Hybrid model intermediate between a laboratory and field study: A humane paradigm shift in feline research

Abstract

Objectives:

Non-surgical contraceptives are under development to provide accessible, affordable and humane alternatives for the management of free-roaming cat populations. The objective of this project was to develop a research approach for promising non-surgical contraceptives using outbred cats in a simulated free-roaming setting, meeting high standards for both animal welfare and scientific rigor.

Methods:

A facility, specially constructed with indoor and outdoor living areas, was approved and regulated as both an animal shelter and a United States Department of Agriculture research facility. Thirty female and five male cats, healthy but at high risk of euthanasia, were recruited from animal shelters and private homes. Guided by a detailed protocol, cats were housed in this facility for up to 18 months after acclimatization. Cats were administered the study product or a placebo, and then entered into a breeding trial. Cats were adopted at the end of the study. A range of methods was used to provide enrichment and balance a natural environment with the need for detailed daily monitoring.

Results:

Primary study results related to contraceptive safety and efficacy are published separately. Achieving a research model that is an intermediate step between a laboratory and an uncontained free-roaming cat colony was complex. Significant learnings shared in this current publication span: the selection of cats; acclimatization to a simulated colony environment; cat behavioral training during the study and in preparation for adoption; disease management; contract staff and volunteer support; and cat behavior throughout a breeding study.

Conclusions and relevance:

This model inspires continued movement away from the paradigm of breeding cats for research and instead sources existing cats at risk for euthanasia. The housing and management of the cats elevates research animals’ quality of life and provides positive post-study outcomes. While not appropriate for every feline research scenario, this hybrid model (between a laboratory and field study) proved to be a practical, humane and reliable scenario for research requiring a simulated real-world environment.

Introduction

The mission of the Alliance for Contraception in Cats & Dogs (ACC&D) is to advance non-surgical fertility control so as to effectively and humanely reduce the number of unwanted cats and dogs worldwide. From 2015–2017 the ACC&D augmented its suite of programs to include a more active research role by exploring the potential of a contraceptive vaccine in free-roaming cats (FRCs).

The gonadotropin-releasing hormone (GnRH)-based vaccine, GonaCon, has shown promise as a contraceptive in several mammalian species. In laboratory studies using purpose-bred female cats from commercial source breeders, one formulation of GonaCon elicited multi-year contraception ¹ and prompted a strong anti-body response.^1,2 What remained unknown was how success in the laboratory might translate to real cats in the real world (see box).

Answering these questions would help determine GonaCon’s practical applicability for use in suppression of fertility in feral cats.

A novel research environment was needed to ask and answer these questions. The ideal scenario was envisioned to exist along the spectrum between a tightly controlled laboratory setting and an uncontrolled population of FRCs or feral cats. The latter was appealing in that it would represent the vaccine’s anticipated target population, but setting up a true field study with FRCs would have been premature and impractical. Containment of the population was necessary to collect data on vaccine efficacy and monitor health; the cats involved would require frequent handling. It was therefore decided to house a mock medium-to-large colony of outbred social cats in a facility that would allow for sufficient monitoring and containment, as well as exposure to a natural indoor–outdoor environment.

It was imperative that this work be conducted in a manner consistent with the ACC&D’s values, demanding equal focus on scientific integrity and animal wellbeing. Study design, scientific protocol and results are addressed in a recently published companion article.3 This present article elucidates what was required to execute a relatively unique research scenario, strategies to ensure heightened animal welfare standards throughout the 16 month study, cat behavior observations, and learnings and practical recommendations. Ultimately, the goal was for this model to serve as a humane and practical alternative to purchasing purpose-bred cats for research, particularly when research aims to reflect natural contexts.

Study description

Study protocol

The written study protocol was designed with the intention to submit results as a pilot study, as part of the regulatory review in the USA. See the companion article for more detail on the protocol and study results. ³

In summary, the 122 page protocol described every aspect of the study conduct, such as: the inclusion criteria used to select animals; environment; food and water sources; study procedures and schedule; data to be collected; laboratory analyses details, including where samples were to be shipped and under what conditions; hypothesis and statistical analyses used to evaluate data; documentation of protocol changes; and housing of study records. A set of study forms was created to record each data point and observation; all forms were dated and signed by the person completing the form as well as by the principal investigator. Protocol changes were documented as protocol amendments, and protocol deviations were also recorded. Periodically, a person not associated with the study conduct reviewed the study documentation (monitoring) for accuracy and completeness. Although the study did not meet all aspects of a Good Clinical Practice (GCP) protocol, because of the lack of formal quality assurance review, the study was conducted very close to GCP standards.

