Abstract

Background:
Stress assessment in working dogs requires a multidimensional approach combining physiological and behavioural indicators. Salivary cortisol is widely used as a non-invasive biomarker of acute stress, while behavioural observations provide complementary information on coping responses.

Aim:
The present exploratory study aimed to assess whether training sessions at the National Training School for Guide Dogs (Florence, Italy) induced measurable stress in guide dog puppies, by evaluating salivary cortisol concentrations and stress-related behaviours at different stages of the training programme.

Methods:
The study included six Labrador Retrievers from the same litter who were monitored from four to 22 months of age between September 2022 and March 2024. Five study days were identified to represent potentially stressful stages of the training programme. On each day, three saliva samples from each dog were collected: before training (T0), immediately after (T1), and 30 minutes later (T2). Cortisol concentrations were measured by radioimmunoassay method. Concurrently, two trained observers recorded stress-related behaviours during 5-minute sessions at T0 and T1, according to ethograms adapted from previous studies.

Results:
Most cortisol values were below the limit of quantification (81.3%; 61/75), with occasional higher values in isolated cases. Behavioural observations showed that stress-related behaviours were rare, with overall very low frequencies throughout the study. No consistent pattern of variation across sampling times (T0–T2) was observed.

Conclusion:
No clear behavioural or measurable endocrine responses to training sessions were detected. The results support the hypothesis that the training methods used, based on positive reinforcement and play, do not adversely affect canine welfare under such conditions. Nevertheless, the limitations of salivary cortisol as a stress biomarker highlight the need for future studies to integrate additional physiological indicators to provide a more comprehensive evaluation of guide dog welfare.

Key words: Assistance dogs; Canine behavior; Saliva; Stress biomarkers; Welfare assessment.