Category Archives: DISCUSSIONS

FRAME’s Online Alternatives Timeline Goes Live

A new online resource aims to collect a range of useful
Three Rs-relevant information in one place
and put it in its true historical context.

FRAME is very excited about the launch of our new website.1 One of its many new features is the FRAME Alternatives Timeline,2 first proposed in the PiLAS supplement of ATLA issue 41(6). The Timeline is to be launched at the World Congress on Alternatives and Animal Use in the Life Sciences, in Prague, Czech Republic (24–28 August 2014). It summarises major advancements made in all fields of alternatives research (see illustration below), including how they relate to the principles of the Three Rs, as outlined originally by Russell and Burch3 and recently reinforced by the European Union.4 It also includes significant moments in both legislative reform and changes in social perceptions, as they relate to alternatives. At its core, we aim to provide a resource for researchers, students, and indeed any other interested individuals, that is content-rich, has ease of access, and is visually appealing.

Timeline screenshot

The current version will be updated dynamically, in order to develop a system that collects, organises and summarises a wide range of previous noteworthy work in replacement, refinement, and reduction, while actively adding new material as it arises. It is integral that the accomplishments of the past are recognised and understood, such that they can benefit those that require them, and furthermore to push progress forward and ensure that vital information is not misplaced. This can be for scientific purposes to advance research, or purely for someone’s own interest to help form their opinion on the alternatives discussion. The Timeline allows the reader to follow the progression of the Three Rs concept through history, from the simplest conceptualisation to the most complex technological innovation.

The ability to make the Timeline available online, with a global reach, is imperative to its functionality. We hope that a global audience — whether from developed or developing nations, or from countries with strict laws or relaxed laws on animal use — can benefit from this resource. As a tool for finding initial and introductory-level information on the beginnings of the shift toward alternatives to animal use in research, Three Rs-based legislation, and public opinion up to contemporary developments, the Timeline sets the stage for further detailed investigation. Links that directly connect the reader with primary source material are provided wherever possible.

The Timeline is one component of a larger picture that is the newly designed FRAME website. FRAME understands the relative importance and value of education, and FRAME is working to deliver accessible tools in one location. Thus, the Timeline will fulfil a number of FRAME’s goals by:

— acting as a review of effective alternatives;
— providing a primary resource for researchers;
— raising the profile of FRAME and the pursuit of the implementation of alternatives;
— providing a database of definitions and ongoing research;
— cross-referencing ineffective animal models and their alternatives;
— identifying when, where and who were involved in formulating the alternative; and
— clearly defining which of the Three Rs is in practice.

In the pursuit of improved science and animal welfare, and in the transition away from animal-based models toward non-animal ones, it is important to appreciate how technology and attitudes have changed over time. Additionally, actively keeping track of new developments and recording them in the Timeline will help many searching for the current trends in alternatives. We hope that you take the time to check out and enjoy the FRAME Alternatives Timeline, as well as the FRAME website as a whole.

If you have any comments or suggestions related to the Timeline or the new website, then we would
really like to hear from you. Email us on
frame@frame.org.uk

Assessing the Effects of Environmental Enrichment on Behavioural Deficits in C57BL Mice

A study on the effects of environmental enrichment on behaviour and cognitive and motor functioning in a standard mouse model and a strain known to have behavioural deficits, suggests that environmental enrichment can positively influence natural functioning and natural behaviour.

 

For many years, rodents have been used as animal models for investigating human diseases such as Alzheimer’s disease (AD) or Huntington’s disease (HD). 1 The mouse genome (specifically that of the C57BL/6 strain) has about 30,000 genes, 99% of which have direct counterparts in humans.2 This makes the mouse a good model for studying the function of human genes, with particular emphasis on disease. Furthermore, mice — with their small size and short generation time — make a very suitable test species.3 Despite the obvious welfare concerns that arise when testing on animals, this practice is likely to continue until reliable and effective alternatives can be employed. In the meantime, it is believed that by enriching the environment  in which a test species is housed, we can take steps toward improving their welfare. Thus, the aim of the present study was to firstly assess whether environmental enrichment (EE) can influence the behaviour of the standard laboratory mouse (here, the C57BL/6 strain) and, if so, whether the effects are strain dependent. Secondly, the goal was to determine whether its close cousin, the C57BL/10 mouse, when housed in a standard environment, would show the motor and cognitive deficits that would actually make it a suitable AD model.

The mouse model

The most widely used mouse strain in behavioural studies is the C57BL/6 (or B6). Alternatively, the C57BL/10 (or B10), which shares a common ancestry with the B6, is used in non-behavioural studies of immunology and inflammation (as a result of stimuli such as prion diseases).4 Thus, B6s and B10s are seemingly identical in appearance and general behaviour, but are used in different studies.4 Observations on the differences of cognition, species-typical behaviour and motor-coordination have shown deficits in B10s.4 In the majority of these tests, B6 mice consistently functioned better. Limited work on B10 mice has suggested deficits in hippocampal functioning and anatomy.5 There is a paucity of literature available on behavioural experiments documenting the B10 behavioural phenotype, and only one paper directly on a comparison of B10 and B6 strains, so most comparisons between the strains are based upon the
research of Deacon et al.4

Abnormalities shown by B10s are possibly representative of some neurodevelopmental and neurodegenerative disorders, such as dyspraxia and AD. Mice expressing different mutant forms of amyloid precursor proteins and/or presenilin-1 may develop functional or cognitive defects resembling the symptoms observed in human AD patients.6 It has been suggested that B10s might be a useful model for aspects of certain neurological disorders, such as autism4 and potentially AD. The latter proposal was tested in the study described in this paper.

Determining the effects of environmental enrichment.

An environment is considered to be ‘enriched’ or not, when it is compared with standard laboratory conditions.7 Standard (S) cages (with bedding and ad libitum access to food and water) are commonly used when housing mice, but it has been shown that enrichment increases brain weight8 and reduces anxiety. Enrichment can be supplied in the form of extra nesting material, plastic tubing, etc. Furthermore, it has been documented that mice (often of the B6 strain), housed in standard cages show impaired and abnormal brain development, repetitive behaviour and an anxious behavioural profile.9

Mice housed in standard cages were hyperactive, with reduced dendrite branching and length. These behavioural and neuronal abnormalities were greatly reduced in environmentally-enriched mice.10 It was found that mice from enriched housing have significantly more neurons (~15%) in the dentate gyrus (part  of the hippocampus) than those of S littermates. Enriched mice were also found to have a larger hippocampal granule cell layer.11

Environmentally-enriched mice also have reduced levels of corticosterone (a hormone associated with anxiogenic situations) following exposure to predator cues such as scent or visual presence.12 Hutchinson et al.13 hypothesised that enriched mice produce less endogenous cortisol and exhibit higher levels of proinflammatory cytokines in response to antigenic immune attacks, suggesting that environmentally enriched mice have a greater anti-inflammatory cytokine profile than their S cohorts.

The study

Housing and test models
We wanted to assess whether the provision of EE could enhance the functioning of B10s to a level similar to that of the more-widely studied B6s. The subjects were 20 female C57BL/6 and 20 female C57BL/ 10 mice (Harlan, Shardlow, Derbyshire, UK). Half of the mice were housed in enriched cages and half in standard cages containing aspen shavings and sizzle-nest bedding (Figure 1c and 1d), for approximately six weeks before the experimentation commenced, and throughout the experimentation. (Figure 1a and 1b),

Figure 1: Environmentally-enriched and standard cages a) Aerial view of environmentally-enriched cage; b) side view of environmentally-enriched cage (show here without bedding); c) aerial  view of standard cage; d) front view of standard cage.
a)

mouse a

 

b)

mouse b

 

c)

mouse c

 

d)

mouse d

 

Food and water were available ad libitum, temperatures were controlled at ~21.1 ±1°C, and a dark cycle commenced at 7pm for 12 hours.
In order to gain a sound idea of whether EE had any effects on mouse behaviour and functioning, a variety of experiments were used. The tester remained ‘blind’ as to which mice were which.

Test methods

The effects of EE include increased socio-positive behaviour (e.g. grooming) and ‘species-typical’ behaviour, and a reduction in agonistic behaviour.14 Therefore we assessed spontaneous semi-naturalistic behaviour, such as digging, grooming, burying objects, climbing, and nest construction. EE has been  attributed to an increase in locomotor and exploratory activity, object exploration and learning ability.14, 15 Therefore, we also wanted to assess  whether EE affected motor skills, so we carried out tests of physical ability, including locomotor activity and strength. These included watching the mouse in an open field (OF) box (assessing how reluctant the mouse was to stay in the open), and assessing the mouse on a horizontal bar (jumping and climbing/balance abilities along static bars). We also carried out  activity tests, by measuring locomotor activity and counting the number of times the mouse crossed back and forth across a boundary.

This logically led to anxiety tests, since environmentally-enriched mice have reduced levels of corticosterone (a hormone associated with anxiogenic  situations) following exposure to predator cues such as scent or visual presence.12 As previously stated, Hutchinson et al.13 hypothesised that environmentally enriched mice produce less endogenous cortisol than their S cohorts. We believed that it was important to test the effects of EE on anxiety levels, so the following behaviours were assessed: a) the willingness of the mouse to exit an enclosed dark space and enter a brightly lit one (light–dark box; Figure 2); b) the willingness of the mouse to explore the open, exposed arms of a maze, as compared to walled arms (the plus-maze [PM]); c) the movement of the mouse in a black–white alley (similar to the light–dark box, where the black section of the alley is where a more anxious mouse will predominantly reside); and d) the movement of the mouse on the ‘hole board’ (a board with holes in it), to test the number of head-dips into a hole, where a confident mouse will exhibit more multiple head-dips than an anxious one. Throughout the trials, faecal boli were counted. Boli or urea are more often excreted by anxious animals. We also tested for hyponeophagia, i.e. the time it takes for a  mouse to investigate a novel food item. The more anxious mouse will take longer to do this.

Figure 2

 

Studies have shown that EE may induce experience- dependent neuroplasticity.16 Thus, cognitive ability trials were also performed. These included tests of mental processes, such as spatial reference and ability to alternate.17 In particular, EE has been shown to mitigate cognitive deficits in a mouse model of AD, with learning and memory deficits (associated with AD) also being ameliorated.18, 19 Mice provided with environmental stimulation were significantly improved in several measures of cognitive performance, with EE strongly modulating the pathological and behavioural progression of AD in the mouse model. This was tested by assessing whether mice would spontaneously investigate one arm of a maze on the first trial, then another arm of the same maze on the second trial, exhibiting memory functioning (spontaneous alternation in the T-maze; Figure 3) and similarly spatial novelty in the Y-maze (Figure 4).

Statistical analysis
For tests with multiple factors (the majority comparing strain and environment), two-way ANOVA was performed. Numerical data, such as the number of faecal boli, were subjected to Chi-Squared analysis. Values of  p ≤ 0.05 were considered statistically significant.

