription drug for its rewarding psychological or physiological effects. Abuse of opioids can occur in the absence of true addiction. Storage and Discard: Ethiqa XR is a Class III opioid. Store in a locked, substantially constructed cabinet according to DEA and local controlled substance guidelines. Discard broached vials after 28 days. Any unused or expired vials must be destroyed by a DEA registered reverse distributor; for further information, call 1-833-384-4729. Physician information: Ethiqa XR injectable suspension is a mu-opioid partial agonist (1.3 mg buprenorphine/mL). In the case of an emergency, provide the physician with the package insert. Naloxone may not be effective in reversing respiratory depression produced by buprenorphine. The onset of naloxone effect may be delayed by 30 minutes or more. Doxapram hydrochloride has also been used as a respiratory stimulant. PRECAUTIONS Mice The safety of Ethiqa XR has not been evaluated in pregnant, lactating, neonatal, or immune-compromised mice. As with other opioids, buprenorphine may cause sedation, decreased blood pressure, decreased heart rate, decreased gastrointestinal mobility, and respiratory depression. Use caution with concomitant administration of Ethiqa XR with drugs that cause respiratory depression. The use of paper or soft bedding for up to 3 days following administration of Ethiqa XR should be considered. Normal mice may exhibit an obtunded response to stimuli up to 4 hours after receiving Ethiqa XR. Buprenorphine is excreted in the feces (see Clinical Pharmacology section below). Coprophagy may lead to ingestion of buprenorphine or its metabolites by mice treated with Ethiqa XR and untreated cage mates. Rats The safety of Ethiqa XR has not been evaluated in pregnant, lactating, neonatal, or immune-compromised rats. As with other opioids, buprenorphine may cause sedation, decreased blood pressure, decreased heart rate, decreased gastrointestinal mobility, and respiratory depression. Use caution with concomitant administration of Ethiqa XR with drugs that cause respiratory depression. Rats may exhibit signs of nausea including pica up to 3 days post-treatment. Rats should be maintained on paper or soft bedding to avoid ingestion of wood chip-type bedding after administration of Ethiqa XR. Pica involving wood chip-type bedding can be lethal in rats. Buprenorphine is excreted in the feces (see Clinical Pharmacology section below). Coprophagy may lead to ingestion of buprenorphine or its metabolites by rats treated with Ethiqa XR and untreated cage mates. ADVERSE REACTIONS Mice No adverse reactions were observed in 20 to 25 gram young adult male and female mice after a single subcutaneous injection of Ethiqa XR at a dose 5 times the indicated dose. Laboratory parameters evaluated in the study included hematology and clinical chemistry; histopathology was also performed. In a second study, adult male and female mice received Ethiqa XR subcutaneously at 5 times the indicated dose for three doses at four day intervals. A surgical procedure was performed on the study mice prior to receiving each of the three doses of Ethiqa XR. Mortality was seen in two male mice after the third surgical procedure and dose of Ethiqa XR (total dose of 49 mg buprenorphine/ kg body weight in 8 days). Weight loss has been observed in mice treated post-procedurally with Ethiqa XR. Rats Adverse reactions were evaluated in 180 to 200 gram young adult male and female rats after a single injection of Ethiqa XR. A surgical procedure was performed on the rats prior to administration of a single dose at the intended dose of 0.65 mg/kg or a single dose of 2, 6 or 10-fold excess dose. Adverse reactions also were evaluated in male and female rats administered 2, 6 and 10 times the intended dose for three doses at four day intervals. A surgical procedure was performed on the rats prior to administration of the first of three doses. Laboratory parameters evaluated in the study included hematology, clinical chemistry, urinalysis, histopathology, and bodyweight. Signs of nausea were observed at all dose levels within 24 hours of the dose. Signs included self-licking, self-gnawing and efforts to eat wood-chip bedding. Mortality was seen in 1 of 36 rats exposed to wood chip bedding. Necropsy revealed the stomach and esophagus were compacted with bedding, the bladder was abnormally distended and the urine contained blood. Mortality was seen in 3 of 222 rats treated with Ethiqa XR due to technical complications with serial bleeding of the jugular vein. For technical assistance, or to report an adverse drug reaction, please call Fidelis Pharmaceuticals LLC at 1-833-384-4729. For additional information about adverse drug experience reporting for animal drugs, contact FDA at 1-888-FDA-VETS or http:// www.fda.gov/AnimalVeterinary/SafetyHealth. CLINICAL PHARMACOLOGY3 Buprenorphine can act as an agonist and antagonist at different classes of opioid receptors. Agonism at the mu opioid receptor and, in some cases, antagonism at the kappa or delta opioid receptors are possible underlying mechanisms for the ceiling effect and bell-shaped dose-response curve of buprenorphine. Studies with knockout mice have shown that the antinociceptive effect of buprenorphine, which is mediated primarily by the mu opioid receptor, is attenuated by the ability of the drug to activate the opioid receptor like (ORL-1) receptor. The drug can be described as a ‘full’ and a ‘partial’ agonist at the same receptor depending on the specific assay. There appears to be no ceiling effect for analgesia, but there is a ceiling effect for respiratory depression. Pharmacokinetic studies with bolus injections of buprenorphine in mice and rats provide similar models. After bolus intravenous administration, plasma levels decline tri-exponentially. The drug is n-deakylated in the liver to norbuprenorphine (NBN), an active metabolite. Studies have shown that glucuronide metabolites of buprenorphine and NBN are also metabolically active, and can approximate or exceed the concentration of the parent drug. Un-metabolized drug excreted in the urine and feces one week after injection was 1.9 and 22.4% of the dose, respectively, and 92% of the dose was accounted for in one week.3 Mice Pharmacokinetic parameters of Ethiqa XR were studied in 6-8 week old male and female Balb/c mice following a single subcutaneous injection of 3.25 mg/kg bodyweight. Clinically significant blood levels were observed up to 72 hours after subcutaneous injection. Rats Pharmacokinetic parameters of Ethiqa XR were studied in 8 week old male and female Fischer rats following a single subcutaneous injection of 0.65 mg/kg bodyweight. Clinically significant blood levels were observed up to 72 hours after subcutaneous injection. HOW SUPPLIED Ethiqa XR is supplied in a multi-use glass vial containing 3.0 mL of injectable drug suspension. Ethiqa XR

3 mL vial

NDC 86084-100-30

U.S. Patent No. 8,461,173 STORAGE INFORMATION Store between 15° and 25°C (59° – 77°F) or refrigerated. DO NOT FREEZE. If stored refrigerated, bring to room temperature before use. Once broached, the multi-dose vial should be discarded after 28 days. REFERENCES 1. Mishra et al., Drug Delivery and Transl. Res, 2:238-253; 2012. 2. Bethune et al., The role of drug-lipid interactions on the disposition of liposome-formulated opioid analgesics in vitro and in vivo. Anesth Analg. 93(4):928-33; 2001. 3. Guarnieri et al., Lab Animal, 41(11): 337-343; 2012. Manufactured for: Fidelis Pharmaceuticals LLC CCIT Incubator 675 US Highway One, Suite B113 North Brunswick, NJ 08902 833-384-4729 www.EthiqaXR.com Fidelis Pharmaceuticals® and EthiqaXR® are registered trademarks of Fidelis Pharmaceuticals LLC, a Delaware Corporation. February 2020

Analgesia Made Easy

Get Up To 3 Days of Post-Procedural Pain Relief With 1 Pharmaceutical-Grade Dose. Ethiqa XR® is the first and only pharmaceutical grade extended-release buprenorphine that’s FDA-affirmed to control post-procedural pain for laboratory mice and rats up to 72 hours with just one injection.

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WARNING: ABUSE POTENTIAL, LIFE-THREATENING RESPIRATORY DEPRESSION, and ACCIDENTAL EXPOSURE Abuse Potential This formulation contains buprenorphine, a high-concentration (1.3 mg/mL) opioid agonist and Schedule III controlled substance with an abuse potential similar to other Schedule III opioids. The high concentration may be a particular target for human abuse. Buprenorphine has opioid properties that in humans may lead to dependence of the morphine type. Abuse of buprenorphine may lead to low or moderate physical dependence or high psychological dependence. The risk of abuse by humans should be considered when storing, administering, and disposing of Ethiqa XR. Persons at increased risk for opioid abuse include those with a personal or family history of substance abuse (including drug or alcohol abuse or addiction) or mental illness (suicidal depression). Because of human safety risks, this drug should be used only with veterinary supervision. Do not dispense Ethiqa XR. Life-Threatening Respiratory Depression The concentration of buprenorphine in Ethiqa XR is 1.3 mg/mL. Respiratory depression, including fatal cases, may occur with abuse of Ethiqa XR. Ethiqa XR has additive CNS depressant effects when used with alcohol, other opioids, or illicit drugs that cause central nervous system depression. Because of the potential for adverse reactions associated with accidental injection, Ethiqa XR should only be administered by a veterinarian or laboratory staff trained in the handling of potent opioids. Important Safety Information for Rats and Mice For Rats and Mice: Only administer Ethiqa XR by subcutaneous injection. Ethiqa XR is not intended for intravenous, intra-arterial, intrathecal, intramuscular, or intra-peritoneal injection. Do not use on mice or rats with pre-existing respiratory deficiencies. Do not keep rats on wood chip-type bedding after administration of Ethiqa XR. Use caution with concomitant administration of Ethiqa XR with drugs that cause respiratory depression. For Humans: Ethiqa XR should only be administered by a veterinarian or laboratory staff trained in the handling of potent opioids. Protective clothing is recommended to avoid direct contact with human skin or mucus membranes which could result in absorption of buprenorphine and adverse reactions. Not for use in humans. For more information, consult the Prescribing Information including the boxed warning located on the next page. ethiqaxr.com 833-EthiqaXR (833-384-4729) © 2021 Fidelis Pharmaceuticals, LLC February 2021 FID-ETH-024

Management/Career & Training

Developing Quality Workforce Training: Thoughtful Course Design By Cheryl Thompson, MAT and Kelly LaRue Brackett, PhD

n the pursuit of providing quality training, one cannot simply rely on textbooks, canned slides or whatever materials were handed down to you from the previous instructor to tell you what to teach. The goals and design of trainings must be thoughtfully developed and reassessed often. Surprisingly, the best place to begin, is at the end. Develop Learning Goals

What do you want your trainees to accomplish by the end of the course? You can probably respond to that question with a single idea, but when your goal is to design robust training, it will be helpful if you can break that idea into at least 3 concepts. Consulting with colleagues will help you to refine the initial idea, but it may be up to you to dissect the idea into its essential parts. For example, our job at The Jackson Laboratory is to teach a genetics course to our Animal Care staff. Our first step was to go to our management team to find out why this topic is of importance to the development of our workforce. After dozens of conversations, the main idea boiled down to the need for our staff to independently make appropriate decisions when choosing which mice to breed and to recognize when something about the breeding doesn’t fall within the expected phenotypes of the progeny. To achieve this functional goal, we broke the idea into three main topics: 1) expected probabilities of offspring in mouse litters 2) Punnett Squares and 3) inheritance of genetic material. The third piece is a pre-requisite to being able to achieve 1 and 2, which are directly related to the overall purpose of the course. To simply list out the topics as I have done above, is not something that is tangible or achievable for trainees. Topics

need to be turned into well-defined statements or learning goals. In education, one of the most widely used tools for developing achievable learning goals is Bloom’s Taxonomy. Unlike species taxonomy which is visualized as an inverted triangle, Bloom’s Revised Taxonomy is a six-leveled triangle with the easiest and simplest cognitive processes at the base (Remembering) and the most difficult and complex intellectual skills at the top (Creating)1 (Figure 1). This hierarchy helps instructors to identify the level of mastery trainees should achieve for each topic. The more practical application of this tool provides educators with lists of specific action verbs to attach to their goal statements, an approach that creates a common expectation for both the instructor and the trainee. It is best to avoid verbs like “know” or “understand”; those terms can be just as intangible as a list of topics. When trying to link action verbs to your goal statements, you may even find that your list of critical topics breaks down even further. For example, we can divide topic 3 into 3a) the division of genetic material during sperm and egg development (i.e., meiosis) and 3b) the nature of and relationship between DNA, genes, chromosomes and genomes. Continuing with 3a as the example, we then used Bloom’s Taxonomy to turn the topic into an actionable statement. We wrote goal statements within the Figure 1. Bloom’s Revised Taxonomy is a 6-leveled triangle with the easiest and simplest cognitive processes at the base (Remembering) and the most difficult and complex intellectual skills at the top (Creating). Image credit: Vanderbilt University Center for Teaching.

