Marmosets: Welfare, Ethical Use, and IACUC/Regulatory Considerations

Abstract

Use of marmosets in biomedical research has increased dramatically in recent years due, in large part, to their suitability for transgenic applications and utility as models for neuroscience investigations. This increased use includes the establishment of new colonies and involvement of people new to marmoset research. To facilitate the use of the marmoset as a research model, we provide an overview of issues surrounding the ethics and regulations associated with captive marmoset research, including discussion of the history of marmosets in research, current uses of marmosets, ethical considerations related to marmoset use, issues related to importation of animals, and recommendations for regulatory oversight of gene-edited marmosets. To understand the main concerns that oversight bodies have regarding captive biomedical research with marmosets, we developed a brief, 15-question survey that was then sent electronically to academic and biomedical research institutions worldwide that were believed to house colonies of marmosets intended for biomedical research. The survey included general questions regarding the individual respondent’s colony, status of research use of the colony and institutional oversight of both the colony itself and the research use of the colony. We received completed surveys from a total of 18 institutions from North America, Europe, and Asia. Overall, there appeared to be no clear difference in regulatory oversight body concerns between countries/regions. One difference that we were able to appreciate was that while biomedical research with marmosets was noted to be either stable or decreasing in Europe, use was clearly increasing elsewhere.

UTILITY OF THE MARMOSET MODEL FOR BIOMEDICAL RESEARCH

The marmosets (Callithrix sp.) are a genus of small monomorphic New World primates. The most frequently used species in biomedical research is the common marmoset (Callithrix jacchus), with some additional species such as black-tufted marmosets (Callithrix penicillata) and white-headed or Geoffroy’s marmosets (Callithrix geoffroyi) being used less frequently. Many factors make the marmoset an attractive model system, including their genetic and physiological similarity to humans; relatively short lifespan, high fertility, rapid maturation, small size, and ease of handling compared with other commonly used nonhuman primate (NHP) models (eg, macaques); social behaviors and communication that resemble those observed in humans; and a human-like social structure consisting of cooperative breeding with shared parenting responsibilities.

Like macaques, marmosets share approximately 93% sequence identity with the human genome¹ and they develop similar age-related conditions as humans including diabetes, cardiovascular disease, and cancer.^2–4 Adult common marmosets average 300–450 g, about the size of a rat. Marmosets are among the shortest-lived anthropoid primates with an average lifespan of approximately 13 years and a maximum lifespan of 15–20 years. They are reproductively competent by approximately 1.5 years of age, routinely produce litters of 2–3 offspring every 5–6 months (giving them the highest fertility of any primate),⁵ and are considered geriatric by 8 years of age.⁶ Their relatively small size and fast life history (compared with other NHPs) represents an advantage in many types of studies, including those involving reproduction, child-rearing, child health, impact of early-life interventions, chronic disease effects, and testing compounds for which only small volumes may be affordably available. Their high fecundity makes them a particularly compelling model for diseases such as Alzheimer’s, Parkinson’s, and other neurodegenerative diseases that would clearly benefit from a transgenic NHP model. Importantly, their rapid life history makes it possible that within the period of a standard 5-year research grant, 3 generations of offspring can be produced; during this time, the founding adult animals will progress through old age. Furthermore, the frequent production of twins and triplets enables study designs that can effectively control for genetic contribution by using siblings in different study groups. Additionally, the similarity to human social structures facilitates use of the common marmoset to model parenting and family effects on child development.

HISTORY OF USE OF MARMOSETS IN BIOMEDICAL RESEARCH

Marmosets have been maintained in captivity for centuries generally as pets.⁷ Prior to the 1960s, research with these animals was mainly focused on behavioral and anthropological studies. In the early 1960s, the use of NHPs as biomedical models increased dramatically with the establishment of the National Institutes of Health (NIH) primate centers program⁸ as did interest specifically in the use of laboratory-housed marmosets to model human disease. Interest in the marmoset model was inspired mainly by both their small size and relative availability and low cost compared with other potential NHP models. Marmoset research in the 1960s covered a broad range of areas, including reproduction, biodefense, and gene therapy.⁷

Since the 1970s, marmosets have been widely used in research related to infectious diseases,^9–11 toxicology,^12,13 drug development,^14–16 neuroscience,^17–19 and transgenics and genome editing^20,21 with growing areas of interest in obesity and aging.^2–4,22,23 In particular, there has been a recent resurgence of interest in the use of New World monkeys in biomedical research driven by several factors: the sequencing of the common marmoset genome,^1,24,25 the development of transgenic marmosets with germline transmission of the transgene,²⁰ their growing popularity as a neuroscience research model,^21,26,27 and the increasing demand for alternatives to Old World primate models.²⁸

Increased demand for marmosets in scientific research studies has perhaps been most acute in neuroscience, where the need to study cognition, behavior, and mental illness in NHP models has grown in part due to recognition of inherent limitations of rodent models in translational studies of human physiology and disease. This couples well with the fact that marmosets are a particularly good model for neuroscience including studies of cognition and the visual and auditory systems due to their shared functional brain architecture and broad behavioral repertoire with humans along with having a lissencephalic (smooth) cortex, which makes it significantly easier to record and manipulate neural activity than in Old World monkeys.¹⁹

