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Abstract

Laboratory animal science is a multidisciplinary branch of science contributing to the humane use of animals in biomedical research and the collection of informative, unbiased and reproducible data from animal experiments (1). Since the 1970s, the concept of the 3Rs (Replacement, Reduction and Refinement) has had a major influence on the field of laboratory animal science. Refinement refers to methods which alleviate or minimise potential pain, suffering or distress, and which enhance animal welfare. The European organisation FELASA (Federation of European Laboratory Animal Science Associations) plays an active role in issuing guidelines in the field of Refinement, and many of its working group reports are published in the journal Laboratory Animals. Refinement is often specifically focussed on certain scientific procedures but it can also be extended to the living conditions of the animals throughout their entire lives (holistic Refinement). In this article I present several examples of how Refinement of procedures and holistic Refinement can improve the welfare of laboratory animals and, at the same time, provide better-quality experimental results.

The 3Rs and FELASA

The concept of the 3Rs (Replacement, Reduction and Refinement) was first described by Russell and Burch in 1959 (2). However, it was not until the 1970s that interest in the 3Rs started to develop. Since then, the 3Rs concept has become a very important one in scientific research and testing involving the use of animals.

FELASA was established in 1978 by twelve national and regional laboratory animal science associations within Europe, with the aim of furthering the implementation of the 3Rs in scientific research and testing. FELASA has a current membership of about 3,000 laboratory animal scientists and has become well-known for providing guidelines on topics such as staff education and health monitoring, which have made a major contribution to the implementation of the 3Rs. FELASAs guidelines are published in the high quality journal Laboratory Animals and are therefore accessible to the scientific community. The guidelines are formulated by working groups of experts with the aim of safeguarding and improving animal welfare while at the same time obtaining valid scientific results.

In the field of education, FELASA guidelines describe the minimum qualifications and competencies that are required of all staff working with laboratory animals in order to guarantee high quality animal care. FELASA has recently begun to accredit teaching and training courses, in order to standardise the quality of education throughout Europe. Similarly, in the field of health monitoring, FELASA has issued guidelines in order to improve the health and quality of laboratory animals, and accreditation of health monitoring programmes by FELASA is currently under development. Various stages of infections by pathogens can differentially influence experimental results (3,4), thereby increasing data variability and hence the number of animals required for obtaining statistically significant results. It is therefore important that high-quality animals, in which pathogens are absent, are used in experimental studies. Accreditation is helping to improve standards in laboratory animal research worldwide and the use of higher quality animals in research may account for the apparent reduction in the number of animals used per scientific paper (5).

The importance of the work of the European laboratory animal science network has also been highlighted by the fact that the European Science Foundation has recently provided additional funding to strengthen this field (COST action B24 Laboratory Animal Science and Welfare). The launch of the UK's National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) in 2004 also represents an important opportunity to advance and promote the concept of the 3Rs, and it is hoped that other countries in the European Union will soon follow this good example.

Refinement of procedures

Animals are used for a variety of scientific purposes. In many cases, Replacement of the use of animals is not yet possible. However, Reduction of animal numbers can be achieved by improved experimental design and statistical analysis, and thorough literature reviews so that unnecessary duplication of animal studies is avoided. Good literature reviews will also contribute to the understanding of how procedures can be optimised and refined.

Refinement techniques aim to minimise any pain, suffering or distress in animals, for example, use of anaesthesia and post-operative analgesia in order to minimise any pain and distress caused by surgical procedures. A recent analysis of biomedical research papers showed that whilst use of analgesics increased from 1992 to 2002, post-operative pain relief for laboratory rodents was provided in only 20% of the 2002 papers surveyed (6). This finding indicates that there needs to be a greater awareness of the importance of Refinement techniques, not only from an animal welfare point of view but also from a scientific standpoint, as Refinement can increase the reliability and validity of experimental results.

Sterile versus non-sterile catheter insertion in rats

It is widely believed that non-sterile surgery in rodents is acceptable, as these animals are able to cope with the introduction of bacteria into the body. However, Popp and Brennan (7) have demonstrated that the use of sterile procedures when implanting catheters in the blood vessels of rats not only improves animal health and welfare but is also advantageous for obtaining reliable experimental outcomes (Table 1).

Of a group of rats that underwent non-sterile surgery, only 50% survived for a period of 25 days after the insertion of the catheter. In contrast, all rats that underwent sterile surgery survived the 25-day post-operative period without complications. After non-sterile surgery, catheters became infected and blocked in 83% of the animals. Bacterial growth can have local effects by inducing a blockage of the catheter, but also systemic effects by causing bacterial infections in other parts of the body, which will influence the immune system. It is essential, therefore, to execute sterile surgical procedures in rodents in order to avoid local and systemic bacterial infections that may interfere with experimental results.

Table 1. Survival and infection rates 25 days after catheter insertion.

