Name: fecal glucocorticoid analysis: non-invasive adrenal monitoring in equids
Uploaded: 2016-04-25T13:00:00
Description: Watch this Scientific Journal Video about Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids at JoVE.com

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.

आचार बयान: क्षेत्र नमूना और पशु विषयों को शामिल प्रक्रियाओं नॉटिंघम ट्रेंट विश्वविद्यालय में पशु, ग्रामीण और पर्यावरण विज्ञान (ARES) के स्कूल द्वारा अनुमोदित किया गया है। 1. मलीय नमूनों के संग्रह नो?...

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+fecal+glucocorticoid+metabolite+measures+in+conservation+biology+research:+considerations+for+application+and+interpretation.">Use of fecal glucocorticoid metabolite measures in conservation biology research: considerations for application and interpretation.</a> General and Comparative Endocrinology. 138, 189-199 (2004).</li></ol>

मलीय corticosterone विश्लेषण घोड़ों में अधिवृक्क गतिविधि की लंबी अवधि के पैटर्न का आकलन करने का एक साधन प्रदान करता है। विधि के गैर इनवेसिव प्रकृति लार और प्लाज्मा विश्लेषण 9 सहित अधिवृक्क गतिविधि का आकलन ?...

विधि वर्णित उद्देश्य आदेश अधिवृक्क गतिविधि का एक गैर-आक्रामक मूल्यांकन प्रदान करने के लिए घोड़े का मल में corticosterone सांद्रता का विश्लेषण करने के लिए। मापने हाईपोथेलेमिक पिट्यूटरी-अधिवृक्क (एचपीए) अक्ष गतिविधि दोनों बंदी और घरेलू प्रजातियों में संभावित प्रतिकूल स्थितियों पर प्रतिक्रिया का अध्ययन करने के लिए एक स्वीकृत दृष्टिकोण है। संदर्भ तकनीक और सबसे व्यापक रूप से इस्तेमाल किया विधि हालांकि, इस तरह मल विश्लेषण के रूप में वैकल्पिक तरीकों का आदेश तनाव रक्त नमूने स्वयं के द्वारा प्रेरित दूर करने और मुक्त करने के लिए लेकर प्रजातियों की निगरानी करने की क्षमता अनुमति देने के लिए विकसित किया गया है रक्त प्लाज्मा 1 का इस्तेमाल होता है। एक प्रतिकूल स्थिति के दौरान, शारीरिक homeostasis बाधित है। रिहा हार्मोन (CRH) जो पूर्वकाल पिट्यूटरी ग्रंथि पर कार्य करता है और adrenocorticotrophic हार्मोन (ACTH) की रिहाई को बढ़ावा corticotrophin मस्तिष्क विज्ञप्ति में हाइपोथेलेमस। ACTH खून में प्रवेश करती है और नकदी छिपाना अधिवृक्क प्रांतस्था को उत्तेजित करता हैविशिष्ट glucocorticoids (जीसी)। Glucocorticoids बारीकी बजाय लगातार सभी ऊर्जा बढ़ राज्यों इसलिए वे अक्सर अन्य तनाव से जुड़े हार्मोन पर 2 वरीयता में मापा जाता है में उत्पादन किया जा रहा तनाव की घटनाओं से जुड़ी हैं। Glucocorticoids घोड़ों में कई अनुकूली प्रभाव के लिए जिम्मेदार हैं। ऊर्जा तेजी से फैटी एसिड और ग्लूकोज के रूप में शरीर में भंडारण साइटों से जुटाए है, ऑक्सीजन की मात्रा बढ़ जाती है, संवेदी समारोह 3 बढ़ाया है और रक्त प्रवाह