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bounds of four of the cognitive hierarchies to see which would match best with the overall functional goal for our workforce: 1. Remember: List the steps of meiosis. 2. Understand: Describe the biological process and outcomes of meiosis. 3. Apply: Sketch the movement of chromosomes during meiosis and division of genetic material in the gametes. 4. Analyze: Contrast the movement of chromosomes during meiosis and mitosis in the generation of genetic diversity. Knowing that this material is a pre-requisite for being able to model genetic crosses in Punnett Squares, establishing the connection between gamete development and the genetic inputs to Punnett Squares is vital. Therefore, the Apply level was chosen as the learning goal for this topic. Most often, I set the Apply or Understand cognitive hierarchies as expectation for a workforce audience, as everything that they learn should be useful within the context of their job duties. The lower-level learning goals are used as landmarks during the course to help build the expectation. The higher-level learning goals are briefly discussed in class, but they are considered beyond the satisfactory expectation.

Align Everything

Establishing learning goals for each of your critical topics provides the framework for designing the rest of your training (Figure 2). For each learning goal, take a single step back to think about how you want to assess achievement in that goal. Your choices in the cognitive hierarchy and specific verbs may easily translate into particular assessment styles or questions. Do your learning goals necessitate a written exam, practical exam, oral exam or projects? Once you’ve decided how you want to assess your students, then think about the essential practices that will prepare them for your criteria of success. What are the small, progressive steps that students need to be able to do in order to accomplish the big picture? Once you determine the practices, you can then decide what instruction you want to provide for them to be ready for practice. Can the background information be delivered via lectures, readings or videos? What materials should they be provided with? Will this be best achieved through in-person or online learning? This process is called Backward Design and for someone who has never done it before, it feels, well, backward! Even though the design is backwards, the actual course runs forward, with instruction preceding practice and sufficient practice occurring before assessment. For example, if our learning goal is “Sketch the movement of chromosomes during meiosis and division of genetic material in the gametes,” we are likely going to assess that on a written exam. The action verb chosen specifies that they need to be able to draw. In order to prepare them for drawing on the exam, we need to practice drawing in class. We demonstrate and provide supervised practice in class in the ways to visually depict chromosomes and their movement in dividing cells. In order to prac-

tice drawing, they first need to know about the structure of chromoLearning somes and the steps Outcomes and final outcomes of meiosis. We choose to provide this instruction through multiple platforms: lecture, textbook Assessment readings and videos. This exercise is not just for de novo trainLearning ings, it can also help Activities you critically evaluate your existing courses and identify all the unnecessary materials, instruction and practicContent es. Removing extraneous “rabbit holes” that do not align with your learning goals will help trainees to focus Figure 2. Course planning flow chart. on the most important concepts and practices in preparation for the ways they will be assessed.

Conclusion

For those of you that have children in primary and secondary school, these course design processes are exactly what their teachers are doing! Although, for teachers, learning goals are most often set by nation or state-wide curriculum. The beauty of workforce training is that we have freedom to decide what skills, practices and knowledge are critical to develop independent employees that produce quality work within each of our specific organizations. Cheryl Thompson, M.A.T. is a Teaching Specialist and Kelly LaRue Brackett, Ph.D. is a Scientific Advisor at The Jackson Laboratory in Bar Harbor, ME. Kelly can be reached at kelly.brackett@jax.org. REFERENCES 1. Armstrong P. [Internet]. 2010. Bloom’s taxonomy. Vanderbilt University Center for Teaching. [Cited 3 September 2021]. Available at: from https://cft.vanderbilt.edu/guides-sub-pages/blooms-taxonomy/ 2. Indiana University. [Internet]. Course planning with backward design. Indiana University eLearning design and Services. [Cited 03 September 2021]. Available at: https://canvas. ucdavis.edu/courses/34528/pages/course-planning-withbackward-design

January 2022 Laboratory Animal Science Professional 41

ENRICHMENT ITEMS

TOYS, TREATS, AND TIPS

Creative Enrichment for Nonhuman Primates By Alyssa Medina

roviding novel and creative enrichments for nonhuman primates improves their mental wellbeing and is thus an important aspect of all husbandry programs. These enrichments are often presented through variations in diet, toys, and environmental features. The challenge with any type of enrichment, whether food, toy, or other device, is that the novelty may diminish, and animals stop interacting with the item as intended. Encouraging nonhuman primates to engage with their enclosure and enrichment devices in new ways may extend the novelty of these items, thereby more efficiently sustaining the mental wellbeing of nonhuman primates. The implementation of creative techniques to reimagine these same enrichment foods and devices is of interest for the community of caregivers that work with these species. Entertainment Variables

I tend to focus on creating enrichment that has more than one variable of entertainment, considering those that are forageable, destructible, and/or visually appealing. This enrichment is provided to the macaques I work with, specifically Macaca mulatta and Macaca fascicularis. I often ask whether the prepared device or treat can provide olfactory or auditory stimulation to the macaques. For example, I originally requested to use paint rollers as a foraging device as they can be covered with seeds, and their ability to roll may motivate play. These paint rollers can be hung vertically or horizontally on the front of an enclosure. Small washers can be hung on the paint rollers, so that they make sounds similar to rattles when hit. The inside of the paint roller may also be stuffed with leafy greens! Their day-long use may provide opportunities for animals to exhibit grooming behaviors, which is especially helpful for high-stress animals. This variability and the cycling out of this toy at random intervals allows for multiple creative and interesting enrichment applications. Recycled paper (which is nontoxic and safe for ingestion) is another great tool to create novel enrichment. It can be covered in peanut butter or honey and laid flat in an enclosure or balled up and placed at the top of an enclosure. I have used craft paper to make “seed packets,” sealed with mashed banana or a paste of flour and water. Colored juice drink powder mixed into these pastes adds flavor and color, which increases interest. These seed packets are great for motivating animals to climb and make use of vertical spaces. Once torn open, seeds and 42 Laboratory Animal Science Professional January 2022

other treats may fall out. This increases the longevity of the experience, as animals will forage for the fallen seeds afterwards. Making origami shapes is another creative way to use paper as an enrichment device. I will often use the balloon or crane shapes to hide seeds or popcorn. Likewise, cardboard is another sturdy but reliable enrichment device. Cardboard tubes smeared and stuffed with treats and blocked by hay make for quick, easily distributable toys. These tubes are good for animals that like to spend time ripping paper and searching for their favorite treats. Fresh produce such as cucumber slices can block the holes of the tubes for a greater challenge. This same method can also be applied to reusable toys. An edible alternative for standard paper is rice paper, which works well to cover holes in enrichment devices and can be dyed with food coloring. Once soaked in water, the rice paper will become slightly sticky, adding texture and an extra challenge. NHPs that had received plain rice paper over enrichment devices, such as crumble feeders, were given a new experience upon coloring them purple (Figure 1)! They behaved as though they were receiving them for the first time. These simple changes can make all the difference in preventing monotony while keeping the comfort of familiarity. Food variety can also cultivate continued interest. Bringing in seasonal fruits and vegetables, as well as changing the kinds of seeds or legumes available, can maintain variability. Pineapples and mangoes are great to use as fresh produce or to incorporate into ice blocks or cubes. They can also be stuffed into Kongs® alongside mashed raspberries or blueberries for added color.

The presentation of produce, like peppers, can change too. “Pepper boats” can be made with slices of pepper smeared with peanut butter and topped with berries or crunchy walnuts (Figure 2). In addition, implementing small changes in enrichment for the nonhuman primate promotes an observably greater interest and excitement for otherwise familiar foods. The important thing to remember is that dried and fresh foods can be mixed around to create different experiences with familiar toys.

interacts with its surroundings. In my group, we continue to devise new experiences for the animals in our care. We consider enrichment to be a superb way for staff to strengthen human-animal bonds while providing ways to occupy time and interest and encourage natural behaviors.

Conclusion

All animal procedures and protocols were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

Enrichment is a critical aspect of nonhuman primate environments and should provide choice in how a primate

Figure 1. Purple dyed rice paper used to create enrichment treat rolls.

Alyssa Medina is an Enrichment Technician in the Gene Therapy Program at the University of Pennsylvania in Philadelphia, PA.

Figure 2. Red bell pepper boats are filled with peanut butter and fruit and or nuts.

Figure 3. Origami shape filled with seeds.

January 2022 Laboratory Animal Science Professional 43

ENRICHMENT ITEMS

TOYS, TREATS, AND TIPS

DIY: Ants on a Log By Kari Buchanan, RLATG

his traditional snack has a variety of options for NHP enrichment. From fruit to cereals, get creative with the “ants” and remember to consider different fillings for the celery log (Figures 1-2). All dietary items should be approved by your veterinarian for the intended species and individual before use.

Instructions

Figure 1. From left to right, celery sticks filled with hummus topped with almonds and sunflower seeds; hummus topped with black olives; hummus topped with cheese balls; peanut butter topped with raisins; cream cheese topped with blueberries; cream cheese topped with peas and corn.

1. Wash the celery sticks and cut each stick in half. 2. Add peanut butter into the concave part of each celery stick and spread. 3. Push raisins into the peanut butter to represent “ants.” Alternatives to peanut butter include: • Nut butters • Cream cheese • Hummus • Other dips or spreads Alternatives to raisins include: • Dried fruit cut into raisin-sized pieces • Peas, corn, olives • Berries • Seeds • Nuts • Goldfish, pretzels • Cereal pieces

Figure 2. Close up of celery sticks filled with hummus topped with almonds and sunflower seeds; hummus topped with black olives; hummus topped with cheese balls.

44 Laboratory Animal Science Professional January 2022

TECH TIPS

Insights in husbandry, enrichment, and new techniques and tactics

The Spiny Web: A Novel Restraint for Spiny Mice By Janna Draper, DVM and Glenn Jackson, DVM, PhD, DACLAM

piny mice, Acomys spp., are used in research for the unique ability to regenerate damaged tissue in addition to being a model for diet-induced type-2 diabetes, physiological adaptions to desert life, and late-gestational development. Spiny mice are members of the Muridae family and classified into the subfamily, Deomyinae. Despite their common name, spiny mice, are more closely related to Gerbillinae (gerbils) as they share a common ancestor.1,2,3 Their natural habitat includes semidry and arid regions of Africa, the Middle East, and Asia. Typical lifespan is 2 to 4 y although in captivity they can live up to 7 y.2,3 They are curious and friendly, but also agile and can excite quickly. Their most notable physical traits include their large spiny hairs along with their fragile skin. Acomys spp. have extremely weak skin that tears in response to handling or attack. Common skin wounds include bite wounds from cage mates and tail degloving (Figure 1).1,2,3 This ability makes handling and restraining Acomys spp. in the laboratory challenging. The purpose of this article is to describe a novel approach for the handling and restraint of Acomys spp. to facilitate examination and short, minor procedures.

Materials and Methods

Cornell University maintains a research colony of spiny mice, approximately 70 animals. They are housed in 24 in × 18 in × 16 in powder-coated galvanized steel cages with wire sides and lids, hamster cages, or in mouse cages depending on research status and cage mate numbers. For short examinations or transferring spiny mice to new cages, a small clear cup can be used. For short proce-

Figure 1. Post-mortem demonstration of tail slip on Acomys spp.

