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.

Etik açıklama: Alan örnekleme ve hayvan denekleri prosedürleri Nottingham Trent Üniversitesi&#39;nde Animal, Kır ve Çevre Bilimi (ARES) Okulu tarafından onaylanmıştır. Dışkı Örneklerinde 1. Koleksiyonu NOT: Dışkı örnekleri ve metanol tutarken eldiven giyilmelidir. Bir hayvan gibi de giyilmelidir bir laboratuvar önlüğü gibi zoonotik hastalık, koruyucu giysiler muzdarip olabileceğini güçlü bir şüphe varsa. <ol><li> dışkılama aşağıdaki (birkaç saat için dakika içinde) en kısa sürede dışkı örnekleri toplamak ve örnek bir torbaya koyun. yaklaşık 10 g toplam ağırlığa sahip bir defecated numunenin üç ila beş Alt-numûnelerin al. zaman ve toplama, bireysel kimlik tarihi ile çanta Etiket ve analize kadar -20 ° C&#39;de örnek dondurmak. </li><li> tüm örnekleri dondurmak, hatayı en aza indirmek için -20 ° C kadar çalışma tamamlandığında sonra tek bir toplu olarak analiz için bir araya örnekleri toplamak. &lt;/ Li&gt; </ol> 2. Dışkı Hormon Ekstraksiyon <ol><li> Oda sıcaklığında çözülmeye dışkı örnekleri bırakın. </li><li> Numuneler el ile kendi örnek torba içinde her numune karıştırılarak homojenize emin olun. </li><li> Her bir numune, ağırlığa tekne bir mikro 0.50 dışkı maddesi (g 0.003 ±) ağırlığında için daha sonra bir 8 ml bir cam şişeye dışkı aktarın. Her bir numune için yeni bir ağırlık teknesi kullanın ve her bir numune arasında% 30 metanol ile cımbız temizleyin. </li><li> 5% 90 ml metanol ile, dışkı maddesinin 0.5 g &#39;ını: su, ve oda sıcaklığında bir orbital çalkalayıcıda O / N sallayın. </li><li> Ertesi sabah, örnek karıştırın ve daha sonra 600 g&#39;de 20 dakika boyunca santrifüj. </li><li> 37 ° C lik bir sıcaklığa ayarlanmış bir su banyosu içinde rafa 16 x 125 mm cam tüpe 2 ve yerine metanol fraksiyonu süzün. Bir çeker ocak içinde hava ile kuruyana kadar buharlaştırın. ekstraksiyon şişeyi yıkayın ve bertaraf için bir klinik atık torbasına kalan dışkı pelet yerleştirin. </li><li> % 100 1 ml metanol dışkı özü yeniden askıya ve 12 x 75 mm2 bir polipropilen tüpü içine transfer. Hemen analiz edilinceye kadar -20 ° C&#39;de kap saklayın. </li></ol> 3. Hormon Analizleri <ol><li> poliklonal kortikosteron CJM006 antiserum kullanarak bir enzim bağlantılı-immunoassay (EIA) ile analizi yürütmek. NOT:. İkinci ÇED (Kortizol R4866 antiserum test edilmiştir, fakat bu (Bölüm 4.1 bakınız) poliklonal kortikosteron CJM006 antiserum ve kortizol R4866 antiserum ve ilgili HRP en CJ Munro, California Üniversitesi tarafından sağlanan biyokimyasal doğrulama adımı gerçekleştirmek için başarısız, Davis, CA </li><li> 1 ile antiserumun seyreltilir: kaplama tamponu (0.05 M NaHCO3, pH 9.6) &#39;de 15,000 tekrar pipet ve 50 ul bir pipet kullanarak, / göz 50 ul, 96 oyuklu bir mikrotitre plakası yükleyin. Bir plaka kapatıcı ile örtün ve plaka, 4 ° C&#39;de O / N inkübe edin. (N, spesifik olmayan bağlayıcı gözenekler temsil kaplanmamış iki kuyu boşSB). </li><li> Kullanımdan hemen önce, bir otomatik mikrotitre plaka yıkayıcısı (0.15 M NaCl,% 0.05 Tween 20 yıkama çözeltisi) kullanılarak, mikro ölçüm levhaları, beş kez yıkayın. </li><li> 1000 ug / - NSB en için oyuk başına 50 ul ve &#39;sıfır&#39; kuyu [EIA tampon maddesi (0.1 M napo 4, 0.149 M NaCl,% 0.1 sığır serum albümini, pH 7.0) içinde, sadece] standartları (kortikosteron, 3.9 plakaları yük kuyu) veya uygun 01:20 ÇED tamponu seyreltilmiş dışkı özü numuneleri (,) bölüm 4.1&#39;e bakınız. Standartlar ve sıfır sırasıyla üç kez ve dört nüsha olarak çalıştırılmalıdır. Sulandırılmış dışkı özü örnekleri ve NSB en nüsha halinde çalıştırılmalıdır. </li><li> 70.000: 50 ul / oyuk ile 1 EIA tamponu içinde seyreltilmiş etiket yaban turpu