Animals in research

By: Adrian Smith, professor in veterinary medicine, secretary of Norecopa.

Introduction 

Few areas in which animals are used create more debate than when they are used as laboratory animals. Animal experiments are often the reverse of good veterinary medicine: animals in the best possible health are treated in a way that makes them sick. Also, the individuals in an animal experiment are relatively uninteresting: their value is in the light that they shed on another target group: other members of their species (e.g. infection experiments in connection with an animal disease outbreak), humans (e.g. animal models of disease) or the environment (e.g. the effects of pollution). However, laboratory animals are living beings with the capacity to feel fear and pain. It is in everyone's best interests to reduce their suffering to an absolute minimum, also because animals that are free from pain and stress will give the most reliable results in an experiment. There are therefore scientific, legal and ethical reasons for treating animals as humanely as possible.  

This article is an attempt to illustrate some of the challenges that users of laboratory animals face. 

Scientific, legal and ethical requirements  

The organisation Scand-LAS has defined laboratory animal science as: The scientific, legally approved and ethically acceptable study of animals for biomedical purposes.

This definition illustrates that plans for animal experiments must be quality assured on at least three different levels: they must be legal; they must be of high scientific quality and they must be ethical. The last of these is, naturally, the most difficult, but scientific standards are not always easy to agree on. Some focus more on "engineering standards" (e.g. requirements for cage size) while others lean towards "functional requirements” (e.g. sufficient space for the animals to thrive).

To weigh the value of an experiment against the cost to the animal, a harm-benefit analysis must be performed[1]. Such analyses are an integral part of the EU Directive on the protection of animals used for scientific purposes (European Commission, 2010). The Directive came into force in the EU on 1 January 2013, and in Norway the requirements have been implemented by a regulation which entered into force on 1 July 2015[2] and an instruction to the Norwegian Food Safety Authority[3]. 

A harm-benefit analysis assumes extensive knowledge of the animals' ability to experience discomfort and pain. The development of welfare indicators to measure this scientifically is a rapidly growing field. It is unfortunately easier to identify negative rather than positive indicators. The principle that animals should be given the benefit of the doubt can usefully be applied here. Various guidelines for the classification of welfare burden on research animals have been developed[4]. Furthermore, the cost in an animal experience (pain and suffering) poses an ethical problem in that it is known that it will occur to certain individuals in the near future, while the benefit of animal research is often more abstract and in the form of increased knowledge and the hope of increased welfare for other animals or humans at some point in the future.  

The launch of the concept of "the 3 R's" (Replacement, Reduction, Refinement) by Russell & Burch (1959) did much to focus attention on the fact that humane research is also good scientific research. The 3R principle has been incorporated in the legislation on animal testing in many countries: 

• Replacement: animal experiments should be replaced with alternatives whenever possible  
• Reduction: the number of animals should be reduced to an absolute minimum consistent with scientific aims, but with a valid experimental design - and preferably with greater scientific yield per animals, if this does not compromise the welfare of the animals. 
• Refinement: experiments that must be performed should be refined to cause the least possible suffering to the animals, and to maximize animal welfare. 

The halving of the number of mammals used in experiments in Norway in the 1980s was probably due to increased attention to the principle of Reduction (Annual reports from the Norwegian Animal Research Authority, in Norwegian only[5]). 

A lesser known but very useful set of guidelines when planning animal experiments is the "3 S's" of Carol Newton (1977): Good Science, Good Sense, Good Sensibilities. It must be permissible to use common sense and to follow one's heart in the absence of scientific data about the harms associated with a procedure. 

Striving for reduction can be a double-edged sword. The sum total of suffering is reduced, of course, but there are two potential risks:  

1. It can be tempting to perform too many interventions per animal.  
2. The number of animals must not become so low that the experiment loses its ability to deliver statistically significant results (De Boo & Hendriksen, 2005). A statistician should therefore undoubtedly be involved very early in the planning of animal experiments in order to help determine the number of animals to be used and how the experiment should be conducted. The development of new statistical methods shows the potential to more than halve the number of animals needed for certain types of research without compromising the quality of the results (for example, Dewi et al., 2014).  

Another tool that is used increasingly is the determination of humane endpoints. Prior to an animal experiment, a discussion takes place between the researchers, animal technicians and managers of the animal facility to identify criteria for terminating the experiment humanely without requiring the animals to continue until death. There should be an end to "counting bodies on Monday morning". A culture of care in the research institution promotes such discussions. 

