Husbandry

Forcibly removing animals from an enclosure can cause injury, stress and take more time. Where possible, perform procedures in the home area and training animals to cooperate with them using positive reinforcement. Because the animals have some control over the situation, by retaining relative freedom of movement, they show reduced stress responses [11,12]. 

Animals may be trained to:

• Cooperate within the social group.
• Present a part of the body for inspection.
• Present a limb for venepuncture.
• Open mouth for oral examination.
• Voluntarily enter a transport cage.

If animals need to be moved out of the home area, this is best done by training to enter a transport or capture cage (see Transport). Whilst not completely stress free for the animal, this method is usually more humane than use of the pole-and-collar.

Young macaques, and animals of known, safe health status, may be hand-caught and held if they are well habituated. Lightweight gloves provide greater sensitivity to pressure applied than thick gauntlets.

• Only handle when strictly necessary (e.g. for veterinary procedures).
• Handle for the minimum length of time.
• Cage squeeze-back mechanisms should be used only where the above cannot be achieved. Animals should be trained to move into the squeeze-back cage using positive reinforcement and the minimum level of restraint applied. Lightly touching the squeeze-back mechanism may ultimately be enough for an animal to cooperate with a procedure. Macaques can be trained to present specific parts of the body to the front of the cage when the squeeze back mechanism is started, reducing the need for repositioning [13,14].

Handling macaques is an advanced skill which should be an essential part of staff training with ongoing competency assessments.

In the image above a macaque is being handled with gauntlets. Animals of unknown health status should be handled with gauntlets for protection from scratches, bites and infections. It is important to ensure that the animal is accustomed to the items; if distress occurs the animals will learn to associate the protective clothing with the handling procedure and become more stressed in future encounters.

• Nets should not be used for capture, except in emergency situations. Use of a net can result in entanglement and injury, and acute stress leading to diarrhoea, rectal prolapse and lacerations. It also leads to acute stress for all members of the group.
• Avoid pole-and-collar capture and restraint. If use of the pole-and-collar is necessary for reasons of staff safety, ensure its use is refined by employing positive reinforcement techniques [15].
• Do not use a squeeze back cage as the home cage, since this can limit structural complexity and enrichment (e.g. wooden branches, swings, hammocks) and the home environment ought to be a secure base for the animal.
• Do not hold or carry a macaque with one hand, holding both arms behind the back; instead macaques should be cradled in both arms. If holding the arms behind the back is necessary for health and safety reasons (e.g. large, non-habituated macaques) take care not to pull the arms backwards as this will be painful and stressful for the macaque, and can lead to dislocation of elbow and shoulder joints or fracture bones [16,17].

Restraint chairs are used for prolonged restraint in a sitting position. Chair restraint can affect the animal’s physiology and compromise animal welfare [17-20]. Use of restraint chairs is a regulated procedure in most countries, requiring approval from the competent authority.

If chair restraint is required, then it should be refined [2]:

• Select calm animals; habituate them to the sensation of sitting in the chair prior to study; reward calm behaviour.
• Train animals to enter the chair from the home enclosure and place their head through the top of the chair [21-23].
• Move occupied chairs carefully and quietly.
• Minimise the duration of restraint; define the maximum length of chair time after discussion with veterinary and animal care staff.
• Attend restrained animals all the time; massage the animal’s legs to maintain proper circulation.
• Where studies allow simultaneous chairing, the presence of a compatible companion animal may ameliorate stress responses [24-28].

Chairs need to be well-designed and suitable in size and operation for the individual animals in order to prevent injury and discomfort [20]. Macaques tend to ‘squat’ rather than sit, so the design needs to reflect this. Useful design features include:

• Minimal confinement, leaving animals room to stretch their limbs; arms should be free (if external catheters are used, the chair design should enable these to be taped out of reach).
• Adjustable sections (e.g base/seat that can be moved up and down, positioners) to accommodate individual physical characteristics; the position and length of the tail, position of the ischial callosities, crown to rump length, limb length and variations in posture, all need to be taken into account.
• Use of anti-friction material (e.g PTFE tape) to prevent chafing.
• Wheels fitted with silent castors.

