Research with Agricultural Animals and Wildlife

Abstract

In fiscal year 2016, agricultural animals such as swine, sheep, goats, and cattle represented 10% of the 820 812 animals used in USDA-regulated research. In addition to traditional agricultural animals, research studies using captive wildlife are becoming increasingly important as human and livestock populations encroach upon, and thus expand interactions with, wildlife populations on the landscape. Optimum healthcare of both livestock and captive wildlife in a research setting requires proper husbandry, management, and veterinary care. Regardless of animal species, proper care and management are essential for animal well-being, valid research data, and the health and safety of animal care personnel. Using wildlife in research presents unique challenges as there is generally limited peer-reviewed research on wildlife welfare, husbandry, and nutrition. Animals often become excited during handling or transport, and care must be taken to avoid injury. When severe injuries do occur, differences may exist in methods of euthanasia. Many wildlife species are evolutionarily programmed to conceal signs of illness, making assessment of their condition difficult; moreover, attending veterinarians are often not as experienced in the care of wildlife as they are in the care of traditional laboratory animals or livestock. These differences are further magnified in the context of wildlife field research. The concepts of replace, reduce, and refine are as valid in livestock and wildlife research as in biomedical research, and investigators should work closely with their Institutional Animal Care and Use Committees to ensure humane animal care. The Institutional Animal Care and Use Committee is centrally important in providing guidelines relative to ethical use of animal subjects for research and can serve as a valuable resource for research accountability.

Introduction

According to the USDA, in fiscal year 2016, agricultural animals such as swine, sheep, goats, and cattle represented 10% of the 820, 812 animals used in USDA-regulated research.¹ In addition to these traditional agricultural animals, controlled research studies using captive wildlife are becoming increasingly important as growing human and domestic animal populations expand their contact with wildlife, resulting in increased numbers of human-livestock-wildlife conflicts.^2,3 Conflicts between wildlife and humans come in many forms, including physical harm to humans, animals, property, crops, or wildlife habitat and the emergence/reemergence of infectious diseases affecting humans, livestock, and wildlife.^2,4,5 As such, information is sorely needed regarding wildlife-livestock-human interactions, disease transmission, and population effects. Acquisition of baseline data and exploration of management strategies targeting specific wildlife or domestic species usually involves preliminary work in controlled environments using captive wildlife or agricultural animal-based models. Based on preliminary data in a controlled setting, studies may then expand to a field environment.^6,7

Regardless of animal species, proper care and management is essential for animal well-being, valid research data, and the health and safety of animal care personnel.⁸ Optimum healthcare of livestock or captive wildlife requires proper husbandry, management, and veterinary care. Animal husbandry practices should be species specific, thus requiring a knowledge of behavior, feeding and nutrition, reproduction, preventative and interventive healthcare and medicine, and environmental enrichment for that species. For example, although cattle and deer are both ruminants and can be maintained on somewhat similar diets of roughage and concentrates, deer are browsers rather than grazers and can benefit from the addition of leaves and soft shoots from woody plants such as small trees and shrubs. The addition of browse to the environment of captive deer can also be an effective environmental enrichment practice.

The term livestock can be applied to a variety of domesticated animals. For our purposes, we limit the use of this term to ungulate species commonly raised on farms for production purposes (e.g., cattle, swine, sheep, goats). In like fashion, the term wildlife may refer to an even broader range of species. Here we limit the use of the term wildlife to those ungulate species commonly involved in diseases that occur at the wildlife, livestock, and human interfaces; these include ungulate species such as deer, elk, bison, and feral swine.

Housing, Husbandry, and Behavioral Requirements

Husbandry may refer to a wide range of animal care aspects, including providing food, water, bedding, housing, and sanitation. Guidelines for housing and husbandry of agricultural research animals can be found in the Guide for the Care and Use of Agricultural Animals in Research and Teaching.⁸ Because they have been domesticated for millennia, most livestock are capable of adaption to a wide variety of environments, ranging from intensive confinement to extensive pastoral settings. Nevertheless, housing of either livestock or wildlife for research purposes can be highly variable and dependent on the purpose of the research, but should attempt to accommodate the behavioral needs of the study species.

