Name: avian semen collection by cloacal massage and isolation of dna from sperm
Uploaded: 2018-02-05T12:30:00.000+00:00
Duration: 7 min 40 s
Description: Watch this Scientific Journal Video about Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm at JoVE.com

Agradecemos a Jose Noguera de asistencia temprana con masaje cloacal, y Jeffrey Kittilson de laboratorio de apoyo. Este trabajo fue apoyado por un premio ND EPSCoR FAR0022429 BJH. Los costos de publicación generosamente fueron apoyados por el Departamento de ciencias biológicas de ganado.

Este protocolo implica sujetos animales vertebrados y ha sido aprobado por el institucional cuidado Animal y el Comité uso (IACUC) en Universidad de estado de Dakota del norte.
1. colección de semen de passerine pájaro mediante masaje cloacal
Nota: El masaje Cloacal es una técnica de colección de semen eficaz reproductivamente activa aves solamente, pero puede realizarse con éxito fuera de la temporada de cría en situaciones de cautiverio apropiadas. Actividad reproductiva debe determinarse para las especies de destino antes de utilizar el masaje cloacal. Esta técnica puede utilizarse en aves silvestres después de captura o en aves en cautiverio.
<ol><li>A partir de un pájaro masculino, (en condición de cría) agarre de bander en la mano no dominante, transferir el pájaro a la mano dominante con la parte ventral, tocando la palma (figura 1).<ol>
<li>Asegúrese de que la cabeza de pájaro es más cercano el borde lateral de la palma. Fijar ligeramente la cabeza y el cuerpo utilizando el pinky, anillo y los dedos medios. Esto deja el pájaro ventilación y cola expuesta.</li>
		<li>Deja las piernas de pájaro suelto o ligeramente restringida utilizando la palma.</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55324/55324fig1.jpg"> Figura 1 : Apretón para masaje cloaca. El pájaro se fija ligeramente en la mano dominante con su lado ventral tocar la palma y la cabeza asegurada por el dedo meñique. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55324/55324fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>
<ol start="2"><li>Coloque el dedo dominante del índice y el pulgar a cada lado del extremo superior de la cloaca y apriete ligeramente los dedos juntos mientras aplica presión ligera hacia la cola ( figura 2). La cloaca se vuelque, exponiendo la mucosa cloacal (rosa interior). Nota: Si el pájaro no está actualmente produciendo semen, protuberancia cloacal puede ser pequeña y la cloaca es difícil de evert. Con experiencia, es fácil distinguir entre las cloacas de los machos reproductivamente activos e inactivos5,11.</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55324/55324fig2.jpg"> Figura 2 : Posición y protuberancia cloacal del dedo. Vuelque la cloaca aplicando ligera presión en el extremo superior (A). En un paseriformes machos reproductivamente activos, la protuberancia cloacal es evidente y la empresa (B). <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55324/55324fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>
<ol start="3"><li>Con la mano no dominante, coloque la almohadilla del dedo índice en el lado dorsal de la base de la cola para estabilizar la mano y luego colocar la punta del pulgar justo por debajo de la cloaca evertido. Nota: La miniatura de un corta y suave es ventajosa para realizar masaje cloacal y protege a las aves de lesiones accidentales.</li>
	<li>Mueva el pulgar no dominante profundamente y cranially en un movimiento repetitivo con presión media, está por debajo de la cloaca. Continúe hasta que el ave eyacule. Dependiendo de la persona, esto normalmente toma entre 1 y varias docena pasadas.</li>
	<li>Tan pronto como el pájaro eyacula, recoger el semen en un tubo de microhematocrito. Repita el masaje cloacal para obtener múltiples eyaculados. Nota: Paseriformes semen es marrón claro y suele tener una consistencia algo espesa. Sustancias oscuras o blanco expulsadas de la cloaca no están semen y pueden contaminar la muestra de semen. La aparición de una pequeña cantidad de sangre en la cloaca o mezclado con el semen generalmente es causado por demasiada presión durante el masaje.</li>
	<li>Para la extracción de ADN, coloque el semen en un tubo de microcentrífuga con 20 μl de 1 x PBS (pH 7.4). Nota: El Semen almacenado en PBS 1 x no es conveniente para la inseminación artificial, pero puede ser utilizado para la extracción de ADN en una fecha posterior cuando se almacena a-80 ° C. El PBS puede ser temperatura ambiente o fría, pero debe estar en estado líquido cuando se deposita la muestra de semen.<ol>
<li>Confirmar la presencia de espermatozoides en la muestra mediante la visualización de una pequeña cantidad de la muestra diluida bajo un microscopio compuesto (figura 3).</li>
	</ol>
</li>
