Monkeys are similar to humans. Not only on the outside but also on the inside. That is because monkeys are genetically related to us. Due to this evolutionary relationship monkeys sometimes are a good model to study human diseases. But only if there is no other way.

Working with monkeys brings a great responsibility. We are responsible for the well being of the animals in our colonies. We continuously seek to conduct research that does not involve animal testing in order to reduce the numbers of animals we work with. In the meantime we accommodate and look after our monkeys with the best possible care.

We do this using the principles of the 3Rs. Refinement, reduction and replacement. Refinement and reduction go hand in hand as Refinement of an animal model will lead to a Reduction of the number of animals per experimental group.

3Rs throughout BPRC



  • Improvement of animal welfare is a continuous process in our institute. BPRC staff take part in (inter)national training programs to remain their high standards and gain new insights.
  • All animals are socially housed.
  • Stress is not good. It affects animals in breeding groups and can even affect the results of an experiment. In order to avoid stress you need to identify stressful events. And for that you need unbiased, objective and reliable parameters to determine stress.
    • Measuring the cortisol levels in hair samples is a method that can provide stress information from an individual animal. By cutting a hair into smaller pieces you can relate the cortisol levels to potential stressful events.
    • We take pictures as an objective measure for alopecia. Alopecia (hairless body parts) can be a sign for acute stress. Caretakers are trained to detect this and to take pictures. Sometimes an animal experiences stress from hierarchy in their breeding group. If that is the case behavioral scientists are notified to monitor the breeding group and if possible take measures.
    • When animals are prepared for housing in an experimental setting they are introduced to a selected cagemate. We can use round the clock camera recordings to monitor their behavior in the absence of a caretaker. This avoids less-compatible pair- housed animals.
  • Positive reinforcement training (PRT). We have trained 25 animal caretakers how to train their animals. They do this twice per week. With this training method we are able to perform certain biotechnical techniques without sedating the animal.
  • All marmosets jump voluntarily on a scale. This way their body weight can be monitored without sedation.
  • All experimentally housed animals were trained to drink from a syringe, thus voluntarily take oral medication.
  • Caretakers spent 15% of their time on (cage)-enrichment. For instance assembling food- puzzels, providing animals with toys or redecorate enclosures.
  • Further improvements were implemented in diet variation, to maximize natural feeding routines.
  • In 2017 an improved version of the ‘Welzijnsevaluaties’ was implemented.
  • New features were introduced in our monkey database for the daily registration of each individual animal.
  • All animals in experiments are observed at least twice a day. During this observation different parameters are ‘scored’. Normally an animal shows a broad variety of natural behaviors. In some models for (infectious) diseases the animal’s behavior changes. This is however a subjective parameter and changes are difficult to observe. Subtle changes during an experiment can provide crucial information. In this case we prefer to measure physical activity with telemetry. These devices register X-Y-Z coordinates of individual animals. If necessary it is also possible to measure body temperature, heartrate, blood pressure. This will lead to further refinement of our animal models.

Optimizing and standardizing in vitro laboratory tests play an important role in the reduction of the animals we work with. Also in 2018 we have implemented new techniques. By using these new conditions, we aim at less variation in laboratory tests that will lead to smaller group sizes in our animal experiments.

Genes play an important role in infections and diseases. We have implemented new techniques to determine the genetic background of animals in the breeding and experimental colonies. This enables us to select (or deselect) appropriate animals to answer particular research questions. For example; we know that certain genes play a role in the development of AIDS after HIV infection. We now know that these genes are also present in monkeys. Selection of animals for an HIV experiment is therefore based on these genes. Proper selection reduces the variation in an experiment and therefore smaller group sizes are required to obtain statistical significant differences.

Statistics at BPRC
One of the hallmarks of good science is statistics. Not only at the end of a proof on concept study to determine whether an HIV-vaccine was successful but also during the design of the study. Therefore, good statistics is part of the 3Rs.

Statistics is often used to determine whether differences in study outcomes are (statistically) significant. This is normally done by rejecting or accepting the null hypothesis, where the null hypothesis states that treatment does not have a significant effect. To do so, the p-value is calculated. If the p-value is below 0.05, the chance that the study outcome arose by chance is smaller than 1 in 20. In that case, the null hypothesis is rejected, supporting the alternative hypothesis that the observed difference was due to the treatment.

But statistical testing is only informative if the study is properly designed. If group sizes are too small a real difference may not be detected and the study will not be informative. If group sizes are large differences will be detected, but at the cost of too many animals. Therefore study design involves, amongst other things, also a so called “power calculation”. The number of animals per group is calculated based on the desired effect of the treatment on the primary outcome (e.g. diseased or not-diseased), the between-animal variation of the treatment effect and the desired power. The desired power is the chance that a real difference, if present, is detected. This is usually set at 80% (i.e. 80 out of 100 studies will yield significant results). Next to the power calculation, the study design also involves methodological topics like randomization of the animals (treatments are allocated by chance) and blinding of observers (treatment is not known). Next to the power analysis, a statistical analysis plan is written before the study is performed. Because monkey studies are often the last step before testing in humans, monkey studies should be designed, performed, analyzed and reported in a similar fashion as clinical trials in humans.

At BPRC statistics also involves analysis of observational data. Marmosets are usually born as twins or triplets. Yet, the mothers’ resources are often not sufficient to raise all three babies. Therefore triplet litters are not desirable. Based on observations made by our colony manager, we investigated whether marmoset mothers that were born as triplets are more likely to produce triplet litters. This proved to be true (Bakker et al 2018 Am J Primatol). The importance of this analysis is that we now can minimize the chance of triplet litters.

