Bird Sexing Workflow

Use molecular biology to tell male and female birds apart

Exotic Birds

Overview

Many birds are sexually monomorphic when young or even when adult, 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:

  • Select appropriate primers for your bird species of interest
  • Extract DNA from feathers or dried blood spots
  • Amplify regions of the bird sex chromosomes Z and W using PCR and an appropriate primer set
  • Visualise and interpret your results using agarose gel electrophoresis

For Primer Set 1 (CHD1F/CHD1R), Primer Set 2 (2550F/2719R), and Primer Set 4 (CHD1LF/CHD1LR), two bands of the appropriate sizes indicate a female bird, and one band indicates a male bird. For Primer Set 3 (P0/P2/3), two or three bands indicate a female bird, and one band indicates a male bird.

Image of agarose electrophoresis gel bird sexing results from domestic pigeons using Primer Set 3 (CHD1LF/CHD1LR). Two bands indicate a female bird, and one band indicates a male bird.

Scientific Background – How does it work?

Birds have two sex chromosomes known as Z and W. These are similar to the 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 amplifying target regions of 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 appropriate primers, 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 gene (present in both males and females) should always be visible – it will only be absent if the DNA extraction or PCR fails. It therefore acts as an individual internal positive control for each specimen.

What does the workflow involve?

Before you start, select an appropriate primer set for your bird species using our guide: Choosing a DNA Primer Set for Bird Sexing. A large proportion of bird species should sex satisfactorily using Primer Set 1 (CHD1F/CHD1R) or Primer Set 2 (2550F/2718R), but some bird species may be problematic, so it is worth checking in advance where possible.

Then:

  1. Sample 2-3 mm of feather tips or a 1×1 mm piece of a dried blood spot into labelled PCR tubes (this is illustrated in the protocols linked below).
  2. Extract DNA from your birds using the protocols for DNA Extraction From Feathers (HotSHOT) or DNA Extraction From Dried Blood Spots (HotSHOT)
  3. Set up and run a PCR using the protocol specific for your chosen primer set:
  4. Visualise your PCR results using agarose gel electrophoresis:
    • Cast a 2% agarose electrophoresis gel, load your PCR products and DNA ladder into it, and run it for 45 minutes for a half-gel run (which should be sufficient for band discrimination), or up to 1 hr 30 for a full length gel for maximum band discrimination
    • Visualise your results using the Bento Lab transilluminator and Gel Imaging Hood, photograph the results with a smartphone camera, and interpret your result with reference to the specific protocol.

What are the possible results of this PCR test?

There are two possible results for most bird species, assuming that appropriate primers are used:

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. For Primer Set 3 (P0/P2/P8) an additional 100 bp larger female-specific band should also be generated, resulting in two or three bands for female birds.

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.

What will you need for this workflow?

The reagents, consumables and equipment you need in order to follow the protocols for DNA Extraction from Feathers, DNA Extraction from Dried Blood Spots, and the Bird Sexing protocols for each primer set are listed below.

Most can be bought from us individually from our online store, or you can buy our Bird Sexing Bundle.

You will need to supply some items yourself: deionised or distilled water (for making up 0.5x TBE buffer from 10x TBE buffer), paper towels to use as a disposable sampling surface and cleaning, and a microwave for melting agarose electrophoresis gels.

Reagents

Consumables

Equipment

Costing Your Bird Sexing Workflow

We’ve put together a spreadsheet calculator to help users of these protocols better understand the costs and resources needed to sex a given number of birds. You can download our spreadsheet calculator here. If you have any questions or feedback about the spreadsheet, please get in touch.

Frequently Asked Questions

If you’re new to DNA sexing, these answers will help you understand why it’s useful, how accurate it is, what you need to get started, and more. Whether you’re wondering about sample collection or costs, this FAQ covers the key points to get started. If you have other questions, get in touch with us.

Why should I use DNA sexing instead of visual methods?

Many bird species are sexually monomorphic, meaning males and females look identical. DNA sexing provides a fast, accurate, and non-invasive way to determine sex without relying on physical traits.

How accurate is DNA bird sexing?

DNA testing is over 99% accurate when performed correctly. It identifies a bird’s sex by detecting differences in genes found on the sex chromosomes.

Which birds can be sexed using this method?

Many bird species can be sexed using this method. The most common approach (based on the CHD1 gene) works for a wide range of species, and we also provide a few alternatives for cases where CHD1 test may not be suitable.

To find out if your bird species is supported, check our searchable guide:
Choosing a DNA Primer Set for Bird Sexing

If your species isn’t listed, or you’re unsure, feel welcome to get in touch with us — we’re happy to offer advice.

What types of samples can be used for testing?

Feathers, blood, or eggshell can all be used for DNA sexing. Feathers are generally the least stressful and easiest to collect, making them a popular choice.

  • Feathers (pulled, not shed)
  • A small drop of blood (from a clipped nail or vein)
  • Eggshell fragments (from recently hatched eggs)

How do I collect a sample safely without harming the bird?

For feathers, gently pluck 2-3 from the chest or underwing. For blood, a tiny drop from a clipped nail is enough. Always follow ethical handling guidelines to minimize stress.

What will I need for the workflow?

We recommend getting our Bird Sexing Bundle and Bento Lab, which will equip you with almost everything you need, apart from a few standard items like water, gloves and paper towel.

All the reagents, consumables and equipment you need in order to follow the DNA Extraction from Feathers and Bird Sexing Protocol are listed below.

Reagents

Consumables

  • 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

Equipment

How much does it cost per sample?

Once you have equipment, the cost can be as low as $2 USD per sample for the materials – depending on which materials you use, and if you’re processing multiple samples at once. We would suggest starting small, and estimating about $5 USD for materials per sample at the very start.

We have put together a spreadsheet calculator if you want to calculate how the costs change with different quantities of samples.

If you have any questions or feedback about the spreadsheet, please get in touch. We are happy to discuss costs via email.

Do I need training to do bird DNA sexing?

You can learn to do it yourself without training.

Bird DNA sexing is a reasonably complex process that involves careful sample collection, DNA extraction, PCR, and gel electrophoresis. If you’re new to molecular biology techniques, some steps can be tricky at first, but many people have successfully learned to do it themselves.

Our resources are designed to be as beginner-friendly as possible, and many users get great results by following them independently. If you get stuck, we’re happy to help by email.

If you prefer hands-on guidance or want to build confidence with expert support, our optional training course walks you through the entire workflow step-by-step, making the learning process smoother and more accessible.

What is the scientific background?

Birds have two sex chromosomes known as Z and W. These are similar to the 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.

Case study: Domestic chicken (Gallus gallus domesticus)

What do the results look like?

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.

There are two primer sets that are commonly used for bird sexing as they can successfully sex birds belonging to many, but not all, taxonomic groupings. These are CHD1F paired with CHD1R, and 2550F paired with 2718R.

When these primers are used to amplify regions of the CHD1-W and CHD1-Z genes from DNA extracted from a female and a male chicken, they produce different amplicons for each sex as can be seen below: two distinct bands for the female specimen, and only one band for the male specimen.

For both primer sets, there are two distinct bands for the female chicken and only one band for the male chicken.

Not found the answer you were looking for?

If we did not answer your question, please contact us directly.