Results

Overall, regardless of strain, environmentally enriched mice showed enhanced functioning, exhibiting greater exploration and reduced anxiety-like behaviour. In tests on anxiety, environmentally enriched mice showed potential reductions in levels of nervousness by making more transits, investigatory head-dips, and having the lowest latency to leave enclosed areas.
Figure 3

In tests of cognition, environmentally-enriched mice also made faster and more correct choices. Environmentally-enriched B10s were found to leap  out of the apparatus with ease, being highly agile and able to escape confinement. Environmentally enriched B10s ran from between the open arms of the PM with high speed and agility (despite higher body mass), and showed increased investigatory behaviour. Both environmentally-enriched strains showed higher levels of burrowing. In species-typical behaviour  tests, latency to start grooming and the number of marbles buried, were improved in the environmentally-enriched B6s. The enrichment of the B6 housing may explain why this group also obtained a higher nesting score, and burrowed more material after two hours than the S B6s. EE may raise B6 performance above that of S B6s. For example, in the holeboard, more investigatory rears (up the sides of the apparatus) and head-dips into the holes were made by environmentally-enriched B6 mice than by S B6 mice.  Environmentally-enriched B10s showed greater exploratory behaviour, and were less anxious than S mice of the same strain. In many cases, environmentally-enriched mice were the highest performers.

Figure 4

Contrary to expectations, the S B6 mice functioned at a lower level relative to S B10s in most, but not all, of the species-typical behaviour tests. The marked deficits of B6s were lower in tests of nesting and burrowing. B6s functioned at a lower level on a number of motor tests, such as the static rods and OF, and were impaired in most tests of anxiety.
Cognitive tests also revealed lower functioning in B6 mice, with B6s making fewer correct alternations in the T-maze, and taking longer to do so. Both S B6 and B10 mice showed deficits in the majority of, but not all, species-typical behaviours (including climbing and burrowing less than their environmentally enriched counterparts), and in motor, anxiety and cognitive tests. Environmentally-enriched mice of both strains appeared to function at the highest levels, with S mice of both strains generally having the longest latencies to leave the closed arms and enter the open arms of the apparatus. Although the B10 brain appeared to be influenced by EE, S B10s  did not show the deficits expected of their strain, suggesting that the B10 would not provide a suitable mouse model for AD. It was also apparent that the behaviour of B10 mice differed significantly from that of B6 mice, suggesting that the strains could not be used interchangeably in trials.

The aim of this study was to assess whether EE influences the behaviour and cognitive and motor functioning of test animals. We used a standard mouse model (B6) and the B10 strain, known to have behavioural deficits. The study aimed to determine whether the B10 mouse housed in an enriched environment would show lesser motor and cognitive deficits, consequently asking whether EE can be influential on brain and behavioural functioning in  the adult B10. Contrary to the results of previous studies, the B10s functioned at a higher level than the B6s in a number of species-typical behaviour, motor, anxiety and cognitive tests. Unlike in previous studies, B10 mice, particularly those that had been raised in an enriched environment, functioned at the highest level of all groups in the majority of tests.

Environmentally-enriched mice generally functioned at higher levels than those of their S counterparts, suggesting that EE may be the cause of raised functioning in environmentally-enriched mice, and of enhanced behaviour in both strains, particularly the B10 strain. This may suggest that EE is not only beneficial to animal welfare, but can also positively  influence natural functioning and natural behaviour.

Ellen J. Coombs
Currently at:
WSPA
222 Gray’s Inn Road
London WC1X 8HB
UK
E-mail: ellenjcoombs@gmail.com
[This work was carried out in the Department of Zoology, Oxford University, UK]

References
1 Smithies, O. (1993). Animal models of human genetic diseases. Trends in Genetics 9, 112–116.
2 Gunter, C. & Dhand, R. (2002). The mouse genome. Nature, London 420, 509.
3 European Commission Workshop (2010). Of mice and men — are mice relevant models for human disease? Outcomes of the European Commission Workshop
‘Are mice relevant models for human disease?’ held in London, UK, on 21 May 2010, 10pp. Brussels, Belgium: Health Directorate, DG Research, European
Commission.
4 Deacon, R.J., Thomas, C.L., Rawlins, J.N.P. & Morley, .J. (2007). A comparison of the behaviour of C57BL/6 and C57BL/10 mice. Behavioural Brain Research 179, 239–247.
5 Wimer, R.E., Wimer, C.C., Chernow, C.R. & Balvanz, B.A. (1980). The genetic organisation of neuron number in the pyramidal cell layer of hippocampal regio superior in house mouse. Brain Research 196,
59–77.
6 Van Leuven, F. (2000). Single and multiple transgenic mice as models for Alzheimer’s disease. Progress in Neurobiology 61, 305–312.
7 Van Praag, H., Kepermann, G. & Gage, F.H. (2000). Neural consequences of environmental enrichment. Nature Reviews Neuroscience 1, 191–198.
8 Henderson, N.D. (1970). Brain weight increases resulting from environmental enrichment: A directional dominance in mice. Science, New York 169, 776–778.
9 Wolfer, D.P., Litvin, O., Morf, S., Nitsch, R.M., Lipp, H-P. & Würbel, H. (2004). Cage enrichment and mouse behaviour. Test responses by laboratory mice are unperturbed by more entertaining housing. Nature, London 432, 821–822.
10 Restivo, L., Ferrari, F., Passino, E., Sgobio, C., Bock, J., Oostra, B.A., Bagni, C. & Ammassari-Teule, M. (2005). Enriched environment promotes behavioural and morphological recovery in a mouse model for the fragile X syndrome. Proceedings of the National Academy of Sciences of the USA 102, 11,557–11,562.
11 Rampon, C., Jiang, C.H., Dong, H., Tang, Y.P., Lockhart, D.J., Schultz, P.G., Tsien, J.Z. & Hu, Y. (2000). Effects of environmental enrichment on gene expression in the brain. Proceedings of the National Academy of Sciences of the USA 97, 12,880–12,884.
12 Roy, V., Belzung, C., Delarue, C. & Chapillion, P. (2001). Environmental enrichment in BALB/c mice: Effects in classical tests of anxiety and exposure to a predatory odor. Physiology & Behaviour 74, 313–320.
13 Hutchinson, E., Avery, A. & Vandewoude, S. (2005). Environmental enrichment for laboratory rodents. ILAR Journal 46, 148–161.
14 Marashi, V., Barnekow, A., Ossendorf, E. & Sascher, N. (2003). Effects of different forms of environmental enrichment on behaviour, endocrinological, and immunological parameters in male mice. Hormones &  Behavior 43, 281–292.
15 Prior, H. & Sachser, N. (1995). Effects of enriched housing environment on the behaviour of young male and female mice in four exploratory tasks. Journal of Experimental Animal Science 37, 57–68.
16 Kempermann, G., Kuhn, G.K. & Gage, F.H. (1997). More hippocampal neurons in adult mice living in an enriched environment. Nature, London 386, 493–495.
17 O’Keefe, J. & Nadel, L. (1978). The Hippocampus as a Cognitive Map, 504pp. Oxford, UK: Clarendon Press.
18 Arendash, G.W., Garcia, M.F., Costa, D.A., Cracchiolo, J.R., Wefes, I.M. & Potter, H. (2004). Environmental enrichment improves cognition in aged Alzheimer’s transgenic mice despite stable beta-amyloid deposition.  NeuroReport 15, 1751–1754.
19 Jankowsky, J.L., Melnikova, T., Fadale, D.J., Xu, G.M., Slunt, H.H., Gonzales, V., Younkin, L.H., Younkin, S.G., Borchelt, D.R. & Savonenko, A.V. (2005). Environmental enrichment mitigates cognitive deficits in a mouse model for Alzheimer’s disease. Journal of Neuroscience  25, 5217–5224.

The Virtual Pharmacology Lab — A Repository of Free Educational Resources to Support Animal-free Pharmacology Teaching

The new repository will support pharmacology practical teaching by
promoting the sharing and re-use of existing pharmacology resources

David Dewhurst and Ross Ward

Teaching pharmacological knowledge, principles and practical skills through student participation in laboratory practical classes is considered important in the majority of university pharmacology degree courses. Many such practical classes use either isolated tissues or in vivo animal preparations. Typically, students will work on a preparation, usually set-up by a technician, and follow a prescribed schedule of experiments designed to achieve specific learning objectives. There has been a tendency for such classes to become less explorative and, in the face of that, some universities have turned to computer based simulations, either to support, or in some instances, replace, laboratory-based  classes. There  have been numerous evaluations of the educational effectiveness of computer-based alternatives and, in the main, the results suggest that they can achieve many of the learning objectives of practical classes that involve the use of animal preparations.1,2,3 Typically, the computer simulations display datatraces, previously recorded from a specific pharmacological preparation, on the computer screen, in a form comparable to that in the real experiment (e.g. contractions of a perfused mammalian heart would be presented on a display which emulates a chart recorder). Data presentation is structured around a series of experiments designed by an experienced and knowledgeable tutor to cover the main learning objectives. The simulations often also contain descriptions of the underlying pharmacology, the preparation, apparatus and methods, and include student tasks and self-assessment questions with feedback, making them potentially suitable for self directed study. They may be used: as direct replacements for animal experiments; to reduce animal use through better preparation of students before participation in a live practical class; to enable students to collect additional data after the live class; and/or as a back-up, should the student miss the class or be unable to collect suitable data. The process of developing a computer simulation involves aggregating various components or learning objects (LOs) by using a software package, and then publishing a compiled, run-time version that renders it non-editable by the user. While this protects the developer from unlawful or inappropriate use of their content, it also prevents teachers from tailoring the programs to their specific needs. The main feedback from users is a desire to be able to modify the content of the programs. It is hoped that the Virtual Pharmacology Lab, by making the individual LOs available to teachers in usable formats, will encourage greater use of alternative methods and further reduce animal use in teaching.

The repository

The Virtual Pharmacology Lab (www.virtualpharmacologylab. com) is an open access repository of quality-assured LOs, designed to support pharmacology practical teaching by promoting the sharing and reuse of existing pharmacology resources. The LOs are the disaggregated components of eleven existing computer-based simulations of practical pharmacology classes developed by one of the authors (brief descriptions at www.sheffbp.co.uk). The disaggregation process has been described previously,4,5 and typically, disaggregation of each computer program  yields 50–100 LOs. Currently, the repository contains >650 LOs, including:

— data-traces: response of a particular tissue preparation to a change in experimental parameters (e.g. administration of a drug/drug combination, electrical stimulation);
— text-files: e.g. a description of an experimental protocol for a particular experiment, the experimental method or the underlying pharmacology;
— images, diagrams and illustrations: usually combined with text to describe the preparation, underlying pharmacology, apparatus and method;
— video: perhaps used to illustrate how a preparation is dissected and set-up in an organ bath, and how pharmacological agent(s) are administered to elicit a tissue response; and
— interactive student tasks, activities and self assessment questions.

