The Housing of Non-Human Primates Used for Experimental Research and Other Scientific Purposes: Issues for Consideration.

Discussion document prepared by the EUPREN Board of Management

1.       Background

Housing of laboratory animals is to be considered at the May 1997 Multilateral Consultation of Parties to the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes. Although the number of non-human primates used for such purposes is relatively small, the issues which need to be addressed in the discussion of their housing are particularly complex. There is, for example, a great diversity of morphological, physiological, psychological and social requirements for the different species of non-human primates used in research. The present paper has been prepared by EUPREN in advance of this Multilateral Consultation, in order to highlight the key issues which need to be considered in any discussion of housing for non-human primates.

It is clear that altering the detail of the European legislation on cage requirements could have wide-ranging implications. While such alterations offer considerable potential to enhance animal well-being, the financial investment required may lead to research being relocated to countries where welfare standards and control are less favourable than in Europe. It is therefore of critical importance to ensure that the legislative guidelines are based upon firm scientific foundations.

Many pre-existing guidelines are somewhat subjective in that they have been formulated on the basis of current practice, economic considerations and good intent. Relatively recent developments in remote and non-invasive monitoring enable the impact of different housing conditions to be assessed more objectively. The underpinning technologies and approaches for such studies were discussed at a EUPREN/EMRG Meeting on "The Implications of Non-invasive and Remote Monitoring Techniques for Non-human Primate Research and Husbandry" (December 1995, Göttingen).

In view of the paucity of fundamental scientific information, there is an urgent need to collate available data, draw up a list of questions which need to be asked and agree upon the collaborative programmes which are required to provide the answers to these questions.

The progress of and recommendations which emerge from this document will be presented during the second EUPREN/EMRG Winter Workshop, "The Implications of Housing and Husbandry for Scientific Quality and Well-being of Non-human Primates".

2.       Factors for Consideration

2.1     Scientific Purpose

The purpose of the particular biological or biomedical research in which non-human primates are involved is an essential underlying consideration. The following example of two extreme situations illustrate the importance of addressing the scientific objectives of the study. In some pharmacological or toxicological investigations, there may be a need to administer test substances, collect blood samples and monitor aspects of metabolism over days. The type of procedures involved and the degree of human intervention required might be expected to require fundamentally different housing from that appropriate to a long term study to investigate hierarchical behaviour in large social groups, with minimal human interaction. Although intuitively one might feel that it is always desirable to provide the largest cage which resources permit, it may well be that the impact of catching and restraining animals for interactive procedures, such as blood sampling, is more marked in animals housed in larger cages or social groups.

It is obvious that the standards and approaches to the provision of housing non-human primates being purpose-bred for subsequent use in research must be no less stringent.

It is proposed that, as a first step, a list should be compiled of the types of scientific and husbandry procedures and associated related scientific rationales, which are currently in use in different areas of research.

Such a list might establish the basis of a categorisation system. This would facilitate discussion of such issues as the scientific justification for single housing could lead ultimately to standardisation of studies involving non-human primates.

2.2     Species Needs and Propensities

As well as factors such as scientific purpose, type and frequency of procedures/human intervention, laboratory housing must be sensitive to species-specific requirements. It is evident that factors such as sexual dimorphism, age, size and group dynamics need to be considered. Of course, captive housing always imposes limitations on the realisation of the behavioural repertoire which would be expected to exist of a particular species in the wild. Nonetheless, it is important to enable animals to exhibit as many aspects of their behavioural repertoire as practicable, within the context of the experimental situation.

While cage floor area and volume are obviously issues which must be considered in addressing species needs and propensities, they must not be considered as the sole determinants of housing quality. The wider considerations of cage design must be discussed and there are potential dangers in overemphasizing floor area and volume. Cage design should encompass features such as usable cage space, furniture, flexibility, configuration, and feeding provision.

2.3     Assessing the Impact of Laboratory Housing

In order to obviate the danger of establishing contraproductive "pseudostandards" which may be adopted uncritically, it is necessary to conduct well defined and tightly focused scientific studies to determine the impact of laboratory housing on the animals. The resulting information will have significant implications for subjective assessment of animal well-being as well as scientific quality.

A co-ordinated multidisciplinary approach will be required because of the inadvisability of reliance upon a single index to assess impact. It is likely that a combination of physiological, endocrinological, behavioural, immunological and biochemical factors will need to be considered . With this in mind, it will be necessary to agree priorities and standardise experimental protocols in order to enable information from diverse sources to be considered together. The establishment and maintenance of an accessible database for relevant existing data and information on proposed work as well as work in progress, will be a key feature.

As well as considering the impact of cage design, it will be important to address the relationship between laboratory housing and procedures conducted for scientific or husbandry purposes. For example, it would be of interest to investigate the implications of training animals to co-operate with experimental procedures or of varying the housing conditions for individual animals in situations where experimental protocols required more restricted caging for a small proportion of the study duration.

Such information would be invaluable in the formulation of meaningful standards of animal care.

Transplantation and xenotransplantation.

Blancho, G.
Nephrology/Clinical Immunology/Transplant Unit
University Hospital Nantes, France

Progress in transplantation is dependent on intensive research using animal models that allow informative data at an vivo level to be obtained. Because of a crucial organ shortage, the strategy to use animal organs to transplant human patients (xenotransplantation) is now more and more envisioned and studied. The major obstacle to succeed in this strategy is the existence of so called natural antibodies that recognize specific epitopes at the surface of the donor endothelial cells and initiate hyperacute rejection. Xenografts may be classified according to the kind of rejection they undergo. The combination is termed discordant when natural xenoantibodies are present, leading to hyperacute rejection within minutes to hours. Alternatively, the combination is termed concordant when they are absent and the natural evolution is an acute vascular rejection after three to four days. In the human situation, the dominant epitopes recognized by natural antibodies appear to be Gal1,3Gal oligosaccharide residues. Although some clinical experiences using nonhuman organs for transplantation into human beings have already been performed, intensive research is still ongoing to learn the mechanisms involved in hyperacute rejection and acute vascular rejection, and to learn how to control them. Different discordant models are very useful, and a classical one is the pig to primate model which mimics closely the potential future clinical combination of pig to human. The primates of interest are the Old World monkeys since like human beings, they do not express Gal1,3Gal epitopes on their endothelial cells, and as a consequence produce natural antibodies against this epitope. Therefore, macaques monkeys and/or baboons would be the most suitable recipients of pig organs in pre-clinical trials. Although primates should be at an immunological level the best candidates to provide organs to humans, they will probably not be utilized for human transplantation for several ethical reasons, and also because of a potential risk of transmission of infectious diseases to humans, especially by retroviruses. In allotransplantation (transplantation between animals from the same species), monkeys are also very useful since they mimic very closely the human situation. Several groups are conducting experiments in allotransplantation with monkeys for the evaluation of efficacy and/or toxicity of different new immunosuppressive drugs. Thus, the need of primates in transplantation will be increasing in the future for a better understanding of the mechanisms involved in the allo and xenorecognition and their responses. However their utilization will most likely remain at a research level without human clinical applications.

