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

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 determine...

<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>

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 border="0" cellpadding="0" cellspacing="0" style="width:572px;" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microhematocrit capillary tubes</td>
			<td style="width:71px;">Fisherbrand</td>
			<td style="width:119px;">22-362566</td>
			<td style="width:167px;">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 height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge tubes</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1x PBS</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td>Used for storing semen samples prior to DNA extraction.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DL-Dithiothreitol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0632</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tris base</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">10708976001</td>
			<td>Any supplier can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EDTA</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">1233508</td>
			<td>Any supplier can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NaCl</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">793566</td>
			<td>Any supplier can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SDS</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">1614363</td>
			<td>Any supplier can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAamp DNA micro kit</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">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.&#160;</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. ...

Research

JoVE Journal

Biology

Harvesting Sperm and Artificial Insemination of Mice

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals.  Peromyscus species are used in a wide range of research ...

Isolation of Genomic DNA from Mouse Tails

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

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 ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

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 ...