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.

Ética declaración: los procedimientos que implican temas de muestreo de campo y animales han sido aprobados por la Escuela de animal, Rural y Ciencias Ambientales (ARES) en la Universidad de Nottingham Trent. 1. Recolección de muestras fecales NOTA: Se deben usar guantes al manipular muestras fecales y metanol. Si existe una fuerte sospecha de que un animal podría estar sufriendo de una enfermedad zoonótica, ropa protectora, como también se debe usar una bata de laboratorio. <ol><li> Recoger las muestras fecales tan pronto como sea posible (dentro de unos pocos minutos a una hora) después de la defecación y colocarlos en una bolsa de muestra. Tomar de tres a cinco submuestras de cualquier muestra defecado con un peso total de aproximadamente 10 g. Etiquetar la bolsa con la hora y fecha de recolección, identificación individual, y congelar la muestra a -20 ° C hasta su análisis. </li><li> Para minimizar el error, congelar las muestras a -20 ° C hasta que el estudio se complete a continuación, recoger las muestras para el análisis en conjunto como un solo lote. &lt;/ Li&gt; </ol> 2. Extracción de la hormona fecal <ol><li> Deje las muestras fecales que se descongele a temperatura ambiente. </li><li> Asegúrese de que las muestras se homogeneizaron mezclando manualmente cada muestra en su propia bolsa de muestra. </li><li> Para cada muestra pesan 0.50 (± 0.003 g) de material fecal en una microbalanza en un barco pesan a continuación, transferir las heces a un vial de vidrio de 8 ml. Use un nuevo barco pesan para cada muestra y limpiar las pinzas con metanol al 30% entre cada muestra. </li><li> Combinar el 0,5 g de materia fecal con 5 ml de metanol al 90%: agua, y agitar O / N en un agitador orbital a temperatura ambiente. </li><li> A la mañana siguiente, la muestra Vortex y centrifugar durante 20 minutos a 600 g. </li><li> Se decanta la fracción de metanol en un tubo de vidrio de 16 x 125 mm 2 y colocar en un estante en un baño de agua a una temperatura de 37 ° C. Se evapora a sequedad del aire en una campana de humos. Enjuague el vial de extracción y colocarlo el sedimento fecal restante en una bolsa de residuos clínicos para su eliminación. </li><li> Vuelva a suspender los extractos fecales en 100%, 1 ml de metanol y transferir a un tubo de polipropileno de 12 x 75 mm 2. Inmediatamente la tapa y se almacena a -20 ° C hasta su análisis. </li></ol> 3. Análisis Hormonal <ol><li> Llevar a cabo el análisis con un inmunoensayo ligado a enzimas (EIA) utilizando antisuero policlonal corticosterona CJM006. NOTA:. Un segundo EIA fue probado (cortisol R4866 antisuero, pero esto no logró alcanzar la etapa de validación bioquímica (ver sección 4.1) El antisuero policlonal corticosterona CJM006 y antisuero cortisol R4866 y el respectivo HRP de son suministrados por CJ Munro, Universidad de California, Davis, CA. </li><li> Diluir el antisuero a 1: 15.000 en tampón de revestimiento (0,05 M NaHCO3, pH 9,6) usando una pipeta repetitiva y una punta de pipeta 50 l, cargar una placa de microtitulación de 96 pocillos con 50 l / pocillo. Cubrir la placa con un sellador de placas e incubar O / N a 4 ° C. Dejar dos pozos no recubiertos para representar los pozos de unión no específica (NSB). </li><li> Inmediatamente antes de su uso lavar las placas de microtitulación cinco veces usando un lavador de placas de microtitulación automático (solución de lavado; 0,15 M NaCl, 0,05% Tween 20). </li><li> Cargar las placas con 50 l por pocillo para la NSB de y pozos [tampón EIA (0,1 M NaPO 4, 0,149 M NaCl, 0,1% de albúmina de suero bovino, pH 7,0) solamente] &quot;cero&quot;, estándares (corticosterona, 3,9 - 1.000 pg / así) o extraer muestras fecales (diluido apropiadamente a las 1:20 tampón EIA, ver sección 4.1). Normas y ceros deben ser manejados por triplicado y cuadruplicado, respectivamente. Diluidas extraer muestras fecales y de NSB deben analizar por duplicado. </li><li> Siga este inmediatamente con la adición de 50 l / pocillo de la etiqueta de rábano picante ᵻ conjugada con peroxidasa, diluida en tampón EIA