Sterile versus non-sterile procedures for the implantation of GM mouse embryos

Genetically modified (GM) mice are produced in order to understand gene function and how genetic diseases arise and can be treated. The process involves the introduction, or deletion, of a gene in the mouse genome. One mechanism by which GM mice are generated is by gene targeting in embryonic stem cells (multi-potent cells which are able to develop into a mouse embryo). The modified stem cells are injected into an early stage "host" embryo and then implanted into the uterus of a female mouse. The modified cells will contribute to the host embryo, which will grow and develop. The resulting mouse will be born with the desired changes to its genome.

Experimental observations have indicated that when implantation of host embryos (two-cell stage embryos into the oviducts) was performed with "clean" instruments, no successful pregnancies resulted. After changing to using sterile procedures (i.e. newly autoclaved surgical instruments for each procedure) and using iodine for disinfecting the surgical wound, many more successful pregnancies resulted (pers. comm., Frederik Dagnaes-Hansen, Associate Professor, Aarhus University, Denmark).

Holistic Refinement

The above examples illustrate how Refinement of surgical procedures can contribute to improved animal health and welfare and to experimental outcomes. However, it is not only the Refinement of a single procedure that is of importance but also the application of Refinement in the context of the whole experiment. Minimising suffering arising from, for example, housing and husbandry can lead to better welfare for the animals and increase the quality of scientific data.

It is expected that the revision of EC Directive 86/609 will include the obligation to enrich the home-cage environment of laboratory animals. In order to successfully achieve enrichment, it is important to understand the characteristics of the animal species one is dealing with, what their essential needs are and how enrichment strategies will interact with the animal and experimental results. Holistic Refinement aims to address and integrate all these aspects in an optimal way in order to achieve successful enrichment. One can consider that successful enrichment has been achieved when the welfare of the animals has been improved and the reliability of the experimental results is maintained or improved.

Adapting housing conditions to species-specific needs reduces aggression and improves breeding success in rats

Several types of rat "houses" are commercially available as enrichment devices. The houses are introduced into the animals cages in order to provide them with shelter and a place to nest. However, many of the commercially available houses are only just wide enough for two rats. In order for group-housed rats to lie together in the shelter, or to build nests in which the young can be together with the mother, a much larger nesting place is needed. Jegstrup et al. (8,9) have, therefore, designed a rat house with dimensions that are based on the average size of nests made by wild rats (Fig 1).

Figure 1. Rat house with dimensions based on the size of wild rats nests.

In a study examining the possible negative influence of dietary pesticides on fertility, two strains of rats (Goto-Kakizaki (GK/Mol) and Brown Norway (BN/HsdCpb)) were provided with the large-sized houses and wood wool as nesting materials (10). GK/Mol was bred for two generations, and BN/HsdCpb was bred once. Access to the larger shelters appeared to give a greatly improved pregnancy rate as compared with reference data obtained from breeding companies (Table 2). This was particularly evident in the BN/HsdCpb strain.

Table 2. A large shelter is associated with improved breeding success in rats.

The rats also exhibited reduced aggression levels (8) and were active in nest building inside the houses throughout the study. Nest building, which is considered an important part of the daily activity pattern of wild rats, was even observed in male rats of three different inbred strains (9). This clearly illustrates that understanding the biology of the species being studied, including their behaviour in nature, will help to form a sound basis for improving the way we keep and use animals in the laboratory.

Species-specific behaviour and restricted feeding

Unrestricted (ad libitum) access to food is considered to have a negative influence on health and welfare, in humans and other animals. Ad libitum feeding leads to obesity, a shorter survival time, kidney and heart diseases, and cancer at an earlier age (11). Restricting food intake (e.g. to 80% of the ad libitum intake) can dramatically improve animal health, welfare and survival, and is considered good veterinary practice (12).

There are other advantages to controlling food intake in animals (13,14). Firstly, controlling the amount of food that each animal receives during an experiment means that less individual variation between animals will occur as compared with ad libitum feeding. Reduced variation in food intake will lead to the use of fewer animals in order to prove the statistical significance of experimental results. Furthermore, it has been shown that when food intake is restricted, animals are more robust and are better able to cope with experimental stressors (12). Therefore, food restriction should be recommended as a standard method in laboratories. Despite this, on the basis of practical and economical reasons, most laboratory rodents are still fed ad libitum .

In long-term toxicological studies (bioassays) in the USA it is standard practice to feed rodents restrictedly, otherwise insufficient animals will survive the necessary 2 year period. For this purpose, animals are housed individually, in order to ensure the same food intake in all animals, yet individual housing conflicts with the desire to house these highly social animals in groups for improved animal welfare. There is, then, a need to develop feeding systems that will guarantee a standardised restricted food intake per individual whilst allowing group-housing.