The housing of non-human primates in the comercial research laboratory.

Brown, C.; Goburdhun, R.; Kirkwood, A. & Robb, D.
Inveresek Research
Tranent EH33 2NE, Scotland

Biomedical safety evaluation studies remain a key factor in the development of novel therapeutic agents in a world in which improved health care remains very high both on political and public agendas. These studies typically require the use of 2 species, one rodent and one non-rodent. Whilst the default non-rodent species is the dog, and despite ever increasing scrutiny of experimental use of non-human primates, monkeys continue to be used in large numbers in safety evaluation studies. Current trends in the development of novel pharmaceuticals, notably biologicals and products derived from biotechnology, suggest this will continue for the foreseeable future. It has been necessary, therefore, to carefully examine the experimental procedures applicable to primates, and to refine these, setting ever higher standards where possible. The conditions in which animals are maintained have received particular attention, with the aim of constructing an enriched environment in which studies may be conducted.

In the authors' laboratory, only purpose-bred animals are used. Group housing of animals (by sex and treatment group) is now routine. Animals are smaller, more homogeneous in origin and specification, and easier to handle and train. This environment not only allows advantages in animal welfare, but provides a better animal, hence a better animal model, for use in safety studies.

This poster elaborates upon procedures for housing and handling macaques in a typical biomedical contract research laboratory, and will illustrate the close harmony that exists between considerations of animal welfare and good scientific practice.

Genetic aspects of colony management.

Bontrop, R.; Noort, R.; Otting N. & Kos, T.
Biomedical Primate Research Centre
Rijswijk, The Netherlands

The Major Histocompatibility Complex (Mhc) of many non-human primates is characterized by a large number of genes that arose by numerous duplications. Some of these genes encode cell surface molecules displaying an extensive degree of allelic variation at the population level. Mhc molecules provide the context for recognition of antigens by T cells and as such play a key role in mounting an immune response. Most vertebrate species possess two major classes of the molecules exhibiting specialized functions. The Mhc class I molecules also referred to as the classical transplantation antigens, bind antigen peptides from intracellular origin. Mhc class I molecules complexed with peptides (from a virus or parasite) may be recognized by CD8+ cytotoxic T cells triggering the lysis of the infected target cells. Mhc class II molecules present peptides of extracellular origin, for instance bacterial antigens to CD4+ T helper cells. Successful activation of T helper cells may result in cytokine release, followed by antibody production or helping cytotoxic T cells to lyse their targets.

At our institute Mhc polymorphisms are also used at the level of colony management. The segregation profiles of polymorphic markers allows one to pedigree animals. By means of this method we control to which extent our non-human primate colonies maintain an inbred or outbred character.

Housing and behavioural testing for long term studies.

Bowditch, A.P.¹, Crofts, H.S.², Muggleton, N.G.¹, Pearce P.C.¹, Prowse S.¹ & Scott, E.A.M.¹
1CBDE Porton Down, Salisbury, UK
²University of Bristol, Bristol, UK

In many studies (including pharmacological and behavioural studies) it is necessary to follow the effects of drugs or the progression of disease states over months to years, using appropriate animal models. The generation of stable performance parameters is critical to the success of such studies, particularly where sensitive behavioural indices are being measured. In order to achieve this, careful consideration must be given to housing and the behavioural testing methods from the outset, such that animals are provided with a stable environment throughout the study.

An approach that has enabled us to achieve success in long term studies, using both marmosets (Callithrix jacchus) and rhesus monkeys (Macaca mulatta), is to combine group housing with home-cage behavioural testing. Marmosets are pair-housed (mixed sex with vasectomised males) and rhesus monkeys (juvenile males) are housed in groups of four. For both species multiple cages units joined by rigid linkers are used. During behavioural training and testing sessions, animals are separated by removing or closing the linkers. The test is presented to each animal by attaching the apparatus (a computer controlled touch-sensitive screen) to the home cage. Performance data that is stable over periods of greater than 12 months have been obtained for both species. The graph below is a typical baseline from one rhesus monkey, showing the number of test sessions required to complete repeated sequences of the behavioural task (an attentional set-shifting paradigm) over a 16 month period.

Further data will be presented, and the benefits of our approach discussed.

Primate housing facilities for pharmaceutical research in Switzerland.

Buerge, T. & Weber, H.
SANDOZ PHARMA Ltd.
Basel, Switzerland

According to Swiss Animal Welfare Legislation primates should be housed in groups. The minimal enclosure area for macaques of the size of rhesus or cynomolgus monkeys for experimental purposes is 15 cubic meters (e.g. a floor area of about 8 square meters and a height of 2 m.). In such an enclosure up to 5 adult animals may be kept with further 2 cubic meters for each additional animal. For experimental purposes the animals may be held in individual cages temporarily but should then have access to an enclosure of the above mentioned area. A housing system which complies with these regulations and still meets the requirements for animal experimentation in pharmacological research will be presented. The system consisting of cages combined with an indoor enclosure was developed on the basis of our experience gained already in the years before the Swiss legislation came into force. It enables the pharmacologists to use monkeys over years for periodically repeated experiments and to keep them in small groups during the intervening period. Only minor problems, chiefly related to aggression and group incompatibility with some animals when they reached sexual maturity have been encountered.

The ethologist's view.

