Heterospecific play, or rat tickling, enhances positive affect towards humans without altering anxiety-related behaviors in female and male Sprague Dawley rats.

Rodent handling methods influence stress, affective state, and behavior in laboratory rodents, which can decrease, but also potentially increase, unwanted variability in outcome measures. Heterospecific play, or tickling, in rats has been proposed as a gentling technique that decreases the stress of handling during experiments, but its effects in adult female and male rats remain unclear. Furthermore, it remains even less understood how tickling might affect subsequent behaviors in experimental tests conducted well after tickling experience. In this study, we examined whether tickling induces positive affect towards experimenters and evaluated subsequent anxiety-like behaviors in adult Sprague Dawley female and male rats. Adult rats underwent either gentle handling via forearm resting or tickling for five consecutive days, with ultrasonic vocalizations recorded during handling sessions. Rats then underwent a human approach test to assess their preference to approach an unfamiliar human hand. Tickling significantly increased the emission of 50-kHz ultrasonic vocalizations and promoted approaches toward an unfamiliar human hand in both female and male rats, indicating enhanced positive affect. In contrast, tickling did not alter anxiety-like behaviors as measured in the open field, elevated zero maze, and light-dark box tests. These findings demonstrate that tickling promotes positive affect and decreased avoidance of human hand interactions in adult female and male rats without influencing subsequent anxiety-related behaviors, supporting its use as a refined gentling technique to reduce handling-related confounds in behavioral research.