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.

倫理文：フィールドサンプリングおよび動物対象を含む手順は、ノッティンガム・トレント大学の動物、農村環境科学（ARES）科で承認されています。 糞便サンプルの1コレクション注：糞便サンプルおよびメタノールを取り扱う際は手袋を着用する必要があります。動物が人獣共通感染症に苦しんでいることができることを強い疑いがある場合は、そのような白衣などの防護服も着?...

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+measurement+of+liquid+and+solid+digesta+retention+in+ruminants,+equines+and+rabbits+given+timothy+hay.">The measurement of liquid and solid digesta retention in ruminants, equines and rabbits given timothy hay.</a> British Journal of Nutrition. 48, 329-339 (1982).</li><li>Goymann, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+monitoring+of+hormones+in+bird+droppings:+biological+validations,+sampling,+extraction,+sex+differences+and+the+influence+of+diet+on+hormone+metabolite+levels.">Non-invasive monitoring of hormones in bird droppings: biological validations, sampling, extraction, sex differences and the influence of diet on hormone metabolite levels.</a> Annals of the New York Academy of Sciences. 1046, 35-53 (2005).</li><li>Touma, C., sachser, N., Mostl, E., Palme, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+sex+and+time+of+day+on+metabolism+and+excretion+of+corticosterone+in+urine+and+feces+of+mice.">Effects of sex and time of day on metabolism and excretion of corticosterone in urine and feces of mice.</a> General and comparative Endocrinology. 130, 267-278 (2003).</li><li>Rattenbacher, S., Mostl, E., Hackl, R., Ghareeb, K., Palme, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+corticosterone+metabolites+in+chicken+droppings.">Measurement of corticosterone metabolites in chicken droppings.</a> British Poultry Science. 45, 704-711 (2004).</li><li>Yarnell, K., Hall, C., Billett, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assessment+of+the+aversive+nature+of+an+animal+management+procedure+using+behavioural+and+physiological+measures.">An assessment of the aversive nature of an animal management procedure using behavioural and physiological measures.</a> Physiology &amp; Behaviour. 118, 32-39 (2013).</li><li>Goymann, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+use+of+non-invasive+hormone+research+in+uncontrolled,+natural+environments:+the+problem+with+sex,+diet,+metabolic+rate+and+the+individual.">On the use of non-invasive hormone research in uncontrolled, natural environments: the problem with sex, diet, metabolic rate and the individual.</a> Methods in Ecology and Evolution. 3, 757-765 (2012).</li><li>Sheriff, M. J., Dantzer, B., Delehanty, B., Palme, R., Boonstra, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+stress+in+wildlife:+techniques+for+quantifying+glucocorticoids.">Measuring stress in wildlife: techniques for quantifying glucocorticoids.</a> Oecologia. 166, 614-619 (2011).</li><li>Watson, R., Munro, C. J., Edwards, K. L., Norton, V., Brown, J. L., Walker, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+versatile+enzyme+immunoassay+for+non-invasive+assessment+of+glucocorticoid+metabolites+in+a+diversity+of+taxonomic+species.">Development of a versatile enzyme immunoassay for non-invasive assessment of glucocorticoid metabolites in a diversity of taxonomic species.</a> General Comparative Endocrinology. 186, 16-24 (2013).</li><li>Touma, C., Palme, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+fecal+glucocorticoid+metabolites+in+mammals+and+birds:+the+importance+of+validation.">Measuring fecal glucocorticoid metabolites in mammals and birds: the importance of validation.</a> Annals of the New York Academy of Sciences. 1046, 54-74 (2005).</li><li>Millspaugh, J. J., Washburn, B. 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糞便のコルチコステロン分析は、ウマにおいて副腎活動の長期的パターンを評価する手段を提供します。方法の非侵襲的な性質は、唾液、血漿分析9を含む副腎活性を評価するために使用される他のサンプリング方法の交絡効果を克服します。自由変動の馬を研究する場合に加えて技術は明確な非侵襲的な利点を有しています。 このメソッドとその適切な利用に?...