There are also a small number of videos depicting laboratory procedures, which were donated by the University of Queensland. The disaggregated learning objects are stored in a database and tagged with descriptive metadata, including author, title, and a brief description. Tagging the data in the repository with meaningful metadata enables users to search the database by keyword, e.g. they could search for all data-traces and other LOs relating to a specific animal preparation or for all LOs relating to the actions of a specific drug or drug combination.

Access

Access to the repository (www.virtualpharmacologylab. com) is open to anyone, and no log-in is required. Use of the LOs is covered by a Creative Commons Licence, granting free use for non-commercial teaching purposes, preventing the creation of derivatives, and requesting suitable acknowledgement of the source of the data. The user — i.e. a teacher or a student — accesses the repository via a web interface (Figure 1).

Dewhurst figure 1

Currently, this contains a welcome message briefly explaining what the repository does and defining the target audience (mainly pharmacology teachers). There is a keyword ‘Search’ facility (box 1), into which a teacher could enter a term (such as an animal species or the name of a drug). The search may then be refined, currently by selecting one of the eleven computer simulation programs (preparations) from which the initial data have been derived (box 2). Once those two options have been selected, clicking ‘Go’ will return the search results. For example, if the search term ‘acetylcholine’ is entered in box 1 (Figure 2) and ‘all preparations’ selected in box 2 (i.e. unrefined search) then 51 LOs are returned — a mixture of data-traces, images, animations, interactive animations, HTML text files, quiz questions, calculations and video. If the user had refined the search by selecting just one of the preparations in box 2, then the number of LOs returned would decrease. For example, a refined search for ‘acetylcholine’ and the preparation ‘Blood pressure (in vivo)’ returns four LOs, and for the preparation ‘Ileum (in vitro)’, the search returns nine LOs.Each LO has a descriptive title indicating to the user what the learning object is and a brief description providing more detail.
dewhurst figure 2

Clicking on the title of one of the LOs shown in Figure 2, e.g. ‘Effect of acetylcholine administered to SCG, electrical stimulation on, in cat nictitating membrane preparation’, leads to a screen display of that LO which, in this instance, is an image of the data-trace (Figure 3).

dewhurst figure 3

For each LO there is a brief description and a web-link (url) to the file in the repository. The url may be embedded, as an active link, in a tutor’s own teaching materials, such as in a document (e.g. a laboratory schedule) or in a PowerPoint presentation, thus enabling that teacher to combine LOs from the repository with their own teaching materials to create a bespoke learning resource. Right-clicking on the image itself (a JPEG file) will expose a menu with several options (e.g. open image; save image; copy image), thus enabling the user to download that file. The display also shows from which preparation the image is derived (in this case ‘cat nictitating membrane’) and a brief summary of the metadata. Users are requested to complete a short questionnaire and the feedback from that will inform future developments.

Conclusion and future developments

The developments described will provide teachers (and students) working in university departments of biomedical/life sciences with free access to a repository of quality-assured, digital LOs, including numerous data sets of traces from animal experiments. It is hoped that making resources available in more granular formats may provide a more acceptable route toward the use of the computer simulations in university practical class teaching. This would be facilitated, if teachers were able to effectively combine LOs from the repository with their own resources, e.g. an image, a set of quiz questions, a short video-clip or an interactive animation, to create a bespoke teaching resource for their students that is tailored to their specific needs. Future developments will depend very much on the level of interest in the repository from the user community. If there is sufficient interest, then the intention is to add more functionality, such as: an easy-to-use upload facility and metadata form; a system for ‘holding’ new LOs pending user-led quality assurance processes; a mechanism for downloading resources (rather than simply embedding a link); a quality-rating system; RSS feed to automatically notify the user community of new content; integration with social media such as Twitter and Facebook; and mobile device compatibility, so that users can access the repository via a Smartphone or tablet computer.

Acknowledgement

The authors would like to thank the Doerenkamp-Zbinden Foundation for their kind financial support of this project.

Author for correspondence:
Professor David Dewhurst
Director of Educational Information Services and Professor of e-Learning
Learning Technology Section
College of Medicine & Veterinary Medicine
University of Edinburgh
Hugh Robson Building
15 George Square
Edinburgh EH8 9XD
UK

E-mail: d.dewhurst@ed.ac.uk

References

1 Knight, A. (2007). The effectiveness of humane teaching methods in veterinary education. ALTEX 24, 91–109.
2 Patronek, G.J. & Rauch, A. (2007). Systematic review of comparative studies examining alternatives to the harmful use of animals in biomedical education. Journal of the American Veterinary Medical Association 230, 37–43.
3 Gruber, F.P. & Dewhurst, D.G. (2004). Alternatives to animal experimentation in biomedical education. ALTEX 21, Suppl. 1, 33–48.
4 Ellaway, R., Dewhurst, D. & Cromar, S. (2004). Challenging the mortality of computer assisted learning materials in the life sciences: The RECAL Project. Bioscience Education E-journal 3, 3–7. Available at: http://www.bioscience.heacademy.ac.uk/journal/vol3/beej-3-7.aspx  (Accessed 01.03.14).
5 Dewhurst, D., Ellaway, R. & Cromar, S. (2005). RECAL: Creating computer-based alternatives using a sustainable learning objects approach. ALTEX 23, Special Issue 2, 54

Ethical Animal Use in Education and Training: From Clinical Rotations to Ethically Sourced Cadavers

The InterNICHE Policy defines the concept of ‘ethically sourced’ material to encourage replacement and enhance the acquisition of knowledge, skills and attitudes.

Nick Jukes

Within the fields of education and training in medicine, veterinary medicine and biological science, students and professionals have access to the wide range of non-animal alternatives that are now available. These replacement methods, usually developed by the teachers and trainers themselves, include models, manikins, mechanical simulators, multi -media software, virtual reality (VR) and student self-experimentation. For zoology and veterinary medicine, hands-on experience of living animals and of animal cadavers, organs and tissue is also necessary. Practical classes in anatomy, pathology, clinical skills and surgery training can benefit considerably from informed curriculum design that uses a combination of non-animal alternative tools and animal based alternative approaches.

Conventional harmful animal use has included the widespread killing of animals for their cadavers, organs and tissue, as well as animal experimentation and other instrumental animal use. The former near ubiquity of such practices, and the normalisation of harm and killing, have contributed to the mistaken belief that these negative interactions with animals are the only ways of working with animals in practical classes. In reality, it is possible to implement methods that can meet teaching objectives effectively, yet at the same time avoid harm completely and, in some cases, provide benefit to individual animals. Within zoology, practical classes that involve the use of animals, but that can be performed in a completely ethical way, include non-invasive field trips with observational work on animals in their ecological context. Within veterinary medicine, they include clinical learning opportunities with animal patients and animal ‘volunteers’, and the use of ‘ethically sourced’ animal cadavers, organs and tissues.1

The humane tradition of student clinical rotations is practised widely in the training of veterinary students, but could be extended considerably. Earlier and greater access to cases in teaching hospitals, and strategic alliances with NGOs for work in shelters and communities, particularly on sterilisation projects, can provide additional valuable hands-on experience. Integrated into the curriculum, they offer authentic scenarios, with positive learning opportunities at many levels. This is in contrast to the contrived experience, involvement in harm, and exposure to the implicit negative lessons from the hidden curriculum, that are presented by animal experiments.

Healthy companion animal ‘volunteers’ can be consenting subjects for non-invasive clinical skills training. They can also be subjects for ‘living anatomy’ lessons, where body painting can allow the study of movement from an anatomical perspective. The participating volunteers receive the reward of food and social interaction.  Operating within the context of healing not harming, such therapeutic work with animal patients and positive interaction with volunteers can validate and encourage the practice of caring as an essential clinical skill.2 These activities can also help to replace harmful animal use in clinical skills and surgery training, by providing a direct and practical alternative.

Animal cadavers, organs and tissues can be used in anatomy and pathology for dissection and for histology classes, and can be preserved with glycerine or formaldehyde, or by the process of plastination. For clinical skills and surgery, they can be used for surgical dissection and for basic and advanced skills practice. Cadavers and organs can also be ‘animated’ by applying perfusion and pulsation, providing opportunities for a wide range of procedural and trauma training.3 In addition, fresh tissue can be used in biochemistry, physiology and pharmacology practical classes.

In its comprehensive Policy on the use of animals and alternatives in education and training,4,5 first published in the book from Guinea Pig to Computer Mouse,6 and available in a updated version online, InterNICHE presents its position and offers guidelines to ensure the effective and fully ethical acquisition of knowledge, skills and attitudes. The Policy includes definitions of alternatives in education and of harm, and presents individual policies on dissection, the sourcing of animal material, work with live animals for clinical skills and surgery training, and field studies. It also addresses the use of animals for the production of alternatives themselves.7

The alternative to the killing of animals for their cadavers, organs and tissues is to obtain them  through ethical means. The Policy introduced and defined the concept of ‘ethically sourced’ material. The strict conditions behind the definition demonstrate how seriously animal harm, including killing, should be considered. The Policy encourages the highest ethical standards, and motivates against the perceived need to compromise. Yet it is also realistic, reflecting sustainable contemporary examples of ethical sourcing, achieved through body donation programmes at progressive veterinary colleges.
Jukes diagram

The flowchart in Figure 1 explores in detail the process of sourcing of cadavers, organs and tissues. It leads the user step-by-step through questions about the animal, its death and other contextual issues, that together guide the assessment of the source. By using the InterNICHE Policy as its base, the source will then be characterised as one of the following: ‘ethically sourced’; ‘acceptable other source’; or ‘not in accordance with Policy’. Summarising the flowchart, the key points are:

 

‘Ethically sourced’

— From a free-living animal (wild, stray or companion animal) that had no freedoms denied
— Not killed for education and training, nor for another purpose
— Died naturally or in an accident, or euthanised for medical reasons
— Client donated, if a companion animal — All other conditions are met

‘Acceptable other source’

— From an animal with some freedoms denied (e.g. from a farm or research facility)
— Not killed for education and training, nor for another purpose
— Died naturally or in an accident, or euthanised for medical reasons
— Considered only when ethically sourced is not feasible
— All other conditions are met

‘Not in accordance with Policy’

— The animal was killed for education and training, or for another purpose
— The animal did not die naturally or in an accident, or was not euthanised for medical reasons

Conventional sources of animal cadavers, organs and tissues include wild caught animals, research facilities, breeders, farms, slaughterhouses, zoos, sporting events, and some animal shelters. The context of instrumental use and the associated freedoms denied and harm caused, mean that any potential ethical element of the use of such sources has been compromised or negated. InterNICHE does not consider material from these sources, including so-called ‘waste’ or ‘surplus’ material, to be ethically sourced. The use of the by-products of killing done at such locations, including for practical or financial reasons, is not acceptable: slaughterhouse material, or animals killed because they are no longer wanted at the end of research or testing, would therefore not be considered replacement alternatives, according to the Policy. Waste chicken ilea taken from the slaughterhouse can indeed help end the purpose-killing of guinea-pigs whose ilea are used in pharmacology preparations, but the resource is dependent on the continued killing of the chickens.

