Physiological parameters.

Monitoring chronic stress in pigs is not only essential in view of animal welfare but is also important for the farmer, given that stress influences the zootechnical performance of the pigs and increases their susceptibility to infectious diseases. To investigate the use of saliva as a non-invasive, objective chronic stress monitoring tool, twenty-four 4-day-old piglets were transferred to artificial brooders. At the age of 7 days, they were assigned to either the control or the stressed group and reared for three weeks. Piglets in the stressed group were exposed to overcrowding, absence of cage enrichment, and frequent mixing of animals between pens. Shotgun analysis using an isobaric labelling method (iTRAQ) for tandem mass spectrometry performed on saliva samples taken after three weeks of chronic stress identified 392 proteins, of which 20 proteins displayed significantly altered concentrations. From these 20 proteins, eight were selected for further validation using parallel reaction monitoring (PRM). For this validation, saliva samples that were taken one week after the start of the experiment and samples that were taken at the end of the experiment were analysed to verify the profile over time. We wanted to investigate whether the candidate biomarkers responded fast or rather slowly to the onset of chronic exposure to multiple stressors. Furthermore, this validation could indicate whether age influenced the baseline concentrations of these salivary proteins, both in healthy and stressed animals. This targeted PRM analysis confirmed that alpha-2-HS-glycoprotein was upregulated in the stressed group after one and three weeks, while odorant-binding protein, chitinase, long palate lung and nasal epithelium protein 5, lipocalin-1, and vomeromodulin-like protein were present in lower concentrations in the saliva of the stressed pigs, albeit only after three weeks. These results indicate that the porcine salivary proteome is altered by chronic exposure to multiple stressors. The affected proteins could be used as salivary biomarkers to identify welfare problems at the farm and facilitate research to optimise rearing conditions.对猪的慢性应激进行监测，不仅关乎动物福利，对于养殖者而言同样至关重要：应激会影响猪的畜牧生产性能，同时提升其感染性疾病易感性。为探究唾液作为非侵入式、客观的慢性应激监测工具的应用潜力，本研究将24头4日龄仔猪转移至人工育雏箱中饲养。仔猪长至7日龄时，被分为对照组与应激组，随后饲养三周。应激组仔猪暴露于饲养密度过高、笼具环境丰容缺失（cage enrichment）以及频繁跨栏混群的环境中。对慢性应激三周后采集的唾液样本采用串联质谱联用同量异位素标记法（iTRAQ）进行鸟枪法蛋白质组分析，共鉴定出392种蛋白质，其中20种蛋白质的浓度存在显著差异。从这20种差异蛋白质中，选取8种采用平行反应监测（PRM）技术开展进一步验证。本次验证阶段采集了实验启动后1周以及实验结束时的唾液样本，以分析目标蛋白质随时间的表达谱变化。本研究旨在探究候选生物标志物对多应激源慢性暴露的响应速率。此外，本次验证还可明确健康与应激状态下的仔猪，其年龄是否会影响上述唾液蛋白质的基础浓度。本次靶向PRM分析证实，应激组仔猪在实验启动后1周及3周时，α2-HS-糖蛋白（alpha-2-HS-glycoprotein）的表达均会上调；而气味结合蛋白（odorant-binding protein）、几丁质酶（chitinase）、腭肺鼻上皮蛋白5、脂笼蛋白-1（lipocalin-1）以及类犁鼻膜蛋白（vomeromodulin-like protein）仅在实验结束时，在应激组仔猪的唾液中呈现低浓度表达。上述结果表明，多应激源的慢性暴露会改变猪的唾液蛋白质组。这些受影响的蛋白质可作为唾液生物标志物，用于识别养殖场的动物福利问题，同时可为优化养殖饲养条件的相关研究提供支撑。