New World Monkeys

New World monkeys originate from tropical rain forests in Central and South America, where they are exclusively arboreal. Their native environment may be hot and humid, and the animals are exposed to sunlight. Ultraviolet light should be supplied if possible, but if not then vitamin D3 must be added to the diet. There are many differences between outwardly similar species of New World monkeys, and the different regimes encountered in captivity are likely to affect their responses in different ways. Animals may thrive in one captive environment, but exhibit poor reproductive capacity or ill health in another very similar environment, or even if moved to a different position in the same room. Social and environmental enrichment appear to play a particularly important role in maintaining the health of these small primates. For more details on marmosets and tamarins see Buchanan-Smith8, on squirrel monkeys see Williams et al.9 and on capuchins see Anderson and Visalberghi10.

Marmoset

The commonest New World species used in research is the common marmoset, Callithrix jacchus. Marmosets are small and relatively easy to keep in the laboratory. For biological data see Table 13.1.

Table 13.1 Useful data: marmoset

Behaviour

Wild marmosets live in family groups of three to eight animals or more, with a well-developed social structure. Males and females pair for life, and both parents care for the young. Individuals show a great deal of interaction, including play and mutual grooming. There is marked diurnal variation in their behaviour, and they are much less active at night.

Housing

Caging for marmosets needs to reflect their behavioural needs. Since they are arboreal they climb well and if startled they flee upwards, not along the ground, so cages for these species should be tall and narrow to reflect this. The stocking density within the room must be kept low, since high stocking densities and overcrowding are associated with increased levels of disease. Marmosets are usually housed in family groups composed of an adult pair and offspring. They are agile and like to climb, and so benefit from branches or other suitable perches within the cage. They will walk along branches on all four feet and use their tails to balance.

Feeding

Marmosets will eat freshly milled New World monkey diet, but benefit from supplements such as pieces of fruit or malt loaf. Diets for New World monkeys usually contain 20–25% protein, and protein should be fed at 3 g/kg of body weight daily. New World monkeys require vitamin D3, which they normally produce during exposure to ultraviolet light. In the laboratory it should be added at 1–2.5 i.u./g of diet. A deficiency in it will predispose to pathological lesions of the bones. Deficiencies in vitamin E, selenium and protein may lead to wasting marmoset syndrome. Adult marmosets eat about 20 g of pelleted diet daily. Water is supplied ad libitum, using bottles or automatic systems. With bottles, vitamins as described above can be added to the water. Drilled wood filled with gum provides good enrichment. Food hoppers and water bottles can be cleaned twice weekly.

Breeding

Marmosets, as all New World monkeys, have oestrous cycles, whereas Old World monkeys have menstrual cycles. Sexual maturity is reached at 14 months; the female is polyoestrous, having cycles every 14–28 days. However, no overt signs of oestrus are seen. If they are kept in large groups with more than one female, only the dominant female will breed. Mating results in gestation lasting 140 days, and twins are usually carried. The fetuses share a placenta with vascular anastomoses, so they share their blood supply and are natural chimeras. After birth, the young are normally weaned at 6 months and stay with their parents until sexual maturity. Typically the males do much of the infant carrying. Females are mated again shortly after parturition and the interbirth interval is 154–178 days. With improved nutrition and health of marmoset colonies, between 20 and 52% of pregnancies may result in triplets. Left alone, the weakest one will drop off at around the third day and die, which raises ethical and welfare issues. The alternatives are rotational feeding, hand rearing or removing one infant for euthanasia. Each of these has certain disadvantages and the management method selected to deal with these extra offspring will depend on the individual circumstances of the colony.

Growth

Marmosets reach their maximum body weight by 20 months of age.

Old World Monkeys

The Old World monkeys all lack prehensile tails and have some degree of callus over the ischial tuberosities. Many have cheek pouches, and all have a narrow nasal septum and opposable thumbs. The Old World monkeys most commonly used in research are Macaca mulatta (rhesus macaque), Macaca fascicularis (long tailed, crab-eating or cynomolgus macaque), Chlorocebus sp. (formerly Cercopithecus sp.) (sometimes called the African green monkey or the vervet monkey) and Papio species (baboons). For biological data for the macaques see Table 13.2. The macaque genus has a wide natural range, extending from Morocco and Gibraltar through the Indian subcontinent up through China and Japan and down through the Philippines, Java and Sumatra. This range includes a wide variation in environmental temperatures and diets, and the macaques have adapted to a variety of ecological niches. This is reflected in normal variations in patterns of behaviour and interactions with humans, for example foraging around urban environments11 to foraging in remote areas.

Table 13.2 Useful data: macaque

Sources, supply and transportation

In Europe the facilities for breeding macaques for research use do not yet supply adequate numbers to match demand. A substantial number of macaques are, therefore, imported into Europe from countries of origin. Since macaques come from a wide range of natural habitats, the genetic backgrounds and responses to experimental procedures will vary for macaques from different populations12. Evidence suggests that when primates are subjected to a stressful event, such as transportation, signs of physiological stress are markedly reduced when in the presence of other individuals13. Juvenile animals may be shipped in pairs within the transport container and the compatibility of paired animals can be ensured by studying the interactions of the animals in their social groups prior to being transported; animals which show affiliation can be assigned to travel together. This approach should extend beyond the transport phase and efforts should be made to ensure that groups which will be housed together at their destination are socially stable and, where possible, from the same breeding group. In this way relationships and social status will have already been established in natal groups, reducing the likelihood and intensity of conflict. This makes future veterinary and behavioural management of the animals easier.