In this case, InterNICHE would instead argue for the use of existing software and the development of immersive VR-based training in such practical classes. There would also be some opportunities for the harvesting of ilea that are ethically sourced, for example from companion animal rodents. Nevertheless, some physiology and pharmacology practical classes just reflect animal experiments performed in science. As significant parts of research and testing continue to move away from animal experimentation toward a new and less-tarnished gold standard, such training at the student level — whether virtual or on real tissue — may also be abandoned. The same is true for the training of laboratory animal scientists and technicians. In the meantime, some training courses already use a range of non-animal alternatives, and in a similar vein to the above, haptic-enabled VR procedural training, along with clinical learning opportunities on animal patients, could achieve considerable replacement in these fields.

Material from animals that do not commonly exist as free-living wild, stray or companion animals may be hard to source ethically. In these cases, deriving cadavers, organs and tissues from sources such as research facilities and farms is an acceptable compromise, when all the other conditions are fully met. This is known as an ‘acceptable other source’ for such material. Importantly, for the characterisation of a material as either ‘ethically sourced’ or ‘acceptable other source’, the animal must still have died naturally or in an accident, or have been euthanised for medical reasons — that is, in response to natural terminal disease or serious non-recoverable injury.

The development of innovative and humane tools and approaches reflects a creativity motivated by the need to better meet teaching objectives and to harness the power of technology to support the learning process. Teachers and trainers who are responsible for curricular design and for the methods employed in practical classes, are encouraged to further implement these progressive methods. Student and trainee access to clinical learning experiences and to ethically sourced cadavers, organs and tissue, can not only help end harmful animal use, but can also demonstrate the potential for positive interactions with animals in education and training. The InterNICHE Policy and the flowchart on sourcing help define the context and present the conditions for the  successful implementation of both non-animal and animal-based replacement alternatives.

 

Nick Jukes
InterNICHE Co-ordinator
98 Clarendon Park Road
Leicester LE2 3AE
UK

E-mail: coordinator@interniche.org

References

1 Martinsen, S. & Jukes, N. (2008). Ethically sourced animal cadavers and tissue: Considerations for education and training. AATEX 14, Special Issue, 265–268.
2 Martinsen, S. (2008). Training the animal doctor: Caring as a clinical skill. AATEX 14, Special Issue, 269–272.
3 Aboud, E., Suarez, C.E., Al-Mefty, O. & Yasargil, M.G. (2004). New alternative to animal models for surgical training. ATLA 32, Suppl. 1, 501–507.
4 Jukes, N. & Chiuia, M. (2003). Policy on the use of animals and alternatives in education and training. In from Guinea Pig to Computer Mouse: Alternative Methods for a Progressive, Humane Education, 2nd edition (ed. N. Jukes & M. Chiuia), pp. 500–508. Leicester, UK: InterNICHE. [Updated Version 3.0 available at www.interniche.org/about/policy.htm]
5 Martinsen, S. & Jukes, N. (2008). From Policy to practice: Illustrating the viability of full replacement. AATEX 14, Special Issue, 249–252.
6 Jukes, N. & Chiuia, M. (2003). from Guinea Pig to Computer Mouse: Alternative Methods for a Prog ressive, Humane Education, 2nd edition, 520pp. Leicester, UK: InterNICHE.
7 Martinsen, S. & Jukes, N. (2012). An ethical scoring system for the production and assessment of alternatives in education and training. ALTEX 1/12, Proceedings of WC8, 399–404.

 

Alternatives to Animal Bioassays for Prions

Combined PMCA and cell assays are being optimised for use
as an effective, or even superior, alternative to
quantitative prion bioassays in laboratory rodents

Prions and prion diseases

Bioassays that involve the use of laboratory animals are often considered to be the ‘gold standard’ for the detection and titration of certain pathogens. This holds particularly true for prions,1, 2 which are the proteinaceous infectious particles that cause fatal neurodegenerative diseases in animals (e.g.  scrapie, bovine spongiform encephalopathy [BSE]) and humans (e.g. sporadic or variant Creutzfeldt–Jakob disease [sCJD, vCJD]). The BSE epidemic and resulting emergence of vCJD in the United Kingdom, and the subsequent occurrence of BSE and vCJD in other countries, have substantially increased the worldwide awareness of prions and their potential risk to public health.

Unlike bacteria, viruses or fungi, prions are pathogens that are devoid of coding nucleic acids. According to the prion hypothesis, they consist essentially of a misfolded and aggregated isoform of  the host-encoded prion protein (PrP).1 Prion-forming conformers of the prion protein are referred to as PrPSc or PrPTSE (‘Sc’ and ‘TSE’ are acronyms for scrapie, and transmissible spongiform encephalopathy, respectively, with the latter being an alternative name for prion diseases).1, 3 The replication of prions shows close similarities to the seeded growth of crystals, and is thought to occur by a mechanism of nucleation- dependent PrP polymerisation,4, 5 i.e. oligomers or polymers of PrPTSE act as nuclei (‘seeds’) that recruit cellular prion protein (PrPC) and incorporate it, in a misfolded form, into their own aggregate structure. When PrPTSE aggregates eventually fall apart into smaller units, this causes a multiplication of PrP particles with proteinaceous seeding activity,  and thereby a further autocatalytic replication of the pathological protein state. According to this concept, the self-replication of prions is based on their biochemical seeding activity, i.e. the ability to convert cellular protease-sensitive prion protein into misfolded, aggregated and often Proteinase K (PK)-resistant PrP (PrPres).

Prion bioassays and the Three Rs

For a long period of time, the infectivity of prions could be quantified only by incubation time interval assays or endpoint titrations in animals.6 Both methods rely on the transmission, often by intracerebral inoculation, of a prion infection that causes cerebral propagation of PrPTSE and, eventually,  neurological disease in the host animal. Mice or Syrian golden hamsters have been the most frequently used test animals in such prion bioassays. Quantitative prion bioassays in small rodents or other laboratory animals usually determine prion titres in terms of the median infective or lethal dose (ID50, LD50) that had been present in the inoculated sample material. One prion ID50 or LD50 is the dose of prions that causes infection (in terms of PrPTSE propagation or symptoms) or fatal disease, respectively, in 50% of the inoculated animals.

According to the concept of the Three Rs, as proposed by Russell and Burch,7 the use of animals in experiments should be replaced by alternative methods whenever possible (replacement), and the number of animals should be reduced to a minimum through good experimental design (reduction). Furthermore, discomfort and stress in any animals used should be minimised (refinement). To comply with these requirements in the field of prion research, particularly in terms of reduction and replacement, has long constituted a challenging task, because of the particular nature and properties of prions.

The quantification of prions by using cell assays has only gradually become feasible during the past few years, and so far, only relatively few cell assays are available for this purpose.8-12 Furthermore, the applicability of such cell assays is often restricted, because they mostly work with just one or a few of the various prion strains that exist under laboratory and real-life conditions. Cell assays for prions typically detect PrPres amplification at a certain time post-inoculation, rather than cytopathic or cytotoxic effects.

Also, the development of cell-free detection methods that would be on a par with animal bioassays for prion titration was impeded for a long period of time by the unconventional chemical composition and replication mechanism of these infectious agents. The identification of seeding-active prion protein as the self-replicating principle of prions, however, has finally given rise to novel approaches for a cell-free biochemical measurement of pathological prion activity. This was possible due to the introduction of an analytical technique called protein misfolding cyclic amplification (PMCA), which mimics nucleation-dependent PrP polymerisation, in an accelerated mode, in the test tube.13Quantitative PMCA, and related techniques, such as RT-QuIC, now permit, at least for specific prion strains, the direct titration of prion-associated seeding activity.14-17 The detection of PrP aggregates or PrPres (by immunohistochemistry of tissue sections, Western blotting of samples, or by other methods) was previously successful in many attempts to provide a qualitative or semi-quantitative surrogate marker for infectious PrP particles.2, 18 However, in some studies (with other combinations of prions strains and host animals), such correlation was not observed,19-22 and the presence of aggregated PrP or PrPres in itself does not provide information about the activity of the protein in terms of biochemical or biological PrP seeding and disease transmission.

Thus, for the assessment of the pathogen load in an unknown prion sample, the following indicators can be tentatively ranked according to their current significance. This produces the following hierarchy in terms of diagnostic value:

1. Lethal dose (as indicated by the transmission of fatal disease to animals).

2. Infective dose (as indicated by the transmission of PrPTSE propagation or disease to animals).

3. Cell culture infective dose (as indicated by the transmission of PrPres propagation to cell cultures).

4. Seeding dose (as indicated by the cell-free propagation of PrPres in PMCA or other cell-free seeding assays).

5. Amount of aggregated PrP / PrPres (as indicated by PrP detection methods).

The biochemical seeding activity and biological cell culture infectivity of prions are incongruent with the infectivity of prions in animals (or humans), so PMCA and cell assay findings cannot be used a priori as a representative for in vivo infectivity. This caveat limits the utility, acceptance and use of alternative methods to animal bioassays in specific areas of prion research, such as the evaluation of reprocessing procedures for medical devices for anti-prion efficacy.

In a recent report on a rationale and methodology for the further reduction and replacement of prion bioassay titrations in laboratory rodents, we have tried to address this problem.

Toward the further reduction and replacement of animal bioassays in prion research by using cell and PMCA assays

 In our article, published in Laboratory Animals, we presented three pairs of PMCA and glial cell assays for different hamster-adapted prion agents (the frequently used 263K scrapie strain, 22A-H scrapie and BSE-H), as well an adaptation of quantitative PMCA to human vCJD prions.23 In this context, we described how to use our PMCA and cell assays for measuring the seeding dose (SD50) and the cell culture infective dose (CCID50), respectively, in a prion test sample. One SD50 represents the dose of prion-associated seeding activity that converts PrPC into PrPres in 50% of PMCA samples, and one CCID50 represents the dose of prions that causes infection (in terms of PrPres propagation) in 50% of inoculated cell cultures. Based on quantitative correlations empirically  established  in reference standards (such as hamster scrapie brain homogenates) between the seeding dose, cell culture infective dose and in vivo infectivity, SD50 and CCID50 values detected in vitro can be tentatively translated into ID50 or LD50 values.