a 1: 70.000. </li><li> Se incuban las placas de microtitulación en la oscuridad durante 2 horas a RT. </li><li> Lavar las placas de microtitulación con solución de lavado 5 veces y se incuban las placas a temperatura ambiente en la oscuridad con 100 l / well de RT sustrato [0,4 mM de 2,2&#39;-azino-di- sal (3-etilbenzotiazolina sulfónico) diamonio, 1,6 mM H 2 O 2, 0,05 M citrato, pH 4,0]. Preparar el sustrato inmediatamente antes del uso. </li><li> Use un espectrofotómetro a una longitud de onda de 405 nm para leer las placas de microtitulación. La incubación de las placas se considera completa cuando la densidad óptica de los pocillos cero llega a 0,8 a 1,0. </li><li> Para asegurar la repetibilidad, utilizar los controles [normas EIA o una muestra biológica diluidas en tampón de EIA para unirse a aproximadamente 30% (estándar EIA), 50% (una muestra biológica, por ejemplo, la piscina de caballo doméstico extracto fecal) y 70% (estándar EIA) ] para demostrar una intra e inter-ensayo coeficientes de variación de &lt;10% y &lt;15%, respectivamente. </li></ol> 4. Enzyme Linked-inmunoensayo: Validación para la especie estudio y sexo <ol><li> Validación bioquímica (paralelismo) <ol><li> Utilizando tampón EIA, lleve a cabo una dilución en serie en agrupada fecal adicionalct, lo ideal es tomado de muestras de alta y baja concentración de la hormona sospechosos de varios individuos (rango: aseado, 1: 2 a 1: 8192) y se ejecutan en el EIA como se describe en la sección 3. NOTA: Un paralelismo se considera logra cuando las diluciones en serie de los extractos fecales producen una curva de desplazamiento paralelo a los resultados curva y lineal de regresión estándar demuestran R2&gt; 0,90, una pendiente&gt; 0,50 y p &lt;0,05. El extracto fecal diluida en serie que se une a alrededor del 50% en la curva estándar se debe considerar la dilución ideal para ejecutar todos los extractos fecales (por ejemplo, 1:20). No hay interferencia en el ensayo considerado cuando los resultados de regresión lineal demuestran R2&gt; 0,90 y p &lt;0,05. </li></ol></li><li> Validación bioquímica (Interferencia) <ol><li> Pico de 200 l de los estándares diluidos en serie (cero, de 3,9 a 1000 ng / pocillo corticosterona) con 200 l de extracto fecal diluidas adecuadamente agrupada [diluida en el 50% de la unión determinada por Parallelism (1:20)] y se ejecutan en el EIA como en la sección 3. Tras gráfico de análisis EIA, la concentración observada menos el fondo (concentración de la muestra cero) frente a la concentración esperada. NOTA: Si la adición de extracto fecal diluida agrupado a las normas no altera significativamente la cantidad esperada y lineales resultados de la regresión produce R2&gt; 0,90, una pendiente cercana a 1,0 y p &lt;0,05. se ha logrado ninguna interferencia con la EIA. </li></ol></li><li> Validación biológica: NOTA: Una demostración de que la EIA elegido tiene la capacidad de medir los cambios de importancia biológica de la hormona de interés [por ejemplo, el aumento de las concentraciones de glucocorticoides siguientes farmacológico (ACTH es a menudo un procedimiento de licencia) o un desafío biológico (por ejemplo, psicológica y / o ambiental; más probable que sea un evento no regulada oportunista)]. <ol><li> Seleccionar, extraer y analizar muestras fecales de las concentraciones de metabolitos de glucocorticoidesmediante el proceso detallado en las secciones 1 a 3 antes y después de un evento desafiante. NOTA: Un desafiante evento será especies específicas. Un procedimiento de ejemplo se proporciona a continuación para el caballo doméstico. Ejemplos completos de eventos desafiantes para los caballos han sido publicados anteriormente incluyendo el uso de gestión 9 y procedimientos de cría de 16. Un aumento significativo en las concentraciones de metabolitos de glucocorticoides después del evento desafiante debe ser observado y confirmado mediante análisis estadístico. Esto debe incorporar en su caso, el efecto de las medidas repetidas, fecha de la muestra e individuales. Se consideró entonces logra la validación biológica. </li></ol></li></ol>

