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

Summary

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.

Abstract

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).

Introduction

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 birds^4,5,6. 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 dec....

Protocol

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.

Results

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 magnification (Figure 3). After extracting DNA from semen samples collected from house sparrows using the protocol described here, eight sa.......

Discussion

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 chemicals^9,13, and semen samples collected from passerines are fairly small (typically a few microlit.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Microhematocrit capillary tubes	Fisherbrand	22-362566	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.
Microcentrifuge tubes	Any
1x PBS	Any		Used for storing semen samples prior to DNA extraction.
DL-Dithiothreitol	Sigma-Aldrich	D0632
Tris base	Sigma-Aldrich	10708976001	Any supplier can be used.
EDTA	Sigma-Aldrich	1233508	Any supplier can be used.
NaCl	Sigma-Aldrich	793566	Any supplier can be used.
SDS	Sigma-Aldrich	1614363	Any supplier can be used.
QIAamp DNA micro kit	Qiagen	56304	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.