AbstractDespite the widely used application of standardized capture-handling protocols to collect blood and assess the physiological stress response, the effect of the actual sampling design (e.g. timing and the number of blood samples) often differs between studies, and the potential implications for the measured physiological endpoints remain understudied. We, therefore, experimentally tested the effects of repeated handling and multiple blood sampling on the stress response in wintering free-living great tits (Parus major). We modified a well-established sampling protocol of avian studies by adding either an additional blood sample or a “sham-manipulation” (i.e. handling associated with the blood sampling procedure without venepuncture), to disentangle the effects of handling stress and blood loss. We combined three different stress metrics along the endocrine-immune interface to investigate the acute short-term stress response: total corticosterone levels (CORT), the heterophil/lymphocyte ratio (H:L), and the Leukocyte Coping Capacity (LCC). Our study provided three key results: i) no relationship between CORT-levels, LCC and H:L, confirming that these three parameters represent different physiological endpoints within the stress response; ii) contrasting dynamics in response to stress by the measured parameters and iii) no difference in stress levels 30 minutes after capture due to one additional blood sampling or handling event. By optimising the sampling design, our results provide implications for animal welfare and planning experimental procedures on stress physiology in passerine species.Summary StatementWhen testing the short-term stress response in free living passerines, both – the scientist and the bird may be better off with a 15-minute stress protocol.
“The stressed bird in the hand”: Influence of sampling design on the physiological stress response in a free-living songbird