Cats

Connections with animal control and sheltering communities helped to identify at-risk cats that were not guaranteed adoption in their current situations (Figure 1). Cats were transferred from four county animal control agencies, one private feline rescue and three private individuals who posted cats for rehoming on a classified advertisements website (Craigslist), yielding a genetically diverse population of cats with largely unknown histories.

Figure 1

(a–d) Connections with animal control and sheltering communities helped to identify at-risk cats that were not guaranteed adoption in their current situations, which resulted in a genetically diverse population of cats with largely unknown histories being recruited into the study

All cats underwent a thorough physical examination at intake, and were given core vaccines ⁴ and parasite treatment/prevention. They were provided with a regular diet and fresh water daily. Two cats received veterinary care for dermatophytosis while in quarantine during the acclimatization phase; they were disease-free at study day 0. Bouts of self-limiting upper respiratory infection and/or diarrhea affected many of the cats during the acclimatization phase; this was largely expected due to the stress of transfer and adjustment to new surroundings The result was that the population represented the desired intermediate step between purpose-bred laboratory cats and an uncontained FRC colony.

Figure 2

(a,b) When inside, cats had access to two stories, with a wrap-around loft and plenty of places to play, socialize or find privacy. (c,d) Outdoors was a large enclosed yard with high, predator-proof fencing and enrichment

Forty-five cats (38 females and seven males) were transferred to the facility as potential candidates for the final study population of 30 queens and five toms. Cats not selected for the study were provided with veterinary care and rehomed through both private adoption and arrangements with adoption agencies in cities (Denver, Colorado and Portland, oregon) that could accommodate cats for adoption without displacing existing homeless animals. Among the queens, the most common reason for exclusion was pregnancy upon intake. Several cats were also rehomed for behavioral reasons; namely, if they did not seem comfortable with other cats and/or with human handling. Among the toms, two were excluded from the study and rehomed before the end of the acclimatization phase due to health and behavioral concerns. For others, removal from the study occurred after integration into the colony due to poor breeding performance or inter-cat conflict (see later).

Regulatory oversight

The facility (Clowder Concepts LLC; a ‘clowder’ being a group of cats) was registered with and licensed by the United States Department of Agriculture (USDA) as a Class R research facility, and all work performed within it adhered to the federal Animal Welfare Act, including establishing and maintaining an active Institutional Animal Care and Use Committee (IACUC). The work also fell under pro forma oversight of the University of Illinois’ IACUC, though the university granted deference to the Clowder Concepts IACUC. Clowder Concepts was also registered as an animal shelter with the Illinois Department of Agriculture due to its animal transfer and adoption activities.

Enrichment and training

Enrichment was prioritized throughout the study to keep the cats physically active and cognitively stimulated, and to encourage species-specific behaviors. the consistent environmental elements, cats were provided with a rich portfolio of both passive (eg, olfactory and visual stimulation) and active inanimate enrichment options (eg, toys and puzzle feeders). Two types of enrichment were rotated in/out of the facility each day so that the cats did not become habituated to any given enrichment item, rendering it no longer novel and therefore defeating its intended purpose. Personnel recorded observations to build an enrichment portfolio for each cat.

Play (Figure 3) and human social enrichment (petting, companionship) were a main-stay throughout the study. Additionally, a training program utilizing desensitization and counter-conditioning techniques helped cats become comfortable with and accustomed to handling and medical procedures, such as being picked up, carried to the examination room, placed on the scales, positioned in the ultrasound tray, sedated and having venipuncture. The same approach was used to prepare cats for activities that would facilitate transition to adoptive homes, such as riding in a carrier, traveling in a car, wearing a collar and walking on a harness. As with enrichment, each cat had a training portfolio, listing which incentives (eg, attention, varied food options) worked for that individual. The training process was well documented in order to optimize each cat’s progress. This enrichment and training produced highly adoptable cats that, based on adopter feedback, have adapted very well to their new homes.

Figure 3

A ball pit was one of the enrichment options provided and was a popular addition with the cats!

