Author Correction: Reproducibility of animal research in light of biological variation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Reproducibility of animal research in light of biological variation.

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.

Correction: Do multiple experimenters improve the reproducibility of animal studies?

[This corrects the article DOI: 10.1371/journal.pbio.3001564.].

Do multiple experimenters improve the reproducibility of animal studies?

The credibility of scientific research has been seriously questioned by the widely claimed "reproducibility crisis". In light of this crisis, there is a growing awareness that the rigorous standardisation of experimental conditions may contribute to poor reproducibility of animal studies. Instead, systematic heterogenisation has been proposed as a tool to enhance reproducibility, but a real-life test across multiple independent laboratories is still pending. The aim of this study was therefore to test whether heterogenisation of experimental conditions by using multiple experimenters improves the reproducibility of research findings compared to standardised conditions with only one experimenter. To this end, we replicated the same animal experiment in 3 independent laboratories, each employing both a heterogenised and a standardised design. Whereas in the standardised design, all animals were tested by a single experimenter; in the heterogenised design, 3 different experimenters were involved in testing the animals. In contrast to our expectation, the inclusion of multiple experimenters in the heterogenised design did not improve the reproducibility of the results across the 3 laboratories. Interestingly, however, a variance component analysis indicated that the variation introduced by the different experimenters was not as high as the variation introduced by the laboratories, probably explaining why this heterogenisation strategy did not bring the anticipated success. Even more interestingly, for the majority of outcome measures, the remaining residual variation was identified as an important source of variance accounting for 41% (CI95 [34%, 49%]) to 72% (CI95 [58%, 88%]) of the observed total variance. Despite some uncertainty surrounding the estimated numbers, these findings argue for systematically including biological variation rather than eliminating it in animal studies and call for future research on effective improvement strategies.

The rearing environment persistently modulates mouse phenotypes from the molecular to the behavioural level.

The phenotype of an organism results from its genotype and the influence of the environment throughout development. Even when using animals of the same genotype, independent studies may test animals of different phenotypes, resulting in poor replicability due to genotype-by-environment interactions. Thus, genetically defined strains of mice may respond differently to experimental treatments depending on their rearing environment. However, the extent of such phenotypic plasticity and its implications for the replicability of research findings have remained unknown. Here, we examined the extent to which common environmental differences between animal facilities modulate the phenotype of genetically homogeneous (inbred) mice. We conducted a comprehensive multicentre study, whereby inbred C57BL/6J mice from a single breeding cohort were allocated to and reared in 5 different animal facilities throughout early life and adolescence, before being transported to a single test laboratory. We found persistent effects of the rearing facility on the composition and heterogeneity of the gut microbial community. These effects were paralleled by persistent differences in body weight and in the behavioural phenotype of the mice. Furthermore, we show that environmental variation among animal facilities is strong enough to influence epigenetic patterns in neurons at the level of chromatin organisation. We detected changes in chromatin organisation in the regulatory regions of genes involved in nucleosome assembly, neuronal differentiation, synaptic plasticity, and regulation of behaviour. Our findings demonstrate that common environmental differences between animal facilities may produce facility-specific phenotypes, from the molecular to the behavioural level. Furthermore, they highlight an important limitation of inferences from single-laboratory studies and thus argue that study designs should take environmental background into account to increase the robustness and replicability of findings.

Systematic assessment of the replicability and generalizability of preclinical findings: Impact of protocol harmonization across laboratory sites.

