Session Seven: Transition of Wild Animals to Captive Settings and Housing Challenges

Moderator: Elaine Kim, Colorado State University

Elaine Kim, senior Institutional Animal Care and Use Committee (IACUC) coordinator at Colorado State University, moderated Session Seven. Kim introduced the speakers: Eileen A. Lacey at the University of California, Berkeley, L. Michael Romero at Tufts University, and Michael Smotherman at Texas A&M University. The speakers shared their experiences using wildlife in the field, in the laboratory, or both. They shared the challenges they encountered with their IACUCs and how they navigated those situations to move forward with their research and/or teaching objectives.

ESTABLISHING CAPTIVE POPULATIONS OF WILD ANIMALS: COMMUNICATING WITH YOUR IACUC

Eileen A. Lacey, a professor of integrative biology at the University of California (UC), Berkeley, discussed establishing captive populations of wild animals in a research setting. She shared her experiences as both a researcher and an IACUC member and offered several suggestions as to how to make the process of establishing such populations more efficient and successful.

Lacey recognized that integrating field and laboratory studies of the same species can be invaluable and can lead to research outcomes that cannot be obtained in either setting alone. Lacey said that field studies offer the benefit of studying organisms in their natural environments (i.e., in the adaptive settings in which they have evolved), but field studies often suffer from a lack of control. Field biologists often have little control over individuals available for study and how those individuals interact with their environments, she said, in contrast to laboratory studies that are conducted in artificial environments and benefit from the ease of controlled experimental manipulation, which is usually required to demonstrate causality between different variables. The clear complementarity of field and laboratory studies suggests that researchers could attempt to integrate these two settings whenever possible, but she recognizes that for many of the natural systems that field biologists study, this is not a practical possibility, explained Lacey.

For those species that can be studied in both the field and the laboratory, such as the naked mole rat, the benefits of this integrated approach can be substantial, Lacey explained. For example, field observations can inform the design of controlled experiments conducted in the laboratory. Laboratory outcomes can be used to improve understanding of what these organisms are doing in their natural environments. Collectively, these benefits suggest that whenever possible, field biologists may gain by bringing their study species into captivity, Lacey said. However, she pointed out that there are significant challenges to bringing wild animals into captivity and determining how the biological requirements of an exotic species are best met in captivity. This may require experimentation or trial and error learning regarding best practices for housing, husbandry, and veterinary care and it is also likely that bringing exotics into captivity will have regulatory implications, Lacey said. She emphasized that it is reasonable to expect that exotic species will require more exceptions to the Guide for the Care and Use of Laboratory Animals (the Guide) and will require approval of new procedures unfamiliar to the IACUC. Deviations from the established guidelines may create an extra regulatory burden for biologists attempting to establish captive populations of exotic species (Sikes 2016), and this extra burden is not limited to potential resistance from IACUCs if they are unwilling to explore new options (NRC 2011), continued Lacey. For housing or husbandry, she explained, it often also requires that researchers take on the extra regulatory burden of demonstrating the efficacy of specific housing or husbandry practices.

To establish captive exotic species populations in the face of these regulatory challenges, Lacey said, communication between IACUCs and researchers is paramount. Lacey has observed that communication often falls into one of two general patterns. Lacey said that first is converting intuition into information. Many field biologists are the experts on the animals that they study, as a result they have a particularly good understanding of what those animals need in a captive setting, but field biologists are often much less prepared to convert that understanding into information: specifically, data that the IACUC can use when evaluating procedures and attempting to approve protocols. The second general form of communication is turning doubts into data. This is aimed at questions or concerns that arise from the IACUC that must be addressed with quantitative information for the IACUC to proceed with protocol approval. Biologists need to be prepared to gather information and share information in a way that is not required of biologists studying more traditional laboratory animal models, Lacey said.

Lacey provided examples that touch on both forms of communication drawn from her own research program. Lacey’s research is focused on the colonial tuco-tucos, a group-living subterranean species of rodent in the South American Genus Ctenomys (Tammone et al. 2021). Lacey said that these animals are endemic to a roughly 1,500-square kilometer area in Southwestern Argentina, all of which are contained within the Parque Nacional Nahuel Huapi in the Neuquen Province. Lacey was drawn to anecdotal reports regarding this animal’s behavior. The dogma has long been that colonial tuco-tucos are solitary, meaning that each adult occupies its own burrow system. Lacey said that, in contrast, early anecdotal reports suggested that the colonial tuco-tucos are social, meaning that multiple adults live together and share the same burrow system.