Deputte, B.L.
European Federation for Primatology
C.N.R.S./UMR 373, Paimpont, France

To maintain primates in captivity for whatever purposes requires to primarily fulfill their social needs. However, in captive environments, these social needs generally result from a compromise between what is known from wild groups and the scientific requirements. To set a compromise implies to reach the optimal one and consequently to scientifically test several possible alternatives. Single-cage housing which should remain an exceptional procedure, has raised the concern of enriching the environment. To put something in a bare cage has been often considered as an environmental enrichment. This is intuitively true; however in some cases it could also yield no improvement at all to the well-being of the monkeys, at a great economic cost. Again a scientific evaluation of what is considered as good from a public point of view, is necessary. I will argue that ethologists have a special and efficient approach to these problems. Ethology is the study of behavior in an evolutionary framework, meaning that the behavior is considered as the output of the integration of the subject-environment interaction. Therefore ethologists always keep in mind what is "natural" for a subject of a given species and so to what extent the monkey is able to cope with a very different environment, given his species-specific constraints. To be considered as beneficial for the subject's well-being the features of an environment have to remain within the subject's range of adaptive mechanisms. I will present two ethological analyses: one experimental, dealing with the effects of an environmental enrichment on the social behavior of a group of captive grey-cheeked mangabeys and another one stressing that, within a given species, biological needs are dependent on the age and the sex of the subjects.

Requirements of biomedical research in terms of housing and husbandry: Neuroscience.

Fuchs, E.
Division for Neurobiology
German Primate Center
Goettingen, Germany

The complexity of the human brain, nervous system and behavior requires the use of non-human primate models to gain a profound understanding of central nervous processes in man. This understanding is a prerequisite for the development of new strategies and approaches to treat brain, nervous system and mood disorders. Therefore, research on non-human primates played a central role in many of the important discoveries in neuroscience in the past and will play an important role in the future.

The presentation will be comprised of three parts. First, a brief review of the role of primates in neuroscience research will be given. Examples will address issues related to the unique contributions made to neurobiology through the research on primates. Part two will address current areas of neuroscience in which there is a need for research in monkeys. Based on these examples, the requirement in terms of housing and husbandry for preclinical research in non-human primates will be discussed in the third part.

Sociophysiological aspects of short-term separation in common marmosets (Callithrix jacchus).

Gerber P.^1,2; Schnell C.R.² & Anzenberger G.¹
1Anthropologisches Institut und Museum der Universität, Zürich
²CIBA Cardiovascular Research Department, Basel, Switzerland

Under captive conditions, common marmosets exhibit a monogamous lifestyle. Social challenges to the integrity of such an individualized pair-bond, as separation from the mate, will trigger coping mechanisms at different proximate levels of behaviour. Although there have been several experimental investigations of the pair-bond in marmosets, no information is currently available on the sociophysiological correlates of this motivational construct.

For this study data were therefore recorded simultaneously at three levels (ethological, sociophysiological and socioendocrinological). This allows monitoring of behaviour in parallel with changes in both physiological control systems, the sympathetic-adrenal-medullary axis and the hypothalamic-pituitary-adrenal-cortical axis. Here only the cardiophysiological responses and changes in locomotor activity to a short-term separation for females and males respectively are presented. An integrated miniaturised telemetry and data acquisition system was used to measure blood pressure (mean arterial blood pressure = MAP) and heart rate (HR) as well as locomotor activity (ACT) in conscious, unrestrained marmosets. Blood pressure, heart rate and locomotor activity were recorded in cyclic runs of 1 min. During each cycle, the animal was measured for 11 s with a sampling rate of 250 Hz. Pair-mates of 6 established pairs were tested. Every animal, female and male, was tested in three replicates. The experiment was divided into three 10-min segments (baseline/separation/reunion). During baseline the pair was left undisturbed in the home cage. For separation both pair-mates were captured but one was returned to the home cage (home cage) whereas the other was transferred to a cage equivalent to the home cage in an unfamiliar room (unfamiliar cage). In the third segment the pair was reunited in the home cage (reunion).

Table: Reactions of females (F) and males (M) during the different segments (home cage/unfamiliar cage/reunion) shown as percentages of increases in mean arterial pressure (MAP), heart rate (HR) and locomotor activity (ACT) compared to baseline.

Females and males showed similar pattern, i.e. in both sexes MAP and HR reached maximum values during 'unfamiliar cage' and ACT was highest during 'reunion'. By comparing results for 'home cage' and 'unfamiliar cage' there were no significant differences in activity, but MAP and HR were clearly higher during 'unfamiliar cage'. This shows, that separation in combination with a transfer to an unfamiliar cage constitutes a particularly stressful event. In other words, a familiar environment can clearly reduce the stress of a separation.

Acknowledgment: This study was supported by a Swiss National Science Foundation Grant (Nr. 82IN-42601).

Housing and caging.

Herrenschmidt, N.*
Centre de Primatologie, Université Louis Pasteur
Strasbourg, France

Good breeding and maintenance procedures for primates in captivity involves the animals being kept in conditions that acknowledge the particular requirements of each species alongside consideration of restrictions required by research protocols and experimental procedures.

Different maintenance structures (cages, outdoor runs, enclosures) will be presented, as will technical aspects pertaining to the materials used, housing systems, environmental enrichment methods, etc.

In view of current pressure from various European consultation exercises to modify Table 9 of Annex A of the Convention, pertaining to cage sizes, propositions by EUPREN in this respect will be presented. These propositions signal an entirely new approach to the problem.

* member of the EUPREN managing board.

Primatological research in Europe; the academic world.

van Hooff, Jan A.R.A.M.
Ethologie & Socio-œcologie Groep
Universiteit Utrecht, The Netherlands

Western Europe takes a second place between Northern America and the Rest of the World with respect to the output of scientific publications in the field of primatology. University institutions are responsible for the majority of this output, although the ratio of university productions versus productions by other institutions and firms differs according to scientific subdiscipline. The same is true within Western Europe. Of the nearly twenty university institutions that do research on primates kept within these institutions some twelve address problems concerning either social behaviour and social organization, or neuropsychology, perception and cognition. Some of these institutions are correspondingly involved in field research concerning (socio-) ecology, population dynamics and genetics. Other topics, namely functional morphology, behavioral development, reproductive physiology, neurophysiology, neuropharmacology, motoric disorders, and virology, each receive attention in only one or two institutions, whereas still other important topics, such as immunology, do not seem to receive explicit attention. The fundamental orientation which prevails in the university institutions forms a worthwhile complement to the more strategic and applied orientations that characterize institutions outside the academic world. The great interest of university primatological groups in the complex social phenomena that characterize primates brings with it an interest in the social aspects of welfare issues, on the one hand, and in the role of social factors in a variety of functions, such as stress effects on cognition and aging. This will be illustrated with a few examples.

