We thank Jose Noguera for early assistance with cloacal massage, and Jeffrey Kittilson for lab support. This work was supported by a ND EPSCoR FAR0022429 award to BJH. Publication costs were generously supported by the NDSU Department of Biological Sciences.

This protocol involves vertebrate animal subjects and has been approved by the Institutional Animal Care and Use Committee (IACUC) at North Dakota State University.
1. Semen collection from a passerine bird using cloacal massage
NOTE: Cloacal massage is an effective semen collection technique on reproductively active birds only, but can be performed successfully outside of the breeding season in appropriate captive situations. Reproductive activity should be determined for target species prior to using cloacal massage. This technique can be used on wild birds upon capture or on birds held in captivity.
<ol>
	<li>Starting with a male bird, (in breeding condition) bander&#39;s grip in the non-dominant hand, transfer the bird to the dominant hand with its ventral side touching the palm (Figure 1).
	<ol>
		<li>Ensure that the bird&#39;s head is nearest to the lateral edge of the palm. Lightly secure the head and body using the pinky, ring, and middle fingers. This leaves the bird&#39;s vent and tail exposed.</li>
		<li>Leave the bird&#39;s legs loose or lightly restrained using the palm.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55324/55324fig1.jpg" /> 
Figure 1: Grip for cloacal massage.&#160;The bird is secured lightly in the dominant hand with its ventral side touching the palm and head secured by the pinky finger.&#160;<a href="//cloudfront.jove.com/files/ftp_upload/55324/55324fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Place the dominant index finger and thumb on either side of the superior end of the cloaca and lightly pinch the fingers together while applying very slight pressure toward the tail ( Figure 2). The cloaca will evert, exposing the cloacal mucosa (pink interior). 
	&#8203;NOTE: If the bird is not currently producing semen, the cloacal protuberance can be small and the cloaca is difficult to evert. With experience, it is easy to differentiate between the cloacas of reproductively active and inactive males5,11.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55324/55324fig2.jpg" /> 
Figure 2: Finger position and cloacal protuberance.&#160;Evert the cloaca by applying slight pressure to the superior end (A). In a reproductively active male passerine, the cloacal protuberance is obvious and firm (B).&#160;<a href="//cloudfront.jove.com/files/ftp_upload/55324/55324fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>With the non-dominant hand, place the pad of the index finger on the dorsal side of the base of the tail to stabilize the hand, and then place the tip of the thumb just below the everted cloaca. 
	NOTE: A short and smooth thumbnail is advantageous for performing cloacal massage and protects the bird from accidental injury.</li>
	<li>Move the non-dominant thumb deep and cranially in a repetitive motion with medium pressure, staying below the cloaca. Continue until the bird ejaculates. Depending on the individual, this typically takes between 1 and several dozen strokes.</li>
	<li>As soon as the bird ejaculates, collect the semen in a microhematocrit tube. Repeat cloacal massage to obtain multiple ejaculates. 
	&#8203;NOTE: Passerine semen is light brown and typically has a somewhat thick consistency. White or dark substances ejected from the cloaca are not semen, and may contaminate the semen sample. The appearance of a small amount of blood on the cloaca or mixed in with the semen is usually caused by too much pressure during massage.</li>
	<li>For DNA extraction, place the semen into a microcentrifuge tube with 20 &#181;L of 1x PBS (pH 7.4). NOTE: Semen stored in 1x PBS is not suitable for artificial insemination, but can be used for DNA extraction at a later date when stored at -80 &#176;C. The PBS can be room temperature or chilled, but should be in a liquid state when the semen sample is deposited.
	<ol>
		<li>Confirm the presence of sperm in the sample by viewing a small amount of the diluted sample under a compound microscope (Figure 3).</li>
	</ol>
	</li>
	<li>Store the diluted semen at -80 &#176;C until extraction.</li>
</ol>
2. Extraction of DNA from bird semen
NOTE: This protocol has been tested with multiple commercial DNA extraction kits, but it has only been successful when used with one DNA kit12. Steps that are modified from the kit protocol are indicated by *.
<ol>
	<li>Prepare a 1 M solution of dithiothreitol (DTT) in water and mix using a vortex.*
	<ol>
		<li>Aliquot the DTT solution and store it at -20 &#176;C until immediately before use. Each sample to be extracted requires 10 &#181;L of 1 M DTT solution.*</li>
	</ol>
	</li>
	<li>Prepare a buffer containing 20 mM Tris-Cl, pH 8.0; 20 mM EDTA; 200 mM NaCL; and 4% SDS. Each sample to be extracted requires 90 &#181;L of this buffer.*
	<ol>
		<li>Store the buffer at room temperature. If it has precipitated, warm it to 56 &#176;C to dissolve.*</li>
	</ol>
	</li>
	<li>Preheat an incubator containing a rocker or shaker to 65 &#176;C.*</li>
	<li>Immediately before beginning the extraction, mix 10 &#181;L DTT solution with 90 &#181;L of the buffer prepared in step 2.2 for each sample to be extracted, e.g. 900 &#181;L buffer plus 100 &#181;L DTT solution if extracting 10 samples. This mixture will be called DTT-buffer.*</li>
	<li>Add 70 &#181;L 1x&#160;PBS to the entire diluted semen sample and mix well using a vortex.*</li>
	<li>Add 100 &#181;L DTT-buffer and 10 &#181;L proteinase K solution to the sample. Vortex for 20 seconds, then incubate in the preheated incubator for 1 hour on a rocker or shaker.*</li>
	<li>Add 200 &#181;L buffer AL and 200 &#181;L fresh EtOH (100%) and vortex for 20 seconds.*</li>
	<li>Transfer the entire sample to a spin column and centrifuge 1 minute at 6,000 x g. Discard the collection tube.</li>
	<li>Place the column on a new tube and add 500 &#181;L prepared buffer AW1. Centrifuge 1 minute at 6,000 x g. Discard the collection tube.</li>
	<li>Place the column on a new tube and add 500 &#181;L prepared buffer AW2. Centrifuge for 1 minute at 6,000 x g. Discard the flow-through and place the column back on the tube.</li>
	<li>Centrifuge 3 minutes at 20,000 x g. Discard the collection tube and place the column on a 1.5 mL microcentrifuge tube.</li>
	<li>Pipet 35 &#181;L of AE buffer directly onto the column membrane and incubate at room temperature for 5 minutes.*</li>
	<li>Centrifuge 1 minute at 14,000 x g. Discard the spin column and store extracted samples at -80 &#176;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>

