Grazie Jose Noguera per intervento tempestivo con massaggio cloacal, e Jeffrey Kittilson per il laboratorio di supporto. Questo lavoro è stato supportato da un premio di ND EPSCoR FAR0022429 a BJH. Spese di pubblicazione sono stati generosamente sostenute dal dipartimento di scienze biologiche di NDSU.

Questo protocollo coinvolge soggetti animali vertebrati ed è stato approvato dal istituzionale Animal Care e uso Committee (IACUC) North Dakota State University.
1. centri di raccolta da un uccello passeriforme utilizzando cloacal massaggi
Nota: Cloacal massaggio è una tecnica di raccolta di sperma efficace sugli uccelli riproduttivo attivi solo, ma può essere eseguita correttamente di fuori della stagione riproduttiva nelle opportune situazioni in cattività. Attività riproduttiva deve essere determinata per specie di destinazione prima di utilizzare massaggio cloacal. Questa tecnica può essere utilizzata su uccelli selvatici al momento cattura o sugli uccelli tenuti in cattività.
<ol><li>A partire con un uccello maschio, (in condizioni di allevamento) presa di Bonetti nella mano non dominante, trasferire l'uccello alla mano dominante con il lato ventrale toccando il palmo (Figura 1).<ol>
<li>Assicurarsi che la testa dell'uccello è più vicino il bordo laterale del palmo della mano. Garantire leggermente la testa e il corpo utilizzando il mignolo, anello e le barrette centrali. Questo lascia l'uccello dello sfiato e coda esposti.</li>
		<li>Lasciare le gambe d'uccello sciolti o leggermente trattenuto usando il palmo.</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55324/55324fig1.jpg"> Figura 1 : Presa per massaggi cloacal. L'uccello è protetto con leggerezza nella mano dominante con il lato ventrale toccando la Palma e la testa, protetta dal dito mignolo. <a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig1large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
<ol start="2"><li>Posizionare l'indice e pollice dominante su entrambi i lati dell'estremità superiore della cloaca e leggermente avvicinare le dita esercitando una pressione molto lieve verso la coda ( Figura 2). La cloaca sarà evert, esponendo la mucosa cloacale (interni rosa). Nota: Se l'uccello non è attualmente in produzione dello sperma, la protuberanza cloacal può essere piccola e la cloaca è difficile da evert. Con esperienza, è facile distinguere tra il cloacas i maschi riproduttivi attivi e inattivi5,11.</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55324/55324fig2.jpg"> Figura 2 : Dito posizione e protuberanza cloacal. Evert cloaca applicando leggera pressione all'estremità superiore (A). In un passeriforme maschio riproduttivo attivo, è evidente la protuberanza cloacale e impresa (B). <a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig2large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
<ol start="3"><li>Con la mano non dominante, posizionare il cuscinetto del dito indice sul lato dorsale della base della coda per stabilizzare la mano e quindi posizionare la punta del pollice appena sotto la cloaca anteverso. Nota: Una miniatura corta e liscia è vantaggiosa per l'esecuzione di massaggi cloacal e protegge l'uccello da un infortunio accidentale.</li>
	<li>Spostare il pollice non dominante cranialmente e profondo in un movimento ripetitivo con media pressione, restando di sotto della cloaca. Continuare fino a quando l'uccello eiacula. A seconda dell'individuo, ciò avviene in genere tra 1 e parecchi dozzina colpi.</li>
