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.

declaração de ética: os procedimentos que envolvem temas de amostragem de campo e animais foram aprovados pela Escola de Animal, Rural e Ciência Ambiental (ARES) na Nottingham Trent University. 1. Recolha de amostras fecais NOTA: Devem ser usadas luvas ao manusear amostras fecais e metanol. Se existe uma forte suspeita de que um animal pode estar sofrendo de uma doença zoonótica, roupas de proteção, como também deve ser usado um jaleco. <ol><li> Recolha das amostras fecais, o mais rapidamente possível (dentro de minutos a algumas horas) após a defecação e colocá-los dentro de um saco de amostra. De três a cinco subamostras de qualquer amostra defecated com um peso total de aproximadamente 10 g. Rotular o saco com o tempo e a data da colheita, identificação individual e congelar a amostra a -20 ° C até à análise. </li><li> Para minimizar o erro, congelar as amostras a -20 ° C até o estudo estar completa, em seguida, reunir as amostras para análise em conjunto como um único lote. &lt;/ Li&gt; </ol> 2. Fecal Hormone Extração <ol><li> Deixar as amostras fecais para descongelar à temperatura ambiente. </li><li> Certifique-se de que as amostras são homogeneizadas por mistura manualmente cada amostra em seu próprio saco de amostra. </li><li> Para cada amostra, pesar 0.50 (± 0,003 g) de material fecal numa microbalança de um barco de pesagem, em seguida, transferir as fezes para um frasco de vidro de 8 mL. Use um novo pesar barco para cada amostra e limpar a pinça com 30% de metanol entre cada amostra. </li><li> Combinar a 0,5 g de material fecal, com 5 ml de 90% de metanol: água, e agitar O / N num agitador orbital, à temperatura ambiente. </li><li> Na manhã seguinte, vortex a amostra e, em seguida, centrifuga-se durante 20 min a 600 g. </li><li> Decantar a fracção de metanol num tubo de vidro de 16 x 125 mm2 e colocar num suporte num banho de água regulado para uma temperatura de 37 ° C. Evaporar a seco usando ar em uma câmara de exaustão. Lavar o frasco de extração e coloque o sedimento fecal restante em um saco de resíduos clínicos para a eliminação. </li><li> Re-suspender os extractos fecais em 100%, 1 ml de metanol e transferir para um tubo de polipropileno de 12 x 75 mm2. Imediatamente PAC e armazenar a -20 ° C até análise. </li></ol> 3. Análise Hormona <ol><li> Realizar a análise com uma enzima ligada-imunoensaio (EIA), utilizando soro policlonal corticosterona CJM006. NOTA:. Um segundo EIA foi testado (Cortisol R4866 anti-soro, mas este não conseguiu alcançar a etapa de validação bioquímica (ver secção 4.1) O anti-soro policlonal corticosterona CJM006 e Cortisol R4866 anti-soro e do respectivo HRP de são fornecidos por CJ Munro, da Universidade da Califórnia, Davis, CA. </li><li> Diluir o anti-soro a 1: 15000 em tampão de revestimento (NaHCO3 0,05 M, pH 9,6) utilizando uma pipeta repetitivo e uma ponta de pipeta 50 ul, carregar uma placa de microtitulação de 96 poços com 50 ul / poço. Cobrir a placa com um vedante de placa e incubar O / N a 4 ° C. Deixar dois poços não revestidos para representar as cavidades de ligação não específica (NSB). </li><li> Imediatamente antes da utilização lavar as placas de microtítulo cinco vezes utilizando um lavador de placas de microtitulação automático (solução de lavagem; 0,15 M de NaCl, 0,05% de Tween 20). </li><li> Carregar as placas com 50 ul por poço, durante a NSB de e poços [tampão EIA (0,1 M NaPO4, NaCl 0,149, 0,1% de albumina de soro de bovino, pH 7,0) somente] &quot;zero&quot;, padrões (corticosterona, 3,9 - 1.000 pg / bem) ou amostras de extracto fecal (apropriadamente diluído em 1:20 tampão EIA, ver secção 4.1). Normas e zeros deve ser executado em triplicado e quadriplicado, respectivamente. Diluídas amostras de extracto fecais e NSB de deve ser executado em duplicado. </li><li> Siga este imediatamente com a adição de 50 ul / cavidade do rótulo ᵻ rábano conjugado com peroxidase diluído em tampão