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.

윤리 문 : 현장 샘플링과 동물 과목을 포함하는 절차는 노팅엄 트렌트 대학의 동물, 농촌 및 환경 과학 (ARES)의 학교에 의해 승인되었습니다. 대변​​ 샘플 1. 컬렉션 참고 : 분변 샘플 및 메탄올을 취급 할 때는 장갑을 착용한다. 동물은 또한 착용해야 실험실 코트와 같은 인수 공통 질병, 보호 복을 앓고 될 수 있다는 강한 의심이있는 경우. <ol><li> 배변 다음 (몇 시간에 분 이내) 가능?...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domesticated+horses+differ+in+their+behavioural+and+physiological+responses+to+isolated+and+group+housing.">Domesticated horses differ in their behavioural and physiological responses to isolated and group housing.</a> Physiology and Behaviour. 143, 51-57 (2015).</li><li>Wielebnowski, N., Watters, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+fecal+endocrine+monitoring+to+conservation+and+behaviour+studies+of+wild+mammals:+important+considerations+and+preliminary+tests.">Applying fecal endocrine monitoring to conservation and behaviour studies of wild mammals: important considerations and preliminary tests.</a> Israel journal of ecology and evolution. 53, 439-460 (2007).</li><li>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+steroids:+guidelines+for+a+practical+application.">Measuring fecal steroids: guidelines for a practical application.</a> Annals of the New York Academy of Sciences. 1046, 75-80 (2005).</li><li>Uden, P., Rounsaville, G. R., Wiggans, G. R., Van Soest, P. 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. 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>

분변 코르티 분석 말 부신 활성의 장기 패턴을 평가하는 수단을 제공한다. 상기 방법의 비 침습적 자연 타액 플라즈마 분석 9 포함한 부신 활성을 평가하기 위해 사용되는 다른 샘플링 방법의 교란 효과를 극복한다. 또한이 기술은 무료에 이르기까지 말을 공부하는 경우 명확한 비 침습적 인 장점이 있습니다. 이 방법의 적절한 사용에 관한 논의하기 위해 몇 가지 주?...

방법 설명 목적은 부신 활성의 비 침습적 평가를 제공하기 위해서 말 대변 코르티 코스 농도를 분석한다. 시상 하부 - 뇌하수체 - 부신 (HPA) 축 활동을 측정하는 것은 포로 및 국내 종 모두에서 잠재적으로 혐오 상황에 대한 반응을 연구하는 허용 방법입니다. 참조 기술과 가장 널리 사용되는 방법은 그러나, 배설물 분석과 같은 다른 방법이없는 범위 종을 감시하는 기능을 채혈 자체에 의해 유도 된 응력을 극복 할 수 있도록하기 위해 개발 된 혈장 (1)의 사용이다. 혐오 상황시, 생리적 항상성이 파괴된다. 앞쪽에 뇌하수체에 작용하여 부 신피질 자극 호르몬 (ACTH)의 방출을 자극 방출 호르몬 (CRH)를 corticotrophin 뇌 릴리스의 시상 하부. ACTH는 혈류를 입력하고 종을 분비하는 부신 피질을 자극s의 특정 글루코 코르티코이드 (GC). 글루코 코르티코이드는 밀접하지 않고 지속적으로 그러므로 그들은 종종 다른 스트레스 링크 호르몬이 이상 환경에서 측정 된 모든 에너지를 높게 한 상태에서 생산되고보다 스트레스가 이벤트에 연결되어 있습니다. 글루코 코르티코이드는 말을 여러 적응 효과에 대한 책임이 있습니다. 에너지가 급속 지방산 및 글루코스의 형태로 본문에 저장 사이트로부터 동원되는 산소 섭취량은 감각 기능 3을 향상 혈류가 이동 4 불필요 영역 감소, 증가된다. 뿐만 아니라 대응 메커니즘으로 작용하는 글루코 코르티코이드에 의한 응력 상승은 다음 스트레스 5 동물을 제조하는 데 도움이된다. 호르몬 혈장 수준과 침을 평가하는 것은 배설물 대책 호르몬의 대사 최종 제품 대사를 측정하지만 실제 순환 호르몬을 측정하는 것을 포함한다. 순환 스테로이드는 리터에 이화된다그들은 장 트랙 6에서 세균성 식물의 효소 활성에 의해 촉진 추가 변경을 거쳐 담즙에 배설하기 전에 이베. 따라서, 혈중 글루코 코르티코이드 지향 면역은 배설물 글루코 코르티코이드의 대사 산물 (7)의 분석에 적합하지 않을 수 있습니다. 찌끼 모음 말 아무런 방해하여 수행 될 수있는 바와 같이, 코르티 대한 대변의 분석은 다수의 환경에서 HPA 활동을 모니터링하기 위해 광범위하게 사용되고있다. 말의 배설물에 상승 코르티 코스 테론은 수술 후 의학적 치료 8시 제한적 하우징 (9)을 포함하여 잠재적 인 혐오 상황에 대한 응답으로보고되었다. 대변 ​​샘플링은 시간이 지남에 풀링 된 글루코 코르티코이드 수준보다는 장기, 만성 또는 계절 패턴 (10)을 모니터링하기위한 적절하고 혈장과 타액에서 제공하는 시간 샘플링에있는 점을 반영한다. 때문에 비 침습적으로방법의 본질은, 샘플 캡처하거나 구속 11 없이도 개인 반복적으로 수집 될 수있다. 샘플링 프로토콜을 계획 할 때, 종 특정 장내 통과 시간이 고려되어야한다. 말에서 소화관 통과 시간 따라서, 부신 반응과 후속 코르티 대사는 HPA 축의 활성화 후 초기 언젠가 대변 검출 할 수 약 18 내지 12 시간이다. 비 침습적 면역 기술을 활용하면 조사중인 종에 대한주의 깊은 검증이 13 필수적이다. 또한, 호르몬 대사 산물의 배설에 성별 차이로 인해 신진 대사 속도와 마우스 (14), 닭 (15) 등 다양한 종으로 배설 코르티 코스 테론의 대사 산물의 종류의 차이에 아마보고되었다. 일에 자세히 설명되어 있습니다로는 분석이 남성과 여성의 국내 말 모두에 사용하기 위해 검증 된이 방법의 일환으로, 따라서 중요전자 프로토콜입니다. 남녀 간의 호르몬 대사의 차이는 해결하지 않고 분석 검증의 일부로 포함 거의되어 아직 데이터 품질에 대한 처벌을 받게됩니다. 이 비 침습적 방법은 국내 말에 부신 활동의 장기 평가를 할 수 있습니다. 프로토콜 세부 분석의 검증 및 분석 기술 자체를 모두.

<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.

국내 말 (N = 16, 8 암말, 8 geldings) 십오년 (± 3)의 평균 연령은 성별과 사회적 고립 (N = 4 말 / 치료)의 증가 수준 4 개의 주택 디자인 대상에 따라 분류 하였다. 밀접하게 그들의 자연 서식지를 시뮬레이션, 무리 환경에서 생활 주택 1 관련된 말. 실내 헛간에 쌍에 살고있는 주택이 포함 된 말. 하우징 (3) 참여 말은 마구간에 있지만, 다른 말과 말의 하우징 (4) 관련 총 격리 시각?...

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.

대변​​ 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

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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대변​​ 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

대변 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링