Marmosets are particularly valuable NHP models for transgenic and genomic editing applications given several factors. First, the efficiency of in vitro fertilization of oocytes is very high, making marmosets highly economical and scalable for generating the number of genetically modified marmosets needed for preclinical evaluation.²⁹ Additionally, gene-edited marmosets can be generated with an intergeneration time and establishment of transgenic lines 2–3 times faster than other NHPs. Much of the current transgenic and genomic editing work in marmosets focuses on neuroscience applications as well. Among the transgenic marmoset models that have been developed are those of stroke,³⁰ polyglutamine disease,³¹ and severe combined immunodeficiency³¹ in addition to models of Parkinson’s disease that were designed to facilitate study of mechanisms associated with the prodromal stage of the disease,³² and models that express genes associated with autism and show behaviors similar to those seen in humans with autism.³³

Their relatively short lifespan makes them a valuable model for the study of aging and chronic diseases such as diabetes. Obesity along with dyslipidemia and altered glucose metabolism leading to hepatomegaly, hepatic steatosis, diabetes, atherosclerosis, cardiomyopathies, and stroke is becoming a more frequent finding in captive marmosets. Age-related increases in insulin resistance have been detected,^22,34 and potential biomarkers of aging have been identified in the plasma metabolome.³⁵ In addition, within 4 months of feeding a high-fat, high-glucose diet, marmosets develop obesity with prolonged hyperglycemia and profound pancreatic islet hyperplasia,³⁴ making them a valuable model for studies of diet-induced obesity.

A major concern in the development of the marmoset biomedical model is their known susceptibility to gastrointestinal disease. Most commonly this is known to present as inflammatory bowel disease–like disease ranging in severity from colitis to more severe forms of chronic lymphocytic enteritis.^36–38 While the cause is unknown, this has been implicated in poor weight gain in young animals³⁹ and excessive weight loss and increased morbidity and mortality in older animals^36,39 and may interfere with captive management and use in research. Nutritional research is underway attempting to better understand the relationship between marmoset intestinal health and diet to improve the utility of the marmoset model.⁴⁰ Recently, a novel gastrointestinal disease in captive marmosets characterized by proximal duodenal obstruction and dilation has been identified.⁴¹ Though the cause is unknown, diet is likely to play a role in this condition aswell.

Marmosets have been used to model the effects of infection caused by numerous viruses, including, but not limited to, hepatitis C,⁴² dengue,⁴³ herpesvirus,⁴⁴ and SARS.⁴⁵ They have also proven to be effective in modeling pathogens relevant to biodefense, including Marburg hemorrhagic fever,^46,47 Ebola hemorrhagic fever,⁴⁷ and Variola virus.⁴⁸ In addition, marmosets have been a very attractive model for preclinical in vivo pharmacology and toxicology studies due to their relatively small body size, which translates to the need for smaller amounts of test compounds, a clear advantage when test material is available in limited quantities.⁴⁹ However, given their small size, blood draw volumes are a concern, particularly if samples are being collected for assessment with assays designed for larger NHP or humans.²³ Average circulating blood volume in a marmoset is 70 mL/kg. Removal of approximately 2 mL of blood (approximately 8% of the total blood volume in a 350-g marmoset) requires at least 1 week of recovery before more blood can be taken. Removal of higher volumes requires longer recovery periods: approximately 2 weeks for removal of 10% (approximately 2.5 mL) of blood volume and a minimum of 4 weeks for removal of 15% (approximately 3.5 mL) of blood volume.⁵⁰ In addition, maximum blood draw volumes must be adjusted to consider age, weight, health, and hydration status.³⁶

ETHICAL CONSIDERATIONS RELATED TO USE OF MARMOSETS IN BIOMEDICAL RESEARCH

The welfare of NHPs used in biomedical research is of extreme importance. Beyond purely ethical considerations related to reducing more than momentary pain, distress, or injury to the absolute minimum, animals that are well-cared for and maintained under conditions that promote optimal animal welfare using evidence-based guidelines make the best research subjects and produce the most replicable results. Consideration of ethics in marmoset research, as with any research, is based on the principles of the 3Rs—replacement, reduction, and refinement—that were developed over 50 years ago by Russell and Burch.⁵¹ Replacement refers to the use of non-animal alternatives such as cell culture or computer simulations, or, if that is not possible, the use of a lower species of animals (eg, use of a mouse model instead of an NHP model). Reduction requires using the fewest number of animals possible while still maintaining a properly powered study. An important caveat with this principle is that excessively reducing the number of animals will result in a study lacking statistical power and therefore of insufficient power to prove the hypothesis because of low animal numbers. Statistically underpowered studies such as these will need to be repeated often, resulting in an overall increase in the number of animals used to address the hypothesis. Proper study design from the onset is critical. While reduction of the actual number of animals used is important, there is often an ethical tug-of-war between the impacts to a single animal in a study vs the cumulative potential impact on all animals in a study. Simply put, is it better for both animal welfare and the scientific objectives to use fewer animals in a study but use them more intensely or to include a large number of animals that are each used in a more minimal way (eg, collecting blood samples monthly over a 1-year period from a group of 10 marmosets vs collecting a blood sample at 1 timepoint from a group of 120 marmosets)? The principle of refinement dictates the implementation of methods that minimize animal pain, distress, and injury and improve welfare. An example of an important recent refinement in marmoset neuroscience work is the adaptation of animals to awake imaging techniques, therefore avoiding the need for long and repeated episodes of anesthesia;^52–55 it should be noted, however, that there could still be stress associated with awake procedures, and appropriate acclimation and monitoring is required.