In order to strengthen the significance of such indirect ID50 or LD50 assessments, we decided to perform combined PMCA and cell assays. PMCA and cell assays represent profoundly different cell-free and cell-based test principles for the biochemical and biological titration, respectively, of prion activity in vitro. Therefore, if these assays independently deliver consistent ID50 or LD50 assessments, then this provides an important methodological safeguard that substantially backs up the overall test reliability, as compared with titrations based on either PMCA or cell assays alone. Furthermore, we proposed to empirically validate our approach of combined PMCA and cell assay measurements. For this purpose, prion titre estimates from a set of different test samples would need to be compared to the actual ID50 or LD50 levels in the respective samples. As recently shown by Pritzkow et al., this could be done, at least partly, by using bioassay data from previous in vivo studies.16

Our approach provides a concept, a methodological platform and a practical roadmap that aim to  establish the combined PMCA and cell assays as an effective, or even superior, alternative to quantitative prion bioassays in laboratory rodents. If successful, this would substantially contribute toward the implementation of the Three Rs. For example, the optimal use of 13 normal hamster brains and slightly more than 2 × 10–6g of 263K scrapie hamster brain tissue in our cell and PMCA assays, is theoretically sufficient to replace the use of 60 bioassay hamsters in the in vivo titration of twelve 263K scrapie samples. However, since our in vitro techniques still require animal tissues, they currently offer only the opportunity for reduction and relative replacement in terms of the Three Rs. It remains to be seen whether further methodological advancements will provide options for the absolute replacement of animal based prion bioassays in the future.

In any case, our assays and other seeding and cell assays can already provide substantial  contributions to replacement. This particularly applies to resolving scientific questions that would previously have required prion titrations in animals, but can now be sufficiently answered by in vitro measurements of seeding activities and/or cell culture infectivities. Finally, PMCA and cell assays are also helpful with respect to reduction, since they can be used to ‘precharacterise’ samples prior to bioassays in animals. This allows careful dose-level or sample selections, and generally improves the practical and statistical planning of animal experiments, in terms of obtaining the information of interest with the smallest possible number of animals.

Conclusions

As the PMCA and cell assays become more versatile and applicable to different prion strains, they will foster the reduction and replacement of animal bioassays in this field of research and testing. Reducing the need for animal experiments that are time-consuming, expensive, restricted in throughput and possibly problematic with respect to their ethics, will meet both the interests of prion researchers and the objectives of the Three Rs.

 

Acknowledgement

The skilful technical assistance of Özhan Demirel is gratefully acknowledged.

Author for correspondence:
Dr Michael Beekes
FG 14–AG 5: Unconventional Pathogens and Their Inactivation
Applied Infection Control and Hospital Hygiene
Robert Koch-Institut
Nordufer 20
13353 Berlin

Germany

E-mail: BeekesM@rki.de

References

1 Prusiner, S.B. (1998). Prions. Proceedings of the National Academy of Sciences of the USA 95, 13,363–13,383.
2 Colby, D.W. & Prusiner, S.B. (2011). Prions. Cold Spring Harbor Perspectives in Biology 3, a006833.
3 Brown, P. & Cervenakova, L. (2005). A prion lexicon (out of control). Lancet 365, 122.
4 Come, J.H., Fraser, P.E. & Lansbury, P.T. (1993). A kinetic model for amyloid formation in the prion diseases: Importance of seeding. Proceedings of the National Academy of Sciences of the USA 90, 5959–5963.
5 Soto, C. (2011). Prion hypothesis: The end of the controversy? Trends in Biochemical Sciences 36, 151–158.
6 Prusiner, S.B. (1987). Bioassays of prions. In Prions — Novel Infectious Pathogens Causing Scrapie and Creutzfeldt-Jakob Disease (ed. S.B. Prusiner & M.P. McKinley), pp. 65-81. San Diego, New York, Toronto: Academic Press.
7 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, 238pp. London, UK: Methuen.
8 Klöhn, P.C., Stoltze, L., Flechsig, E., Enari, M. & Weissmann, C. (2003). A quantitative, highly sensitive cell-based infectivity assay for mouse scrapie prions. Proceedings of the National Academy of Sciences of the USA 100, 11,666–11,671.
9 Mahal, S.P., Demczyk, C.A., Smith, E.W., Klöhn, P.C. & Weissmann, C. (2008). Assaying prions in cell culture: The standard scrapie cell assay (SSCA) and the scrapie cell assay in end point format (SCEPA). Methods in Molecular Biology 459, 49–68.
10 Edgeworth, J.A., Jackson, G.S., Clarke, A.R., Weissmann, C. & Collinge, J. (2009). Highly sensitive, quantitative cell-based assay for prions absorbed to solid surfaces. Proceedings of the National Academyof Sciences of the USA 106, 3479–3483.
11 Arellano-Anaya, Z.E., Savistchenko, J., Mathey, J., Huor, A., Lacroux, C., Andreoletti, O. & Vilette, D. (2011). A simple, versatile and sensitive cell-based assay for prions from various species. PLoS One 6, e20563.
12 Bian, J., Napier, D., Khaychuck, V., Angers, R., Graham, C. & Telling, G. (2010). Cell-based quantification of chronic wasting disease prions. Journal of Virology 84, 8322–8326.
13 Saborio, G.P., Permanne, B. & Soto, C. (2001). Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature 411, 810–813.
14 Chen, B., Morales, R., Barria, M.A. & Soto, C. (2010). Estimating prion concentration in fluids and tissues by quantitative PMCA. Nature Methods 7, 519–520.
15 Wilham, J.M., Orru, C.D., Bessen, R.A., Atarashi, R., Sano, K., Race, B., Meade-White, K.D., Taubner, L.M., Timmes, A. & Caughey, B. (2010). Rapid endpoint quantitation of prion seeding activity with sensitivity comparable to bioassays. PLoS Pathogens 6, e1001217.
16 Pritzkow, S., Wagenführ, K., Daus, M.L., Boerner, S., Lemmer, K., Thomzig, A., Mielke, M. & Beekes, M. (2011). Quantitative detection and biological propagation of scrapie seeding activity in vitro facilitate use of prions as model pathogens for disinfection. PLoS One 6, e20384.
17 Makarava, N., Savtchenko, R., Alexeeva, I., Rohwer, R.G. & Baskakov, I.V. (2012). Fast and ultrasensitive method for quantitating prion infectivity titre. Nature Communications 3, 741.
18 Aguzzi, A. & Calella, A.M. (2009). Prions: Protein aggregation and infectious disease. Physiological Reviews 89, 1105–1152. 19 Lasmezas, C.I., Deslys, J.P., Robain, O., Jaegly, A., Beringue, V., Peyrin, J.M., Fournier, J.G., Hauw, J.J., Rossier, J. & Dormont, D. (1997). Transmission of the BSE agent to mice in the absence of detectable abnormal prion protein. Science 275, 402–405.
20 Race, R., Meade-White, K., Raines, A., Raymond, G.J., Caughey, B. & Chesebro, B. (2002). Subclinical scrapie infection in a resistant species: Persistence, replication, and adaptation of infectivity during four passages. Journal of Infectious Diseases 186, Suppl. 2, S166–S170.
21 Barron, R.M., Campbell, S.L., King, D., Bellon, A., Chapman, K.E., Williamson, R.A. & Manson, J.C. (2007). High titres of transmissible spongiform encephalopathy infectivity associated with extremely low levels of PrPSc in vivo. Journal of Biological Chemistry 282, 35,878–35,886.
22 Doby, K. & Barron, R. (2013). Dissociation between transmissible spongiform encephalopathy (TSE) infectivity and Proteinase K-resistant PrPSc levels in peripheral tissue from a murine transgenic model of TSE disease. Journal of Virology 87, 5895–5903.
23 Boerner, S., Wagenführ, K., Daus, M.L., Thomzig, A. & Beekes, M. (2013). Towards further reduction and replacement of animal bioassays in prion research by cell and protein misfolding cyclic amplification assays. Laboratory Animals 47, 106–115.

FRAME’s Alternatives Timeline Project

FRAME’s proposed online resource
on alternatives will be easy to access,
visually appealing and content rich

The use of animals in experiments is well documented. Its history and evolution can be traced through to contemporary techniques. Centuries ago, the ancient Greeks and Romans first began exploratory surgery on animals, to discover the inner workings of living things. Notably, the Roman physician, Galen of Pergamon (ca 130–201AD), investigated the complexities of the respiratory, cardiovascular and nervous systems by dissecting living animals. He has been subsequently labelled the father of vivisection.1  By the 17th and 18th centuries, experimentation on animals was popularised within a wide variety of disciplines. With it, the debate originated as to whether these techniques were morally, religiously and scientifically right. Defenders of vivisection believed that the purpose of animals on Earth was to serve man in whatever means necessary.

The effectiveness of these techniques, however, has been questioned for hundreds of years. Protests, official inquiries, scientific studies and debates have been ongoing since before the turn of the 20th century. The first public debate on the topic took place in Florence in 1863, while the first anti-vivisection organisations emerged in Britain in 1875.2 Many academics, and a large proportion of the general public, have not been convinced of the validity, economic rationale and necessity of experimentation on animals. Arguably, the most important question that has been raised is, “Are animal models representative of human systems?”

Those that argue in favour of vivisection and its relative necessity stand firm on its foundation of past ‘successes’. Claims that much modern advancement in drug development and medicine would not have been possible without experimentation on animals are strengthened, because of the depth of the well from which to draw successes. In other words, due to the large quantity of animal experiments that have been performed — some of which have led to notable discoveries — there is enough evidence to support the methodology. It is only in recent decades that the debate regarding the relative scientific correlation between species, i.e. the results of a non-human study being relevant to humans, has gained momentum.

There are a number of reasons behind this. First, with the ever-expanding availability and transfer of knowledge through the internet and other media, public awareness of the use of experimental animals has brought the topic into the spotlight. As governments and businesses are put under growing pressure from the public to provide proof and/or explanations for the necessity of animal use in experiments, more and more questions are being asked. Second, the development of more universal directives to protect animals has advanced the examination of such animal use. The European Union, for instance, has put in place mandatory uniformity across nations that previously varied significantly in their approach to animal welfare — for example, Directive 2010/63/EU and Regulation (EC) No 1223/2009.3, 4 Third, and almost certainly the most important reason, is the growth in available alternatives to the use of animals. By questioning whether animals are effective models of human systems, we can then ask; “If not, then what should we be using instead?”

Before Russell and Burch first outlined the concept of the Three Rs in detail in 1959,5 scientists and researchers had already developed successful alternatives to the use of animals in experiments. At FRAME, it has come to our attention that although these alternatives exist, it is relatively difficult and time-consuming to discover and/or learn about them. What if there was one tool that a student, researcher or member of the public could utilise to effectively search for and learn about past alternatives, ones that are available now, and those proposed for the near future? FRAME believes that is it imperative to accurately define and present this history as an interactive timeline. This timeline will fulfil a number of goals by:

— acting as a review of effective alternatives;

— providing a primary resource for researchers;

— raising the profile of FRAME and the pursuit of the implementation of alternatives;

— providing a database of definitions and ongoing research;

— cross-referencing ineffective animal models and their alternatives;

— clearly defining when and where the alternative was formulated, and who were involved and;

— clearly defining which of the Three Rs are in practice.