Housing animals used for infectious disease research may require complex biocontainment facilities, whereas research on behavior, reproduction, or nutrition may be conducted in outside pens or paddocks with fewer biosecurity concerns. High-level biocontainment housing design is driven by requirements to contain the pathogen of interest as well as animals that may be infected with this pathogen.⁹ These biocontainment requirements often limit the ability to modify animal housing to reflect a more “normal” environment. This is especially true for wildlife that may vary considerably from livestock species in physiology, body size, or behavior.

Housing or caging is one of the most important features of an animal’s environment. A primary factor in determining the ideal housing/caging is species behavior in terms of its environment. For example, high containment room flooring for large animals is often made of rubberized material instead of concrete to improve animal comfort and foot health. Swine are naturally motivated to root with their snout and can inflict serious damage to rubberized floors. Providing an area containing sanitized soil where pigs can root or dig may not only decrease damage to flooring but also provide a source of environmental enrichment. Some prey species such as deer may benefit from containment room designs that allow for hiding from visual contact with personnel. This can be accomplished through the use of visual barriers such as netting over gating or fencing that provide a sense of concealment, while still allowing for observation through the netting by animal care personnel.

An effective, albeit labor intensive means of acclimating animals to containment is hand-rearing young animals inside an enclosure similar to containment housing. For example, hand-rearing white-tailed deer fawns that are to be housed in high-level containment is an effective way to decrease animal stress and avoid animal injuries.¹⁰ A critical component of this practice, however, is ready access to newborn fawns, as socialization to humans decreases when handling is delayed even by several days.¹¹ An animal’s response to handling is dependent on previous experiences as well as innate temperament¹²; therefore, the sooner after birth contact with humans is initiated, the greater the likelihood the young will develop a positive response to humans. These early handling experiences (positive and negative) can have long-lasting effects.¹³

A strong understanding of livestock and wildlife behavior supports and promotes better science. It behooves investigators and animal care personnel to understand all aspects of behavior including the effects of stress, disease, social organization, and food consumption as they lead to well-founded research results while improving overall animal well-being. Wildlife often have very different fight or flight responses than livestock, which can dramatically affect animal handling practices and equipment. Animal handlers should have a full understanding of the flight zones of the species they are handling.⁸

It is critical that investigators and animal care personnel become familiar with species-specific behavior, both normal and abnormal. As many of the wildlife studied are prey species in nature, display of weakness due to sickness or injury is detrimental to their survival,¹⁴ which may delay recognition of animals in need of medical attention. In group housing it is especially difficult to recognize signs of illness (e.g., decreased food consumption) in an individual animal until weight loss becomes noticeably visible.

Recommendations for livestock housing may be inappropriate for wildlife, and guidelines on housing, such as appropriate room or pen size, may not be readily available for many wildlife species. Consideration should be given to the behavior, physiology, and activity of the species, and the environmental conditions from which the animals originated.¹⁵ Helpful resources for information on housing captive wildlife include publications such as the Journal of Mammalogy published by the American Society of Mammologists, the ILAR Journal published by the Institute for Laboratory Animal Research, the Journal of the American Association for Laboratory Animal Science and Laboratory Animal Science Professional, both published by the American Association for Laboratory Animal Science, as well as other peer resources familiar with the species of interest.