peroksidaz bağı ᵻ hemen takip etmektedir. </li><li> Oda sıcaklığında 2 saat boyunca karanlıkta mikrotitre inkübe edin. </li><li> 100 ul ile karanlıkta, oda sıcaklığında plakalar yıkama çözeltisi ile 5 kez mikrotitre plakalar, yıkama ve inkübe / welRT substrat [0.4 mM 2,2&#39;-azino-di- (3-etilbenztiyazolin-sülfonik asit) diamonyum tuzu, 1.6 mM H2O 2, 0.05 M sitrat, pH 4.0] l. kullanımdan hemen önce alt-tabakanın hazırlanması. </li><li> mikrotitre plakaları okumak için 405 nm bir dalga boyunda bir spectrophometer kullanın. Sıfır kuyu optik yoğunluk ile 1.0 0.8 ulaştığında levha kuluçka tamamlandıktan olarak kabul edilir. </li><li> Tekrarlanabilirlik sağlamak için, yaklaşık% 30 (EIA standardı),% 50 (biyolojik numune örneğin, yerli at dışkı ekstresinin havuz) ve% 70 (EIA standardı) bağlamak için ÇED tamponunda seyreltilmiş kontrolleri [ÇED standartları ya da biyolojik bir örnek kullanınız ] sırasıyla &lt;% 10 değişim ve &lt;% 15 oranında bir içi ve inter-analiz katsayılarını göstermek için. </li></ol> 4. Enzim Linked-immunoassay: Çalışma Tür ve Sex Doğrulama <ol><li> Biyokimyasal Doğrulama (Paralellik) <ol><li> ÇED tampon kullanarak, toplanmış fekal ekstra bir seri seyreltme gerçekleştirmekct, ideal olarak birkaç bireylerden şüphelenilen yüksek ve düşük hormon konsantrasyonu örneklerinden alınan (aralık: temiz, 1: 8192: 2 ile 1) ve 3. bölümde açıklandığı gibi ÇED çalıştırın. NOT: Dışkı özler seri seyreltme standart eğri ve lineer regresyon sonuçlarına bir deplasman eğrisi paralel verim BIR paralellik sağlanmıştır kabul edilir R2&gt; 0.90, bir yamaç&gt; 0.50 ve p &lt;0.05 göstermektedir. Standart eğri üzerinde% 50 civarında bağlayan seri seyreltilmiş dışkı özü tüm dışkı özler çalıştırmak için ideal bir seyreltme düşünülmelidir (örneğin, 1:20). lineer regresyon sonuçları R2&gt; 0.90 ve p &lt;0.05 göstermek zaman tahlil üzerinde düşünülen hiçbir girişim yoktur. </li></ol></li><li> Biyokimyasal Doğrulama (Girişim) <ol><li> % 50 sulandırılmış uygun toplanmış seyreltilmiş dışkı ekstresi [200 ul paralleli tarafından belirlenen bağlanma ile (3.9 1000 ng / çukur kortikosteron sıfır) seri olarak seyreltilmiş standartların 200 ul Spikesm (1:20)] ve ÇED analiz arsa ardından bölüm 3&#39;teki gibi ÇED üzerinde çalışan gözlenen konsantrasyon eksi beklenen konsantrasyonuna karşı arka plan (sıfır numunenin konsantrasyonu). NOT: standartlara seyreltilmiş toplanmış fekal ekstresinin eklenmesi önemli ölçüde miktarı beklenen ve lineer regresyon sonuçlarını değiştirmez vermezse R 2&gt; 0.90, 1.0 ve p &lt;0.05 yakın bir eğim verir. EIA ile hiçbir girişim elde edilmiştir. </li></ol></li><li> Biyolojik Doğrulama: NOT: Seçilen ÇED ilgi hormon biyolojik ilgili değişiklikleri ölçmek yeteneğine sahip bir gösteri [örneğin, bir farmakolojik aşağıdaki glukokortikoid konsantrasyonlarda artış ya da biyolojik bir meydan okuma (ACTH genellikle lisanslı bir işlemdir) (örneğin, psikolojik ve / veya çevre; bir fırsatçı düzensiz olay olması daha muhtemeldir)]. <ol><li> Seçin, özü ve glukokortikoid metabolit konsantrasyonları için dışkı örnekleri analizönce ve zorlu bir olaydan sonra 3 - bölümlerde 1 detaylı süreci kullanarak. NOT: zorlu bir olay özgü türler olacaktır. Bir örnek yöntemi yerli at için aşağıda verilmiştir. Atlar için zorlu olaylarla dolu örnekler yönetimi 9 ve hayvancılık prosedürleri 16 kullanımı dahil, daha önce yayınlanmıştır. zorlu olayın ardından glukokortikoid metabolit konsantrasyonlarında anlamlı bir artış gözlendi ve istatistiksel analiz kullanılarak onaylanmalıdır. Bu, tekrarlı ölçümlerde, örnek tarih ve bireysel etkisini uygun olan yerlerde içermelidir. Biyolojik doğrulama sonra elde sayılır. </li></ol></li></ol>