• Water should be available ad libitum.
• Provide both dry and wet pellets – dry pellets help to maintain good dental hygiene while wet pellets, moistened using warm water, fruit juice of non-milk-fat milkshake, are more palatable [2].
• If feeding insects to macaques in captivity, feed the insects on a calcium rich diet for 2-3 days before use [31].
• Supplement commercially available, dry monkey pellets with fresh foods, including:
  
  - Fruits and vegetables, especially leafy greens [2]. Tough, fibrous foods will help to maintain good dental hygiene.
  
  - Animal protein (e.g. live insects).
  
  - Seeds and nuts.

See Food-based for additional information and videos on food presentation.

• Feed several times a day.
• Provide a choice of food at any given feeding time.
• Less preferred foods can be provided in the morning, when hunger is at its greatest.
• Plan a feeding roster, so that different foods are provided on consecutive days.
• Predictable feeding times can elicit stereotypies, agitation, aggression , inactivity and coprophagy [32-34]. Avoid food anticipation by varying feeding time between days. Precede feeding with a reliable signal, such as a buzzer or bell, when feeding at random times [35,36].
• Scatter food around the enclosure so that all animals can access it.
• Provide food at various heights within the enclosure.
• Suspend foods by skewing on wire or wood and hanging from ceilings or walls.
• Use enrichment items, such as foraging substrates and puzzles.
• Provide whole foods as well as food cut up in smaller portions.
• Make a fine-grain forage mix to keep animals searching for food for longer periods of time.

Primates require a varied nutritional content in their diet (see Diet). Energy requirements may differ between individuals based on species, age, sex, body weight, health, reproductive status and environment (i.e. activity levels as determined by the type of enclosure) [37]. A juvenile macaque weighing 2kg requires approximately 80-90g (200kcal/kg/day) daily, whereas an adult of 8kg needs 110-190g (100kcal/kg/day) daily; lactating and pregnant females have a higher daily energy requirement (125kcal/kg/day) [31].

• Group living can make it more difficult to ensure that all animals receive adequate amounts of food. Careful monitoring at feeding time will help to determine if specific individuals require supplemental feeding.
• Macaques may eat in excess of their basic energy needs, so obesity is a risk in these species [2]. Regular weighing can help to maintain healthy weights.
• Remember that food treats used for positive reinforcement training need to be considered as part of the nutritional content of the diet.

For recommendations on the general nutritional requirements for non-human primates, and macaques in particular, see the following references. You can also consult a primate nutrition specialist.

• Nutrient Requirements of Non-human Primates
• The Merck Veterinary Manual
• Wolfensohn & Honess 2005 (information from McCance and Widdowson’s The Composition of Foods integrated dataset)

Zootrition® is a useful tool in estimating nutritional content of diets.

Predictability of general husbandry procedures has been shown to be important for animal welfare. For example, whilst feeding times at a macaque facility typically occur within a given time frame, this event can sometimes be delayed (e.g. by emergency events or staff shortages). This causes a semi-predictable event to become unpredictable, resulting in a significant, negative behavioural and physiological changes in the animals (i.e. stress) [38].

Predictability is invariably linked with the concept of control; control can be gained by animals in a predictable environment, and providing an element of control can make an environment more predictable. Predictability of an event can be both temporal (the event occurring consistently at the same time of the day) and signalled (the event preceded by a reliable signal) [39]. The combination of the two is more effective than the use of them individually in reducing stress and anticipatory behaviours [40].

Giving animals a signal that an event, whether positive or negative, is about to take place can reduce their long-term anticipation of it. Signals can be used for husbandry procedures, such as feeding or cleaning, animal-human interactions (e.g. habituation and training), and for the arrival of an aversive event (e.g. veterinary or experimental procedure). Something as simple as a specific sound or coloured card can be used to signify a given event; animals will quickly associate the two. So in the above example, if feeding time is kept as consistent as possible and always combined with a signal, then the effect of any delay in temporal predictability could be mitigated by the use of signalled predictability.

