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 (innerhalb von Minuten bis zu einigen Stunden) nach einer Darmentleerung und legen Sie sie in einen Probenbeutel. Dauern drei bis fünf Unterstichproben von jeder defecated Probe mit einem Gesamtgewicht von ca. 10 g. Beschriften Sie die Tasche mit dem Datum und Uhrzeit der Sammlung, individuelle ID und gefrieren die Probe bei -20 ° C bis zur Analyse. </li><li> Zur Minimierung der Fehler, einfrieren alle Proben bei -20 ° C, bis die Studie abgeschlossen ist, die Proben zusammen für die Analyse als eine einzelne Charge dann sammeln. &lt;/ Li&gt; </ol> 2. Fecal Hormon Extraktion <ol><li> Lassen Sie die Kotproben bei RT auftauen. </li><li> Stellen Sie sicher, dass die Proben durch manuelles Mischen jede Probe in einem eigenen Probenbeutel homogenisiert werden. </li><li> Für jede Probe in einem Boot dann wiegen auf einer Mikrowaage 0,50 (± 0,003 g) von Fäkalien wiegen übertragen die Fäkalien auf ein 8 ml Glasfläschchen. Verwenden Sie eine neue wiegen Boot für jede Probe und reinigen Sie die Pinzette mit 30% Methanol zwischen jeder Probe. </li><li> Kombinieren Sie die 0,5 g Fäkalien mit 5 ml 90% Methanol: Wasser, und schütteln O / N auf einem Orbitalschüttler bei RT. </li><li> Am nächsten Morgen, Strudel der Probe und dann für 20 Minuten bei 600 g zentrifugiert werden. </li><li> Man dekantiert die Methanolfraktion in eine 16 x 125 mm Glasrohr 2 und in einem Gestell in einem Wasserbad auf eine Temperatur von 37 ° C eingestellt. Dampfe in einem Abzug mit Luft bis zur Trockenheit. Spülen Sie die Extraktion Fläschchen und legen Sie die restlichen fäkalen Pellets in einer klinischen Abfallbeutel zur Entsorgung bringen. </li><li> Re-suspend die Stuhlextrakten in 100%, 1 ml Methanol und Transfer in ein 12 x 75 mm 2 Polypropylenröhrchen. Sofort verschließen und bei -20 ° C bis zur Analyse. </li></ol> 3. Hormonanalyse <ol><li> Führen Sie die Analyse mit einem Enzym Linked-Immunoassay (EIA) polyklonalen Corticosteron CJM006 Antiserum. . HINWEIS: Eine zweite EIA (Cortisol R4866 Antiserum getestet wurde, aber dies konnte die biochemische Validierungsschritt (siehe Abschnitt 4.1) Die polyklonalen Corticosteron CJM006 Antiserum und Cortisol R4866 Antiserum und die jeweilige HRP des von CJ Munro, University of California geliefert werden, zu erreichen, Davis, CA. </li><li> Verdünnen Sie das Antiserum bei 1: 15.000 in Beschichtungspuffer (0,05 M NaHCO3, pH 9,6) eine sich wiederholende Pipette und eine 50 ul Pipettenspitze, legen Sie eine 96-Well Mikrotiterplatte mit 50 &amp; mgr; l / Vertiefung. Die Platte mit Abklebefolie und bebrüten O / N bei 4 ° C. Verlassen zwei Vertiefungen, die nicht-spezifischen Bindungs ​​Vertiefungen darzustellen ungestrichen (NSB). </li><li> Unmittelbar vor dem Gebrauch waschen Sie die Mikrotiterplatten fünfmal eine automatisierte Mikrotiterplatte Wascher (Waschlösung, 0,15 M NaCl, 0,05% Tween 20). </li><li> Legen Sie die Platten mit 50 &amp; mgr; l pro Vertiefung für die NSB und &quot;Null&quot; Brunnen [EIA - Puffer (0,1 M NaPO 4, 0,149 M NaCl, 0,1% Rinderserumalbumin, pH 7,0) nur], Normen (Corticosteron, 3,9 - 1000 pg / gut) oder Fäkalextrakt Proben (in geeigneter Weise um 1:20 EIA-Puffer verdünnt, siehe Abschnitt 4.1). Standards und Nullen sollten in dreifacher Ausfertigung und vierfacher Ausfertigung, jeweils ausgeführt werden. Verwässertes Fäkalextrakt Proben und NSB sollte doppelt durchgeführt werden. </li><li> Folgen Sie diese sofort mit der Zugabe von 50 &amp; mgr; l / Vertiefung des Etiketts Meerrettichperoxidasekonjugat ᵻ in EIA - Puffer verdünnt auf 1: 70.000. </li><li> Inkubieren der Mikrotiterplatten im Dunkeln für 2 h bei RT. </li><li> Waschen Sie die Mikrotiterplatten mit Waschlösung 5 mal und Inkubation der Platten bei RT im Dunkeln mit 100 ul / well der RT - Substrat [0,4 mM 