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The authors have nothing to disclose.

Dışkı kortikosteron analizi atlarda adrenal aktivitenin uzun süreli desen değerlendirmek için bir araç sağlar. Yöntemin non-invaziv doğa tükürük ve plazma analizi 9 olmak üzere adrenal aktivitesini değerlendirmek için kullanılan diğer örnekleme yöntemleri karıştırıcı etkilerini ortadan kaldırmaktadır. Buna ek olarak teknik ücretsiz değişen atları okuyan eğer net bir non-invaziv bir avantaja sahiptir. Bu yöntemle ve uygun kullanımıyla ilgili gör?...

yöntem tarif amaçları adrenal aktivite non-invaziv bir değerlendirme sağlamak amacıyla at dışkısı kortikosteron konsantrasyonları analiz etmek. hipotalamus-hipofiz-adrenal (HPA) eksen aktivitesinin ölçülmesi esir ve yerli türler hem potansiyel caydırıcı durumlara tepki incelemek için kabul edilmiş bir yaklaşımdır. Referans tekniği ve en yaygın olarak kullanılan yöntem, ancak bu tür dışkı analizi gibi alternatif yöntemler bilgilerini kadar tür izleme olanağı kan örneği kendisinin neden olduğu stres üstesinden gelmek ve sağlamak amacıyla geliştirilmiştir kan plazması 1 &#39;in kullanımı olup. caydırıcı bir durum sırasında, fizyolojik homeostasis bozulur. ön hipofiz bezi üzerinde hareket eder ve adrenokortikotropik hormon (ACTH) salınımını uyarır salgılatıcı hormon (CRH) kortikotropin beyin sürümlerde hipotalamus. ACTH kan dolaşımına girer ve türünden salgılaması için adrenal korteksi uyarırs, belirli glukokortikoidler (GC). Glukokortikoidler yakından oldukça tutarlı nedenle genellikle diğer stres bağlantılı hormonların üzerinde 2 tercih ölçülen tüm enerji artan devletler üretilen daha stresli olaylarla bağlantılıdır. Glukokortikoidler atların birçok adaptif etkileri sorumludur. Enerji hızlı yağlı asitler ve glukoz şeklinde vücuda depolama sitelerinden mobilize, oksijen alımı sensör fonksiyonu 3 geliştirilmiş ve kan akımı hareket 4 için gerekli olmayan alanlara azaltılır, artar. Yanı sıra bir başa çıkma mekanizması olarak hareket olarak, Glukokortikoid stres kaynaklı artış da sonraki stresör 5 için hayvan hazırlamak için yardımcı olabilir. Hormon plazmadaki düzeyleri ve tükürük değerlendirilmesi gaita önlemler hormon metabolik son ürünü metabolitleri ölçmek, ancak gerçek dolaşan hormonu ölçüm içerir. Sirkülasyon steroid l katabolize edilenonlar bağırsak parça 6 içinde bakteri florasının enzimatik faaliyetleri kolaylaştırdı ileri değişikliklere uğrarlar safra için atılımı önce Iver. Bu nedenle, kan glukokortikoid yönelik bağışıklık dışkı glukokortikoid metabolitlerin 7 analizi için uygun olmayabilir. Dışkı toplanması, at için bir rahatsızlık ile gerçekleştirilebilir gibi, kortikosteron için dışkı analizi, koşullar, bir dizi HPA etkinliğini izlemek için yaygın olarak kullanılmaktadır. Atların dışkı içinde yükseltilmiş kortikosteron ameliyat sonrası tedavisi için veteriner hekimliğinde 8 boyunca ve kısıtlayıcı yuva 9 da dahil olmak üzere, potansiyel olarak caydırıcı