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

Area of Science:

• Laboratory animal science
• Experimental design
• Behavioral research

Background:

• The life sciences face a reproducibility crisis, partly due to over-standardized experiments yielding idiosyncratic results.
• Systematic heterogenisation, introducing variation within experiments, is proposed to enhance robustness and external validity, especially in animal studies.
• Identifying practical heterogenisation factors is crucial for implementing this approach.

Purpose of the Study:

• To investigate the impact of testing time on the reproducibility of behavioral differences between mouse strains.
• To evaluate time of day as a potential heterogenisation factor in laboratory animal science.

Main Methods:

• Behavioral testing of C57BL/6J and DBA/2N mice at different times of day (morning, noon, afternoon).
• Analysis of strain-specific differences in anxiety, exploration, and learning.
• Simulation approach to assess the effect of including multiple testing times on reproducibility.

Main Results:

• Significant variations in behavioral strain effects (anxiety, exploration, learning) were observed depending on the time of day.
• The time of day significantly impacted the reproducibility of behavioral differences between the two mouse strains.
• Systematic inclusion of two testing times in simulations substantially improved reproducibility.

Conclusions:

• Time of day is an effective and easily manageable heterogenisation factor for improving the reproducibility of single-laboratory studies.
• Systematic variation of testing times can enhance the robustness of research findings in laboratory animal science.
• Addressing experimental design and conduct is critical for overcoming the reproducibility crisis.