Figure 1: Important characteristics of FRAME’s new alternatives timeline

Ease of access: a well thought-out design to promote efficiency, in a format that anyone can use.

Visually appealing: a contemporary design that is as pleasing to look at as it is to use.

Content rich: this will be the most important component of the timeline. Many timelines are limited in the sense that they are not dynamic in their content. We envision a timeline that will display as much or as little content as the user wishes.

Evidently, there are a number of benefits to this project. We are unaware of any other resource that brings together so many essential components to progress and inform the field of alternatives to animals in experimentation. We envision a timeline that, at its core, balances ease of access, appealing visuals, and rich content. This coincides perfectly with the proposed launch of FRAME’s new website design. A well-constructed and maintained interactive timeline will provide an opportunity to better understand where alternative research began, its progress and current trends.

The timeline will focus on each of the Three Rs and present key dates related to their use or adaptation. There is a considerable volume of information available about major advancements in replacement, reduction, and refinement, yet this information is scattered and not easily connected. The alternatives timeline will collect and compartmentalise this information in one source. Science promotes evolution and avoids anything static. Why should we be content with techniques that have changed very little for hundreds of years and rarely yield any usable results? As we move forward in the 21st century, we need to continue to advance and make the most of available technology. The FRAME alternatives timeline will use the power of the internet to connect everyone in the world to a single modern resource. We believe that it is vital that this information be made available to everyone, in a medium that can produce the maximum amount of support and clarity.

The alternatives timeline will provide a platform for anyone with an interest in improved science and animal welfare, who wish to gain a better understanding of how to shift research focus away from animal models and toward non-animal ones. Many scientists who actively use animals claim that they would prefer not to if they had the option. By simply searching this easy-to-use interactive timeline, a researcher would be able to discover available alternative techniques in seconds, including a concise line drawing each technique back to its genesis with accurate dating, a precise description of the technique with all uncommon words defined, and links to further information (i.e. relevant studies). This could prove to be an invaluable tool in the first step in many new transitions away from animal experimentation, while solidifying those transitions that already have taken place.

The proposed alternatives timeline is in the planning stages, and we welcome feedback from any potential users or other interested parties with regard to its content and its layout, in order to make the end result as informative as we possibly can.

Kevin Coll

FRAME
96–98 North Sherwood Street
Nottingham NG1 4EE
UK

E-mail: kevin@frame.org.uk

 

References

1 Rupke, N.A. (1987). Vivisection in Historical Perspective, p. 15. Kent, UK: Croom Helm Ltd.
2 Rupke, N.A. (1987). Vivisection in Historical Perspective, p. 2. Kent, UK: Croom Helm Ltd.
3 Anon. (2010). Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. Official Journal of the European Union L276, 20.10.2010, 33–79.
4 Anon. (2009). Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products (text with EEA relevance). Official Journal of the European Union L342, 22.12.2009, 59–209.
5 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, 238pp. London, UK: Methuen.

Animal Experimentation in the UK: Probing Beyond the Rhetoric

The amended Animals (Scientific Procedures) Act 1986 (ASPA) came into force on 1 January 2013, in compliance with the requirements of Directive 2010/63/EU on the protection of animals used for scientific purposes, amid a flood of rhetoric about the uniquely high standards of laboratory animal care and use in the UK.

A few weeks later, the British Union for the Abolition of Vivisection (BUAV) sent Imperial College London a dossier containing allegations about what were claimed to be a wide range of infringements of the ASPA at the College. The Home Office, which is the government department responsible for the administration of the ASPA, immediately began an enquiry, and Imperial College, to its great credit, set up its own enquiry, by an independent committee, under the chairmanship of Professor Steve Brown, Director of the Medical Research Council’s Mammalian Genetics Unit at Harwell.

The remit of the committee was “not to investigate the specific allegations made by the BUAV”, but “to undertake a broad and detailed examination of all aspects of animal experimentation at the College facilities”. The committee interviewed a wide range of personnel, obtained a large number of documents, and visited the facility that was the focus of the BUAV’s allegations, and concentrated its deliberations on four areas: a) the Animal Welfare and Ethical Review Body (AWERB) process; b) the operations of the Central Biomedical Services (CBS); c) training and competency assessment; and d)culture, leadership and management.

On 10 December 2013, the committee produced a severely critical 38-page report,1 in which it concluded that the College’s animal research facilities are understaffed, under-resourced, and operating without adequate systems for training, supervision, management and ethical review.

The AWERB process was considered to be “not fit for purpose” and in need of “wholesale reform”, and required “a new senior administrative appointment … to manage the process”. The committee found the facilities to be “well equipped”, with “a high quality of animal husbandry”, but there was room for considerable improvement “in terms of operational structures and standards, communication and working practices, as well as the mechanisms for reporting animal welfare concerns”, which would “have a substantive impact upon animal welfare and the Three Rs”. However, “the provision for training, supervision and competency assessment” was “ad hoc”, with “little evidence of effective mechanisms for sharing information and best practice”. They found “a strong emphasis on process and procedural issues to the detriment of focus on improvements in the Three Rs”, with “relatively limited opportunities for interactions that bring together and promote ideas and developments for the Three Rs between diverse groups”, and that, “overall, a culture of whole teams working together was lacking”.

The committee made 33 recommendations, and emphasised the need for “a clear restatement of the key role of the Named Veterinary Surgeon (NVS) and the Named Animal Care and Welfare Officer in animal welfare and the Three Rs, along with a clear route for escalation of concerns to the AWERB”. Again to its credit, the College “accepted all the recommendations”, admitted that there was “significant scope for improvement”, and said that it “will now move quickly to implement the recommendations”.2 The need for this investigation leads to two important questions. First, why was such a situation allowed to develop at Imperial College, one of the world’s most prestigious universities? Why were the Certificate Holder, the senior academics, the project and personal licence holders, the NVS, the Home Office Inspector and others, not performing their duties up to even the minimal standards required of them?

The second question, is that: if this situation could be allowed to develop at Imperial College, in what other institutions do similar problems exist and are similarly low standards considered acceptable? Professor Paul Flecknell, a member of the committee and director of the research animal facilities at Newcastle University, said that, while the report was specific to Imperial College, “every institution will pick up something we’ve said and think, ‘we should take more note of that’ ”.3 The Home Office, and the equivalent authorities in other countries, should insist that they do just that.

 

References

1 Anon. (2013). Independent Investigation into Animal Research at Imperial College London, 38pp. London, UK: Imperial College. Available at: http://brownreportinfo/wp-content/uploads/2013/12/Brown-Report-Final-EMBARGOED-0001GMT-10-12-132.pdf.

2 Jones, J-P. (2013). Imperial responds to animal research investigation report. London, UK: Imperial College. Available at: http://www3.imperial.ac.uk/newsandeventspggrp/imperialcollege/newssummary/news_9-12-2013-18-21-18.

3 Cressey, D. (2013). Report slams university’s animal research. Nature News, 10 December 2013. Available at: http://www.nature.com/news/report-slams-universitys-animal-research-1.14329.

Time for Reconsideration

Laboratory animals have long been used as surrogates for human beings, because it has been considered acceptable to expose them to conditions and procedures which would not be considered ethical, if applied to ourselves. Initially, the focus was mainly on gaining a better understanding of how the body’s cells, organs and systems function and are controlled, and how failures of one kind or another can lead to pathological conditions. Then, from about the middle of the last century, animals came to be used more and more in tests to determine the effects of exposure to chemicals and chemical products — in attempts to determine information of direct relevance concerning the efficacy of drugs and vaccines and/or the adverse effects of chemicals and various kinds of chemical products, in the hope of predicting likely effects in humans, as a basis for appropriate action.

However, attitudes toward the reliance on this approach are now changing, with the increasing recognition that the knowledge gained from animal studies cannot be expected to have direct relevance to humans, which has uncertain, and even dangerous, consequences. This problem is particularly acute for the pharmaceutical industry, which is in a state of crisis, because of the late withdrawal of new drugs as a result of lack of efficacy or unacceptable side-effects not detected during preclinical testing, despite the application of highly expensive and seemingly sophisticated testing in animals. There are two main reasons for these difficulties. Firstly, functions and controls in animals and humans tend to be very different in detail, however similar they may appear to be on the surface. Animals are highly adapted to their individual and specific lifestyles and environments.

During evolution, species separate and diverge from common ancestors, based on these adaptations, which tend not to involve the emergence of something totally new, but which rely instead on modifications of what was already there. This has profound implications for attempts to model human diseases in animals, especially since, in view of the absence of sufficient knowledge about what is being modelled, it is impossible to judge whether or not the model has any value. One response to this unsolvable problem of species differences is the attempt to humanise animals by transferring human genes into their genomes, in the hope of simulating effects and responses in humans. However, that can be considered naïve, since the manipulation of complex networks of interacting controls, which are not well understood and which will inevitably differ considerably in animals and humans, is likely to produce information which cannot be interpreted with confidence, and which may be dangerously misleading. Perhaps introducing human genes in animals will have an outcome analogous to the unforeseen and unwanted effects of introducing rabbits into Australia!

The second insuperable problem is that the ‘human’ being to be modelled in animals doesn’t actually exist. Human polymorphism leads to infinite variety, and there are many, many sub-populations within the overall human population, which will differ in their susceptibilities to disease and in their responses to chemicals and chemical products, including drugs. One resultof this is that a drug which is highly effective in one patient can be lethal to another. Similarly, a chemical which has no effect in some individuals, could result in a highly allergic response in others. The only way forward is to recognise that the modern, but still developing, techniques of cell biology and molecular biology, combined intelligently with the vast information storage and computational systems which are now available, should be applied directly to human material in vitro and ex vivo, and, with strict ethical controls, to human volunteers.

Carefully planned and executed, this approach could take account of human polymorphism, past or concurrent diseases or therapies, and the differential effects of age, occupation, geographical location, lifestyle and exposure to medicines and other chemicals. In the pharmaceutical industry, for example, the ‘one drug suits all’ concept has been overthrown, and therapies in the future will involve personalised medication, where the treatment will be designed specifically for the individual patient. A contribution to this development could be the Personal Genome Project, which began at Harvard University in 2005. The aim is to recruit people willing to freely donate their individual genome sequences and associated medical and other biologically-relevant data, for use for research purposes.

The data are to be placed within the public domain and made freely available to researchers, so that they can test their hypotheses about the relationships between genotype, phenotype and environment — as well as susceptibility to disease and responses to therapies. A related initiative was launched in the UK on 7 November 2013, with the hope of persuading 100,000 individuals to agree to take part. While this kind of initiative may appear to offer the solution to some problems, it also opens up new ones, including quality control of the data (which will come from diverse sources of uneven quality), protection of the privacy of the data donors, and management of the commercial exploitation of what is donated.

