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.

Ethik-Anweisung: Verfahren beteiligt Feld Probenahme und Tier Themen wurden von der Schule für Tier, ländliche und Umweltwissenschaften (ARES) an der Nottingham Trent University genehmigt. 1. Sammlung von Kotproben HINWEIS: sollten Handschuhe getragen werden, wenn Stuhlproben und Umgang mit Methanol. Wenn es einen starken Verdacht, dass ein Tier aus einem Zoonose, Schutzkleidung wie Laborkittel leiden könnten, sollten auch getragen werden. <ol><li> Sammeln Sie die Kotproben so schnell wie möglich (...

<ol>
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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. 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Fecal Corticosteron Analyse stellt ein Mittel zur bei Pferden langfristig Muster der Nebentätigkeit zu beurteilen. Die nicht-invasive Art des Verfahrens überwindet die verwirrende Wirkung anderer Stichprobenverfahren verwendet , um adrenale Aktivität einschließlich Speichel und Plasmaanalyse 9 beurteilen. Darüber hinaus hat die Technik eine klare, nicht-invasive Vorteil, wenn freilaufenden Pferde zu studieren. Es gibt mehrere wichtige Punkte in Bezug auf diese Methode und ihr...

Das beschriebene Verfahren Ziele Corticosteron-Konzentrationen in equine Kot, um eine nicht-invasive Beurteilung von adrenalen Aktivität bereitzustellen zu analysieren. Hypothalamus-Hypophysen-Nebennieren-Achse (HPA) Aktivität zu messen, ist ein akzeptierter Ansatz, um die Reaktion auf potenziell aversiven Situationen sowohl in Gefangenschaft und Haustierarten zu untersuchen. Die Referenztechnik und die am weitesten verbreitete Methode ist die Verwendung von Blutplasma 1 jedoch alternative Methoden wie Stuhlanalyse wurden entwickelt, um sich durch Blutentnahme induziert , den Stress zu überwinden und ermöglichen die Fähigkeit , frei lebenden Arten zu überwachen. Während einer aversiven Situation wird physiologische Homöostase gestört. Der Hypothalamus im Gehirn Freisetzungen Releasing Hormon (CRH), Corticotropin, die auf der vorderen Hypophyse wirkt und stimuliert Freisetzung von adrenocorticotropen Hormons (ACTH). ACTH gelangt in den Blutkreislauf und regt die NNR specie absonderns spezifische Glucocorticoide (GC). Glucocorticoide sind eng mit belastenden Ereignissen verknüpft und nicht in allen Energie erhöhte Staaten konsequent produziert deshalb werden sie häufig bevorzugt , gemessen über andere Stress verbunden Hormone 2. Glucocorticoide sind mehrere adaptive Effekte bei Pferden verantwortlich. Energie schnell von Speicherstellen im Körper in Form von Fettsäuren und Glukose mobilisiert wird, wird die Sauerstoffaufnahme erhöht, sensorische Funktion 3 erhöht und der Blutfluss zu den Bereichen verringert nicht erforderlich , für die Bewegung 4. Neben dem Einsatz als Bewältigungsmechanismus wirkt, kann der Stress induzierten Anstieg der Glukokortikoide auch das Tier für die nächste Stressor helfen 5 vorzubereiten. Beurteilung der Hormonspiegel im Plasma und Speichel beinhaltet jedoch die tatsächliche zirkulierenden Hormons Messung der Stoffwechselprodukte in den Faeces Maßnahmen die metabolische Endprodukt des Hormons gemessen wird. Zirkulieren Steroide sind in der l katabolisiertIver vor Ausscheidung in die Galle , wo sie weitere Veränderungen durch die enzymatischen Aktivitäten der Bakterienflora im Darm Spur 6 erleichtert unterziehen. Daher auf Blut Glucocorticoide gerichtet Immunoassays kann nicht für die Analyse von Stuhl Glukokortikoidmetaboliten 7 geeignet sein. Als fäkale Sammlung kann das Pferd ohne Störung durchgeführt werden, die Analyse der Faeces für Corticosteron, umfassend zu überwachen, HPA-Aktivität in einer Reihe von Umständen verwendet wurde. Erhöhte Corticosteron im Kot von Pferden hat als Reaktion auf potenziell aversiven Situationen berichtet worden 9 während der postoperativen Behandlung Veterinär 8 und in restriktiver Gehäuse einschließlich. Fecal Probenahme spiegelt eine gepoolte Glucocorticoid - Ebene im Laufe der Zeit , statt den Zeitpunkt Probenahme von Plasma und Speichel bot es angemessen für die Überwachung der langfristigen machen, chronische oder saisonale Muster 10. Aufgrund der nicht-invasiveNatur des Verfahrens Proben 11 ohne die Notwendigkeit für die Aufnahme oder Rückhalte wiederholt für einen einzelnen gesammelt werden. Allerdings müssen bestimmte Darmtransitzeit Arten berücksichtigt werden, wenn ein Probenahmeprotokoll zu planen. Bei Pferden ist gut Laufzeit ca. 18 Std 12 daher, Neben Reaktion und anschließender Corticosteron - Metaboliten in den Kot einen Tag nach der ersten Aktivierung der HPA - Achse erfasst werden. Wenn nicht-invasive Techniken Immunoassay verwendet eine sorgfältige Validierung für die Spezies untersucht 13 von wesentlicher Bedeutung. Darüber hinaus Geschlechtsunterschiede in der Hormon Ausscheidung der Metaboliten wurden in Stoffwechselrate und die Art des Corticosteron - Metaboliten ausgeschieden in verschiedenen Arten , darunter Mäuse 14 und Hühner 15 aufgrund von Unterschieden berichtet wahrscheinlich ist. Es war daher wichtig, als Teil dieses Verfahrens, daß der Assay für die Verwendung sowohl in männlichen und weiblichen Hauspferde validiert wurde wie detailliert in the Protokoll. Dieser Unterschied in der Hormonmetabolismus zwischen den Geschlechtern hat Konsequenzen für die Datenqualität noch ist es selten als Teil der Assayvalidierung angesprochen und einbezogen. Diese nicht-invasive Methode ermöglicht langfristige Beurteilung der Nebentätigkeit bei domestizierten Pferden. Die Protokolldetails sowohl die Validierung des Assays und der Assay-Technik selbst.

<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.

Hauspferde (n = 16, 8 Stuten, 8 Wallache) mit einem mittleren Alter von 15 Jahren (± 3) wurden nach Geschlecht gruppiert und zu vier Gehäusebauformen mit zunehmenden sozialen Isolation unterworfen (n = 4 Pferd / Behandlung). Das Gehäuse 1 beteiligt Pferde in einer Herde Umgebung leben, eng ihrem natürlichen Lebensraum zu simulieren. Das Gehäuse 2 beteiligt Pferde paarweise in einer Indoor-Scheune leben. Das Gehäuse 3 beteiligten Pferde allein in Ställen untergebracht, aber mit Sic...

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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.

Fecal Glucocorticoid-Analyse: Nicht-invasive Monitoring-Adrenal in 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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