Salivary corticosterone: an appropriate alternative for total and free blood corticosterone in birds?

Corticosterone is widely used as a physiological indicator of stress in birds. Blood sampling remains invasive, encouraging the search for alternative methods that minimize the impact of sample collection. Among these, saliva has recently been proposed as a promising matrix that may reflect the free, biologically active fraction of corticosterone. Still rarely tested and validated in avian species, the present study aimed to evaluate the feasibility and validity of salivary corticosterone measurements in barn owl (Tyto alba) nestlings by comparing them with total and free corticosterone concentrations in plasma under both baseline and stress-induced conditions. Blood and saliva samples were collected sequentially at baseline and after standardized handling stress. Total plasma and salivary corticosterone concentrations were quantified using an enzyme immunoassay, while free plasma corticosterone was indirectly estimated from corticosteroid-binding globulin capacity determined by a radioligand-binding assay. A slight increase in salivary corticosterone was detected following stress exposure, suggesting though that the peak may occur later than the 30-minute post-stress sampling time point used in our protocol. Salivary corticosterone was positively associated with plasma corticosterone, showing the strongest correlations with stress-induced total plasma corticosterone and with baseline free plasma corticosterone. These results support salivary corticosterone as a reliable alternative to plasma corticosterone, while being less invasive in certain aspects, as it avoids the pain, hematoma and infection risks associated with venipuncture. Some limitations still exist, including the relatively long collection time and the minimal sample volume required, making such a procedure challenging or even impossible in smaller species. Additional research is needed to accurately characterize the amplitude and timing of the salivary stress response and to eventually explore shorter sampling protocols, which could further reduce invasiveness.