Funding for the production of this manuscript was provided by Nottingham Trent University. The authors wish to thank the University yard manager, Anna Gregory for the use of her horses and provision of fecal samples for use in the protocol. Thanks also to Chester Zoo Wildlife Endocrinology Laboratory for use of their facilities.

伦理学声明:涉及野外采样和动物个体程序已通过动物，农村与环境科学（ARES）在诺丁汉特伦特大学学院。 1.粪便样本的收集注:手套应处理粪便样本和甲醇时佩戴。如果有一个强烈怀疑的动物能够从一种人畜共患疾病，防护服是患如白大褂也应佩戴。 <ol><li>尽快收集粪便样本（内分几个小时）以下的排便，将它们放入一个样品袋中。采取任何大便样品三至五子样本与大约10克总重量。标记的时间和收集，个别ID的日期的袋子，在-20冻结样品℃直至分析。 </li><li>为了尽量减少错误，冻结所有样品在-20 °C，直到研究完成，然后收集样品一起分析，单批&lt;/ LI&gt; </ol> 2.粪便激素提取<ol><li>离开粪便样品在室温下解冻。 </li><li>确保该样品通过在其自己的样品袋各样品手工混合均匀。 </li><li>对每个样品称重0.50粪便物质的（±0.003克）上微量天平在称量舟然后粪便转移到8毫升的玻璃小瓶中。使用新的权衡船每个样本与每个样品之​​间30％的甲醇清洁镊子。 </li><li>结合0.5克粪便物质的用5毫升90％甲醇:水，并在在室温下轨道摇床摇动O / N。 </li><li>第二天早晨，涡旋样品，然后在600克离心20分钟。 </li><li>滗在设定为37℃的温度下的水浴齿条甲醇馏分到一个16×125 平方毫米的玻璃管和地点。蒸发在通风橱用空气至干。冲洗提取小瓶和剩余的粪便颗粒放入一个临床废物袋处置。 </li><li>重悬的粪便提取物在100％，1毫升甲醇和转移到一个12×75毫米2的聚丙烯管中。马上帽并储存在-20℃直至分析。 </li></ol> 3.激素分析<ol><li>开展与使用皮质酮多克隆抗血清CJM006的酶联免疫分析（EIA）的分析。 注意:第二次环评进行了测试（皮质醇R4866抗血清，但未能达到生化验证步骤（见第4.1节）多克隆皮质CJM006抗血清皮质醇R4866抗血清和相应的HRP的是由CJ蒙罗，加州大学提供，戴维斯，CA. </li><li>在1稀释的抗血清:使用重复吸管和50微升枪头在包被缓冲液（0.05M碳酸氢钠，pH值9.6）15,000，装入96孔微量滴定板与50μl/孔。盖上板密封该板，并在4℃孵育O / N。假两件未涂井代表非特异性结合井（NSB）。 </li><li>使用前立即用自动微量滴定板垫圈（0.15 M氯化钠，0.05％吐温20洗涤溶液）洗涤微量滴定板五次。 </li><li>加载板，每孔50微升为NSB的和"零"的孔[EIA缓冲液（0.1M磷酸钠4，0.149摩尔NaCl，0.1％牛血清白蛋白，pH 7.0）中仅]，标准（皮质酮，3.9 - 1000皮克/好）或粪便提取物样品（在1:20 EIA缓冲液适当稀释，见4.1）。标准和零点应一式三份，并一式四份分别运行。稀释粪便提取物样品和NSB的应一式两份运行。 </li><li> 70000:立即加入50微升/孔的EIA缓冲液稀释至1标签辣根过氧化物酶结合物ᵻ按照此。 </li><li>孵育在黑暗中微量滴定板在室温2小时。 </li><li>用洗涤液在室温在暗处用100微升洗涤微量滴定板5次，并培育板/ WEL升的RT衬底[0.4mM的2,2&#39;-连氮基-二- （3-乙基磺酸）二铵盐，1.6毫的 H 2 O 2，0.05M的柠檬酸盐，pH为4.0]中。准备基底在使用前立即。 </li><li>使用分光光度计在405nm的波长读取微量滴定板。板的温育被认为完成时，零孔的光密度达到0.8至1.0。 </li><li>以确保可重复性，使用在EIA缓冲液稀释到结合在约30％（EIA标准），50％（生物样品如国内马的粪便提取物的池）和70％（EIA标准）的控制[EIA标准或生物样品]分别展示出的&lt;10％的变化和&lt;15％的区域内和测定间​​的系数。 </li></ol> 4.酶联免疫法:为验证研究物种和性别<ol><li>生化验证（并行） <ol><li>使用EIA缓冲池化粪额外进行连续稀释CT，最好从疑似高，低浓度的激素样取自几个人（范围:整齐，1:2至1:8192）和第3节中描述的EIA运行。 注意:被视为实现了并行时粪便提取物的连续稀释产生平行于标准曲线和线性回归结果的位移曲线展示R2&gt; 0.90，坡度&gt; 0.50和P &lt;0.05。该标准曲线上结合约50％的序列稀释的粪便提取物应该被认为是理想的稀释运行所有粪便提取物（ 例如，1:20）。没有对测定考虑当线性回归结果表明R2&gt; 0.90和p &lt;0.05的干扰。 </li></ol></li><li>生化验证（干扰） <ol><li>穗200微升的连续稀释的标准（零，3.9到1000纳克/孔皮质酮）与200微升适当稀释汇集粪便提取物[在50％稀释的结合而paralleli确定SM（1:20）和环评作为运行在第3节继EIA分析图观察到的浓度减去背景（零样品浓度）与预期的浓度。 注:如果在加入稀释汇集粪便提取物与标准不显著改变量预期和线性回归的结果产生，R 2&gt; 0.90，接近1.0且p &lt;0.05的斜率。环评无干扰已经实现。 </li></ol></li><li>生物验证: 注:所选择的环境影响评价具有测量感兴趣的激素生物相关变化的能力演示[ 例如，在以下药理糖皮质激素浓度上升（ACTH往往是有执照的程序）或生物攻击（ 例如，心理和/或环境;更可能是一种机会不管制事件）。 <ol><li>选择，提取和分析粪便样本对糖皮质激素代谢物浓度使用1节详述的过程 - 前一个具有挑战性的事件之后3。 注:一个具有挑战性的活动将是具体的物种。的示例方法是针对国内马下面提供。马具有挑战性的活动全部例子已经包括使用管理9和饲养过程16先前公布。糖皮质激素代谢物浓度显著上升后的富有挑战性的活动必须遵守并利用统计分析证实。这必须包括在适当情况下，重复测量，采样日期和个人的影响。生物验证，然后视为实现。 </li></ol></li></ol>

