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Reproducibility of animal research in light of biological variation

An Author Correction to this article was published on 08 June 2020

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Abstract

Context-dependent biological variation presents a unique challenge to the reproducibility of results in experimental animal research, because organisms’ responses to experimental treatments can vary with both genotype and environmental conditions. In March 2019, experts in animal biology, experimental design and statistics convened in Blonay, Switzerland, to discuss strategies addressing this challenge. In contrast to the current gold standard of rigorous standardization in experimental animal research, we recommend the use of systematic heterogenization of study samples and conditions by actively incorporating biological variation into study design through diversifying study samples and conditions. Here we provide the scientific rationale for this approach in the hope that researchers, regulators, funders and editors can embrace this paradigm shift. We also present a road map towards better practices in view of improving the reproducibility of animal research.

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Fig. 1: Context-dependent treatment effect.

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Acknowledgements

The Swiss National Science Foundation (IZSEZ0_184010) provided funding to B.V. for the workshop ‘Variation in in vivo experiments: the norm of reaction and reproducibility’. B.V., H.W. and M.J.K. were funded by the European Union Horizon 2020 research and innovation programme and the European Federation of Pharmaceutical Industries and Associations (Innovative Medicines Initiative, IMI2, grant agreement no. 777364, European Quality in Preclinical Data). A.F. thanks Linnaeus University for funding. H.S. was supported by the German Research Foundation (INST 215/543-1, 396782608). I.J. was funded by the Swiss National Science Foundation (310030_179254).

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Glossary

Biological variation

The variation of phenotypes in a population of organisms. It is the result of genetic variation, environmental influences on the organism and gene × environment interactions.

Confirmatory studies

Studies designed to test specific hypotheses about the existence of a relationship or effect, its direction and magnitude using inferential statistical methods. The hypotheses are based on previous knowledge of the study system.

Exploratory studies

Studies designed to probe for relationships or treatment effects of novel interventions without specific hypotheses about the direction and size of effects. The outcome of an exploratory study is a descriptive account of the observed effects.

External validity

The extent to which findings can be generalized to the desired inference space of animals (including humans) and/or other environmental conditions.

Gene × environment interactions

These subsume the non-additive joint effect of genetic and environmental influences on the development of the phenotype. As a consequence, environmental influences can have different effects on the phenotype depending on the organism’s genotype or genes can have differential effects depending on features of the environment.

Genotypes

Organisms’ hereditary information as encoded in the genome.

Heterogenization

The deliberate augmentation of systematic or random biological variation in the study population.

Inference space

The range of organisms and environmental contexts for which the inference of an experiment is valid.

Internal validity

Refers to whether the effects observed in a study are due to manipulation of the independent variables and not some other, unknown factors.

Norm of reaction

A property of a genotype describing how an environmental factor affects the development of the phenotype. It can be conceptualized as a function mapping expected phenotypic trait values onto environmental parameter values.

Phenotype

The sum of an organism’s observable characteristics or traits, including its morphological, biochemical and physiological processes, behaviour and responses to external stimulation and treatments.

Phenotypic plasticity

The extent to which an organism changes its phenotype in response to environmental influences.

Random noise

Also known as measurement error, refers to unexplained variability in the data. It affects the variation but not the size of an experimental treatment effect.

Reproducibility

The ability to produce similar results by an independent replicate experiment using the same methodology in the same or a different laboratory.

Robustness

The ability of an organism to maintain a functioning phenotype under varying environmental conditions. It also refers to the stability of a response to an experimental treatment in the face of variation in environmental conditions.

3Rs principles

The guiding principles for a responsible approach to experimental animal research. They imply that a study involving the use of animals should be conducted only if the intended outcome cannot be achieved by use of no or non-sentient animals (replace), fewer animals (reduce) or procedures that are less harmful or improve animal well-being (refine).

Scientific rigour

As defined by the US National Institutes of Health, this means “the strict application of the scientific method to ensure robust and unbiased experimental design, methodology, analysis, interpretation and reporting of results. This includes full transparency in reporting experimental details so that others may reproduce and extend the findings”.

Standardization

The practice of minimizing both technical and biological variation in the study outcomes by identifying and controlling sources of variation that are believed to be putative confounders. Standardization can aim at aspects of the environment in which a study is conducted (environmental standardization), aspects of the study subjects (phenotype standardization) or aspects of how procedures and interventions are conducted and how measurements are taken (operational standardization).

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Voelkl, B., Altman, N.S., Forsman, A. et al. Reproducibility of animal research in light of biological variation. Nat Rev Neurosci 21, 384–393 (2020). https://doi.org/10.1038/s41583-020-0313-3

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