TO HANDLE OR NOT TO HANDLE? A PREREGISTERED STUDY ASSESSING THE IMPACT OF CUP HANDLING ON MOUSE BEHAVIOUR

Is animal handling a neutral preparatory step in behavioural neuroscience, or an underestimated experimental variable shaping behavioural outcomes? Although tunnel and cup handling are increasingly recommended as refinement strategies, the behavioural consequences of commonly used handling methods remain incompletely characterised across assays. Here, we systematically compared tail and cup handling in mice in a preregistered multi-assay study (Fig.1). We assessed spontaneous approach-avoidance behaviour towards the handler using the voluntary interaction test (VIT), anxiety and exploratory activity in the open field test (OFT) and elevated plus maze (EPM), motivational and affective state in the sucrose splash test (SPL), object interaction and memory in the novel object recognition test (NOR), and sociability in the social interaction test (SIT).

Figure 1: Experimental Design

As expected, cup handling strongly promoted voluntary approach behaviour towards the handler during the VIT. However, handling did not alter anxiety measures in the OFT, self-care behaviour in the SPL, or sociability in the SIT. While standard anxiety measures were largely unaffected in the EPM, handling decreased protected stretch attend postures (PSAP) and increased head dips in the open arms, suggesting increased exploratory drive in anxiogenic environments. Similarly, during NOR, cup handling increased object exploration during both familiarisation and test phases; however, tail-handled animals explored the objects less and failed to acquire a novel object preference. Contrary to expectations of broad anxiolytic effects, we observed subtle shifts in exploratory behaviour to novel stimuli and anxiogenic environments, underscoring routine handling as a critical experimental factor rather than a neutral procedural detail.