Agradecemos Jose Noguera assistência precoce com massagem cloacal, e Jeffrey Kittilson para laboratório de apoio. Este trabalho foi apoiado por um prêmio de ND EPSCoR FAR0022429 a BJH. Custos de publicação foram generosamente apoiados pelo departamento de ciências biológicas da NDSU.

Este protocolo envolve assuntos animais vertebrados e foi aprovado pelo Comitê de uso (IACUC) da North Dakota State University e institucional Cuidado Animal.
1. coleta de sêmen de uma ave passeriforme da família usando esfregaços massagem
Nota: Esfregaços massagem é uma técnica de coleção de sêmen eficaz sobre aves reprodutivamente ativas somente, mas pode ser realizada com sucesso fora da época de reprodução em situações apropriadas em cativeiro. Atividade reprodutiva deve ser determinada para a espécie-alvo antes de utilizar a massagem esfregaços. Esta técnica pode ser usada em aves selvagens após captura ou em pássaros mantidos em cativeiro.
<ol><li>Começando com um pássaro masculino, (em condição de reprodução) aderência do bander na mão não dominante, transferir o pássaro para a mão dominante com seu lado ventral, tocando a palma (Figura 1).<ol>
<li>Certifique-se de que a cabeça da ave é o mais próximo a borda lateral da palma da mão. Seguro levemente a cabeça e o corpo usando o mindinho, anel e os dedos do meio. Isso deixa do pássaro respiradouro e cauda exposta.</li>
		<li>Deixe as pernas do pássaro solto ou levemente comedido, usando a palma da mão.</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55324/55324fig1.jpg"> Figura 1 : Aperto para massagem esfregaços. O pássaro está seguro levemente na mão dominante, com seu lado ventral, tocando a palma e cabeça garantidas pelo dedo mindinho. <a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig1large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
<ol start="2"><li>Coloque o dominante o dedo indicador e o polegar de cada lado da extremidade superior da cloaca e levemente belisca os dedos juntos, aplicando uma pressão muito leve em direção a cauda ( Figura 2). A cloaca será evert, expondo a mucosa cloacal (interior cor de rosa). Nota: Se o pássaro não está atualmente produzindo sêmen, a protuberância cloacal pode ser pequena e a cloaca é difícil de evert. Com a experiência, é fácil diferenciar entre as cloacas de machos reprodutivamente ativas e inativas5,11.</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55324/55324fig2.jpg"> Figura 2 : Dedo posição e protuberância cloacal. Evert cloaca aplicando pressão ligeira à extremidade superior (A). Em um ativo reprodutivamente masculino ave passeriforme da família, a protuberância cloacal é óbvia e empresa (B). <a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig2large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
<ol start="3"><li>Com a mão não dominante, coloque a almofada do dedo no lado dorsal da base da cauda para estabilizar a mão e em seguida, coloque a ponta do polegar logo abaixo da cloaca de sempre. Nota: Uma miniatura curta e Lisa é vantajosa para a realização de massagem cloacal e protege o pássaro de lesão acidental.</li>
	<li>Mova o polegar não-dominante cranialmente e profunda em um movimento repetitivo com média pressão, ficando abaixo da cloaca. Continue até o pássaro ejacula. Dependendo do indivíduo, isto normalmente leva entre 1 e várias dezenas de cursos.</li>
	<li>Assim que o pássaro ejacula, colete o sêmen em um tubo de Microhematócrito. Repita a massagem esfregaços para obter múltiplos ejacula. Nota: Passeriformes sémen é castanho claro e normalmente tem uma consistência um pouco grossa. Substâncias brancas ou escuras, expelidas da cloaca não estão sémen e podem contaminar a amostra de sêmen. A aparência de uma pequena quantidade de sangue na cloaca ou misturado com o sêmen é geralmente causado por muita pressão durante a massagem.</li>
	<li>Para a extração de DNA, coloca o sêmen em um tubo de microcentrifugadora com 20 µ l de 1X PBS (pH 7,4). Nota: Sêmen armazenado em PBS 1x não é apropriado para a inseminação artificial, mas pode ser usado para a extração de DNA em uma data posterior quando armazenado a-80 ° C. O PBS pode ser temperatura ambiente ou refrigerados, mas deve estar no estado líquido, quando a amostra de sêmen é depositada.<ol>
<li>Confirme a presença de espermatozoides na amostra exibindo uma pequena quantidade da amostra diluída sob um microscópio composto (Figura 3).</li>
	</ol>
</li>
	<li>Armazene o sêmen diluído a-80 ° C até a extração.</li>
</ol>2. extração de DNA do sêmen do pássaro
Nota: Este protocolo foi testado com vários jogos comerciais de extração de DNA, mas só faz sucesso quando usado com um DNA kit12. Etapas que são modificadas desde o protocolo do kit são indicadas por *.
<ol><li>Preparar uma solução 1m de ditiotreitol (DTT) em água e misture usando um vortex.*<ol>
<li>Alíquota da solução de TDT e armazená-lo a-20 ° C até imediatamente antes da utilização. Cada amostra a ser extraído requer 10 µ l de solução de TDT * 1m</li>
	</ol>
</li>
	<li>Preparar um buffer contendo 20 mM Tris-Cl, pH 8.0; 20 mM EDTA; 200 mM NaCL; e 4% SDS. Cada amostra a ser extraído exige 90 µ l desta buffer.*<ol>
<li>Armazene o buffer em temperatura ambiente. Se ele se precipitou, aquecê-lo a 56 ° C, para dissolve.*</li>
	</ol>
</li>
	<li>Pré-aqueça uma incubadora contendo um balancim ou agitador a 65 ° c</li>
	<li>Imediatamente antes de iniciar a extração, misture a solução de TDT 10 µ l com 90 µ l de buffer preparado no passo 2.2 para cada amostra a ser extraído, por exemplo, 900 buffer de µ l além de 100 µ l TDT solução se extraindo 10 amostras. Esta mistura será chamada TDT-buffer.*</li>
	<li>Adicionar 70 µ l 1X PBS para a amostra de sêmen diluído inteiro e misture bem, usando um vortex.*</li>
	<li>Adicione 100 µ l tampão-DTT e solução de proteinase K 10 µ l de amostra. Vórtex por 20 segundos, então incubar na incubadora pré-aquecido por 1 hora em um balancim ou shaker.*</li>
	<li>Adicionar 200 µ l de buffer AL e 200 µ l EtOH fresco (100%) e vórtice para encerrar 20</li>
	<li>Transferir a amostra inteira para uma coluna de rotação e centrifugar 1 minuto em 6.000 x. g. descarte o tubo de coleta.</li>
	<li>Coloque a coluna em um tubo novo e juntar 500 µ l preparado tampão AW1. Centrifugar 1 minuto em 6.000 x. g. descarte o tubo de coleta.</li>
	<li>Coloque a coluna em um tubo novo e juntar 500 µ l preparado tampão AW2. Centrífuga para 1 minuto em 6.000 g. x descartar o escoamento e coloque a coluna de volta no tubo.</li>
	<li>Centrifugar a 3 minutos a 20.000 x g. descarte o tubo de coleta e colocar a coluna em um tubo de microcentrifugadora de 1,5 mL.</li>
	<li>Pipeta 35 µ l de AE buffer directamente sobre a membrana da coluna e incubar a temperatura ambiente por 5 minutes.*</li>
	<li>Centrifugar 1 minuto a 14.000 x. g. descartar a coluna de rotação e armazenar amostras extraídas a-80 ° C.</li>
</ol>