In many cases, the objective of an animal experiment will be a crucial factor in deciding whether it is appropriate to use animals. In few areas is this more evident than when animals are considered for use in teaching. Products such as audiovisual aids, 3D models and simulators can be fully valid alternatives to animal experiments for some groups of students. They can also be useful in training persons who will go on to perform animal experiments, by providing a dry-run opportunity. Procedures such as blood sampling, injections and some surgical procedures can be carried out without subjecting living animals to poor treatment. Databases are available with information about thousands of such products (for example, the NORINA database). 

There is considerable political and societal interest in statistics on the number of laboratory animals used in individual countries. Norway uses quite a large number of animals per inhabitant. Around two million animals are currently used for research purposes (See: https://norecopa.no/legislation/statistics), which corresponds to a fifth of the total quantity used in the entire EU. Over 95% of these are fish, due primarily to research and development work in the aquaculture industry. Is a high number of research animals problematic? Is it perhaps merely indicative of a high level of research activity in the country or that it is necessary to conduct research on large numbers of animals at a time? What is most important, of course, is to avoid the use of animals unnecessarily and to subject them to as little welfare burden as possible.

Test articles, test systems and reporting of animal experiments  

Animal experiments must be quality assured to ensure that the results are reproducible and can be transferred to the target group. There are currently three forms of validity used in animal research: the ‘three Vs’ (construct validity, internal validity and external validity).  

Quality assurance includes an analysis of the most critical points in an experiment, where any deviations from the protocol could cause major problems.  

Quality assurance also entails characterisation of the "test system" (the animals) and the "test article" (the substance or procedures to which the animals will be exposed). Such characterisation makes it easier to replicate an experiment and obtain the same results in other laboratories. This may be necessary to verify the results or to establish an animal model in another facility. Studies have shown that researchers are generally much better at characterising (and thus standardising) test articles than test systems (Smith et al., 1997; Kilkenny et al., 2009). This is not surprising: It is easier to describe the chemicals used in an experiment than the animals. The problem is that by far the greatest potential for variation in an experiment lies within the animals, given their complex biological systems. This variation arises partly due to the animals' intrinsic characteristics (e.g. genotype, health status) and partly due to environmental influences (e.g. temperature, water quality, feed, housing conditions, effects of other animals and humans around them). A survey conducted by the science journal Nature among 1,500 researchers showed that over half of them were not able to reproduce their own research results and over 70% were unable to reproduce the results of other researchers in their laboratories (Baker, 2016). Much attention has been devoted to this ‘reproducibility crisis’, as it is called, and much written about its causes (see https://norecopa.no/concerns).  

To try to rectify this, guidelines have been developed for both the planning of animal experiments (for example, Smith et al., 2017; 2018a) and reporting on them in the literature (for example, Hooijmans et al., 2010; Percie du Sert et al., 2020). Many journals have adopted these guidelines, but there are signs that they are not always followed diligently (Baker et al., 2014; Leung et al., 2018; Hair et al., 2019).  

Laboratory animal science is in its youth. The learning curve is still steep, especially regarding work with those animal species with which we have the least experience. This is particularly true of fish, which make up over 95% of all laboratory animals in Norway. The varying degrees of "bambi factor" among different species have also influenced the pace of research efforts aimed at improving animal experiments. Recent research showing that fish too have the capacity to feel pain (Sneddon, 2019) has, however, contributed to an increased focus on the welfare of fish species.

Challenges in housing laboratory animals

The optimal housing of laboratory animals should receive careful attention early on in the planning of an experiment. Both the number of animals per group, and the interactions between the animals and their environment, will have a major impact on factors such as the correct statistical analysis of data from the experiment. For the animals themselves, housing conditions can also be critical. Housing social animals (e.g. many rodent species and schooling fish) in abnormally small groups will be stressful for them. Most animal species will not be happy in a "single room". Research suggests that mice housed alone can develop symptoms that in humans would be interpreted as signs of depression (Kalliokoski et al., 2014). However, housing animals in groups is not always positive for everyone: some may be bullied, and they can seem relieved to be removed from the group. It is therefore important to keep an eye on the establishment of the hierarchy. 

An increase in the number of laboratory animals in a facility must be accompanied by a proportional increase in human resources to take care of them. The question "How many animals can a technician look after in the course of a week?" should ring alarm bells. Animals must be inspected daily for signs of illness, pain and distress. A professional dilemma can arise when we try to enrich the animals’ environment: they can then hide behind the objects placed in the cage or tank. Or do the animals have a need for privacy? This is particularly important for breeding colonies. This requires a good knowledge of the species and regular observation of the individuals.  