Lacey made her first trip to Argentina in 1991 and established a study site on the western banks of the Limay River and has maintained an ongoing annual program of field research ever since. Lacey said that annual efforts in the field consist of an extensive mark-recapture program coupled with intensive radio telemetry. By combining these data, Lacey’s research team gathered robust information regarding spatial relationships among known individuals in their study population from which they can draw inferences regarding social relationships. Given that these animals are subterranean and spend the vast majority of their time underground, Lacey shared that there are marked constraints on how much of their behavior her research team can observe in a field setting. These constraints, specifically, the inability to observe the animals directly in the field, led Lacey to establish a captive population of colonial tuco-tucos on the UC Berkeley campus in 1996. Lacey said there have been challenges because this species had not previously been housed in captivity and they had to start from scratch to develop appropriate procedures for housing the animals and providing them with a sustainable captive diet. This is the only place that this species is housed in captivity, and they have attempted to recreate a borough system environment in the laboratory. Lacey’s team worked with veterinarians at UC Berkeley to address the specific health needs of the species, such as appropriate maintenance of their cheek teeth. The program has been successful and the population has grown from an initial 12 animals to more than 150 with regular reproduction occurring every year in the laboratory.

Lacey offered suggestions for success based on her experiences establishing a captive population of colonial tuco-tucos. At multiple points during her work with captive colonial tuco-tucos, it was necessary to convert intuitive understanding of these animals into information that the IACUC could use and evaluate, turning doubts raised by the IACUC into data for them to evaluate proposed practices and procedures. For example, Lacey discussed her experience developing an appropriate lighting regime for colonial tuco-tucos housed in captivity. When these animals were brought to the laboratory, one of the immediate conditions that was given by IACUC was that the colonial tuco-tucos had to be housed under full illumination, such that any animal was visible at any moment, Lacey said; but this created a different lighting environment in the laboratory versus in the field. As someone familiar with these animals, this was a concern and resulted in a potential conflict between what seemed best to Lacey, given the biology of these animals and what the IACUC was willing to allow.

Lacey designed a study to determine whether, if given a choice, captive colonial tuco-tucos prefer to nest in less versus more illuminated portions of their burrow systems using two transparent plastic acrylic nest boxes, one clear and one red. The red nest box decreased the level of illumination in one nest

but still allowed for ready visual detection of the animals. According to Lacey, the results of the study showed that there was a significant tendency for the animals to be found more often in the red nest box, which was not surprising to Lacey given the biology and natural subterranean habitat of colonial tuco-tucos. As a result of this study, Lacey was given IACUC permission to use red nest boxes as part of the artificial burrow systems that they constructed. The study Lacey undertook to find that subterranean rodents prefer less illuminated areas seemed simple, but Lacey needed to undertake the study to convert her intuitive understanding of what colonial tuco-tucos needed into information and data that her IACUC could use to justify approval for non-standard housing.

Lacey suggested that researchers attempting to establish captive populations of exotic species need to be prepared to do extra work to demonstrate what is best for their animals. This strategy does not reduce the regulatory burden of housing exotics, but the process of establishing captive populations will likely proceed more smoothly and more efficiently. As a final comment, Lacey noted that conducting studies such as the illumination experiment with colonial tuco-tucos can offer important educational and collaborative benefits. Many of these types of projects provide opportunities to engage students in basic research, and it may also be possible to engage the animal care staff, Lacey elaborated. While establishing exotics in captivity may require some additional work, those efforts can be advantageous and do more than just provide data to IACUC committees, Lacey said.

To conclude, Lacey said bringing exotic species into captivity is challenging due to the biology of the animals and regulatory burden that does not occur when establishing populations of traditional laboratory animal models. There are several strategies for increasing the efficiency of establishing captive populations, including effective communication with IACUCs and efforts to collect data for the purpose of validating the best captive conditions for exotic animals. Lacey shared that the major benefits of these efforts are producing better research and gaining a richer understanding of the organisms being studied.