EIA para 1: 70000. </li><li> Incubar as placas de microtitulação no escuro durante 2 horas à temperatura ambiente. </li><li> Lavam-se as placas de microtitulação com uma solução de lavagem 5 vezes e incuba-se as placas à TA no escuro com 100 ul / WELl de substrato RT [0,4 mM de 2,2&#39;-azino-di- sal (ácido 3-etilbenztiazolino sulfónico) de diamónio, 1,6 mM de H 2 O 2, citrato 0,05 M, pH 4,0]. Prepara-se o substrato imediatamente antes do uso. </li><li> Usar um espectrofotômetro no comprimento de onda de 405 nm e ler as placas de microtítulo. A incubação das placas é considerada completa quando a densidade óptica dos poços de zero atinge 0,8 a 1,0. </li><li> Para assegurar a reprodutibilidade, utilizar controlos [normas EIA ou uma amostra biológica diluída em tampão EIA para se ligar a cerca de 30% (padrão EIA), 50% (uma amostra biológica, por exemplo, piscina de cavalo doméstico extracto fecal) e 70% (padrão EIA) ] para demonstrar uma intra- e inter-ensaio coeficientes de variação de &lt;10% e &lt;15%, respectivamente. </li></ol> 4. Enzyme Linked-imunoensaio: Validação para as espécies Estudo e Sexo <ol><li> Biochemical Validation (Paralelismo) <ol><li> Usando tampão EIA, execute uma diluição em série em pool fecal adicionalct, de preferência proveniente de amostras suspeitas de alta e baixa concentração de hormônio de vários indivíduos (intervalo: puro, 1: 2 a 1: 8192) e executado no EIA como descrito na seção 3. NOTA: Um paralelismo é considerada alcançada quando as diluições em série de extractos fecais produzir uma curva de deslocamento paralelo com os resultados da curva de regressão linear e padrão demonstram R2&gt; 0,90, uma inclinação&gt; 0,50 e p &lt;0,05. O extracto fecal diluídos em série que liga-se a cerca de 50% na curva padrão deve ser considerada a diluição ideal para executar todos os extractos fecais (por exemplo, 1:20). Não há nenhuma interferência considerado no ensaio, quando os resultados de regressão linear demonstram R2&gt; 0,90 e p &lt;0,05. </li></ol></li><li> Biochemical Validation (Interferência) <ol><li> Pico de 200 ul de padrões diluídos em série (zero, 3,9 a 1000 ng / poço de corticosterona) com 200 ul do extracto fecal apropriadamente diluído reunidas [diluído a 50% de ligação determinadas por ParalleliSM (01:20)] e executado no EIA como na secção 3. A seguir trama análise EIA a concentração observada menos o fundo (concentração da amostra zero) em função da concentração esperada. NOTA: Se a adição de extrato fecal reunidas diluída com padrões não altera significativamente os resultados da regressão quantidade esperada e lineares produz R 2&gt; 0,90, uma encosta perto de 1,0 e p &lt;0,05. Nenhuma interferência com o EIA foi alcançado. </li></ol></li><li> Validação Biológica: NOTA: A demonstração de que o EIA escolhido tem a capacidade de medir as mudanças biologicamente relevantes da hormona de interesse [por exemplo, o aumento da concentração de glicocorticóides na sequência de um farmacológico (ACTH é muitas vezes um procedimento licenciado) ou desafio biológico (por exemplo, psicológica e / ou ambiental; mais provável que seja um acontecimento desregulada oportunista)]. <ol><li> Selecione, extrair e analisar amostras fecais para as concentrações de metabólitos de glicocorticóidesusando o processo descrito nas secções 1-3 antes e depois de um evento desafiador. NOTA: Um evento desafiador será espécies específicas. Um método de exemplo é fornecido abaixo para o cavalo doméstico. Exemplos completos de eventos desafiadores para os cavalos têm sido publicados anteriormente, incluindo o uso de gestão 9 e maneio procedimentos 16. Um aumento significativo nas concentrações de metabolitos de glucocorticóides após o evento desafiador devem ser observadas e confirmadas por meio de análise estatística. Esta deve incorporar, onde apropriado, o efeito de medidas repetidas, data da amostra e individuais. validação biológico é então considerado alcançado. </li></ol></li></ol>