Communication between shipper and recipient should ensure the animals’ favoured and acceptable foods can be available on arrival. A change in diet can affect the gut flora and will add to the animals’ stress. A gradual change to a new diet over 4–5 days will reduce the incidence of diarrhoea. There may also be disease risks associated with the transportation of primates, both between the animals and handling staff and between consignments of animals, particularly if the health status of the animals is unknown.

Breeding

There are several types of captive breeding systems for primates, including free-ranging island colonies, semi-free-ranging corral colonies and enclosure or pen pairs or harems. A description of each is provided in reference 14. For further details on breeding see Wolfensohn15 and Kirkwood and Stathatos16.

In female rhesus monkeys puberty occurs at 2–3 years and 3–4 years in males. For cynomolgus monkeys puberty occurs at 3–4 years in both sexes. Rhesus monkeys have a menstrual cycle lasting 28 days, and breed seasonally from September to January. Cynomolgus monkeys are non-seasonal breeders, with a menstrual cycle of 31 days. Old World monkeys have sexual skin, which may swell and change colour to indicate the animal’s readiness to breed. Ovulation in macaques is indicated by changes in the female’s behaviour, olfactory cues and swelling and reddening of the perineal skin. The size and colour of the sexual skin indicates the occurrence of ovulation, and may also be associated with attractiveness in the context of mate choice, both of females to males and vice versa (e.g. in rhesus macaques17). In females the anogenital region is frequently the primary site of sexual skin, but it may occur in other areas, such as the side of the thigh in rhesus macaques. Sexual skin in male primates may also be dramatic and brightly coloured (e.g. the blue of the scrotum of the vervet monkey, Chlorocebus aethiops). In addition to visual signals of reproductive state, communication via auditory and olfactory routes is extremely important in many primate species. Gestation lasts 164 days in the rhesus and 167 days in the cynomolgus; and one infant is usually born. Dependence on the mother for nutrition gradually wanes between 3 and 6 months of age and the infant becomes independent for foraging and travelling by about 1 year of age. However, psychological support and protective intervention from the mother will extend well into the juvenile years.

The mother will terminate suckling at a point determined by the metabolic independence of the offspring, this being related to the offspring’s body weight at birth and at weaning: the ‘metabolic weaning weight’. Natural weaning is not an abrupt event but a process lasting several months. It is during this time, and shortly after, that the young begin to learn from their mother and to refine food-selection, -acquisition and -processing skills18,19. After the first year, juvenile animals, especially males, become more and more involved in peer-group activities.

Opinions differ on the ideal time for separating offspring from their mothers20,21,22,23. However, if young monkeys are reared with an inappropriate social background they will show indicators of poor welfare such as a restricted repertoire of behaviours, an abnormal activity pattern, inadequate social behaviour and/or abnormal behaviours such as self-injury. Therefore it is recommended that weaning (meaning the physical separation from the mother) should not occur until after 12 months of age. Tracking the performance of animals during training within both the breeding facility and the experimental facility will help to identify individuals that produce offspring that are more likely to be easily trainable and to cooperate with research protocols. This will contribute, when coupled with the highest welfare standards in housing and husbandry, to a reduction in the stress of primates maintained for research.

Housing

The physical environment in which captive primates are housed contributes significantly to the physical and psychological health of the animals. Replication of the natural environment in the captive situation as far as possible ensures that the animals’ health is maximized. Otherwise, although there may be plentiful provision of food and effective disease control, there may be a reduction in activity leading to boredom and obesity with resultant poor health.

Existing recommendations for primate housing are generally based solely on the weight of an animal without reference to linear dimensions, and the sizes of cages are sometimes too small to meet the behavioural needs of the animals, providing neither adequate space for exercise nor room for environmental enrichment24. The assumption is made that heavier individuals require more space than lighter ones, although in practice light, young animals are usually more active than the heavier adults7. Rather than basing cage size on parameters such as body weight or crown–rump length it is better to use performance standards which assess the ability of the animal to express its species-typical behaviours and which will vary depending on species, age, sex, individual temperament, group composition and dynamics25. It is important to appreciate that most recommendations for cage sizes represent minimum dimensions for animals of all ages. The duration of stay of the animal is also relevant. Where primates are to be kept for a period of years the necessity for housing them in an environment that satisfies their needs is even more important than for those animals that will only stay for a short while. There is an ethical obligation to house and maintain animals used in research in the best conditions that can be provided. Establishments wishing to demonstrate best practice should endeavour to produce conditions for their animals which provide key safe features of a naturalistic environment that will contribute to encouraging a range and frequency of behaviours as close to the natural situation as possible. So, as ‘best practice’ evolves it is necessary to continually re-evaluate whether the facilities and their operation and management can be improved.

Visual barriers are important in group-housing and multiple escape routes provide opportunities to avoid attacks and also prevent dominant individuals from restricting access of subordinates to other parts of the cage26. Where individuals may need to be treated individually, as in some toxicology experiments, areas with a partition to allow separation or varying degrees of social contact is required. There should flexibility of use incorporated the design of the housing.

