Crossing the comfort zone: rabbit appeasing pheromone enhances acclimation to a novel environment

Appeasing pheromones are known to help animals cope with challenging and stressful situations, facilitating acclimation to diverse environments and contexts across a wide range of species. As prey animals highly sensitive to environmental stressors, rabbits are particularly good candidates to benefit from such interventions and represent a relevant species for research in this field. This study evaluated the behavioral effects of topical skin application of Rabbit Appeasing Pheromone (RAP) in rabbits exposed to a novel environment, using an Open-Field (OF) test conducted over two consecutive days. Twenty-eight rabbits were randomly divided into two groups, balanced for sex, neutering status, and parity. Behavioral parameters were assessed using both behavioral observation software and automated tracking software to determine the effects of RAP on locomotion, exploration, and grooming. During the first Open-Field test (OF1), all rabbits exhibited a behavioral profile characterized by increased locomotion. During the second exposure (OF2), this pattern shifted toward reduced locomotion and increased stationary behaviors. Rabbits exhibited increased grooming frequency during OF2, regardless of treatment. Grooming duration showed a numerical increase in the placebo group during OF2; however, this increase did not reach statistical significance. The treatment effects were most evident in measures of explorative behaviors, with RAP-treated rabbits showing faster entry into the central zone and spending more time there in both tests. In addition, a statistical tendency was observed for RAP-treated rabbits to enter the arena voluntarily sooner than placebo-treated rabbits. Overall, these results highlight the substantial role of novelty-driven locomotor activity in rabbits, along with the distinct behavioral effects of RAP on their interaction with the novel environment. In particular, the spatial use of the arena by RAP-treated rabbits across both tests is consistent with reduced anxiety-related responses to a novel environment and its repeated exposure. These findings support the potential role of topical RAP application in facilitating rabbits' behavioral acclimation to novel and potentially challenging situations.