Specific requirements of Macaca species.

van Hooff, Jan A.R.A.M.
Ethologie & Socio-œcologie Groep
Universiteit Utrecht, The Netherlands

Primates are interesting as models in research for two main reasons: First, their phylogenetic relatedness to our own species makes them relevant as heuristic models in certain areas of physiological, neuro-physiological, neuro-ethological, and psychological research. Secondly, most primates display complex social organizations; therefore, social processes and the role of social variables as factors form a specific domain of interest. This has as a consequence that in maintaining primate populations for research, one has to pay special attention to the role of sociality as a welfare factor. It also means that adequate social conditions may be important for valid research in fields which not specifically social.

Especially with respect to social phenomena, one has to realize that primates show enormous interspecific (as well as intraspecific) variations. Macaques have always been one of the most accessible taxonomic groups for research purposes. However, a macaque is not a langur is not a callithrix is not an ape. Adequate knowledge of the social organization and the socio-ecology should be at the basis of any program of keeping these animals in captivity. In this presentation the variation in social strategies that result in particular social organizations will be dealt with, as well as the specific character of 'the macaque', and some specific complications that arise from their socio-sexual organization with respect to a responsible maintenance in captivity will be treated.

Viral diseases and zoonoses of primate origin.

Hunsmann, G. & Nisslein, T.
German Primate Center
Goettingen, Germany

Biomedical research has a need to use non-human primates in cases where the information required or the necessary predictability of results for the development of medications or vaccines cannot be obtained in another species.

Viral infections of primates are employed in such experiments potentially dangerous for both animals and men, since early diagnostic and therapeutic measures have not yet been developed and prophylactic vaccines cannot be applied or are not available. Major viral diseases of man may threaten primates both in captivity and in the wild. Moreover, viruses not causing diseases in their natural non-human primate hosts may spread and induce diseases in human populations. Five modes of transmission of viruses among non-human primates and humans can be discriminated: (1) intraspecies, (e.g. Ebola Reston virus), (2) interspecies among non-human primates (e.g. Simian Immunodeficiency Virus), (3) interspecies from non-human primates to man (e.g. Herpes virus simiae or Marburg virus), (4) interspecies transmission from man to non-human primates (e.g. measles or hepatitis A virus), and finally (5) interspecies transmission via arthropod vectors (Yellow fever virus).

Safety precautions to avoid such transmission will be summarised briefly.

General aspects of primate keeping and husbandry.

Kaumanns, W.
German Primate Center
Goettingen, Germany

In Europe about 25.000 primates in about 200 species and subspecies are kept in almost 600 institutions in 40 countries. About 85% of the primates are living in zoos; 15% in research institutions. The latter cover a small spectrum of species only. Most European primate populations are too small and demographically unsuitable to serve as models and reservoirs for wild populations. The zoo community therefore has made efforts to improve the situation by evaluating the captive populations and by carrying out various management programs. They are organized above the level of individual institutions and provide concepts and considerations which might be helpful for the management of non-zoo primate populations, too. The genetic management of small populations is an important topic within this framework.

Recently developed more complex approaches to the establishment of selfsustaining captive populations of wild animals however put more emphasis on the integration of basic concepts and findings of Evolutionary biology like "Adaptation", "Adaptive potential", "Fitness", etc. According to this approach the relevant entity for management considerations would be the individual phenotype of an organism, which includes its genotype and characters which are acquired via learning. Management programs have to produce "adaptive phenotypes" which in order to contribute to an adaptive population furthermore have to show a certain degree of phenotypic variability.

To develop these concepts further and to assess their practical consequences for colony management requires a multidisciplinary approach which refers to genetical, physiological, behavioural and other aspects of primates. It has to refer to the fact that captive primates live under more or less restricted conditions which ideally should still fit with their adaptive potential. A science based approach to primate keeping here meets Conservation biology which has to deal with reduced wild populations suffering from habitat destruction and the potential of which to cope to this has to be assessed.

Measuring psychological well-being: a methodological approach using heart rate telemetry, space utilization and behavioral time budgets in common marmosets.

Kerl, J.
Ethologische Station Sennickerode, Institute of Anthropology
University of Goettingen, Germany

The captive environment of a non-human primate (NHP) provides all parameters to the animal that influence its well-being. Even though many investigations on environmental enrichment for NHP's have been published the question still remains how the animal is influenced by the inanimate environment. In studies on Callithrix jacchus some sensitive parameters of the animals reaction to experimental environmental change (cage-size and cage-equipment) were found: the mean heart rate, space utilization and behavioral time budgets. The mean day heart rate responded to variations in cage-size when the cages were equipped very simply but not when they were richly equipped. The mean night heart rate responded only to cage size, irrespectively of its equipment. The most useful heart rate parameter in detecting influences of cage-size and cage-equipment is the difference between the mean day and the mean night heart rate. Via quantification of the space utilization the places for feeding devices and sleeping boxes were proved to be wrong in the simply equipped cages in comparison to the situation when additional feeding devices and sleeping boxes were offered in the same-sized enriched cages. Furthermore the calculated behavioral time budgets showed to be sensitive especially in detecting differences between the responses of the marmosets to different cage equipments. The problems of calculating behavioral time budgets as well as the lack of standardization in order to obtain comparable results are discussed.

Supported by DFG grant Ro 356/14-1

Ethological studies of well-being in two species of macaques after transition from single cages to housing in social groups.