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The authors have nothing to disclose.

análise de corticosterona fecal fornece um meio de avaliar os padrões de longo prazo da atividade adrenal em cavalos. A natureza não-invasiva do método supera os efeitos de confusão de outros métodos de amostragem utilizados para avaliar a atividade adrenal incluindo saliva e no plasma análise 9. Além disso, a técnica tem uma vantagem não-invasivo claro se estudando cavalos livres que vão. Há vários pontos-chave para discussão em relação a este método e seu uso ad...

O método descrito pretende analisar concentrações de corticosterona em fezes equina, a fim de fornecer uma avaliação não-invasiva da actividade supra-renal. Medir a atividade do hipotálamo-pituitária-adrenal (HPA) é uma abordagem aceite para estudar a resposta a situações potencialmente aversivas em ambas as espécies em cativeiro e domésticos. A técnica de referência e o método mais amplamente utilizado é o uso de um plasma sanguíneo no entanto, métodos alternativos, tais como a análise fecal foram desenvolvidas de modo a ultrapassar a tensão induzida por amostragem de sangue em si e permitem que a capacidade de monitorar as espécies que vão livre. Durante uma situação de aversivo, homeostase fisiológica é interrompido. O hipotálamo no cérebro libera hormônio liberador de corticotrofina (CRH), que atua na glândula pituitária e estimula a liberação de hormônio adrenocorticotrófico (ACTH). ACTH entra na corrente sanguínea e estimula o córtex adrenal a secretar speciede s glicocorticóides específicos (CG). Os glicocorticóides estão intimamente ligados a eventos estressantes ao invés de ser consistentemente produzidos em todos os estados de energia elevada, portanto, eles são muitas vezes medidos em preferência sobre outros de estresse ligado hormônios 2. Os glucocorticóides são responsáveis ​​por diversos efeitos adaptativos em cavalos. A energia é rapidamente mobilizados a partir de locais de armazenagem no corpo sob a forma de ácidos gordos e de glucose, o consumo de oxigénio é aumentada, a função sensorial é reforçada 3 e o fluxo sanguíneo é reduzido em áreas não necessárias para o movimento 4. Além de atuar como um mecanismo de enfrentamento, o aumento induzido stress na glicocorticóides também pode ajudar a preparar o animal para a próxima estressor 5. Avaliando os níveis de hormona no plasma e na saliva envolve a medição da hormona em circulação real no entanto, a medição dos metabolitos nas fezes medidas do produto final metabólico da hormona. esteróides circulantes são catabolized na lIver antes da excreção para a bílis onde sofrem mais alterações facilitados pelas atividades enzimáticas da flora bacteriana intestinal dentro da faixa 6. Portanto, imunoensaios dirigidos a glucocorticóides no sangue pode não ser adequado para análise de metabolitos fecais 7 glucocorticóides. Como recolha de fezes pode ser levada a cabo sem perturbar o cavalo, a análise de fezes para corticosterona, tem sido amplamente utilizado para monitorizar a actividade HPA num número de circunstâncias. Corticosterona elevada nas fezes de cavalos tem sido relatada em resposta a situações potencialmente aversivas, nomeadamente durante o tratamento veterinário pós-operatório 8 e em habitação restritiva 9. Amostragem fecal reflete um nível de glicocorticóides agrupada ao longo do tempo, em vez de o ponto de amostragem de tempo oferecido pelo plasma e saliva tornando-o adequado para o acompanhamento a longo prazo, crónica ou padrões sazonais 10. Devido à não-invasivanatureza do método, as amostras podem ser colhidas repetidamente para um indivíduo sem a necessidade de captura ou de contenção 11. No entanto, as espécies de tempo específico trânsito intestinal deve ser levado em conta no planejamento de um protocolo de amostragem. Em cavalos, tempo de trânsito intestinal é de cerca de 18 h, por conseguinte, 12, resposta adrenal e metabolitos subsequentes de corticosterona pode ser detectado nas fezes um dia após a activação inicial do eixo HPA. Ao utilizar técnicas de imunoensaio não-invasivos uma validação cuidadosa para a espécie a ser investigada é essencial 13. Além disso, as diferenças de sexo na excreção de metabolitos da hormona foram relatados provavelmente devido a diferenças na taxa metabólica e o tipo de metabólito excretado corticosterona em várias espécies, incluindo ratos, 14 e 15 galinhas. Foi, portanto, importante como parte deste método que o ensaio foi validado para uso em cavalos domésticos masculinos e femininos, como está detalhado no the protocolo. Essa diferença no metabolismo hormonal entre os sexos tem consequências para a qualidade dos dados, no entanto, raramente é abordada e incluído como parte da validação do ensaio. Este método não-invasivo permite a avaliação de longo prazo da atividade adrenal em cavalos domésticos. Os detalhes do protocolo tanto a validação do ensaio e da técnica de ensaio em si.

<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.

cavalos domésticos (n = 16, 8 éguas, 8 capões), com uma idade média de 15 anos (± 3) foram agrupados de acordo com o sexo e submetido a quatro projetos de habitação com níveis crescentes de isolamento social (n = 4 cavalo / tratamento). Habitação 1 envolveu cavalos que vivem em um ambiente de rebanho, simulando intimamente seu habitat natural. Habitação 2 envolvidos cavalos que vivem em pares em um celeiro interior. Habitação 3 cavalos envolvidos alojado sozinho em estábul...

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 Análise de glicocorticóides: Non-invasive Monitoring Adrenal em eqüídeos

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 visualizações.  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.

Vídeo: 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.

Medição de cortisol em 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 ...

Bioquímica

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.

Extração de cortisol de matrizes de Sturgeon Fin e Jawbone

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) ...

Ambiente

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;

Medindo níveis de stress biofísicos e psicológicos após visitação a três locais com diferentes níveis de natureza

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

Comportamento

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.

Fornecendo o enriquecimento ambiental significativo e medindo o cortisol do saliva nos porcos alojados no revestimento slatted

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.

Thermal Imaging para estudo de estresse não-invasiva em aves Unrestrained

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

Neurociência

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.

Um novo método: Super selectiva Adrenal amostragem venosa

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

Medicina

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.

A Triagem procedimento químico para Glucocorticoid Sinalização com um Sistema Reporter Zebrafish Zergling luciferase

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

Biologia

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.

Extração e Análise de Cortisol de cabelo humano e do macaco

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.

Microscopia Confocal para medir três modos de dinâmica de poros de fusão em células adrenal chromaffin

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.

Cultura primária de células Adrenocortical rato e ensaios das funções reguladora

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