Environmental enrichment is essential for captive non-human primates to meet their ethological and psychological needs27. It should provide the animals with the opportunity to carry out a sufficiently varied daily programme of activity. The actual enrichment provided will vary, but there is now a large literature offering a wide variety of options. The effectiveness of many is reviewed in Honess and Marin25. Opportunities for the animals to exercise a full locomotory repertoire should be provided in the living area. To allow this, the cage must be sufficiently large and contain adequate furnishings. Cage furniture itself can provide opportunities for a wide repertoire of behaviour. Perches, ladders, swings, plastic chains, car tyres, etc. are all of value, and allow the animals to utilise the three-dimensional space and have plenty of places to sit without having to squabble. This allows a social hierarchy to develop with less aggressive encounters.

Foraging is a natural and desirable behaviour which can occupy a significant period of time, reducing boredom and the incidence of stereotypic behaviours. Therefore, a substrate such as woodchips, hemp or shavings should be provided in which forage can be scattered. Hay, straw or other material such as shredded paper may be provided for environmental enrichment. Devices to encourage foraging (such as puzzle feeders) have also proved effective. Provision of foraging opportunities in an adequate amount of space is a way of offering a socially sanctioned method of establishing the hierarchy that does not result in significant wounding and injury. Some simple and practical methods include the use of wire grids over food hoppers, ice cubes of frozen juice, frozen grapes or melon cubes, food on top of the enclosure to encourage animals to forage upwards through the mesh, wrapping or boxing of treats, empty diet bags or cardboard boxes, and foraging boxes full of hay or sawdust28. Novelty is important so the items should be changed frequently to avoid habituation and a programme of rotation of items of environmental enrichment should be recorded. Reducing the incidence of aggressive encounters by resorting to long-term single housing of animals is not acceptable; the environment and the group composition need to be actively managed to ensure maintenance of social stability. A key component in a successful strategy is to start with the right compatible grouping, which may require coordination and effective communication with the source breeding colony which establishes the groups at weaning. Training animals to cooperate with carers and experimenters also helps to enrich the animal’s life and reduces handling stress29. It is vital to remember that the animal’s principal purpose is for a scientific procedure and the animal model must be valid in terms of normality and reproducibility. The environmental enrichment programme should be reviewed to consider any effect (positive or negative) on the quality of the scientific data generated.

Physical environment

The environmental sensitivity of primates varies considerably between species, some being more tolerant of environmental change than others. In the wild, rhesus macaques (M. mulatta) occur across a wide range of altitudes from sea level to 3000 m30, indicating a tolerance of a substantial temperature range. Generally the temperature that will be comfortable for staff will also be optimal for the animals. Relative humidity levels should be maintained between 40 and 70%, but extensive daily washing, such as pressure-hosing, may result in surfaces which remain permanently wet, resulting in elevated humidity levels and also presenting a potential health risk to the animals as many pathogens remain viable in a warm, wet environment. Under certain experimental regimes and containment levels it may be important to ensure negative air pressure in the animal rooms.

Where access to daylight is not available a lighting regime of 12 h light/12 h dark is typically provided, in which case efforts should be made to replicate dawn and dusk gradations in light intensity. In the wild, primates rely on the ultraviolet radiation in daylight to produce vitamin D3. Care should be taken to ensure that artificial light is provided using daylight spectrum tubes or bulbs, rather than standard fluorescent lighting. This is of particular importance where there may be a familial predisposition to metabolic bone disease or a deficit in dietary vitamin D331. Care should be taken in the positioning and intensity of artificial lighting in interior accommodation to avoid reflective glare off polished metal surfaces.

High noise levels may intimidate animals and have a negative effect on their psychological well-being. Primates are highly social animals and one of their primary modes of communication is vocal. Dominance can be asserted through vocalisations and therefore care needs to be taken in placement of overly assertive animals in proximity to those that may be negatively affected by vocal bullying. If cages are constructed using metal sheeting and heavy meshing with metal furniture, such as swings, noise production can be substantial. The use of materials such as wood will help reduce noise, as will flooring covered with sound dampening forage substrate such as wood chips. People working around the primates can generate considerable noise. It may be reassuring for the animals to hear the voices of familiar care staff but not all staff are viewed as friendly by the animals and their voices may, particularly when loud, produce a response suggesting stress or anxiety. Staff should be encouraged to keep voices down and to work around the animals in a way that reduces unnecessary loud noises, such as avoidance of slamming doors or crashing trolleys against walls.

Laboratory primates are often kept in cages or rooms, but some larger species may be kept in outside pens or corrals, with free access to a heated indoor area. There is a perception that there is a welfare advantage to all primates associated with the provision of external accommodation. However, it is the quality of the accommodation and the handling of the animals which matters most in terms of animal welfare and outdoor accommodation is not necessarily in itself a benefit. External accommodation carries with it risks from exposure to wildlife and disease transmission. The facility must be fit for purpose but there should be no underlying assumption that outside living areas are somehow automatically better32. A well-designed facility is critical to be able to deliver a successful animal care programme and behavioural management.