	<li>Non appena l'uccello eiacula, raccogliere lo sperma in un tubo di microhematocrit. Ripetere cloacal massaggio per ottenere più eiacula. Nota: Lo sperma passeriforme è marrone chiaro e in genere ha una consistenza piuttosto densa. Bianche o scure sostanze espulsione dalla cloaca non sono lo sperma e potrebbero contaminare il campione di sperma. L'aspetto di una piccola quantità di sangue sulla cloaca o misto in con lo sperma è solitamente causato da troppa pressione durante il massaggio.</li>
	<li>Per estrazione del DNA, è possibile inserire lo sperma in un tubo del microcentrifuge con 20 µ l di 1X PBS (pH 7.4). Nota: Semen memorizzati in 1X PBS non è adatto per l'inseminazione artificiale, ma può essere utilizzato per l'estrazione di DNA in un secondo momento quando conservato a-80 ° C. Il PBS può essere a temperatura ambiente o refrigerati, ma dovrebbe essere in uno stato liquido quando viene depositato il campione di sperma.<ol>
<li>Confermare la presenza di spermatozoi nel campione visualizzando una piccola quantità del campione diluito sotto un microscopio composto (Figura 3).</li>
	</ol>
</li>
	<li>Conservare lo sperma diluito a-80 ° C fino all'estrazione.</li>
</ol>2. estrazione di DNA da sperma di uccello
Nota: Questo protocollo è stato testato con più kit commerciali di estrazione del DNA, ma ha solo avuto successo quando viene utilizzato con un DNA kit12. Passaggi che vengono modificati dal protocollo kit sono indicati da *.
<ol><li>Preparare una soluzione 1 M di ditiotreitolo (DTT) in acqua e mescolare usando un vortex.*<ol>
<li>Aliquota della soluzione DTT e conservare a-20 ° C fino a immediatamente prima dell'uso. Ogni campione deve essere estratto richiede 10 µ l di soluzione di 1m DTT</li>
	</ol>
</li>
	<li>Preparare un tampone contenente 20 mM Tris-Cl, pH 8.0; 20 mM EDTA; 200 mM NaCL; e 4% SDS. Ogni campione deve essere estratto richiede 90 µ l di questa buffer.*<ol>
<li>Conservare il tampone a temperatura ambiente. Se è precipitato, riscaldarlo a 56 ° C per dissolve.*</li>
	</ol>
</li>
	<li>Preriscaldare un incubatore contenente un rocker o agitatore a 65 ° c. *</li>
	<li>Immediatamente prima di iniziare l'estrazione, mescolare 10 µ l di soluzione DTT con 90 µ l di tampone preparata al punto 2.2 per ciascun campione da estrarre, ad esempio 900 µ l di tampone più 100 µ l di soluzione DTT se l'estrazione di 10 campioni. Questa miscela si chiamerà DTT-buffer.*</li>
	<li>Aggiungere 70 µ l 1X PBS per il campione di sperma diluito intero e mescolare bene con un vortex.*</li>
	<li>Aggiungere 100 µ l DTT-buffer e 10 µ l di soluzione di proteinasi K al campione. Vortice per 20 secondi, quindi Incubare in incubatrice preriscaldato per 1 ora su un bilanciere o shaker.*</li>
	<li>Aggiungere 200 µ l tampone AL e 200 µ l fresco EtOH (100%) e vortex per 20 seconds.*</li>
	<li>Trasferire l'intero campione a una colonna di spin e centrifugare a 1 minuto a 6.000 x g. gettare la provetta di raccolta.</li>
	<li>Posizionare la colonna su un tubo nuovo e aggiungere 500 µ l preparati buffer AW1. Centrifugare 1 minuto a 6.000 x g. gettare la provetta di raccolta.</li>
	<li>Posizionare la colonna su un tubo nuovo e aggiungere 500 µ l preparati buffer AW2. Centrifugare per 1 minuto a 6.000 g. x eliminare il flusso continuo e posto la colonna posteriore sul tubo.</li>
	<li>Centrifugare per 3 minuti a 20.000 x g. gettare il tubo di raccolta e posizionare la colonna in una microcentrifuga da 1,5 mL.</li>
	<li>Dispensare 35 µ l di AE buffer direttamente sulla membrana di colonna e incubare a temperatura ambiente per 5 minuti. *</li>
	<li>Centrifugare 1 minuto a 14.000 x g. gettare la colonna di spin e conservare i campioni estratti a-80 ° C.</li>
</ol>