Personnel

In addition to the principal investigator, the project employed a full-time manager (BS, Animal Sciences) and two part-time staff (University of Illinois Animal Sciences students); these individuals provided daily care, facility maintenance and data management. They also coordinated and supervised a large contingent of undergraduate volunteers, many studying on a pre-veterinary track and in the animal welfare club; this latter group conducted the bulk of the training and enrichment activities described above. Individualized attention was the cornerstone of each cat’s wellbeing, and sustaining this level of attention would have been difficult without student engagement.

Behavior observations

The study protocol required documentation of observed estrous and mating behaviors; personnel were provided with clear parameters of what constituted these behaviors.

The study started during the non-breeding season and males were added at the time of year when breeding generally resumes in cats. Females were monitored monthly by ultrasound for pregnancy and spayed between 0 and 8 days following pregnancy diagnosis, and always prior to an estimated day 45 of gestation.

Initially, the population of 30 females lived in harmony, including during the period in which males were introduced at the beginning of the breeding season. However, 7 months into the study, inter-cat aggression emerged within a week of when cats were returned to the colony following spay surgeries. Severe, persistent aggression initiated by spayed cats became a considerable welfare issue. The cause of the behavior change is unknown, since aggression was not observed following sterilization in previous studies (JF Levy, unpublished data). In contrast to the previous studies, all of the spay surgeries were performed when cats were pregnant, which may have contributed to behavior change in some previously unrecognized manner. Inter-cat aggression following the spaying of pregnant cats in FRC colonies, animal shelters and private homes is not a generally recognized risk. The aggression was managed by the separation of cats when indicated and declined as the population dwindled due to adoptions. Postoperative pain was not believed to be a factor in the aggression because multimodal analgesia was used, and cats remained sociable with their caregivers.

All male cats were assessed for fertility parameters (semen quantity, morphology and motility) prior to introduction to the females. It was observed that libido and breeding performance varied among individual males. To ensure sufficient breeding activity, five different males were introduced to the population one at a time over the course of the study. Three males were removed for poor breeding performance. one showed no sexual interest in the queens but exhibited predatory-like aggression toward them. Another showed appropriate libido when first introduced, but after several weeks of failed mounting attempts, the cat’s libido declined. The third tom was a highly efficient breeder for 1 month, then became fixated on just one female cat (which was already pregnant) for 1 month, and thereafter displayed no breeding behaviors. In the second month this cat became aggressive toward select females, repeatedly attacking one female in particular; during this time, the entire female population also targeted the male with collective aggression.

Other behavior conflicts among the males, and between males and females, followed no discernible pattern. For most of the study, the toms had to be rotated into the population one at a time. Two toms were occasionally fractious when being separated from the population, and only certain personnel were able to handle them. The final two males peacefully coexisted with each other and the females, with no rotation necessary for the last 3 months of the study. This coincided with fewer females in estrus, and fewer cats in the population overall.

A surprising observation was the exhibition of strong mate preferences, with males being far more discriminating than females. All females in estrus allowed a male to mount, whereas it was not uncommon for males to completely ignore certain females displaying signs of estrus. Even when those females asserted themselves by stalking and rolling in front of the males, the males were frequently observed stepping over the females and walking away. Males occasionally became fixated on certain females and would stalk them, even after the female became pregnant and was spayed. Several of the females also regularly mounted other females. one tom repeatedly tried to mount another tom while they were co-housed prior to introduction. This led to fighting, separate housing and eventual removal from the study.

Lessons learned

The project resulted in many learning opportunities. The following core recommendations may facilitate and optimize future endeavors.

Behavior

The study yielded three primary behavior-related learnings. First, the emphasis on socialization, enrichment and training for life after the study seemed to produce highly adaptable and adoptable cats that, based on adopter feedback, transitioned extremely easily into ‘forever’ homes. Industry guidelines are generally non-specific regarding what constitutes ‘enrichment’ for confined cats, and the time that should be allotted to enrichment activities.

We are confident that the time devoted to enrichment for individual cats, and the extent of indoor and outdoor enrichment options they were offered, exceeded industry standards. We support this heightened level of enrichment for the wellbeing of and long-term outcomes for cats, although we recognize it was made possible by the support of volunteer personnel. Secondly, our results suggest an opportunity that a future study of this type engage a skilled feline behaviorist to implement low stress training programs ⁵ to condition cats to study procedures, especially venipuncture. Though not the norm, personnel performing venipuncture occasionally resorted to sedation in order to collect enough blood for analysis. Formal training of personnel and veterinarians prior to the study’s initiation could offer greater consistency.