	<li>Almacenar el semen diluido a-80 ° C hasta la extracción.</li>
</ol>2. extracción de ADN de semen de aves
Nota: Este protocolo ha sido probado con múltiples kits comerciales de extracción de ADN, pero sólo ha tenido éxito cuando se utiliza con un ADN kit12. Pasos que se modificarán el protocolo del kit se indican con *.
<ol><li>Preparar una solución de 1 M de Ditiotreitol (DTT) en agua y mezclar con un vortex.*<ol>
<li>Alícuota de la solución DTT y almacenar a-20 ° C hasta inmediatamente antes del uso. Cada muestra se extrae requiere 10 μl de 1 M TDT solution.*</li>
	</ol>
</li>
	<li>Preparar una memoria intermedia que contiene 20 mM Tris-Cl, pH 8.0; 20 mM EDTA; 200 mM de NaCL; y 4% SDS. Cada muestra se extrae requiere 90 μl de este buffer.*<ol>
<li>Almacenar el buffer a temperatura ambiente. Si ha precipitado, caliéntelo a 56 ° C para dissolve.*</li>
	</ol>
</li>
	<li>Precalentar una incubadora que contiene un eje de balancín o coctelera a 65 ° C.*</li>
	<li>Inmediatamente antes de comenzar la extracción, mezclar 10 μl TDT solución con 90 μl del buffer preparado en el paso 2.2 para cada muestra se extrae, por ejemplo, 900 μl de tampón más solución DTT 100 μl si extraer 10 muestras. Esta mezcla se llama TDT-buffer.*</li>
	<li>Añadir 70 μl 1 x PBS a la muestra de semen diluido todo y mezclar bien con un vortex.*</li>
	<li>Añada 100 μl tampón de TDT y 10 μl proteinasa K solución de la muestra. Vórtex por 20 segundos, luego incubar en la incubadora precalentada durante 1 hora en un eje de balancín o shaker.*</li>
	<li>Añadir 200 μL de tampón AL y 200 μL EtOH fresco (100%) y agitar por 20 segundos.*</li>
	<li>Transferencia de toda la muestra a una columna de spin y centrifugar 1 minuto a x 6.000 g. Deseche el tubo de la colección.</li>
	<li>Coloque la columna en un nuevo tubo y agregar 500 μl preparado de tampón de AW1. Centrifugar 1 minuto a x 6.000 g. Deseche el tubo de la colección.</li>
	<li>Coloque la columna en un nuevo tubo y agregar 500 μl preparado de tampón de AW2. Centrifugue durante 1 minuto a 6.000 x g. Deseche el flujo y el lugar la columna de nuevo en el tubo.</li>
	<li>Centrifugar 3 minutos a 20.000 x g. Deseche el tubo de la colección y coloque la columna en un tubo de microcentrífuga de 1,5 mL.</li>
	<li>Pipeta 35 μl de AE directamente sobre la membrana de la columna de búfer e incubar a temperatura ambiente durante 5 minutos.*</li>
	<li>Centrifugar 1 minuto a 14.000 x g. descarte la columna spin y almacene extraídas muestras a-80 ° C.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forensic+application+of+DNA+'fingerprints'.">Forensic application of DNA 'fingerprints'.</a> Nature. 318 (12), 577-579 (1985).</li><li>Criscuolo, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantitative+PCR+assay+for+measurement+of+avian+telomeres.">Real-time quantitative PCR assay for measurement of avian telomeres.</a> J. Avian Biol. 40, 342-347 (2009).</li><li>Lovette, I. J., Fitzpatrick, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cornell Lab of Ornithology Handbook of Bird Biology, 3rd Edition. , (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> QIAamp DNA Micro Handbook. , (2014).</li><li>Carter, K. L., Robertson, B. C., Kempenaers, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+differential+DNA+extraction+method+for+sperm+on+the+perivitelline+membrane+of+avian+eggs.">A differential DNA extraction method for sperm on the perivitelline membrane of avian eggs.</a> Mol. Ecol. 9, 2149-2154 (2000).</li><li>Birkhead, T. R., Buchanan, K. L., Devoogd, T. J., Pellatt, E. J., Catchpole, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Song,+sperm+quality+and+testes+asymmetry+in+the+sedge+warbler.">Song, sperm quality and testes asymmetry in the sedge warbler.</a> Anim. Behav. 53, 965-971 (1997).</li><li>L&#252;pold, S., Calhim, S., Immler, S., Birkhead, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+morphology+and+sperm+velocity+in+passerine+birds.">Sperm morphology and sperm velocity in passerine birds.</a> Proc. R. Soc. B. 276, 1175-1181 (2009).</li><li>Samour, J. H., Smith, C. A., Moore, H. D., Markham, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semen+collection+and+spermatozoa+characteristics+in+budgerigars+(Melopsittacus+undulates).">Semen collection and spermatozoa characteristics in budgerigars (Melopsittacus undulates).</a> Vet. Rec. 118, 397-399 (1986).</li><li>Stelzer, G., Crosta, L., B&#252;rkle, M., Krautwalt-Junghanns, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attempted+semen+collection+using+the+massage+technique+and+semen+analysis+in+various+psittacine+species.">Attempted semen collection using the massage technique and semen analysis in various psittacine species.</a> J. Avian Med. Surg. 19 (1), 7-13 (2005).</li></ol>