Potential sources of stress during transportation include [48]:

• Handling.
• Separation from group.
• Isolation.
• Confinement.
• Loading and unloading.
• Movement and vibrations during transportation.
• Physical stress from maintaining balance.
• Unfamiliar surroundings.
• Changes in temperature, humidity and light/dark regime.
• Food/water restrictions, either voluntarily or involuntarily.
• New staff and other macaques.
• New housing and care protocols.

Minimising the reaction to stressors, using desensitisation (see Habituation and training), is important for animal welfare reasons.

Macaques can be trained using positive reinforcement techniques to enter a transport container on request [17,21,49,50]:

• Allow the macaques to investigate the containers without restricting their movements. Place the container inside or adjacent to the home enclosure and allow free exploration. Food rewards can help to encourage investigation, particularly inside the containers.
• Present the container on repeated days until macaques enter the container quickly and calmly. Reward calm behaviour in the container.
• Progressively close the door or slide to the container so that macaques become accustomed to the action, and then become accustomed to remaining inside for a food reward while the door closes. Macaques may initially flee when you reach for the door. This is a normal response. This procedure will take several training sessions.
• Increase the length of time the macaque is held inside the container. Provide rewards to minimise stress.

Journeys between establishments should be planned in advance. A journey plan should be agreed between the provider and recipient. This should list the responsibilities of the individuals involved and the events at each stage of transport.

The following factors should be taken into consideration [48]:

• Documentation for travel outside the facility (e.g. individual history file).
• Fitness of the animals for travel.
• Number of animals being transported.
• Design and construction of the container.
• The amount of space given to each animal.
• Where the animals can be transported as compatible pairs.
• The environmental conditions within the container.
• The quality of the substrate, food and water provided.
• Labelling of containers and vehicles (e.g. ‘Live animals on board’; ‘This way up’).
• Means of monitoring animal welfare during transportation.
• The route of travel is the shortest route the best route?
• Duration of travel minimise total journey time where possible.
• Number of stops, or changes between transportation vehicles minimise these where possible.
• The attitude, experience and training of personnel handling and transporting the animals (including the driver of the vehicle).
• Contingency plans, in the case of delays to shipments, problems with the chosen route, vehicle breakdown, unexpected demands from border officials, and death during transit (including emergency euthanasia).
• Health checking on arrival.
• The time and protocol necessary to help the animal adjust at the end of the journey.
• Monitoring of the animals in their new surroundings.

Details of transport containers for international transport of macaques are given in the IATA Live Animals Regulations, Swallow et al. 2005.

Most forms of quarantine are intended to protect human health through screening and treatment of the animals, or to detect disease in the animals, but the process can have a considerable impact on animal welfare. On arrival, imported macaques are likely to be disoriented and fearful of their new surroundings, and stressed by isolation from former social companions, coupled with fear of unfamiliar animals and humans in close proximity. Some exporting and importing centres, set up to deal with large batches of macaques, house the animals singly in small cages with little or no enrichment. Negative experiences and behavioural pathologies that develop during quarantine periods can have long-term effects and may counter the value of refinement and enrichment efforts applied subsequently.

Opportunities to refine quarantine [2] include:

