The 3R principle

The 3R principle, i.e., Replacement, Refinement and Reduction is the model that all scientists are required to refer to when designing and carrying out studies on an animal. From a legal point of view, the 3R principle is at the basis of European legislation dedicated to the protection of animals used in scientific experiments.


Whenever possible, researchers endeavour to replace animal models with alternative methods in their experiments.

Alternative methods to be preferred include computational models, cellular models (also called in vitro models), organoids, engineered tissues and studies on human volunteers. At the University of Ferrara, most research groups deal with in vitro and ex vivo research.


When their use is necessary, animals must be reared and housed in controlled conditions that ensure their physical and mental well-being.

Each stage of research involving animals is designed to safeguard animal welfare as far as possible, to minimise the use of invasive or painful procedures, and anaesthetics and analgesics are used wherever possible.

An important principle that researchers are obliged to follow concerns the choice of animal species to be used in research. In fact, the law prescribes that if the results are the same, the species with the least encephalisation, the least neurological development, should be chosen. Thus, when it comes to studying the basic mechanisms of tumour development, studying small rodents will often be sufficient, whereas to understand how the brain controls the hand, it becomes essential to use animals that use their fingers independently, such as lower primates. Sometimes the scientific question behind the research concerns social interaction disorders such as autism. In such cases, an animal species with the capacity for social interaction should be used. Rodents can often be used successfully and there are also strains of animals genetically 'sick' of syndromes similar to human autism.

In conclusion, at the basis of the choice of animal species there is always the search for strategies to reduce animal discomfort as much as possible through refinement, also because stress and discomfort can negatively influence the results of scientific research. It is therefore in the interest of all researchers to apply the principle of refinement.


Researchers must design experiments in such a way that they answer the scientific questions posed while involving as few animals as possible. This is done, for example, by making good use of statistics and collecting as much information as possible from each individual animal.

An example of the application of the principle of reduction that concerns the University of Ferrara is the decision to reduce the number of animals kept on farms. At the European Mouse Mutant Archive (EMMA) in Rome, a number of mouse embryos have recently been preserved, which will remain healthy and viable for several decades after freezing and will only be thawed when needed. This strategy avoids the continual breeding of mouse colonies not needed for experimentation. 

Another example of the principle of reduction, applied at national level, is the possibility of obtaining frozen tissues made available by other research groups by consulting a special ministerial database, thus avoiding sacrificing additional animals.