2,2&#39;-Azino-di- (3-ethylbenzthiazolinsulfonsäure) diammoniumsalz, 1,6 mM H 2 O 2, 0,05 M Citrat, pH 4,0]. Vorbereiten des Substrats unmittelbar vor der Verwendung. </li><li> Verwenden Sie ein Spektrophotometer bei einer Wellenlänge von 405 nm der Mikrotiterplatten zu lesen. Inkubation der Platten wird als vollständig betrachtet, wenn die optische Dichte der Null Vertiefungen 0,8 bis 1,0 erreicht. </li><li> Um sicherzustellen , Wiederholbarkeit, verwenden Kontrollen [EIA - Standards oder einer biologischen Probe , verdünnt in EIA - Puffer bei etwa 30% (EIA - Standard) zu binden, 50% (eine biologische Probe zB Pool von Hauspferd Fäkalextrakt) und 70% (EIA - Standard) ] intra- und inter-Assay-Variationskoeffizienten von &lt;10% und &lt;15% bzw. zu demonstrieren. </li></ol> 4. Enzyme Linked-Immunoassay: Validierung für die Studie Spezies und Sex <ol><li> Biochemische Validation (Parallelism) <ol><li> Mit EIA Puffer, führen Sie eine serielle Verdünnung auf gepoolten fäkale Extract, im Idealfall genommen von Verdacht auf hohe und niedrige Hormonkonzentration Proben von mehreren Personen (Bereich: ordentlich, 1: 2 bis 1: 8192) und auf der EIA laufen, wie in Kapitel 3 beschrieben. ANMERKUNG: Eine Parallelität erreicht betrachtet, wenn die Reihenverdünnungen von Stuhlextrakten eine Verschiebungskurve parallel zu der Standardkurve und der linearen Regressionsergebnisse ergeben zeigen R2&gt; 0,90, eine Steigung&gt; 0,50 und p &lt;0,05. Die serielle verdünnte Fäkalextrakt , die rund 50% auf die Standardkurve bindet sollte die ideale Verdünnung betrachtet werden alle Stuhlextrakten zu laufen (zB 1:20). Es gibt keine Störungen auf den Assay betrachtet, wenn die lineare Regressionsergebnisse zeigen, R2&gt; 0,90 und p &lt;0,05. </li></ol></li><li> Biochemische Validation (Interferenz) <ol><li> Spike 200 &amp; mgr; l seriell verdünnte Standards (Null, 3,9 bis 1000 ng / well Corticosteron) mit 200 ul der entsprechend verdünnten gepoolt Fäkalextrakt [bei 50% verdünnt paralleli bestimmten Bindungsm (1:20)] und laufen auf der EIA wie in Abschnitt 3 EIA Analyseplot Nach der beobachtete Konzentration minus der Hintergrund (Konzentration der Nullprobe) im Vergleich zu der erwarteten Konzentration. HINWEIS: Wenn die Zugabe von verdünnter gepoolt Fäkalextrakt Standards nicht wesentlich ändern , um die Menge zu erwarten und die lineare Regression Ergebnisse liefert R 2&gt; 0,90, eine Steigung nahe bei 1,0 und p &lt;0,05. Keine Interferenz mit der EIA wurde erreicht. </li></ol></li><li> Biologische Validierung: Hinweis: Eine Demonstration , dass die gewählte EIA die Fähigkeit zur Messung der biologisch relevanten Änderungen des Hormons von Interesse hat [zB der Anstieg der Glukokortikoid - Konzentrationen nach einer pharmakologischen (ACTH ist oft ein lizenzierter Verfahren) oder biologische Herausforderung (zB psychologische und / oder Umwelt, eher eine opportunistische unregulierten Ereignis)] zu sein. <ol><li> Wählen Sie, zu extrahieren und zu analysieren Kotproben für Glucocorticoid MetabolitkonzentrationenVerwendung des Verfahrens in Abschnitten ausführlich 1-3 vor und nach einem schwierigen Fall. HINWEIS: Eine anspruchsvolle Veranstaltung artspezifisch sein wird. Ein beispielhaftes Verfahren wird im Folgenden für das Hauspferd zur Verfügung gestellt. Ausführliche Beispiele für anspruchsvolle Veranstaltungen für Pferde wurden zuvor einschließlich der Verwendung von Management - 9 und Haltungsverfahren 16 veröffentlicht. Ein signifikanter Anstieg der Glucocorticoid-Metabolitenkonzentrationen nach dem herausfordernden Ereignis muss anhand einer statistischen Analyse beobachtet und bestätigt werden. Diese müssen über die gegebenenfalls die Wirkung der wiederholten Messungen, Proben Datum und individuell. Biologische Validierung wird angenommen, dann erreicht. </li></ol></li></ol>