The influence of protocol standardization between laboratories on their replicability of preclinical results has not been addressed in a systematic way. While standardization is considered good research practice as a means to control for undesired external noise (i.e., highly variable results), some reports suggest that standardized protocols may lead to idiosyncratic results, thus undermining replicability. Through the EQIPD consortium, a multi-lab collaboration between academic and industry partners, we aimed to elucidate parameters that impact the replicability of preclinical animal studies. To this end, 3 experimental protocols were implemented across 7 laboratories. The replicability of results was determined using the distance travelled in an open field after administration of pharmacological compounds known to modulate locomotor activity (MK-801, diazepam, and clozapine) in C57BL/6 mice as a worked example. The goal was to determine whether harmonization of study protocols across laboratories improves the replicability of the results and whether replicability can be further improved by systematic variation (heterogenization) of 2 environmental factors (time of testing and light intensity during testing) within laboratories. Protocols were tested in 3 consecutive stages and differed in the extent of harmonization across laboratories and standardization within laboratories: stage 1, minimally aligned across sites (local protocol); stage 2, fully aligned across sites (harmonized protocol) with and without systematic variation (standardized and heterogenized cohort); and stage 3, fully aligned across sites (standardized protocol) with a different compound. All protocols resulted in consistent treatment effects across laboratories, which were also replicated within laboratories across the different stages. Harmonization of protocols across laboratories reduced between-lab variability substantially compared to each lab using their local protocol. In contrast, the environmental factors chosen to introduce systematic variation within laboratories did not affect the behavioral outcome. Therefore, heterogenization did not reduce between-lab variability further compared to the harmonization of the standardized protocol. Altogether, these findings demonstrate that subtle variations between lab-specific study protocols may introduce variation across independent replicate studies even after protocol harmonization and that systematic heterogenization of environmental factors may not be sufficient to account for such between-lab variation. Differences in replicability of results within and between laboratories highlight the ubiquity of study-specific variation due to between-lab variability, the importance of transparent and fine-grained reporting of methodologies and research protocols, and the importance of independent study replication.

The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research.

Reproducible science requires transparent reporting. The ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) were originally developed in 2010 to improve the reporting of animal research. They consist of a checklist of information to include in publications describing in vivo experiments to enable others to scrutinise the work adequately, evaluate its methodological rigour, and reproduce the methods and results. Despite considerable levels of endorsement by funders and journals over the years, adherence to the guidelines has been inconsistent, and the anticipated improvements in the quality of reporting in animal research publications have not been achieved. Here, we introduce ARRIVE 2.0. The guidelines have been updated and information reorganised to facilitate their use in practice. We used a Delphi exercise to prioritise and divide the items of the guidelines into 2 sets, the "ARRIVE Essential 10," which constitutes the minimum requirement, and the "Recommended Set," which describes the research context. This division facilitates improved reporting of animal research by supporting a stepwise approach to implementation. This helps journal editors and reviewers verify that the most important items are being reported in manuscripts. We have also developed the accompanying Explanation and Elaboration (E&E) document, which serves (1) to explain the rationale behind each item in the guidelines, (2) to clarify key concepts, and (3) to provide illustrative examples. We aim, through these changes, to help ensure that researchers, reviewers, and journal editors are better equipped to improve the rigour and transparency of the scientific process and thus reproducibility.

Reporting animal research: Explanation and elaboration for the ARRIVE guidelines 2.0.

Improving the reproducibility of biomedical research is a major challenge. Transparent and accurate reporting is vital to this process; it allows readers to assess the reliability of the findings and repeat or build upon the work of other researchers. The ARRIVE guidelines (Animal Research: Reporting In Vivo Experiments) were developed in 2010 to help authors and journals identify the minimum information necessary to report in publications describing in vivo experiments. Despite widespread endorsement by the scientific community, the impact of ARRIVE on the transparency of reporting in animal research publications has been limited. We have revised the ARRIVE guidelines to update them and facilitate their use in practice. The revised guidelines are published alongside this paper. This explanation and elaboration document was developed as part of the revision. It provides further information about each of the 21 items in ARRIVE 2.0, including the rationale and supporting evidence for their inclusion in the guidelines, elaboration of details to report, and examples of good reporting from the published literature. This document also covers advice and best practice in the design and conduct of animal studies to support researchers in improving standards from the start of the experimental design process through to publication.

Reproducibility of preclinical animal research improves with heterogeneity of study samples.

Single-laboratory studies conducted under highly standardized conditions are the gold standard in preclinical animal research. Using simulations based on 440 preclinical studies across 13 different interventions in animal models of stroke, myocardial infarction, and breast cancer, we compared the accuracy of effect size estimates between single-laboratory and multi-laboratory study designs. Single-laboratory studies generally failed to predict effect size accurately, and larger sample sizes rendered effect size estimates even less accurate. By contrast, multi-laboratory designs including as few as 2 to 4 laboratories increased coverage probability by up to 42 percentage points without a need for larger sample sizes. These findings demonstrate that within-study standardization is a major cause of poor reproducibility. More representative study samples are required to improve the external validity and reproducibility of preclinical animal research and to prevent wasting animals and resources for inconclusive research.

The ARRIVE guidelines 2.0: updated guidelines for reporting animal research.