46 Laboratory Animal Science Professional January 2022

Figure 2. Spiny web restraint. (A) Cover dominant hand with hair net, (B) reach over and surround Spiny mouse, (C) close hand with hair net, and (D) gather up extra hair net material with non-dominant hand and make a fist with extra material in dominant hand.

dures such as subcutaneous injections, blood collection or microchipping, spiny mice are typically anesthetized using isoflurane. To decrease the risks from handling and avoid use of inhalant anesthesia during examination of clinical veterinary cases, we assessed the feasibility of different materials including towels, leather gloves, shoe covers, and hair nets to find a novel restraint device. Of these options, we found that the hair net was the ideal solution as it allowed simple low stress restraint, easy examination, and minor procedures. The development of this technique was done as part of routine veterinary care and was not part of any research protocol. The ideal method for utilizing the hair net is as follows; (A) cover dominant hand with hair net, (B) reach over and lower your hand with hairnet to surround the spiny mouse, (C) close the hand with the hair net, (D) gather up the extra hair net material with your non-dominant hand and make a fist with the extra material in dominant hand (Figure 2 A-D). The final restraint should look similar to a holding a bouquet with the extra hair net material acting as the “stems” of the bouquet and

Figure 3. Subcutaneous injection through spiny web (A) and hindlimb removed from spiny web for lateral saphenous blood collection (B).

the spiny mouse sitting on top of the hand acting as the “flower.” This restraint technique allows for an easy physical examination as the hair net material is relatively thin and easy to see through. Furthermore, appendages can be removed from the hair net either through the bottom of the hair net or by creating a small hole. Short procedures, including subcutaneous injection and blood collection can be easily performed with two people when using this restraint method. For subcutaneous injection, the needle can be passed directly through the hair net material. For blood collection, a hindlimb can be removed from the hairnet and a small amount of blood can be collected from the lateral saphenous (Figure 3 A-B).

Conclusion

Spiny mice are a unique species of rodent that are gaining popularity in the laboratory setting for multiple purposes thanks to their unique biology. Their spiny hair and weak skin make typical rodent restraint techniques of limited utility. In an attempt to refine handling and restraint, we investigated multiple materials and found a hair net was an ideal solution. The hair net provides a low cost, easy handling and disposable restraint device that can be used for examinations and procedures on spiny mice. This technique may be useful in restraint of other rodents that have fragile skin, such as Mus pahari.4

Janna Draper, DVM, is a third year Resident in the Laboratory Animal Medicine Residency at Cornell University, Ithaca, NY.

Glenn Jackson, DVM, PhD, DACLAM, is a Veterinarian at the Center for Animal Resources and Education at Cornell University and is the Director of Cornell University’s Laboratory Animal Medicine Residency, Ithaca, NY. REFERENCES 1. Gaire J, Varholick JA, Rana S, Sunshine MD, Doré S, Barbazuk WB, Fuller DD, Maden M, Simmons CS. 2021. Spiny mouse (Acomys spp.): an emerging research organism for regenerative medicine with applications beyond the skin. NPJ Regen Med 6:1-6 2. Haughton CL, Gawriluk TR, and Seifert AW. 2016. The biology and husbandry of the african spiny mouse (Acomys cahirinus) and the research uses of a laboratory colony. J Am Assoc Lab Anim Sci 55:9-17. 3. Pinheiro G, Prata DF, Araújo IM, Tiscornia G. 2018. The African spiny mouse (Acomys spp.) as an emerging model for development and regeneration. Lab Anim 52:565-576. 4. Pratt CH, Potter CS, Kuiper RV, Karst SY, Dadras SS, Roopenian DC, Sundberg JP. 2017. Skin fragility in the wild-derived, inbred mouse strain Mus pahari/Ei J. Exp Mol Pathol 102:128-132.

January 2022 Laboratory Animal Science Professional 47

TECH TIPS

Insights in husbandry, enrichment, and new techniques and tactics

Development and Implementation of a Positive Reinforcement Training Program for Nonhuman Primates in Biomedical Research By Selena M. Guerrero-Martin, Samuel Brill, Bess W. Carlson, Melanie L. Graham, Lydia M. Hopper, and Kelly Metcalf Pate

onhuman primate (NHP) models are essential to advance biomedical science due to their high translational value. The husbandry and research practices utilized in working with NHPs influence the validity and reliability of data obtained from research studies4, 5, 8 and the animals’ welfare.2, 7 Positive reinforcement training (PRT) is one refinement shown to alleviate stress associated with laboratory sampling, enhance the reliability of data collection, and increase safety of animal care personnel.6 PRT is based on principles of operant conditioning, an associative learning process used to modify frequency of a behavior through pairing it with a stimulus. Specifically, positive reinforcement increases likelihood of a behavior’s occurrence on a specified cue by pairing it with a reward.3 Despite clear benefits of PRT for NHPs and biomedical research, investigators often view the investment in personnel expertise and time required to train animals as deterrents towards adopting PRT. We provide an example of how this framework could be implemented with NHPs in research settings based on our own experience in building a new PRT program.

Key Concepts in PRT

Framework for Developing a PRT Program

Case Study: Voluntary Presentation for Subcutaneous Injections

The first steps towards developing a PRT program are to identify opportunities for animal model refinements that can be achieved using PRT and to define the program goal(s). Examples of goals include alleviating stress, providing cognitive enrichment, and/or teaching specific behaviors relevant to the research protocol. Once the goals of the training program are determined, a comprehensive shaping plan should be developed for goal behavior(s), defining sequential steps to train the intermediate and goal behaviors (see Figure 1). This plan should outline small steps towards the goal to increase the likelihood of animals having success each attempt, a definition of success for each step, and information for the trainers to provide consistency during the training process. For example, the plan should define the cues to signal for a behavior, the type of reinforcement used for each step, and precisely what behavior the animal must perform to receive reinforcement at each step. Formalizing a shaping plan ahead of time allows for easy tracking and evaluation of each animal’s progress, identification of any required modifications of the plan, and maintaining consistency across multiple team members. 48 Laboratory Animal Science Professional January 2022

When training an animal to perform a behavior the first step is to ensure that they associate preferred rewards with a bridge that acts as secondary reinforcer. Bridges include a clicker, whistle, or verbal cue given immediately after the animal performs the desired behavior. Different rewards should be available based on perceived value of reward. If possible, preference testing for each animals’ favorite treats can be conducted prior to initiation of training. Once the animal has learned that the bridge signals reward, shaping of desired behaviors can begin through a series of training sessions following the shaping plan. Generally, shorter sessions of 5 to 10 minutes, one to two times a day per animal have been shown to be most effective in fostering learning progression.1 Importantly, each behavior must have an associated vocal or visual cue that the trainer gives to ask for the behavior. Thus, the aim of training is not only to foster the desired behavior but that the animal should perform it primarily on cue. In this way, different behaviors can be associated with different cues allowing for a repertoire of behaviors to be trained and requested.

The initial goal for our research group was to utilize PRT to mitigate a common stress that occurs in our research, both to reduce stress as a potential confounding variable and enhance animal welfare. As subcutaneous injections are done daily for most animals on our studies, we chose to train for voluntary presentation for injection. We defined this goal behavior as a macaque pushing the side of its thigh against the front of the cage and remaining still during a needle poke and fluid injection. This behavior was broken down into six stages for the shaping plan (Figure 1). Socially housed rhesus and pigtail macaques were initially acclimated to the trainer and target trained to touch a specific object on cue (Figure 1A; Image 1), trained and acclimated to separate from their cage mate (Figure 1B), and desensitized to the movement of the squeeze back (Figure 1C). These habituation steps reduced stress and allowed animals to focus solely on the training interaction. Finally, animals were taught to present the side of their hip to the trainer (Figure 1D; Image 2) and to hold still for a needle stick (Figure 1E) followed by a fluid injection (Figure 1F).

Figure 1: Example Training Plan for Voluntary Subcutaneous Injections. The macaques were required to perform each step in the following series on the first cue 80% of the time (e.g., 4 out of 5 times in a row) to proceed through a subsequent stage. Each behavior (e.g., target, separate, side and hold) was given a specific associated verbal and hand cue and the animal was reinforced first with a verbal bridge and then with the reward (i.e., food reward) for performing it on command. Macaques were first acclimated to the presence of the trainers, and target trained to touch a specific item handing on the outside of the cage on cue (A; See Image 1). Macaques were then acclimated to separation from their cage mate (B) and to movement of the squeeze back cage, reducing the space available by 75% (C). This was done for the safety of the macaques, to limit the space they could jump back if frightened and any potential for physical injury as a result. The next step in the shaping plan was to train animals to turn sideways and lean their hip against the front of the bars (D; See Image 2). This behavior was captured by moving the target away from the front of the monkey and providing positive reinforcement as the animal turns sideways towards the new target position. At this point the behavior “hold” was introduced, to train animals to remain still in a particular position. Macaques were then acclimated to a needle stick, first by using a blunted needle and then moving to a sharp one (E). Finally, macaques were trained to hold for injections of sterile fluid subcutaneously (F). The number of sessions needed for each animal to complete each stage varied based on the number of other macaques in the animal room that had already undergone the training program, suggesting social learning aids in the rate of training progress; however, in general acclimation to the squeeze back and holding for the needle stick took the greatest number of sessions. We have completed this process with juvenile rhesus and pigtailed macaques, all socially housed, but the principles of the training plan development should extend to other species.

January 2022 Laboratory Animal Science Professional 49

Lessons Learned from Implementing a PRT Program

Figure 1A, Image 1: Example of “Target” Behavior.

Establishing a PRT program at a biomedical research institution requires patience and resilience on the part of the NHPs and the humans involved; however, there are resources available, and steps that can be taken, to help alleviate the uncertainty that can occur during the building phase. We highly recommend reaching out to colleagues who have previously developed PRT programs to benefit from their experiences, while also recognizing that the process and timeline will look different at each facility, across species, and study goals. We included general tips and tricks based on the lessons we have learned throughout this process in Table 1. We believe that the implementation of the behavioral training program in our research group has provided meaningful benefits. The level of physical effort needed to administer the daily injections to our macaques, as well as the emotional toll from restraining resistant NHPs, has decreased greatly for the people involved in the animal work. The time needed to complete the daily injections is also reduced significantly when working with trained compared to untrained animals. We have noted a calmer environment in our macaque rooms, with trained animals displaying more affiliative and less fear behavior towards humans. While the impacts of the training program on the scientific data generated from our NHP model are still being evaluated, the change in the quality of the interactions between humans and NHPs involved in the work have been significant enough for us to continue implementing the training program and encouraging others to consider similar refinements. All animal work was approved by the Johns Hopkins University Institutional Animal Care and Use Committee. Selena Guerrero-Martin, BS, is a PhD candidate at the Johns Hopkins School of Medicine in Baltimore, Maryland.

Samuel Brill, BS, is a DVM-PhD student at the Colorado State University in Fort Collins, Colorado. Bess Carlson, BS, is an independent contractor in Baltimore, Maryland.

Melanie Graham, MPH, PhD, is the Robert and Katherine Goodale Chair in Minimally Invasive Surgery, an Associate Professor of Surgery, the Director of the Preclinical Research Center and an Associate Professor of Veterinary Population Medicine at the University of Minnesota in St. Paul, Minnesota. Lydia M. Hopper, PhD, is the Director of Behavioral Management and an Associate Professor of Molecular and Comparative Pathobiology at Johns Hopkins School of Medicine in Baltimore, Maryland.

Kelly A. Metcalf Pate, DVM, PhD, is the Director of the Division of Comparative Medicine and the Dorothy W. Poitras Associate Professor of Biological Engineering at the Massachusetts Institute of Technology in Cambridge, Massachusetts. Figure 1D, Image 2: Example of “Side” and “Hold” Behavior.