Los autores declaran que no tienen intereses financieros que compiten.

Describimos un método simple y confiable para la recogida de semen de aves pequeñas y medianas en condición activa reproductivamente y extracción de ADN de esperma aviar.
El protocolo de extracción de ADN de semen descrita es modificado a partir de un kit para la simplicidad, pero fue refinado para el uso en semen aviar. Espermatozoides son resistentes a la lisis por productos químicos de extracción estándar9,13, y las muestras de semen recogidas de paseriformes son bastante pequeñas (típicamente de unos pocos microlitros). Estos factores presentan retos que se superan con éxito mediante una concentración adecuada de la TDT y la incubación a 65 ° C. Usando este método, hemos aislado constantemente cantidades utilizables de ADN para el análisis de qPCR de longitud de los telómeros de la esperma (normalmente entre 40-250 ng/μl, dependiendo del volumen de eyaculado).
TDT se ha utilizado anteriormente para extraer ADN de esperma aviar en combinación con una reacción de fenol/cloroformo13. Reacciones del fenol/cloroformo son eficaces para aislar ADN, pero son más lentos, son más peligrosos y requieren de una campana de humos. Una limitación notable de nuestro protocolo de extracción es que sólo ha tenido éxito cuando se utiliza con una marca y el tipo de kit de extracción, posiblemente debido al pequeño volumen de muestra de partida. Muestras de semen muy pequeña (menos de 1 μl) o las muestras con densidad baja de la esperma pueden prevenir extracción exitosa de ADN debido a la insuficiente ADN presente en la muestra, pero no hemos encontrado este problema cuando se realiza masaje cloaca en las aves en condiciones de cría . Para evitar este problema, masaje cloacal puede repetirse inmediatamente para recoger múltiples eyaculaciones que pueden combinarse para la extracción.
Masaje cloacal ha estado en uso durante décadas y en muchas especies1,4,5,6,7,15,16,17y es un medio rápido y eficaz de recogida de semen de aves en muchos casos. Para estudios con aves silvestres o inexpertas que pueden capturar, es más eficaz que los métodos de recopilación que confían en convencer a un pájaro macho copular con un elemento y depositar su semen voluntariamente4. Otro método de colección de semen es por la disección de los testículos o glomera seminal, pero esto no es una opción para fines de conservación o muestreo repetitivo4,14. La limitación primaria de masaje cloacal es la dificultad en el aprendizaje de la técnica sin demostración. Descripciones escritas son a menudo insuficientes para entender y aprender a implementar el proceso. Ilustrando el masaje cloacal en detalle, esperamos reducir el tiempo de aprendizaje y dificultad para aquellos que deseen aprender la técnica.