<ol><li>Briskie, J. V., Montgomerie, R. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/1349186">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/pubmed?term=((Telomeres+and+life+histories%3A+the+long+and+the+short+of+it%5BTitle%5D)+AND+%22Ann.+N.Y.+Acad.+Sci%22%5BJournal%5D)">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/pubmed?term=((Epigenetics+of+the+male+gamete%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">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="https://academic.oup.com/ps/article-abstract/16/1/19/1564432?redirectedFrom=fulltext">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="https://www.jstor.org/stable/4510381?seq=1#page_scan_tab_contents">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="https://www.jstor.org/stable/4089416?seq=1#page_scan_tab_contents">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="https://link.springer.com/article/10.1007/s00265-009-0871-x">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://onlinelibrary.wiley.com/doi/10.1111/jai.12070/abstract">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="https://www.nature.com/articles/318577a0">Forensic application of DNA 'fingerprints'.</a> Nature. 318 (12), 577-579 (1985).</li>
<li>Criscuolo, F., et al. <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1111/j.1600-048X.2008.04623.x/abstract">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.birds.cornell.edu/homestudy/handbook-of-bird-biology">Cornell Lab of Ornithology Handbook of Bird Biology, 3rd Edition</a>. , Wiley-Blackwell, Princeton University Press. ISBN 9781118291047 (2016).</li>
<li><a target="_blank" href="https://www.scribd.com/document/54238221/QIAamp-DNA-Micro-Handbook">QIAamp DNA Micro Handbook</a>. , Third edition (2014).</li>
<li>Carter, K. L., Robertson, B. C., Kempenaers, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+differential+DNA+extraction+method+for+sperm+on+the+perivitelline+membrane+of+avian+eggs%5BTitle%5D)+AND+%22Mol.+Ecol%22%5BJournal%5D)">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.sciencedirect.com/science/article/pii/S0003347296904236">Song, sperm quality and testes asymmetry in the sedge warbler.</a> Anim. Behav. 53, 965-971 (1997).</li>
<li>Lüpold, S., Calhim, S., Immler, S., Birkhead, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sperm+morphology+and+sperm+velocity+in+passerine+birds%5BTitle%5D)+AND+%22Proc.+R.+Soc.+B%22%5BJournal%5D)">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/pubmed?term=((Semen+collection+and+spermatozoa+characteristics+in+budgerigars+%28Melopsittacus+undulates%29%5BTitle%5D)+AND+%22Vet.+Rec%22%5BJournal%5D)">Semen collection and spermatozoa characteristics in budgerigars (Melopsittacus undulates).</a> Vet. Rec. 118, 397-399 (1986).</li>
<li>Stelzer, G., Crosta, L., Bürkle, M., Krautwalt-Junghanns, M. E. <a target="_blank" href="https://www.researchgate.net/publication/232663050_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>

Gli autori dichiarano di non avere nessun concorrenti interessi finanziari.

Descriviamo un metodo semplice e affidabile per la raccolta dello sperma da uccelli di piccoli e medi in stato riproduttivo attivo ed estrazione di DNA da sperma aviaria.
Il protocollo di estrazione del DNA dello sperma descritto viene modificato da un kit per semplicità, ma è stato perfezionato per l'uso su sperma aviaria. Gli spermatozoi sono resistenti alla lisi di estrazione standard prodotti chimici9,13, e campioni di sperma rac...

Gli uccelli sono soggetti ideali per studi su qualità dello sperma e concorso1, sperma del telomero dinamica2, epigenetica3e argomenti simili, come essi sono ampiamente utilizzati nella ricerca biologica e dello sperma può essere facilmente campionato utilizzando cloacal massaggi. Cloacal massaggio è una tecnica manuale dolce, non invasiva per la raccolta dello sperma da uccelli4,5,6. Campioni ripetuti possono essere ottenuti facilmente e attrezzi speciali non sono necessari, che lo rende semplice da eseguire nel campo o nel laboratorio. Anche se cloacal massaggi sono stato in uso per decenni, è difficile da imparare dalla scritta descrizioni disponibili. Questa pubblicazione è intesa a ridurre il tempo e l'incertezza coinvolti nell'apprendimento massaggi cloacal.
Lo sperma raccolto da uccelli utilizzando cloacal massaggio può essere utilizzato immediatamente per motilità valutazione4,7 o8di inseminazione artificiale, o congelato per altri usi come imaging avanzato e l'estrazione del DNA. Campioni di liquido seminale da uccelli passeriformi sono piccoli ma contengono sperma densamente imballato. Il DNA è estratto usando un kit di estrazione commerciale per semplicità, con modifiche per superare le caratteristiche protettive specializzate di sperma9. Dopo l'estrazione, i telomeri dello sperma possono essere misurati 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