Marmosets, like all NHP, are intelligent and have complex social needs. This must be considered in designing all aspects of a marmoset research program, including both standard husbandry practices and experimental design. For example, long-term single-housing should be avoided if possible, as it interferes with study results by altering such factors as behavior and activity. However, the social environment may also interfere with required testing. For example, related to their highly social nature, attempts to engage marmosets in cognitive testing while animals have access to others in their family group can be challenging.⁵⁶

A specific consideration regarding captive marmoset welfare concerns cage size. Required cage sizes are based on floor space; however, these highly arboreal animals preferentially use vertical space,⁵⁷ and therefore exemptions to regulations based on floor space to allow more vertical as opposed to horizontal space are often beneficial. Other ethical considerations regarding current marmoset research are a result of the specific types of research for which they are being used. For example, neuroscience protocols often involve restraint and food or water regulation to facilitate the animals’ compliance with cognitive evaluations, and nutrition and obesity studies may require single housing to allow close monitoring of dietary intake. These must be clearly and carefully evaluated by regulatory oversight committees to consider all aspects of the 3Rs.

MARMOSET RESEARCH REGULATIONS

Here we briefly review the regulatory framework in countries representing the current major users of marmosets in biomedical research.

In the United States, the animal research oversight system is composed of a combination of activities mandated by law or required as a condition of funding and activities that an individual or institution comply with voluntarily as part of their commitment to excellence in research.⁵⁸ Marmosets are considered a “covered species” (warm-blooded animals used in research except birds, rats of the genus Rattus, and mice of the genus Mus bred for research), and therefore all research with them in the United States is regulated by the Animal Welfare Act and its regulations which is administered by the Animal and Plant Health Inspection Service of the US Department of Agriculture (USDA). The Animal Welfare Act explicitly follows the 3Rs and establishes certain institutional responsibilities, including the appointment of a local Institutional Animal Care and Use Committee (IACUC) that reviews research projects, a program of adequate veterinary care, training of personnel, recordkeeping, and reporting. Any institution using covered species in their research program must be licensed by the USDA for this work to ensure that laboratory animals are provided with appropriate care and veterinary support meeting USDA standards. As part of this registration, institutions are required to submit an annual report stating how many regulated animals were used at their institution and if any painful experiments were conducted. Public Health Service (PHS)-funded (eg, NIH, Centers for Disease Control and Prevention) research programs are further required to follow the PHS Policy on Humane Care and Use of Laboratory Animals,⁵⁹ which requires that institutions follow the regulations set forth in The Guide for the Care and Use of Laboratory Animals⁶⁰ and to have a written assurance document to this effect that has been approved by thePHS.

Canada has explicitly adopted the 3Rs as the set of principles to guide the ethical evaluation of animal use.⁶¹ The Canadian Council on Animal Care (CCAC) is the national organization that has the responsibility for overseeing the care and use of animals in science through adherence to their Guide to the Care and Use of Experimental Animals,⁶² and CCAC certification is required for all institutions that receive funding from the federal granting agencies for animal-based studies. Much like in the United States, each CCAC-certified institution must have at least 1 local animal care committee responsible for overseeing all aspects of animal ethics and care at the institution, and this committee must review and approve all animal-based research before it begins.

In the United Kingdom, use of all “protected species” (defined as any living vertebrate other than human and any living cephalopod) in research that “may have the effect of causing that animal pain, suffering, distress or lasting harm” is controlled nationally by the Animals (Scientific Procedures) Act 1986 Amendments Regulation 2012. This legislation operates by use of a licensing system, administered and regulated by the Home Office’s Animals in Science Regulation Unit. There are 3 tiers of licensing that must be attained to initiate research. First, one must obtain an establishment license for the place where the work is to be performed. This license ensures suitable facilities, based on The Home Office Code of Practice, are available for both animal research and maintenance and requires establishment of an Animal Welfare and Ethical Review Body, whose role is similar to that of the IACUC in the United States. The next step is attainment of the project license, granted for a maximum of 5 years, which is similar to the IACUC protocol in the United States. As part of this review, NHPs, including marmosets, are viewed as species of special consideration and can only be used if the purpose of the research cannot be achieved using other species. The origin of the marmosets is also important: only purpose-bred offspring of animals born in captivity (ie, F2 generation or later) are allowed to be used unless an exception is granted by the Secretary of State. Before a project license application is formally submitted to the Animals in Science Regulation Unit to be considered for a harm-benefit analysis, it is reviewed by the establishment’s Animal Welfare and Ethical Review Body, who advise the establishment license holder whether to support the application from a local institutional perspective. Finally, a personal license to ensure that the person performing regulated procedures is suitably qualified to do so and has the necessary and appropriate training must be obtained (ie, the person is declared competent to perform a certain procedure and is “re-examined” at least every 5 years, but generally annually). Specific to marmoset research, UK guidelines for floor space and cage height are more generous than those in the United States. While US regulations determine cage size based solely on animal weight, European regulations include the following in consideration of their cage size requirements: adult size of the animals, the size of the group, and sufficient space to create a complex and challenging environment.⁶³ The European regulations also focus on vertical space over floor space for these arboreal animals.