The social structures of both livestock and wildlife species must be considered. Wild or domestic ungulates that are social in nature should be housed in compatible groups to minimize stress. In some cases, human interaction may serve as a substitute for contact if animals are to be housed individually.⁸ Social behaviors of some species are seasonally variable, and accommodations must be made accordingly. For example, during breeding season male ungulates may behave much more aggressively toward other male conspecifics and caretakers than they would at other times of year. Appropriate adjustments may include modification of pen arrangements, separation of individuals, extra caution when entering pens, minimizing handling, and removal of antlers if feasible.¹³

Restraint and Handling

Regardless of species, human-animal interactions should be respectful, calm, considerate, and careful.⁸ Calm, respectful mannerisms produce animals that are easier to work with compared with those handled roughly or disrespectfully.⁸ A low stress handling environment is optimal, and keeping noise levels low aides in reducing stress, although, in some situations it may be helpful to habituate animals to some level of handling-related noises and the sound of human voices.¹⁶ Habituating animals to human presence and handling is also key for decreasing the risk of potentially injurious flight responses.

Positive behavior modification and training can be implemented both prior to containment housing and inside containment. A simple positive behavior training practice is provision of sweet feed or other treats to cattle, or grooming when they are restrained in the head catch of a cattle chute, then releasing the animal with no further procedures. These training sessions should occur multiple times prior to any sample collections; then at the time of sampling, cattle move through the chute with less apprehension and agitation. A long acclimation period prior to initiation of a study, although costly in the sense of time and facility usage, allows for such positive behavior training, thereby creating calmer animals and safer working conditions while producing more externally valid data. Agitation and fear can also be reduced by providing nonslip flooring, avoiding sudden movements, and moving animals using the least amount of pressure required.⁸

Moving and handling wildlife such as deer is best done in a calm, quiet manner, using specialized handling equipment designed for restraining deer, such as a drop-floor chute.¹⁰ The equipment, methods, or attitudes typically used for handling cattle will likely result in injuries (e.g., abrasions, lacerations, fractures) or other stress-induced conditions such as capture myopathy or abomasal ulcers when handling deer.¹⁷ If prolonged restraint or multiple handling events over a short period of time are required, chemical restraint should be considered as an alternative or as a supplement to manual restraint. An excellent resource for chemical immobilization of wildlife can be found in reference books such as the Handbook of Wildlife Chemical Immobilization.¹⁸ In addition, peer-reviewed literature in various scientific journals describes numerous effective, reversible anesthetic protocols for specific species.^19–23 Even when using species-specific restraint devices or chemical immobilization, injuries may occur. Due to the natural stoicism of wildlife, injuries that are not readily visible after a single handling event will become grossly apparent after multiple handling events.

Feeding

Generally, the nutritional needs for livestock are met by following the guidelines set forth by organizations such as the National Research Council. Most adult animal diets are formulated for maintenance; therefore, adjustments to the diets may be required for breeding colonies. Newborn and younger animals are fed to meet their maintenance and normal growth requirement. At times, nutritional requirements can be dependent on the objective of the project, for example, nutrition studies comparing diets in domestic animals. Occasionally, the project length influences feeding practices. For example, ad libitum feeding for a short-term swine project can provide nutritional needs and serve as environmental enrichment to prevent boredom, which could lead to destructive rooting behaviors. Feeding for maintenance vs ad libitum feeding for long-term projects may be required where space constraints and handling equipment limitations exist. Many feed companies, especially those manufacturing feed for zoos, provide nutritional guidelines for captive deer in states of growth, lactation, and maintenance. Many zoos also have nutritionists trained in feeding wildlife in captive settings.

Enrichment

Environmental enrichment is an important component of husbandry for both wildlife and livestock and can be categorized as social, occupational, or nutritional.⁸ The goal of enrichment is to provide species-appropriate environmental, social, and cognitive stimulation. Good environmental enrichment will result in animals that are less aggressive or fearful and more peaceful.⁸ Lack of stimulation in most biocontainment settings can result in boredom and development of stereotypic behaviors, which can negatively impact research results. Environmental enrichment strategies can range from simple acts such as spreading feed on the floor to simulate grazing behaviors to more complex measures such as providing visual barriers or objects such as puzzle feeders, scented toys or other novel objects, music, or skin brushes. One must be cautious of enrichment objects involving fibrous or plastic components that might be consumed or contain sharp edges.