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The authors have nothing to disclose.

análisis corticosterona fecal proporciona un medio de evaluar los patrones de largo plazo de la actividad suprarrenal en los caballos. La naturaleza no invasiva del método supera los efectos de confusión de otros métodos de muestreo utilizados para evaluar la actividad suprarrenal incluyendo la saliva y el plasma análisis 9. Además, la técnica tiene una clara ventaja no invasivo si el estudio de los caballos que van libres. Hay varios puntos clave para discutir con respecto...

Los objetivos método descrito para analizar las concentraciones de corticosterona en las heces de equino con el fin de proporcionar una evaluación no invasiva de la actividad suprarrenal. La medición de la actividad (HPA) eje hipotálamo-hipofisario-suprarrenal es un método aceptado para estudiar la respuesta a situaciones potencialmente aversivas en ambas especies en cautividad y domésticas. La técnica de referencia y el método más ampliamente utilizado es el uso de plasma de la sangre 1 Sin embargo, los métodos alternativos tales como el análisis fecal se han desarrollado con el fin de superar el estrés inducido por el muestreo de sangre en sí y permitir la capacidad de seguimiento de las especies que van libres. Durante una situación aversiva, la homeostasis fisiológica se altera. El hipotálamo en el cerebro libera hormona liberadora de corticotropina (CRH) que actúa sobre la glándula pituitaria anterior y estimula la liberación de la hormona adrenocorticotrópica (ACTH). ACTH entra en el torrente sanguíneo y estimula la corteza adrenal para secretar especieS glucocorticoides específicos (GC). Los glucocorticoides están estrechamente vinculados a los acontecimientos estresantes en lugar de ser producido constantemente en todos los estados elevados de energía, por lo tanto a menudo se miden en preferencia sobre otras hormonas de estrés ligado 2. Los glucocorticoides son responsables de varios efectos adaptativos en caballos. La energía se movilizó rápidamente desde los lugares de almacenamiento en el cuerpo en forma de ácidos grasos y glucosa, el consumo de oxígeno se incrementa, la función sensorial se potencia 3 y el flujo sanguíneo se reduce a las zonas no necesarias para el movimiento 4. Además de actuar como mecanismo de defensa, el aumento inducido por el estrés de glucocorticoides también puede ayudar a preparar a los animales para el siguiente factor de estrés 5. La evaluación de los niveles de hormonas en el plasma y saliva implica la medición de la hormona circulante actual, sin embargo, la medición de los metabolitos en las medidas de heces el producto final metabólico de la hormona. esteroides circulantes son catabolizados en el liver antes de su excreción en la bilis a la donde se someten a cambios adicionales facilitadas por las actividades enzimáticas de la flora bacteriana en el tracto intestinal 6. Por lo tanto, los inmunoensayos dirigidos a los glucocorticoides en la sangre no pueden ser adecuados para el análisis de los metabolitos de glucocorticoides fecales 7. Como recogida fecal puede llevar a cabo sin perturbación para el caballo, el análisis de heces para la corticosterona, se ha utilizado ampliamente para controlar la actividad HPA en un número de circunstancias. Corticosterona elevada en las heces de los caballos ha sido reportada en respuesta a situaciones potencialmente aversivos incluso durante el tratamiento veterinario postoperatorio 8 y 9 en la vivienda restrictiva. Toma de muestras fecales refleja un nivel de glucocorticoides combinado con el tiempo en lugar de en el punto de muestreo en tiempo ofrecido por el plasma y la saliva por lo que es apropiado para el seguimiento a largo plazo, crónica o patrones estacionales 10. Debido a la no invasivanaturaleza del método, las muestras se pueden recoger en varias ocasiones para un individuo sin la necesidad para la captura o la restricción 11. Sin embargo, las especies de tiempo específico de tránsito intestinal se debe tener en cuenta al planificar un protocolo de muestreo. En los caballos, tiempo de tránsito intestinal es de alrededor de 18 hr 12, por lo tanto, la respuesta adrenal y posteriores metabolitos de corticosterona se pueden detectar en las heces un día después de la activación inicial del eje HPA. Cuando se utilizan técnicas de inmunoensayo no invasivos una validación cuidadosa de la especie objeto de la investigación es esencial 13. Además, las diferencias de sexo en la hormona de excreción de los metabolitos se han reportado probablemente debido a las diferencias en la tasa metabólica y el tipo de metabolito excretado corticosterona en diversas especies, incluyendo ratones 14, 15 y pollos. Por ello es importante como parte de este método que el ensayo fue validado para su uso tanto en los caballos domésticos masculinos y femeninos como se detalla en la THprotocolo de correo. Esta diferencia en el metabolismo de las hormonas entre los géneros tiene consecuencias para la calidad de los datos, sin embargo, rara vez se aborda y se incluye como parte de la validación de un ensayo. Este método no invasivo permite una evaluación a largo plazo de la actividad suprarrenal en los caballos domésticos. Los detalles del protocolo tanto en la validación del ensayo y de la técnica de ensayo en sí.