• Review the necessity for each quarantine episode (and the number and type of tests requested) and reduce the number where possible (e.g. by direct import to a registered quarantine facility in the user establishment; by gathering greater information about the disease control requirements of regulators).
• Pair or group-house animals during quarantine. If in the event of individual clinical or subclinical disease cases, near neighbours or the entire room will be treated in the same way, then single housing is not justified. For animals from the same breeding colony, it should be possible to form new pairs among juveniles fairly quickly, or better still, arrange for animals to be pre-paired or pre-grouped at the colony of origin.
• If single housing is unavoidable for justifiable veterinary or human health reasons, limit this to as short a time as possible (and ensure animals can see and hear their conspecifics). Explore ways of expediting test results.
• Provide sufficient space for a good amount of environmental enrichment to allow and encourage a wide range of behaviours. Capturing animals in larger cages should not be a problem if they are adequately habituated to humans early in life (although the stress of shipment may make them less cooperative). If concerned about ease of sanitisation, focus on a ‘softer’ environment and disposable materials, such as hay, wood, cardboard and (non-printed) paper, which can be easily replaced. For animals previously living under natural conditions, such enrichments may afford more familiarity and comfort value than artificial objects or devices.
• Provide visual barriers to calm frightened animals. Panels or strips of muslin, burlap or opaque plastic have been used successfully as visual barriers on the outside of enclosures.
• Reduce cage-related noise. Macaque raised in outdoor settings appear to be particularly sensitive to acoustic stress.
• If a prolonged period of quarantine is required in the importing country, and the health status of animals has been defined, it may be possible to commence positive reinforcement training before the end of the quarantine period. This will be easier if the animals have been exposed to habituation and positive reinforcement techniques (e.g. training them to accept food from the hand) at the exporting centre (see Habituation and training).

Habituating animals to research, veterinary and husbandry procedures will help to reduce the fear and distress such activities may cause [2,14,51]. Signs of stress include urinating or passing faeces, facial expressions of fear and alarm vocalisations (see Expressions and Vocalisations). Macaques should be habituated to humans and procedures as early in life as possible, while still in their family group. The presence of habituated adults will help to reassure younger individuals. Strategies for habituating animals include [2]:

• Spending time with the monkeys each day.
• Minimising noise in the vicinity of the animals.
• Talking to the animals in a calm voice.
• Positioning food and water near the front of enclosures to encourage animals to approach and avoid fear withdrawals to the back of the enclosure.
• Interacting with the animals in positive manner (e.g. feeding from the hand, eliciting play) so that the animals do not associate staff with only negative events.
• Improving the visibility from the enclosure of human activity and approach via, for example, use of windows, verandas, mirrors and widely-spaced bars.

When habituating animals to procedures, develop a slow build up to the complete procedure. For example, prior to injecting an unrestrained animal, training should first take place to accustom the macaque to:

• The syringe.
• Presenting the appropriate body part.
• Accepting being touched with a needleless syringe to the appropriate body part.

These steps should be progressive and always positively rewarded. Also reward after the completion of the entire procedure, in order to ensure that the training session finishes on a positive note. It is helpful to developing a plan in advance, showing the steps for shaping the behaviour to the desired end goal (successive approximation).

The aim of training is to shape a behaviour so that the animal actively responds in a way that is desired by the trainer for the purpose of an activity. Training macaques to cooperate with scientific, husbandry and veterinary procedures can have significant benefits for animal welfare, research and staff, especially when combined with socialisation, habituation and desensitisation [52-54].

Training can be used for:

• Encouraging cooperation with husbandry procedures, including moving between cages, weighing, temperature recording, scanning of microchips, inspection of body parts, injections, and topical application of medicines.
• Encouraging cooperation with scientific procedures and reducing associated stress, which will help to improve the quality of the research data.
• Enriching the captive experience.
• Developing trust bonds between the macaques and humans.

Adapted from Prescott & Buchanan-Smith 2007.

• Habituation: The waning of a response as a result of repeated stimulation but not fatigue.
• Socialisation: The process by which macaques learning to successfully interact with members of their own species (conspecifics) and humans.
• Desensitisation: Systematically pairing positive reward with an uncomfortable or aversive experience or stimulus in order to reduce any associated fear or anxiety response. 
• Training: The shaping of the behaviour of a macaque so that it responds in a way that is desired by the trainer. 
• Positive reinforcement: The process of increasing the frequency of a desired behaviour by introducing a rewarding stimulus (e.g. food treat) contingent on its performance. 
• Negative reinforcement: The process of increasing the frequency of a desired behaviour by removing a noxious stimulus (e.g. the cage squeeze-back mechanism) contingent on its performance. 
• Positive punishment: The process of decreasing the frequency of an undesirable behaviour by introducing a noxious stimulus on its performance (e.g. verbal command “No!”).
• Successive approximation: The process of rewarding small steps that will eventually lead to performance of the desired behaviour. 

