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.

Ethiek statement: procedures waarbij de bemonstering en dierlijke veld onderwerpen zijn goedgekeurd door de School of Animal, Rural en Environmental Science (ARES) aan de Nottingham Trent University. 1. Het verzamelen van fecale monsters LET OP: Handschoenen moeten gedragen worden bij de behandeling van fecale monsters en methanol. Als er een sterk vermoeden dat een dier zou kunnen lijden aan een zoönose, beschermende kleding, zoals een laboratoriumjas ook worden gedragen. <ol><li> Verzamel de fecale ...

<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>

Fecale corticosteron analyse geeft een evaluatieinstrument langdurige patronen van bijnieractiviteit bij paarden. De niet-invasieve karakter van de methode overwint de verstorende effecten van andere bemonsteringsmethoden gebruikt om de adrenaline-activiteit met inbegrip van speeksel en plasma-analyse 9 te beoordelen. Daarnaast heeft de techniek een duidelijk non-invasieve voordeel als het bestuderen van loslopende paarden. Er zijn een aantal belangrijke punten te bespreken met be...

De beschreven werkwijze beoogt corticosteron concentraties in paarden ontlasting analyseren om een ​​niet-invasieve evaluatie van adrenale activiteit. Meten hypothalamus-hypofyse-bijnier (HPA) as activiteit een geaccepteerde aanpak van de reactie op potentieel aversieve situaties zowel gevangen en huisdiersoorten bestuderen. De referentietechniek en de meest gebruikte methode is het gebruik van bloedplasma 1 hebben echter alternatieve werkwijzen zoals fecale analyse ontwikkeld om de stress veroorzaakt door bloedafname zichzelf te lossen en kan bijhouden loslopende species. Tijdens een aversieve situatie wordt fysiologische homeostase verstoord. De hypothalamus in de hersenen releases corticotrophine releasing hormoon (CRH), die werkt op de hypofyse en stimuleert de afgifte van adrenocorticotroop hormoon (ACTH). ACTH in de bloedbaan en stimuleert de adrenale cortex specie scheidens specifieke glucocorticoïden (GC). Glucocorticoïden zijn nauw verbonden met stressvolle gebeurtenissen in plaats van constant geproduceerd in alle energie verhoogde staten daarom zijn ze vaak gemeten in voorkeur ten opzichte van andere stress-hormonen gekoppeld 2. Glucocorticoïden zijn verantwoordelijk voor verschillende adaptieve effecten bij paarden. Energie snel gemobiliseerd uit opslagplaatsen in het lichaam in de vorm van vetzuren en glucose wordt zuurstofopname verhoogd, sensibiliteit versterkt 3 en de bloedstroom wordt verminderd tot niet nodig voor beweging 4. Evenals als een coping-mechanisme, kan de stress veroorzaakte stijging van Glucocorticoïden ook helpen om het dier te bereiden op de volgende stressor 5. Het beoordelen hormoonspiegels in het plasma en speeksel omvat het meten van de werkelijke circulerend hormoon echter meten metabolieten in de feces meet de metabole eindproduct van het hormoon. Circulerende steroïden worden gekataboliseerd in de lIver vóór excretie in de gal, waar ze verdere veranderingen vergemakkelijkt door de enzymatische activiteiten van de bacteriële flora in de darm nummer 6 ondergaan. Daarom kunnen immunotesten gericht bloed glucocorticoïden niet geschikt voor de analyse van fecale metabolieten glucocorticoïde 7 zijn. Zoals fecale verzameling met geen verstoring van het paard kan worden uitgevoerd, analyse van feces voor corticosteron, wordt veelvuldig gebruikt om HPA te monitoren in een aantal omstandigheden. Verhoogde corticosteron in de uitwerpselen van paarden is gemeld in reactie op potentieel aversieve situaties zoals bij postoperatieve veterinaire behandeling 8 en 9 beperkende behuizing. Fecal bemonstering weerspiegelt een gepoolde glucocorticoïden niveau na verloop van tijd in plaats van het moment bemonstering aangeboden door plasma en speeksel waardoor het geschikt is voor het toezicht op de lange termijn, chronische of seizoenspatronen 10. Vanwege de niet-invasieveaard van de werkwijze kan herhaald worden monsters verzameld voor een individu zonder de noodzaak voor het vangen of bevestigingssysteem 11. Er moet echter soortspecifiek gut transittijd rekening worden gehouden bij de planning van een sampling protocol. Bij paarden gut reistijd is ongeveer 18 uur 12 derhalve bijnierrespons gevolgd corticosteron metabolieten kunnen worden in de feces gedetecteerd één dag na de eerste activering van de HPA-as. Bij het ​​gebruik van niet-invasieve immunoassaytechnieken zorgvuldige validatie van de soort onderzochte essentieel 13. Bovendien zijn sexeverschillen bij hormoon metabolietenuitscheiding is waarschijnlijk gemeld als gevolg van verschillen in metabolisme en een type corticosteron metaboliet in verschillende soorten, waaronder muizen 14 en 15 kippen. Het was dus belangrijk onderdeel van deze werkwijze dat de test werd gevalideerd voor gebruik in zowel mannelijke als vrouwelijke gedomesticeerde paarden zoals beschreven in The protocol. Dit verschil hormoon- tussen geslachten nog gevolgen voor datakwaliteit is zelden gericht en als onderdeel assay validatie. Deze niet-invasieve methode maakt het mogelijk lange termijn evaluatie van de adrenaline-activiteit in de binnenlandse paarden. Het protocol gegevens zowel de validatie van de test en de test techniek zelf.

<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.

Gedomesticeerde paarden (n = 16, 8 merries, 8 ruinen) met een gemiddelde leeftijd van 15 jaar (± 3) werden gegroepeerd op basis van geslacht en onderworpen aan vier woningen ontwerpen met toenemende mate van sociaal isolement (n = 4 paard / behandeling). Behuizing 1 betrokken paarden leven in een kudde milieu, op de voet te simuleren hun natuurlijke habitat. Behuizing 2 betrokken paarden wonen in paren in een inpandige schuur. Behuizing 3 betrokken paarden alleen gehuisvest in stallen, ...

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.

Fecale Glucocorticoïd Analyse: Niet-invasieve bijnier Toezicht op Equids

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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