Policy on animal research

Hasselt University has chosen to conduct a transparent policy on animal research. This was ratified by signing the statement in support of animal research and a transparent approach. In terms of transparency, already a few initiatives were taken, including a lecture about animal experiments at the senior university, presentations for secondary schools, participation in the re-place platform and interviews in several media. 

At Hasselt University, we mainly conduct fundamental research to unravel how diseases develop, what might be the underlying causes and which factors determine the disease progression. In addition, there is translational or applicable research, in which potential treatments and therapies are investigated.

No. Since 2004 it is forbidden within the EU to test cosmetic products by means of animal experiments. Starting from 2009 it is prohibited to test cosmetic ingredients in animals in the EU member states. Since 2013 there is even a ban on the import of cosmetic products and ingredients that were tested on animals elsewhere in the world. Since 2011 animal experiments for the development of tobacco products are forbidden as well.

Alternative methods are methods which do not require the use of animal experiments. If validated alternative methods are established that can answer the research question, it is prohibited to perform an animal experiment. Alternative methods can be divided into several categories:

• In silico: These are experiments performed by means of a computer model. This model can predict chemical reactions. The model is, of course, limited to the knowledge embedded within the model.
  
  
• In chemico: This is research done based on the chemical and physiological characteristics (e.g. toxicity) of products.
  
  
• In vitro: These are experiments in cell and tissue cultures, performed in test tubes. In this kind of research, the direct interactions on a cellular level can be investigated.

Also at UHasselt, alternative methods are always used as much as possible if they exist. At UHasselt, new alternative methods are also being actively developed.

More complex interactions (e.g. the effect of a product on the spatial memory in Alzheimer’s disease) can only be investigated in an in vivo model (living animal).

An experimental animal is an animal that is being used for scientific purposes. However, not all animal species are considered to be experimental animals. Only vertebrates and squids are considered to be experimental animals since it is known that these species can sense pain stimuli. Within the vertebrates, we have the following classes: fish, birds, amphibians, reptiles, and mammals. Humans are not considered to be experimental animals. Free living larval forms and mammalian embryos from the third trimester of the pregnancy are included. At Hasselt University, we mainly work with rodents.

An animal experiment is the use of an experimental animal for scientific purposes or for education and training, in which the animal experiences as much discomfort as or more discomfort than from the prick of a needle according to good veterinary practices. The creation of genetically modified organisms and the breeding of genetically modified animals that experience discomfort(s) due to their genetic modification(s), are both considered to be animal experiments and therefore need the approval of the Ethical Committee Animal Experiments. The solely killing of animals for in vitro use of tissues is not an animal experiment. Whenever an animal is euthanized in order to obtain tissues or organs for an experiment, the animal is an experimental animal but the experiment is not an animal experiment.

Replacement means that animal experiment free alternative must be used where possible. In case it is necessary to work with animals, one should use the lowest evolved species possible. This way, for example, pigs will never be used when it is possible to perform a study in mice.

Reduction will make sure the lowest number of animals is used while still sufficient in order to obtain statistically relevant results.

Refinement aims to improve the welfare of the animals. This can, for example, be done by using pain medication and anaesthesia for surgical procedures, enrichment of the cages (e.g. gnawing cubes, nesting material, adjusted bedding material) but also by housing conditions (e.g. group housing of social animals) and by means of humane endpoints.

At Hasselt university, we also value a 4th R, namely Responsibility. Each researcher and animal caretaker has to take his or her responsibility in order to guarantee animal welfare by means of good caretaking and treatment of the animals. In addition, we also have an animal welfare body that stands by to provide advice. In case (welfare) problems occur, the animal welfare body will investigate how the problems arose, how they can be solved and how they can be prevented in the future.

As instructed by legislation, rodents need to be purchased from recognized vendors. These vendors breed the animals with the aim to be used for scientific purposes.

In contrast to, for example, rodents that are available in general pet stores, lab mice and rats are being bred in a hygienic and very strictly controlled environment. The air in the housing rooms is filtered, the cages and bottles but also the drinking water and food are being sterilized and the personnel has to undergo a washing and changing procedure before entering the animal housing rooms. These hygienic measures are also applied in the research facilities to prevent the animals from picking up infections. When an animal is contaminated with an infection, this can have an impact on the research and of course, it can be detrimental to animal welfare. These are two important reasons to protect our animals against bacteria, viruses, and parasites.

