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.

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.

Structural enrichment for laboratory mice: exploring the effects of novelty and complexity.

Providing structural enrichment is a widespread refinement method for laboratory rodents and other animals in captivity. So far, animal welfare research has mostly focused on the effect of increased complexity either by accumulating or combining different enrichment items. However, increasing complexity is not the only possibility to refine housing conditions. Another refinement option is to increase novelty by regularly exchanging known enrichment items with new ones. In the present study, we used pair-housed non-breeding female C57BL/6J and DBA/2N mice to investigate the effect of novelty when applying structural enrichment. We used a double cage system, in which one cage served as home cage and the other as extra cage. While the home cage was furnished in the same way for all mice, in the extra cage we either provided only space with no additional enrichment items (space), a fixed set of enrichment items (complexity), or a changing set of enrichment items (novelty). Over 5 weeks, we assessed spontaneous behaviors, body weight, and extra cage usage as indicators of welfare and preference. Our main results showed that mice with access to structurally enriched extra cages (complexity and novelty) spent more time in their extra cages and complexity mice had lower latencies to enter their extra cages than mice with access to the extra cages without any structural enrichment (space). This indicates that the mice preferred the structurally enriched extra cages over the structurally non-enriched space cages. We found only one statistically significant difference between the novelty and complexity condition: during week 3, novelty mice spent more time in their extra cages than complexity mice. Although we did not detect any other significant differences between the novelty and complexity condition in the present study, more research is required to further explore the potential benefits of novelty beyond complexity.

Improving reproducibility in animal research by splitting the study population into several 'mini-experiments'.

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.

Differential Effects of Serotonin Transporter Genotype on Anxiety-Like Behavior and Cognitive Judgment Bias in Mice.

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.

Heterogenising study samples across testing time improves reproducibility of behavioural data.

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.

Have I been here before? Complex interactions of age and test experience modulate the results of behavioural tests.

Recently, a discussion about the reproducibility of results from behavioural phenotyping experiments has emerged. A huge emphasis has therefore been put on the identification of those factors that might limit the reproducibility of behavioural data. As a comprehensive phenotypic characterisation can involve testing of the same animal repeatedly over a specific time period, the aim of the present study was to systematically investigate effects of two potentially confounding variables, age of the animals and test experience. For this purpose, the behaviour of 48 male C57BL/6 J mice of two different ages (9 vs. 13 weeks) was assessed in a battery of common behavioural tests measuring anxiety-like and exploratory behaviour (Elevated Plus Maze, Dark-Light test, Open Field test, Novel Cage test). While half of the mice of each age group was naïve to the test battery, the other half had experienced the same tests before. Besides main effects of both age and test experience on anxiety-like and exploratory behaviour, the analysis also revealed profound interactions between these factors. More precisely, an effect of age was apparent in experienced but not in naïve mice. Furthermore, the effect of previous test experience was more pronounced in older than in younger mice. These findings clearly demonstrate that experimental factors, such as age and test experience, can influence behavioural data not just additively, but also in a complex, interactive way. To provide robust and reproducible results, it is thus fundamental to consider such factors systematically in the study design.