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Tips and Tricks for Developing and Implementing a Behavioral Training Program Set realistic expectations for the development and implementation of the program (e.g., timeline for completion, types of behaviors to be initially trained, number of animals that can be trained). Choose a limited number of defined behaviors to start with that are high yield for meeting the goals of the program. Encourage investigators whose studies may benefit from behavioral training to include material and personnel support in grant applications. Introduce researchers to other researchers who have successfully implemented behavioral training programs at their institution and set up one-on-one meetings to discuss this work. Reach out to others who have successfully designed and implemented a behavioral training program at their institution for support. Recognize that these people and their macaques are more experienced with the process and do not be discouraged if your animals are not doing all the same behaviors from day one. If possible, recruit an experienced trainer to help start the program. Whether or not this is possible, review the basics of macaque behavior and the principles of operant conditioning with the team early in the process, and ensure that all team members understand and follow the shaping plan so that all trainers interacting with the animals are using the same cues. Start with a training team of two trainers, if possible. Two can encourage each other through the process, provide training feedback, and ensure that animals can receive regular training sessions throughout the week. Video recording of training sessions can also provide a way to evaluate the training process and ensure consistency across trainers. If possible, as animals are initially being trained, keep the same trainer-macaque pairs working together to prevent any slight differences in human training style from impacting the macaque's clarity and ability to learn. Once behaviors are solidly established, they should be reinforced with other trainers as well. Be patient with the process. The training plan will be completed more quickly as additional animals are put through the process. Do not start with macaques that are expected to be trained next week. Maintain comprehensive records of individual animals’ training progress and the version of the shaping plan used for each animal. This will help the training team evaluate their own progress as trainers and will allow for a better understanding of which parts of the shaping plan may need to be adjusted to increase the program's efficiency and success. Iteratively evaluate training progress to assess the effectiveness and efficiency of the training plan. Table 1. Tips and Tricks for Developing and Implementing a Behavioral Training Program

Glossary of Common Terminology Used in Behavioral Training Bridge

A signal that marks the instant in time when an animal successfully completes a desired behavior

Capture

A training technique in which a trainer waits for a behavior to happen and then rewards it when it does

Cue

An indicator to an animal to perform a specific behavior

Desensitization

Use of PRT to change an anxiety or fear behavior response to a neutral or relaxed response by progressively exposing an animal to an aversive stimulus

Negative Punishment

Removal of a reward item to decrease the occurrence of an undesirable behavior; akin to a “time-out” from positive reinforcement

Negative Reinforcement

Removal of a noxious stimulus immediately following a behavior to increase the likelihood that the behavior is performed when cued

Operant Conditioning

An associative learning process used to modify frequency of a behavior through pairing it with a stimulus

Positive Punishment

Application of negative stimulus to reduce the occurrence of an undesirable behavior; should only be used in behavior training to prevent imminent risk to humans or NHPs

Positive Reinforcement

Presentation of a reward item immediately following a behavior to increase the likelihood that the behavior is performed when cued

Primary Reinforcer

An item that serves as a reward to an animal due to the natural, biologic need of the animal for the item i.e., food, water

Secondary Reinforcer

A stimuli or object that has been conditioned to be reinforcing to an animal due to their association with a primary reinforcer i.e., bridge

Shaping

A process of reinforcing successively closer approximations of the target behavior to eventually achieve a desired behavior

Target Train

A behavior in which an animal is taught to touch a certain body part to an object or area of interest on cue

Table 2. Glossary of Common Terminology Used in Behavioral Training

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REFERENCES 1. Fernström AL, Fredlund H, Spångberg M, Westlund K. 2009. Positive reinforcement training in rhesus macaques-training progress as a result of training frequency. Am J Primatol 71(5):373-9. 2. Graham ML, Rieke EF, Mutch LA, Zolondek EK, Faig AW, Dufour TA, Munson JW, Kittredge JA, Schuurman HJ. 2012. Successful implementation of cooperative handling eliminates the need for restraint in a complex non-human primate disease model. J Med Primatol 41(2): 89-106. 3. Graham ML. 2017. Positive Reinforcement Training and Research, p 187-200. In: Schapiro SJ, editor. Handbook of Primate Behavioral Management. Boca Raton (FL): CRC Press. 4. Guerrero-Martin SM, Rubin LH, McGee KM, Shirk EN, Queen SE, Li M, Bullock B, Carlson BW, Adams RJ, Gama L, Graham DR, Zink C, Clements JE, Mankowski JL, Metcalf Pate KA. 2021. Psychosocial Stress Alters the Immune Response and Results in Higher Viral Load During Acute SIV Infection in a Pigtailed Macaque Model of HIV. J Infect Dis jiab252. Epub

ahead of print. 5. Hocum Stone L, Oppler SH, Nugent, JL, Gresch S, Hering BJ, Murtaugh MP, Hegstad-Davies RL, Ramachandran S, Graham ML. 2021. Serum cytokine profiles in healthy nonhuman primates are blunted by sedation and demonstrate sexual dimorphism as detected by a validated multiplex immunoassay. Sci Rep 11: 2340. 6. Laule GE, Bloomsmith MA, Schapiro SJ. 2003. The use of positive reinforcement training techniques to enhance the care, management, and welfare of primates in the laboratory. J Appl Anim Welf Sci 6(3): 163-173. 7. Prior H, Blunt H, Crossman L, McGuire A, Stow R, Sewell F. 2021. Refining Procedures within Regulatory Toxicology Studies: Improving Animal Welfare and Data. Animals 11(11): 3057. 8. Shirasaki, Y., Yoshioka, N., Kanazawa, K., Maekawa, T., Horikawa, T. and Hayashi, T. 2013. Effect of physical restraint on glucose tolerance in cynomolgus monkeys. J Med Primatol 42: 165-68.

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52 Laboratory Animal Science Professional January 2022

Illuminated Injection Cones

January 2022 Laboratory Animal Science Professional 53

TECH TIPS

Insights in husbandry, enrichment, and new techniques and tactics

Common Marmoset Cross-fostering: Tips for Success! Jennifer Haupt, DVM, DACVS-LA, DACLAM; Khia Thompson; Morgan Jamiel

ommon marmosets (Callithrix jacchus) are increasing in demand for nonhuman primate biomedical research studies. However, despite their increasing popularity, little is published about neonatal clinical assessment and care. Neonatal mortality in marmosets is variable and can be as high as 62% in primiparous mothers. Mortality is also impacted by litter size, with triplet litters having only 40% of infants born surviving to weaning. Increasing infant survival is essential for colony expansion and is vital for developing transgenic marmosets born via assisted reproductive technology. Options for managing large litters or valuable infants needing additional support include rotational hand-rearing, full nursery rearing, or fostering to a lactating dam. Rotational rearing is where one infant per day may be removed from the group, and formula-fed then returned to the group at the end of the day. Full nursery rearing involves complete feeding with formula, with the infant being maintained in an incubator immediately adjacent to its home cage. The goal is to return the infant to a social group by three months of age. Fostering is recommended if the infants are in good condition and there is another dam in the colony that is nursing one infant and has given birth within three weeks. Fostering is a preferred option for many primates to reduce the occurrence of stereotypical and abnormal behaviors often seen with nursery-reared animals. There are very few descriptions about fostering success in the common marmoset. The only citation about success rates is a Figure 2. Successful acceptance of foster infant.

paper from 1979 from Hearn et al. that described only a 33% success rate (2/6 attempts) without explaining the techniques or evaluating potential reasons for success or failure. At MIT, we maintain a breeding colony to primarily support the development of transgenic marmoset models. Approximately 75-100 infants are born from natural mating, artificial insemination, and embryo transfer each year. We currently support infants with rotational feeding and fostering to promote infant survival if deemed needed. This project aimed to assess our program's success in fostering marmoset infants to promote infant survival.

Methods

Figure 1. Infant "cuddle" method to allow foster infant gain scent of the foster family.

54 Laboratory Animal Science Professional January 2022

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

MARMOSET INFANT FOSTER ASSESSMENT AND PROTOCOL Date:_________________ Infant ID:_____________________ Date of Birth: _____________________ Infant Sex: � Male � Female

Genetics: � WT � TG

Foster Dam: ________________ Lactating: � Yes � No

Number of animals in cage: ________ Number of nursing infants: _____________Infant age(s): ___________ 1. Reason for foster attempt: � Lack of nursing by dam (no weight gain) � Rejected/found dropped � Cesarean section � Other: ________________________________ 2. Assess for wounds prior to attempting foster. 3. Body weight: ___________ grams. Note: infants <24 grams have poor prognosis for survival. 4. Make sure the infant is stable before attempting foster. Provide supplemental feeding(s) and potentially SQ fluids before attempting foster. 5. Place infant in warmed incubator and attempt to have infant gain scent of foster family- options: • Bathe infant in warm, dilute betadine solution. Allow to completely dry. • Wrap infant in dirty hammock or other cage item from the foster group home cage. • Allow infant to stay with other infant from foster family home cage for at least 30 minutes. 6. To minimize stress to foster family, it is recommended to separate out juveniles/young adults from the parents for introduction. Gradually introduce infants including the foster to the family by place foster infant in the nest box and allow family to take foster infant before giving back other infants. • Note: Place a divider and dog bed in the cage to help protect the infant in case of a fall. Time for family to pick up infant: _____________ Family member picking up infant: ___________________ 7. Minimize the number of people observing the foster attempt as this could stress out the foster family. 8. Make regular assessments of the foster group – infant dynamic (every 30 minutes): Date/Time

Family Member Carrying Infant

Behavior (nursing, cleaning, aggression, vocalizations)

9. Re-assessment of the infant is dictated by the nursing status of the foster group and the health of the infant at the time of foster. It is advisable to re-weigh the infant the morning following fostering to ensure the infant is gaining weight. 10. Failed foster attempts are noted by aggression toward an infant or dropping of the infant. At this point, the infant should be euthanized. Final Outcome: � Successful � Euthanized – Reason: _____________________________ � Found Dead Signature and date: _________________________________ _______/____/______

Figure 3. Marmoset infant assessment and foster protocol form for evaluating infants, methods for introduction to foster family, and monitoring after foster attempt.

of introduction to the family (bathing, incubator with other infants), and success/failure rates (survival to weaning, notes for the reason of failure). Our protocol for introducing infant fosters was developed using tips and tricks from other marmoset colonies. Infants are stabilized before the foster attempt, including supplemental feedings and subcutaneous fluids if indicated. Attempts are made to have the infant gain scent of the foster family, including wrapping the infant in a soiled cage item from the foster group home cage or allowing the infant to stay in the incubator with the foster family's infant(s), also known as the "cuddle method" (Figure 1). For introducing the infant to the foster family, any method to minimize stress is encouraged. This may include separating juveniles or young adults from the parents for introduction, followed by a gradual introduction of the infants. To reduce injury if the infants are dropped, a cage divider and dog bed (or other soft pad) is placed in the cage to help protect the infant in case of a fall. Regular observation of the foster groups is performed to ensure the infants are being carried by the foster family (Figure 2). Re-assessment of the infant is advisable by re-weighing the infant the morning following fostering to ensure the infant is gaining weight. Data was collected using our “Marmoset Infant Foster Assessment and Protocol” form (Figure 3) in addition to medical records. Chi-square analysis and Fisher Exact Test were used to evaluate differences.

Overall Infant Survival

Twenty-one foster attempts involving 19 infants were evaluated in this study. 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 (60-90 days).

Reason for Foster Impacted Success Rates and Infant Survival The reason for foster attempt was significantly different between successful and failed foster attempts, with all failures occurring in infants born via cesarean section (- = 0.05). All infants fostered because of poor weight gain or considered high value (potential transgenic from assisted reproductive technology or born from a genetically modified parent) survived to weaning.

Infant Age Influences Foster Success and Survival

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 (juveniles or subadults) in the foster cage, number, or age of nursing infants with foster dam and introduction method on infant survival or foster success.

