In light of the hotly discussed ‘reproducibility crisis’, a rethinking of current methodologies appears essential. Implementing multi-laboratory designs has been shown to enhance the external validity and hence the reproducibility of findings from animal research. We here aimed at proposing a new experimental strategy that transfers this logic into a single-laboratory setting. We systematically introduced heterogeneity into our study population by splitting an experiment into several ‘mini-experiments’ spread over different time points a few weeks apart. We hypothesised to observe improved reproducibility in such a ‘mini-experiment’ design in comparison to a conventionally standardised design, according to which all animals are tested at one specific point in time. By comparing both designs across independent replicates, we could indeed show that the use of such a ‘mini-experiment’ design improved the reproducibility and accurate detection of exemplary treatment effects (behavioural and physiological differences between four mouse strains) in about half of all investigated strain comparisons. Thus, we successfully implemented and empirically validated an easy-to-handle strategy to tackle poor reproducibility in single-laboratory studies. Since other experiments within different life science disciplines share the main characteristics with the investigation reported here, these studies are likely to also benefit from this approach.

The ongoing debate on the reproducibility crisis in the life sciences highlights the need for a rethinking of current methodologies. Since the trend towards ever more standardised experiments is at risk of causing highly idiosyncratic results, an alternative approach has been suggested to improve the robustness of findings, particularly from animal experiments. This concept, referred to as “systematic heterogenisation”, postulates increased external validity and hence, improved reproducibility by introducing variation systematically into a single experiment. However, the implementation of this concept in practice requires the identification of suitable heterogenisation factors. Here we show that the time of day at which experiments are conducted has a significant impact on the reproducibility of behavioural differences between two mouse strains, C57BL/6J and DBA/2N. Specifically, we found remarkably varying strain effects on anxiety, exploration, and learning, depending on the testing time, i.e. morning, noon or afternoon. In a follow-up simulation approach, we demonstrate that the systematic inclusion of two different testing times significantly improved reproducibility between replicate experiments. Our results emphasise the potential of time as an effective and easy-to-handle heterogenisation factor for single-laboratory studies. Its systematic variation likely improves reproducibility of research findings and hence contributes to a fundamental issue of experimental design and conduct in laboratory animal science.

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.

In humans, the short allele of a common polymorphism in the serotonin transporter (5-HTT) gene is associated with a higher risk to develop depression and anxiety disorders. Furthermore, individuals carrying this allele are characterized by negative judgment biases, as they tend to interpret ambiguous information in a more pessimistic way. 5-HTT knockout mice, lacking the 5-HTT gene either homo- or heterozygously, provide a widely used model organism for the study of symptoms related to human anxiety disorders. In the present study, we aimed to prove the anxiety-like phenotype of the 5-HTT mouse model, and to investigate whether 5-HTT genotype also causes differences in judgment bias. While our results confirm that homozygous 5-HTT knockout mice display highest levels of anxiety-like behavior, it was decreased in heterozygous mice. Against our expectations, we did not detect differences in the animals’ judgment bias. These results indicate that at least in mice the association between 5-HTT genotype and judgment bias is not straightforward and that other factors, including multiple genes as well as environmental influences, are implicated in the modulation of judgment biases. More research is needed to gain further insights into their function as potential endophenotypes for psychopathology.

Individuals differ in the way they judge ambiguous information: some individuals interpret ambiguous information in a more optimistic, and others in a more pessimistic way. Over the past two decades, such “optimistic” and “pessimistic” cognitive judgment biases (CJBs) have been utilized in animal welfare science as indicators of animals’ emotional states. However, empirical studies on their ecological and evolutionary relevance are still lacking. We, therefore, aimed at transferring the concept of “optimism” and “pessimism” to behavioral ecology and investigated the role of genetic and environmental factors in modulating CJB in mice. In addition, we assessed the temporal stability of individual differences in CJB. We show that the chosen genotypes (C57BL/6J and B6D2F1N) and environments (“scarce” and “complex”) did not have a statistically significant influence on the responses in the CJB test. By contrast, they influenced anxiety-like behavior with C57BL/6J mice and mice from the “complex” environment displaying less anxiety-like behavior than B6D2F1N mice and mice from the “scarce” environment. As the selected genotypes and environments did not explain the existing differences in CJB, future studies might investigate the impact of other genotypes and environmental conditions on CJB, and additionally, elucidate the role of other potential causes like endocrine profiles and epigenetic modifications. Furthermore, we show that individual differences in CJB were repeatable over a period of seven weeks, suggesting that CJB represents a temporally stable trait in laboratory mice. Therefore, we encourage the further study of CJB within an animal personality framework.

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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.

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