Novel application of CO2 sensor technology for breath-by-breath assessment of stress and energetics in unrestrained free-living animals

动物如何管理它们的时间和精力对它们的繁殖成功以及进化有影响。因此，测量动物的压力和能源成本，具体到不同的行为和环境，是很重要的，能够预测动物将如何应对人类引起的干扰和环境变化。然而，现有的方法都存在伦理问题和技术缺陷。在标准的呼吸测量（测量O2消耗和/或CO2产生）中，动物被限制在室内或戴上面具，这可能会限制运动行为的表达，就像在野外一样。在野外，记录仪可以连接到动物身上或植入动物体内，以测量与代谢率相关的变量，例如心率或动态身体加速度（首先需要在实验室中使用呼吸测量法进行校准）。另一种现场选择是将同位素注入动物的血液中，然后对血液进行采样以量化二氧化碳的产生。压力水平也可以通过采集血液进行激素分析来评估。所有这些野外方法都需要捕获动物（通常是重新捕获），已知会引起应激反应，有些是侵入性的。同位素方法的时间分辨率较低，虽然加速的时间分辨率较高，但加速是由于运动引起的，不能说明动物静止的时期，因此不能说明基础成本，或体温调节、孵化或压力的成本。测量CO2浓度的技术已经取得了令人难以置信的进步，非色散红外CO2光谱（NoDIS）传感器现在能够将浓度解析为0.1 ppm并以高频率记录（例如，20 Hz）。在我们最近在风洞中进行的“尾流呼吸测量”的概念验证工作中，我们证明了可以通过在动物的顺风处放置开放路径NoDIS（LICOR的Li 7500 https：//www.licor.com/env/products/eddy_covariance/LI-7500DS）来监测动物压力和通气率。随着时间的推移，呼出的CO2或羽流的尾流给出峰值的信号响应，提供逐呼吸分辨率。我们证明了可以测量鸟类对应激源的快速反应和从运动中恢复，并且，在理论上，这可以通过整合完整的CO2唤醒来计算代谢率。我们这项研究的主要目的是将唤醒呼吸测量的高时间分辨率带到现场，并探索定量静止的自由生活动物的压力和能量学的潜力，例如，孵卵冬眠栖息晒太阳如果这起作用，将打开许多门来研究动物的代谢节律和任何改变这些节律的因素，人为干扰、城市化、生态旅游、资源可用性、温度（与全球变暖有关）甚至疾病（例如，蝙蝠的白色噪声综合征）。我们的具体目标是：1）进行气体模拟实验，以优化巢箱中逐呼吸分辨率的现场设置条件。2)在优化的巢箱设置条件下，试验测量圈养鸽子通风和代谢率的方法，并测量它们对感知到的威胁和新物体的反应。3)采取现场的方法来量化孵化仓鸮Tyto alba的压力和能量，与牲畜接近度（已知会引起压力），噪音，光线和温度有关。4)用不同体型的狗在野外进行代谢率的测定试验，用涡度相关技术测定CO2通量和代谢率。这将涉及使用LiCOR声波风速计和智能通量系统，将联合收割机风速和风向数据与二氧化碳数据结合起来。

How animals manage their time and energy has implications for their reproductive success and, therefore, evolution. Measuring animal stress and energy costs specific to different behaviours and in relation to the environment is, therefore, important to be able to predict how animals will respond to human-induced disturbance and environmental change. However, methods available to do this each come with ethical issues and technical drawbacks.In standard respirometry (measuring O2 consumption and/or CO2 production), animals are confined to chambers or fitted with a mask which can limit expression of movement behaviour as it would be in the wild. In the field, loggers can be either attached to, or implanted in, animals to measure variables that correlate with metabolic rate, such as heart rate or dynamic body acceleration (first needing calibration with respirometry in the lab). Another field option is to inject isotopes into the blood of animals and later sample blood to quantify CO2 production. Stress levels can also be assessed by sampling blood for hormone analysis. All of these field methods require animal capture (and often recapture), known to cause a stress response, and some are invasive. Temporal resolution is low for the isotope method, and while it is high for acceleration, acceleration is due to movement, and cannot account for periods when animals are still and therefore basal costs, or the costs of thermoregulation, incubation or stress. Technology for measuring CO2 concentration has advanced incredibly, with non-dispersive infrared CO2 spectroscopy (NoDIS) sensors now able to resolve concentrations as 0.1 ppm and record at high frequency (e.g., 20 Hz). In our recent proof-of-concept work for 'wake respirometry' in wind tunnels, we demonstrated that it is possible to monitor animal stress and ventilation rates by positioning an open path NoDIS (Li7500 from LICOR https://www.licor.com/env/products/eddy_covariance/LI-7500DS) downwind of animals. The wake of exhaled CO2, or plume, over time, gives a signal response of peaks, providing breath-by-breath resolution. We demonstrated that rapid responses to stressors and recovery from exercise in birds could be measured, and that, in theory, this could be developed to calculate metabolic rate by integrating the full CO2 wake.Our principal aim is of this research is to take the high temporal resolution of wake respirometry to the field and explore the potential to quantity stress and energetics of free living animals that are stationary, e.g., incubating, hibernating, roosting, basking. If this works, many doors will be opened to investigate metabolic rhythms of animals and any factors that change those rhythms e.g., anthropogenic disturbance, urbanisation, ecotourism, resource availability, temperature (with links to global warming) and even disease (e.g., white noise syndrome in bats).Our specific objectives are to: 1) conduct gas simulation experiments to optimise field set up conditions for breath-by-breath resolution in nest boxes. 2) Under optimised nest box set up conditions, trial the method for measuring ventilation and metabolic rates of captive pigeons and measure their responses to a perceived threat and novel object. 3) Take the method to field to quantify stress and energetics in incubating barn owls Tyto alba in relation to livestock proximity (known to cause stress), noise, light and temperature. 4) Trial measuring metabolic rates in open air using dogs of different body size and the eddy covariance technique to measure CO2 flux and metabolic rate. This will involve using a LiCOR sonic anemometer and smart flux system to combine wind speed and direction data with CO2 data.