<ol>
	<li>Briskie, J. V., Montgomerie, 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+size+and+sperm+competition+in+birds.">Sperm size and sperm competition in birds.</a> Proc. R. Soc. B. 247, 89-95 (1992).</li><li>Monaghan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeres+and+life+histories:+the+long+and+the+short+of+it.">Telomeres and life histories: the long and the short of it.</a> Ann. N.Y. Acad. Sci. 1206, 130-142 (2010).</li><li>Carrell, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics+of+the+male+gamete.">Epigenetics of the male gamete.</a> Fertility and Sterility. 97 (2), 267-274 (2012).</li><li>Burrows, W. H., Quinn, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+collection+of+spermatozoa+from+the+domestic+fowl+and+turkey.">The collection of spermatozoa from the domestic fowl and turkey.</a> Poultry Science. 16 (1), 19-24 (1937).</li><li>Wolfson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cloacal+protuberance:+a+means+for+determining+breeding+condition+in+live+male+birds.">The cloacal protuberance: a means for determining breeding condition in live male birds.</a> Bird-Banding. 23 (4), 159-165 (1952).</li><li>Kast, T. L., Ketterson, E. D., Nolan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+ejaculate+quality+in+dark-eyed+juncos+according+to+season,+stage+of+reproduction,+and+testosterone+treatment.">Variation in ejaculate quality in dark-eyed juncos according to season, stage of reproduction, and testosterone treatment.</a> The Auk. 115 (3), 684-693 (1998).</li><li>Helfenstein, F., Podevin, M., Richner, H. <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,+swimming+velocity,+and+longevity+in+the+house+sparrow+Passer+domesticus.">Sperm morphology, swimming velocity, and longevity in the house sparrow Passer domesticus.</a> Behav. Ecol. Sociobiol. 64, 557-565 (2010).</li><li>Purchase, C. F., Earle, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modifications+to+the+IMAGEJ+computer+assisted+sperm+analysis+plugin+greatly+improve+efficiency+and+fundamentally+alter+the+scope+of+attainable+data.">Modifications to the IMAGEJ computer assisted sperm analysis plugin greatly improve efficiency and fundamentally alter the scope of attainable data.</a> J. Appl. Icthyol. 28, 1013-1016 (2012).</li><li>Gill, P., Jeffreys, A. J., Werrett, D. 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>

Os autores declaram que eles têm não tem interesses financeiro concorrente.