Differences Between Livestock and Wildlife

Using wildlife in research presents unique animal welfare challenges. There is generally limited peer-reviewed research on wildlife welfare including the effects of capture, handling, and confinement. As mentioned previously, many wildlife species are evolutionarily programmed to conceal signs of illness, making assessment of their condition difficult.¹⁴ Wildlife species tend to become more excited during handling or transport vs domestic species of animals and care must be taken to avoid injury. Conditioning of animals to handling practices and use of experienced animal care personnel help decrease stress and handling-related injuries. Husbandry, nutrition, environment, and other differences may exist in general animal care. Attending veterinarians are often not as experienced in the care of wildlife as they are in the care of traditional laboratory animals or livestock,²⁴ and differences in medical, surgical, chemical immobilization, and other health-care related areas must often be addressed. Differing methods of euthanasia may exist as well, for which there are resources such as Guidelines for the Euthanasia of Nondomestic Animals²⁵ and the AVMA Guidelines on Euthanasia²⁶

Field Studies

The inclusion of a section on Field Investigations in the most recent Guide for the Care and Use of Laboratory Animals signals the awareness of regulators that there is an increase in the interest of wildlife research and that field studies play an important role.²⁷ Some activities, such as identification, sedation, anesthesia, surgery, transportation, or euthanasia, may be similar for animals used in laboratory, captive, or field environments.²⁷ Nevertheless, fundamental differences between free-ranging wildlife, captive wildlife, and livestock will affect concepts of study design and animal care, use, and handling.^28,29 Wildlife field studies are often designed to evaluate observational aspects of biology, ecological relationships, population density, behavior, reproduction, or other topics. Other studies are more invasive, requiring capture, tagging, blood sampling, radio transmitter placement, or even surgery.

Standard guidelines and regulations designed for livestock or captive wildlife research may require modification to accommodate the challenges of wildlife field research. A primary objective of regulation of all animal research is to minimize animal suffering, and deviation from this norm requires justification. However, specific guidelines for the handling of free-ranging wildlife can be difficult to formulate. The basic goals of ensuring humane treatment, limited distress, or pain must take into consideration the species, age, behavior, effect of stress, fight or flight responses, and reproductive status as well as geographical, environmental, and seasonal factors.^28,30 Capture and handling of a wild animal, by its very nature, introduces measurable changes in behavior, physiology, stress, and fear, and includes the possibility of death.³¹ Under the best of field conditions, capture alone may result in some level of mortality. Furthermore, it has been suggested that any work with free-ranging wildlife has the ability to negatively impact not just the individual animal but the study population as well. Members of Institutional Animal Care and Use Committees (IACUC) reviewing protocols on wildlife field studies must be prepared to consider modifications in care and use guidelines to account for species specificity and associated challenges of wildlife field studies, including an estimate of anticipated mortality and a plan for responding to greater than expected mortality.^28,31 Finally, it is highly recommended to consult with experts regarding behavior and ecology of the target species before conducting such studies.

Procurement

Livestock and wildlife research subjects must be true representatives of the species intended for study. Potential sources should be evaluated and inspected for the quality of animals they supply.²⁸ All animals should be examined by expert personnel before procurement, and prior to study initiation, to avoid confounding effects caused by disease, genetics, malnutrition, parasitism, or other factors. The research facility should have sufficient capacity, the proper equipment, and staff expertise to adequately manage these animals for research.²⁸

All animals must be acquired lawfully. When acquiring both livestock and wildlife offsite, all permitting and testing requirements by appropriate agencies must be met. These requirements are of particular importance when animals are imported from another state or country. The state veterinarians, USDA Area Veterinarians in Charge, and officials in state wildlife and natural resources departments can be contacted for information regarding certificates of inspection, brand inspections, or permitting requirements. Many states require permits to transport wildlife through their state or have specific restrictions associated with certain species. In the event that a state has no specific requirements regarding transportation of a species through their state, the state and federal authorities should still be contacted as a courtesy. When migratory birds or endangered species are to be acquired, or potentially affected in any way by the intended research, federal authorities such as the US Fish and Wildlife Service must be contacted and appropriate permits secured.