durumlara cevap olarak bildirilmiştir. Fekal örnekleme zamanla toplanmış glukokortikoid düzeyi ziyade uzun vadeli, kronik veya mevsimsel desenleri 10 izlenmesi için uygun hale plazma ve tükürük tarafından sunulan zaman örnekleme noktasını yansıtır. Nedeniyle non-invaziv içinYöntemin niteliği, numuneler yakalama veya bağlama 11 gerek kalmadan bir birey için, art arda toplanabilir. Bir numune alma protokolü planlama Ancak, türe özgü bağırsaktan geçiş süresi dikkate alınmalıdır. Atlarda, bağırsaktan geçiş süresi bu • durumda, böbrek üstü tepkisi ve daha sonra kortikosteron metabolitleri HPA ekseninin ilk aktivasyon bir gün sonra dışkı ile saptanabilir yaklaşık 18 saat 12&#39;dir. Non-invaziv immunoassay teknikleri kullanılarak zaman araştırılan türler için dikkatli bir doğrulama 13 esastır. Buna ek olarak, hormon metabolit boşaltım cinsiyet farkları nedeniyle metabolizma hızı ve fareler 14. ve tavuk 15 de dahil olmak üzere çeşitli türlerin atılır kortikosteron metaboliti türü farklılıkları muhtemelen bildirilmiştir. th ayrıntılı gibi deney erkek ve dişi, evcil atlar uygulamalarında kullanmak için geçerli olan bu yöntemin bir parçası olarak, bu nedenle önemlie protokolü. Cinsiyetler arasındaki hormon metabolizmasında Bu fark ele alınması ve tahlil doğrulama parçası olarak dahil nadiren edilir henüz veri kalitesi için sonuçları vardır. Bu non-invaziv yöntem yerli atlar adrenal aktivitenin uzun süreli değerlendirmesini sağlar. protokol ayrıntıları testin geçerliliği ve tahlil tekniği kendisi hem de.

<table><tbody><tr><td>Corticosterone antibody &amp;amp; HRP kit</td><td>Coralie Munro - UC Davis</td><td></td><td>No longer available through UC Davis - please see Arbor Assays</td></tr><tr><td>Cortisol antibody &amp;amp; HRP kit</td><td>Coralie Munro - UC Davis</td><td></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>Harmful 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&amp;reg; pipettes - multipack option 2</td><td>VWR</td><td></td><td></td></tr><tr><td>Pipette - 1,000 &amp;micro;l</td><td>VWR</td><td></td><td></td></tr><tr><td>Pipette - 200 &amp;micro;l</td><td>VWR</td><td></td><td></td></tr><tr><td>Pipette - 20 &amp;micro;l</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.

Yurtiçi atları (n = 16, 8 kısrak, 8 geldings) 15 yıl (± 3) yaş ortalaması ile cinsiyete ve sosyal izolasyon (n = 4 at / tedavi) artan seviyeleri ile dört konut tasarımları tabi göre gruplandırıldı. yakından doğal yaşam simülasyonu, bir sürü ortamında yaşayan Konut 1. katılan atlar. kapalı ahırda çiftler yaşayan Konut 2 dahil atlar. Konut 3 tutulan atlar ahırlarda ancak diğer atlar ve atlar konut 4 dahil toplam izolasyon görsel temas yalnız ev sahipliği yap...

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.

Fekal Glukokortikoid Analizi: Tek tırnaklılarda in Non-invaziv Adrenal İzleme

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.6K Görüntüleme.  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.