Las aves son sujetos ideales para estudios relacionados con la calidad del esperma y competencia1esperma telomere dinámica2, epigenética3y temas similares, como son ampliamente utilizados en la investigación biológica y esperma puede probarse fácilmente usando cloacal masaje. Masaje cloacal es una técnica manual suave, no invasiva para la recogida de semen de aves4,5,6. Muestras repetidas pueden obtenerse fácilmente y herramientas especiales no se necesitan, lo que facilita llevar a cabo en el campo o en laboratorio. Aunque masaje cloacal ha estado en uso durante décadas, es difícil aprender de las descripciones por escrito disponible. Esta publicación se pretende reducir el tiempo y la incertidumbre implicados en el aprendizaje de masaje cloacal.
Semen de aves con masaje cloacal puede ser utilizada inmediatamente para movilidad evaluación4,7 o8de la inseminación artificial, o congelado para otros usos como proyección de imagen avanzada y extracción de ADN. Muestras de semen de las aves paseriformes son pequeñas pero contienen esperma densamente. Se extrajo ADN utilizando un kit comercial de extracción para la simplicidad, con modificaciones para superar las características de protección especializadas de esperma9. Después de la extracción, se pueden medir los telómeros de esperma utilizando qPCR10.

<table><tbody><tr><td>Microhematocrit capillary tubes</td><td>Fisherbrand</td><td>22-362566</td><td>Any supplier can be used. Smaller tubes may be more effective for collection of small semen samples, such as from very small passerine birds like chipping sparrow.</td></tr><tr><td>Microcentrifuge tubes</td><td>Any</td><td></td><td></td></tr><tr><td>1x PBS</td><td>Any</td><td></td><td>Used for storing semen samples prior to DNA extraction.</td></tr><tr><td>DL-Dithiothreitol</td><td>Sigma-Aldrich</td><td>D0632</td><td></td></tr><tr><td>Tris base</td><td>Sigma-Aldrich</td><td>10708976001</td><td>Any supplier can be used.</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>1233508</td><td>Any supplier can be used.</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>793566</td><td>Any supplier can be used.</td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>1614363</td><td>Any supplier can be used.</td></tr><tr><td>QIAamp DNA micro kit</td><td>Qiagen</td><td>56304</td><td>Qiagen QIAamp DNA micro kit is recommended specifically. This DNA extraction protocol has been attempted with other commercial kits but with little success. The reasons for this are unknown to us.&amp;nbsp;</td></tr></tbody></table>

avian semen collection by cloacal massage and isolation of dna from sperm

Colección de semen puede ser útil para una amplia gama de aplicaciones, incluyendo estudios que involucran la calidad del esperma, esperma telomere dinámica y epigenética. Las aves son temas ampliamente utilizados en la investigación biológica y son ideales para estudios con muestras de esperma repetidas. Sin embargo, pocos recursos están disponibles para aquellos que deseen aprender a recoger y extraer ADN de esperma aviar. Aquí describimos cloacal masaje, una técnica manual suave, no invasiva para la recogida de esperma aviar. Aunque esta técnica se establece en la literatura, puede ser difícil de aprender de las descripciones disponibles. También ofrecemos información para la extracción de ADN de semen aviar utilizando un kit de extracción comercial con modificaciones. Masaje cloacal puede utilizarse fácilmente en cualquier pájaro macho tamaño pequeño a mediano en condición reproductiva. Después de colección, el semen puede ser utilizado inmediatamente para los ensayos de motilidad, o congelado para la extracción de ADN siguiendo el protocolo descrito en este documento. Este protocolo de extracción fue refinado aviar de los espermatozoides y se ha utilizado con éxito en muestras recogidas de varias especies de paseriformes (Passer domesticus, Spizella passerina, Haemorhous mexicanusy migratorius de Turdus ) y un columbid (Columba livia).