CHALLENGES OF WILDLIFE ADJUSTING TO CAPTIVITY: NEGOTIATING WITH THE IACUC

L. Michael Romero, a professor of biology at Tufts University with 30 years of experience studying the vertebrate stress response in wildlife both in the field and in the laboratory, discussed the various challenges that wildlife species have in adjusting to captivity and how the IACUC might cope with those challenges.

According to Romero, one important feature for bringing wild animals into captivity is the introduction of those animals to a novel stressful environment. Romero and a former graduate student published a review paper exploring how animals cope with the introduction to captivity (Fischer and Romero 2019), which examined changes in body weight, as shown in Figure 7-1.

Romero said that many of the studies reported a decrease in bodyweight at first due to the introduction to a stressful captive environment, but over time some acclimated, and had the same mass as they did prior to capture, with a few overcompensating. The take-home message, said Romero, is that species, when brought into captivity, can both loose or gain weight; some may never recover their mass, which creates some IACUC challenges because body weight is often one of the major ways in which the health of an animal can be judged. For example, Romero elaborated, if an animal is heavier than it used to be, then there is a larger buffer in those animals before body weight loss will create a problem, but for others body weight loss can become an issue much earlier than anticipated. Romero suggested that IACUCs take this kind of feature into account, but it is not always clear which species are going to show an increase, decrease, or stay at the same weight as when they were brought into captivity.

In that same review paper, Romero looked at the changes in the primary vertebrate stress hormone, glucocorticoids, as animals were introduced to captivity, as shown in Figure 7-2. Romero noted that a majority of studies report an increase in glucocorticoids as these animals are having to cope with a new stressful captive environment. Romero reported that as time progresses up to about 6 weeks to 2–3 months, the majority of species still show an increase in glucocorticoid levels; there are more species that show no change; and even a few where the glucocorticoids are below what they were in the wild. He said

that more species show long-term changes, which suggests that most species do not fully acclimate to captive conditions, even over long periods of time (Fischer and Romero 2019). According to Romero, the bottom line is that captive animals are physiologically different from domesticated individuals and from their free-living congeners, and will probably never completely adapt to captive conditions, which creates challenges for IACUCs.

One challenge for IACUCs is dealing with sample sizes, with one of the charges of an IACUC being to reduce the number of animals, Romero said; but minimizing sample sizes is not always appropriate for studies on wild animals. For example, Romero said, wild animals are often patchily distributed across their environment, and this means that there is unpredictable trapping success. Romero recounted being unsuccessful with trapping for weeks and sometimes even months and then suddenly, 100–200 animals will immediately come into the trap. He said that these kinds of studies are opportunistic. Romero stated that biologists might be able to complete 5 or 10 years of studies in just a few days where they could not trap the animals at all for another 4 or 5 years. Romero said that trying to explain this to IACUCs and getting those sample sizes approved is often difficult; and bringing those animals into captivity is also of concern to a lot of the animal care facilities because capturing animals is different from ordering purpose-bred animals, Romero explained.

For instance, if a researcher wants another batch of laboratory rodents, they can simply have them ordered and the animal care staff know exactly when they are arriving. Romero said that when he needs to capture animals, he often has no idea when or how many animals will be brought in on a specific day. A second challenge with sample sizes is that having more individuals often results in better science, Romero noted. For example, many ecologists are interested in understanding the survival rates of a species, the movement patterns, the reproductive outputs, or the yearly patterns, said Romero. For each one of these, the data could be better with a higher number of sample sizes. The idea is not necessarily to have as few as possible, but to have as many as possible. Romero emphasized that wildlife studies are trying to quantify variation, whereas most laboratory studies on laboratory animals are trying to control variation. He added that it is the difference between experiments that test hypotheses versus describing populations that often take place with wildlife studies and coping with these different kinds of sample sizes is often a challenge for IACUCs.

Additionally, bringing wild animals into captivity can create unanticipated challenges, Romero noted. Normal husbandry is often quite stressful to these animals that are unaccustomed to routine cage changes every couple of days. For some species, live prey is required to stimulate feeding behaviors. A good example of this are fish that require live prey because otherwise there is an absence of the appropriate stimuli for successful foraging. It is unclear whether the live prey animals are themselves subject to animal use regulations and covered by IACUCs, as they are often vertebrates but are not actually the subject of the research, Romero said. IACUCs have yet to produce a satisfactory answer to this question.