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Positron emission tomography–computed tomography (PET-CT) is a visualization technique that combines anatomic localization (x-ray) and functional imaging (nuclear medicine). In hospitals PET-CT is already widely used during the diagnosis and treatment of cancer. But quite recently PET-CT also found its way to biomedical research with animals.

PET-CT offers many advantages over traditional techniques. First, PET-CT is non-invasive. Results from blood tests, biopsies/swabs or cells washed out of the organ of interest can be indicative for infection, they are often poor indicators for actual disease manifestations. Biopsies only provide information of the tissue in the biopsy but often not of the entire organ. The combination of x-ray and specific radioactive probes allows screening of the entire body. This minimizes the discomfort of the animals and provides you a much broader view.

In addition PET-CT offers the opportunity to visualize disease progression or therapeutic response over time (longitudinal). This is particularly relevant when critical organs need to be studied, like lungs or brains. PET-CT in combination with 18F-Fluorodeoxyglucose (FDG) as imaging agent is well-established and commonly used in both animals and humans. FDG visualizes the glucose metabolism in the body and shows increased signal in areas with inflammatory activity. This makes FDG PET-CT highly sensitive for detecting for instance tuberculosis and influenza lesions in the lungs.

BPRC already started to use PET-CT in 2017. Initially only in our tuberculosis research but currently we are investigating the options to apply this state of the art technique also for other research programs. In the near future PET-CT will not only lead to new scientific insights but also increase the translational value of our animal models as PET-CT can also be applied to patients.

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In 2009 BPRC-researchers developed an new in vitro assay to test drugs for it’s anti-malaria activity. This assay replaces the use of monkeys. Last year we tested 33 new potential anti- malaria drugs with this assay. Before 2009, 33 monkeys would have been necessary to test these 34 compounds. So far, BPRC tested 999 drugs with the animal-free assay.


3Rs Alternatives Unit BPRC



When a disease cannot be studied in humans, scientists can induce experimental diseases in animals. The more closely the animal species is related, the better the disease processes resemble that of humans. Although this approach has led to many important discoveries and new therapies, this approach also may have an impact on the welfare of animals. We are fully aware of our responsibility to society and animals and we are only allowed to use animals when there are no other -alternative- methods available. Alternative methods are categorized along the principle of the 3Rs of Replacement, Reduction and Refinement, all of which have a place within BPRC. When alternative methods are available, Dutch law obliges researchers to use the alternatives and forbids the use of animals. However, not many of such methods are available yet. Rather than waiting, BPRC is actively testing and developing alternative methods. The 3Rs are implemented in the research of every department, as well as in the separate Unit Alternatives.

Developing in vitro methods for the central nervous system

In the Western world, society is gradually aging and more and more people suffer from age-related diseases. Many of these diseases, like Alzheimer’s disease, Parkinson’s disease and multiple sclerosis affect the central nervous system. BPRC works with animal models for each of these diseases. In vitro methods to complement, refine, reduce and finally replace the use of animals in such models are therefore highly relevant. Over the years we have successfully developed methods to study individual brain cells in test tubes. In 2018 we have made significant progress towards the development of in vitro methods that even better resemble non-activated cells tissue. In addition we have started an initiative to cultivate stem cells with the aim to generate 3D organoids, mini brains, in a dish.

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European initiatives: VAC2VAC and TRANSVAC2

Vaccines are the biggest success story of biomedical science. They have changed human life expectancy dramatically. Animals are not only used during the development phase of vaccines, but also during the production and quality control phase. We participate in European initiatives that aim to reduce animal use in both phases.

Vaccines are typically produced in batches. Every batch undergoes the same strict series of quality controls, involving many animal experiments, before it is released. The European VAC2VAC initiative tries to change this. The idea is that every new batch of a vaccine should not be treated as an entirely new entity, but rather as one of a series. This implies that every new batch only needs to be similar to the previous batch, possibly circumventing animal testing. To prove similarity between batches, animal-free methods are used. By adding our panel of in-house engineered cell lines to the consortium we characterized several different vaccines and batches in 2018. Our results demonstrate that different batches are indeed highly similar according to our tests. Together with the tests that are developed in other European labs, this initiative should lead to abandoning animal testing in vaccine batch release. The European TRANSVAC2 initiative is stimulating innovative vaccine approaches. We contribute by making our library of bioassays available to the European research community. In 2018 we have started research on the mechanisms that affect vaccine and adjuvant efficacy when immunization is done via the skin.

Developing an adjuvant without adverse effects

Adjuvants are formulations, which upon administration lead to non-specific immune stimulation. They are often used to stimulate immune responses directed against pathogens (for vaccination studies) or against components of the body itself (in animal models for human auto- immune diseases like multiple sclerosis). Some adjuvants are notorious for their adverse effects. Most notable is complete Freund's adjuvant (CFA), which causes inflammation of the skin accompanied by granuloma formation in non-human primates. It is however still being widely used in many animal species because of a lack of alternative. Using our bioassays, we have developed a new adjuvant in house, MiMyc. In 2018, MiMyc was tested in a small in vivo experiment and proved to be a potent adjuvant without causing adverse effects. If MiMyc can replace CFA in animal models for auto-immune diseases this would represent a considerable refinement. We aim to test this in the coming year.