आंदोलन 4 के लिए आवश्यक नहीं क्षेत्रों के लिए कम हो जाती है। साथ ही एक मुकाबला तंत्र के रूप में काम कर के रूप में, Glucocorticoids में तनाव प्रेरित वृद्धि भी अगले तनाव 5 के लिए पशु तैयार करने में मदद कर सकता है। प्लाज्मा में हार्मोन का स्तर और लार का आकलन हालांकि वास्तविक घूम हार्मोन को मापने, हार्मोन के चयापचय अंत उत्पाद मल उपायों में चयापचयों मापने शामिल है। परिसंचारी स्टेरॉयड एल में catabolized रहे हैंपित्त, जहां वे आंतों 6 ट्रैक के भीतर बैक्टीरिया वनस्पति के enzymatic गतिविधियों द्वारा सुविधा आगे के परिवर्तन से गुजरना करने में उत्सर्जन से पहले iver। इसलिए, रक्त glucocorticoids की ओर निर्देशित immunoassays मल glucocorticoid चयापचयों 7 के विश्लेषण के लिए उपयुक्त नहीं हो सकता। के रूप में मल संग्रह घोड़े के लिए कोई अशांति के साथ किया जा सकता है, corticosterone के लिए मल का विश्लेषण, परिस्थितियों की संख्या में HPA गतिविधि पर नजर रखने के लिए बड़े पैमाने पर इस्तेमाल किया गया है। घोड़ों के मल में ऊंचा corticosterone पोस्ट ऑपरेटिव पशु चिकित्सा उपचार 8 दौरान और प्रतिबंधात्मक आवास 9 में शामिल संभावित प्रतिकूल स्थितियों के जवाब में सूचना दी गई है। मल के नमूने एक जमा glucocorticoid बजाय इसे लंबे समय तक, क्रोनिक या मौसमी पैटर्न में 10 की निगरानी के लिए उपयुक्त बनाने के समय प्लाज्मा और लार द्वारा की पेशकश नमूने में बिंदु समय के साथ स्तर को दर्शाता है। गैर इनवेसिव के कारणविधि की प्रकृति, नमूने कब्जा या संयम 11 के लिए आवश्यकता के बिना एक व्यक्ति के लिए बार-बार एकत्र किया जा सकता है। हालांकि, विशिष्ट प्रजाति पेट पारगमन समय था जब एक नमूना प्रोटोकॉल की योजना बना ध्यान में रखा जाना चाहिए। घोड़ों में, पेट पारगमन समय के आसपास 18 घंटा 12 इसलिए, अधिवृक्क प्रतिक्रिया और बाद में corticosterone चयापचयों HPA अक्ष की प्रारंभिक सक्रियण के एक दिन बाद मल में पाया जा सकता है। जब गैर-आक्रामक प्रतिरक्षा तकनीक का उपयोग प्रजातियों के लिए एक सावधान सत्यापन जांच की जा रही आवश्यक 13 है। इसके अलावा, हार्मोन मेटाबोलाइट उत्सर्जन में लिंग भेद चयापचय दर और corticosterone मेटाबोलाइट चूहों 14, और मुर्गियों 15 सहित विभिन्न प्रजातियों में उत्सर्जित के प्रकार में मतभेद के कारण शायद सूचित किया गया है। इसलिए यह इस पद्धति है कि परख दोनों पुरुष और महिला घरेलू घोड़ों में उपयोग के लिए मान्य किया गया था के हिस्से के रूप में महत्वपूर्ण था, क्योंकि वीं में विस्तृत हैई प्रोटोकॉल। लिंग के बीच हार्मोन चयापचय में यह अंतर यह शायद ही कभी किया जाता है को संबोधित किया और परख सत्यापन के हिस्से के रूप में शामिल अभी तक डेटा की गुणवत्ता के लिए परिणाम है। यह गैर इनवेसिव विधि घरेलू घोड़ों में अधिवृक्क गतिविधि की लंबी अवधि के मूल्यांकन की अनुमति देता। प्रोटोकॉल विवरण दोनों परख के सत्यापन और परख तकनीक ही।

