Fækal Glukokortikoid Analyse: Non-invasive Adrenal Overvågning i dyr fra hestefamilien
Summary
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.
Abstract
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.
Introduction
Fremgangsmåden beskrevet formål at analysere corticosteron koncentrationer i heste afføring for at tilvejebringe en ikke-invasiv vurdering af adrenal aktivitet. Måling hypothalamus-hypofyse-binyre (HPA) aksen aktivitet er en accepteret metode til at studere reaktion på potentielt afskrækningsmiddel situationer i både i fangenskab og tamdyr. Henvisningen teknik og den mest anvendte metode er brugen af blodplasma 1 har imidlertid alternative metoder såsom fækalt analyse blevet udviklet for at overvinde stress induceret af blodprøveudtagning selv og tillade muligheden for at overvåge fritgående arter.
Under et afskrækningsmiddel situation er fysiologisk homøostase forstyrret. Hypothalamus i hjernen frigiver corticotrophin frigørende hormon (CRH), som virker på den forreste hypofyse og stimulerer frigivelsen af adrenokortikotropt hormon (ACTH). ACTH ind i blodbanen og stimulerer binyrebarken til at udskille specieDette findes specifikke glukokortikoider (GC). Glukokortikoider er tæt knyttet til stressende begivenheder snarere end at blive konsekvent fremstilles i alle energirelaterede øget stater de er derfor ofte målt i præference frem for andre stress forbundet hormoner 2. Glukokortikoider er ansvarlig for flere adaptive effekter i heste. Energi er hurtigt mobiliseres fra opbevaringssteder i kroppen i form af fedtsyrer og glucose, er iltoptagelse øges, sensorisk funktion er forbedret 3 og blodgennemstrømningen er reduceret til områder, der ikke er nødvendige bevægelse 4 for. Samt at fungere som en coping mekanisme, kan stress inducerede stigning i Glukokortikoider også bidrage til at forberede dyret til næste stressor fem.
Vurdere hormon niveauer i plasmaet og spyttet involverer måling af faktiske cirkulerende hormon dog måle metabolitterne i fæces foranstaltninger den metaboliske slutprodukt af hormonet. Cirkulerende steroider kataboliseres i lIver før udskillelse i til galden, hvor de gennemgår yderligere ændringer lettes af de enzymatiske aktiviteter af bakterieflora i tarmsystemet spor 6. Derfor kan immunoassays rettet mod blod glukokortikoider ikke være egnet til analyse af fækale glukokortikoid metabolitter 7.
Som fækalt samling kan udføres uden forstyrrelse af hesten, analyse af afføring for corticosteron, er blevet anvendt i vid udstrækning til at overvåge HPA-aktivitet i en række omstændigheder. Forhøjede corticosteron i fæces af heste er blevet rapporteret som reaktion på potentielt afskrækningsmiddel situationer, herunder ved postoperativ dyrlægebehandling 8 og i restriktiv hus 9. Fækal sampling afspejler et pooled glukokortikoid niveau over tid snarere end det tidspunkt, prøveudtagning, der tilbydes af plasma og spyt gør det hensigtsmæssigt, at overvågning af langsigtet, kroniske eller sæsonbestemte mønstre 10. På grund af den ikke-invasivkarakter af fremgangsmåden kan indsamles prøver gentagne gange for en individuel uden behov for indfangning eller fastholdelsesanordning 11. Dog skal der tages artsspecifik gut transittid i betragtning, når man planlægger en prøvetagningsprotokol. Hos heste gut transittiden er omkring 18 timer 12 derfor kan detekteres adrenal respons og efterfølgende corticosteron metabolitter i fæces én dag efter første aktivering af HPA-akse.
Når der anvendes ikke-invasive immunoassayteknikker er afgørende 13 en omhyggelig validering for de arter, der undersøges. Desuden har kønsforskelle i hormon metabolitudskillelse blevet rapporteret sandsynligvis på grund af forskelle i metabolisk hastighed og typen af corticosteron metabolit udskilles i forskellige arter, herunder mus 14 og høns 15. Det var derfor vigtigt som en del af denne metode, at analysen er valideret til brug i både mandlige og kvindelige indenlandske heste som er detaljeret i the-protokollen. Denne forskel i hormon stofskifte mellem kønnene har konsekvenser for datakvalitet men det er sjældent rettet til, og indgår som en del af analysen validering.