Reproducible science requires transparent reporting. The ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) were originally developed in 2010 to improve the reporting of animal research. They consist of a checklist of information to include in publications describing in vivo experiments to enable others to scrutinise the work adequately, evaluate its methodological rigour, and reproduce the methods and results. Despite considerable levels of endorsement by funders and journals over the years, adherence to the guidelines has been inconsistent, and the anticipated improvements in the quality of reporting in animal research publications have not been achieved. Here, we introduce ARRIVE 2.0. The guidelines have been updated and information reorganised to facilitate their use in practice. We used a Delphi exercise to prioritise and divide the items of the guidelines into 2 sets, the 'ARRIVE Essential 10,' which constitutes the minimum requirement, and the 'Recommended Set,' which describes the research context. This division facilitates improved reporting of animal research by supporting a stepwise approach to implementation. This helps journal editors and reviewers verify that the most important items are being reported in manuscripts. We have also developed the accompanying Explanation and Elaboration document, which serves (1) to explain the rationale behind each item in the guidelines, (2) to clarify key concepts, and (3) to provide illustrative examples. We aim, through these changes, to help ensure that researchers, reviewers, and journal editors are better equipped to improve the rigour and transparency of the scientific process and thus reproducibility.

The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research.

Reproducible science requires transparent reporting. The ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) were originally developed in 2010 to improve the reporting of animal research. They consist of a checklist of information to include in publications describing in vivo experiments to enable others to scrutinise the work adequately, evaluate its methodological rigour, and reproduce the methods and results. Despite considerable levels of endorsement by funders and journals over the years, adherence to the guidelines has been inconsistent, and the anticipated improvements in the quality of reporting in animal research publications have not been achieved. Here, we introduce ARRIVE 2.0. The guidelines have been updated and information reorganised to facilitate their use in practice. We used a Delphi exercise to prioritise and divide the items of the guidelines into 2 sets, the "ARRIVE Essential 10," which constitutes the minimum requirement, and the "Recommended Set," which describes the research context. This division facilitates improved reporting of animal research by supporting a stepwise approach to implementation. This helps journal editors and reviewers verify that the most important items are being reported in manuscripts. We have also developed the accompanying Explanation and Elaboration document, which serves (1) to explain the rationale behind each item in the guidelines, (2) to clarify key concepts, and (3) to provide illustrative examples. We aim, through these changes, to help ensure that researchers, reviewers, and journal editors are better equipped to improve the rigour and transparency of the scientific process and thus reproducibility.

The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research.

Reproducible science requires transparent reporting. The ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) were originally developed in 2010 to improve the reporting of animal research. They consist of a checklist of information to include in publications describing in vivo experiments to enable others to scrutinise the work adequately, evaluate its methodological rigour, and reproduce the methods and results. Despite considerable levels of endorsement by funders and journals over the years, adherence to the guidelines has been inconsistent, and the anticipated improvements in the quality of reporting in animal research publications have not been achieved. Here, we introduce ARRIVE 2.0. The guidelines have been updated and information reorganised to facilitate their use in practice. We used a Delphi exercise to prioritise and divide the items of the guidelines into 2 sets, the "ARRIVE Essential 10," which constitutes the minimum requirement, and the "Recommended Set," which describes the research context. This division facilitates improved reporting of animal research by supporting a stepwise approach to implementation. This helps journal editors and reviewers verify that the most important items are being reported in manuscripts. We have also developed the accompanying Explanation and Elaboration document, which serves (1) to explain the rationale behind each item in the guidelines, (2) to clarify key concepts, and (3) to provide illustrative examples. We aim, through these changes, to help ensure that researchers, reviewers, and journal editors are better equipped to improve the rigour and transparency of the scientific process and thus reproducibility.

Authorization of Animal Experiments Is Based on Confidence Rather than Evidence of Scientific Rigor.