Husbandry regimes that may be more labour-intensive but have a reduced impact on the animals should be used. These will have other benefits; for example, reduced aggression and fewer traumatic injuries, and easier training producing a more valid research model.

Feeding/watering

Food

In the wild many primates choose to spend a substantial amount of their time foraging, and anything that may restrict this should be regarded as potentially detrimental to their welfare. It is better to provide small portions of easily accessible pelleted food with an unpredictable schedule, providing the remainder as forage to satisfy the animal’s behavioural needs, rather than to provide the quantity required to satisfy the daily nutritional requirements in just one feed. Tables of the nutrient contents of foods commonly used to feed primates are published by the National Research Council33.

An overview of energy requirements and how these are calculated is given in chapter 4 of Wolfensohn and Honess7. This energy requirement is met by a combination of commercial primate diet, a selection of fruit and vegetables and forage mix, but variety is essential and when adding any supplements such as fruit, care must be taken not to upset the nutrient balance. Amounts fed will also need to be adjusted according to levels of activity and weight gain/loss must be evaluated regularly.

Supplements are generally given for environmental enrichment or as part of positive reinforcement training, rather then primarily for nutrition but may have the undesired effect of distorting the balance of the nutrition provided. It is therefore important to use items that are nutritionally complete or which are high in moisture and low in calories – such as fresh fruit and vegetables – rather than offering energy-dense but nutritionally incomplete foods such as nuts and raisins. Since fresh fruit and vegetables are 80–90% moisture, if these make up less than 40% of the wet weight of the diet they will be providing less than 10% of the total dietary dry matter and therefore will minimally distort the nutrient balance.

Animals should be fed according to their individual needs, and these will depend on species, age, sex, physiological and reproductive status, health and environmental conditions. Growth can be measured in various ways such as body weight, crown–rump length, limb length or head circumference. The linear measurements are not distorted by accumulations of body fat in the way that body weight may be. Growth rate will vary depending on genetic background, maternal nutrition in pregnancy and lactation, availability of supplementary weaning foods and rearing practices. Both sexes show an adolescent growth spurt, particularly males.

Water

The amount of water drunk to satisfy physiological needs will depend on many factors such as the water and electrolyte content of the food eaten, the ambient temperature and humidity, and activity level of the animal. The mean total water intake in cynomolgus monkeys has been found to be 76±35 ml/kg per day for males and 100±51 ml/kg per day for females, of which drinking water comprised 50±33 ml/kg per day for males and 49±48 ml/kg per day for females, with other water being derived from food34. Even moderate restriction of water availability will reduce food consumption.

Health Monitoring, Quarantine and Common Diseases

When monkeys are brought into a facility basic information about them is necessary in order to be able to design the quarantine programme and establish the health-screening programme, which will be affected by the source of the animals. Details should be obtained of the groups in which they were kept and how long they had been established. If they were weaned at less than 12 months they will be more likely to be stressed and more likely to be carrying subclinical infections, which may then become a clinical problem when they arrive. Information should be obtained on the standards of care in the source establishment and the background health status of the source colony, taking note of the time period over which this been established. During the quarantine period incoming monkeys need to be isolated from existing stock. It is vital to set aside enough time to allow for observation of these new animals, building up a picture of each one’s normal behavioural repertoire, so that changes due to illness or experimental stress may be spotted rapidly. The behaviour of juvenile long-tailed macaques does not settle to pre-transport levels even 1 month after international transport and relocation to a new unit35.

Quarantine can have a considerable impact on animal welfare, particularly if there is single housing, small cage sizes and a lack of enrichment, isolation from former social companions, or fear of unfamiliar animals and humans in close proximity. If quarantine is required it is important to avoid unnecessary constraints on the animal’s husbandry and care.

Health-screening programme

It is preferable to prevent diseases rather than to have to deal with them when they arise. A number of infectious diseases of primates are potentially zoonotic so health-screening programmes cannot only improve animal health and welfare but will also contribute to the occupational health programme. The health programme should also take into account any necessity to screen staff to prevent spread of infections from humans to monkeys.

Monkeys can carry a number of potentially serious infectious diseases: some bacterial, ranging from salmonellosis to tuberculosis, and some viral. The UK Advisory Committee on Dangerous Pathogens has made particular recommendations with respect to simian herpesvirus (Herpesvirus simiae, B-virus), and simian retroviruses36. A health-screening programme should be drawn up for every facility holding primates which should take account of the source of the animals, the use to which they are put (e.g. breeding/experimental, long term/short term, immunology/neuroscience). For the general principles of health monitoring see Chapter 5 in this volume and for more detail on primate health screening see Wolfensohn15 and Wolfensohn and Honess7. There are many diseases which should be included in a regular screening programme as discussed above. Full details on the diseases of primates and their management can also be found in Bennett et al.37 and Wolfe-Coote38.

Diseases

Infectious diseases

The potential of primates to carry serious zoonotic diseases should not be underestimated. Captive-bred animals of known health status are less of a risk, and these comprise the majority of animals used. Monkeys are susceptible to many diseases carried by humans, such as colds and flu, tuberculosis, measles and many others. Therefore it is important to exclude casual visitors or at least wear a suitable face mask to reduce the risk of spreading such infections to the monkeys if appropriate occupational health surveillance has not been established. Power hoses promote the formation of aerosols, which may allow transmission of zoonotic diseases, so suitable protective clothing must be worn when operating these.