Similar to the system in the United Kingdom, the Netherlands requires an establishment license and a project license to perform animal research. The establishment license is applied for through the Netherlands Food and Consumer Product Authority, and the project license is issued by the Central Animal Testing Committee (Centrale Commissie Dierproeven) after recommendation from an Animal Ethics Committee. The Experiments on Animals Act describes purposes for which animal testing may be performed and includes requirements regarding welfare issues such as availability of appropriate expertise and housing and the caveat that research may only be performed with animals specifically bred for that purpose. This act requires establishment of an institutional animal welfare body (Instantie voor Dierenwelzijn) that ensures that all experiments with animals are carried out in accordance with the 3Rs.

The Science Council of Japan issued Guidelines for the Proper Conduct of Animal Experiments in 2006 as a result of a 2005 amendment of the Law for the Humane Treatment and Management of Animals and the request of several government agencies. Both the amended law and the guidelines required adherence to the 3Rs. While establishment of local IACUCs is recommended by the Science Council of Japan Guidelines, unlike in the United States, the institutional director is ultimately responsible for oversight and approval of all animal experiments at their institution, and the IACUC serves as advisory to the director.⁶⁴

IACUC CONCERNS REGARDING RESEARCH WITH MARMOSETS: RESULTS OF AN INTERNATIONAL SURVEY

In an attempt to understand the main concerns that oversight bodies have regarding captive biomedical research with marmosets, we developed a brief, 15-question survey. The survey included general questions regarding the individual respondents’ colony, status of research use of the colony, and IACUC oversight of both the colony itself and the research use of the colony. This survey was sent electronically to academic and biomedical research institutions worldwide that were believed to house colonies of marmosets intended for biomedical research. The list of institutions we contacted was drawn from the authors’ collective knowledge in addition to the mailing list from the Marmoset Working Group, which is a collaboration between the National Primate Research Centers, the NIH–Office of Research Infrastructure Programs/Division of Comparative Medicine, and colleagues at various academic institutions. The purpose of this group is to strengthen communications, leverage system-wide resources, and facilitate sharing of information and best practices across institutions housing common marmosets.

Forty institutions were contacted. Of these, 50% completed and returned the survey. Two of the surveys indicated that the institution did not currently house marmosets for biomedical research. Of the 18 surveys completed from institutions housing marmosets for biomedical research, 14 were from institutions in North America, 2 in Europe, and 2 in Asia. Individuals completing the survey self-identified as a researcher (n = 13), veterinarian (n = 5), and colony manager (n = 2). One individual identified as both a veterinarian and a researcher, and another individual identified as both a colony manager and a researcher. All 18 respondents indicated that they had colonies of common marmosets (Callithrix jacchus). In addition, 1 colony also included populations of black-tufted marmosets (Callithrix penicillata) and white-headed or Geoffroy’s marmosets (Callithrix geoffroyi). Overall colony size was 140 ± 35 (mean ± SEM) with a range from 10 to 600 individuals.

To understand current and predicted trends in marmoset research, we posed questions regarding the general types of research being performed at each institution, if use of the colony was increasing or decreasing, and if there were plans to alter the size of the colony. We specifically asked if each institution was involved in the following types of research: aging, infectious diseases, neuroscience, and reproduction. Respondents were also able to enter additional areas of research in an “other” category. A detailed breakdown of type of research by region is presented in Figure 1. The most common area of research was neuroscience. Eight of the 18 (44%) institutions indicated that they were involved in research in more than 1 specific area. Of the remaining 10 institutions that indicated they were performing research in only 1 specific area, 9 were involved in neuroscience research. At the remaining institution, the marmosets were participating in research focused solely on oncology. Of particular note, 3 institutions from Canada indicated that only neuroscience research was currently being conducted with their marmoset colonies. In response to the question about the perceived trend in marmoset research at their institution, 78% (14/18) indicated that research use of their marmoset colonies was increasing, 5% (1/18) indicated that it was decreasing, and 17% (3/18) indicated that the level was stable (Figure 2). Although this is admittedly a small sample, the responses imply that while worldwide use of marmosets in biomedical research is increasing, use in Europe is stable or declining (Figure 2). In response to these changes in the level of use of the colonies, 56% of colonies indicated that they plan to increase their colony size by acquiring new animals and 27% indicated that this is something they are considering. The remaining 17% of respondents, the 2 European colonies and 1 Canadian colony, indicated that they did not plan to increase their colonysize.

Figure 1.

Stack plot indicating types of research being conducted by region by the 18 respondent institutions. Institutions may be participating in more than 1 type of research. Other included obesity and oncology.

