Vocal Signatures of Stress Relief: Effects of Appeasing Harness and Synthetic Pheromone on Puppy Whine Acoustics in Separation Context (Canis familiaris)

In mammals, loud, high-pitched, and harsh-sounding calls typically accompany heightened emotional arousal, particularly during distress such as separation. However, whether subtle arousal reductions can be detected through acoustic analysis within a single negative context remains unclear. We investigated whether source-related acoustic parameters of puppy whines reflect arousal modulations induced by calming interventions during maternal separation. Thirty-five eight-week-old Beagle puppies were recorded under four conditions combining synthetic appeasing pheromone and a pressure harness. Vocal behavior, activity, whine duration, and intensity, did not significantly differ across treatments, suggesting interventions did not suppress separation-related vocal responses. Nevertheless, calming products selectively altered acoustic parameters known to index arousal in dog vocalizations. Puppies receiving combined treatments produced whines with lower fundamental frequency (fo) and reduced fo variability, while pheromone exposure increased call tonality, reflected by reduced jitter and shimmer and elevated harmonics-to-noise ratios. Spectral entropy remained unchanged, possibly because the proportion of whines containing nonlinear phenomena did not vary across conditions. Reductions in fo, fo variability, and acoustic roughness are consistent with established correlates of lower arousal in mammals, suggesting source-related vocal parameters sensitively capture subtle arousal shifts even when overt vocal behavior remains stable, supporting their use as bioacoustic indicators for evaluating welfare interventions.