糖皮质激素粪分析：在马属动物无创监测肾上腺
Summary
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.
Abstract
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.
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Corticosterone antibody & HRP kit | Coralie Munro – UC Davis | NA | No longer available through UC Davis – please see Arbor Assays |
| Cortisol antibody & HRP kit | Coralie Munro – UC Davis | NA | No longer available through UC Davis – please see Arbor Assays |
| Corticosterone synthetic standard hormone | Sigma Aldrich | 50-23-7 | Harnful if ingested or with skin contact. Use in fume cupboard |
| Cortisol synthetic standard hormone | Sigma Aldrich | 15087-01-1 | Harnful if ingested or with skin contact. Use in fume cupboard |
| Methanol | Sigma Aldrich | 67-56-1 | Irritant. Use in fume cupboard |
| Sodium Bicarbonate | Sigma Aldrich | 144-55-8 | Irritant |
| Sodium Carbonate Anhydrous | Sigma Aldrich | 497-19-8 | Irritant |
| Sodium Phosphate Dibasic | Sigma Aldrich | 7558-79-4 | Irritant |
| Sodium Phosphate Monobasic | Sigma Aldrich | 10049-21-5 | Irritant |
| BSA | Sigma Aldrich | 9048-46-8 | Irritant |
| Tween 20 | Sigma Aldrich | 9005-64-5 | Irritant |
| Citric Acid | Sigma Aldrich | 77-92-9 | Irritant |
| ABTS | Sigma Aldrich | 30931-67-0 | Irritant |
| Hydrogen Peroxide 30% | Sigma Aldrich | 7722-84-1 | Irritant |
| Sodium Chloride | Sigma Aldrich | 7647-14-5 | Irritant |
| Buffer capsules – pH 4 | VWR | 332732B | |
| Buffer capsules – pH 7 | VWR | 332742D | |
| Buffer capsules – pH 10 | VWR | 332762H | |
| Hydrochloric Acid | Sigma Aldrich | 435570 | Irritant. Use in fume cupboard |
| Sodium Hydroxide | Sigma Aldrich | S5881 | Irritant |
| Analytical balance | Fisher Scientific | BFS-525-010A | |
| Air compressor | |||
| Centrifuge | |||
| Computer +printer | |||
| fridge-freezer | |||
| Drying apparatus | |||
| +tubing | |||
| Flammable liquid storagecabinet | VWR | 649-002 | |
| Fume cupboard | |||
| Hot-plate stirrer | VWR | 640-282 | |
| Microplate reader | VWR | ||
| Microplate washer | VWR | ||
| pH meter | VWR | ||
| Eppendorf Research® pipettes – multipack option 2 | VWR | ||
| Pipette – 1000ul | VWR | ||
| Pipette – 200ul | VWR | ||
| Pipette – 20ul | VWR | ||
| Repeater pipette | VWR | ||
| Pipette filler | VWR | ||
| Orbital shaker | Progen Scientific | ||
| Sonicator | Hilsonic | ||
| Vortex | VWR | ||
| Warm water bath | |||
| Water purification system | Millipore |
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