Nós descrevemos um método simples e confiável para coleta de sêmen de aves pequenas e médias em condição reprodutivamente ativa e extrair DNA de esperma aviária.
O protocolo de extração de DNA de sémen descrito é modificado a partir de um kit para manter a simplicidade, mas foi refinado para uso em sêmen aviária. Espermatozoides são resistentes à Lise por extração padrão produtos químicos9,13, e amostras de sêmen c...

Pássaros são temas ideais para estudos que envolvem a qualidade do esperma e competição1, esperma telômero dinâmica2, epigenetics3e tópicos semelhantes, como são amplamente utilizados em pesquisas biológicas e esperma pode ser degustadas facilmente usando esfregaços massagem. Esfregaços massagem é uma técnica manual suave, não-invasivo para a coleta de sêmen de aves4,5,6. Amostras repetidas podem ser obtidas facilmente e sem ferramentas especiais são necessárias, tornando simples para executar no campo ou no laboratório. Apesar de esfregaços massagem tem sido usado por décadas, é difícil de aprender com os escritos descrições disponíveis. Esta publicação destina-se a reduzir o tempo e incerteza envolvidos em aprender massagem esfregaços.
Sêmen coletado de aves usando esfregaços massagem pode ser usado imediatamente para avaliação de motilidade4,7 ou inseminação artificial8, ou congelado para outros usos, tais como de imagem avançada e extração de DNA. Amostras de sêmen de aves da ordem Passeriformes são pequenas mas contenham esperma densamente. DNA é extraído usando um kit de extração comercial para manter a simplicidade, com modificações para superar as funções de protecção especializadas de esperma9. Após a extração, telômeros de esperma podem ser medidos usando 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

Colheita de sémen pode ser útil para uma vasta gama de aplicações, incluindo estudos que envolvem a qualidade do esperma, esperma telômero dinâmica e epigenética. Pássaros são assuntos amplamente utilizados na investigação biológica e são ideais para estudos que envolvam amostras repetidas de esperma. No entanto, alguns recursos estão atualmente disponíveis para aqueles que desejam aprender a coletar e extrair DNA de esperma aviária. Aqui descrevemos esfregaços massagem, uma técnica manual suave, não-invasivo para a coleta de esperma aviária. Embora esta técnica é estabelecida na literatura, pode ser difícil de aprender com as descrições disponíveis. Nós também fornecemos informações para extrair DNA de sémen aviária usando um kit de extração comercial com modificações. Esfregaços massagem pode ser usada facilmente em qualquer pequeno para médio porte masculino ave condição reprodutiva. Após a coleta, o sêmen pode ser usado imediatamente para ensaios de motilidade ou congelado para extração de DNA, seguindo o protocolo descrito neste documento. Este protocolo de extração foi refinado por esperma aviária e tem sido utilizado com sucesso em amostras coletadas de diversas espécies de passeriformes (Passer domesticus, Spizella passerina, Haemorhous mexicanuse Turdus migratorius ) e um columbid (Columba livia).

Massagem de esfregaços e DNA extração usando que o protocolo descrito foi realizado em várias espécies de passeriformes e um columbid (tabela 1). A presença de espermatozoides em amostras de sêmen recolhidas pela massagem esfregaços foi confirmada pela visualização de uma pequena quantidade das amostras diluídas sob um microscópio composto em ampliação de 400 X (Figura 3). Depois de extrair o DNA de amostras de sêmen coletados ...

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 descreve esfregaços massagem, uma técnica não-invasiva manual para coleta de sêmen de aves e extração de DNA de esperma aviária usando um kit de extração comercial com modificações.

Coleta de sêmen aviária por esfregaços massagem e isolamento do DNA de esperma

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

avian-semen-collection-cloacal-massage-isolation-dna-from

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Biology

15.9K visualizações.  North Dakota State University. Este protocolo descreve esfregaços massagem, uma técnica não-invasiva manual para coleta de sêmen de aves e extração de DNA de esperma aviária usando um kit de extração comercial com modificações.

Vídeo: 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.

Uma abordagem padronizada para a purificação multiespecífica de células germinais masculinas de mamíferos por dissociação mecânica do tecido e citometria de fluxo

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

Biologia do Desenvolvimento

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.

Manipulação e tratamento de macho Europeu enguias (Anguilla anguilla) para maturação Hormonal e criopreservação 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.

Enriquecimento de espermatócitos de Pachytene e Spermatids de testes de camundongo usando equipamento de laboratório padrão

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.

Isolamento de células spermatogênicas murinas usando um corante de ligação de DNA permeável celular-permeável violeta

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.

Isolamento e In vitro A ativação do 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 ...

Biologia

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.

Transgênicos Rodent Ensaio para Quantificação celular germinativas masculinas Mutant Frequency

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.

Coleção de esperma de qualidade diferencial usando centrifugação gradiente de densidade

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.

Coleção gamete e fertilização 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 correntes elétricas através da membrana plasmática de espermatozóides 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.