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

Cortisol Measurement in Koala (Phascolarctos cinereus) Fur

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial species, the koala (Phascolarctos cinereus), faces prolonged exposure to anthropogenic-induced stressors and assessment of chronic stress in wild populations is urgently warranted. One of the most effective ways to measure chronic stress is through analyzing the glucocorticoid hormone cortisol in hair or fur, as it supports physiological and behavioral responses. This laboratory validation study aims to test current techniques to validate an optimal hormone extraction method to be used as a non-invasive measure of cortisol in koala fur. It is recognized that using non-invasive techniques to measure stress hormones is preferred over traditional, invasive techniques due to their ideal practical and ethical standpoints. Additionally, it is comparatively easier to acquire fur from koalas than it is to acquire samples of their blood. This study used samples of koala fur acquired from the Adelaide Koala and Wildlife Hospital to run a number of hormone extraction techniques in an attempt to validate an optimal cortisol extraction method. Results showed that 100% methanol provided the most optimal solvent extraction compared to 100% ethanol or 100% isopropanol based on parallelism results. In conclusion, this method of cortisol extraction from koala fur provided a reliable non-invasive assay that could be used to study chronic stress in koalas.

Cortisol Measurement in Koala Phascolarctos cinereus Fur

We present a protocol to determine the optimal extraction solvent to measure cortisol from koala fur. The solvents used in this protocol are methanol, ethanol and isopropanol. Determining an optimal extraction solvent will aid in reliably measuring fur to determine the impact of chronic stress on koalas.

Koalak'da Kortizol Ölçümü (Phascolarctos cinereus) Kürk

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial ...

Biyokimya

Cortisol Extraction from Sturgeon Fin and Jawbone Matrices

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) and quantify any differences in fin cortisol levels among three main sturgeon species. Fins were harvested from 19 sacrificed sturgeons including seven beluga (Huso huso), seven Siberian (Acipenser baerii), and five sevruga (A. stellatus). The sturgeons were raised in Iranian farms for 2 years (2017-2018), and cortisol extraction analysis was conducted in South Korea (January-February 2019). Jawbones from five H. huso were also used for cortisol extraction. Data were analyzed using the general linear model (GLM) procedure in the SAS environment. The intra- and inter-assay coefficients of variation were 14.15 and 7.70, respectively. Briefly, the cortisol extraction technique involved washing the samples (300 &#177; 10 mg) with 3 mL of solvent (ultrapure water and isopropanol) twice, rotation at 80&#160;rpm for 2.5 min, air-drying the washed samples at room temperature (22-28 &#176;C) for 7 days, further drying the samples using a bead beater at 50 Hz for 32 min and grinding them into powder, applying 1.5 mL methanol to the dried powder (75 &#177; 5 mg), and slow rotation (40 rpm) for 18 h at room temperature with continuous mixing. Following extraction, samples were centrifuged (9,500 x g for 10 min), and 1 mL supernatant was transferred into a new microcentrifuge tube (1.5 mL), incubated at 38 &#176;C to evaporate the methanol, and analyzed via enzyme-linked immunosorbent assay (ELISA). No differences were observed in fin cortisol levels among species or in fin and jawbone cortisol levels between washing solvents. The results of this study demonstrate that the sturgeon jawbone matrix is a promising alternative stress indicator to solid matrices.

In this study, we present a protocol for cortisol extraction from the fin and jawbone of sturgeon species. Fin and jawbone cortisol levels were further examined by comparing two washing solvents followed by ELISA assays. This study piloted the feasibility of jawbone cortisol as a novel stress indicator.

Mersin Balığı Fin ve Çene Kemiği Matrislerinden Kortizol Ekstraksiyonu

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) ...

Çevre

Measuring Biophysical and Psychological Stress Levels Following Visitation to Three Locations with Differing Levels of Nature

Visitation to natural environments has been linked to psychological stress reduction. Although most stress-related research has relied on self-report formats, a growing number of studies now incorporate biological stress-related hormones and catalysts, such as cortisol and &#945;-amylase, to measure levels of stress. Presented here is a protocol to examine the effects on levels of biophysical and psychological stress following visitation to three different locations with differing levels of nature. Biophysical and self-reported psychological stress levels are measured immediately upon entering the selected locations and just prior to the visitors leaving the site. Using a &#34;drool&#34;&#160;method, the biophysical measure consists of 1-2 mL samples of saliva provided by study subjects upon entry to one of three study locations. As prescribed by extant literature, the saliva is collected within a 45 minute time frame following the end of the visitor&#8217;s engagement at the location. Following saliva collection, the samples are labeled and transported to a biological lab. Cortisol is the biophysical variable of interest in this study and measured using an ELISA process with a TECAN plate reader. To measure self-reported stress, the Perceived Stress Questionnaire (PSQ), which reports levels of worry, tension, joy, and perceived demands. Data are collected at all three sites in the late afternoon through early evening. When compared across all three settings, stress levels, as measured by both the biological markers and self-reports, are significantly lower after visitation to the most natural setting.