Sources of livestock and captive wildlife include institutionally maintained breeding herds, flocks, or colonies; purchased animals from outside commercial sources (e.g., livestock producers, private contractors, sale barns, auction houses); or in some cases, animals procured from the wild. There are multiple advantages for maintaining on-site breeding colonies, herds, or flocks as a source of subject animals. On-site breeding records allow maintenance of specific gene lines for research projects and elimination of animals with genetics that are inferior relative to physiology or behavior (i.e., aggression, stress, flight or fight response).¹³ Because age, gender, health status, preventive care, and nutritional histories are known for on-site breeding colonies, study design and scheduling are improved. Control over reproductive status, nutritional status, preventative health, and rearing conditions ensure that animals are of optimal age and physiological condition when selecting research subjects. Animals reared on-site tend to be better habituated to housing, handling, and human contact, which decrease animal stress and the potential for injury or death. In the case of high-stress wildlife such as white-tailed deer, on-site hand raising of offspring produces human-acclimated subjects that have a reduced flight response.¹⁰ Disadvantages to maintaining on-site breeding populations include space and feed costs; maintenance of pastures, pens, or confinement areas; and labor costs for daily care, veterinary care, and hand-rearing young if required. Introduction of out-bred animals may be required periodically to prevent occurrence of genetic bottlenecks, which may create a population of animals that is not truly representative of the species of interest.

Animals procured from private producers are often healthier than those purchased from other sources, such as livestock sale barns, and information on the breeding history and husbandry is often known or more reliable compared with sale barn records. However, locating contractors capable of supplying adequate numbers of animals of a specific age or gender can be challenging. Some contractors piece together requested groups of animals by purchasing animals from multiple sources. As a last resort, such a practice may be necessary, but doing so increases the risk of disease introduction among animals from dissimilar backgrounds. Lack of source information on herd health status, health maintenance programs, and potential exposure to organisms that can cause severe illness or confound research efforts (e.g., prior exposure to Mycobacterium avium subsp. paratuberculosis for animals intended for Mycobacterium bovis studies) are some of the distinct disadvantages of using such procurement sources. Inspection of all animals procured from outside sources and testing for species-related diseases or conditions should be performed prior to purchase. Quarantine of such animals should be done in an area that is isolated from any susceptible on-site animals.

Wildlife purchased from private vendors are often raised for game farms or slaughter; in general, such animals have been minimally handled and maintain their normal flight responses. Capture from the wild is sometimes the only option available for obtaining wildlife for research purposes. An advantage of this strategy is that animals are true representatives of the wild populations of interest. Disadvantages include the lack of control related to age, reproductive, and health status, and the drastic difference in the behaviors of wild-caught animals compared with their captive-reared counterparts.

When wild-caught animals are to be used for research, a safe, secure method of capture, transport, and housing must be provided. Likewise, appropriate diet and optimal thermoregulatory conditions must be considered as well as the potential presence of endo- and external-parasites and animal or zoonotic transmissible pathogens.³² Simple requirements such as how to provide feed and water, remove waste, and move the animals within the holding facility must be addressed and in place prior to procurement. Housing substrates and bedding should approximate the natural environment when possible. If animals are to be held indoors, flooring should be constructed of materials that drain well, can be easily cleaned, provide good footing, and be devoid of slats, drains, or areas where toes or hooves could get caught or injured.

Care must be taken during transport of wildlife, even when farm-raised, to minimize stress and fractiousness that may result in injury. Adequate ventilation must be provided, and actions must be taken to prevent extremes of temperature and humidity above or below the animals’ normal thermoregulatory zone.²⁸ Minimizing crowding, noise, and excessive movement and keeping the transport carrier darkened may decrease stress in some species. When possible, transport over long distances should be avoided.