Recommendations

Overall, foster success was higher in our colony of marmosets than previously reported and should be considered as a viable method to increase infant survival. These results reveal that infant age was the most critical factor related to foster success, rather than foster dam experience and age of other nursing infants. Based on these findings, infants born via cesarean section may benefit from being nursery reared for at least 24 hours before foster attempt to increase the rate of success beyond what was reported in this study. Acknowledgments: Our entire marmoset team supported this project as part of our ongoing improvement for increasing the care and welfare of common marmosets in our care. Animal work was performed at the Massachusetts Institute of Technology under an approved IACUC protocol. Jennifer Haupt, DVM, DACVS-LA, DACLAM is the Chief of Surgical Resources at the Massachusetts Institute of Technology Division of Comparative Medicine.

Khia Thompson is the Assistant Supervisor of Large Animal Technical Services at MIT DCM. Morgan Jamiel was the Supervisor of Large Animal Technical Resources at MIT DCM at the time of this study and is currently at In Vivo Project Supervisor at Satellite Bio.

January 2022 Laboratory Animal Science Professional 55

TECH TIPS

Insights in husbandry, enrichment, and new techniques and tactics

Management of a Water Outage in a Large Rodent Facility By Cynthia Lockworth, DVM, DACLAM; Andrew T. Pesek, Jr., BS, RLATG, CMAR

ood, water, and clean cages make up the 3-legged stool of rodent husbandry, as all are essential. Recently, we were faced with a significant dilemma that would undermine this balance, as our facility was faced with an unavoidable water outage that would affect thousands of cages that were dependent on an automated watering system. A Leaking Drinking System

Several years ago, our North Campus animal facility at MD Anderson Cancer Center moved into a new vivarium, replete with an automated watering system, new equipment, and all that is needed for a stateof-the-art facility. Unexpectedly, the water lines associated with our animal drinking system began to leak. Leaking occurred so often that sections of pipe required frequent replacement in the interstitial space. This occurred because, at the time that this new facility was constructed, the piping joints were connected using a new process that made placement more efficient, but the longterm results were failures along the line that occurred continuously and with increasing frequency. Finally, we were informed that our entire rodent drinking water supply line needed to be replaced, a process that would culminate in at least 4 days in which no water would be delivered to our entire North Campus rodent colony. This was a formidable task, as we had to find an alternative means to supply water to nearly 20,000 cages of mice. We developed a management plan for the water replacement to include water replacement selection, roll-out logistics, personnel roles and duties during the replacement period, personnel training, research community communication, rodent health monitoring, and restoration upon completion.

Figure 1 A-F. Day 1; gel application (A). Day 1; exploration of gel (B). Day 1; acclimation to gel (C). Day 2; gel pouch openings filled with bedding and minimal loss of gel (D). Day 3; debris covers and fills the pouches making visual assessment difficult. There is an average of 30% overall reduction in gel (E). Day 4; increased debris and inability to visualize gel with an average of 60% reduction in gel (F).

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exchanges or entry of bedding material into consideration. Thus, we determined that this should be examined further. We performed a trial on the 2 gel options with different strains to assess use and consumption: CD-1, C57BL/6, and nudes. During this trial, we also assessed the gel application process, consumption of gels by the mice, duration of gels in the cage environment, and the appearance of gels associated with bedding material. Figure 2. Gels were cut with a large ‘X’ followed by the removal of a triangular section prior to placement into cages.

Figure 3. Personnel trained in preparation for the water outage.

Identifying Options

We considered several water replacements options. Prefilled water bottles would not work, as we only had ~6700 bottles and sipper tubes on hand and would need to purchase an additional 13,000; moreover, there were associated problems including labor, storage, and refills, if needed. We considered prefilled carboys, but these were not practical as they are highly labor intensive, and refilling would need to be performed at another campus. Water filled pouches were considered as they would involve minimal labor for placement and would be easy to monitor; however, we would need to purchase disposable valves with the prefilled pouches, and our cages would also require adaptors at great expense to the institution. Lastly, we considered gels. Gels are 98% water, come in 8 oz pouches at less than $3 per pouch, and have been effectively used by small facilities on a small scale on an emergency basis. They have also been proven in rodent transport and for hydration support for years. Initial concerns with gels included the labor-intensive process of gel application which would require every cage to be opened, and, of course, health monitoring for signs of dehydration. We compared 2 gel products. Each contained 220 g of gel. Assuming that each mouse consumes approximately 4-7 g per day, and a full cage of 5 mice consumes 35 g per day, theoretically a gel pouch could last for 6.5 d. However, this does not take desiccation from air

Gel Performance

We found that both gels performed well (Figure 1). All 3 strains consumed the gels adequately. Within 24 h of gel application, bedding covered the open surface of the pouches; however, this did not hinder mouse access to gels. We noted that there was an acceleration of gel loss over time, likely due to acclimation by the mice and desiccation from our corncob bedding. We noted that the great amount of debris that accumulated in the pouches would make visualization difficult, so there would need to be an even greater emphasis on good health checks and monitoring by personnel. The gel lasted through day 5; therefore, we planned to change out gel pouches on day 4, should the outage be extended. Lastly, we determined that a simple “X” cut of the gels was not adequate for smaller mice access to the gel, so we removed a triangular portion to ensure ease of access (Figure 2). We selected the gel that was most firm in texture, rectangular in shape, and was easiest to cut and handle, as this would be important to technicians cutting the gels over many hours. To confirm that we could deploy all gels on day 1, we performed a timed trial. From this, we determined that it took 26 sec per cage for 1 person, and 14 sec per cage with 2 people. Thus, two person teams would be most efficient for gel deployment. Moreover, based upon this trial, we could place all gels in a 6–8-h period.

Communication is Key

Communication with the research community is pivotal to the success of such a large endeavor. We shared important information involving the water outage using multiple formats including email, hallway monitors and flyers throughout the facility. In preparation for the outage, training for our personnel included gel preparation, box cutter use, gel application, gel visualization, rodent health monitoring, management plan for hydration concerns, change-out plans, and miscellaneous associated tasks (Figure 3). We also ensured that all personnel practiced cutting gels and received cut resistant gloves recommended by EH&S. Additionally, we provided our personnel with a hydration algorithm which gave them a visual outline of the manJanuary 2022 Laboratory Animal Science Professional 57

Figure 4. Technician Hydration Algorithm: Our final management plan for technician-identified dehydration was provided to technicians in an algorithm, cage-side, to assist with the steps needed if/when they were to identify dehydrated mice.

Establishing the Rollout Plan

Figure 5. Carts with gels were pre-stated allowing work to begin immediately Monday morning.

Figure 6. Teams of two personnel working together to prepare gels and place them into cages. Teamwork!

agement plan steps for technician identified dehydration (Figure 4).

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Paradoxically, treatment for dehydration was a quandary, as we typically treat dehydration with gels; therefore, we settled on the use of 76A gel cups which provide both hydration and nutrition support. Our rollout plan for the outage consisted of 3 phases. Pre-outage, we increased cage change-outs and staged equipment and materials (Figure 5). On Monday of the outage, the water was turned off and all gels were deployed by personnel into rodent cages (Figure 6). On Tuesday through Thursday, technicians focused on health checks as their primary task, with spot changes later in the day as needed. On Friday morning, water was restored. Technicians reconnected recoil hoses to racks and toggled valves to resume normal water flow into the watering valves. During the post-outage week, routine change-outs resumed, at which time, gels were removed, and water was tested. Upon restoration of the automated water supply, we determined that it was necessary to assess our overall management of this event in order to apply lessons learned to future events of this magnitude and scope. To evaluate our process, we collected data on dehydration cases, treatments, deaths, gel pouches applied and replaced, and water bottles used. Health technicians were interviewed for feedback. Husbandry personnel and the research community were administered an anonymous survey. Lastly, cost and labor analyses were performed.

Figure 7. Data collected from facility management software to examine death trends prior to and after the outage demonstrated no appreciable increase during the outage. Death rates were comparable or lower than our sister campus rates.

Figure 8. Total numbers of dystocia cases were low on all weeks evaluated. There was a slight increase during the week of the water outage, however, the same increase was noted at our sister campus. This suggests that there was not an increase in dystocia cases related to the outage.

To assess dehydration cases, we quantified the number of 76A gel and water bottle applications that occurred and determined that 3% of cages were provided with supplementation. We surmised that while some cases of dehydration were likely, some instances of hydration treatments were administered out of precaution or as a result of researcher request.

and supplies were 2% and 0.2%, respectively. We also performed a labor analysis, noting that we had 36 personnel actively on the floor during the day of gel application, and a total of 67.7 h of overtime, primarily incurred during the preparation process.

Evaluating the Data

Data was collected from facility management software to look for death trends prior and after the outage and compared with our sister facility that did not undergo a water outage. There appeared to be no appreciable increase in deaths during the outage (Figure 7). Similarly, dystocia was evaluated as a sign of stress but there were no notable increases in cases during the outage (Figure 8). Anonymous questionnaire results from our personnel suggested that they felt prepared for the outage, were comfortable with their dehydration assessment skills, and felt safe during the process. Anonymous questionnaires to the research community indicated that they were well informed of the outage. Responses indicated that 22% felt that their research was significantly impacted by the outage, whereas 78% felt it was not affected. Responses suggested that 7% felt that the health of their animals was impacted, while 93% felt that it was not. There were also comments expressing concerns that weight loss had occurred in some nude mice. We performed a cost and labor analysis. We determined that the majority of cost was a result of gels, as gels comprised 97.8% of total expenses, whereas labor

Conclusion

In the future, if we were to have a similar event requiring water replacement, we would consider preplacement of bottles on select active breeders, weanlings, and nude mice, as these populations were most likely to be noted as needing additional hydration support. We may also consider a non-experimental nude mouse trial in advance to assess for difference in hydration via water bottles versus gels to validate potential concerns with this strain. Our overall impression, supported by our analyses, was that the process went exceptionally well. We prepared extensively, our teams worked well together, and execution ran as smoothly as possible. The use of gels has now been demonstrated to be effective on a large scale and should be considered as an option for use in any size facility to support a water outage or as part of a disaster plan. Cynthia Lockworth, DVM, DACLAM, is an Associate Professor and Section Chief in the Department of Veterinary Medicine & Surgery at MD Anderson Cancer Center in Houston, TX. Andrew T. Pesek, Jr., BS, RLATG, CMAR, is the Associate Director of the Animal Facility in the Department of Veterinary Medicine & Surgery at MD Anderson Cancer Center in Houston, TX.

January 2022 Laboratory Animal Science Professional 59

TECH TIPS

Insights in husbandry, enrichment, and new techniques and tactics

Confirming Sexual Maturity in Male Cynomolgus Macaques by Urinary Spermatozoa Microscopic Examination By Christyann Rosales, BS, RALAT; Courtney Sands, DVM

ynomolgus macaques (Macaca fascicularis) are used widely in multiple aspects of research including the development and testing of biological compounds for therapeutic uses. In order to evaluate safety thoroughly, these medications often require studies performed in sexually mature animals. At this time, there are few options available to confirm sexual maturity in male macaques. Electroejaculation and collection of seminal fluid are invasive and require either anesthesia or prolonged acclimation. Testicular volume is, at best, a surrogate and does not reliably confirm that the animal is sexually mature and producing sperm. It was hypothesized that spermatozoa could be found in a urine sample post ejaculation, thereby verifying sexual maturity.

Materials and Methods

Materials used to collect and examine the urine sample were: • 3 ml syringe • Microscope • Microscope slide • Cover slip • Kimwipes • Tube rack

Figure 2. Placing urine sample on slide.

Figure 1. Samples were collected via 3ml syringe and identified under a microscope.