The authors declare that they have no competing financial interests.

We describe a simple and reliable method for collecting semen from small and medium birds in reproductively active condition, and extracting DNA from avian sperm.
The described semen DNA extraction protocol is modified from a kit for simplicity, but was refined for use on avian semen. Sperm are resistant to lysis by standard extraction chemicals9,13, and semen samples collected from passerines are fairly small (typically a few microlit...

Birds are ideal subjects for studies involving sperm quality and competition1, sperm telomere dynamics2, epigenetics3, and similar topics, as they are widely used in biological research and sperm can be easily sampled using cloacal massage. Cloacal massage is a gentle, non-invasive manual technique for collecting semen from birds4,5,6. Repeated samples can be obtained easily and no special tools are required, making it simple to perform in the field or lab. Although cloacal massage has been in use for decades, it is difficult to learn from the available written descriptions. This publication is intended to reduce the time and uncertainty involved in learning cloacal massage.
Semen collected from birds using cloacal massage can be used immediately for motility assessment4,7 or artificial insemination8, or frozen for other uses such as advanced imaging and DNA extraction. Semen samples from passerine birds are small but contain densely packed sperm. DNA is extracted using a commercial extraction kit for simplicity, with modifications to overcome the specialized protective features of sperm9. After extraction, sperm telomeres can be measured using 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