Acclimation

Wildlife may be slower than livestock to adapt to their new surroundings and nutritional sources; therefore, extended habituation periods combined with astute observation by caretakers is required. The length of this period varies among species and should not be strictly dictated by the study design. The individual and collective animal behaviors, maintenance of weight, adaptation to captive diet, and responses to behavioral modification and training relative to the study must be considered. At intake, or prior to start of the study, a wellness check should be performed to assess the health of the study subjects. Administration of antiparasitic agents, vaccinations, collection of pre-study blood samples, or other requirements should be performed at this time.

Record Keeping

Record keeping is critical for good science. Records should be maintained with the intent to serve as documentation of best care and practices for inspection agencies, or peer-review resources, as well as for future guidelines for animal maintenance and study. Institutional animal records begin at procurement and conclude when the individual or group is removed from the study or euthanized. When animals are reared on-site, records begin at birth and should include breeding history if possible. Records for animals procured via a vendor, sale, or auction should be initiated when the animal is transferred into the possession of the institution. Copies of certificates of inspection, permits for transportation, bills of sale, prior disease testing, health status assessment, and prior husbandry information are examples of items that may be added to the record at this time. When wildlife are collected from the wild, records should include collection and transport permits, documentation of trapping location and methods, use of chemical immobilization, monitoring data collected while the animal was immobilized, and any other information pertinent to the health status of the animal. Recorded transportation information may include the method and dates of transport, weather conditions (e.g., temperature, relative humidity, heat index), access to food or water, and other such information that may be valuable should some intra- or post-transport event occur involving an individual or a group of subjects. Use of temperature and humidity data loggers inside the transport trailer can ensure that animals were maintained within conditions that allow optimal thermoregulation.

Veterinary records should be maintained on individuals beginning at intake and should include health certificates, pretransport disease test or health evaluation results (e.g., brucellosis test results, compete blood cell counts, serum chemistry panel, etc.), vaccinations, or other relevant information. On-site health assessments, disease monitoring, vaccinations, deworming, treatments, or surgeries should be documented in an individual animal record. Identification of each individual must be documented. Identification methods for livestock and wildlife typically include ear tags, micro-chips, collars, tattoos, and brands. Choice of identification should be based on the behavior of the species. For example, white-tailed deer and cattle will often chew on tags in the ears of their cohorts, rendering the tags unreadable or, in some cases, removing the tag entirely. Use of multiple identification methods (e.g., tagging both ears, microchipping) will negate loss of identification. Unique individual markings (e.g., spots, stripes, scars, physical attributes) may be documented for identification as well. Additional identifying information should include gender, approximate age (if exact age is unknown), reproductive status, and any other information that may be relevant to the maintenance of the animal or the study.

Once procured, daily observational data relative to feed intake, health status, and appearance should be recorded by animal care staff. In pasture or herd housing situations, evaluation of individuals may be difficult; however, staff should still document the status of the housing conditions, make note of food consumption, and spend time evaluating the general status of animals within the group. Additional records may include sources, nutrient analyses, and storage conditions of feed and hay, pasture maintenance and rotation, documentation of pen cleaning, water source cleaning or changing, and any housing repairs. Health monitoring, tuberculosis testing, rabies vaccination, Brucella antibody testing, training, and continuing education records for animal care staff and the attending veterinarian may be considered relevant to record keeping in some institutions. If the institute maintains a controlled breeding herd, sire and dam information should be documented to develop genetic data that may influence study design. This level of detail may not be possible with breeding herds of captive wildlife; however, an effort should be made to approximate this level of record keeping.

Accountability

Accountability in animal research is vital and involves all topics described above, as well as transparency related to all aspects of animal care and welfare. Investigators conducting animal research should understand that research quality is affected if animals are not well cared for, treated humanely through good handling practices, and judicious endpoints used to prevent undue suffering.