Figure 2.

Stack plot indicating the change in amount of marmoset research being performed by region at the 18 respondent institutions. The survey asked the general question: Is marmoset research at your institution increasing, decreasing or staying the same? No specific time frame was indicated.

The survey asked how marmoset research at each institution is reviewed and approved specifically questioning if this was done by a general institution-wide IACUC or other oversight board, or if the institution had an IACUC or other oversight board assembled specifically for oversight of marmosets or primates in general. Eighty-nine percent (16/18) of respondents indicated that marmoset research at their institution was reviewed and approved by an institution-wide committee. The 2 remaining institutions indicated that marmoset research at their institution is reviewed and approved by a marmoset- or primate-specific oversight board.

We next asked if the IACUC or oversight board reviewing marmoset research at their institution had specifically questioned or commented on any of a broad array of topics in their review of marmoset research protocols. The topics we specifically asked about were overall utility of the marmoset model, appropriateness of the marmoset model for the specific area of research being performed, the number of animals requested to be used in studies, the use and form of social housing employed, cage structure, the environmental enrichment plan, type of diet, sanitization procedures employed (including for the room, cage, cage furniture, and enrichment), the specific procedures planned as part of the research protocol, the overall number of either a specific procedure or all procedures in the protocol, the amount of blood to be drawn overall or in a single draw, the use of anesthesia for procedures as opposed to working with animals under conscious restraint, and the anesthesia and analgesia plans associated with the planned research. All topics were of interest to the review committees overseeing research at the respondent institutions (Figure 3) with no particular topic standing out as being of particular concern or of no concern.

Figure 3.

Stack plot indicating responses to the general subject of questions asked, by region, by institutional review committees regarding marmoset research protocols.

In addition to asking if these topics were ever questioned with respect to marmoset research at their institution, respondents had the opportunity to provide comments on the types of concerns expressed by their oversight committees with respect to each specific topic. Regarding the utility of the marmoset model, 7 (39%) respondents indicated that this was an area of concern for their oversight body and was discussed in the protocol or research application form and at related meetings. Two respondents (11%) specifically indicated that their institution required careful and detailed justification indicating why marmosets vs other animal models (ie, rodent or other NHP) were required for the proposed research.

The appropriateness of the marmoset model for the specific area of research being proposed was noted as being questioned at 9 (50%) of the respondent institutions. Comments regarding this question included that the appropriateness of the marmoset model was questioned, particularly regarding the social and welfare impact of genetically modified marmosets; 1 institution had to specifically address why rodents were not a more appropriate model for the specific research; and another institution noted often receiving questions regarding whether a larger NHP would be a better model. One respondent commented that the quality of the marmoset model for COVID-19 research was specifically questioned, leading to denial of the use of marmosets for that particular research project. One institution indicated that both utility and appropriateness of the marmoset model were heavily discussed when they initiated their first marmoset research protocol.

A total 61% of respondents indicated that the number of marmosets proposed to be included in studies was a concern of their oversight body. One Asian respondent noted that this is always a concern, especially with NHP work, and others indicated that the discussion of animal numbers based on the principal of the 3Rs is an important part of the protocol for the oversight committee. One respondent indicated that details regarding how the number of animals was calculated along with specific animal information (eg, age, sex, identification number, cage, and room location) are required in the protocol application. Respondents from 2 institutions in the United States indicated that their IACUCs express concern over the small number of animals included in studies and that they need to provide clear justification that the small sample size will result in significant findings.

According to respondents, issues surrounding social housing were a concern at 61% of the institutions. All respondents indicated that social housing was the preferred situation but that there are times when that is not possible (ie, due to lack of an appropriate partner or experimental situations). One respondent indicated that without an approved justification for special housing needs, social housing is the standard situation and is decided by the veterinarians. One respondent stated that they were required to submit reports to their oversight board every 6 months giving the number of marmosets that were ever singly housed, the period for which they were singly housed, and the reason for the single housing. The goal of this reporting was reportedly to try to minimize all single housing unless required for veterinary reasons. A common theme articulated was that oversight bodies expressed concern any time marmosets were housed singly and required strong justification in these situations or for any special housing situations.

Cage structure was specifically noted to be a concern of their regulatory oversight body by respondents from 8 institutions (44%) and something that was questioned during routine inspections. Two respondents noted that special cage structures and accessories need to be scientifically justified at their institution and failing justification, caging needs are determined by the veterinarians. One respondent indicated that their IACUC expressed safety concerns regarding cages that were modified for specific research. Two institutions described use of cage sizes that did not technically conform to regulatory standards. One institution noted that they have a specific IACUC-approved exemption to allow family units to stay together, allowing offspring to remain in the family unit up to 1.5 years of age but with a smaller floor space per animal than what is required by The Guide or the Animal Welfare Act Regulations. They specifically justified this exemption by adding perches to the cages and referencing the large cage volume resulting from the height of their cages. Similarly, another institution has a specific IACUC-approved exemption to allow marmosets to be housed in cages that technically have less floor space than required by The Guide and Animal Welfare Act Regulations but overall have more cubic space for these arboreal animals.