The purpose of this paper is to identify changes in stress levels after visitation to three different settings and to describe the methods used in identifying stress levels based on measures of salivary cortisol, &#945;-amylase, and a psychological self-report instrument.&#160;

Doğanın farklı seviyeleri ile üç yerlere ziyaretten sonra biyofizik ve psikolojik stres seviyeleri ölçme

Visitation to natural environments has been linked to psychological stress reduction. Although most stress-related research has relied on self-report ...

Davranış

Providing Meaningful Environmental Enrichment and Measuring Saliva Cortisol in Pigs Housed on Slatted Flooring

As pork is the most consumed meat worldwide, the welfare of animals in the swine industry has increasingly become a major public concern, which imposes a substantial pressure coming from customers, legislators and other stakeholders, to make management changes to improve the well-being of these animals. Several studies have demonstrated that providing environmental enrichment to pigs allows them to express their natural behavior, such as rooting and exploring, as well as nesting prior to farrowing, and is associated with reduced stress and improved production and welfare. However, many considerations should be taken into account when providing environmental enrichment, such as the type of floor, drainage and sewage systems, the pigs&#39; stage in life, the material, as well as its hanging method, height and location within the pen. The objectives of this paper are (1) to give methodologic information on how to provide a relatively simple and practical meaningful environmental enrichment for pigs which are housed on slatted floors during the different stages of their life, and (2) to demonstrate how to collect saliva samples for the measurement of cortisol concentrations, as a biomarker for acute stress. Protocols include information regarding the use of jute, cotton ropes, straw in racks, as well as chewable silicone sticks devices as environmental enrichment in pens of farrowing and lactation, weaners and finishers. In addition, the use of cotton rope for a non-invasive saliva samples collection for cortisol concentrations analysis is detailed. The protocols provided are relevant for professionals aiming to improve and monitor animal welfare, in both research and industrial swine farming.

This protocol demonstrates how to provide practical meaningful environmental enrichment for pigs which are housed on slatted flooring during the different stages of their lives, and how to collect saliva samples in a non-invasive manner for the measurement of cortisol concentrations, as a biomarker for acute stress.

Slatted Döşeme de Housed Domuzlarda Anlamlı Çevresel Zenginleştirme ve Ölçüm Tükürük Kortizol Sağlanması

As pork is the most consumed meat worldwide, the welfare of animals in the swine industry has increasingly become a major public concern, which imposes a ...

Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood flow that results in a net influx of blood to key organs and an increase in core temperature. This stress-induced hyperthermia is used to assess stress. However, measuring core temperature is invasive. As blood flow is redirected to the core, the periphery of the body can cool. This paper describes a protocol where peripheral body temperature is measured non-invasively in wild blue tits (Cyanistes caeruleus) using infrared thermography. In the field we created a set-up bringing the birds to an ideal position in front of the camera by using a baited box. The camera takes a short thermal video recording of the undisturbed bird before applying a mild stressor (closing the box and therefore capturing the bird), and the bird&#8217;s response to being trapped is recorded. The bare skin of the eye-region is the warmest area in the image. This allows an automated extraction of the maximum eye-region temperature from each image frame, followed by further steps of manual data filtering removing the most common sources of errors (motion blur, blinking). This protocol provides a time series of eye-region temperature with a fine temporal resolution that allows us to study the dynamics of the stress response non-invasively. Further work needs to demonstrate the usefulness of the method to assess stress, for instance to investigate whether eye-region temperature response is proportional to the strength of the stressor. If this can be confirmed, it will provide a valuable alternative method of stress assessment in animals and will be useful to a wide range of researchers from ecologists, conservation biologists, physiologists to animal welfare researchers.

There is a need for a non-invasive assessment of stress. This paper describes a simple protocol using thermal imaging to detect a significant response in eye-region temperature in wild blue tits to a mild acute stressor.

Termal Görüntüleme olmayan invaziv Yarı Kuşlar Stres Çalışması için

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood ...

