Environmental Exposure to the Common Trunk of Mammalian Appeasing Pheromone Modulates Social Behavior and Reduces Fight Wounds in Male Mice

Abstract

Aggression among group-housed male laboratory mice poses significant challenges for animal welfare and scientific outcomes. Semiochemicals, such as mammalian appeasing pheromones, have shown potential in modulating social behaviors in various species. This study aimed to evaluate the effects of a synthetic Common Trunk (CT) of mammalian appeasing pheromone on social behavior, aggression, and welfare indicators in adult male mice. Specific Pathogen Free (SPF) male RjOrl:SWISS mice (8-12 weeks old) were housed in open-top cages and exposed via environmental passive diffusion to either a 2% synthetic CT pheromone formulation or a placebo (excipient only). Behavioral tests included the Elevated Plus Maze (EPM) and the Resident-Intruder (RI) test. Clinical welfare assessments and hematobiochemical analyses were also performed. Behavioral testing revealed minimal differences between groups, except for-in animals exposed to the CT-significantly fewer unsupported rearings, both in number (p = 0.0284) and duration (p = 0.0184), suggesting reduced vigilance (EPM Test) and shorter upright posture durations (p = 0.0031), a behavior linked to social signaling during conflict (RI test). Welfare assessments and risk-based analyses indicated that mice exposed to the CT of the appeasing pheromone displayed more agonistic but less violent confrontations, with fewer visible lesions. The protective effect was most pronounced during early group housing of unfamiliar adult males, with significantly fewer injuries in treated mice during the first (p = 0.0215) and second week (p = 0.0329). Treated mice also showed higher serotonin levels (p = 0.0295), suggesting reduced aggressiveness in line with observed behavioral outcomes. Exposure to the CT appeared to improve social dynamics and reduce escalation of aggression in male mice, supporting its potential as a refinement tool in laboratory housing practices.

Keywords: aggression; fight wounds; laboratory mouse; mammalian appeasing pheromone; prosocial behaviors.

Figure 1

Study timeline, including duration, testing windows, and placements of diffusers. Groups A and B refer to treatment groups, with A being Placebo and B being CT. Progressive numbers (1, 2, and 3) indicate subsequent batches placed over time. EPM indicates Elevated Plus Maze and RI indicates Resident–Intruder Test.

Figure 2

Effect of treatment on visible fights (a) and squeaks (b). ** 0.001 ≤ p-value < 0.01; * 0.01 ≤ p-value < 0.05.

Figure 3

Effect of treatment on number of mice with fight lesions (a) and proportion of mice with lesions over the 4 weeks of habituation (all treatments) (b). ** 0.001 ≤ p-value < 0.01; * 0.01 ≤ p-value < 0.05; “t” statistical trend (0.05 ≤ p-value < 0.1). Means with standard errors (SEs) are shown.

Figure 4

(a) (Week 1) and (b) (Week 2): Effect of treatment on number of mice with lesions in the first and second week post regrouping. * 0.01 ≤ p-value < 0.05. Means with standard errors (SEs) are shown.

Figure 5

(a) (Duration) and (b) (Occurrence): Effect of treatment on unsupported rearing duration and occurrence. * 0.01≤ p-value < 0.05RI Test. Means with standard errors (SEs) are shown.

Figure 6

Effect of treatment on upright posture duration. ** 0.001 ≤ p-value < 0.01. (Left): binary part of the hurdle model (number of individuals with a non-zero upright posture duration); (Right): positive part of the hurdle model (proportion of time spent upright among individuals with non-zero duration). Means with standard errors (SEs) are shown.

Figure 7

Effect of treatment on Serotonin plasma levels, ng/mL. * 0.01 ≤ p-value < 0.05. Means with standard errors (SEs) are shown.