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The authors have nothing to disclose.

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...

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.

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, ...

fecal-glucocorticoid-analysis-non-invasive-adrenal-monitoring

Research

JoVE Journal

Biology

9.6K Aufrufe.  Nottingham Trent University. 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.

Serie: Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Mouse Adrenal Chromaffin Cell Isolation

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex.  The digestion of the medulla into single chromaffin cells is then demonstrated.

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated.

Maus Adrenal Chromaffin Zellisolation

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This ...

Forschung

Biologie

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Labeling hESCs und hMSCs mit Eisenoxid-Nanopartikel für Non-Invasive in vivo-Tracking mit MR-Bildgebung

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Labeling Stammzellen mit fluoreszierenden Farbstoffen zur nicht-invasiven Detektion mit Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

Nicht-invasive 3D-Visualisierung mit Sub-Mikron-Auflösung mit Synchrotron-Röntgen-Tomographie

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

Eine reversible, nicht-invasive Methode für Airway Resistance-Messungen und bronchoalveolären Lavage Sampling in Mäuse

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Langfristige letale Toxizität Test mit dem Krustentier Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Schnelle Genetische Analyse von epithelial-mesenchymale Signaling Während Haarregeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Eine Stoff Screening Verfahren für die Glucocorticoid-Signalisierung mit einem Zebrafisch-Larve Luciferase-Reportersystems

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Who is Who? Non-invasive Methods to Individually Sex and Mark Altricial Chicks

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal welfare guidelines, non-invasive techniques should be preferred whenever applicable. In our group, we work on different species of song birds in the lab and in the field, and we successfully apply non-invasive methods to sex and individually mark chicks. This paper presents a comprehensive non-invasive tool-box. Sexing birds prior to the expression of secondary sexual traits requires the collection of DNA-bearing material for PCR. We established a quick and easy method to sex birds of any age (post hatching) by extracting DNA from buccal swabs. Results can be obtained within 3 hours. For individual marking chick's down feathers are trimmed in specific patterns allowing fast identification within the hatching order. This set of methods is easily applicable in a standard equipped lab and especially suitable for working in the field as no special equipment is required for sampling and storage. Handling of chicks is minimized and marking and sexing techniques are non-invasive thereby supporting the RRR-principle of animal welfare guidelines.

This protocol provides a convenient set of methods, which enables extremely fast, easy, non-invasive, reliable and low-cost, molecular sex determination of birds and their non-invasive, quick, safe and easily recognizable marking shortly after hatching. Only limited handling of chicks is required. This convenient toolbox of methods complies entirely with the RRR-guidelines.

Wer ist wer? Nicht-invasive Methoden zur Individuell Sex und Mark Nesthocker Chicks

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal ...

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.

LDL-Cholesterin-Aufnahme-Assay mit Live Cell Imaging-Analyse mit Zelle Systemüberwachung

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...