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The authors have nothing to disclose.

粪便皮质酮分析提供了评估在马肾上腺活性长期型态的手段。该方法的非侵入性克服了用于评估肾上腺活性，包括唾液和血浆分析9其他采样方法混杂影响。此外，该技术具有如果学习自由测距马匹的明确的非侵入性的优势。 有几个关键点对本方法及其适当的使用进行讨论。在协议中的一个关键步骤是测定在质询和适当抗体的选择品种的验证。如果所使用的抗体与结?...

所描述的方法的目的来分析，以便提供肾上腺活动的非侵入性的评价在马粪便皮质酮浓度。测量下丘脑 - 垂体 - 肾上腺轴（HPA）的活动是一个公认的方法来研究在两个俘虏和国内品种可能厌恶的情况下的响应。参考技术和最广泛使用的方法是使用血浆1然而，替代的方法如粪便分析人为了克服由采血本身引起的应力，并允许监测自由测距物种的能力得到了发展。 在厌恶的情况下，生理动态平衡被打乱。在大脑释放促肾上腺皮质激素作用于垂体前叶，刺激促肾上腺皮质激素（ACTH）的释放释放激素（CRH）下丘脑。促肾上腺皮质激素进入血液，刺激肾上腺皮质分泌物种的具体糖皮质激素（GC）。糖皮质激素有密切联系的应激事件，而不是因此它们优先于其他应激激素的链接通常2测量的所有能量提高国家被一致产生。糖皮质激素是负责马匹数自适应的效果。能量被迅速地从存储位点中的脂肪酸和葡萄糖的形式体动员，氧摄取量的增加，感觉功能增强3和血流减少到没有必要移动4个区域。以及用作应对机构，在糖皮质激素的应力引起的上升也可能有助于制备动物为下一个压力源5。 评估血浆激素水平和唾液涉及测量实际循环激素然而，测量代谢物在粪便措施的激素的代谢终产物。循环类固醇分解代谢的L到，这里它们经受肠道6内菌群的酶活性促进进一步的变化胆汁排泄之前艾弗。因此，针对血糖皮质激素的免疫测定可能不适合用于粪便的糖皮质激素的代谢产物7的分析。 作为粪便收集可以没有干扰对马进行，粪便为皮质酮的分析，已被广泛用于监测在许多情况HPA活性。在马的粪便升高皮质酮已经报道响应于潜在厌恶情况，包括在手术后的兽医治疗8和限制性壳体9。粪便采样反映一段时间内汇集糖皮质激素水平，而不是由血浆和唾液提供实时采样使它更适合监测长期，慢性或季节性模式10点。由于非侵入性该方法的性质，样品可反复，而不需要对捕获或约束11的个体收集的。然而，在规划一个采样协议时物种特异性肠道转运时间，必须考虑到。在马，肠道运输时间大约是18小时12因此，肾上腺反应和随后的皮质酮代谢物可在粪便中一天HPA轴的初始激活后检测。 当使用非侵入性免疫分析技术正在调查该物种进行仔细验证是必需的13。此外，在激素代谢物的排泄性别差异已可能是由于在代谢率和在不同物种包括小鼠14，和鸡15排出皮质酮代谢物的类型的不同的报道。因此，有作为此方法的测定法验证在男性和女性国内马使用的一部分重要的，因为是在第详细Ë协议。男女之间的激素代谢这种差异对数据质量的后果，但它很少讨论并作为实验验证的一部分。 这种非侵入性方法允许在国内马肾上腺活性长期评估。该协议的细节在测定两者的验证和测定技术本身。

<table><tbody><tr><td>Corticosterone antibody &amp;amp; HRP kit</td><td>Coralie Munro - UC Davis</td><td></td><td>No longer available through UC Davis - please see Arbor Assays</td></tr><tr><td>Cortisol antibody &amp;amp; HRP kit</td><td>Coralie Munro - UC Davis</td><td></td><td>No longer available through UC Davis - please see Arbor Assays</td></tr><tr><td>Corticosterone synthetic standard hormone</td><td>Sigma Aldrich</td><td>50-23-7</td><td>Harnful if ingested or with skin contact. Use in fume cupboard</td></tr><tr><td>Cortisol synthetic standard hormone</td><td>Sigma Aldrich</td><td>15087-01-1</td><td>Harmful if ingested or with skin contact. Use in fume cupboard</td></tr><tr><td>Methanol</td><td>Sigma Aldrich</td><td>67-56-1</td><td>Irritant. Use in fume cupboard</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma Aldrich</td><td>144-55-8</td><td>Irritant</td></tr><tr><td>Sodium Carbonate Anhydrous</td><td>Sigma Aldrich</td><td>497-19-8</td><td>Irritant</td></tr><tr><td>Sodium Phosphate Dibasic</td><td>Sigma Aldrich</td><td>7558-79-4</td><td>Irritant</td></tr><tr><td>Sodium Phosphate Monobasic</td><td>Sigma