Thirdly, we would recommend having a skilled behaviorist available to address intercat aggression, as was observed primarily after the cats were spayed. There are limited published data on inter-cat aggression in colonies, including laboratory and feral/free roaming; further research on this topic is warranted.

Cat selection and colony assembly time

As noted above, research into this contraceptive is inspired, at least in part, by the need for humane management tools for unowned FRCs. The project therefore aimed to study an appropriate target population while also providing at-risk cats with a positive alternative to dying in a shelter. The approach was determined and executed with significant reflection on the ethics involved, as well as a review of the legal framework surrounding animal care and use.

The most salient legal consideration was that of pound seizure, the process whereby pounds and shelters sell or release dogs and cats to research laboratories for experimentation, research or teaching. ⁶ Pound seizure is expressly prohibited in some states and required in others. Some states delegate the decision on pound seizures to local authorities, and some do not address the issue at all.

Review of relevant legislation in the state of Illinois, where this project was conducted, did not result in a clear directive for the proposed project’s sourcing of cats. After consulting with appropriate authorities, Clowder Concepts was ultimately registered as both a USDA research center and an animal shelter (licensed through the Illinois Department of Agriculture). The authors are not aware of any other facilities with this dual classification. There is tremendous variation among state laws and regulatory structures around animal care and use, so a review of any location’s legal structure would be a prerequisite to establishing a similar facility. It is likely that this approach would be considered illegal in some jurisdictions.

Some of the cat and colony challenges encountered in the study were anticipated, such as not all males being interested in breeding, and a high risk of infectious diseases. We included some latitude for these issues, such as starting with more males and females than were needed, and having enrollment criteria and a quarantine protocol to mitigate disease transmission.

other challenges were more difficult to navigate. Cats at most risk were in shelters lacking adequate resources including veterinary care. In order to admit a sufficient number of cats meeting the breeding age, sex and health requirements for the study during the planned acclimatization period, multiple shelters were recruited to supply cats. Though project personnel were involved in the selection of cats, and the study protocol required transfer of healthy-appearing cats, human error during the fast-paced intake period resulted in two cats being admitted to the facility with dermatophytosis. This led to unanticipated medical treatment needs and a prolonged period before a suitable cohort of healthy cats was assembled. Upper respiratory infection and diarrhea were also challenges within the population, though it is expected that their prevalence was at least in part due to the stress of transport and changes to the cats’ living environment.

Based on this experience, we would recommend that selection and intake protocols, as well as personnel training and support, be strengthened to ensure that cats are free of contagious conditions prior to entering the facility. We would also recommend selecting cats newly admitted to shelters rather than ones with longer stays and more exposure to shelter-acquired infections.

The study timeline allowed roughly 9 weeks between the first day of cat intake and study day 0 (treatment day). This timeline was too short to accommodate the cats’ unknown medical conditions, variable incubation periods of different pathogens, and the likelihood of feline herpesvirus reactivation due to stress. The result was a prolonged period of treating upper respiratory infection, diarrhea and dermatophytosis prior to study day 0. As discussed above, a stricter approach to cat selection and intake, as well as more experienced personnel, may shorten this period. While a more generous timeline would allow more time for acclimatization, an increased length of stay may not be in the cats’ best interest. When planning future studies, the timing must be carefully considered; the sea-sonality of cats’ estrous cycling is an inflexible component that must be balanced against other factors such as facility/personnel management and cat availability.

This example illustrates the tension created by blending a research with a shelter environment. Best practices for either setting may very well be in conflict with the other. Decreasing length of stay is a primary goal for any well-managed shelter, yet here a longer length of stay is proposed in order to achieve project goals. In this case, one could also argue that purpose-bred cats, raised in and accustomed to the research environment, would have adjusted better to the project setting.

In addition to the population’s physical health, a more generous timeline would also facilitate conditioning of cats to study procedures. The protocol was front-loaded with successive blood collections, which were arguably the most stressful procedures performed during the study. Since a procedure conditioning program was not built into the acclimatization period, some cats developed an aversion that would prove difficult to reverse.

When planning a project outside of a traditional research infrastructure, sufficient attention should be devoted to logistical needs typically met by research institutions. These would include assembling supplies, establishing pay-roll and procurement procedures, drafting animal transfer contracts, negotiating legal and insurance agreements, implementing facility protocols, and ensuring compliance with appropriate local, state and federal regulatory agencies.