The key points for developing a successful training program include [3,53,54]:

• Always use positive reinforcement techniques. If negative reinforcement is necessary (e.g. where the scientific procedures are aversive and time is limited), always balance this with positive reinforcement [55]. Limited use of negative reinforcement can ensure the animals have a successful training session overall.
• Give the reward immediately after performance of the desired behaviour (or as near as possible); behaviours consistently paired with receiving a signal (click or whistle) and/or food reward are more likely to be performed again.
• Start with the basics – typically training animals to approach the hander and to station at a specific location for further training.
• Use vocal commands paired with hand gestures – this multi-modal approach has proven to be successful.
• Slowly develop the desired behaviour. Reward the behavioural steps which approach the desired end goal behaviour. Complicated behaviours should be broken down into manageable smaller steps.
• Recognise that some animals learn faster than others, and that training will not necessarily always produce consistent results between individuals. Temperament testing may be valuable [56,57].
• Do not continuously push an unsuccessful command. If the animal does not cooperate or understand what is required of it, revert back to a previously learnt command to rebuild confidence.
• Always remain calm. Ignore undesirable behaviour.
• Always finish a training session on a positive note, even if you have to go back to basics in order to do so.
• It is important not to interrupt someone else’s training session. This can disturb the animals and halt the flow of progress.

Have realistic expectations of training time and likely outcomes. Here we present examples of training in macaques for a variety of purposes and the length of time taken to achieve the goal.

• Reinhardt (1997) Training non-human primates to cooperate during handling procedures: a review
• Prescott et al. (2005) Resources for developing and implementing training programmes
• Bloomsmith (2012) Positive reinforcement animal training in primate laboratories
• Perlman et al. (2012) Implementing positive reinforcement animal training programs at primate laboratories

For further articles and advice on training non-human primates, see the Training animals page of the NC3Rs website.

Behavioural management is the combined use of positive reinforcement techniques and enrichment to develop, direct or alter behaviour [61]. It is particularly applied to the management of abnormal behaviours (see Signs of poor welfare).

It is important to have both short and long term goals as part of any behavioural management strategy. This ensures that care staff are proactive rather than reactive. Responding to behavioural problems after they have developed is less successful than the management of behaviour to in order to avoid the development of these problems [61].

Behavioural management should be part of everyday husbandry. This is maintained through the application of:

• Positive reinforcement training.
• Enrichment.
• Social housing and management of compatible animals.
• Socalisation and habituation.
• Recognition of individual needs.

Short-term goals of behavioural management may include:

• Reducing boredom that day.
• Occupying animals to reduce a conflict.
• Reducing the agitation that may be elicited by a veterinary inspection.

Long-term goals of behavioural management may include:

• Developing trust between care staff/researcher and animals.
• Habituating animals to transportation cages.
• Achieving voluntary cooperation with veterinary and research procedures.
• Improving how the animals cope with stress.
• Creating stable groups [62,63].

Cynomolgus macaques perching in position

Managing abnormal behaviour

If an abnormal behaviour does require management, a considered and systematic approach is required. Following these steps will aid in this process [61].

1. Collect data:

• When does the behaviour occur?
• When did the behaviour first occur or when was it first observed?
• What are the circumstances surrounding the behaviour?
• Does the behaviour consistently occur during a specific event?
• What is the social status of the animal? Is the animal trying to seek attention, either negative or positive?

2. Develop a hypothesis. This will help you focus your data and the potential explanation for the occurrence of the behaviour, thus improving the likelihood of actively managing the problem.

3. Identify specific behavioural goals. It is important to be realistic during this step. It is not always possible to completely eradicate an unwanted behaviour, but through careful management you may be able to reduce the occurrence or severity of it.

