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.