Cloacal de masaje y extracción de DNA usando que el protocolo descrito ha llevado a cabo en varias especies de passeriformes y uno columbid (tabla 1). La presencia de espermatozoides en las muestras de semen recogidas por masaje cloacal fue confirmada mediante la visualización de una pequeña cantidad de las muestras bajo un microscopio compuesto con 400 aumentos (figura 3). Después de extraer ADN de muestras de semen recogidas de gorriones, utilizando el protocolo descrito aquí, ocho muestras se corrieron en electroforesis en gel con una escalera de DNA (tres muestras pueden verse en la figura 4). Distintas bandas sin manchar fueron vistos en los ocho de los carriles de la muestra, que indica éxito aislamiento de ADN de alto calidad. Resto de las muestras extraída mediante este protocolo se evaluó la concentración de ADN utilizando un espectrofotómetro (ver tabla 1).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55324/55324fig3.jpg"> Figura 3: esperma de dos pequeños Passeriformes. Estos espermatozoides se obtuvieron muestras mediante masaje cloacal de un Pinzón de la casa (Haemorhous mexicanus), izquierda y un gorrión (Passer domesticus), derecha, atrapados en Fargo, ND en 2016. Las muestras fueron cada uno diluidas en 20 μl PBS y vistas bajo un microscopio compuesto a 1.000 X y aumento de 400 X, respectivamente. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55324/55324fig3large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55324/55324fig4.jpg"> Figura 4 : ADN gel electroforesis de la DNA de la esperma. Tres muestras de la DNA extraída de gorrión semen correr en un gel con una escalera de ADN. Tenga en cuenta la presencia de distintas bandas, que indica la extracción exitosa de ADN de alta calidad. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55324/55324fig4large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td></td>
			<td>Gorrión común (Passer domesticus)</td>
			<td>Chipping sparrow (Spizella passerina)</td>
			<td>Pinzón de la casa (Haemorhous mexicanus)</td>
			<td>Petirrojo americano (Turdus migratorius)</td>
			<td>Paloma (Columba livia)</td>
		</tr>
<tr>
<td>Número de muestras recolectadas</td>
			<td>156</td>
			<td>3</td>
			<td>2</td>
			<td>2</td>
			<td>2</td>
		</tr>
<tr>
<td>Número de muestras extraídas</td>
			<td>90</td>
			<td>3</td>
			<td>2</td>
			<td>2</td>
			<td>2</td>
		</tr>
<tr>
<td>Tasa de éxito de colección de semen</td>
			<td>98%</td>
			<td>75%</td>
			<td>100%</td>
			<td>67%</td>
			<td>67%</td>
		</tr>
</tbody></table>Tabla 1: especies y muestras recogidas y extrae hasta la fecha. En el momento de publicación, se han procesado muestras utilizando el protocolo descrito de 4 especies de passeriformes y 1 columbid. Tasa de éxito de colección de semen se refiere al porcentaje de cada uno para que con éxito se obtuvo una muestra sobre el total tratado.

Watch this Scientific Journal Video about Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm at JoVE.com

Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

Este protocolo describe cloacal masaje, una técnica manual no invasivo para la recogida de semen de aves y la extracción de ADN de esperma aviar utilizando un kit de extracción comercial con modificaciones.

Colección de Semen aviar por masaje Cloacal y el aislamiento de ADN de esperma

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. ...

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Nanyang Technological University

Research

JoVE Journal

Biology

15.9K vistas.  North Dakota State University. Este protocolo describe cloacal masaje, una técnica manual no invasivo para la recogida de semen de aves y la extracción de ADN de esperma aviar utilizando un kit de extracción comercial con modificaciones.

Video: Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Un enfoque estandarizado para la purificación multiespecífica de células germinales masculinas de mamíferos por disociación mecánica de tejidos y citometría de flujo

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Investigación

Biología del Desarrollo

Handling and Treatment of Male European Eels (Anguilla anguilla) for Hormonal Maturation and Sperm Cryopreservation

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and maturation. As of yet, weekly injections of human chorionic gonadotropin (hCG) have proved to maturate males after just 5-6 weeks of treatment, producing high volumes of high-quality sperm during several weeks. In addition, sperm cryopreservation protocols using different extenders, cryoprotectants and cooling and thawing times have been previously described for European eel. Here, we show that Tanaka&#180;s extender solution can be directly used for fertilization or for cryopreservation, making unnecessary the usage of different types of solutions and dilutions. Furthermore, the use of methanol as a cryoprotectant makes this protocol easy to use as methanol has low toxicity and does not activate the sperm. The sperm does not need to be cryopreserved immediately after the addition of the cryoprotectant, and it can be used long after being thawed. Moreover, sperm motility is still high after thawing although it is lower than that of fresh sperm. The aim of this work is to show the best available protocol for European eel handling, maturation, and sperm cryopreservation.

Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation

Protocols for European eel maturation and sperm cryopreservation have been improved over the last years. This article describes the best protocol available using human chorionic gonadotropin (hCG) for inducing maturation and methanol as cryoprotectant.

Manejo y tratamiento del hombre europeo anguilas (Anguilla anguilla) de maduración Hormonal y criopreservación de esperma

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and ...

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.

Presented here is a protocol for enriching pachytene spermatocytes, round spermatids, and elongating spermatids from adult mouse testes using a discontinuous bovine serum albumin density gradient with standard laboratory equipment.

Enriquecimiento de espermaocitos y espermatídos de los testículos de ratón que utilizan equipos de laboratorio estándar

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete ...

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.

Aislamiento de células espermatogénicas murinas usando un tinte de unión de ADN permeable a células violetas-excitado

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

El aislamiento y la In vitro La activación de Caenorhabditis elegans El esperma

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Biología

Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro positive selection assay to measure in vivo mutations induced in a transgenic &#955;gt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.

De novo mutations in the male germline may contribute to adverse health outcomes in subsequent generations. Here we describe a protocol for the use of a transgenic rodent model for quantifying mutations in male germ cells induced by environmental agents.

Transgénicos Roedor Ensayo para la cuantificación de la célula germinal masculina Mutant Frecuencia

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for ...

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

Recogida de esperma de calidad diferencial mediante centrifugación de gradiente de densidad

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish species is that it possesses interfertile cave and river-dwelling populations. This enables the genetic mapping of heritable traits that were fixed during adaptation to the different environments of these populations. While this species can be maintained and bred in the lab, it is challenging to both obtain embryos during the daytime and create hybrid embryos between strains. In vitro fertilization (IVF) has been used with a variety of different model organisms to successfully and repeatedly breed animals in the lab. In this protocol, we show how, by acclimatizing A. mexicanus to different light cycles coupled with changes in water temperature, we can shift breeding cycles to a chosen time of the day. Subsequently, we show how to identify suitable parental fish, collect healthy gametes from males and females, and produce viable offspring using IVF. This enables related procedures such as the injection of genetic constructs or developmental analysis to occur during normal working hours. Furthermore, this technique can be used to create hybrids between the cave and surface-dwelling populations, and thereby enable the study of the genetic basis of phenotypic adaptations to different environments.

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

In vitro fertilization is a commonly used technique with a variety of model organisms to maintain lab populations and produce synchronized embryos for downstream applications. Here, we present a protocol that implements this technique for different populations of the Mexican tetra fish, Astyanax mexicanus.

Gamete Collection y Fertilización In Vitro de Astyanax mexicanus

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish ...

Recording Electrical Currents across the Plasma Membrane of Mammalian Sperm Cells

Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for electrophysiologists for many decades. The method known as "spermatozoan patch clamp" was introduced in 2006. It has enabled the direct recording of ion channel activity in whole-cell and cell-attached configurations and has been instrumental in describing sperm cell physiology and the molecular identity of various calcium, potassium, sodium, chloride, and proton ion channels. However, recording from single spermatozoa requires advanced skills and training in electrophysiology. This detailed protocol summarizes the step-by-step procedure and highlights several 'tricks-of-the-trade' in order to make it available to anyone who wishes to explore the fascinating physiology of the sperm cell. Specifically, the protocol describes recording from human and murine sperm cells but can be adapted to essentially any mammalian sperm cell of any species. The protocol covers important details of the application of this technique, such as isolation of sperm cells, selection of reagents and equipment, immobilization of the highly motile cells, formation of the tight (Gigaohm) seal between a recording electrode and the plasma membrane of the sperm cells, transition into the whole-spermatozoan mode (also known as break-in), and exemplary recordings of the sperm cell calcium ion channel, CatSper, from six mammalian species. The advantages and limitations of the sperm patch clamp method, as well as the most critical steps, are discussed.

<meta charset="utf8"></head><body>Registro de corrientes eléctricas a través de la membrana plasmática de espermatozoides de mamíferos

This protocol describes how to perform electrical recordings from mammalian sperm cells in a whole-cell configuration, with the goal of directly recording ion channel activity. The method has been instrumental in describing the electrophysiological profiles of several sperm ion channels and helped to reveal their molecular identity and regulation.