Finally, many animals exist in natural habitats that are quite dirty, so the clean environments of laboratories are not necessarily the best environment for these animals. For example, Romero outlined, the African clawed frog (Xenopus laevis) has often been used in developmental studies for many years,

but its original habitat is quiet streams and stagnant water in Africa. After all these years, Romero said, they are currently held in laboratory settings and have probably adapted to clean water conditions, but if researchers brought animals directly from the field into laboratory housing systems, the frogs would probably have been happier remaining in their natural environment.

Other characteristics or IACUC requirements of laboratory settings, Romero said, can conflict with species needs. As a model for bringing other taxa into captivity, Romero shared his personal experience and concern about bird-specific husbandry after bringing wild birds into captivity. One example given was that many facilities use fluorescent lights, which have a natural flicker rate, but birds have a higher visual acuity than most mammals and many species can detect that fluorescent-like flicker. Romero said that the flicker rate of fluorescent lights can impact bird behavior. Another example he said was that many facilities would prefer using plastic perches for birds because these are easier to clean than wooden perches. However, experience has shown that many wild birds housed with plastic perches end up with severe problems with their feet and thus wooden perches are better to use. A third example was the challenge of keeping rooms clean when birds molt and drop all their feathers and then replace them. All those feathers being dropped at the same time inside a room can create massive problems for animal husbandry staff.

Another problem is the use of gloves, Romero said; IACUCs would like them to use gloves, both to protect the birds from them, and a way to protect them when handling the birds. For many years they used gloves with their wild birds, Romero explained. The problem is that many of bird species being used have sharp claws on the ends of their feet that would get stuck inside the latex of the gloves. Romero said that one of two things would happen: they would rip the glove to shreds, in which case it was no longer serving as an effective barrier; or the bird would get its claws hooked and trapped inside the latex and the bird would struggle, twist, and break its leg. They had several birds early on that broke their legs and at that point they decided not to use gloves while handling them.

Finally, Romero discussed other issues related to IACUCs. According to Romero, in most facilities, many researchers are using rodents, and there are many training modules available but for only those rodent species. Romero said that conducting surgery on birds is a completely different issue than surgery on rodents; however, these training issues are not covered by most IACUCs.

In conclusion, Romero emphasized that it is important to remember that captive wild animals are not physiologically equivalent to free-living animals, and that individuals may or may not acclimate to captive conditions. Romero continued that species differ in their housing needs and procedural requirements and the best information available to IACUCs are the taxon-specific guides.

COMPLIANCE CHALLENGES FOR CAPTURING, TRANSFERRING, AND KEEPING WILD BATS IN CAPTIVITY FOR RESEARCH AND TEACHING

Michael Smotherman, professor of biology at Texas A&M University, neurophysiologist, and behavioral ecologist with more than 10 years on the IACUC, discussed compliance challenges for capturing, transferring, and keeping wild bats in captivity. Smotherman briefly discussed the capture and transport of wild bats; primary enclosures and space needs, specifically related to bats; heterothermy and how it influences regulations for temperature and humidity control in facilities; and managing torpor and hibernation and how they influence keeping bats in captivity while experimenting on hibernating bats. Smotherman referred to the following resources: the Guide (NRC 2011), the Guidelines of the American Society of Mammalogists for the Use of Wild Animals in Research (Sikes 2016), and the Guidelines for the Humane Transportation of Research Animals (NRC 2006).

Smotherman illustrated common methods to catch bats—by hand, with a hand net, a harp trap attached to the side of a house, or a mist net—and noted that most investigators will put all of these methods on their animal use protocol in anticipation of needing more than one method when the opportunity arises. Once bats have been captured and placed in a small cage, they can be transported to a facility, Smotherman said, and for short car trips, cages can be placed in a secondary container and then

placed in an air-conditioned area of the car. For overnight trips, bats can be removed from the car and given access to water and food and placed back in the car the next day.

Acclimating wild bats to captive environments has multiple challenges, Smotherman said, especially in the first few days. While fruit bats eat quickly and are easier to acclimate, insect-eating bats display species and individual differences in their willingness to eat hand-fed mealworms. Nutritional supplementation is often needed, which can extend acclimatization time, and bats that do not eat within 48 hours of capture often need to be either re-released or euthanized. Smotherman recommended releasing bats back to the site where they were captured if state law allows.