A Toolbox for Animal Welfare Education

A new online resource, aimed at qualified vets
and veterinary science students,
is being developed by WSPA

Ellen Coombs

The Tertiary Education Toolbox1 is the newest addition to the Animal Mosaic website,2 and is a new supporting component of the tertiary education programme currently being developed by the World Society for the Protection of Animals (WSPA).

WSPA’s approach to tertiary (i.e. post-compulsory) education is known as Advanced Concepts in Animal Welfare (ACAW). It involves working with veterinary colleges and animal science training institutions around the world, to ensure that animal welfare is included within the teaching curriculum and delivered effectively throughout their faculties. The Toolbox adds an extra dimension to WSPA’s newly launched 3rd edition of the Concepts in Animal Welfare (CAW) syllabus.3 Both components are designed to assist with the teaching of animal welfare to tertiary level audiences.

The Toolbox is a comprehensive collection of resources, designed to supplement and accompany the CAW modules, and consists of an online collection of high-quality animal welfare teaching tools. The online collection currently hosts materials from a large consortium of organisations, each providing resources that have been handpicked to fit into the respective Tools, Videos or Links sections. Contributors to the site include organisations, academic institutes and NGOs from around the world, such as the World Organisation for Animal Health (OIE), the British Veterinary Association (BVA), and the Federation of Veterinarians of Europe (FVE).

The environment is readily searchable and userfriendly, and can easily be accessed by cohorts and learners of varying abilities. Once users have decided which section they want to use, they can refine resources with great accuracy. The structure of this virtual resource mirrors a library or a catalogue, where users can refine by Subject Area, Category, Language and even Region. The Subject Area and Category options are numerous and varied, with the number of different language articles increasing rapidly. The 3rd edition of the CAW resource is already available in Chinese, and Spanish and Portuguese versions will become available soon.

Users are provided with a choice of subjects and categories to select from, with a multiple selection function available to allow visitors to quickly and accurately find what they are looking for. The Toolbox hosts a plethora of resources on a variety of subjects designed to aid and support qualified veterinarians, veterinary science lecturers and tertiary-level veterinary students. Users can search through a wide range of resources tagged to subjects such as Behaviour, Legislation, Codes and Standards, Professional Skills, Slaughter and Euthanasia, and many more. An extra refinement box allows users to search by animal category, e.g. Companion animals, Farm animals, etc. Examples of tools that are currently hosted on the Toolbox include:
— canine and feline Acute Pain Scales (Colorado State University);
— general guidelines for vets, such as The Vet
– verinarians Animal Welfare Toolkit (New Zealand Veterinary Association) and the FVE’s European Code of Conduct and Veterinary Act;
— links to open access articles in journals such as ATLA and BioMed Central;
— information for vets on a wide range of subjects, including legislation (e.g. the OIE’s Guidelines on
Veterinary Legislation) and how to recognise abuse in humans and in animals;
— a virtual fetal pig dissection (Whitman College); and
— emergency case simulators (Royal Veterinary College).

The resources are high-quality and wide-ranging, to support a variety of subjects and real-life scenarios that veterinary students and qualified veterinarians might encounter. Alongside the CAW resource, the Toolbox can provide a platform for Continuing Professional Development (CPD) for graduates and for practising vets.
Website landing page

One purpose of the Toolbox is to gather resources which encourage the use of alternatives to live animal use in educational contexts. Teaching students via non-harmful alternatives (computer simulators, high-quality videos, models, and surgical simulators) is not only beneficial from an animal welfare perspective, but it can also be beneficial from a learning perspective. Studies of veterinary students were reviewed, by comparing learning outcomes generated by non-harmful teaching methods with those achieved through harmful animal use.4 Of papers analysed that were assessing the efficacy of teaching methods, 45.5% demonstrated superior learning outcomes when more humane alternatives were used, 45.5% demonstrated equivalent learning outcomes compared to when less-humane (or even harmful) teaching methods were used, and only 9.1% demonstrated inferior learning outcomes.4
Toolbox toolsThere are also the added benefits of using humane teaching methods, such as cost and time savings for students and faculties, better repeatability of exercises, increased student confidence and satisfaction, experience of the successful combining of clinical skills with ethics early in the curriculum, and compliance with animal use legislation.5 The Toolbox hosts simulators, case studies, virtual dissections, and links to other sites featuring alternatives to animal experimentation (such as Find Your Dissection Alternative, from Animalearn). It is anticipated that it will host many more similar resources in the future. The website access comes with the added benefit of being open to all, as well as free to all, and it requires no sign-up or academic affiliation. The available information is constantly being updated, as guidelines for best practice develop. Animal Mosaic also hosts a useful community forum, where users can join discussions and topics of interest. The site has recently been launched in Spanish.6
Students and educators are becoming ever-more confident in seeking out and using web-based materials to supplement traditional learning methods. However, users are bombarded with such a huge selection of resources, which can make sorting high-quality resources from low-quality resources a time-consuming task. As an educator, it can seem daunting to incorporate animal welfare education (AWE) into lesson plans or lectures. As a student, it can seem overwhelming to find additional high-quality and reliable resources to strengthen studies outside the classroom. The Toolbox has performed some of the preliminary hard work, and has gathered all the best animal welfare education resources from a wide range of organisations and individuals and put them in one comprehensive resource library. These resources can be used independently or alongside our Concepts in Animal Welfare resource.

Ellen Coombs
WSPA
222 Grays Inn Road
London WC1X 8HB UK
E-mail: EllenCoombs@wspa-international.org

References
1 http://www.animalmosaic.org/education/tertiaryeducation/
toolbox/default.aspx (Accessed 16.10.13).
2 http://www.animalmosaic.org/ (Accessed 16.10.13).
3 http://www.animalmosaic.org/education/tertiaryeducation/
advanced-concepts-in-animal-welfare/
default.aspx (Accessed 16.10.13).
4 Knight, A. (2007). Humane teaching methods prove efficacious within veterinary and other biomedical education. AATEX 14, Special Issue, 213–220.
5 Knight, A. (2007). The effectiveness of humane teaching methods in veterinary education. ALTEX 24, 91–109.
6 http://www.mosaicoanimal.org/ (Accessed 16.10.13

The Current Status of Laboratory Animal Ethics in South Africa

An overview of the successes and current challenges
in implementing and raising awareness
of the Three Rs in South Africa

Bert Mohr

In South Africa, the humane care and use of nonhuman animals for scientific and teaching purposes is governed by the widely accepted ethical framework of the Three Rs — i.e. the Replacement of animals by non-animal models where possible, the Reduction of the number of animals used to the minimum required to yield valid scientific results, and the Refinement of scientific procedures and animal care standards in order to limit the potential for pain, suffering, distress or lasting harm, thus improving animal wellbeing. This article outlines the successes and current challenges in implementing and raising awareness of the Three Rs in South Africa. The South African National Standard The South African Medical Research Council first published guidelines on ethical considerations for the use of animals in research in 1979,2 and in subsequent revisions of this document in 1987,3 1993 4 and 2004,5 in order to sensitise biomedical scientists, research institutions, their Animal Ethics Committees (AECs) and animal care staff, to the interests and welfare of research animals.

The first national code for the use of animals for scientific and teaching purposes was produced in 1990 by the Department of Agriculture.6 This code was superseded in 2008 by the current South African National Standard (SANS) for the care and use of animals for scientific purposes (SANS 10386:2008).7 Produced by the South African Bureau of Standards, the SANS is primarily based on the Australian code of practice for the care and use of animals for scientific purposes (1997),8 and on the European convention for the protection of vertebrate animals used for experimental and other scientific purposes (1986).9

The purpose of the SANS is to ensure the ethical and humane care and use of all animals involved in scientific or teaching activities in South Africa, encompassing all aspects of the care and use of animals in medicine, biology, agriculture, industry, veterinary, wildlife, and other animal sciences, including animal use in research, teaching, field trials, product testing, diagnosis, the production of biological substances, and environmental studies.7 The SANS covers all live non-human vertebrates and higher invertebrates (e.g. advanced cephalopods and decapods), including their embryonated eggs, and fetuses, where an integrated nervous system is evident.7 The aims of the SANS are: to establish uniform minimum national standards for animal care and use, based on international standards; to emphasise the responsibilities of researchers, teachers and institutions that use animals; to ensure that the use of animals is always appropriately justified by the formal review of all scientific and teaching protocols by institutional AECs; to ensure that animal welfare is appropriately considered; to ensure adherence to the core ethical principles of the Three Rs; and to promote the development of Three Rs methodology.7

Though not a legal prerequisite, the majority of South African research institutions have adopted the SANS. The responsibility to ensure compliance with the SANS rests with institutional AECs, whose members are required to regularly inspect all animal holding areas and laboratories, to ensure compliance.7 AECs are also responsible for ensuring that scientific studies and teaching activities are audited in relation to compliance with the submitted protocol.7

The establishment of the SANS has had a major impact on elevating the standards of animal care and use in South Africa. Major advances in the fields of animal science and welfare in recent years are reflected in the Australian code for the care and use of animals for scientific purposes (2013),10 and in the European directive on the protection of animals used for scientific purposes (2010).11 As these represent the international standards on which the SANS is based, it is likely that the SANS will soon be updated, in order to continue to meet international best practice recommendations.

Institutional Animal Ethics Committees
Formal ethical review processes for the use of animals in research and teaching have been broadly implemented in South African universities and research institutions since the mid-1970s, as a result of the appointment of institutional AECs.5 AECs in South Africa are decentralised, i.e. each institution’s AEC operates according to its own policies. However, institutional AECs are required to establish formal terms of reference and standard operating procedures, in order to ensure that all animal care and use within their institution is conducted in compliance with the SANS, incorporates the principles of the Three Rs, and provides adequate welfare and proper justification for the use of the animals.7

The membership of AECs is defined by the SANS, with the requirement that each AEC should comprise at least four categories of members — i.e. veterinarians (or in special cases, persons with comparable expertise), persons with substantial recent experience in the use of animals in scientific studies or teaching, representatives from independent animal welfare organisations, and independent persons who have not conducted scientific studies or teaching that involved the use of animals.7 The balance between categories must be maintained. Staff members responsible for the breeding and care of animals are also required to attend AEC meetings. Additional members (e.g. bioethicists, statisticians, animal behaviour scientists, etc.) may also be appointed. Written proposals for scientific studies and teaching activities may be considered and approved only at quorate AEC meetings.7

Usually, the independent animal welfare representatives on South African AECs include members of the Animal Ethics Unit of the National Council of Societies for the Prevention of Cruelty to Animals (NSPCA). The NSPCA’s Animal Ethics Unit, established in 2001, actively promotes the Three Rs in research and teaching, by: the involvement of their members in the review of research and teaching protocols by institutional AECs; performing routine inspections of animal facilities nationally; and identifying areas for improvement in legislation and national standards that govern animal research.12

All AECs that evaluate protocols where the research could impact on human health, must be registered with the National Health Research Ethics Council (NHREC).13 The functions of the NHREC include: setting norms and standards for conducting research on humans and animals; determining guidelines for the functioning of health research ethics committees; registering and auditing health research ethics committees; adjudicating on complaints about the functioning of health research ethics committees; instituting disciplinary action against persons who are in violation of any norms, standards, or guidelines for the conduct of research in terms of the National Health Act;13 and advising on ethical issues concerning research in South Africa.