Ljungberg, T.¹; Westlund K.¹ & Ryden, L.²
1Department of Zoology, University of Stockholm
²Laboratory Animal Unit, University of Uppsala, Sweden

Two species of macaques (M. mulatta and M. fascicularis) have been used for several decades in biomedical research at the University of Uppsala, Sweden. The monkeys were previously housed in single cages and enrichment routines were not ordinarily used. Infants of M. mulatta were weaned early, kept separated from the mother and often hand-fed by the keepers. These monkeys thus became, at least partially, imprinted on man and this routine was deliberately used for facilitating handling. During recent years, the housing of the macaques has been changed and larger cages and group-housing has been introduced. Interestingly, different strategies have been used for the M. mulatta and fascicularis. For the fascicularis, cages with a floor area of 2.8-3.6 m2 and with a height of 2.25 m were used. In these cages, 2-4 animals, tested for compatibility, formed a group. Enrichment material was provided. For the mulatta, larger cages were constructed and each of six cages consisted of a 100 m2 outdoor area and 30 m2 indoor area. No enrichment material except a row of wooden shelves on the walls of the indoor area was provided. The groups at the time of study consisted of between 3-8 animals, with a single male present in each group. Ethological studies of social interactions, affiliative and aggressive behaviours and time-budgets were performed for both species using previously developed ethograms and focal sample technique. Each animal was the focal individual for 80-100 minutes. The social interactions between the M. fascicularis in their small groups were characterized by low levels of aggression and social grooming at normal levels including all individuals. Time-budget studies showed effect of enrichment in that foraging times were high and abnormal behaviours were almost absent. The results for the M. mulatta were radically different. Over all, the animals were very inactive. The groups were not well functioning, with certain individuals in each group being the main victims of the observed aggression and being without social grooming relationships. High levels of auto-grooming was often seen in these animals. Stereotypes or other forms of abnormal behaviours were seen in almost all animals. We also studied the occurence of reconciliatory behaviour, a behaviour previously described in great detail in the rhesus monkey. In 4 of the 5 rooms studied, reconciliatory behaviours were absent. Our results show that the change from keeping macaques in single cages to keeping them in groups has to be done with awareness of the advantages and problems associated with different forms of group-housing. It is not recommended to on a trial-and-error basis try to create new, large social groups with e.g. adult rhesus macaques, especially when the animals have a history of early weaning, social separation and imprinting on man. It is not enough to just give macaques more space, this has to be combined with adequate knowledge of primate social behaviour and an enrichment program.

Resocialisation of rhesus macaques.

Louwerse, A.L.¹; Kos, T.² & van Hooff, J.A.R.A.M.¹
1Department of Comparative Physiology, Ethology & Socio-ecology, University of Utrecht
²Biomedical Primate Research Centre, Rijswijk, The Netherlands

The new legislation on housing and husbandry of non-human primates forces research institutes to house formerly single caged experimental animals in social groups. Due to the poor ethological expertise in medical research centres and because singly housed experimental animals lack social skills, the first resocialisation projects were rather unsuccessful.

Last summer the B.P.R.C. collaborated with the Ethology department of the Utrecht University on a program to resocialise adult experimental animals. An effort has been made to create such groups, not only to increase the well-being of the monkeys, but also to prevent the importation of non-human primates from breeding centres in source countries. Guidelines were established in order to form stable social breeding groups. Important aspects of these guidelines were species specific behavioural information and ethological training of biotechnical personel. Using this protocol, the first new breeding group was established, existing of 7 adult unrelated females and one male.

Behavioural observations made of this first resocialisation suggest that collaboration between medical and behavioural research institutes will contribute to the formation of more of these stable social breeding groups in the future.

Where does primate research go from here?

Mahouy, G.
Institut d'Hématologie, Hôpital Saint-Louis
Paris, France

Because of their close phylogenic relationship to men, non-human primates are of great value, often indispensable, for the investigation of a number of human health problems. They will continue to play a key role in crucial areas of biomedical research. On the other hand, it is also clear that the welfare of these intelligent animals with complex behaviour and social structure must be respected and safeguarded.

It was the aim of this EUPREN/EMRG Workshop to discuss the requirements of the different research fields, as well as the requirements of the primate species on housing and husbandry. It remains that one of the major current problems concerns the supply of non-human primates. There is a strong trend toward the use of purpose-bred instead of wild-caught primates in scientific procedures. It is expected that a ban on the importation of wild-caught monkeys could be adopted within few years by certain developed countries, for example by the European Union members. Most of scientists share these views. They wish to use purpose-bred primates because these animals have a greater homogeneity, a better control of the sanitary status, less stress (which is often a source of non predictive variation in research) and ensure wild life protection. However, such a ban should be acceptable as long as sufficient purpose-bred primates are available.

The solution is clearly to develop breeding programmes in user's countries, especially in the European Community. EUPREN has certainly a key role to play. But we must also encourage breeding in source countries, where several primate breeding facilities already exist. A positive step might be the establishment of some sort of accreditation programme for these primate breeders. The different aspects of these problems will be discussed.

Magnetic resonance imaging (MRI) and histopathology analysis during experimental autoimmune encephalomyelitis (EAE) in marmosets: characterisation of MR-detectable lesions in the course of EAE.

Massacesi, L.¹; Mueller, H.J.²; Bauer J. ³; Groen, B.⁴; Melchers, B.⁴; Klaas, N.²; Bontrop, R.⁵; Lassmann, H.³ & 't Hart, B.⁵
1Dept. of Neurological and Psychiatric Sciences, University of Florence, Italy
²Dept. of In Vivo NMR Spectroscopy, Bijvoet Center of Biomolecular Research  
  Utrecht University, The Netherlands
³Institute of Neurology, Vienna University, Austria
⁴Dept. of Pharmacology, TNO, Rijswijk, The Netherlands
⁵Dept. of Immunobiology, Biomedical Primate Research Centre, Rijswijk, The Netherlands

A relapsing-remitting form of EAE, highly representative of Multiple Sclerosis (MS), can be induced in the New World monkey species Callithrix jacchus (CJ) by immunization with myelin antigens (Massacesi et al., 1995). The various types of lesions of the central nervous system (CNS), that are induced in this experimental model, can easily be characterized histopathologically and be recognized by MRI using a high magnetic field equipment. Considering that the histopathological correlates of MS lesions recognized by MRI in the CNS have still been poorly characterized, the form of EAE induced in CJ's represents a precious instrument as to shed light on this problem.

In this study, animals affected with chronic relapsing-remitting EAE, have been examined by brain MRI several months from the onset of neurological symptoms, by using standardized sequences for the study of CNS lesions. The T1, T1 + Gadolinium and T2 axial sections of the brain of living anaesthetized animals have been studied with a MR 4,7 Tesla imager. The animals have been sacrified immediately after the image acquisition and a new MRI brain scanning has been performed on formalin-fixed brains for a precise three dimensional localization of the lesions that were examined histopathologically. The MRI scanning has shown lesions of different types, spread through the white matter. As in MS; T2 lesions, both enhancing and non-enhancing in T1, have been observed. The histopathological analysis of MRI lesions shows a microscopical pattern of lesions differing for intensity and composition: they include perivascular and parenchimal infiltrates of mononucleated inflammatory cells, myelin loss with preservation of correspondent axons and astrogliosis.