According to Smotherman, the biggest challenge with keeping bats in captivity is providing them with an appropriate primary enclosure. Bats require ample room to fly and stay healthy, but there are a few other issues to consider. For biosecurity purposes, an ante-chamber or a second door reduces the risk of bats escaping from the facility. While bats share the same flight space, they need multiple roosting sites. Bats also require a platform to land and access their food. Bats require rough or irregular surfaces for perching and for breaking up echoes, creating a more naturalistic environment for their echolocation systems, in contrast with the smooth, impermeable surfaces specified in laboratory animal guidelines.

Smotherman asked How much space do bats need and what is appropriate? Most of the time Smotherman takes as much room as possible when building a facility (as shown in Box 7-1) and noted that a limitation is how much space is available for the flight cage. The flight cage will contain bat houses or areas where the bats spend their day when the lights are on, Smotherman said. Bats are hypersocial during this period and tend to cluster in groups, on the roof, or go in a bat house, he said. At night, bats become solitary. Therefore, according to Smotherman, a bat facility needs to accommodate both extremes: one room for bats to fly around and another smaller place to feel comfortable.

According to Smotherman, when transporting bats, it is reasonable to put 25 bats into a small cage for a short period of time because they are going to clump together in a dense group and remain inactive until they are taken out of the cage. For longer-term transport, a bigger space is required for flying, and the bats need different houses, perches, and additional feeding platforms to prevent competition for the same food and water. The daily health inspection of individuals becomes challenging in a flight cage with multiple bat houses. This can be resolved by pit tagging or tattooing every bat, so each one is individually tracked.

Smotherman asked What are the appropriate environmental conditions for bats? In general, bats are more flexible than rodents, and consequently housing temperature is rarely a big issue for facilities and animal use protocols (Stones and Weibers 1965). This flexibility derives from the fact that bats are heterothermic. Most species, including neotropical fruit bats, utilize a facultative daily torpor and may undergo seasonal hibernation, but only if temperatures remain low for an extended period. During daily torpor, bats become inactive, and they lower their body temperature to meet ambient temperatures; this influences surveillance and monitoring. Smotherman said that they also group huddle to minimize energy expenditures during torpor and use flying at night to raise body temperatures. Active bats may prefer warmer temperatures and high humidity, but they possess the physiological, social, and behavioral mechanisms to allow them to thrive at lower body temperatures. Sometimes, temperature and humidity

manipulations are part of the experimental protocol, and they can influence experimental outcomes (Soriano et al. 2002). In these instances, heterothermy becomes a protocol issue because the environmental conditions for the experiment may need to vary beyond what is typically considered acceptable for other small animals.

Experiments, specifically dealing with torpor and hibernation, may require special conditions that warrant appropriate deviations from the usual guidelines, Smothernam said. Hibernation is studied in a variety of different animals, but bats introduce a few quirks. Smotherman illustrated how environmental chambers are used at Bucknell University by DeeAnne Reeder and Ken Field to study hibernation in 13-lined ground squirrels (Ictidomys tridecemlineatus). However, the protocols are different for bats because they need to be housed socially even though they are in hibernation. Unlike bats, ground squirrels can be housed individually. Individual bats cannot be inspected daily because it will arouse them and interrupt their hibernation. Instead, video cameras can be used to remotely monitor activity levels inside the environmental chambers without disturbing the bats. Smotherman said that this permits less frequent opening of the chambers. Smotherman advised against leaving live mealworms in the chambers for any extended period because they will either die or crawl and make a mess, but he noted to always provide water.

Smotherman concluded his presentation by stating that flight and sociality necessitate some modest deviations from the normal guidelines, but these are usually quite manageable if left up to the judgment of the IACUC. Heterothermy raises its own issues, but in many ways, it is an advantage that bats are more flexible than other mammals and a researcher can utilize that. The Guide does not address or make exceptions for the needs of heterothermic animals, specifically, but it leaves room for deviations that are consistent with the needs of bats.

Lastly, Smotherman noted biosecurity and surveillance will be challenging with socially housed flying mammals, but a well-designed primary enclosure and a good personnel training program mitigates many problems, as shown in Box 7-2.