Animals used in experiments should wherever possible be from herpes B-virus-free colonies, particularly where animals are handled under conditions of close contact, in long-term studies or for neurological surgery, or for work where a latent infection may be reactivated, such as those involving psychological stress or immunosuppression.

Enteric disease may be caused by a number of factors including pathogenic organisms. Monkeys will pass loose stools if they are frightened, or if there are changes in their environment or diet. Stress, such as after weaning, transportation, surgery or due to changes in social hierarchy, can also cause subclinical infections to become clinical, so any case of diarrhoea should be investigated to eliminate infectious causes. Zoonoses such as Shigella, Salmonella, Campylobacter and Yersinia are common bacterial pathogens. Husbandry and environmental factors may also contribute to the development of enteric disease, as will viruses such as rotavirus, retroviruses and haemorrhagic viruses, and parasites such as Giardia spp., Trichomonas spp., Entamoeba histolytica, Cryptosporidium spp. and Balantidium spp., and the metazoans Strongyloides spp. and Trichuris spp.39. Therefore, prompt and accurate diagnosis of the aetiology of the enteritis is important so that a specific therapy can be instituted.

Husbandry-related diseases

A management strategy for dealing with such problems as fight injuries or nutritional imbalances that may be encountered with increasing use of foraging and group housing should be developed and built into any primate health-management programme. The benefit of social housing is that the environment is dynamic, unpredictable and variable so there is little habituation, but there are increased risks of infection, wounding and competition for food. With good management strategies, such as appropriate housing, these risks can be minimised but not altogether removed.

Clinical problems may be brought about by the increasing provision of forage mix in addition to normal pelleted diet. Although the forage mixes are all well balanced nutritionally, this assumes that the monkey will not pick out its favourite bits and leave the rest, so the provision of forage has to be carefully managed to prevent deficiencies. By introducing an element of choice the diet may become unbalanced and problems, such as rickets in juvenile animals, may arise from this. Social rank may be associated with obesity40 since the dominant animal determines feeding time and the subordinates eat afterwards, depending on spatial distribution of food and the mix of food types. Dietary restriction (but without essential nutrient deficiencies) will increase survival and delay the onset of degenerative ageing conditions.

Health and safety

Particular care must be taken when handling primates because of the danger of transmitting potentially zoonotic diseases. Appropriate protective clothing must be worn. For quarantine animals, this should include cap, gown, mask, boots and gloves. For other animals, gloves and protective gowns may be sufficient but there will be local safety rules for each institution which must be followed. These rules will take into account the origin of the animal and the results of health screening.

Laboratory Procedures

Handling and restraint

All primates can and will, on occasion, bite. They are very quick, surprisingly strong and will snatch and grab at such things as jewellery and loose clothing. Injuries can be prevented by knowledge of the particular species and the individual, including knowledge and interpretation of the posture and expression of the animal. The responses of different species of primates to environmental stressors varies and this is reflected in their response to minor procedures such as routine capture. When compared with rhesus, cynomolgus macaques tend to be more stressed41, and the animals’ responses to stress can have implications for research42.

Restraint for any purpose and whether for short duration or longer may induce fear and stress responses, such as physical resistance to handling, alarm vocalisations, defensive threatening and aggression, urination and defecation, and there are also likely to be physiological responses that will increase unwanted data variability25,43. These fear and stress responses should be recognised as indicative of a serious welfare problem that must be properly addressed. It may be that restraint itself can be avoided. Habituation and socialisation, together with training animals to cooperate with procedures using positive reinforcement techniques, can obviate the need for physical or chemical restraint.

If restraint is required to control a primate during a scientific procedure then the method used should provide the least level of restraint for the minimum frequency and duration necessary consistent with achieving the experimental aims. It should protect both primate and personnel from harm and should avoid causing unnecessary distress or discomfort.

Restraint chairs are used to support primates in a sitting position when it is deemed necessary to restrain them for prolonged periods, such as for single-cell recording within the central nervous system or chronic infusion when they may be required to remain in one position for several hours. Chair restraint can affect the animal’s physiology44 and can severely compromise their welfare45,46.

In order to carry out a full examination of a primate it may be necessary to sedate it in order to reduce the risk of injury to the handler, to reduce the stress to the animal and also to enable the examination to be carried out thoroughly to yield the maximum possible information. A ‘restraint’ cage may be used in which the back is pulled gently forward or the front pushed gently backwards. However, most animals find this procedure very stressful. They may be netted47 but this is also significantly stressful and most animals will not respond well to this method of capture. It is much better to spend time training the monkey to present itself for the procedure, which can then be carried out without stress to animal or handler. Macaques can quite quickly and easily be trained to present their hindquarters for injection, or present a limb for blood sampling43,48,49,50.

Even if a macaque is sedated it should be held by the upper arms, to keep its face away from the handler. Even better is to train the animal for manual capture and restraint since sedatives themselves are stressors13. Many experimental procedures require gaining access to the individual animal but primates can readily be trained to cooperate even when they are living in social groups. Training is encouraged by the use of positive reinforcement and they can also be trained using voice commands, which will not distort their nutrition.