As with other NHPs maintained in captivity for biomedical research, environmental enrichment is an important component of captive marmoset care. Thirty-nine percent of institutions responding to the survey indicated that their environmental enrichment program was of particular interest to the IACUC or oversight body, with several respondents stating that any specialized enrichment needs to be scientifically justified and that the environmental enrichment program is reviewed during routine inspections. Responses regarding diet were similar. Thirty-nine percent of respondents indicated that routine diet fed to their marmosets was of concern to their oversight committees and was something discussed during routine inspections. As with environmental enrichment, specialized diets need to be scientifically justified and approved by the oversight committee. Two respondents indicated that their IACUC or oversight body had concerns related to food and water restriction protocols frequently used in neuroscience research.

Only 28% of respondents indicated that their IACUC or oversight board had concerns or questions related to sanitization processes, with 1 respondent commenting that their committee is always concerned with the frequency of cage changes and that in general they do not understand marmoset scent-marking practices. Marmosets are known to scent mark frequently both in the wild and in captivity.^65–68 Unlike Old World monkeys, New World monkeys possess an intact accessory olfactory system,⁶⁹ and olfactory communication is particularly important for them. Studies have found significantly increased levels of scent-marking behavior either when animals are placed in novel, unscented cages or when clean, unscented materials such as branches are added to cages.^70,71 While cages, cage furniture, and enrichment devices should clearly be cleaned to provide adequate sanitation, frequent removal of all scents could lead to abnormally high frequencies of scent marking.⁷² It is likely important for marmoset welfare that familiar scents are not totally removed from the captive environment during routine cleaning, perhaps only sanitizing or replacing one-half of the branches each time.^72,73

Sixty-seven percent of respondents indicated that their institutional regulatory body had questions or concerns related to the specific procedures to be performed with the marmosets as part of their different research protocols. This topic appears to be of most concern, with the highest percentage of our respondents indicating that their review committee questioned or commented on these areas of their protocols. In general, respondents stated that all procedures must be clearly described in protocols and approved by the IACUC or other regulatory committee and that committees often had comments and suggestions specifically for certain surgical procedures. In addition, respondents noted committee interest in acclimation to handling and positive reinforcement techniques and concern with the use of certain potentially harmful substances— including radioisotopes, chemical carcinogens, and heavy metals—in the marmosets. One respondent expressed concern that many of the standard operating procedures that the IACUC uses as a reference are written as general NHP procedures and are not necessarily appropriate, as written, for marmosets. Similarly, another respondent indicated that at their institution, their IACUC tends to view procedures, specifically craniotomy approaches, in terms of macaque standards. A respondent indicated that at their institution, the IACUC will not approve surgical procedures to remove cranial implants so that animals can serve as breeders after they have completed their involvement in the specific research protocol. Of concern for colony management, 1 respondent indicated that their oversight body does not believe vasectomy to be a necessary surgery and that their inability to receive approval for vasectomies impacts social housing options.

The overall number of either a specific procedure or all procedures in the protocol was noted as an issue for 8 (44%) respondents. As with specific planned procedures, the actual number of procedures per animal and often the frequency of procedures must be included in an animal use protocol and reviewed and approved by the oversight committee. One respondent indicated that their IACUC questioned the number of major procedures each animal can physically withstand over a lifetime, and another respondent indicated that their oversight committee questioned the number of imaging and anesthesia episodes an animal could undergo.

Thirty-nine percent of respondents indicated that their oversight committees are concerned with blood draw volumes, both the amount of blood to be drawn overall and in a single draw. One respondent stated that this was consistently monitored and questioned by their oversight board.

Given marmosets’ small body size and lack of zoonotic concerns, it is possible to perform certain procedures on them without the use of sedation/anesthesia, which may be considered a refinement under the principle of the 3Rs, though potential stress associated with awake procedures and appropriate acclimation must be considered. For many areas of research as well as animal welfare, this can be a large advantage over working with Old World monkeys. Devices and techniques have been designed to allow for successful awake restraint of marmosets for a range of procedures, including blood sampling,^74,75 body composition imaging,^22,76,77 ultrasound imaging, various neuroendocrine procedures,⁷⁸ and magnetic resonance imaging.^52–55 Although these are proven techniques, 2 respondents indicated that their oversight bodies still express concern over procedures being conducted without the use of anesthesia. Overall, 33% of respondents indicated that the use of anesthesia for procedures as opposed to working with animals under conscious restraint was a concern for their oversight committee. In addition to questions regarding the ability to perform procedures on awake marmosets, 1 respondent indicated that their committee requires them to use sedation to reduce any potential stress associated with in-house transport.

Thirty-nine percent of respondents indicated specific sedation, anesthesia, and analgesia plans associated with planned research was a concern of their IACUC or other oversight body. All indicated that detailed sedation, anesthesia, and analgesia plans must be included in their animal care and use protocols, and these must be reviewed and approved by their oversight committee. One respondent stated that ventilator/respirator circuit function and safety was of concern for their IACUC, and another indicated that their committee was not supportive of the use of injectable agents for mid-length procedures, preferring the use of inhalant anesthesia for all procedures.