Accumulating evidence indicates high risk of bias in preclinical animal research, questioning the scientific validity and reproducibility of published research findings. Systematic reviews found low rates of reporting of measures against risks of bias in the published literature (e.g., randomization, blinding, sample size calculation) and a correlation between low reporting rates and inflated treatment effects. That most animal research undergoes peer review or ethical review would offer the possibility to detect risks of bias at an earlier stage, before the research has been conducted. For example, in Switzerland, animal experiments are licensed based on a detailed description of the study protocol and a harm-benefit analysis. We therefore screened applications for animal experiments submitted to Swiss authorities (n = 1,277) for the rates at which the use of seven basic measures against bias (allocation concealment, blinding, randomization, sample size calculation, inclusion/exclusion criteria, primary outcome variable, and statistical analysis plan) were described and compared them with the reporting rates of the same measures in a representative sub-sample of publications (n = 50) resulting from studies described in these applications. Measures against bias were described at very low rates, ranging on average from 2.4% for statistical analysis plan to 19% for primary outcome variable in applications for animal experiments, and from 0.0% for sample size calculation to 34% for statistical analysis plan in publications from these experiments. Calculating an internal validity score (IVS) based on the proportion of the seven measures against bias, we found a weak positive correlation between the IVS of applications and that of publications (Spearman's rho = 0.34, p = 0.014), indicating that the rates of description of these measures in applications partly predict their rates of reporting in publications. These results indicate that the authorities licensing animal experiments are lacking important information about experimental conduct that determines the scientific validity of the findings, which may be critical for the weight attributed to the benefit of the research in the harm-benefit analysis. Similar to manuscripts getting accepted for publication despite poor reporting of measures against bias, applications for animal experiments may often be approved based on implicit confidence rather than explicit evidence of scientific rigor. Our findings shed serious doubt on the current authorization procedure for animal experiments, as well as the peer-review process for scientific publications, which in the long run may undermine the credibility of research. Developing existing authorization procedures that are already in place in many countries towards a preregistration system for animal research is one promising way to reform the system. This would not only benefit the scientific validity of findings from animal experiments but also help to avoid unnecessary harm to animals for inconclusive research.

Context-dependent individual differences in playfulness in male rats.

Play has been proposed as an indicator of positive emotions and welfare in higher vertebrates. This study investigated playfulness in male rats by exploring its consistency across motivational states (with/without prior short social isolation) and two age points at early and late adolescence. Twenty-four male Lister Hooded rats housed in cages of four underwent two play tests: conspecific Play-in-Pairs and Tickling by the experimenter, which were compared with play in the home cage and basal anxiety levels. Play-in-Pairs measures were consistent across age and motivational states, and were independent from anxiety. Positively valenced vocalizations in the Tickling test were also consistent across age, yet were negatively related to anxiety. Play-in-Pairs and Tickling play contexts, as well as social and solitary play types, were unrelated. Therefore, this study supports the existence of consistent individual differences in playfulness in rats, and suggests that different play contexts and types represent motivationally distinct systems.

Heterogeneity of animal experiments and how to deal with it.

Heterogeneity of study samples is ubiquitous in animal experiments. Here, we discuss the different options of how to deal with heterogeneity in the statistical analysis of a single experiment. Specifically, data from different sub-groups (e.g. sex, strain, age cohorts) may be analysed separately, heterogenization factors may be ignored and data pooled for analysis, or heterogenization factors may be included as additional variables in the statistical model. The cost of ignoring a heterogenization factor is an inflated estimate of the variance and a consequent loss of statistical power. Therefore, it is usually preferable to include the heterogenization factor in the statistical model, especially if the heterogenization factor has been introduced intentionally (e.g. using both sexes). If heterogenization factors are included, they can be treated either as fixed factors in an analysis of variance design or sometimes as random effects in mixed effects regression models. Finally, for an appropriate sample size estimation, it is necessary to decide whether to treat heterogenization factors as nuisance variables, or whether the experiment should be powered to be able to detect not only the main effect of the treatment but also interactions between heterogenization factors and the treatment variable.

Evidence of HARKing in mouse behavioural tests of anxiety.

Over the last decades, behavioural tests in animals, especially rodents, have been a standard screening method to determine the mechanisms of action and efficacy of psychopharmacological compounds. Yet, recently the reproducibility of some of these tests has been questioned. Based on a systematic review of the sensitivity of mouse behavioural tests to anxiolytic drugs, we analysed behavioural outcomes extracted from 206 studies testing the effect of diazepam in either the open-field test or the hole-board test. Surprisingly, we found that both the rationale given for using the test, whether to detect anxiolytic or sedative effects, and the predicted effect of diazepam, anxiolytic or sedative, strongly depended on the reported test results. The most likely explanation for such strong dependency is post hoc reasoning, also called hypothesizing after the results are known (HARKing). HARKing can invalidate study outcomes and hampers evidence synthesis by inflating effect sizes. It may also lead researchers into blind alleys, and waste animals, time and resources for inconclusive research.