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.

بيان الأخلاق: الإجراءات التي تنطوي على أخذ العينات الميدانية والحيوان موضوعات تم اعتمادها من قبل مدرسة الحيوان، الريفية والعلوم البيئية (آريس) في جامعة نوتنغهام ترنت. 1. جمع عينات البراز ملاحظة: يجب ارتداء القفازات ?...

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يوفر تحليل البراز كورتيزون وسيلة لتقييم أنماط طويلة المدى لنشاط الغدة الكظرية في الخيول. طبيعة غير الغازية من طريقة تتغلب على آثار الخلط من طرق أخذ العينات الأخرى المستخدمة لتقييم نشاط الغدة الكظرية بما في ذلك اللعاب والبلازما تحليل 9. وبالإضافة إلى ذلك الأسلو...

أهداف الطريقة الموصوفة لتحليل تركيز الكورتيزون في البراز الخيول من أجل توفير تقييم غير الغازية للنشاط الغدة الكظرية. قياس النشاط (HPA) محور الغدة النخامية، الغدة الكظرية هو نهج قبولهم للدراسة الاستجابة لحالات يحتمل أن تكون مكره في كل من الأنواع في الأسر والمحلية. تقنية الإشارة وطريقة الأكثر استخداما هو استخدام بلازما الدم 1 ومع ذلك، الطرق البديلة مثل تحليل البراز وضعت من أجل التغلب على الإجهاد الناجم عن اخذ عينات من الدم نفسها والسماح القدرة على رصد الأنواع الطليقة. أثناء وضع مكره، تعطل التوازن الفيزيولوجي. منطقة ما تحت المهاد في الدماغ النشرات المنمية القشرية الافراج عن هرمون (CRH) الذي يعمل على الغدة النخامية الأمامية ويحفز الافراج عن الهرمون الموجه لقشر الكظر (ACTH). ACTH يدخل مجرى الدم، ويحفز قشرة الغدة الكظرية لإفراز مسكوكةق السكرية محددة (GC). ترتبط ارتباطا وثيقا السكرية الأحداث المجهدة بدلا من أن تنتج باستمرار في جميع ولايات الطاقة المتزايدة ولذا غالبا ما يتم قياسها في تفضيل اكثر من غيرها من الإجهاد ترتبط الهرمونات 2. السكرية هي المسؤولة عن العديد من الآثار التكيف في الخيول. وحشدت الطاقة بسرعة من مواقع التخزين في الجسم في شكل من الأحماض الدهنية والجلوكوز، وزيادة كمية الأوكسجين، مما يعزز وظيفة حسية (3) وانخفاض تدفق الدم الى مناطق غير الضرورية لحركة 4. وكذلك بوصفها آلية التعامل، وارتفاع التوتر الناجم في السكرية قد يساعد أيضا على إعداد الحيوان للالضغوطات المقبل 5. تقييم مستويات الهرمون في البلازما واللعاب ينطوي على قياس هرمون تعميم الفعلي ومع ذلك، وقياس نواتج الأيض في التدابير البراز المنتج النهائي التمثيل الغذائي للهرمون. وcatabolized المنشطات المتداولة في لترإيفر قبل الإفراز في لالصفراء حيث أنها تخضع لتغييرات أخرى بتسهيل من الأنشطة الإنزيمية من النباتات البكتيرية داخل مسار الأمعاء 6. لذلك، المناعية الموجهة نحو السكرية في الدم قد لا تكون مناسبة لتحليل نواتج الأيض جلايكورتيكود البرازية 7. كما يمكن أن يتم جمع البراز من دون أي إزعاج للحصان، وتحليل البراز للكورتيزون، وقد استخدم على نطاق واسع لمراقبة نشاط HPA في عدد من الظروف. تم الإبلاغ عن كورتيزون ارتفاع في البراز من الخيول استجابة لحالات يحتمل أن تكون مكره بما في ذلك أثناء العلاج البيطري بعد العملية 8 وفي السكن تقييدا ​​9. تعكس أخذ العينات البرازية مستوى جلايكورتيكود المجمعة مع مرور الوقت بدلا من نقطة في أخذ العينات في الوقت التي تقدمها البلازما واللعاب مما يجعله مناسبا لرصد المدى الطويل المزمن أو الأنماط الموسمية 10. ويرجع ذلك إلى غير الغازيةطبيعة الأسلوب، والعينات ويمكن جمع مرارا وتكرارا للفرد دون الحاجة إلى التقاط أو ضبط النفس 11. ومع ذلك، يجب أن تؤخذ أنواع محددة وقت عبور القناة الهضمية في الاعتبار عند التخطيط لبروتوكول أخذ العينات. في الخيول، والوقت الأمعاء عبور حوالي 18 ساعة 12 وبالتالي، يمكن الكشف عن استجابة الغدة الكظرية والأيض كورتيزون لاحقة في البراز بعد يوم واحد تفعيل الأولي للمحور HPA. عندما تستخدم تقنيات المناعية غير الغازية التحقق من صحة دقيق لهذه الأنواع يجري التحقيق ضروري 13. وبالإضافة إلى ذلك، تم الإبلاغ عن الفروق بين الجنسين في هرمون الأيض إفراز ربما بسبب الاختلافات في معدل الأيض ونوع الأيض كورتيزون تفرز في مختلف الأنواع بما في ذلك الفئران 14، والدجاج (15). ولذلك كان من المهم كجزء من هذا الأسلوب الذي تم التحقق من صحة فحص للاستخدام في كل من الذكور والإناث الخيول المحلية كما هو مفصل في البروتوكول البريد. هذا الاختلاف في عملية الأيض الهرموني بين الجنسين له عواقب على نوعية البيانات إلا أنه نادرا ما يتم معالجتها وتضمينها كجزء من التحقق من صحة الفحص. وتسمح هذه الطريقة غير الغازية تقييم المدى الطويل من النشاط الغدة الكظرية في الخيول المحلية. تفاصيل البروتوكول كل من المصادقة على فحص وتقنية فحص نفسها.

<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.

الخيول المحلية (ن = 16، 8 الأفراس، 8 الحيوانات المخصية) تم تجميع مع متوسط ​​أعمارهم 15 عاما (± 3) وفقا لنوع الجنس وتعرض لأربعة تصاميم المساكن مع مستويات متزايدة من العزلة الاجتماعية (ن = 4 حصان / العلاج). الإسكان 1 تشارك الخيول الذين يعيشون في بيئة القطيع، م...

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.

برازي تحليل جلايكورتيكود: رصد الغدة الكظرية غير الغازية في الخيليات

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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