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. Birds are widely used subjects in biological research and are ideal for studies involving repeated sperm samples. However, few resources are currently available for those wishing to learn how to collect and extract DNA from avian sperm. Here we describe cloacal massage, a gentle, non-invasive manual technique for collecting avian sperm. Although this technique is established in the literature, it can be difficult to learn from the available descriptions. We also provide information for extracting DNA from avian semen using a commercial extraction kit with modifications. Cloacal massage can be easily used on any small- to medium-sized male bird in reproductive condition. Following collection, the semen can be used immediately for motility assays, or frozen for DNA extraction following the protocol described herein. This extraction protocol was refined for avian sperm and has been successfully used on samples collected from several passerine species (Passer domesticus, Spizella passerina, Haemorhous mexicanus, and Turdus migratorius) and one columbid (Columba livia).

Cloacal massage and DNA extraction using the described protocol has been performed on several passerine species and one columbid (Table 1). The presence of sperm in semen samples collected by cloacal massage was confirmed by viewing a small amount of the diluted samples under a compound microscope at 400X&#160;magnification (Figure 3). After extracting DNA from semen samples collected from house sparrows using the protocol described here, eig...

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

This protocol describes cloacal massage, a non-invasive manual technique for collecting semen from birds, and DNA extraction from avian sperm using a commercial extraction kit with modifications.

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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Biology

15.9K Views.  North Dakota State University. Dit protocol beschrijft cloaca massage, een niet-invasieve handmatige techniek voor het verzamelen van sperma van vogels, en DNA-extractie van aviaire-sperma met behulp van een commerciële winning kit met wijzigingen.

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.

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

Developmental Biology

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for ...

U-Shaped Horizontal Swimming Technique for Preparing High-Quality Sperm with Low DNA Fragmentation Index

Human semen is a complex mixture comprising progressively motile spermatozoa, non-progressively motile spermatozoa, immotile spermatozoa, cell debris, and viscous seminal plasma. High-quality sperm refers to progressively motile spermatozoa with normal morphology, which often exhibit a lower DNA fragmentation index (DFI) and higher fertilization potential. Preparing high-quality sperm is a critical step in human-assisted reproductive technology. The traditional sperm preparation method, discontinuous density gradient centrifugation (DGC), is time-consuming and labor-intensive. Repeated centrifugation can damage sperm DNA, thereby affecting subsequent fertilization and embryo development. This study introduces a U-shaped horizontal swimming (UHS) method for preparing intracytoplasmic sperm injection (ICSI) sperm, which significantly eliminates the detrimental effects of&#160;centrifugation on sperm DNA.&#160;The UHS method involves creating a UHS lane using a fertilization medium within an ICSI operating dish. A 10 &#181;L fertilization medium microdroplet is placed at the starting point on the left side of the UHS lane to hold the semen. Two additional 10 &#181;L fertilization medium buffer droplets are positioned at intervals along the left middle section of the lane, with all droplets connected by fertilization medium. The dish is then covered with culture oil and incubated overnight at 37 &#176;C with 6% CO2 to equilibrate. Subsequently, 3 &#181;L of semen is added to the microdroplet at the starting point. High-quality spermatozoa swim to the right side of the UHS lane, facilitating their suction into the ICSI injection needle. Dead sperm, cell debris, and other viscous impurities largely remain at the initial point or in the buffer droplets. We simultaneously processed 21 semen samples using both UHS and DGC techniques and compared their DFI. The results demonstrated that the DFI in the DGC group was 5.5% &#177; 3.2%, whereas the DFI in the UHS group was 1.7% &#177; 1.1%. The difference between the two groups was statistically significant (P &#60; 0.05).

This protocol describes a method for screening high-quality sperm with a low DNA fragmentation index by utilizing sperm motility characteristics and thigmotaxis. The method employs a U-shaped horizontal swimming lane, enabling high-quality sperm to reach the opposite side through buffer droplets, while dead sperm, cell debris, and impurities are excluded.

Human semen is a complex mixture comprising progressively motile spermatozoa, non-progressively motile spermatozoa, immotile spermatozoa, cell debris, and ...

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

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