Macaques can be trained to enter a transport container and other methods include catching animals in a nest box or transport container, or use of a pole and collar. These methods all have advantages and disadvantages and a useful discussion of the welfare implications of the various techniques is contained in Rennie and Buchanan-Smith51. Whatever the method of capture, macaques should be safely restrained and their body weight supported when they are carried.

Training

Training animals to cooperate with scientific, veterinary and husbandry procedures helps to reduce the stress that may be caused to both the animal and the laboratory staff. Primates are not domesticated animals and contact with humans can be extremely stressful, especially where the primates are not in control of the level and intensity of that contact, which is a particular problem in the laboratory14. Habituating and socialising captive primates to the presence (sight, sound, smell) and behaviour of humans as early as possible in their lives is essential. Otherwise, both animal welfare and the science can be seriously affected. Early habituation reduces any fear or distress the animals may experience when confronted with new situations as adults. It also allows staff to observe behaviour patterns which are relatively unaffected by their own presence, and this helps them to assess the welfare of the animals more effectively.

There should be a formal habituation and socialisation programme, based on the principles of positive reinforcement52. In addition, all staff that come into contact with the animals should understand the need to ensure that their own actions make a positive and consistent contribution to the habituation and socialisation programme, and everyone should be trained accordingly. However, in order to start a programme of training with laboratory primates it is first necessary to train the people to communicate and interact positively with primates through recognition and interpretation of primate signals, and to help the primates to respond positively to humans through habituation and socialisation.

Training methods should be based on positive reinforcement techniques, which reward desired behaviour, since this method of training is considered to be the most humane53,54. Negative reinforcement should only be used when positive alternatives have been exhausted (e.g. training for aversive procedures), and it should only be used in combination with positive reinforcement.

Positive reinforcement training refers to any form of training that is based on rewards rather than punishment. The animal is rewarded with some treat, such as a morsel of food, for a job well done. Negative reinforcement is the opposite of positive reinforcement and involves a punishment for less than favourable performances and usually implies some kind of fear, pain or discomfort for the animal being trained. Another way to get an animal to perform a task is to motivate it by withholding food or water and it has to perform the task in order to obtain food or water. As the intake of water is essential for survival, water deprivation is a stronger motivator than food deprivation and will achieve faster results since an animal will become physiologically compromised quicker with water deprivation than with food deprivation. Access to fluid in these conditions cannot be described as ‘positive reward’.

Fluid regulation may simply involve strict scheduling of the access to water, or may involve restriction in which the total amount of water is strictly controlled, so that thirst becomes the motivator for performance of certain tasks. With limited access to water, food consumption also decreases and these protocols are often associated with weight loss and associated stress. On such protocols the amount of fluid consumed and a hydration assessment should be recorded daily for each animal. Variables that can be used to assess hydration status include the body weight, food intake, skin turgor, urine output, moistness of faeces, general appearance and demeanour. It is important to evaluate the health and welfare of each individually55. Animals should generally be given free access to water for some period of time on days when there are no experimental sessions33. There is currently much debate over the use of water restriction paradigms56–59.

Administration of substances, blood sampling

Some recent reviews have been published which provide detailed advice on refinements for the administration of substances and of sampling60–63. Factors to consider include whether the particular individual or species is the most appropriate for the study or whether it is too easily stressed by handling and requires further time to acclimatise to the procedures. Pre-screening should be conducted to determine whether the particular animal’s temperament is conducive to training and to performing the tasks required. The use of sedatives may or may not reduce stress13, but may confound the experiment and not be feasible. If possible animals should be trained to cooperate with the procedures to avoid the need for physical restraint. Staff should be competent to carry out the procedure and post-administration monitoring.

Telemetry

Telemetry is frequently used in studies involving primates, including those carried out to fulfil the requirements of regulatory bodies. Telemetry is widely viewed as benefiting science and animal welfare because it can reduce stress caused to animals (e.g. by restraint), enable reductions in animal numbers and provide indicators of animal well-being to help implement humane end points. However, telemetry can require invasive procedures such as implantation surgery, single housing and use of jackets, which can cause pain and distress. Although telemetry is often described as a refinement, this will not be the case unless the technique itself has been fully refined. The use of telemetry may also increase the time for which an individual animal is used on procedure and the cumulative severity must therefore be taken into account64.

Detailed advice on refinements in telemetry can be found in Morton et al.65 and Hawkins et al.66 including maintaining stable group housing, and behavioural pre-screening to ensure that individuals are suitable for projects and are not implanted unnecessarily.

Anaesthesia, analgesia and post-operative care

Before anaesthesia primates should have food withheld for at least 6 h, and for Old World monkeys it is important to check that the cheek pouches are empty to ensure that the animal does not choke on recovery. A selection of drugs that may be used to sedate primates to facilitate handling are listed in Table 9.3g. Veterinary advice should be sought on the use of these but some further information is provided by Foster et al.67 and Sun et al.68. In the UK, all these drugs are prescription-only medicines and therefore must only be used under veterinary direction.

