Many birds are sexually monomorphic, which means that their sex can not be identified based on appearance. Instead, DNA testing can be used based on male and female birds having a different combination of the bird sex chromosomes Z and W.
This collection of bird sexing workflows shows how to extract DNA from feathers, and how to identify the sex of birds using PCR and gel electrophoresis. Two bands indicate a female bird, one band indicates a male bird.
Are you interested in DIY bird sexing?
We are passionate about making the power of PCR available to anyone. Identifying the sex of birds can be essential for bird owners, breeders, conservationists, and many others.
If you are interested in carrying out this workflow for yourself, we would love to hear from you! You can find all of the resources and information on these pages, and if you have questions, we are happy to answer them.
What will you need for this workflow?
The reagents, consumables and equipment you need in order to follow the DNA Extraction from Feathers and Bird Sexing Protocol are listed below.
- HotSHOT DNA Extraction Kit
- 5x HOT FIREPol® Blend Master Mix Ready To Load
- PCR Grade Water
- Bird Sexing Primer Mixes
- Agarose Tablets
- GelGreen® DNA Stain
- 100 bp DNA Ladder
- 10X TBE buffer (to be diluted to 0.5X)
- Deionised water (not included)
- Plucked feather(s)
- Disposable scalpels or razor blades (not included)
- Nitrile gloves (not included)
- Paper towel (not included)
- 2-200μl Pipette Tips (1 rack)
- PCR tubes
- Bento Lab
- Glass beaker or conical flask (not included)
- 2-20μl Adjustable Pipette
- 20-200μl Adjustable Pipette
Scientific Background – How does it work?
Birds have two sex chromosomes known as Z and W. These are similar tothe X and Y sex chromosomes in humans – each inherited from one parent. Male birds have two copies of the Z chromosome (ZZ) and females have one copy of the Z chromosome and one copy of the W chromosome (ZW). This is the opposite of the situation to humans, where most biological females have two copies of the X chromosome (XX) and most biological males have an X and a Y chromosome (XY).
This PCR workflow involves the CHD1 (Chromodomain Helicase DNA Binding Protein 1) genes, which are present on the W and Z sex chromosomes of birds as two variants – CHD1-W (on the female-specific W chromosome) and CHD1-Z (present in males and females on the Z chromosome). These genes are homologous (almost identical in structure and function) but contain introns (DNA that is removed by RNA splicing before it is translated into mature RNA) that usually differ in length between sexes within a species and between species.
This difference in length allows both sexes to be determined using a simple assay based on intron length differences. Once DNA has been extracted, regions within the CHD1-Z and CHD1-W (if present) genes can be amplified using the polymerase chain reaction (PCR), and the amplified DNA visualised on an electrophoresis gel. If only one band is present, this suggests that only one CHD1 coding variant is present (CHD1-Z), which would be expected for a male with a ZZ chromosome pair. If two bands are present, this suggests that both CHD1-Z and CHD1-W genes are present, which would be expected for a female with a ZW chromosome pair.
The advantage of amplifying both variants (if present) together is that the band produced by the CHD1-Z should always be visible – it will only be absent if the DNA extraction or PCR fails. It therefore acts as an individual positive control for each specimen.
What are the possible results of this PCR test?
There are two possible results for most birds:
Female: One copy of the CHD1-Z gene is present on the Z chromosome, and one copy of the CHD1-W gene is present on the W chromosome. The target amplified regions of these genes are generally different in length and will be visible on an electrophoresis gel as two distinct bands.
Male: Two copies of the CHD1-Z gene are present – one on each of the Z chromosomes. The amplified regions of these genes are almost always identical in length and will be visible on an electrophoresis gel as a single band.
Case study: Domestic chicken (Gallus gallus domesticus)
There are two primer sets that are commonly used for bird sexing as they can successfully sex birds belonging to the majority of the 10 avian orders. These are CHD1F paired with CHD1R and 2550F paired with 2718R. When the experiment is run using DNA extracted from a female and a male chicken, we see the following amplicons on the electrophoresis gel: