Stage 2 Registered Report: Refinement of tickling protocols to improve positive animal welfare in laboratory rats

Background: Rat tickling (heterospecific play between rats and the human hand), can promote positive affect and improve laboratory rat welfare. However, individual variation, particularly in response to the standard tickling protocol involving frequent pinning, may limit its effectiveness. Following hypothesis registration (https://f1000research.com/articles/11-1053/v2), we aimed to refine the protocol by testing responses to different amounts of pinning during playful handling (PH), a flexible form of tactile interaction resembling juvenile rough and tumble rat play. Methods: Juvenile male and female Wistar rats received six daily 30-second sessions of PH with 0, 1, or 4 pins per session. A non-PH control involved a motionless hand. The primary response variables were the count and variability of 50 kHz ultrasonic vocalisations (USVs), which are measures of positive affect in rats. Additional exploratory outcomes included behavioural and physiological responses in the elevated plus maze and open field tests. Results: All PH treatments increased 50 kHz USVs compared to controls, confirming their positive effect. Males showed no difference in USVs across pinning conditions, while females vocalised more in treatments with minimal or no pinning. Except for the highest pinning treatment (P4), females produced more USVs than males across all conditions, including controls. However, reducing pinning did not reliably decrease USV variability or alter behavioural and physiological outcomes. Conclusions: Applying 50 kHz USVs as a measure of positive affect, these results confirm that tickling induces positive affect in juvenile laboratory rats. We report the novel finding of sex differences in response to tickling. Females preferred tickling where the amount of pinning is minimised relative to males that showed no preference across tickling treatments.