Denne ikke-invasiv metode giver udtryk vurdering af adrenal aktivitet længe i indenlandske heste. Detaljerne protokol både validering af analysen og analysen teknik selv.
Protocol
Representative Results
Discussion
Fækal corticosteron analyse giver et middel til at vurdere langsigtede mønstre af adrenal aktivitet i heste. Den ikke-invasive karakter af metoden overvinder forstyrrende virkninger af andre prøvetagningsmetoder anvendes til at vurdere adrenal aktivitet, herunder spyt og plasma-analyse 9. Derudover har teknikken en klar ikke-invasiv fordel, hvis studere fritgående heste.
Der er flere vigtige punkter at diskutere vedrørende denne metode og dens hensigtsmæssig brug. Et kritisk…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Corticosterone antibody & HRP kit | Coralie Munro – UC Davis | NA | No longer available through UC Davis – please see Arbor Assays |
| Cortisol antibody & HRP kit | Coralie Munro – UC Davis | NA | No longer available through UC Davis – please see Arbor Assays |
| Corticosterone synthetic standard hormone | Sigma Aldrich | 50-23-7 | Harnful if ingested or with skin contact. Use in fume cupboard |
| Cortisol synthetic standard hormone | Sigma Aldrich | 15087-01-1 | Harnful if ingested or with skin contact. Use in fume cupboard |
| Methanol | Sigma Aldrich | 67-56-1 | Irritant. Use in fume cupboard |
| Sodium Bicarbonate | Sigma Aldrich | 144-55-8 | Irritant |
| Sodium Carbonate Anhydrous | Sigma Aldrich | 497-19-8 | Irritant |
| Sodium Phosphate Dibasic | Sigma Aldrich | 7558-79-4 | Irritant |
| Sodium Phosphate Monobasic | Sigma Aldrich | 10049-21-5 | Irritant |
| BSA | Sigma Aldrich | 9048-46-8 | Irritant |
| Tween 20 | Sigma Aldrich | 9005-64-5 | Irritant |
| Citric Acid | Sigma Aldrich | 77-92-9 | Irritant |
| ABTS | Sigma Aldrich | 30931-67-0 | Irritant |
| Hydrogen Peroxide 30% | Sigma Aldrich | 7722-84-1 | Irritant |
| Sodium Chloride | Sigma Aldrich | 7647-14-5 | Irritant |
| Buffer capsules – pH 4 | VWR | 332732B | |
| Buffer capsules – pH 7 | VWR | 332742D | |
| Buffer capsules – pH 10 | VWR | 332762H | |
| Hydrochloric Acid | Sigma Aldrich | 435570 | Irritant. Use in fume cupboard |
| Sodium Hydroxide | Sigma Aldrich | S5881 | Irritant |
| Analytical balance | Fisher Scientific | BFS-525-010A | |
| Air compressor | |||
| Centrifuge | |||
| Computer +printer | |||
| fridge-freezer | |||
| Drying apparatus | |||
| +tubing | |||
| Flammable liquid storagecabinet | VWR | 649-002 | |
| Fume cupboard | |||
| Hot-plate stirrer | VWR | 640-282 | |
| Microplate reader | VWR | ||
| Microplate washer | VWR | ||
| pH meter | VWR | ||
| Eppendorf Research® pipettes – multipack option 2 | VWR | ||
| Pipette – 1000ul | VWR | ||
| Pipette – 200ul | VWR | ||
| Pipette – 20ul | VWR | ||
| Repeater pipette | VWR | ||
| Pipette filler | VWR | ||
| Orbital shaker | Progen Scientific | ||
| Sonicator | Hilsonic | ||
| Vortex | VWR | ||
| Warm water bath | |||
| Water purification system | Millipore |
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