Induction (and maintenance) of anaesthesia can be by intravenous infusion of propofol. Barbiturates are associated with respiratory depression and resultant hypercapnia. Primates should be intubated, having first sprayed the larynx with local anaesthetic to prevent larygospasm. Prior to intubation, administration of oxygen by face mask is advisable. Maintenance of anaesthesia using isoflurane or sevoflurane is most satisfactory and gives a good quality of recovery. During anaesthesia it is essential to monitor the vital signs including temperature, blood pressure, oxygen and carbon dioxide levels and maintain physiological stability with administration of fluids. Post-operatively, analgesia can be provided with opioids or non-steroidal anti-inflammatory drugs. Post-operative gastritis and vomiting can be controlled using metoclopramide. Selection of the appropriate regime will also depend on the area of scientific investigation as some anaesthetics may affect the area under study69.

Husbandry post-surgery

Animals may need to be allowed to recover separately in the immediate post-operative period. However, it has been successfully demonstrated that surgically treated animals can be pair or group housed soon after recovery70 and they should be returned to their social group as soon as possible.

Interrupted subcuticular sutures, with the possible additional use of tissue adhesives, should be sufficient to maintain the integrity of a surgical wound such that removal of sutures by the individual or cage mates is not a problem. Adequate perioperative analgesia will prevent animals from paying untoward attention to their own wound sites and attracting the attention of others. It has also been shown that primates with surgical implants, for example cranial implants, can successfully be kept in social groups70. For work on extremities and limbs primates will tolerate bandaging to provide protection and support of the affected area provided the dressing is carefully applied.

When planning housing for the recovery period, remember that primates may climb before they are fully recovered from anaesthesia, and may fall and injure themselves. A purpose-built recovery enclosure may be used or a temporary reduction in the height of the home area may be implemented. If a recovery enclosure is used, it should be constructed so that the primate does not have to be physically caught in order to be returned to the home cage, or animals should be trained to enter and leave a transport cage. Animals should be allowed to recover in proximity to others from social group, not in social isolation. If surgery involves producing neurological lesions, the home area may need to be adapted to take account of any possible long-term side effects. If single housing following surgery can be justified, individual animals should have visual, auditory and if possible tactile contact with other primates.

Welfare

There are a number of parameters that can be used to assess welfare, which may be behavioural, physiological or biochemical (for more information see Chapter 4). The most readily accessible and commonly used index for assessing well-being in primates is behaviour.

Natural behaviour

All the primate species commonly used in research are highly social animals. Old World primates are typically female kin-bonded groups with the distribution of males superimposed onto that of the females. In the wild the group structure varies according to differences in the quality and density of resources and incidence of disease and level of predation. Knowledge of the normal social structure (including how groups are constituted and dominance hierarchies established) should be used to inform how they should be group housed. This is important in determining group size and structure, defining socialisation programmes, managing aggression, promoting natural behaviour and optimising reproductive success71.

Primates live in social hierarchies in which there are inevitable conflicts and fights will occur in which some animals may be injured or occasionally even killed. A singly housed animal may be in excellent physical health, but single housing is a major stressor and the animal will suffer the stress of social deprivation, and such animals frequently show a poor behavioural repertoire with abnormal and even self-harming behaviours and do not have well-developed coping skills. In nature, many primates live in fairly large groups (around 50 in a troop for macaques) and while putting a small group of animals or a pair together will give them company, there appears to be a higher incidence of fights and unsettled behaviour in these than in larger groups, even if floor space per monkey is lower in the latter case. A pair is not a normal social group.

The components of a wild macaque’s diet typically come in small aliquots (e.g. seeds, buds and flowers), dispersed over a wide area, so the process of gathering them is very time consuming and demands much energy. The daily activity cycle reflects this as a macaque troop typically spends the morning foraging, followed by a rest period, then another bout of food gathering through the afternoon, finally moving to a sleeping site for grooming and sleep. Of the two macaque species commonly used in research, the cynomolgus monkey spends more of its time in the trees whereas the rhesus is a ground-dwelling monkey that forages on the floor and takes to the trees for safety and to sleep. Macaques also appear to enjoy playing in water and, although they do not actively swim, they will forage for food in shallow water and use water for thermoregulation.

Primates communicate in a variety of ways using sounds, facial expressions and postural changes. Many signals can be easily interpreted. For example, shaking a tree is a sign of aggression which, in a captive environment, translates to rattling the cage. A low-grade threat can be communicated in some primates with a brief ‘eyebrow flash’ which in humans is an indication of recognition and may form part of ritualised flirting behaviour. Raised eyebrows combined with lip-smacking are used as a social appeasement signal in macaques. Other facial expressions may communicate quite different meanings to non-human primates than to humans. For example, the grimace, or silent bared-teeth face, indicates uncertainty or submission in many primates but, as a smile in humans, can be seen as an expression of friendliness. Another common source of confusion is the human ‘stare’. A person may stare at an animal out of interest, but most primates will interpret this as an aggressive threat and respond accordingly. This ‘miscommunication’ can be a problem when people who have no experience of interacting with primates visit animal units.