The present results indicate that the EAE induced in marmosets represents a unique tool for the characterization of inflammatory MRI lesions of the brain. The characterization of MRI lesions in diseases histopathologically identical (as the EAE induced in marmosets and the MS), will contribute to clarify both the pathogenesis of lesions and the pathophysiology of symptoms. In addition we foresee that this method will substantially contribute to MRI scanning acceptance as valid end-point in studies on the natural history of MS and on the efficacy of therapies.

Sleep studies in pair-housed and singly-housed marmosets: preliminary observations.

Pearce P.C.¹; Crofts, H.S.²; Wilson S.²; Dickson C.¹; Muggleton, N.¹& Scott, E.A.M.¹
1Medical Countermeasures, CBD Porton Down, Salisbury, UK
²Psychopharmacology Unit, University of Bristol, Bristol, UK

Until recently the methodology has not been readily available for monitoring EEG during sleep in unrestrained primates. Radiotelemetry techniques have been developed at CBD Porton Down for long-term monitoring (>12 months) of the cortical electroencephalogram (EEG) in freely moving marmosets (Pearce et al. 1996, Proc. US Biosci. Rev. In press). Freedom from restraint allows measurement of patterns of sleep architecture with minimal disturbance to the animal. This has provided an opportunity to collect data on sleep patterns from both single- and pair-housed marmosets.

In order to determine sleep patterns, EEG has been analysed in association with observations made from time-lapse infra-red video recordings. The resultant sleeping patterns are similar to those seen in man, in that stages of orthodox (slow wave) sleep and paradoxical (REM, Rapid Eye Movement) sleep are seen in cycles, but these cycles are shorter and occur much more frequently. Marmosets spend more time in light sleep than humans and REM sleep occurs more frequently later in the night. REM sleep periods do not appear to increase in length as they do in humans.

Sleep patterns from both single- and pair-housed marmosets will be presented and compared.

Immunology: Vaccines.

Perini, L.
Biocine S.p.A.
Siena, Italy

In the vaccine field, with the studies of Sabin in the 50s, the pharmaceutical industry started to use non-human primates for the production and control of the live poliomyelitis vaccine for oral use (OPV). For vaccine production, the ideal non-human primate model was found to be Cercopithecus aethiops (AGM) which presents a reduced kidney contamination by foreign viruses in comparison with Macaca mulatta. Nevertheless, Macaca mulatta and Macaca fascicularis are more suitable for vaccine safety control; they were used in the neurovirulence test (NVT) to check that the product had not reacquired neurotropic components which can cause vaccine paralysis in children.

Initially, all non-human primates used by the industry and by the national control authorities (Health Ministries) were wild-caught animals from Africa, Philippines, Indonesia and India, with severe exploitation of natural populations. It was only in 1970 that captive breeding centres, able to supply quality animals, came into consideration.

In Italy and Europe, contrary to the United States of America, breeding centres are continually talked about, but no-one (industry, university, European Community members) takes the initiative to financially support this objective. As regards this idleness, Biocine (Sclavo until 1992) decided in 1989 to start breeding non-human primates, investing in facilities in the United States. The use of non-human primates originating from their own breeding centres, despite being more expensive, has resulted in remarkable improvements in OPV production and in a reduction of animals' use. However, airline restrictions (i.e. embargo) to transport non-human primates, originating from breeding centres, makes the future of the European vaccine industry uncertain and it will, in fact, eventually be excluded from the polio business.

Research and animal welfare needs when studying social learning: the case of a colony of captive common marmosets.

Queyras, A.; Scolavino, M. & Vitale, A.
Section of Comparative Psychology, Lab. di Fisiopatologia di Organo e di Sistema
Istituto Superiore di Sanità, Roma, Italy

It's not always the case that research and welfare of the animals cannot coexist. It is very important for a researcher starting a study in captivity, to consider that each species has a series of behavioural patterns typical of the species itself. Therefore, it is necessary to decide in advance what kind of questions he wants to ask and then, in relation to such questions, to choose the most suitable species to be studied. In our case, the choice of the common marmoset (Callithrix jacchus), a small New World monkey, led us to recreate, in the space we were allowed for, the natural family group typical of this species: the adult pair with their twin offspring. The physical environment in which the animals live is obviously very important too, so there are many ecological and ethological implications to consider to make life in laboratory as much tolerable as possible. The common marmoset is an arboreal species and shows related behavioural patterns concerning, for example, the making and following marking of holes on tree trunks. Then, cage enriched with branches, and other wooden surfaces, are very suitable for these animals considering their biology. The main interest of the primatological group of the Section of Comparative Psychology at the Istituto Superiore di Sanità of Rome, is on the effects of the social context on the response of individuals to environmental novelties. In particular, in our first research we wanted to observe the animals' responses to new objects, focalizing our attention on the behaviour of the adult females, in relation to food-related new tasks. Then, another research concerned the social transmission of food preferences in relation to the particular social repertoire of our study species. In these researches we considered both our experimental aims, and the animals' requirements. For example, the use of new tasks and new foods as a part of an experimental protocol, represent also an environmental enrichment for the captive animals, and a good possibility to break the monotony of laboratory life. Furthermore our experiments, considering the social nature of the study species, never contemplated an individual tested in isolation. In conclusion, the ethical considerations in conceiving our experimental protocols are important both for the welfare of the animals, and for the quality of our research.

Training and monitoring of animals.