When using restricted feeding schedules, it is important to do this in accordance with species-specific needs (14). For example, rabbits in the wild mainly forage late in the afternoon and during the night. When laboratory rabbits were fed restricted amounts of food in the morning, they started to behave abnormally by drinking too much water and they showed increased food conversion (15). They also showed more stereotyped behaviour (i.e. biting the bars, and scratching the floor, of their cage) as compared with ad libitum feeding (16). However, when the rabbits were fed a restricted amount of food in the late afternoon, a normal water intake and a significantly reduced frequency of stereotyped behaviour were observed as compared with ad libitum feeding or feeding a restricted amount of food in the morning (16). These data indicate that feeding restrictedly at an appropriate time point improved the welfare of the rabbit and, potentially, reduced data variability and improved the reliability of the experimental results.

If certain animals start to drink more water and/or exert stereotypic behaviour, this will result in increased data variability, which will in turn lead to a need to use more animals in order to obtain statistically significant results. With regard to the reliability of the results, increased water intake will, for example, interfere with kidney function measurements. Stereotyped behaviour will automatically influence behavioural observations, and if an animal exerts a high level of stereotypic physical activity this will at least interfere with energy and muscle metabolism.

Holistic Refinement during kidney physiology studies in dogs

Beagle dogs have been used to study the influence of the level of dietary sodium intake on physiological blood pressure regulation in the conscious, awake state. During the experiments, the dogs were individually fed a standardised amount of low-sodium food at 2 pm each day. The dogs were housed in groups except for the days when experiments were performed (once every 14 days).

Before being able to take reliable blood pressure measurements the animals must undergo an extensive training program lasting several months (Fig 2). Effective training results in a low resting blood pressure and heart rate during experimentation, indicating that the animals are not experiencing stress during the procedure. Blood pressure and heart rate immediately rise to higher levels, when the animals experience stressful situations.

Figure 2. A dog that has been trained to stand in a sling during blood pressure measurements.

Large variations in the levels of urine sodium excretion were observed for the experimental group. In order to explain this finding, the behaviour of the dogs was observed remotely. Video recordings of the dogs during the night revealed that they could throw up food as long as 8 hours after eating a meal, and that other dogs would eat the vomited food, resulting in the large variations in urine sodium excretion. Vomiting food is a natural behaviour for dogs in the wild, as this is how pups obtain their food from the parents. Pups will "ask" for food from a parent by gently pushing their nose in the mouth of their parent. The parent then vomits and the pup will eat. However, this behaviour is not usual for adult dogs.
 
A dog behaviour specialist examined all the laboratory procedures with the research staff and advised on a general training and enrichment programme, in addition to the training needed for the experimental purposes (Table 3). The behaviour specialist concluded that, as there was no real harmony between the individual dogs in the group, the animals overtly caused stress to one another. Stress reduces gastrointestinal activity, which may explain how the dogs were able to throw up as long as 8 hours after eating a meal. The new training and enrichment programme included elements specifically focussed on encouraging the dogs to function harmoniously as a group. In addition, interactions with all laboratory personnel were increased, including outside of the experimental setting (Figs 3-11).

Table 3. Details of old and new dog training programmes.

Figure 3. The dogs were socialised with other dogs and staff each morning, when the animals were moved from their night quarters to the running area.

Figure 4. The dogs were taken for walks outdoors.

Figure 5. Staff and dogs went on training courses.

Figure 6. Once in a while, food was provided in dog toys to encourage the animals to work for their food.

Figure 7. Smelling boxes were used to provide olfactory enrichment for the dogs and consisted of a metal box with holes in the sides.

Figure 8. Regularly providing different items inside the boxes (e.g. meat) stimulated the dogs with new smells. The boxes cannot be opened by the dogs, thereby preventing ingestion of the contents.

Figure 9. Lots of toys were provided to stimulate the dogs to play.

Figure 10. The dogs were given massages, once in a while, which increases the production of happiness hormones in both dogs and their handlers and improves confidence on both sides.

Figure 11. In between experiments, dogs were allowed to holiday on a farm, where they have access to an outdoor running area.

Experiments that were performed after the new training and enrichment programme was implemented showed that the individual urine sodium excretion levels measured in the morning were as expected based on the individual food intake levels at 2 pm the previous day. In addition, the variation in urine sodium excretion levels was significantly reduced. Urine sodium excretions, measured every 30 minutes over a period of about 3 hours, showed that individual levels in the unstable dog group ranged from 2-27 µmol/min (Fig 12), whereas they ranged from 1-3 µmol/min in the socially stable group (Fig 13). The new training and enrichment programme therefore dramatically reduced the variation in the experimental results, increased the validity of the experimental results and improved the welfare of all the dogs in the colony. Moreover, it increased the job satisfaction of the personnel training the dogs and satisfied the scientific needs of the researchers.

Figure 12.Urine sodium excretion for the unstable dog group.

Figure 13. Urine sodium excretion for the stable dog group.

Conclusion

In this article I have presented several examples of how Refinement can improve the welfare of laboratory animals and, at the same time, provide better-quality experimental results. More scientific research is needed in order to define how Refinement should be executed to ensure genuine improvements in animal welfare while simultaneously maintaining or improving the quality of science.

References

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All views and opinions expressed in this article are those of the author and do not necessarily reflect the views and opinions of the NC3Rs.