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The process of confirming the presence of sperm in urine requires few materials and showed to be time sensitive for veterinary technicians. Urine sample collection is performed at the beginning of the day before husbandry staff members wash the animals' cages. The veterinary technician collects the urine sample from an easy to obtain, flat surfaced cage pan using a 3mL syringe (Figure 1). When collecting samples for multiple animals, staff can place samples in a tube rack to hold them in a vertical position promoting any sperm present to concentrate at the tip of the syringe. After urine samples from all animals have been collected, they are inspected microscopically. A few drops of the collected urine sample are placed on a microscope slide which is covered with a cover slip (Figure 2). Excess urine is wiped off the slide with kimwipe and the sample is

the primary collection method to confirm sexually mature males. On average within an 8-month period of time, we found that only 13% of males were being confirmed by finding seminal fluid in the pan versus 87% of the animals being confirmed by collecting a urine sample (Figure 4). The highest number of animals that were found within one week increased from 9 to 12 animals, all found from a urine sample, with a total of 15 animals being monitored each week. Spermatozoa was identified by both urine and seminal fluid on each animal but urine was found most of the time when checking the pan. As a result, collecting a urine sample (preferably fresh) in lieu of ejaculate via electroejaculation refines the process, confirms sexually maturity in animals at a faster pace, reduces contact time between veterinary technician and animal, positively impacts animal welfare by reducing potential stress, while ensuring that we provide a viable animal model for critical research studies. Figure 3. Spermatozoa present in urine samples.

Figure 4. Urine vs seminal confirmations by week. A total of 15 animals were checked each week and sperm was identified by either urine or seminal fluid depending on which sample technician would find in cage pan. Once sperm was identified by either seminal fluid or sperm animal was confirmed. New animals were added each week to be able to check 15 each week.

evaluated using a microscope at CP-ACHROMAT 40x//0,65 (Figure 3). This process is repeated for each sample collected. If no sample is provided in the early hours, the veterinary technician can attempt another sample collection at the end of the day.

Results

Prior to validating a technique for confirming sexual maturation in male cynomolgus macaques via urine spermatozoa microscopic examination, veterinary technicians were relying on the presence of ejaculate in the animal’s cage, which proved to be an inconsistent and time-consuming process. If ejaculate was not present in the morning, veterinary technicians would return to the animal’s cage at a later point in the day in hopes that an ejaculate sample was available. Even with repeated checks, veterinary technicians, on average, only collected 1-2 ejaculate samples a month. Once the urine collection process for confirmation was proven to work, collecting urine, became

Conclusion

In conclusion, we found that confirming sexual maturity in male Cynomolgus macaques via the presence of spermatozoa in urine, improved time efficiency and the overall sexual maturity confirmation rate. The findings from using this novel technique refined the process of confirming an animal to be sexually mature through visualization of sperm in seminal fluid. Seminal fluid was not always present or visible in the cage pan while a urine sample could be collected daily. Collecting urine indirectly from an animal that is willingly able to provide a sample made this process much more efficient for the technician and less invasive for the animal, thus reducing potential stress. While not suitable for quantitative or qualitative analysis, urine collection is an efficient, noninvasive screening tool to assist in the determination of sexual maturity. This new method improved the procedure significantly and accelerated findings by more than 70%, providing an ideal animal model for crucial animal research studies at a faster pace. We confirmed more animals in less time enhancing efficiency throughout various departments but most importantly refining a method that successfully improves animal welfare. This refinement project was performed on an IACUC approved training protocol. Acknowledgments: Special thanks to the help and support of Charles River Houston veterinary team. Thank you to Dr. Abraham Robinson and Dr. Courtney Sands for providing mentorship. Thank you to Dr. Courtney Sands for introducing me to the AALAS National Meeting and encouraging me to prepare a poster presentation and then turning it into this article. Finally, I’d like to thank Roberto Ramirez for finding the most sperm! Christyann Rosales, BS, RALAT, is a Veterinary Technician at Charles River Laboratories, Houston, TX.

Courtney Sands, DVM, is a Clinical Veterinarian at University of Houston, Houston, TX.

January 2022 Laboratory Animal Science Professional 61

TECH TIPS

Insights in husbandry, enrichment, and new techniques and tactics

Interpreting Macaque Facial Expressions and Body Behavior in Preparation for Social Housing By Stephanie Tomeczko, LAT

ocial housing non-human primates is considered the default housing in a biomedical research setting and is essential for their well-being. Non-human primates that have an established bond have decreased stress and increased species specific behaviors. Many biomedical research institutions house non-human primate housing in indoor cages allowing for one to two compatible partners. This article will help those who are new to working with non-human primates. Interpreting macaque facial expressions and body behavior is important for beginning the process of pair housing two animals. Rhesus macaque (Macaca mulatta) and cynomolgus macaque (Macaca fascicularis) have many facial expressions and body postures, and each one has a specific meaning. These expressions and postures can be categorized into different temperaments which are useful for future social pairings. There are 4 main categories that allow us to classify temperament for individuals, which are neutral, affiliative, dominant (agonistic), and submissive (fearful). To determine an animal’s temperament, we look at facial expression, including the position of ears, eyes, eyebrows, mouth, and body posture. These are the most common and easily observable expressions and postures. The first category that an animal can be classified is neutral (Figure 1). An animal is considered neutral when the ears, eyes, and eyebrows are relaxed, and the mouth is closed. The neutral body position is observed when an animal is sitting or lying down. An animal that shows neutral traits can usually be paired with either a dominant or submissive temperament animal. Problems do not usually occur when pairing a neutral animal. The second category we look for is affiliative (Figure 2). When observing the face, the main expression seen is lip-smacking. This is rapid movement of the lips paired with making a smacking sound. Affiliative body posture includes presenting the hindquarters to another individual in the room. If an animal is sitting towards the front and facing forward in its home cage, the animal is likely affiliative. Affiliative qualities in an animal are a good sign that this animal will successfully pair. This animal is properly displaying all signs of understanding a hierarchy in a social setting.

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Figure 1. Neutral cynomolgus macaque male with relaxed facial expression and body posture.

Figure 2. Affiliative rhesus macaque female presenting hindquarters while standing at the front of the cage.

The third temperament category is dominant (agonistic) (Figure 3). The main facial expression displayed is the open mouth threat. With this display, the animal’s eyes are focused straight forward. They do not avert the stare. The mouth is wide open baring the canines, and the eyebrows and ears are directed forward. An open mouth threat can also be seen combined with the animal charging towards the front of the cage. The body appears tense and alert. When pairing a highly dominant animal, it is best to select a submissive partner. Sometimes an affiliative temperament will also be successful. Pairing 2 animals that have a clear dominant/submissive hierarchy ensure that the 2 temperaments do not conflict. The last temperament category is submissive (fear) (Figure 4). Fear grimacing is an easily observed facial expression. When a macaque fear grins, the animal pulls their lips far back over their teeth showing gums and teeth. Yawning can also be viewed as a stress response. A stress yawn can be confused with a tired yawn. These two behaviors can be differentiated based on context. Things to notice are high room activity, loud noises, or new people in the room. A common posture in submissive animals is staying as far back in the cage as possible. A submissive animal feels the safest in the darkest and Figure 4. Submissive cynomolgus macaque female fear grinning while staying at the farthest part of the cage.

farthest corner of a cage allowing them to hide, thus avoiding visual interaction with other animals in the room. The behavior and well-being of a submissive animal should be monitored closely during the initial stages of pairing. While there is a clear hierarchy, a submissive animal could be overly submissive or fearful towards their more dominant partner. As a result, submissive animals may lose body weight during the pairing process due to stress-induced reduction in appetite. Body weight should be monitored throughout the pairing process. If changes in weight occur, the pair should be re-evaluated. These facial expressions and body postures along with other indicators (age, sex, body size) can help determine if 2 animals may be compatible for social housing. The temperament categories mentioned above are the ones that our facility has found to be the most observed. They are also the quickest and easiest to learn to recognize, especially for employees who do not have a lot of experience working with macaques. For more information regarding macaque behavior, please reference https://www.nc3rs.org.uk/macaques/ macaques/behaviour-and-communication

Figure 3. Dominant cynomolgus macaque male not breaking eye contact with an open mouth threat and stiff body posture.

Stephanie Tomeczko, LAT, is an Animal Behaviorist at the University of Illinois-Chicago in Chicago, IL.

January 2022 Laboratory Animal Science Professional 63

TECH TIPS

Insights in husbandry, enrichment, and new techniques and tactics

Refining Enrichment Toy Interactions in Common Marmosets through Usage Assessment Tamara Aird, BA, MA, and Peter J. Pierre, PhD

nimals in captivity benefit from enrichment toys that facilitate species-typical behavior. For example, exudate feeders mimic natural behavior as wild common marmosets often spend up to half of total foraging time gouging trees for gum and sap.5 Apart from species-typical foraging behavior, a large body of the literature addresses enriching captive marmoset environments through structural and housing adaptations to promote activity and exploration.6,8,10 These studies simulate vertical elements and climbing substrates in the captive environment and are often composed of gnawable wooden elements.1,4 However, relatively few studies address the extent to which marmosets engage with manipulable enrichment toys intended to invite curiosity and sustain interaction. 3,9 To bridge this gap in the literature, we evaluated the time budgets of novel and familiar toys by opportunistically sampling pairs of marmosets in our colony. We offered movable and stationary

toys, as well as toys with or without a food element, to examine how frequently the animals interacted with the toys (Figure 1).

Methods

Forty common marmosets (Callithrix jacchus) ranging in age from 2 to 10 years participated in this study. All animals were housed in mixed sex pairs. Housing enclosures had nest boxes and extended vertical elements that allowed for the placement of wooden dowels simulating “natural” branch substrates that encourage climbing and natural gouging behaviors. The animals received ad libitum water and were fed a standard diet (Mazuri Callitrichid High Fiber Diet, 5MI6) twice daily with daily apportionment of supplemental protein through foods such as wax worms, peanuts, cottage cheese. Every animal had daily access to toys that are rotated every two weeks for sanitization and novelty. Addition-

Figure 1. Movable (left) and stationary (right) wooden toys (panel A). The movable toy was clipped to the side of the cage and the stationary toy was bolted to the floor. Fruit was impaled on a metal skewer and frozen. The skewer was clipped to the side of the cage (panel B). The mirror toy had reflective surfaces that rotated on an axis when manipulated. It was attached firmly to the cage wall (panel C).

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Figure 2. Time budgets of toy interaction. Percentages of duration and type of contact with a stationary or movable wooden block toy (panel A), a food skewer (panel B) and a mirror toy (panel C). Panel A: Animals interacted more with the stationary toy (2%) as compared to the movable wooden block toy (<1%), and the stationary toys had a higher gouge frequency than movable toys (inset, F(1, 46) = 7.91, p<.01). Panel B: Animals interacted for 31% of the total duration sampled; however, the food was consumed, on average, 10 minutes into the session and little interaction occurred afterword. Panel C: Animals interacted for 4% of the total duration sampled. Interaction was characterized by short intermittent bouts. Half of the interactions with this toy were visual inspections.

al enrichment is provided in the form of destructible and foraging items at least once a day. Some examples include boxes, paper, ice blocks, and frozen food inside toys. All procedures were approved by the University of Wisconsin-Madison’s Institutional Animal Care and Use Committee. Behavioral observations were recorded an hour or more after morning feeding for a duration of 1hr. Data presented represents a time-budget of the first 20 min of these sessions. A one-way analysis of variance compared cumulative gouging frequency data between the stationary and movable wooden block toy.

Wooden Block Toys

Movable and stationary toys made out of wood were offered to marmosets simultaneously to determine if the animals have a preference to interact with stationary toys over movable toys. Movable toys were clipped to the side of the cage from the top of the toy, leaving the toy free-hanging. Stationary toys were bolted at the base to a shelf in the cage, leaving no room for movement (with the exception of movable components of each individual block). Neither of these toys were novel to our colony. We measured interaction with the toys by observing video recorded sessions to determine how frequently the marmosets made contact with the toys. Additionally, we recorded the total number of gouges on each toy. Although the animals spent only 2% of the total observable time engaging with either toy, the marmosets showed a preference for the stationary toys. The stationary toys allowed marmosets to engage in more gouging behavior, as opposed to the movable wooden toy (¯X = 9.96 vs ¯X= 2.30, F (1,46) = 7.91, P <.01, Figure 2, panel A).