MAJOR REGULATORY OVERSIGHT CONCERNS: SURVEY RESULTS

The final questions of the survey addressed major themes related to the regulatory oversight of marmoset use in biomedical research. We specifically asked 3 open response–style questions: (1) what appear to be the major concerns of the IACUC or oversight board related to marmoset research at your institution? (2) Does the IACUC or oversight board appear to have similar concerns related to other NHP research at your institution? And (3) Does the IACUC or oversight board appear to have similar concerns related to other animal research at your institution?

In response to the first question, 5 respondents (28%: 1 from Asia, 1 from Europe, 3 from North America) indicated that overall, their oversight bodies had no major concerns with their marmoset research and were in general supportive of their research protocols. The remaining respondents noted various major concerns expressed by their oversight boards (Table 1). These concerns ranged from the public perception of participating in NHP research, to general animal welfare concerns and more specific issues related to justification for the animal model, animal availability, housing and environmental enrichment practices, and how marmosets cope with the research performed. There was no single major concern that was mentioned by a majority of respondents, and no clear regional differences were apparent. The most frequently identified theme was the low number of animals available for research both from vendors and within respondents’ colonies and associated concerns regarding genetic diversity.

Table 1.

Noted Major Concerns of the IACUC or Oversight Boards Related to Marmoset Research

Major concerns noted	Where noted
Public perception	NAm
Overall animal welfare	NAm
Why marmosets instead of rodents	Asia
Challenges working with small sized animals	NAm
Fragility of marmosets	NAm
Low numbers of animals available for use	NAm
Lack of genetic diversity	NAm
Lack of availability from vendors	NAm
Status of breeding colonies	NAm
Housing conditions	NAm
Single housing	Europe
Social enrichment	NAm
Appropriate procedures for marmosets	Asia, NAm
Difficult post-procedure recoveries	NAm
Genetic manipulation	NAm

As noted in response to the open survey question: What appear to be the major concerns of the IACUC or oversight board related to marmoset research at your institution? NAm, North America.

In response to the second major question–does the IACUC or oversight board appear to have similar concerns related to other NHP research at your institution?–28% of respondents indicated that they had only marmosets at their institution or that currently no other primate research was being conducted at their institution, 39% indicated that in general at their institution marmoset research received the same type and level of concern as did research with other primate species and the remaining 33% indicated that there may be some differences between review and oversight of marmoset protocols vs those in other NHPs (Table 2). Specific reasons (Table 2) for the potential differences in oversight included:

Table 2.

Response to ‘Does the IACUC or Oversight Board Appear to Have Similar Concerns Related to Other NHP research at your Institution?’

Response	Number of Respondents	Where Noted
Only marmosets or no other primate research	5/18 (28%)a	Asia, Europe, NAmc
Same level of concern as other primates	7/18 (39%)a	Asia, Europe, NAm
Different level of concern from other primates	6/18 (33%)a	NAm
Marmoset is new species	2/6 (33%)b	NAm
Macaques are default	2/6 (33%)b	NAm
Concerns specific to marmoset	1/6 (17%)b	NAm
Concerns specific to population types	1/6 (17%)b	NAm

^aNumber of respondents indicating this response/total number of respondents (% total respondents)

^bNumber of respondents indicating this response/number of respondents indicating that at their institution marmosets receive a different level of concern than other primates (% institutions with different levels of concern).

^cNAm represent North America.

1. Their institution considered the marmoset to be a “new” species, and therefore staff training and expertise with marmosets was more limited than with other NHPs, potentially leading to some differences in review and oversight practices,

2. Macaques are used as the default reference at their institutions as the majority of the focus of their institution and their institutional oversight body is on macaquework,

3. The concerns of their institutional oversight body with marmoset work (ie, lack of availability of marmosets from vendors that could be used to increase genetic diversity in their colony and the small size of the animals making them a challenging surgical model) were specific to marmosets and therefore did not apply to other NHP protocols at their institution,and

4. The concerns of their oversight body differed between their NHPs due to issues specific to each population (eg, the age and health of the animals).

For the third question asked—Does the IACUC or oversight board appear to have similar concerns related to other animal research at your institution?–44% of respondents indicated either that they did not know the answer or that their institution did little or no research with non-primate species so they were unable to answer the question. Three respondents indicated that all animal use protocols undergo the same review process regardless of the species. Five respondents indicated that while marmoset research protocols undergo the same review process as protocols for use of other species, the oversight bodies seem generally more strict with primate research than non-primate research, though 1 respondent indicated that while this is true for macaque research at their institution, marmoset protocols do not generally seem to garner the same level of scrutiny. The remaining 2 respondents simply indicated that no, their oversight bodies do not appear to have similar concerns with other animal research at their institution. These “no” responses appear to be related to the specific, marmoset-focused concerns expressed by the oversight body (eg, small population size).

