Abstract
Physiological biomarkers offer valuable insights into the relationships between physiological responses and ecological conditions, serving as powerful tools to assess the health and resilience of wild populations. Among them, leucocyte profiles are particularly informative indicators of individual condition, immune function and stress response. However, several aspects of blood sampling protocols can introduce bias into leucocyte measurements, potentially leading to misinterpretation of results. Here, we investigated the effects of capture-induced stress and within-individual repeated blood sampling on leucocyte profiles in two avian model species: free-ranging Spotless Starlings (Sturnus unicolor) and captive Red-legged Partridges (Alectoris rufa). Baseline samples were collected immediately after capture, and post-stress samples were obtained after keeping birds in a cotton bag for 15–120 min. Repeated-sampling effects were evaluated through a between-subjects comparison, contrasting individuals sampled only once (after 15–100 min) against the second sample of individuals sampled twice (before and after 15–100 min). We found that capture-induced stress significantly affected leucocyte measures. Total leucocyte counts decreased significantly 60 min after capture in both species. Also, in starlings, eosinophils decreased after 45 min, whereas in partridges, heterophils decreased after 30 min and lymphocytes showed an initial drop followed by an increase after 90 min. The heterophil-to-lymphocyte (H:L) ratio remained stable in starlings but declined 30 min after capture in partridges. These capture-stress effects were not confounded by repeated sampling, although repeated sampling altered absolute values of lymphocyte proportions in starlings but had no effect in partridges. Overall, our findings show that leucocyte profiles are influenced by sampling protocol conditions (i.e., capture stress, time since capture, and previous venipuncture), and that these effects may vary subtly among species. We recommend collecting blood samples as early as possible after capture in order to avoid potential biases. If immediate sampling is not possible, sampling within 15 min appears to minimize bias introduction across leucocyte parameters, at least in Spotless Starlings and Partridges. Nevertheless, recording the exact time elapsed between capture and sampling is advisable for statistically controlling this variable a posteriori. Future studies should address the ecological and evolutionary factors shaping interspecific differences in stress-related variations in leucocyte counts.