Usually, the animals are euthanized at the end of the study so that the researchers can further investigate the brain, spinal cord, heart or other tissues.

Figure 2: Microscopic photograph of spinal cord tissue from a mouse used in research on multiple sclerosis. After staining, different cell structures and cell types become visible. In this photograph: immune cells in green or red and their nuclei in blue.

Not all experimental animals are necessarily in pain. However, in animal experiments, all animals suffer some sort of discomfort. Discomfort can include several types of suffering, not only pain but also stress, fear, loneliness, and lasting injuries.

The discomfort will be scaled into four categories: mild, moderate, severe and terminal. The determination of this classification is based on the severity, duration, and frequency of the discomfort.

• Mild: Short-lasting mild discomfort
  Example: A study in which the animals receive a dietary supplement via the food and a few blood samples are taken.

• Moderate: Short-lasting moderate discomfort or long-lasting/frequent mild discomfort
  Example: A surgical procedure under general anaesthesia with adequate pain medication or multiple blood samples.

• Severe: Short-lasting severe discomfort or long-lasting/frequent moderate discomfort
  Example: A tumour induction to test a new cancer treatment or multiple surgical procedures under general anaesthesia with adequate pain medication.

• Terminal: The experiment is performed under general anaesthesia, from which the animal will not recover.
  Example: The animal receives an anaesthetic overdose. Right before the animal dies, when it is sedated and does not feel any pain, tissue samples are taken. This technique is only performed in case it is not possible to use tissues from dead animals.

It is prohibited by law to perform animals experiments in which the animals will suffer a long-lasting severe discomfort. In order to prevent long-lasting discomfort, humane endpoints are being used.

We try to avoid that the animals suffer pain as much as possible. We do this, of course, because this is the right thing to do from an ethical point of view but also because pain can have an effect on test results. Therefore, the researchers are trained in pain recognition in order to detect signals of pain in time and treat it with painkillers.

All animals are monitored daily. These are the experimental animals as well as the animals used for breeding purposes. At weekdays, this daily monitoring is performed by animal caretakers. On bank holidays or during the weekend, we have a rotation schedule in which the animal caretakers, as well as the researchers, are responsible for the daily monitoring. During this monitoring, it is checked whether the animals have sufficient amount of food and water at their disposal. When necessary, the food and drinking water are refilled. In addition, it is checked whether there are any sick or wounded animals. When this occurs, the responsible researcher and animal welfare body are contacted. They will decide, in mutual conversation, how the animal needs to be taken care of and followed up and whether it needs to be euthanized. Also during the weekend and on bank holidays, this treatment and follow-up or the euthanasia needs to be conducted immediately.

Furthermore, the responsible researcher will daily (or multiple times per day) monitor the animals in his/her experiment in detail and document any possible clinical signs. For each individual animal, there is an online welfare diary in which aberrant behaviour and/or physical abnormalities are documented. Each note in the welfare diaries is automatically reported to the animal welfare body, which in turn will decide upon the treatment, follow-up and possible euthanasia of the animal. After each study, an evaluation is done, in which we investigate how the welfare of animals in future experiments can be improved.

In addition, the external veterinarian performs an inspection every three months. Finally, every five years, each lab is being inspected by the Animal welfare office of the Flemish department of Environment.

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Humane endpoints are predetermined indications on which animals are being removed from the animal experiment in order to prevent that the animal experiences more discomfort than strictly necessary for the experiment. For example, an animal can be euthanized when it loses too much body weight.

More information can be found on this website.

Personnel working with the animals needs to have followed an adjusted education and is obligated to follow permanent training routines at least on a yearly basis.

Animal caretakers who are responsible for the elementary caretaking of the animals (cleaning and disinfecting rooms, cages and containers; providing bedding material, water and food to the animals; transporting animals; handling animals) need to have followed a theoretical education of at least four hours.

Animal caretakers who are responsible for the special caretaking of the animals (post-operative care; monitoring the optimal environment of the animals; competent assistance in the euthanasia of animals) need to follow an education of 25 hours theory and practice with testing.

Persons who carry out the animal experiment need to have followed an education of at least 40 hours with testing of knowledge.

