The 3 R’s

Replacement. Reduction. Refinement. Also known as the 3 R’s.

On the face of it, the 3 R’s form a fairly straightforward guide to limit the amount of suffering endured by animals in your experiments. However, these are stepping stones to quite indepth process for advancing technologies and rigorous planning, as defined by the following on the NC3R’s website1:

  • Replacement – Accelerating the development and use of models and tools, based on the latest science and technologies, to address important scientific questions without the use of animals
  • Reduction – Appropriately designed and analysed animal experiments that are robust and reproducible, and truly add to the knowledge base
  • Refinement – Advancing animal welfare by exploiting the latest in vivo technologies and by improving understanding of the impact of welfare on scientific outcomes

These are major points that I think about frequently when planning and performing animal experiments.

Before you plan an experiment

Replacement would seem quite straightforward for someone who works on the mouse neural system, in that it’s not something in my control, so not to worry about it. And while it is true that I rarely have intentions to work in non-animals systems, that doesn’t mean it’s irrelevant.

Really, this needs addressing at the most fundamental level, before I even plan an experiment, ie. is the scientific question I want to answer relevant to a whole-animal neural system. Does it require the use of an animal to answer this question?

For example, I have often used relatively unknown neuropeptide agonists in my work; if I wanted to know more about the intracellular signalling mechanisms these agonists use, it would be both unethical and a waste of time, money and animals to test this on live brain slices (which I use for patch clamping). Instead, one would use a cultured cell line, such as HeLa cells.

Robust and reproducible

Reduction is an interesting one. It’s easy to think, well I’ll just use fewer animals in my experiment. However, this misses the key point of “robust and reproducible” experiments. What if you used fewer animals and didn’t see an effect? Is that because there is no biological effect of your treatment, or is it because you didn’t have enough animals in your study to show a statistical effect? This is where power analyses come in to play: they help you plan a robust study without using an unnecessary number of animals.

It is also important to think of optimising your study design to produce the most statistical power (eg. using crossover studies and repeated measures ANOVA) and to negate the need of repeating studies in the future. Even during an experiment, I am conscious of this metric, because I am always trying to reduce the variability in a study (for example, by reducing animal stress) in order to improve the power and numbers needed for future studies.

Improve data quality and impact

Refinement is really where it’s at for me. I spend a lot of time optimising techniques and, more recently, developing technology, to improve experimental conditions. The thing is, when your mice are unhappy and stressed, they will not behave naturally and your data will be more variable. So it makes sense, from both a pragmatic point of view and for animal welfare, to refine your experiments as best you can.

Refinement can include anything from your study design, acclimatisation of the animals and their housing conditions to advances in technology allowing better data to be collected.

Technology and the 3 R’s

Ever since my PhD, I have been interested in the use of technology to produce better data, and improve animal welfare along the way. I was particularly keen on the use of telemetry to obtain high quality physiology data while minimising the stressful environment. Since then, I’ve been interested in using AAV’s to target neurone populations of interest, and then more advanced technologies including optogenetics and fibre photometry.

In addition to improving the animal welfare in a single experiment, these more advanced technologies can provide more impactful data with deeper insights, which means fewer studies need to be performed to provide a clear picture of the biology in question.

I have also become very interested in developing in vivo technologies myself to improve on aspects that I know impact negatively on animal welfare, for example trying to perform fibre-free optogenetics to limit a lot of the negative aspects of those experiments (such as the need to have head-tethered animals in open cages during experimentation).

A 3 R’s framework

The 3 R’s provide an excellent framework with which to approach animal research in a way that aims to be as ethical as possible. And in fact, I would argue that we are morally bound to consider such questions whenever we intend to perform experiments on animals.


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