<table>
	<tbody>
		<tr>
			<td>Corticosterone antibody &#38; HRP kit</td>
			<td>Coralie Munro - UC Davis</td>
			<td>NA</td>
			<td>No longer available through UC Davis - please see Arbor Assays</td>
		</tr>
		<tr>
			<td>Cortisol antibody &#38; HRP kit</td>
			<td>Coralie Munro - UC Davis</td>
			<td>NA</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>Harnful 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&#174; pipettes - multipack option 2</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette - 1000ul</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette - 200ul</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette - 20ul</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.

घरेलू घोड़ों (एन = 16, 8 घोड़ी, 8 geldings) 15 साल (± 3) का एक मतलब उम्र के साथ लिंग और सामाजिक अलगाव (एन = 4 घोड़ा / उपचार) के बढ़ते स्तर के साथ चार आवास डिजाइन के अधीन अनुसार वर्गीकृत किया गया। हाउसिंग 1 शामिल ह?...

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.

मलीय Glucocorticoid विश्लेषण: Equids में गैर-आक्रामक अधिवृक्क निगरानी

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

The overall goal of this procedure is to demonstrate how adrenal activity can be assessed non-invasively in equines through the extraction and analysis of fecal glucocortocoid metabolites. This method can help us answer key questions in the area of welfare assessment by looking at the physiological impacts of husbandry protocols on domestic horses, all the way through to wild populations of African zebras. The main advantage of this technique is it offers a non-invasive option for measuring the long-term adrenal activity in both domestic and free-ranging horses.On the day of the experiment, after thawing at room temperature, manually homogenize the fecal samples by mixing within each individual sample bag. Then, for each sample, measure out the appropriate amount of feces on a micro balance in a clean weigh boat, and transfer the samples into individual eight milliliter glass vials. Next, mix five milliliters of 90%methanol in water with each sample, and shake the samples overnight on an orbital shaker at room temperature.The following morning, vortex the samples and spin them down. At the end of the centrifugation, decant the methanol fractions into individual 16 by 125 millimeter glass tubes. Then briefly place the tubes in a 37-degree Celsius water bath, followed by evaporation of the methanol under air in a fume cupboard.When all of the methanol has dispersed, resuspend the fecal extracts in one milliliter of 100%methanol. Then transfer the extracts into individual 12 by 75 millimeter polypropylene tubes, cap the tubes, and store the samples at minus 20 degrees Celsius until their analysis. Before beginning the analysis, use a repetitive pipette with a 50 microliter pipette tip to load 50 microliters of polyclonal corticosterone CJM006 antiserum, diluted in coating buffer, per well, in a 96 well microtiter plate.Then cover the plate with a plate sealer and incubate the antiserum overnight at four degrees Celsius. The next day, immediately before beginning the analysis, use an automated microtiter plate washer to wash the plate five times with wash solution. Then load the nonspecific binding and zero wells with 50 microliters of enzyme-linked immunoassay, or EIA buffer, per well, the standard wells with the appropriate concentrations of corticosterone, and the experimental wells with the fecal extract samples diluted in EIA buffer.After loading the last sample, immediately add 50 microliters of horseradish perodixase conjugate diluted in EIA buffer to each well, and incubate the microtiter plate in the dark for two hours at room temperature. At the end of the incubation, wash the plate with wash solution five times. Then incubate the samples with 100 microliters per well of freshly prepared, room temperature substrate in the dark at room temperature, with periodic reading of the plate at 405 nanometers on a spectrophotometer.The incubation is considered complete when the optical density of the zero wells reaches 0.8 to 1.0. To perform a biochemical validation for parallelism, perform a serial dilution on pooled fecal extracts, ideally taken from suspected high-and low-hormone concentration samples from several individuals. Then run the samples on the EIA as just demonstrated.A parallelism is considered achieved when the serial dilutions of the fecal extracts yield a displacement curve parallel to the standard curve, and the linear regression results demonstrate an R squared greater than 0.9, a slope greater than 0.5, and a P less than 05. To perform a biochemical validation for interference, spike 200 microliters of serial diluted standards with 200 microliters of the appropriate pooled fecal extract, diluted at the 50%binding as determined by the parallelism assay. Then run the samples on the EIA as demonstrated.Following the EIA analysis, plot the observed concentration minus the background concentration of the zero sample versus the expected concentration. No interference is considered achieved if the addition of diluted pooled fecal extract to the standards does not significantly alter the amount expected, and linear regression results yields R squared greater than 0.9, a slope close to one, and P less than 05. To perform a biological validation, run samples extracted before, and after a challenging event on the EIA as just demonstrated.The biological validation is a critical step for the validation of EIA, and is considered achieved when a significant rise in glucocorticoid metabolites is observed after a challenging event. The data in graph A and B demonstrate the biochemical validation of adult male and female domestic horses, by demonstrating parallelism with a corticosterone standard curve. No biochemical validation with the cortisol EIA was achieved, as there was no parallelism between the pooled fecal extract and the cortisol standard curve.These data from challenged male and female domestic horses demonstrate biological validation, as the concentration of fecal corticosterone increased as the level of isolation increased in these animals. Indeed, the horses in the isolated housing situation exhibited significantly higher levels of fecal corticosterone, compared to all of the other housing designs. So assessing hormone concentrations in saliva or plasma usually involves us looking at the native hormone, but when we're assessing it in fecal material, we're looking at a breakdown of that product and looking at the metabolic end products of that hormone.Indeed, fecal sampling offers a pooled glucocortocoid concentration over time, rather than the single point reflected by saliva or plasma. This makes it more appropriate for measuring long term, chronic or seasonal adrenal activity. So as fecal sample collection is non-invasive to the animal, it's been used extensively to monitor equine adrenal activity in numerous natural and experimental circumstances.When running EIA, it is important to minimize the time of loading the microtiter plate to under 10 minutes, as longer times will result in drift. It is also important to ensure that duplicate samples have a coefficient of variation of less than 10%and controls have an intra-and inter-assay coefficient variation of 10%and 15%respectively. So after watching this video, you should have a really good understanding of how to extract fecal glucocortocoid metabolites, analyze those extracts in an enzyme immunoassay, and also perform the required validation steps.

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