Other challenges with animal experiments

Are the animals suffering?

EU Directive 2010/63 (European Commission, 2010) sets an absolute limit in cases where animals are subjected to severe pain over time. How can this be measured? Qualitative scoring systems that provide so-called ordinal data (e.g. on a scale from 1–10) can be devised, but these leave much room for individual interpretation unless every step can be precisely defined. The best option is systems that use continuous data (numbers such as weight and length). Score sheets are also useful aids (Smith et al., 2018b). Recently, several research groups have developed systems for measuring animals' suffering based on their facial expressions: so-called "Grimace Scales" (Mogil et al., 2020). 

How specific must researchers be?

Another dilemma occurs when scientists are very specific in their ordering of laboratory animals. This can lead to suppliers increasing their animal breeding in order to fulfil the requests. The number of surplus animals that must be killed will therefore increase and, even though these animals do not show up in a country's statistics, the numbers can be large. These animals will have lived under conditions that are at least as unnatural as those in an animal facility.  

What is to be done with the animals?  

Norwegian legislation does not specify maximum housing durations for different animal species. Should animals with long biological lifespans be used for as long as possible? One example is the ‘fistulation’ of larger domesticated animals, such as cows, by which a permanent opening is made through the skin to the stomach or intestines, similar to the operation used to attach a colostomy bag in humans. The opening makes it possible to extract degradation products from various areas of the digestive system to gain a better understanding of where and how nutrients are broken down. Direct pain is mostly limited to a short post-operative phase. Can these animals then be used for years, or should there be a limit to how long they can be used for experiments? Replacing them means performing surgery on new animals, with a new potential for pain. The ‘cost’ to the animal can be reduced to some extent through such measures as allowing the animal access to pasture. 

Almost all animal experiments end with the animal being killed, whether this is necessary for the experiment or not. Animals in research facilities can end up in lifelong quarantine because of import regulations. In some cases, it is possible to use the animals in new experiments or to transfer them to adoptive homes. These alternatives must be considered carefully well ahead of the experiment.  

Reuse of laboratory animals also creates ethical dilemmas. From what we know today about the memory capacities of animals (including fish), this is potentially a very stressful practice. It can be tempting for several reasons to use animals again, especially in a country like Norway where most laboratory animals are imported. Attacks on airlines have in some cases led to imported animals being subjected to longer transport times through transit countries, because the airlines that fly direct will not carry them. Which is worse: the stress associated with transporting and acclimatising new laboratory animals, or keeping animals longer that are already accustomed to the animal facility? Is being a laboratory animal so far removed from the animal's natural life that an individual's stay in an animal facility should be kept to an absolute minimum, or can we create liveable conditions in which the animals may even thrive? 

Some might say that we do not perform experiments on humans, even if it would benefit society, and should therefore not do so on animals. They would also point out the paradox that we justify animal experiments because the animals show roughly the same responses as humans, while at the same time subjecting these animals to treatment that would be completely unacceptable for our own species. Regardless of personal opinions, the fate of the animals must be discussed thoroughly when planning animal experiments. There may be many different considerations.  

Who will decide?  

Animal testing requires a dialogue among all stakeholders in animal research: government bodies, industry, the academic community, and animal welfare organisations. In this way, differences of opinion can be discussed in a civilised manner and consensus sought. This will also promote the application of the 3 R's. National consensus platforms that work according to this principle have been established in Europe, as well as 3R centres that work to promote replacement, reduction and refinement of animal experiments. Norway's national platform is called Norecopa.

This article has been translated from Norwegian by Louisa Lyon, Akasie språktjenester AS (2015). Updates in 2022 were translated by Totaltekst.

References

[1] https://norecopa.no/more-resources/harm-benefit-assessment

[2] https://lovdata.no/dokument/SF/forskrift/2015-06-18-761

[3] https://norecopa.no/media/6332/endelig-instruks-for-forsoksdyrforvaltningen.pdf

[4] https://norecopa.no/more-resources/severity-classification

[5] https://www.mattilsynet.no/dyr_og_dyrehold/dyrevelferd/forsoksdyr

Further reading: 

National Committee for Research Ethics in Science and Technology, Ethical Guidelines for the Use of Animals in Research (2018)

Smith, A.J. Guidelines for planning and conducting high-quality research and testing on animals. Lab Anim Res 36, 21 (2020).