The experiment leader, which is the researcher responsible for the animal experiment, has to follow a basic course in laboratory animal science of 80 hours. This course is also provided by our university. The following topics are extensively discussed in the course:

• Introduction and history of animal experiments
• Ethics of animal experiments
• Alternatives in animal experiments (principle of the 3Rs)
• National and European legislation
• Biology of the different species used in animal experiments
• Reproduction (techniques) and genetics
• Pathology in experimental animals and health screening and management
• Procedures and good laboratory practices
• Management of health risks (hygiene, safety of personnel, waste  management,…)

Thereafter, all personnel is obligated to follow permanent training at least once a year.

Genetically modified organisms, called GMOs, are organisms whose genetic material (DNA) is altered.

For example, pieces of DNA can be cut out (knock-out), as in the “Apolipoprotein E receptor knock-out mouse”.
In this mouse, the Apolipoprotein E receptorgen is cut out. Due to the absence of this receptor, cholesterol transport is disturbed. Because of this, the animals develop high cholesterol levels in the blood.

Figure 3: Female Apolipoprotein E receptor knock-out mouse with her litter.

In addition, pieces of DNA can also be added (knock-in), as in the “CX3-CR1-eGFP mouse”.
In this mouse, a green fluorescent protein (eGFP) is added to the CX3-CR1 gene, which is present in microglia, a special cell type of the central nervous system. Therefore, these microglia have a green colour when looked at from under a microscope.

Figure 4: Male CX3-CR1-eGFP mouse.

Figure 5: Microscopic photograph of tissue from a CX3-CR1-eGFP mouse. By labeling the DNA of the mouse with a green fluorescent protein (eGFP), the microglia, a specific cell type of the nervous system, have a green color. Additional manual staining was performed to visualize the blood vessels in this photograph with a red fluorescent color.

Even human DNA can be added as in the “APP/PS1 mouse”.
In this case, two human mutations were applied to the DNA of the mouse. These mutations were found in patients diagnosed with Alzheimer’s disease. Therefore, the animals will develop a similar disease process. At a later age, they will encounter memory problems.

The majority (96%) of our research is performed in mice, 4% is performed in rats.

For at least 95% of the human genes, a similar variant is found in mice. Furthermore, there are a lot of genetically modified mouse lines available. It is even possible to introduce human genes in the DNA of mice in order to investigate specific diseases. In addition to this, mice are relatively cheap, they have a short reproduction cycle and they are rather small, which makes them easy to house.

In 2022, 2808 animals were used in animal experiments at Hasselt University. The majority of these animals were mice.

For each experiment, a statistical analysis is done a priori in order to determine the number of animals that is needed. We strive to obtain a statistically relevant result while using the lowest possible number of animals.

Figure: Number of experimental animals at Hasselt university in 2022.

According to law, the safety of new products first needs to be investigated in animals before the products can be tested in humans. Besides this legal framework, there is of course also an ethical aspect which plays a big role in this debate.

For the development of new products, medicines or therapies, it is important to first investigate the underlying mechanisms of the disease. This is so called fundamental research. This research is mainly conducted in vitro but sometimes it is also done by means of an animal experiment because complex processes cannot always be simulated in a petri dish. At the end of the study, the experimental animal is usually euthanized in order to further investigate the tissues and organs. This last step is ethically impossible in humans.

The European Union has written its first Directive in 1986. The last revision dates from 2010 (2010/63/EU). Based on this Directive, the Belgian government has written law texts. These were implemented and translated to practices by means of Royal Decrees. The most recent Royal Decree (KB 2013-05-29) was revised in 2017 in the Decree of the Flemish government (BVR 2017-02-17).

Mice and rats are social animals, which means that in nature, they live in groups. Therefore, the animals are also group housed in our university, unless this is not possible within the experiment. The bottom of the cage is covered with bedding material and cage enrichment (nesting material, play tunnels, gnawing cubes) is present. In addition, the animals have free access to food and water.

Figure 9: Group housing mice with cage enrichment at Hasselt University (side view).

Figure 10: Group housing rats with cage enrichment at Hasselt University (top view).

Mice and rats are also nocturnal animals, which means they are mainly active at night. When researchers perform a behavioural test, this is done during the active phase (at night). To prevent that researchers would have to test their animals during the night, the day/night cycle of these animals is reversed. In these housing rooms, the lights are on during the night, while in the daytime it is dark.

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