<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.

caballos domésticos (n = 16, 8 yeguas, 8 castrados) con una edad media de 15 años (± 3) se agruparon de acuerdo con el género y se sometió a cuatro diseños de las viviendas con el aumento de los niveles de aislamiento social (n = 4 caballos / tratamiento). 1 vivienda involucrada caballos que viven en un ambiente de la manada, que simula estrechamente su hábitat natural. Viviendas 2 involucrada caballos que viven en parejas en un granero de interior. Viviendas de 3 caballos que par...

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.

Análisis de glucocorticoides fecales: Monitoreo suprarrenal no invasiva en los équidos

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 vistas.  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.

Medición de cortisol en Koala (Phascolarctos cinereus) Piel

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

Investigación

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.

Extracción de cortisol de las matrices de aleta de esturión y mandíbula

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) ...

Medio 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;

Medición de los niveles de estrés biofísico y psicológico después de la visita a tres lugares con diferentes niveles de naturaleza

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

Comportamiento

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.

Proporcionar un enriquecimiento ambiental significativo y medir el cortisol de saliva en cerdos alojados en pisos de lama

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.

Imágenes Térmicas para estudiar el estrés de forma no invasiva en ilimitados 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 ...

Neurociencias

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.

Un método novedoso: Muestreo venoso suprarrenal selectivo súper

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.

Un procedimiento de detección química de glucocorticoides Señalización con un sistema indicador de pez cebra Larva luciferasa

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

Biología

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.

Extracción y análisis de cortisol de Derechos Humanos y de mono de pelo

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.

Microscopía confocal para medir tres modos de dinámica de poros de fusión en células cromafines suprarrenales

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.

Cultivo primario de células adrenocorticales rata y análisis de funciones Steroidogenic

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