Food Skewer

The food skewer is a long stainless steel rod that fruit was impaled on then frozen to increase the effort required to remove the food. The skewer was movable, as it was hung from the top of the toy to the side of the cage with a clip. We analyzed 20 min video sessions of marmosets beginning immediately after the toy was placed in the cage to determine how they were interacting with the toys. The interactions we assessed were visual inspection, contact (with either mouth or hands), and manipulation (actively interacting with the toy). A fourth category of joint interaction described 1 or both animals engaging in 2 or more interactions at once (for example, 1 animal visually inspecting the toy while the other animal made contact, or one animal both contacting and manipulating the toy at the same time). We found that the marmosets interacted with the toy in some fashion for 31% of the observed time. The marmosets interacted with the skewer toy until the food was gone, which was, on average, 10 min into the session. Interaction after the food was consumed was minimal (Figure 2, panel B).

Mirror Toys

The mirror toy had reflective surfaces that rotated on an axis when manipulated and was firmly attached to the cage wall. Data on interactions with mirror toys were collected identically to the food skewers. These toys did not contain any food, but were novel to the marmosets in our colony. We found that the marmosets spent 4.5% of the observed time interacting with the mirror toys. Half of all total interactions with the mirror toys were visual inspections (Figure 2, panel C). January 2022 Laboratory Animal Science Professional 65

Conclusion

It is not surprising that the marmosets in our colony interacted more with stationary toys than they did with movable toys being that stationary toys encouraged natural gouging behavior from a stable substrate. The skewer toy promoted interaction only until the food source was gone, with little interaction with the toy afterward. This suggests the animals were not interested in interacting with the toy alone, implying limited utility in terms of sustaining curiosity and manipulation. Wild common marmosets spend up to 55% of time foraging and feeding, so it is possible that the toys without food are not directly encouraging their innate behaviors.7 Furthermore, the material of the food skewer (metal) and the mirror toy (plastic) limits the marmosets’ ability to gouge into the toy, unlike that of the wooden block toys that the marmosets did interact with when stable. Therefore, future directions will be to develop novel toys from materials that support gouging or extractive foraging behaviors from stable platforms that our observations suggest is likely to be preferred. In general, the marmosets spent a little of the total observed time interacting with toys and spent a significant amount of time out of view. Some of this was due to the fact that we placed the focus of our recording equipment on the toys rather than the environment as a whole. However, it may also suggest that marmosets’ normative interactive strategies are characterized by short duration and frequency of interaction. We will continue to develop behavioral sampling techniques that are more inclusive of entire cage activity and social aspects of interaction for future studies. Both social contagion in response to auditory stimuli11 and food sharing has been reported in marmosets.2,12 Thus, within the context of socially housed animals, short duration interactions with toys does not necessarily reflect disinterest. Rather, further development of measurement strategies encompassing whole environment interaction in the assessment of enrichment for these roving samplers is required. Acknowledgments: We greatly acknowledge Lyndsey Ace, Jennifer Sullivan, Michele Rosga, Kavya S. Ayalasomayajula, and Atulya C. Reddy for data collection and design. The project was supported by Wisconsin National Primate Research Center Grant ODO11106.

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Tamara Aird, BA, MA, is a Research Technician in the Behavior Services Unit at the Wisconsin National Primate Research Center in Madison, WI. Peter J. Pierre, PhD, is an Associate Scientist and Behavior Sciences, Unit Head at the Wisconsin National Primate Research Center in Madison, WI.

REFERENCES 1. Bjone SJ, Price IR, McGreevy PD. 2006. Food distribution effects on the behaviour of captive common marmosets, Callithrix jacchus. Anim Welf. 15:131-140. 2. Burkart JM, Fehr E, Efferson C, van Schalk CP. 2007. Other-regarding preferences in a non-human primate: Common marmosets provision food altruistically. PNAS. 104(50): 19762-19766. 3. de Rosa C, Vitale A, Puopolo M. 2002. The puzzle-feeder as feeding enrichment for common marmosets (Callithrix jacchus): a pilot study. Lab Anim. 37:100-107. 4. Kitchen AM, Martin AA. 1999. The effects of cage size and complexity on the behaviour of captive common marmosets, Callithrix jacchus jacchus. Lab Anim. 30: 317-326. 5. Lacher TE, Fonseca GAG, Alves C, Magalhaes-Castro B. 1984. Parasitism of trees by marmosets in central Brazilian gallery forest. Biotropica. 16:202-209. 6. Majolo B, Buchanan-Smith HM, Bell J. 2003. Response to novel objects and foraging tasks by common marmoset (Callithrix jacchus) female pairs. Lab Anim. 32(3): 32-38. 7. Pinheiro HLN, Pontes ARM. 2015. Home range, diet, and activity patterns of common marmosets (Callithrix jacchus) in very small and isolated fragments of the Atlantic Forest of Northeastern Brazil. Intern J Ecol, DOI: 10.1155/2015/685816. 8. Roberts RL, Roytburd LA, Newman JD. 1999. Puzzle feeders and gum feeders as environmental enrichment for common marmosets. J Am Assoc Lab Anim Sci: Contemporary topics 38(5): 27-31. 9. Sambrook TD, Buchanan-Smith HM. 1996. What makes novel objects enriching? A comparison of the qualities of control and complexity. Lab Primate Newslett 35, 1–4. 10.Vignes S, Newman JD, Roberts RL. 2001. Mealworm feeders as environmental enrichment for common marmosets. J Am Assoc Lab Anim Sci: Contemporary topics 40(3): 26-29. 11.Watson CF, Caldwell CA. 2010. Neighbor effects in marmosets: Social contagion of agonism and affiliation in captive Callithrix jacchus. Am J of Primatol 72: 549-558. 12.Werdenich D, Huber L. 2002. Social factors determine cooperation in marmosets. Anim Behav 64: 771-781.

AALAS Crossword By Kari Buchanan, RLATG

Nonhuman Primates

1 2

5 6

11 12

16 17

21 22

ACROSS

DOWN

2 A ______ is the primary means of identification in nonhuman primates. 4 Nonhuman primates that originate in South and Central America are referred to as what type of monkeys? 9 Staring and showing of ______ can be signs of aggression in nonhuman primates. 12 A _______ tail is a distinguishing feature of new world monkeys. 14 A method of movement that uses the arms to swing from branch to branch is called: 15 NHPs generally display signs of aggression and submission through _______ expressions and posturing. 17 The tuberculin for a tuberculosis test is administered by __________ injection into the eyelid of the monkey. 19 Working with NHPs is associated with risk of physical injuries and ________ disease transmission. 20 It is essential that the diet for monkeys contain an adequate amount of what vitamin to prevent scurvy? 21 What is the scientific name for the Rhesus macaque? 22 Keeping new primates isolated from others is order to monitor for diseases is called: 23 A group of nonhuman primates is called a(n) ________. 24 The disease carried by rhesus, cynomolgus, and other macaques that can be fatal to humans is commonly called:

1 What NHP is no longer used in biomedical research, but still used in environmental and conservation research in the wild? 3 Nonhuman primates that originate in Africa and Asia are referred to as what type of monkeys? 5 What blood collection site usually required a "blind stick" in nonhuman primates? 6 Originally from the Atlas Mountains and the Rif Mountains of Morocco, the ______ _______ population in Gibraltar is the only wild monkey population on the European continent. 7 Upon arrival at a facility, NHPs should have a(n) _________ period before they are used in research studies. 8 What is the scientific name for the genus of squirrel monkeys? 10 _______ restraint uses poles, collars, and chairs to restrain NHPs during research procedures. 11 ______ _______ are a distinctive feature of old world monkeys. 13 Providing toys, food treats, foraging devices, and environmental stimuli to promote psychological wellbeing is called: 16 _______ are the order of mammals which include humans and monkeys. 18 How many permanent teeth do new world monkeys have? 23 What is the scientific name for the genus of capuchins?

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AALAS CONNECTION

2021 Member Survey of AALAS Educational Resources By Nicole Duffee, DVM, PhD, and Brenda Johnson, MS

he AALAS Educational Resources Committee (ERC) oversees the development of training resources such as the ALAT, LAT, and LATG Training Manuals, and other educational materials. In the summer of 2021, the ERC sent a survey by email to all AALAS members to gather feedback on: • • • • • •

Technician Training Manuals Technician and Regulatory Workbooks AALAS in a Flash and Regulatory Flash Cards Techniques Training Books Technician Training Shorts Webinar Recordings

The ERC would like to thank those 185 members who completed the survey. Respondents indicated they had primary responsibilities at their facilities in management, husbandry, clinical care, and training. All reported using some AALAS educational resources over the past 12 months, and the majority were moderately to highly satisfied with the current educational resources. (see Figure 1) The survey’s questions drilled down into what each educational resource needed to continue being effective. For the printed materials, the two most frequent choices for respondents who used each resource were to produce a digital version and to make no change in the content.

Figure 2. The top 2 choices by survey respondents on their recommendations for future versions of the print-based AALAS educational resources.

(see Figure 2) When asked if a print or digital format would be preferred for viewing AALAS training materials, over 90% of the respondents indicated that they would like to have both print and digital formats. Of the resources that are already digital, (Techniques Training Shorts and webinar recordings) the two top responses were “no change needed” and “to provide more topics.” On all resources, many respondents offered individualized comments, and the ERC is considering how to address these. As digital products were a consistent preference across all respondents, the ERC is glad to announce the good news that AALAS is currently starting work on creating a Digital Hub! A Digital Hub will provide the means to distribute via the Web digital versions of AALAS resources, which is not currently feasible on the existing AALAS website. When the Digital Hub is launched next year, AALAS members will be able to access more digital content. The ERC plans to use this as an opportunity to offer more resources in a digital format. Stay tuned for future updates! Nicole Duffee, DVM, PhD, is the Director of Education and Scientific Affairs at AALAS.

Figure 1. Survey respondents who were moderately to completely satisfied with AALAS educational resources.

68 Laboratory Animal Science Professional January 2022

Brenda Johnson, MS, is the Coordinator of Education and Scientific Affairs at AALAS.