Legislation
While there are currently no specific laws that regulate the care and use of animals for scientific or teaching purposes in South Africa, the SANS,7 though itself not a law, must be read in conjunction with South African legislation that pertains to animals,14-23including the Animals Protection Act.18 Of special relevance to institutions that use animals for research and teaching are the Veterinary and Para-Veterinary Professions Act,15 and the Rules relating to the practising of the para-veterinary professions of laboratory animal technologists20 and veterinary nurses,23 which specify the professional services associated with these and other para-veterinary professions. All persons who are not registered with the South African Veterinary Council (SAVC) as veterinary or para-veterinary professionals, but who perform some of the services of these professions, e.g. general anaesthesia, clinical procedures or surgery on animals, are required to obtain authorisation from the SAVC in order to be permitted to do so. In such cases, a veterinarian is required to verify the competence of these persons, who, in turn, are required to work under the direct or indirect supervision of a veterinarian. The Chair of the AEC also needs to confirm that all personnel are competent. Institutions are responsible for ensuring, through their AECs, that their use of animals complies with these legal requirements.7 The Medicines and Related Sub stances Control Act19 governs the use of scheduled substances and drugs in research and teaching that involves animals.

The National Health Act 13 prescribes that all animal research that could impact on human health, requires ethical approval from a research ethics committee in South Africa that is registered with the NHREC. This Act defines ‘health research’ to include any research which contributes to the knowledge of the biological, clinical, psychological or social processes in humans; improved methods for the provision of health services; human pathology; the causes of disease; the effects of the environment on the human body; the development or new application of pharmaceuticals, medicines and related substances; or the development of new applications of health technology.13 In contrast to the stipulations of the SANS,7 there is currently no legal requirement to obtain formal ethical approval for scientific activities or teaching that involve animals in South Africa, unless the research could impact on human health.13 The NSPCA was founded in 1955, to provide a forum to bring uniformity to welfare legislation and standards in South Africa.12 The NSPCA administers the Societies for the Prevention of Cruelty to Animals Act,21 with its inspectors authorised in terms of the Animals Protection Act,18 among others.22 The NSPCA Animal Ethics Unit identifies areas for improvement in the laws and standards that govern animal research.12

Teaching and training
In order to ensure the optimal implementation of the Three Rs, adequate training is required for all perentific and teaching purposes. These include persons who perform clinical or experimental procedures, breed and care for animals; scientists and teachers who use animals; AEC members; and those who set policy.
Laboratory Animal Technologists (LATs) are the paraveterinary professionals specifically trained in the theoretical and practical aspects of the humane care and use of animals for scientific purposes in South Africa.20 National training courses for LATs were initiated in 1973, resulting in a three-year correspondence diploma course in laboratory animal technology, presented by the Technikon RSA from 1981 onwards, including practical training modules at accredited research animal facilities. Reduced government subsidy for animal facilities in the early 1990s led to a decreased demand for qualified personnel, with the subsequent discontinuation of the LAT diploma course in 1997.24 While there are currently only 20 registered LATs in South Africa, the field of animal science is constantly expanding. The re-establishment of a national training course for LATs is being considered, in order to address this deficiency in trained personnel.

The South African Association for Laboratory Animal Science (SAALAS),24 established in 1978, actively promotes education and training in laboratory animal science in South Africa. Links with the UK’s Institute of Animal Technology (IAT) enabled many South Africans who perform the services of LATs, to enrol on the international IAT correspondence course. Following completion of the IAT course, practical training in required techniques enables such persons to be registered with, or authorised by, the SAVC to perform the services of a LAT. SAALAS aims to establish accredited national facilities for practical training, in order to promote recognised standards of proficiency for all laboratory animal personnel, including continuing education courses for LATs. The recent establishment of a veterinary Master’s degree in laboratory animal science at the Faculty of Veterinary Science, University of Pretoria, presents a major advance in the field of laboratory animal science in South Africa.

The training of researchers who engage in animal research is conducted at the institutional level, with several institutions offering in-house training in animal ethics and care, such as the Introductory Course in Laboratory Animal Science at the University of Cape Town. The competency-based practical training of researchers in clinical and experimental techniques is similarly conducted at the institutional level, in order to meet legislative requirements for animal research.7,15,20,23 SAALAS aims to establish accredited national courses for the training of researchers, in order to promote recognised standards of competency in clinical and experimental techniques. Training for South African AEC members is currently being planned by the Southern African Research and Innovation Management Association (SARIMA), an association for research and innovation managers in higher education, science councils, private research institutions, and industry, thus aiding the implementation and raising awareness of the Three Rs in South Africa. Courses aimed at increasing awareness of the Three Rs among researchers and AEC members are also presented at the national level by the NSPCA, e.g. the 2012 series of workshops on the use of non-animal models to replace the use of live animals in some research and teaching activities.

The national perspective
While it is estimated that at least 100,000 non-human animals are used annually for scientific and teaching purposes in South Africa, no formal reporting requirement or mechanism for compiling national statistics currently exists. It appears likely that the NSPCA’s Animal Ethics Unit, whose members serve on the majority of South African AECs, could in future provide an avenue for collecting and collating such statistics, in order to objectively monitor national animal usage trends. Though there is currently no systematic audit of institutional AECs in South Africa, the NHREC is in the process of registering and auditing all research ethics committees that evaluate protocols where the research could impact on human health,13 thus contributing toward standardising the nature and quality of animal ethical review processes in South Africa.

Auditing mechanisms for the remaining AECs, i.e. those that do not evaluate protocols that pertain to human health, remain to be developed, in order to ensure compliance with the SANS7 and applicable legislation. Due to its physical separation from developed countries and their closely-regulated standards for animal care and use, many South African institutions depend significantly on accessing international best practice recommendations for implementing the Three Rs, such as the information available on the websites of the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs); the Federation of European Laboratory Animal Science Associations (FELASA); Digital Resources for Veterinary Trainers; the IAT; the Fund for the Replacement of Animals in Medical Experiments (FRAME); and Understanding Animal Research (UAR).

Conclusion
South Africa serves as an established reference point for the biomedical sciences in the African continent.25 The highest ethical standards are thus paramount, in order to ensure the optimal implementation of the Three Rs in scientific activities and teaching involving animals across Africa. Although South African laws and standards should be subject to further development, they are unambiguous in terms of setting the required norms for scientific activities and teaching involving animals, based on internationally-accepted moral values. The primary challenge facing animal research in South Africa is confirming compliance with these laws and standards. The training of AEC members, biomedical scientists, animal care staff and policy-makers will help to address this challenge. It is contemplated that SAALAS will play an active role in the training and continuing education of laboratory animal science personnel.

There are currently no specific funding resources available for promoting research into the Three Rs in South Africa. The establishment of a South African equivalent to the UK’s NC3Rs would present a major step toward improving the implementation of the Three Rs on the continent. Appropriate subsidy of research animal facilities remains critical, to maintain appropriate standards.

Dr Bert Mohr
Research Animal Facility; and the
Hatter Institute for Cardiovascular Research in
Africa, Department of Medicine
Faculty of Health Sciences
University of Cape Town
Private Bag X3
Observatory 7935
South Africa
E-mail: bert.mohr@uct.ac.za

References and notes
1 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, 238pp. London, UK: Methuen.
2 South African MRC (1979). Guide to Ethical Considerations in Medical Research. Cape Town, South Africa: South African Medical Research Council.
3 South African MRC (1987). Ethical Considerations in Medical Research. Cape Town, South Africa: South African Medical Research Council.
4 South African MRC (1993). Guidelines on Ethics for Medical Research. Cape Town, South Africa: South African Medical Research Council.
5 South African MRC (2004). Guidelines on Ethics for Medical Research: Use of Animals in Research and Training, 62pp. Cape Town, South Africa: South African Medical Research Council.
6 South African DAFF (1990). National Code for Animal Use in Research, Education, Diagnosis and Testing of Drugs and Related Substances, 3pp. Pretoria, South Africa: Department of Agriculture, Forestries & Fisheries, Republic of South Africa.
7 SABS (2008). South African National Standard: The Care and Use of Animals for Scientific Purposes, 1st edn (SANS 10386:2008), 232pp. Pretoria, South Africa: South African Bureau of Standards, Standards Division.
8 National Health and Medical Research Council (1997). Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, 6th edition 1997, 83pp. Canberra, Australia: National Health and Medical Research Council.
9 Council of Europe (1986). European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (ETS 123). Available at: http://conventions.coe.int/Treaty/en/ Treaties/Html/123.htm (Accessed 16.09.13). Strasbourg, France: Council of Europe.
10 National Health and Medical Research Council (2013). Australian Code for the Care and Use of Animals for Scientific Purposes, 8th edition 2013, 90pp. Canberra, Australia: National Health and Medical Research Council.
11 Anon. (2010). Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes. Official Journal of the European Union L276, 20.10.2010, 33–79.
12 National Council of Societies for the Prevention of Cruelty to Animals website: http://www.nspca.co.za/ (Accessed 16.09.13).
13 National Health Act. Act No. 61 of 2003.
14 Animal Health Act. Act No. 7 of 2002.
15 Veterinary and Para-Veterinary Professions Act. Act No. 19 of 1982.
16 Animal Diseases Act. Act No. 35 of 1984.
17 Fertilizers Farm Feeds Agricultural Remedies and Stock Remedies Act. Act No. 36 of 1947.
18 Animals Protection Act. Act No. 71 of 1962.
19 Medicines and Related Substances Control Act. Act No. 101 of 1965.
20 South African DAFF (1997). GN 1445 of 3 October 1997: Rules Relating to the Practising of the ParaVeterinary Profession of Laboratory Animal Technologist, 4pp. Pretoria, South Africa: Department of Agriculture, Forestries & Fisheries, Republic of South Africa.
21 Societies for the Prevention of Cruelty to Animals Act. Act No. 169 of 1993.
22 Performing Animals Protection Act. Act No. 24 of 1935.
23 South African DAFF (1991). GNR. 1065 of 17 May 1991: Rules Relating to the Practising of the Profession of Veterinary Nurse, 2pp. Pretoria, South Africa: Department of Agriculture, Forestries & Fisheries, Republic of South Africa.
24 South African Association for Laboratory Animal Science website: http://www.saalas.org/ (Accessed 16.09.13).
25 Anon. (2013). Times Higher Education World University Rankings 2012–2013. Available at: http://www.timeshighereducation.co.uk/world-universityrankings/ (Accessed 16.09.13). P51