この方法は、副腎活性の非侵襲的評価を提供するために馬の糞便中のコルチコステロン濃度を分析することを目的とします。視床下部 - 下垂体 - 副腎（HPA）軸の活性を測定すると、キャプティブと国内の両方の種において潜在的に嫌悪事態への対応を研究するための一般に認められた手法です。参考技術と最も広く使用される方法は、しかしながら、例えば、糞便分析などの別の方法は、遊離の範囲の種を監視する能力を採血自体によって誘発されるストレスを克服し、可能にするために開発されてきた血漿1の使用です。 嫌悪状況の間に、生理的恒常性が破壊されています。下垂体前葉に作用し、副腎皮質刺激ホルモン（ACTH）の放出を刺激ホルモン放出ホルモン（CRH）の副腎皮質脳のリリースでは視床下部。 ACTHは血流に入り、種を分泌する副腎皮質を刺激しますsの特定のグルココルチコイド（GC）。グルココルチコイドは密接ではなく、一貫したがって、彼らはしばしば他のストレスリンクのホルモン2の上に優先的に測定されているすべてのエネルギーの高まりの状態で製造されるよりも、ストレスの多いイベントにリンクされています。グルココルチコイドは、馬のいくつかの適応効果を担当しています。エネルギーが急速に酸素摂取量を増加させ、脂肪酸およびグルコースの形で体内の貯蔵部位から動員され、感覚機能は、3と、血流を向上さ動き4に必要でない領域まで低下します。同様に対処メカニズムとして機能するように、グルココルチコイドの応力誘起される上昇はまた、次のストレッサー5のために動物を準備するのを助けることができます。 血漿および唾液中のホルモンのレベルを評価することは、糞便中の代謝物の測定は、ホルモンの代謝最終産物を測定し、しかし実際の循環ホルモンを測定することを含みます。循環ステロイドはリットルに異化されています彼らは腸のトラック6内細菌叢の酵素活性によって容易にさらなる変化を受ける胆汁に排泄前にアイバー。したがって、血液グルココルチコイドに向けイムノアッセイは、糞便、グルココルチコイド代謝物7の分析に適していないかもしれません。 糞便収集が馬に乱れを行うことができるように、コルチコステロンのための糞の分析は、多くの状況でHPA活性をモニターするために広く使用されています。馬の糞便中の上昇コルチコステロンは、術後獣医の治療8の間にあり、限定ハウジング9に含む潜在的嫌悪状況に応じて報告されています。糞便サンプルは、時間をかけてプールされたグルココルチコイドレベルではなく、血漿および唾液長期的に監視することが適切で作る、慢性または季節的パターン10によって提供される時間サンプリング点を反映しています。非侵襲性に起因し方法の性質は、サンプルを捕捉または拘束具11を必要とせずに、個々の繰り返し収集することができます。サンプリングプロトコルを計画する際しかし、種特異的腸通過時間を考慮しなければなりません。馬では、腸の通過時間は約18時間12したがって、副腎応答およびその後のコルチコステロン代謝物は1日HPA軸の初期起動後に糞便中に検出することができます。 非侵襲的なイムノアッセイ技術を利用する場合検討さ種について注意深く検証は13不可欠です。また、ホルモンの代謝物の排泄の性差が原因で、マウス14、鶏15を含む様々な種に排泄コルチコステロン代謝物の代謝速度とタイプの違いにおそらく報告されています。アッセイは目に詳述されているように男性と女性の国内の馬の両方で使用するために検証されたそのため、この方法の一部として重要でした電子プロトコル。男女間のホルモン代謝におけるこの違いは、アドレス指定されず、アッセイの検証の一部として含まれているまれまだデータ品質に対する影響を有しています。 この非侵襲的方法は、国内のウマにおける副腎活動の長期評価を可能にします。プロトコルの詳細分析の検証およびアッセイ技術自体の両方。

<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.

国内馬（N = 16は、8頭の牝馬、8去勢馬）15年（±3）の平均年齢と性別に応じてグループ化され、社会的孤立のレベルの増加と4住宅のデザインを施した（n = 4の馬/処理）しました。密接に彼らの自然の生息地をシミュレートし、群れ環境に住んでいる住宅1関与馬。屋内納屋でペアに住んでいる住宅2関与馬。ハウジング3関与馬は厩舎ではなく、他の馬と馬のハウジング4?...

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.

糞便グルココルチコイド分析：ウマにおける非侵襲性副腎モニタリング

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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