Aldrich</td><td>10049-21-5</td><td>Irritant</td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>9048-46-8</td><td>Irritant</td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>9005-64-5</td><td>Irritant</td></tr><tr><td>Citric Acid</td><td>Sigma Aldrich</td><td>77-92-9</td><td>Irritant</td></tr><tr><td>ABTS</td><td>Sigma Aldrich</td><td>30931-67-0</td><td>Irritant</td></tr><tr><td>Hydrogen Peroxide 30%</td><td>Sigma Aldrich</td><td>7722-84-1</td><td>Irritant</td></tr><tr><td>Sodium Chloride</td><td>Sigma Aldrich</td><td>7647-14-5</td><td>Irritant</td></tr><tr><td>Buffer capsules - pH 4</td><td>VWR</td><td>332732B</td><td></td></tr><tr><td>Buffer capsules - pH 7</td><td>VWR</td><td>332742D</td><td></td></tr><tr><td>Buffer capsules - pH 10</td><td>VWR</td><td>332762H</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Sigma Aldrich</td><td>435570</td><td>Irritant. Use in fume cupboard</td></tr><tr><td>Sodium Hydroxide</td><td>Sigma Aldrich</td><td>S5881</td><td>Irritant</td></tr><tr><td>Analytical balance</td><td>Fisher Scientific</td><td>BFS-525-010A</td><td></td></tr><tr><td>Air compressor</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td></td></tr><tr><td>Computer +printer</td><td></td><td></td><td></td></tr><tr><td>fridge-freezer</td><td></td><td></td><td></td></tr><tr><td>Drying apparatus</td><td></td><td></td><td></td></tr><tr><td>+tubing</td><td></td><td></td><td></td></tr><tr><td>Flammable liquid storagecabinet</td><td>VWR</td><td>649-002</td><td></td></tr><tr><td>Fume cupboard</td><td></td><td></td><td></td></tr><tr><td>Hot-plate stirrer</td><td>VWR</td><td>640-282</td><td></td></tr><tr><td>Microplate reader</td><td>VWR</td><td></td><td></td></tr><tr><td>Microplate washer</td><td>VWR</td><td></td><td></td></tr><tr><td>pH meter</td><td>VWR</td><td></td><td></td></tr><tr><td>Eppendorf Research&amp;reg; pipettes - multipack option 2</td><td>VWR</td><td></td><td></td></tr><tr><td>Pipette - 1,000 &amp;micro;l</td><td>VWR</td><td></td><td></td></tr><tr><td>Pipette - 200 &amp;micro;l</td><td>VWR</td><td></td><td></td></tr><tr><td>Pipette - 20 &amp;micro;l</td><td>VWR</td><td></td><td></td></tr><tr><td>Repeater pipette</td><td>VWR</td><td></td><td></td></tr><tr><td>Pipette filler</td><td>VWR</td><td></td><td></td></tr><tr><td>Orbital shaker</td><td>Progen Scientific</td><td></td><td></td></tr><tr><td>Sonicator</td><td>Hilsonic</td><td></td><td></td></tr><tr><td>Vortex</td><td>VWR</td><td></td><td></td></tr><tr><td>Warm water bath</td><td></td><td></td><td></td></tr><tr><td>Water purification system</td><td>Millipore</td><td></td><td></td></tr></tbody></table>

