An essential part of the design of any experimental method involves consideration of the "3 R's", first described in The Principles of Humane Experimental Technique by Russell and Burch (1959).
The 3 R’s are:
Reduction – Minimize the number of animals used to smallest number needed to obtain statistically relevant results.
Refinement – Use techniques that reduce the incidence or severity of pain and/or distress in animals.
Replacement – Substitution of non-animal methods or material or a lower species that may be less sensitive to pain and distress.