Raccolta di sperma può essere utile per una vasta gamma di applicazioni, tra cui studi su qualità dello sperma, sperma del telomero dinamiche e l'epigenetica. Gli uccelli sono soggetti ampiamente usati nella ricerca biologica e sono ideali per studi condotti su campioni di sperma ripetuti. Tuttavia, poche risorse sono disponibili per coloro che desiderano imparare a raccogliere ed estrarre DNA da sperma aviaria. Qui descriviamo cloacal massaggio, una tecnica manuale dolce, non invasiva per la raccolta dello sperma aviaria. Anche se questa tecnica è stabilita nella letteratura, può essere difficile da imparare dalle descrizioni disponibili. Forniamo anche informazioni per l'estrazione del DNA da aviaria sperma usando un kit di estrazione commerciale con modifiche. Massaggi cloacal possono essere facilmente utilizzato su qualsiasi uccello maschio medio-piccole in condizione riproduttiva. Dopo il prelievo, lo sperma può essere utilizzato immediatamente per saggi di motilità, o bloccato per l'estrazione del DNA seguendo il protocollo descritto nel presente documento. Questo protocollo di estrazione è stato perfezionato per aviaria sperma ed è stato utilizzato con successo su campioni raccolti da diverse specie di passeriformi (Passer domesticus, Spizella passerina, Haemorhous mexicanuse Turdus migratorius ) e un columbid (Columba livia).

Cloacal massaggi e DNA estrazione utilizzando che il protocollo descritto è stato eseguito su diverse specie di passeriformi e uno columbid (tabella 1). Visualizzando una piccola quantità di campioni diluiti sotto un microscopio composto a 400 ingrandimenti (Figura 3) è stata confermata la presenza di spermatozoi nel seme campioni raccolti da massaggio cloacal. Dopo l'estrazione del DNA dai campioni di sperma raccolto da passeri della came...

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

Questo protocollo descrive cloacal massaggio, tecnica manuale non invasiva per la raccolta di sperma da uccelli ed estrazione di DNA da aviaria dello sperma usando un kit di estrazione commerciale con modifiche.

Raccolta dello sperma aviaria Cloacal massaggi e isolamento di DNA da sperma

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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Universidad de Cartagena UDEC

Research

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Biology

16.0K Views.  North Dakota State University. Questo protocollo descrive cloacal massaggio, tecnica manuale non invasiva per la raccolta di sperma da uccelli ed estrazione di DNA da aviaria dello sperma usando un kit di estrazione commerciale con modifiche.

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 approccio standardizzato per la depurazione multispecie di cellule germinali maschili di mammiferi mediante dissociazione tissutale meccanica e citometria del flusso

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

Ricerca

Biologia dello sviluppo

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.

Gestione e trattamento di maschio anguilla europea (Anguilla anguilla) per maturazione ormonale e crioconservazione degli spermatozoi

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.

Arricchimento di spermatociti pachytene e Spermatidi da test del mouse utilizzando attrezzature di laboratorio standard

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 delle cellule spermatogeniche murine utilizzando un colorante legante del DNA permeabile alle cellule violetto

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 Attivazione di 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.

Transgenici Roditore Assay per quantificare Maschio cellule germinali Mutant Frequenza

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.

Raccolta di sperma di qualità differenziale mediante centrifugazione in gradiente di densità

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.

Collezione Gamete e Fecondazione in vitro di 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>Registrazione delle correnti elettriche attraverso la membrana plasmatica degli spermatozoi dei mammiferi

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.