fecal glucocorticoid analysis: non-invasive adrenal monitoring in equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

国产马（N = 16，8匹母马，8阉马）15年（±3），平均年龄按性别，进行到四房的设计与社会隔离（N = 4马/治疗）的增加水平分组。房屋1参与马生活在畜群环境，密切模拟其自然栖息地。房屋2匹涉及生活在对在室内谷仓。房屋3参与马马厩但视觉接触到其他马匹和马匹房屋涉及4完全隔离单独放置。 暴露于每种处理是在一个随机的...

Watch this Scientific Journal Video about Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids at JoVE.com

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析:在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

fecal-glucocorticoid-analysis-non-invasive-adrenal-monitoring

Research

JoVE Journal

Biology

9.6K 次观看.  Nottingham Trent University. Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

视频: Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Cortisol Measurement in Koala (Phascolarctos cinereus) Fur

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial species, the koala (Phascolarctos cinereus), faces prolonged exposure to anthropogenic-induced stressors and assessment of chronic stress in wild populations is urgently warranted. One of the most effective ways to measure chronic stress is through analyzing the glucocorticoid hormone cortisol in hair or fur, as it supports physiological and behavioral responses. This laboratory validation study aims to test current techniques to validate an optimal hormone extraction method to be used as a non-invasive measure of cortisol in koala fur. It is recognized that using non-invasive techniques to measure stress hormones is preferred over traditional, invasive techniques due to their ideal practical and ethical standpoints. Additionally, it is comparatively easier to acquire fur from koalas than it is to acquire samples of their blood. This study used samples of koala fur acquired from the Adelaide Koala and Wildlife Hospital to run a number of hormone extraction techniques in an attempt to validate an optimal cortisol extraction method. Results showed that 100% methanol provided the most optimal solvent extraction compared to 100% ethanol or 100% isopropanol based on parallelism results. In conclusion, this method of cortisol extraction from koala fur provided a reliable non-invasive assay that could be used to study chronic stress in koalas.

Cortisol Measurement in Koala Phascolarctos cinereus Fur

We present a protocol to determine the optimal extraction solvent to measure cortisol from koala fur. The solvents used in this protocol are methanol, ethanol and isopropanol. Determining an optimal extraction solvent will aid in reliably measuring fur to determine the impact of chronic stress on koalas.

科蒂醇测量在考拉 （菲斯科拉托斯西尼鲁斯） 毛皮

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial ...