Schnell, C.R.¹ & Gerber P.²
1Research Dept., CIBA-Pharma, Basel, Switzerland
²Anthropologisches Institut und Museum der Universität, Zürich, Switzerland

Many areas of animal research requires interaction between the animal and the research staff and this often involves handling. Such interactions can cause marked changes to commonly measured biological parameters such as serum hormones, tissue metabolites levels, heart rate and blood pressure. It is now well accepted that acclimation to handling (or gentling) can have a significant impact on the quality of data measured in reducing variance and increasing the significance level of observed changes. Moreover, training (or gentling) is of special importance in experimental studies involving non-human primates. It has been reported in the literature that monkeys unaccustomed to experimental situation exhibit external behavioral manifestations of excitation and develop considerable less obvious changes in a number of physiological functions. However, there are relatively few published data on the effects of handling and gentling in non-humans primates. This can be related to the difficulties encountered by animal researcher in obtaining meaningful basal control values (which are pivotal for such studies) without disturbing the animal involved (for example by restraint, catheterisation, isolation or presence of the experimenter)

The use of pre-invasive implantable radiotelemetry has revolutionized the collection of physiological data under stress-free conditions. It is now possible to measure accurately haemodynamic and electrical parameters in conscious and unrestrained monkeys. This enabled us to record 'normal' baseline data for blood pressure and heart rate in different non-human primate species, maintained under a range of housing and husbandry conditions.

Recent results obtained in our laboratory will be used to demonstrate the pronounced acute and chronic effects on cardiovascular parameters of singly or repeated oral administration procedures in marmosets. Advantages, as well as limitations, of alternative "low-stress" ways of dosing will be presented. It is frequently suggested that training is not "cost-effective". Data from our own studies have demonstrated unequivocally the impact of housing/husbandry practices on physiological parameters and pharmacological outcome. Remote and pre-invasive monitoring provide opportunities for such assessments and further studies are urgently needed to enable "best-practice" to be identified using objective criteria.

Specific requirements of Callitrichidae species.

Scott, L.
CBDE Porton Down
Salisbury, UK

Marmosets (genus Callithrix) and tamarins (genus Saguinus), belong to the Callitrichidae family of small arboreal diurnal New World primates which originate in the Amazon basin. There are marked species differences in size, dentition, genital morphology and behavioural repertoire. The most widely studied member of the genus Callithrix, the common marmoset (Callithrix jacchus) breeds well in captivity and is currently used extensively in biological and biomedical research. Colonies of other callitrichids, notably Saguinus oedipus, S. nigricollis, S. fuscicollis and S. labiatus, are also maintained for highly specialised areas of research, particularly in the fields of immunology, virology and oncology.

Common marmosets have a diverse behavioural repertoire. The marmoset hand is clawed with modified nails and the thumb is not opposable. As sap feeders, they are specialist tree-gougers and ano-genital scent marking is an important feature of their complex social behaviour. Marmosets also exhibit context and gender specific vocalisation and have colour vision.

This talk will concentrate upon the species propensities of marmosets which must be addressed in laboratory housing and husbandry and suggestions will be made about how these requirements can be provided within the constraints of research environments.

A reference programme for biomedical research with primates.

Thomas, A.W.
Dept. Parasitology, Biomedical Primate Research Centre
Rijswijk, The Netherlands

Unique requirements are associated with the use of non-human primates (NHP) for biomedical research and development, both in terms of housing and husbandry, and in terms of research expertise. The most effective use of this limited resource is therefore in principle obtained through centres that specialise in NHP. However, because such primate centres are limited in number but are distributed worldwide, they have historically remained rather isolated. This has tended to discourage innovation, and has restricted the transfer and implementation of technologies that improve and enhance experimental outcome. This is not ethically acceptable in a world where increasing emphasis is being placed on the refinement, reduction and replacement of experimental work with all animals.

What is needed is the focussed and responsive technical development and diffusion of innovative and improved solutions to the particular problems of NHP research.

A Reference Programme will allow the focussed development of standards and common tools through cooperative work between diverse, geographically distant facilities, and ensure optimal alignment between the needs of the research community and the need for continually improving standards of animal welfare. The development of a wide ranging set of standards has been called for in a joint EUPREN and PVEN paper the elements of which are shown below.

Colony health and quality
Virological, bacteriological, fungal and parasitological testing/screening
Implementation of welfare standards
Hematology and clinical chemistry - Normal & Pathological values

Colony characterisation and development
MHC typing
DNA fingerprinting/other genetic analyses
Genetic conservation of laboratory primates
Breeding - Systems for in and out breeding per species
Breeding - Reproduction technology

Research fundamentals
Cytokines
Hormones
Blood groups and MHC
CD markers
Techniques for cellular immunology
Reference stocks - Species and strain types adapted/relevant to primates
Pathology - Tissue & Serum Bank
Ethics
Experimental techniques
Non-invasive monitoring of physical and behavioural responses

Training
Improve capabilities of technical staff
Develop academic skills and expertise through collaborative research

The elements described above can be seen as cutting horizontally through many research disciplines. One such is Parasitology, and I shall take this as an example of how a Programme can be developed through collaboration between such discrete vertical disciplines.

Nutricion and feeding systems of laboratory primates.

Thornhill, A.
Special Diets Services
Witham, England

Nutrition of laboratory Primates can be divided into two broad categories, New World Diets and Old World Diets. This paper will describe some of the nutritional differences between these two groups and the guidelines that SDS, as feed manufacturers, use when formulating the diets.

Feeding systems vary widely between country, institution and species held. Some of the more common methods of feeding primates will be described together with successful regimens to ensure the animals receive a balanced diet.

Finally, enhancement of the animals environment is a key issue and feeding can play a major role in helping to alleviate abnormal stereotypic behaviour. An outline of some of the feeding techniques available to enrich an animal's living space will be given.

Ethical proposals for the use of non-human primates.

Vitale, A.
Section of Comparative Psychology, Lab. di Fisiopatologia di Organo e di Sistema
Istituto Superiore di Sanità, Roma, Italy.

The evolutionary proximity between the human species and the other primates carries with it sentimental and evocative, as well as scientific, consequences. This is especially the case also because monkeys and apes are in many ways considered an appropriate model for the study of humans. Robert Hinde has indicated that the study of non-human primates can contribute to an understanding of human species in two principal ways. The first one is a better knowledge in such areas as anatomy, pathology and physiology, thanks to shared characteristics between human and non-human primates. The second is the application of methodologies and principles derived from behavioural studies of non-human primates to human ethology. However, thanks to the complexity of the nervous system of non-human primates, which allows for the attribution of feelings and emotions, these animals occupy a special niche in the concerns of people who care for the well-being of animals. For example, it must be cited the effort, promoted by Peter Singer and Paola Cavalieri, in including the apes in the "community of equals". It follows that the scientist who has chosen the non-human primates as his favourite animal model is influenced by all his cultural, political and scientific background and expectations but, at the same time, the scientific community and the society demand, rightly so, an adequately humane treatment of the study subjects. It will be proposed here that the solution to a possible dichotomy between for/against use of non-human primates in bio-medical experimentation can be not the choice of one of the two extreme positions, but a more conscious approach to the experimental work, through the identification of the scientist as a moral and social, as well as scientific agent.

