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  • Writer's pictureDirk

3 R's - Replacement, Reduction and Refinement

Updated: Dec 14, 2019

​Early in science there was no questioning, if killing (or sacrificing) individuals was necessary for achieving any kind of scientific purpose, it was okay. You will find millions of sacrificed individuals in museums, dark cellars of universities, and freezers around the world from that time. A colleague of mine, when genetics was about allozymes, killed more than 10.000 butterflies and i myself thought it necessary to sacrifice hundreds of individuals for my own research. I had remorse at that time, but working with allozymes asked for fresh tissue in high quantities so that enzymes would be active and plenty enough to be seen on cellulose acetate plates. The misuse of specimens by science brought (with all rights) animal welfarers to push forward what is know as the 3Rs (Replacement, Reduction and Refinement), first defined by the scientists William Russell and Rex Burch in 'The Principles of Humane Experimental Technique' (1959). In 2010 (50 years after), the Directive 2010/63/EU on the protection of animals used for scientific purposes includes an explicit reference to the 3Rs principle. And also the first consideration of animal welfare took 27 years to find its entry to EU legislation.

What are the 3Rs?


Replacement can be defined as methods, strategies or approaches which do not involve the use of live animals.

Replacement may be achieved through a number of tools or their combinations including

  • in vitro systems using tissues, whole cells or parts of cells

  • systems based on biochemical approaches, i.e. using synthetic (macro)molecules as proxies of (reactive) toxicity targets. Such methods are referred to as "in chimico"

  • computer-based models and approaches – often termed in silico

  • use of 'omics' technologies (e.g. transcriptomics, proteomics and metabonomics)

  • non-testing approaches such as 'read-across' technique


The concept of reduction covers any approach that will result in fewer animals being used to achieve the same objective, including maximising the information obtained per animal, reducing the number of animals used in the original procedure and/or limiting or avoiding the subsequent use of additional animals.

The number of animals can also be reduced by performing procedures on animals more than once, where this does not detract from the scientific objective or result in poor animal welfare. However, the benefit of reusing animals should always be balanced against any adverse effects on their welfare, taking into account the lifetime experience of the individual animal. As a result of this potential conflict, the reuse of animals should be considered on a case-by-case basis.


Today, the term refinement signifies the modification of any procedures or husbandry and care practices from the time the experimental animal is born until its death, so as to minimise the pain, suffering and distress experienced by the animal and enhance its well-being.

When an animal experiences pain, suffering or distress, there are often accompanying physiological changes which may increase the variability of scientific results. Refinement therefore is also likely to improve data quality and contribute to Reduction.

It is not always easy to develop an appropriate protocol for the 3Rs and it takes time. We have now published a methods paper on how to isolate fungal strains from minimal invasive amphibian tissue to respect the 3Rs.

From the paper

Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi

Fisher M.C., Ghosh P., Shelton J.M.G., Bates K., Brookes L., Wierzbicki C., Rosa G.M., Farrer R.A., Aanensen D.M., Alvarado-Rybak M., Bataille A., Berger L., Böll S., Bosch J., Clare F.C., A. Courtois E., Crottini A., Cunningham A.A., Doherty-Bone T.M., Gebresenbet F., Gower D.J., Höglund J., James T.Y., Jenkinson T.S., Kosch T.A., Lambertini C., Laurila A., Lin C.-F., Loyau A., Martel A., Meurling S., Miaud C., Minting P., Ndriantsoa S., O’Hanlon S.J., Pasmans F., Rakotonanahary T., Rabemananjara F.C.E., Ribeiro L.P., Schmeller D.S., Schmidt B.R., Skerratt L., Smith F., Soto-Azat C., Tessa G., Toledo L.F., Valenzuela-Sánchez A., Verster R., Vörös J., Waldman B., Webb R.J., Weldon C., Wombwell E., Zamudio K.R., Longcore J.E., Garner T.W.J. (2018) . Scientific Reports 8, 7772.

The ability to isolate and culture both Bd and Bsal has played a key role in their discovery and by catalysing research into their pathogenesis and virulence15,16,17, phenotypic characteristics18,19,20 and a wealth of experimental studies on epidemiologically relevant parameters21,22,23. Longcore et al.24 first isolated Bd from infected amphibians by modifying techniques used to isolate other chytrids25. Longcore cleaned small (<0.5 mm dia) pieces of Bd-infected leg and foot skin by wiping them through agar and then placed skin pieces onto a clean plate of nutrient agar containing penicillin G and streptomycin. This method worked well for isolating from dead animals sent by courier from North and Central America. The method, however, requires euthanizing potentially healthy animals if their infection status was unknown. Further, it is difficult to perform these techniques in remote regions that lack suitable laboratory facilities, and the lethal sampling of amphibians may be contraindicated if the species is endangered, protected or located in protected areas.

We confronted this issue in a 2008–2014 project funded by BiodivERsA ( – RACE: Risk Assessment of Chytridiomycosis to European amphibian biodiversity26. One of the objectives of this project was to adjust the original protocol of Longcore et al.24 to (i) reduce the need to kill adult amphibians, (ii) improve rates of chytrid isolation by allowing the use of more animals, (iii) develop protocols that enabled isolation in a field setting, and, (iv) integrate the data into the GPS-smartphone enabled epidemiological software application Epicollect27,28.

#Chytrid #Amphibians #Experiments #Isolationpathogens

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