科研

生物化学

Cortisol Extraction from Sturgeon Fin and Jawbone Matrices

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) and quantify any differences in fin cortisol levels among three main sturgeon species. Fins were harvested from 19 sacrificed sturgeons including seven beluga (Huso huso), seven Siberian (Acipenser baerii), and five sevruga (A. stellatus). The sturgeons were raised in Iranian farms for 2 years (2017-2018), and cortisol extraction analysis was conducted in South Korea (January-February 2019). Jawbones from five H. huso were also used for cortisol extraction. Data were analyzed using the general linear model (GLM) procedure in the SAS environment. The intra- and inter-assay coefficients of variation were 14.15 and 7.70, respectively. Briefly, the cortisol extraction technique involved washing the samples (300 &#177; 10 mg) with 3 mL of solvent (ultrapure water and isopropanol) twice, rotation at 80&#160;rpm for 2.5 min, air-drying the washed samples at room temperature (22-28 &#176;C) for 7 days, further drying the samples using a bead beater at 50 Hz for 32 min and grinding them into powder, applying 1.5 mL methanol to the dried powder (75 &#177; 5 mg), and slow rotation (40 rpm) for 18 h at room temperature with continuous mixing. Following extraction, samples were centrifuged (9,500 x g for 10 min), and 1 mL supernatant was transferred into a new microcentrifuge tube (1.5 mL), incubated at 38 &#176;C to evaporate the methanol, and analyzed via enzyme-linked immunosorbent assay (ELISA). No differences were observed in fin cortisol levels among species or in fin and jawbone cortisol levels between washing solvents. The results of this study demonstrate that the sturgeon jawbone matrix is a promising alternative stress indicator to solid matrices.

In this study, we present a protocol for cortisol extraction from the fin and jawbone of sturgeon species. Fin and jawbone cortisol levels were further examined by comparing two washing solvents followed by ELISA assays. This study piloted the feasibility of jawbone cortisol as a novel stress indicator.

皮质醇从鱼翅和爪骨基质提取

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) ...

环境科学

Measuring Biophysical and Psychological Stress Levels Following Visitation to Three Locations with Differing Levels of Nature

Visitation to natural environments has been linked to psychological stress reduction. Although most stress-related research has relied on self-report formats, a growing number of studies now incorporate biological stress-related hormones and catalysts, such as cortisol and &#945;-amylase, to measure levels of stress. Presented here is a protocol to examine the effects on levels of biophysical and psychological stress following visitation to three different locations with differing levels of nature. Biophysical and self-reported psychological stress levels are measured immediately upon entering the selected locations and just prior to the visitors leaving the site. Using a &#34;drool&#34;&#160;method, the biophysical measure consists of 1-2 mL samples of saliva provided by study subjects upon entry to one of three study locations. As prescribed by extant literature, the saliva is collected within a 45 minute time frame following the end of the visitor&#8217;s engagement at the location. Following saliva collection, the samples are labeled and transported to a biological lab. Cortisol is the biophysical variable of interest in this study and measured using an ELISA process with a TECAN plate reader. To measure self-reported stress, the Perceived Stress Questionnaire (PSQ), which reports levels of worry, tension, joy, and perceived demands. Data are collected at all three sites in the late afternoon through early evening. When compared across all three settings, stress levels, as measured by both the biological markers and self-reports, are significantly lower after visitation to the most natural setting.

The purpose of this paper is to identify changes in stress levels after visitation to three different settings and to describe the methods used in identifying stress levels based on measures of salivary cortisol, &#945;-amylase, and a psychological self-report instrument.&#160;

测量对三个自然水平不同的地点进行访问后的生物物理和心理压力水平

Visitation to natural environments has been linked to psychological stress reduction. Although most stress-related research has relied on self-report ...