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U Y G S E C M N A E T M N I A X U O Y G L OM GE AN MM AA S C MUN ITY E UNIT XCHA NIT EXCH CHAN CHANGE AALA E AALAS C AALAS CO COM OMM U X MMUN MUNITY E Y E C E T M I O Y G S M N C T N I A X U S E M CO NG GE AAL CHA OMM MMUN MMUNITY AALA S CO ALAS XCHA LAS C CHAN CHANGE TY EX AALA E AALAS C AALAS CO GE A NITY E CO E AA TY EX MUNI N I X U G S E M A N E M N A H U O Y G L ANGE M A T E N O MM AA S C EXC CH NG UNI HANG XCHA AALA E AALAS C AALAS CO ITY EX OMM UNITY XCHA XCHANGE Y EXC Y EXC E N C E T M I U Y G S M NITY E N T M N I A U O G L E IT OM GE AN MM AA S C CHA MMUN MMUNITY MMUN N EXCH CHAN AALA E AALAS C AALAS CO NGE TY EX S CO MUNI ITY EX ITY EXCHA CHANGE AS CO S COMMU UNITY G L S CO M N E M N A A U O L G M A A M C A N H A O X N M A E L S S E C C U H A A A O G X Y E A L L S E M C A N T C EX E AA HA ITY OM UNI E AA AALA E AALAS HANG CHANGE UNITY Y EXC LAS C AS COMM MMUN HANG CHANGE HANG X Y EXC ANG E AA L S CO LAS COMM OMMUNIT E C T H I A A G X X Y L N E E C A N T I A U X A Y Y E N C E A T M H I A U Y G S EX C M N A E T M N I A X UNIT U O G L E OM MM AA S C CHA NGE UNITY HAN MMUN UNITY AALA E AALAS C AALAS CO ITY EX OMM U XCHA XCHANGE Y EXC N E C E T M I AS CO AS COMM U Y G S M N T M N I A U MM ANG AAL S CO LA CHA MUN ITY E COM AL NGE H A O X N M A A E L S E C C U H A A O G X Y E L S E M C A N T C A G M LA S NI EX E AA HA ITY GE A AALA AALAS CO S COMMU E AA HANG CHANGE UNITY Y EXC MMUN CHAN EXCHANG CHANGE HAN X Y EXC E LA S CO LAS COMM OMMUNIT E C T I A A G X Y L N E A N T I A U X A Y Y T AA MUN NGE ITY E XCH UNIT GE A LAS C AS COMM E A N M E ITY MUNI M N A H U Y O N G A M A T C M I U C N H O X C UN OM Y E AAL OMM ALAS AS C XCHA XCHANGE L C E E A TY EX I A S Y G LAS C LAS COMM OMMUNIT MM N A E T A N I U GE AAL MM ANG S CO TY E CHA MUN C AA EXCH MUNI CHAN CHANGE AALA E AAL ITY EX ALAS AS CO ALAS COM X M Y L N E A E NGE T I O A U Y G N C E A T M I U EX G S M AN NG GE E A OMM MMUN MMUNITY AALA S CO EXCH CHAN EXCHA XCHA CHAN EXCHANG AALA E AALAS C AALAS CO O ITY EX UNITY ANGE Y C N E TY EX T M H I I U S Y G C M N N T A M N I X U U L O G E AA MM AN MM MUN ITY COM NGE UNITY AS C XCHA EXCH COM ALAS AS CO AS COMM XCHA XCHANGE NGE AAL MMUN MUNITY E Y L E A T I O A Y MM N C A E T I ALAS U S GE MM ANG AAL S CO ITY E MUN COM XCHA AALA EXCH CHAN CHANGE AALA E AA ALAS AS CO ALAS COM X MMUN MUNITY E Y L E A E T O I A ANGE Y G C N E A T S G EX M MU HAN UNI GE ANG E A AALA CHAN EXCH AS CO ALAS COM AS COMM OMMUNITY UNITY EXC EXCH CHAN EXCHANG ITY EX ANGE NGE AAL Y L N C A H TY EX T M I I A U Y S C M N N A E T M X I A U U O E G L IT OM GE AN MM AA S C CHA OMM MMUN MMUNITY MMUN EXCH CHAN AALA E AALAS C AALAS CO NGE TY EX S CO MUNI ITY EX ITY EXCHA CHANGE AS CO S COM UNITY S CO G L M N AALA E A M N A U L O G M A A A M C N H C HA ALA OM GE E A LAS S CO MUN TY EX ITY EX Y EXC Y EXCHAN GE A AALA E AALAS C LAS COM E A HANG ANGE AA MUNI MMUN OMMUNIT T CHAN EXCHANG C I COM NGE A H O X G N A A S E C C N U H A X Y A E L S E M C T C H I A A G X X M Y Y L S N E E C A N T I O A A U X A Y Y L E N S C MM AA E A MU NGE ITY UNIT UNIT EXCH AALA HANG CHANGE AS CO ALAS COM AS COMM OMMUN OMM XCHA MMUN MMUNITY E L E C A O G X Y E A N T C I X A S N ITY GE S C CH AAL E A TY E AALA AALAS CO S COMMU AALA MMUN S CO TY EX CHAN EXCHANG CHANGE MUNI MUNI AALA E NGE ITY EX ALA AS CO ALAS COM GE X A M Y L N E A E N T H I O A U A Y G C N C E A T M H N G S C A A E NG UNI OM MMU TY EX AALA EXCH CHAN EXCHANG CHANGE OMM XCHA LAS C S CO MUNI ITY EX UNITY ANGE NITY E EX AALA E AALAS C LAS COM E AA TY EX TY M H I I U Y G C M N N E T M N I X U U O G E A M N M AA CO NG HAN AS C OMM OMMU XCH NITY A MMU E L S E C C COM U H A A O G X Y L S E M C A N T C I A A X A M O UN GE S C LAS CH NITY AAL E A TY E AALA S C TY EX E AA E AALAS C LAS COMM S COMMU CHAN EXCHANG CHANGE MUNI MUNI AALA E NGE COM ITY EX A A G X A M Y L S N E A E N T H I O A A U A Y G L C N C A E T M H N I X A U G G S E C A M N A E M N N A H U O Y G L A A M T E C M I C AA EX OM XCH XCH LAS S CO MUN CHAN EXCHANG UNITY ANGE NITY E NITY E AALA E AALAS C LAS COM E AA TY E TY EX M H I I U Y G C M N N E T M N I X U U O G E A M N U O Y G MM AN AA MM S C CH S CO LAS COMM OMMUNIT EXCH CHAN AALA E AALAS C AALAS CO NGE TY EX OMM C MUNI ITY EX ITY EXCHA CHANGE A UNITY G LAS C AS S M N A E M N A A U O G L A A M E M C N A H O X G N A M A E L S E C C U H A A O G X Y E L S E M C A N T C I A A OM UN ANG GE LAS CHA NITY AAL E A TY EX EXCH E AA E AALAS C LAS COMM S COMMU MUNI ITY EX TY EXCHAN EXCHANG HANG CHANGE G N M C N U O X A E M C M LA S NI CH AA ITY ANG ITY EX UNITY AALA AALAS CO S COMMU E AA TY EX NGE UNITY EXCH MMUN MMUN MUNI HANG CHANGE XCHA E LA S CO LAS COMM OM E C UNITY A A G X Y L E M A N T I A AS CO ALAS COM X A M Y IT GE S C CH CO AA E A TY E MUN A AALA MMUN TY EX CHAN EXCHANG CHANGE MUNI COM ALAS NGE MUNI AALA NGE ITY EX ALAS AS CO ALAS COM GE A X A M Y L N E A E N T H I O A U A Y G C N C A E T M H N I X U G S E A M N A E M N A N H U O Y G L EXC A M T E C M I C N A EX CH OM XCHA ANG E A LAS S CO MUN CHA TY EX UNITY EXCH EXCHANG ANGE AA NITY E AALA E AALAS C LAS COM TY EX T M I I U Y M N N E T MUNI M I U U O G N M AA MU CO HAN AS C OMM XCH OMM OM NITY ANG M E L S E C C C U H A O A G X Y S L S E M C A N T C I A A A X A M Y L S IT O UN GE S C CH AAL E A TY E AALA LAS C AS COMM MMUN AALA TY EX CHAN EXCHANG CHANGE MUNI ANGE ANGE AA MUNI AAL NGE ITY EX AL AS CO ALAS COM X M A Y L N E A E T H O I A U Y G C N C A E T M H N I X E 2022 Laboratory Science UN HA MMU E A C69 MAnimal C O M January I T YProfessional ALAS HANG CHANGE Y EXC O X G N ANG A M S E C C N U H A X O Y A E L S C E M T C ITY XCH ANG Y EX E AA COM TY EX NITY AALA E AALAS MUNI I G S N M E N A U O G L A MMUN OMMUNIT MMUNITY E NITY EXCH U MM AA S C XCH CHAN EXCHANG C AALA AALAS CO LAS COMM COM NGE MMU S CO NITY E TY EX ALAS ITY S XCHA CHANGE A E N E A AALA AALAS CO S COMMU A MUNI L U Y G M A E T E M I N A O X G E C UN HA NG A E OM HAN AALA OMM ALAS UNITY XCHA Y EXC ANGE GE A LAS C HANG

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AALAS COMMUNITY EXCHANGE

Enter a place where AALAS members can exchange knowledge, curate content, incubate ideas, and network with other professionals. Your AALAS membership provides access to ACE, the AALAS Community Exchange. Log in today!

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AALAS CONNECTION

Calling all GLAS Applicants!

pplication forms and instructions are now available online for the AALAS Grants for Laboratory Animal Science (GLAS) program! The GLAS program provides two types of research grants: the standard grant, of up to $50,000 for studies with a sound hypothesis and preliminary data; and the small grant, for up to $7,500 for innovative or pilot studies. Applications are due on February 1. The GLAS mission is to enhance scientific knowledge in laboratory animal health and welfare through research and to promote collaborative efforts by the AALAS membership within the broader scientific community. The GLAS program contributes to the AALAS mission to advance responsible laboratory animal care and use to benefit people and animals in such areas as environmental conditions, housing and enrichment, pain and distress, health and welfare, euthanasia, and advancements in animal care and use. Since the first grants were awarded in 2007, the GLAS program has awarded 86 grants totaling $1,684,062. As a result of the GLAS program, a total of 361 cited presentations, publications, and posters stemming from GLAS-sponsored projects have been produced and are listed on the AALAS website. Through these outcomes, AALAS has had a visible role in advancing research and knowledge in laboratory animal science.

The GLAS program requires that the principal investigator be an AALAS member at any level, but it is not restricted geographically. GLAS awards have been made to recipients in Canada, Spain, and Sweden. The GLAS application form and details about the GLAS program, application instructions, and an application tutorial may be viewed at www.aalas.org/glas. This website also has recordings of the AALAS Scientific Advisory Committee’s perspectives and suggestions to prospective GLAS candidates on how to craft a successful GLAS application. The recordings are the panel discussion and Q&A session from last year’s National Meeting. If you have a proposal to improve laboratory animal health and welfare in research, we hope you will submit your idea in a GLAS application!

The 2022 AALAS Election: Is it Time for You to Lead?

he 2022 Nominations Committee, chaired by Dr. Kim Saunders, is looking for our next AALAS leaders. We need your time, energy, and ideas to advance and improve our association and the many programs and benefits we offer to members.

Are you ready for a leadership role?

• Do you want to influence current and future AALAS programs? • Do you want to give back to your professional association? • Do you want to get a sense of satisfaction from helping AALAS members reach their goals? • Do you want to interact with people who will enrich your life? • Do you want to gain experience, learn new skills, network and demonstrate your work ethic? • Do you want to gain more leadership skills? • Do you want to work with and learn from the best association staff anywhere? If you answered yes to any of these questions, then you should consider running for one of our open positions! The ballot for the 2022 AALAS Election has the following open positions: • Vice President-Elect • At-Large Trustee Seat 3: commercial member or an individual 70 Laboratory Animal Science Professional January 2022

• • • • • • • •

working in the pharmaceutical industry with international experience At-Large Alternate Trustee Seat 3: runner-up automatically appointed to alternate District 4 Alternate Trustee District 5 Trustee District 6 Trustee District 6 Alternate Trustee District 7 Alternate Trustee District 8 Trustee District 8 Alternate Trustee

The qualifications for running for a leadership psition can be found on the AALAS website’s nominations page (https://www.aalas. org/leadership/leadership-nomination) along with details about the application process. Candidates are required to submit an application, candidate agreement, nominating petition (Trustees/Alternates), and CV (Vice President-Elect). If you are need more information about the time commitment or other required duties, please reach out to former or current Executive Committee Members, Trustees, or the AALAS office at info@aalas. org.

AALAS Crossword Answers Nonhuman Primates

G 2 R T A E A 4 T N A P 12 13 P R E H E N S N R I 19 Z O O N O T I C H 21 M A 22 Q U A R A N T I N E N T

T T O O L D E WW O R L D O 11 R C 14 S I L E H B 16 D E P E R K I P M O A C A C A M U L A T T C E H S E 24 H E R P E S B V I R

F 10 B A S T E E T H M A C A M E 15 R A C H I A T I O N F A C B L M R H 17 18 A I I N T R A D E R M A R M R H L N 20 Y A I I V I T A M I M T R E C A I T I A 23 C O Y N C O L A N S E Q I B U S X U E S

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