4. Initiate training and enrichment strategies. Some behavioural abnormalities require one or the other, or a combination of both. Remember that training of the care staff is also required in these situations, to avoid unconsciously reinforcing the behaviour.

5. Consider the results of your strategies from both a short- and long-term perspective. The initial management may have an immediate effect but the behaviour may reoccur later. It is important that the management strategy does not become complacent because of quick results or frustrated by the lack of results in the early stages.

6. Adjust the method of approach if necessary. If the applied management does not elicit results, consider another approach to the problem. This may mean collecting new data, rethinking your hypothesis and using alternative training and/or enrichment strategies

Monitoring and managing the health status of a macaque colony is important for human safety, as macaques can carry and transmit pathogens to humans (and vice versa); deaths have been attributed to Macacine herpesvirus 1 and Filovirus [64]. Fatal epizootic outbreaks like tuberculosis, simian haemorrhagic fever and simian retrovirus D have posed serious health risks to primate workers in the past.

Avoiding disease is also important to maintain the animals in good health; for instance minimising the risk of enteric infections such as Shigella. Maintaining appropriate health status also depends on the purpose of the colony. For example, screening for sexually transmitted diseases will prevent possible detrimental effects to the offspring and is most relevant to breeding establishments; whereas other microorganisms can cause subclinical disease which can alter experimental data (although less is known about this compared to other laboratory animal species, such as rodents) [65].

At present, no single recognised health control programme exists for laboratory non-human primates, but there are initiatives to harmonise existing protocols (e.g. FELASA guidelines; see also CDC/NIH guidelines).

The design of a health monitoring protocol is complex, requires careful consideration of which agents to screen for, and should be tailored to each facility. Appropriate statistical methods need to be applied to determine numbers of samples and frequency of sampling. Sampling protocols will depend on the prevalence of a microorganism, its biology and epidemiology (e.g. shedding patterns) and other information to be obtained. An appropriate testing method needs to be chosen depending on the desired specificity, sensitivity and predictive values of the technology, and the detail of the health monitoring protocol.

Managing a colonies health status includes ensuring appropriate facility management (e.g. security/restricted access, personal protective equipment, sanitation, waste handling), equipment (e.g. cage washer), personnel management (e.g. standard operating procedures, staff training) and a comprehensive veterinary care program (e.g. management of a quarantine facility).

If an animal needs to be sedated for sample collection (e.g. blood), its body weight and other relevant information, such as body condition score, body mass index (BMI), alopecia score and the outcome of a physical exam (see Health indicators), should be recorded. Individual records should be kept for the lifetime of the animal. All results of diagnostic tests, including the testing protocol and method, should be kept for the duration of the research establishment.

To reduce the number of sedations, health checks should be combined with sedations or general anaesthetics performed for another reason (e.g. experimental procedure) wherever possible. Conducting post-mortem examinations will provide additional valuable information on a colonies health status.

Regular reviews of the colony health monitoring protocol and long-term record keeping is crucial for detecting changes early and managing interventions effectively.

The decision on what PPE to wear, will be based on several factors including the health status of the colony (e.g. Macacine herpesvirus 1, tuberculosis) and the vaccination status of the animals and humans (e.g. measles; Tb), as well as expertise of staff working with them [69-70]. Human health considerations (e.g. altered immune status due to intake of medication) and other risk factors, such as pregnancy or illness, should be considered. Liaison with the local occupational health service is advisable [71].

The benefits of the PPE (e.g. use of face masks to protect the animals from respiratory infection from staff) need to be balanced against the potential disadvantages (e.g. reduced level of communication between staff and animals because facial expressions are obscured) [72,73].

For animals used in scientific procedures, the nature of the work (e.g. immunology research) might also impact on the design of the health monitoring programme and thus affect the choice of PPE (e.g. transmission of commensal human skin flora, such as Staphylococcus, to instrumented animals; or prevalence of MRSA [Methicillin-resistant Staphylococcus aureus] to allow optimal management of surgical protocols) [74].

Written by Dr Caroline Bergmann, University of Oxford, with assistance from the NC3Rs.