Facility and study design: the role of shelter medicine principles

Although the study was intended to replicate a ‘real world’ scenario (ie, FRCs), practicality required the participation of cats subject to prior shelter containment and confined in a relatively dense indoor–outdoor environment. Given that, shelter medicine principles, such as those summarized in the Association of Shelter Veterinarians’ Guidelines for Standards of Care in Animal Shelters, ⁷ should have more strongly guided conceptualization and construction of the research space in order to mitigate disease transmission. The facility did not have a true isolation area in terms of air exchange. Certain materials inside the facility (eg, wood, carpeted platforms), as well as the entire outdoor enclosure, could not be sanitized. Expectations for isolation/ sanitation criteria should be addressed prior to construction or selection of a research site.

Another improvement would be to increase the number of separation areas. The two small rooms proved insufficient for this study; at a minimum two more rooms would have been helpful, especially during the periods of cat intake, behavioral conflicts and surgical recovery. At least a portion of these separation areas should include complete segregation with anterooms to prevent cross-contamination.

Personnel

The cats’ enrichment and training, high levels of sociability, and ease in adjusting to adoptive homes after the study was thanks in large part to student volunteer personnel (Figure 4). However, when any project relies heavily on volunteers, it is important to ensure that personnel requirements and expectations are realistic. It is common for people to get excited about participating in a project and overcommit at the start. Given this study’s heavy reliance on students, maintaining numbers during holiday breaks was also a challenge.

Figure 4

The cats’ enrichment and training, high levels of sociability and ease in adjusting to adoptive homes after the study were thanks in large part to the involvement of student volunteers

Many personnel struggled emotionally with cats being adopted, despite knowing that it was the planned and best outcome. This emotional experience is not unlike seeing beloved foster pets return to the shelter or transition to a new home. The study also created a community and a social outlet that was lost when the project concluded, despite ongoing activity on social media for those involved in the study. It is possible that the sense of loss was exacerbated by the fact that this study was planned to continue for 5 years and instead was ended 1 year after vaccination due to lack of sufficient contraceptive effect to justify a longer observation period. Given the emotions experienced in this study, we would recommend that projects build out training and support to increase personnel resilience.

Plan and budget for veterinary support

The amount of veterinary support required to build and maintain a healthy colony, and to execute the project itself, was underestimated. The study initially involved veterinary professionals who were employed elsewhere and provided their services on a part-time basis as an extension of that employment. When it became clear that more veterinary expertise was needed, the decision was made to contract with two relief veterinarians and two relief veterinary technicians for onsite work, and also to establish a relationship with a local veterinary clinic, where six different associate veterinarians saw several cats for the diagnosis and treatment of various condi-tions. This patchwork approach to veterinary coverage was problematic because different opinions were sometimes offered on the most appropriate course of action. This inconsistency was confusing for project personnel and often resulted in an inefficient and occasionally ineffective program of care. Thus, it was concluded that a multifaceted study lasting approximately 1 year requires consistent veterinary support.

The ‘hybrid’ research model

Achieving a research model that is an intermediate step between a laboratory study and an uncontained FRC colony is complex and requires consideration of animals, study environment and study protocol.

As discussed earlier, we believe that the random-source population was assembled and cared for in a way that best represented the target population of cats for this contraceptive, while remaining both humane and ethical. The latter criterion included monitoring for health issues and treating with prompt veterinary care, providing a regular diet of wet and dry food, and providing core vaccines and parasite treatment/prevention.

Creating an optimal hybrid study environment was complex, as there is inherent tension between simulating field conditions and optimizing the health of a colony of 30 or more cats. For example, it can be argued that study protocols that were important for the cats’ safety and wellbeing, such as bringing animals inside at night to avoid predation, permitting cats to stay indoors during the day if they wished, and offering extensive enrichment and socialization, were at odds with what a cat would experience in field conditions.

Lastly, implementing a study protocol appropriate for regulatory submission for potential product approval for commercialization requires meeting certain standards for monitoring safety and efficacy. This included having cats that were comfortable with human handling and without medical conditions that could interfere with evaluating the vaccine.

Key Points

• While this model will not be appropriate for every feline research scenario, it inspires continued movement away from the paradigm of breeding cats for research, and toward one that elevates research animals’ standard of care and provides a humane alternative for at-risk shelter cats when ethically justifiable.

• The authors welcome inquiries and opportunities to share more detailed information and guidance with parties interested in pursuing similar endeavors.