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...

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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.

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	<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. ...

The overall goal of this procedure is to collect semen from male birds and isolate DNA from the semen samples. This method can be used to answer key questions in behavioral and evolutionary ecology related to sperm competition and epigenetic inheritance. The main advantage of this technique is that it is easy to use.Cloacal massage can be performed quickly in the field or lab, and the DNA extraction method uses a commercially available kit with modifications. This technique can be used on wild or captive birds during the breeding season, and in certain captive situations it can be effective year round. Take hold of the reproductively active male with a bander's grip in the non-dominant hand.Then transfer it to the dominant hand with its ventral side touching the palm and with its head at the palm's lateral edge. Now lightly secure the head and body using the pinky, ring and middle fingers. The bird's vent and tail are now exposed.The legs can be left loose or lightly restrained using the palm. Next evert the cloaca by placing the dominant index finger and thumb on either side of its superior end and lightly pinching while applying light pressure towards the tail. The pink cloacal mucosa should clearly appear unless the bird is not producing semen.With experience it is easy to differentiate between the cloacas of reproductively active and inactive males. To collect semen, place the pad of the index finger of the non-dominant hand on the dorsal side of the base of the tail for stability, and then place the tip of the thumb just below the everted cloaca. A short smooth thumbnail is strongly recommended.Now move thumb deep and cranially in a repetitive motion with medium pressure, staying below the cloaca until the bird ejaculates. This can take just one stroke to several dozen strokes. As soon as the bird ejaculates, collect the viscous light brown semen into a microhematocrit tube.White or dark substances are contaminants, while any amount of blood indicates that too much pressure was applied. As needed, repeat the massage to obtain multiple ejaculates. For DNA extraction, transfer the sample to a micro centrifuge tube with 20 microliters of PBS.To confirm the presence of sperm in the sample, view a small amount of the diluted sample under a compound microscope at 400X magnification. Then until needed store the sperm samples at minus 80 degrees celsius. This protocol is a modification of a commercial kit.Have prepared 10 microliter aliquots of one moler DTT in water, stored at minus 20 degrees celsius. Just before beginning the DNA extraction, mix an aliquot of DTT with an aliquot of tris chloride for each sample. Next add 70 microliters of PBS to each sample and vortex the mixtures.Then add one DTT buffer mixture and 10 microliters of proteinase K solution to each sample. To mix the samples, vortex them for 20 seconds. Now load the samples into a 65 degree celsius incubator on a shaker, and incubate the samples for an hour with gentle agitation.After the incubation, add 200 microliters of buffer AL and 200 microliters of fresh 100%ethanol, and vortex the samples for 20 seconds. Then transfer each sample to a spin column and centrifuge the samples for one minute at 6, 000G. Discard the collection tube and place the columns onto new tubes.Add 500 microliters of buffer AW1 to the column, and spin the columns again. Again discard the collection tube and place the columns onto new tubes. Now add 500 microliters of buffer AW2 and repeat the spin cycle.Next discard the flowthrough, and return the spin column to the centrifuge for three minutes of centrifugation, now at 20, 000G. Then discard the collection tube and place the column on a micro centrifuge tube. Now directly onto the columns membrane, add 35 microliters of buffer AE and let the solution elute the DNA for five minutes at room temperature.Then move the elusion buffer with the DNA into the micro centrifuge tube using a 60-second spin. Now discard the columns and store the DNA at minus 80 degrees celsius. The described cloacal massage and DNA extraction methods have been used successfully on several passerin species and on one Columbid.When a gel electrophoresis of the extracted DNA has distinct bands without smearing, this indicates a successful isolation of high quality DNA. This example is of DNA from house sparrow semen. After watching this video you should have a good understanding of how to gently collect semen samples from reproductive male birds using cloacal massage, and how to extract DNA from those semen samples using an extraction kit.Once mastered, cloacal massage can be done in just seconds if performed properly.

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