Nörobilim

A Novel Method: Super-selective Adrenal Venous Sampling

Primary aldosteronism (PA) and subclinical Cushing&#39;s syndrome (SCS) are conditions in which the adrenal glands autonomously produce excessive amounts of aldosterone and cortisol, respectively. The conventional adrenal venous sampling (cAVS) method collects blood samples from both adrenal central veins and is useful for identifying the laterality of excess hormone production in a unilateral lesion(s), as documented in PA cases. In cAVS, plasma cortisol concentrations (PCCs) are used to normalize plasma aldosterone concentrations (PACs). A novel &#34;super-selective&#34; adrenal venous sampling (ssAVS) method was developed using a micro-catheter, which collects blood samples from adrenal tributary veins (TVs). PACs in ssAVS samples do not require PCC normalization because samples contain a limited amount of systemic venous blood, if any. The ssAVS method enabled segmental lesion(s) to be detected in both adrenal glands, which may be treated by bilateral adrenalectomy, thereby sparing lesion-free segment(s). Right and left adrenals typically have three TVs each, i.e., the superior, lateral, and inferior TVs in the right adrenal as well as the superior-median, superior-lateral, and lateral TVs in the left adrenal. In the ssAVS method, specific parent catheters and a technique to handle them are required, and have been described herein. Furthermore, ssAVS results from three cases of PA are presented: bilateral aldosterone-producing adenoma (APA) (Case #1), left APA and right possible cortisol-producing adenoma causing SCS (Case #2), and idiopathic hyperaldosteronism in which bilateral adrenal segments produced excessive amounts of aldosterone (Case #3). The ssAVS method is not difficult for expert angiographers, and, thus, is recommended worldwide to treat PA cases for which cAVS does not represent a viable surgical treatment option.

&#39;Super-selective&#39; adrenal venous sampling (ssAVS, also called segmental adrenal venous sampling: sAVS)&#160;was performed using a micro-catheter to identify adrenal segment(s) that produce excessive amounts of hormones. The ssAVS technique was described, cases in which adrenal segmental lesion(s) were identified by ssAVS were presented, and the usefulness of ssAVS in future adrenal research was discussed.

Bir roman yöntemi: Süper seçici Adrenal Venöz örnekleme

Primary aldosteronism (PA) and subclinical Cushing&#39;s syndrome (SCS) are conditions in which the adrenal glands autonomously produce excessive amounts ...

Tıp

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.

Bir Zebra balığı Larva Lusıferaz Muhabir Sistemi ile glukokortikoid Signaling&#39;den için bir Kimyasal Tarama Prosedürü

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

Biyoloji

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT &#34;point samples&#34; obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.
In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair&#39;s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.

Cortisol (CORT) accumulates in the growing hair shaft of humans and nonhuman primates. We describe methods for extracting and analyzing hair CORT with high precision and sensitivity. Measurement of hair CORT is particularly well-suited for assessing chronic stress over periods of weeks to months.

Ekstraksiyon ve İnsan ve Maymun Saç gelen Kortizol Analizi

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and ...

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal microscopy was used in combination with patch-clamp recording to detect three fusion modes in primary culture bovine adrenal chromaffin cells. The three fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving shrinkage of the fusion-generated &#937;-shape profile until it merges completely at the plasma membrane.
To detect these fusion modes, the plasma membrane was labeled by overexpressing mNeonGreen attached with the PH domain of phospholipase C &#948; (PH-mNG), which binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at the cytosol-facing leaflet of the plasma membrane; vesicles were loaded with the fluorescent false neurotransmitter FFN511 to detect vesicular content release; and Atto 655 was included in the bath solution to detect fusion pore closure. These three fluorescent probes were imaged simultaneously at ~20-90 ms per frame in live chromaffin cells to detect fusion pore opening, content release, fusion pore closure, and fusing vesicle size changes. The analysis method is described to distinguish three fusion modes from these fluorescence measurements. The method described here can, in principle, apply to many secretory cells beyond chromaffin cells.

This protocol describes a confocal imaging technique to detect three fusion modes in bovine adrenal chromaffin cells. These fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving fused vesicle shrinkage.

Adrenal Kromafin Hücrelerinde Üç Füzyon Gözenek Dinamiği Modunu Ölçmek için Konfokal Mikroskopi

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

Sıçan Adrenokortikal hücreleri ve deneyleri Steroidogenic fonksiyonların birincil kültür

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...