The design and statistical analysis of animal experiments.

Abstract

Research scientists are strongly urged to read all of the papers in this issue if they want to improve the quality and quantity of their research and ensure that their papers are accepted in high-quality journals. There is always room for improvement; some experiments are performed quite badly (Festing 1994, 2000; Festing et al. 2002; Roberts et al. 2002). The main problem seems to be poor training in statistics and a general horror among some biologists of anything to do with mathematics. However, experience shows that most scientists have a keen interest in experimental design and are not nearly as frightened of statistical methods that are planned at the same time as their experiments. For this reason, I believe that courses for research workers should emphasize design aspects. After all, if an experiment is well designed, it is relatively easy to get help with the statistical analysis; but if it is incorrectly designed, it may be impossible to extract any useful information from it. Animal experiments are usually done to discover something about the biology of, for example, the species, strain, or sex being studied and indirectly to infer something about humans or other target species. Thus, laboratory animals are usually used as “models” of some other species. The use of models involves the following three distinct steps: (1) choice of a suitable model, based on our current knowledge about disease processes in the target species and in potential model organisms; (2) one or more experiments to indicate how the model responds to any applied treatments; and (3) consideration of the relevance of the results for the target species. This issue of ILAR Journal is concerned with the second step, the design and analysis of experiments using animals that have already been chosen as likely to be informative indirectly about the target species. Anyone planning an animal experiment should first consider the important ethical issues involved. A good framework for this consideration is to use Russell and Burch’s “3Rs” (Russell and Burch 1959), namely, whether the animal model can be replaced by a less or nonsentient alternative such as an insect, nematode, cell culture, or computer simulation. If not, the possibility of refining the experiment in an effort to minimize pain and distress for each individual should be considered. Animals should be housed in good conditions, free of pathogens. Surgical techniques should use appropriate anesthesia and analgesia, and humane endpoints should be used. Finally, the number of animals should be reduced to the minimum required to achieve the scientific objectives of the experiment. Reduction can be achieved by choosing the most appropriate animal model (Herman 2002), allowing for the fact that it is not always necessary for the models to mimic the human condition exactly (Elsea and Lucas 2002), and by good experimental design and statistical methods, as outlined in this issue. All animal models are subject to biological variation as a result of genetic and nongenetic variation and the interaction between them. Even genetically identical littermates will vary to some extent as a result of chance developmental effects, social hierarchy, and unequal exposure to environmental influences. Good experimental design aims to control this variation so that it does not obscure any treatment effect, with the statistical analysis being designed to extract all useful information and take into account any remaining variation. A first step is to review likely sources of variation (Howard 2002). Species, strains, sexes, and individuals differ as a result of genetics; however, there are also many environmental influences that can either bias the results in some way or can contribute so much “noise” that it is impossible to identify the effects of any experimental treatments. Some of this variation is even introduced by the experimenter in the form of measurement error. One of the most serious causes of interindividual variation is clinical or subclinical disease, but fortunately (at least in the case of rats and mice), so-called “specific pathogen-free” animals have been available for many years. This availability does not mean that the problem has disappeared. Barriers break down due to new and existing pathogens somehow gaining entry, particularly when investigators exchange strains of animals; however, at least this source of variation is well Michael F. W. Festing, M.Sc., Ph.D., D.Sc., CStat., BIBiol., is a Senior Research Scientist at the MRC Toxicology Unit, University of Leicester, UK.