Despite the widely used application of standardized capture-handling protocols to collect blood and assess the physiological stress response, 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 concentrations (Cort), the heterophil/lymphocyte ratio (H:L), and the Leucocyte 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 physiological stress levels 30 min 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.

尽管广泛使用标准化的捕获处理协议来收集血液和评估生理应激反应，但实际的采样设计（例如，时间和血样数量）在研究之间经常不同，并且对测量的生理终点的潜在影响仍然未充分研究。因此，我们通过实验测试了反复处理和多次采血对越冬自由生活的大山雀（Parus major）应激反应的影响）。我们通过添加额外的血样或“假操作”（即，在没有静脉穿刺的情况下与采血程序相关的处理）修改了完善的禽类研究采样方案，以消除处理压力和失血的影响。我们沿内分泌-免疫界面结合了三种不同的压力指标来研究急性短期压力反应：总皮质酮浓度 (Cort)、异嗜细胞/淋巴细胞比率 (H:L) 和白细胞应对能力 (LCC)。我们的研究提供了三个关键结果：i) Cort 水平、LCC 和 H:L 之间没有关系，证实这三个参数代表压力反应中的不同生理终点；ii) 对比测量参数对压力的反应动力学和 iii) 由于一次额外的血液采样或处理事件，捕获后 30 分钟生理压力水平没有差异。通过优化抽样设计，我们的结果为动物福利和计划对雀类物种胁迫生理学的实验程序提供了启示。