The seminal manuscript of Russell and Burch³³ introduced the concept of the three Rs (i.e., replacement, reduction, refinement). In simplest definitions, replacement refers to the use of nonsentient materials to replace living animals whenever appropriate after it has been proven that replacement will function as an accurate model for the living animal. Reduction refers to proper design of experiments using the lowest numbers of animals necessary to still retain statistical power. Refinement refers to, but is not limited to, reducing stress/distress in subject animals to the lowest possible level. Newton’s concept of the 3 Ss (i.e., good science, good sense, good sensibilities) are complimentary tools that improve accountability of animal research as well.³⁴ Good science involves obtaining the most useful information possible in the shortest time while using the fewest subjects possible. Good sense equates to common sense in “using the right animal for the right reason” to answer the pertinent scientific questions. Good sensibilities value empathy as a means for recognizing stress, distress, discomfort, and/or pain in research subjects. For example, if some aspects of a study result in pain or distress if applied to a human subject, it may be realistic to assume that an animal might also experience distress or pain.³⁴

Reduction and good science must not compromise the quality of the experimental design, rigor, or statistical power of the study. These concepts allow scientists to gather data using a minimum number of animals; however, sufficient animal numbers are required for generation of significant results and precise statistical analyses and to prevent the need to repeat experiments and, consequently, use even more animals. Replacement and good sense should serve as guides when determining the validity, justification, and translatability of animal model studies.³⁴ For example, two species may have different metabolic or immunologic responses to disease challenge; therefore, substitution of one animal for another may not be appropriate and constitutes “wasting” the lives of these surrogate animals. Refinement and good sensibilities are prerequisite for humane treatment of animals in research but must not reach a level of anthropomorphism that impedes the ability to conduct such research.

The concepts of replace, reduce, and refine are just as valid in wildlife research as in biomedical research,³⁵ but the responses to the questions may differ, especially in wildlife field studies. For example, to study behavior of a particular species, replacement with a non-animal model or similar species will not suffice. Wildlife studies are typically designed for research of a specific species. Other wildlife, captive-raised, or domestic surrogates may exhibit different behavior, physiological responses, and ecological niche requirements and are therefore not scientifically acceptable.³⁶ In certain situations, however, it may actually be appropriate to substitute with livestock or laboratory animal models to generate preliminary or “proof-of-concept” data before initiating studies in a larger, less tractable species.

Reduction of animal numbers may not be possible due to the outbred nature of most livestock and wildlife, the study design, environmental factors, and other challenges related to wildlife field research.³⁶ When the study hypothesis addresses new questions or is subject to the proclivities of the environment and the study subject’s natural behavior, exact numbers of individuals required for sampling may be unknown at the beginning of the study. Use of too few animals to answer the study hypothesis can be considered just as egregious as the use of too many animals if an insufficient number of animals are sampled and an inadequate amount of data is collected.²⁸ Use of a power analysis may serve as a guide to estimate the minimum and maximum number of animals necessary to achieve statistical significance.²⁸ When working with rare or elusive species, it may not be possible to obtain animal numbers that reach statistical significance. In such unavoidable situations, careful consideration must be given to choosing a statistical significance level that reduces the chance of rejecting the presence of a difference when there actually is a difference (i.e., failing to reject a null hypothesis when it is false; Type 2 error).

In all animal research, refinement of the methods employed to gather data should be used whenever possible. Use of minimally invasive handling and sampling techniques should be considered during study design development and be included in the IACUC review process. Consideration must be given to potential behavioral and physiological responses of livestock or wildlife, and modifications of capture and release strategies, anesthetic and analgesic regimens, and other handling expectations of the study may require modification to ensure animal safety.

Increased research and reporting transparency has been suggested as a means to address public resistance to animal experimentation.³⁷ Peer-to-peer transparency in research is a means to ensure that valid rigorous scientific methods and humane animal practices are followed. Some research institutions and peer-reviewed journals have instituted policies requiring posting of study summaries and/or raw data in open access repositories as a means to increase confidence in animal research regulation and conduct. Research studies should be designed and implemented with the idea in mind that any peer should be able to repeat the work and that scrutiny by peers or the public may occur.