Welfare implications in biomedical research.

van Vlissingen, J.M.F.
Biomedical Primate Research Centre
Rijswijk, The Netherlands

An animal experiment is legally defined at the level of a single animal, in case its welfare is affected negatively in any way for scientific or experimental purposes. In the ethical evaluation of proposed animal experiments, three major aspects are considered:

• the protocol and its scientific significance (fundamental or applied) and quality
• the number of animals involved, related to the scientific design
• the anticipated discomfort for the animals.

The discomfort should be evaluated at the level of the individual animal, in an integrated way, including its past, present, and future. The acquisition (either husbandry in captivity or captivating) affect animal welfare, as well as shipment and other major changes of environment. Also, in experimental use, husbandry requirements limit the fulfillment of the animals' social and psychological needs. Single caging, even in cages of adequate size, implies social isolation from conspecifics. Lack of environmental stimuli may cause chronic stress and some animals seek stereotypic behaviour as a coping strategy. Proximity of people may have positive (enrichment) or negative (aversive response) effects. Experimental procedures are another source of discomfort. Procedures may cause pain, fear and illness. An additional complication is the necessity for sedating or anaesthetizing primates for handling in many cases, ever for procedures that would only require physical restraint in other species. Physical restraint is similarly stressful and only acceptable for a short period of time. Primates possess complex psychological faculties. This may help them to cope more effectively, but it may also enhance predictability of events in a stressful way. Short-term pain and pathological processes caused by experimental procedures can mostly be treated effectively, but chronic pain or disablement have no easy solutions. The value and the longlivedness of individual animals implies that animals may be kept for a long period of time to be used for different experiments sequentially. This re-use policy helps to save on the number of animals bred and kept for research, and may express respect for the life of an individual animal, but there are major drawbacks as well. The degree of standardization of the animals may decrease to an extent that complicates the interpretation of experimental research or the experimental design may require more animals to reach conclusive results. In addition, the animals suffer accumulated discomfort, and for this latter reason legal limitations to re-use were defined. The problems domains are related to breeding, husbandry, and transportation for the acquisition of animals. Related to experimental use, moving, housing, handling, experimental procedures and effects of experimental treatments are added. Short-term stress can be coped with, short term pain can be alleviated, but long-term stress or pain are hard to deal with and should therefore be prevented. Priorities should be defined to address the most urgent problems first: acquisition (breeding near the site of use), husbandry (environmental enrichment), standardization (definition and maintenance of the quality of animals used for research- genetics, microbiology, behaviour, individual history), refinement of experimental techniques, definition of humane end points.

Survey on the use of primates in the pharmaceutical industry.

Weber, H.
Basel, Switzerland

Non-human primates are used both in research (drug discovery) as well as in drug development, when close relationship to humans is required for reliable predictability. Beside the pharmaceutical industry, the vaccine companies are other important users of primates, both as organ donors for the production of vaccines as well animals for the testing of safety and efficacy. Whereas in the phase of drug development as well as in the safety tests for vaccines fairly large numbers of animals are needed, in the phase of drug discovery fewer animals that are usually kept for prolonged periods of time and used repeatedly in individual experiments are required.

Research areas in which non-human primates are frequently used are psychopharmacology, neurodegeneration, infectious diseases, cardiovascular pharmacology, lung function, and metabolism. An increasing demand is observed for research on the immune system, and for testing pharmaceuticals, produced on the basis of biotechnical methods. The most commonly used species are macaques; in recent years the interest has concentrated on the cynomolgus instead of the rhesus monkey. Other frequently used species are vervets, marmosets, squirrel monkeys and occasionally also baboons.

The European Pharmaceutical Industry has come up with a position paper, in which the interest in purpose-bred animals is stated in view of good scientific practice. It is expected that these animals are of better health standards than those caught from the wild. However, some concern exists about the possibilities of obtaining sufficient numbers of purpose-bred animals in batches with limited age and weight range and the desirable quality in the near future.

Requirements of biomedical research in terms of housing and husbandry: Pharmacology and Toxicology.

Woolley, A.P.A.H.
Ciba Pharmaceuticals
Stamford Lodge, Wilmslow, UK

The objective of toxicology and pharmacology studies is to detect change or variation from normal and to interpret the significance of such change. For this purpose it is important that "normal" is defined adequately. The usual practice is to define normality by means of historical control data and by control of as many variables as possible to achieve a consistent model in which experimental hypotheses may be tested. Detection of change related to the experimental procedure in use is made more difficult by confounding factors, such as abnormal behaviour, injury, pre-existing disease or parasitism, stress or pathological abnormality arising from these factors. In order to meet the objectives of toxicology or pharmacology studies these factors need to be eliminated or minimised as far as possible. Most of these factors may be attributable to the use of wild-caught animals housed singly in small cages.

For toxicology and pharmacology, housing and husbandry systems for non-human primates (NHPs) should be conceived so as to allow the control of as many external variable as possible while facilitating the recording of experimental observations and measurements. The housing should allow easy identification and capture of animals, clear observation and recording of data and be easy to maintain hygienically. Husbandry should recognise the special needs of NHPs, in terms of diet, environment and stress. Over recent years the housing of NHPs has evolved from single to group housing, with consequently larger and more complex caging. Generally the only adverse impact on data quality has been on food and water consumption data; however, these were not particularly reliable in singly housed NHPs and may be assessed indirectly by other means. There have been clear subjective improvements in data quality or variability which may be attributable to better housing and husbandry techniques; for example these include reduced mortality in marmosets due to wasting syndrome and reduced behavioural problems in baboons or macaques. Although it would seem to be common sense that such an approach should lead to better data, it is actually difficult to prove in a quantitative manner, for instance in terms of clinical pathological data. In general it is reasonable to suggest that high quality housing and husbandry are reflected in high quality data, due to the use of a more normal and less variable animal.