行为学

Providing Meaningful Environmental Enrichment and Measuring Saliva Cortisol in Pigs Housed on Slatted Flooring

As pork is the most consumed meat worldwide, the welfare of animals in the swine industry has increasingly become a major public concern, which imposes a substantial pressure coming from customers, legislators and other stakeholders, to make management changes to improve the well-being of these animals. Several studies have demonstrated that providing environmental enrichment to pigs allows them to express their natural behavior, such as rooting and exploring, as well as nesting prior to farrowing, and is associated with reduced stress and improved production and welfare. However, many considerations should be taken into account when providing environmental enrichment, such as the type of floor, drainage and sewage systems, the pigs&#39; stage in life, the material, as well as its hanging method, height and location within the pen. The objectives of this paper are (1) to give methodologic information on how to provide a relatively simple and practical meaningful environmental enrichment for pigs which are housed on slatted floors during the different stages of their life, and (2) to demonstrate how to collect saliva samples for the measurement of cortisol concentrations, as a biomarker for acute stress. Protocols include information regarding the use of jute, cotton ropes, straw in racks, as well as chewable silicone sticks devices as environmental enrichment in pens of farrowing and lactation, weaners and finishers. In addition, the use of cotton rope for a non-invasive saliva samples collection for cortisol concentrations analysis is detailed. The protocols provided are relevant for professionals aiming to improve and monitor animal welfare, in both research and industrial swine farming.

This protocol demonstrates how to provide practical meaningful environmental enrichment for pigs which are housed on slatted flooring during the different stages of their lives, and how to collect saliva samples in a non-invasive manner for the measurement of cortisol concentrations, as a biomarker for acute stress.

提供有意义的环境富集和测量唾液皮质醇在猪安置在板状地板

As pork is the most consumed meat worldwide, the welfare of animals in the swine industry has increasingly become a major public concern, which imposes a ...

Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood flow that results in a net influx of blood to key organs and an increase in core temperature. This stress-induced hyperthermia is used to assess stress. However, measuring core temperature is invasive. As blood flow is redirected to the core, the periphery of the body can cool. This paper describes a protocol where peripheral body temperature is measured non-invasively in wild blue tits (Cyanistes caeruleus) using infrared thermography. In the field we created a set-up bringing the birds to an ideal position in front of the camera by using a baited box. The camera takes a short thermal video recording of the undisturbed bird before applying a mild stressor (closing the box and therefore capturing the bird), and the bird&#8217;s response to being trapped is recorded. The bare skin of the eye-region is the warmest area in the image. This allows an automated extraction of the maximum eye-region temperature from each image frame, followed by further steps of manual data filtering removing the most common sources of errors (motion blur, blinking). This protocol provides a time series of eye-region temperature with a fine temporal resolution that allows us to study the dynamics of the stress response non-invasively. Further work needs to demonstrate the usefulness of the method to assess stress, for instance to investigate whether eye-region temperature response is proportional to the strength of the stressor. If this can be confirmed, it will provide a valuable alternative method of stress assessment in animals and will be useful to a wide range of researchers from ecologists, conservation biologists, physiologists to animal welfare researchers.

There is a need for a non-invasive assessment of stress. This paper describes a simple protocol using thermal imaging to detect a significant response in eye-region temperature in wild blue tits to a mild acute stressor.

热成像学习压力非侵入性的无拘无束的小鸟

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood ...

神经科学

A Novel Method: Super-selective Adrenal Venous Sampling

Primary aldosteronism (PA) and subclinical Cushing&#39;s syndrome (SCS) are conditions in which the adrenal glands autonomously produce excessive amounts of aldosterone and cortisol, respectively. The conventional adrenal venous sampling (cAVS) method collects blood samples from both adrenal central veins and is useful for identifying the laterality of excess hormone production in a unilateral lesion(s), as documented in PA cases. In cAVS, plasma cortisol concentrations (PCCs) are used to normalize plasma aldosterone concentrations (PACs). A novel &#34;super-selective&#34; adrenal venous sampling (ssAVS) method was developed using a micro-catheter, which collects blood samples from adrenal tributary veins (TVs). PACs in ssAVS samples do not require PCC normalization because samples contain a limited amount of systemic venous blood, if any. The ssAVS method enabled segmental lesion(s) to be detected in both adrenal glands, which may be treated by bilateral adrenalectomy, thereby sparing lesion-free segment(s). Right and left adrenals typically have three TVs each, i.e., the superior, lateral, and inferior TVs in the right adrenal as well as the superior-median, superior-lateral, and lateral TVs in the left adrenal. In the ssAVS method, specific parent catheters and a technique to handle them are required, and have been described herein. Furthermore, ssAVS results from three cases of PA are presented: bilateral aldosterone-producing adenoma (APA) (Case #1), left APA and right possible cortisol-producing adenoma causing SCS (Case #2), and idiopathic hyperaldosteronism in which bilateral adrenal segments produced excessive amounts of aldosterone (Case #3). The ssAVS method is not difficult for expert angiographers, and, thus, is recommended worldwide to treat PA cases for which cAVS does not represent a viable surgical treatment option.