DIFFERENCES BETWEEN CONTINENTS

Unfortunately, given the small number of responses from non-US countries, we were unable to form a clear understanding of any differences in oversight body concerns with marmoset research between continents. While the small number of responses clearly impacts our interpretation, we can say that based on the responses we did receive, there were no obvious difference in IACUC or oversight body concerns between countries. The 1 difference that we were able to appreciate with these survey responses was that while biomedical research with marmosets was noted to be either stable or decreasing in Europe, use was clearly increasing in Asia and North America. Another difference noted based on the regulations is that European guidelines for floor space and cage height are more generous than those in North America. While US regulations determine cage size based solely on animal weight, European regulations include the following in consideration of their cage size requirements: adult size of the animals, the size of the group, and sufficient space to create a complex and challenging environment.⁶³ The European regulations also appropriately focus on vertical space over floor space for these arboreal animals.

IMPORTATION OF ANIMALS, SPERM, OVA, AND EMBRYOS

One option for increasing marmoset populations is to import from source countries. This is particularly challenging for several reasons. First, an export permit from the country of origin must be acquired. Moreover, only purchase of purpose-bred >F2 animals is permitted. In addition, in response to pressure from animal rights groups, many commercial airlines will no longer transport animals to be used for research, creating logistical transportation challenges and increased costs.⁷⁹ There are likely to be additional regulations associated with both the origin and destination locations that must be adhered to as well. A further concern associated with importation of sperm, ova, and embryos is standardization of techniques to successfully acquire, store, and ship materials. There are efforts underway to address many of these issues, but at this time there are no easy ways to overcome these challenges.

RECOMMENDATIONS FOR REGULATORY OVERSIGHT OF GENE-EDITED MARMOSETS

Regulation of gene-edited animals requires additional consideration. The purpose of genetic modification is often to produce a disease model that more closely resembles the human condition or to create model tools for specific uses (eg, animals that are modified to have specific neurons labeled for imaging). This has the potential to cause both intended phenotypic changes that may be harmful and unintended harm. The intended phenotypic changes may come directly from modeling the human condition of interest. For example, if a gene in humans known to be related to Parkinson’s is edited in marmosets, the outcome should be a marmoset that exhibits the symptoms of Parkinson’s. With this model, there is also the potential for unintended outcomes related to the unknown, off-target negative effects of the manipulation. Both outcomes are of concern and should be carefully evaluated by regulatory bodies according to the 3Rs. Oversight of this work may need to include specifically designed assessment tools such as disease- or condition-specific scoring tools and endpoint determinations and enhanced reporting to institutional oversight bodies, and should also include evaluation of the breeding program required to support transgenic research. A clear and thorough understanding of the human presentation and progression of the disease or condition being modeled may provide valuable insight into specialized care that the animal model may require.⁸⁰

FUTURE OF MARMOSET RESEARCH REGULATION

There are some perceived overall trends in research animal oversight that have an impact on marmoset work. Specifically, there has been increasing involvement of the lay public in issues related to animal research regulation with increasing use of the legal system to gain access to research records⁸¹ coupled with a failure of many to appreciate the importance of basic science research and a reductionist approach to the 3Rs that primarily focuses on replacement.⁵⁸ In an effort to support a reductionist approach, the emerging field of animal rights law has focused on “personhood” claims for animals.^79,81 In the United States, an appreciation of the increasing administrative burden associated with animal research regulation⁷⁹ has led to guidance from federal regulatory bodies that allows for streamlining of some processes associated with changes to approved research. For marmoset research specifically, as interest in the use of marmoset models particularly for transgenics and neuroscience continues to grow, so will the sizes of the colonies needed to support this work. Maintenance of larger colonies requires additional considerations such as the impact of cage and room density on animal welfare and the availability of appropriately trained staff. Growing interest in marmoset models also dictates a need to establish welfare-based approaches to management throughout the life cycle. There is also the potential that with increasing numbers of marmosets being used in biomedical research, scrutiny from groups opposed to this research will increase.

CONCLUSIONS

Marmosets have been used in biomedical research for decades, but their use has increased dramatically in recent years. With this increased interest, there is a crucial need to both understand the regulatory environment and to better address issues related to the use of marmosets in research. There needs to be enhanced appreciation for the fact that marmosets are different from macaques and may require adaptation of some of the regulations as well as specifically trained personnel. Existing expertise should be made available to, and utilized by, those working to establish and maintain new colonies. This is being addressed in part by the Marmoset Working Group, collaboration established in early 2019 between multiple institutions with broad-ranging marmoset expertise. One goal of this group is to make a comprehensive set of marmoset-specific information available online to the marmoset community, including protocols and standard operating procedures for common techniques, housing, and enrichment. There must also be a carefully considered and coordinated plan to increase the availability of genetically diverse marmoset populations for biomedical research. In the United States, this is the goal of a new initiative of the NIH with particular focus on animals for neuroscience research. In conclusion, with the growing use of marmosets in biomedical research, care must be taken to address welfare, ethical use, and regulatory considerations specifically and appropriately for this unique species.

Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA; Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, Wisconsin, USA; Biomedical Primate Research Centre, Rijswijk, the Netherlands; University of Cambridge, Cambridge, United Kingdom; Primate Research Institute, Kyoto University, Kyoto, Japan; Department of Life Sciences, Texas A&M University, San Antonio, Texas, USA; and Population Health, Texas Biomedical Research Institute, San Antonio, Texas,USA.