Assessment of welfare

Primates are highly intelligent, sentient and social animals with a complex range of physical as well as psychological needs; therefore both physical and psychological well-being should be considered when assessing welfare. There is no doubt that they experience pain and a range of negative psychological states such as anxiety, apprehension, fear, frustration, boredom and depression72, as well as a range of positive states. Mood can also be affected by social status72. It should be assumed that procedures that cause pain and distress in humans will probably cause pain or distress in other primates73,74. A monkey in pain may show a generally miserable appearance, and may adopt a huddled position or crouch with head forwards and arms across the body. It will tend to refuse food and drink and to avoid companions, although monkeys that are unwell may attract extra attention from cage mates, varying from social grooming to attack. Vocalization is more likely to indicate anger than pain.

The health should be evaluated regularly and records kept and a programme to review and improve physical and psychological health applied where necessary. Evaluation of the psychological condition of the animal should take into account its physical health, its behavioural repertoire and an assessment of its coping skills when presented with novelty. The latter will evaluate normal inquisitive and explorative behaviour or any abnormal response and how long the animal takes to return to baseline level of behavioural repertoire. All staff–animal interactions should be based on an understanding of species-typical behaviour patterns and communication systems, such that these are interpreted correctly and responded to appropriately.

Clinical scoring systems are usually used for monitoring of single incidents but there should also be some assessment of the cumulative suffering and the lifetime experience of the animal. This will include direct suffering from the procedure as well as contingent suffering75 as a result of transport, housing, the environment and injuries from caging or conspecifics, to name just a few examples. The life span of primates, compared with other laboratory animal species, is relatively long. They are valuable animals, have long breeding lives and are likely to be used in long-term experiments. Some primates may therefore be housed in the laboratory for many years. In such circumstances there are particular welfare issues that must be addressed65,66. A welfare assessment matrix can be used to give an estimation of cumulative suffering and a pictorial view of welfare76.

Physical health

The first step in assessment of the monkey’s physical health is to observe the animal in its home cage and evaluate its appearance, behaviour and general demeanour. In order to do this it is vital that the observer has some experience of the animal (both the species and the individual) to be able to judge whether the animal is exhibiting a normal behavioural repertoire. Just like humans some individual non-human primates exhibit behavioural patterns that are specific for that individual, but do not necessarily reflect poor well-being. Only after completing the examination from a distance and noting the behavioural responses should the animal be caught to enable it to be examined more closely. Catching it will markedly affect its behaviour, whether or not sedatives are used, which it why it is important to make a full evaluation first. For a full description of clinical assessment of a monkey see Wolfensohn and Honess7 and Smith et al.55.

The animal’s weight should be recorded and its body condition scored. It is assessed by palpating the monkey over its thoracic and lumbar vertebrae (at the level of the last rib) and making a judgement as to the amount of fat and muscle covering the bony prominences of the vertebrae and giving a quantitative score. Condition scoring at weaning and during the post-weaning period, combined with regular weight measurements, is important to ensure that the animal is receiving adequate nutrition and growing properly.

Psychological health

This may be assessed by behavioural monitoring and by monitoring conditions such as alopecia77. When assessing welfare using behavioural parameters it is important to assess the animals at various times. The animal’s behaviour should be noted on the approach of the observer. Observations should then be repeated after the animal has had time to habituate to the presence of the observer. The use of space and structures, any self-maintenance behaviour, and social behaviour, as well as the animal’s disposition and interaction with observers, are all recorded.

An awareness of the normal behavioural repertoire for the species is important to enable provision of appropriate care and management and for assessing their physical and psychological well-being. Knowledge of the behaviour of individual animals is also essential since primates show a high degree of individuality. Any deviations from normal must also be recognised to enable evaluation of refinements such as changes in cage design or environmental enrichment.

The advice of a primate behaviour specialist is invaluable in training both care staff and research staff to recognise and interpret deviations from normal behaviour and to assist in developing programmes to ensure good psychological health.

Euthanasia

The principles of euthanasia are set out in Chapter 8 in this volume and in Close et al.78,79, American Veterinary Medical Association 80 and Wolfensohn32, all of which refer to the animal welfare concerns with respect to carrying out of euthanasia. Having to kill a primate may be a more emotionally challenging experience for staff than having to kill some other species. Staff who carry out this procedure must not only be competent in the appropriate methods, but it is essential that they receive good training, adequate supervision and any necessary empathetic support.

The most common method for killing primates is an overdose of an anaesthetic, such as sodium pentobarbitone (at 100 mg/kg) administered intravenously. For larger primates it will be necessary to sedate the individual before attempting an intravenous injection.

When primates are euthanased, every effort should be made to make full use of their tissues and blood, particularly if this will minimise the number of animals having to be used. This requires good mechanisms for communication to match supply and demand both within and between establishments. Establishing tissue banks and data-exchange networks is one means of coordinating, optimising, reducing and refining primate use. In the USA www.primate.wisc.edu/pin/achieves this objective and in Europe EUPRIM-NET offers a tissue and gene bank (www.euprim-net.eu/).

Record keeping

All primates should be uniquely identifiable, for example by microchip or tattoo. A file or ‘passport’ giving full details on each individual primate should be maintained. It should keep the details of their biography enabling a full picture of previous experiences to be established. This should be sent with the animal if it is moved between institutions, together with general information on the establishment of origin, such as details of animal care and routine procedures, in order to assist in continuity of care and to ensure successful acclimatisation to the new facility. Good record keeping is essential for retrospective review and for the assessment of cumulative severity.

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