&#39;Super-selective&#39; adrenal venous sampling (ssAVS, also called segmental adrenal venous sampling: sAVS)&#160;was performed using a micro-catheter to identify adrenal segment(s) that produce excessive amounts of hormones. The ssAVS technique was described, cases in which adrenal segmental lesion(s) were identified by ssAVS were presented, and the usefulness of ssAVS in future adrenal research was discussed.

一种新的方法: 超选择性肾上腺静脉取样

Primary aldosteronism (PA) and subclinical Cushing&#39;s syndrome (SCS) are conditions in which the adrenal glands autonomously produce excessive amounts ...

医学

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

对糖皮质激素信号A化学筛选程序与斑马鱼幼虫的荧光素酶报告系统

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

生物学

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT &#34;point samples&#34; obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.
In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair&#39;s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.

Cortisol (CORT) accumulates in the growing hair shaft of humans and nonhuman primates. We describe methods for extracting and analyzing hair CORT with high precision and sensitivity. Measurement of hair CORT is particularly well-suited for assessing chronic stress over periods of weeks to months.

提取与分析皮质醇从人与猴的毛

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and ...

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal microscopy was used in combination with patch-clamp recording to detect three fusion modes in primary culture bovine adrenal chromaffin cells. The three fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving shrinkage of the fusion-generated &#937;-shape profile until it merges completely at the plasma membrane.
To detect these fusion modes, the plasma membrane was labeled by overexpressing mNeonGreen attached with the PH domain of phospholipase C &#948; (PH-mNG), which binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at the cytosol-facing leaflet of the plasma membrane; vesicles were loaded with the fluorescent false neurotransmitter FFN511 to detect vesicular content release; and Atto 655 was included in the bath solution to detect fusion pore closure. These three fluorescent probes were imaged simultaneously at ~20-90 ms per frame in live chromaffin cells to detect fusion pore opening, content release, fusion pore closure, and fusing vesicle size changes. The analysis method is described to distinguish three fusion modes from these fluorescence measurements. The method described here can, in principle, apply to many secretory cells beyond chromaffin cells.

This protocol describes a confocal imaging technique to detect three fusion modes in bovine adrenal chromaffin cells. These fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving fused vesicle shrinkage.

共聚焦显微镜测量肾上腺嗜铬细胞融合孔隙动力学的三种模式

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

大鼠肾上腺皮质细胞的原代培养及非甾体功能的测定

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

糖皮质激素粪分析:在马属动物无创监测肾上腺

摘要

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此视频中的章节

科蒂醇测量在考拉（菲斯科拉托斯西尼鲁斯）毛皮

皮质醇从鱼翅和爪骨基质提取

测量对三个自然水平不同的地点进行访问后的生物物理和心理压力水平

提供有意义的环境富集和测量唾液皮质醇在猪安置在板状地板

热成像学习压力非侵入性的无拘无束的小鸟

一种新的方法: 超选择性肾上腺静脉取样

对糖皮质激素信号A化学筛选程序与斑马鱼幼虫的荧光素酶报告系统

提取与分析皮质醇从人与猴的毛

共聚焦显微镜测量肾上腺嗜铬细胞融合孔隙动力学的三种模式

大鼠肾上腺皮质细胞的原代培养及非甾体功能的测定

糖皮质激素粪分析:在马属动物无创监测肾上腺

摘要

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此视频中的章节

科蒂醇测量在考拉 （菲斯科拉托斯西尼鲁斯） 毛皮

皮质醇从鱼翅和爪骨基质提取

测量对三个自然水平不同的地点进行访问后的生物物理和心理压力水平

提供有意义的环境富集和测量唾液皮质醇在猪安置在板状地板

热成像学习压力非侵入性的无拘无束的小鸟

一种新的方法: 超选择性肾上腺静脉取样

对糖皮质激素信号A化学筛选程序与斑马鱼幼虫的荧光素酶报告系统

提取与分析皮质醇从人与猴的毛

共聚焦显微镜测量肾上腺嗜铬细胞融合孔隙动力学的三种模式

大鼠肾上腺皮质细胞的原代培养及非甾体功能的测定

科蒂醇测量在考拉（菲斯科拉托斯西尼鲁斯）毛皮