Particularly relevant to wildlife field studies, the investigator is accountable to the group or agency that exercises stewardship over wildlife. In the United States, individual state governments and Native American nations have jurisdiction over the fish and wildlife within their borders. In most states, authority for the management of wildlife is assigned to state fish and wildlife agencies.²⁵ In some states, the department of agriculture has jurisdiction over some captive wildlife species. These agencies have legal authority to control killing, capturing, monitoring, or processing for any reason. The federal government has authority regarding disturbance of sensitive wildlife such as migratory birds (Migratory Bird Treaty Act; 16 U.S.C. 703-712) and endangered species (Endangered Species Act; 16 U.S.C. ch. 35 § 1531 et seq.).

In all types of animal research, the IACUC is centrally important in providing guidelines and controls relative to ethical use of animal subjects for research and can serve as a valuable resource for research accountability.³⁸ All institutions conducting research on livestock or wildlife must establish an IACUC that is in compliance with federal, state, and local laws, regulations, and policies. In the United States, an institution solely conducting food/fiber research with agricultural species and without government funding would not be required to have an IACUC unless there was a state or local requirement to do so. Resources available for regulations regarding the establishment of an IACUC include The Institutional Animal Care and Use Committee Guidebook,³⁹ including Appendix B; US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training, Public Health Service Policy on Humane Care and Use of Laboratory Animals⁴⁰; the Guide for the Care and Use of Agricultural Animals in Research and Teaching;⁸ and the Animal Welfare Act.⁴¹ Some animal species are exempted from the Animal Welfare Act; however, this does not exempt application of IACUC guidelines for humane care of those species when used for research.

In the United States, according to National Institutes of Health Office of Laboratory Animal Welfare Guidelines, IACUCs should comprise no less than 5 individuals. These individuals must include a doctor of veterinary medicine with training or experience in laboratory animal science, a scientist experienced in animal research, a person whose primary concerns are nonscientific, and an individual who is not affiliated with the institution or an immediate family member of someone affiliated with the institution.^8,28 An IACUC that reviews wildlife or livestock research protocols should consider including a scientist experienced in wildlife research or wildlife management or an individual familiar with livestock handling and husbandry. The IACUC should meet at scheduled intervals for the purpose of approving/disapproving protocols; investigating concerns, complaints, or reports of noncompliance; supporting and monitoring humane and appropriate animal care and use; and performing required institutional functions required by laws and regulations (e.g., semiannual inspections of study areas, facilities, animal housing areas; submitting a written report summarizing the institution’s compliance with established guidelines).²⁷

Animal care and support staff should be cognizant of all requirements for the humane care and use of research animals and the mechanism for reporting adverse events or issues of protocol deviation. These individuals often have more contact with research subjects than investigators or administrators, and institutions should provide training to empower their capability to perform their duties. Training programs may be developed by the institution; however, to prevent bias, training offered by external sources, such as The American Association for Laboratory Science should be considered. An important resource for training of support staff, The American Association for Laboratory Science can provide educational opportunities and materials as well as administer certification programs for animal care professionals, managers, and investigators.

Conclusions

There continues to be a great need for research in both livestock and wildlife species. Ever-increasing interactions among livestock, wildlife, and humans create situations that demand a knowledge base that can support management of the negative outcomes such interactions frequently produce. In the context of infectious diseases that affect livestock, wildlife, and humans, research towards understanding disease pathogenesis, transmission, and diagnosis can require studying species not commonly used in most research settings. These types of research settings require not only compliance with existing guidelines and regulations pertaining to traditional animal research but also an enhanced awareness of the unique welfare and husbandry needs and of individual wildlife species as well as the requirements of agencies that have jurisdiction over wildlife. As investigators work through the challenges posed by such research, it will be important for them to work closely with their corresponding IACUC. Likewise, it will be important for IACUCs to be open to novel manners in which the principles of replace, reduce, and refine are applied to this important area of research.

References