Zoo Biology 26:425–431 (2007)

BRIEF REPORT

Sexing a Wider Range of Avian Species

Based on Two CHD1 Introns With
a Unified Reaction Condition
Lih-Chiann Wang,1,2 Chi-Tsong Chen,3 Hsiao-Yuan Lee,2 Shou-Hsien Li,4
Jihn-Tsair Lir,5 Shin-Chien Chin,2 Chang-En Pu,3 and Ching-Ho Wang1
1
Graduate Institute of Veterinary Medicine, Department of Veterinary Medicine,
National Taiwan University, Taipei, Taiwan
2
Taipei Zoo, Taipei, Taiwan
3
Scientific and Technical Research Center, Ministry Justice Investigation Bureau,
Hsin-Tien City, Taipei, Taiwan
4
Department of Biology, National Taiwan Normal University, Taipei, Taiwan
5
Chung-Shan Institute of Science and Technology, Taipei, Taiwan

Identifying the sex of a bird is important to ensure successful breeding strategies

and effective conservation programs. Sex may be identified from the intron size of
the CHD1 gene located on the avian sex chromosomes Z and W. However,
because of the great nucleotide diversity across different avian species, no given
intron is in widespread use without ambiguous results. Complicated modifications
of the reaction condition are required to suit different species. Two CHD1 introns
were used with a unified reaction condition in this study to simplify the procedure.
Consequently, genders of 73 avian species covering 19 families were successfully
identified based on this two-intron approach. This means the ability to sex a wider
range of avian species using a simplified procedure, greatly assisting in population
management at zoos. Zoo Biol 26:425–431, 2007. c 2007 Wiley-Liss, Inc.

Keywords: intron; CHD1; sexing

Correspondence to: Ching-Ho Wang, Graduate Institute of Veterinary Medicine, Department of

Veterinary Medicine, National Taiwan University, 1 Sec. 4, Roosevelt Road, Taipei, Taiwan.
E-mail: chingho@ntu.edu.tw
Received 28 July 2006; Revised 16 April 2007; Accepted 4 May 2007
DOI 10.1002/zoo.20149
Published online 20 July 2007 in Wiley InterScience (www.interscience.wiley.com).

r 2007 Wiley-Liss, Inc.

426 Wang et al.

INTRODUCTION
Information about an individual bird’s sex is important, for ecologic and
evolutionary studies and also for appropriate breeding strategy and conservation
program implementation [Fridolfsson and Ellegren, 1999]. A bird’s sex can be
determined based on the size difference of the CHD1 intron between chromosome Z
and W (CHD1Z and CHD1W, respectively) [Griffiths et al., 1998; Kahn et al., 1998;
Fridolfsson and Ellegren, 1999]. Although there are several introns in the CHD1
gene, none of them alone can identify the sex of all species because of the nucleotide
diversity [Montell et al., 2001]. The major problem with a single CHD1 intron is the
ambiguous results that appear on the running gels for some species [Fridolfsson and
Ellegren, 1999; Ito et al., 2003]. A number of modifications have been implemented
for each individual species to decrease the confusion, including different primer pairs
spanning the same intron, different polymerase chain reaction (PCR) annealing
temperatures, different magnesium ions, or agarose gel concentrations [Jensen et al.,
2003].
We used two CHD1 introns with a unified reaction condition for all species to
decrease the ambiguity and simplify the procedure. A wider range of avian species
was then successfully sexed based on the two-intron approach, which will greatly
assist in population management at zoos.

MATERIALS AND METHODS

Samples for sex identification including muscle, blood, and feathers were
collected by the Taipei Zoo. Muscle tissues of avian carcasses were obtained during
necropsy. The internal reproductive organs of each individual was checked and
recorded. Blood and feathers were collected during health checkups. The sex of some
living individuals was confirmed using endoscopy or behavior observations.
Genomic DNA was extracted by incubating 0.1 g muscle, 100 mL blood, or a
single feather with 20 mL proteinase K (Amresco, Solon, OH) and 500 mL digestion
buffer (Amresco) at 561C overnight, then followed by 3  phenol/chloroform
isolation. The DNA was precipitated with 70% ethanol and dissolved in 100 mL
1  TE buffer (Amresco).
Two pairs of primers, 1237L/1272H [Kahn et al., 1998] and 2550F/2718R
[Fridolfsson and Ellegren, 1999] targeting two different CHD1 introns, were used in
this study. PCR was carried out in a reaction volume of 25 mL containing 0.2 mM
dNTP, 0.2 mM each primer, 5 mL genomic DNA, 1.25 units Super Thermal Gold
DNA polymerase (Promega, Madison, WI), and 2.5 mL 10  Gold ST PCR buffer
(Promega). The thermal profile for amplification was 951C for 5 min, 35  (951C for
45 sec, 501C for 45 sec, and 721C for 45 sec), 721C for 5 min. PCR products were
separated in 2.5% agarose gels (Gibco, Grand Island, NY), run in 1  tris/borate/
ethylenediaminetetraacetic acid buffer with 0.5 mg/mL ethidium bromide (Gibco) at
120 V for 2 hr, and visualized under ultraviolet light.

RESULTS
Table 1 shows a summary of sex identification employing intron 1237L/1272H
and intron 2550F/2718R. Among the 80 tested avian species, the genders of 63

Zoo Biology DOI 10.1002/zoo

 TABLE 1. Summary of sex identification by intron 1237L/1272H and intron 2550F/2718R

Intron 1237L/ Intron 2550F/

Familya Genus and speciesa Common namea 1272H 2718R Confirmed method
Spheniscidae Aptenodytes patagonicus King Penguin 1 1 Behavior
Spheniscus demersus African Penguin 1 1 Behavior
Ardeidae Nycticorax nycticorax Black-crowned Night-heron 1 1 Necropsy
Gorsachius melanolophus Malaysian Night-heron 1 1 Necropsy
Ciconiidae Leptoptilos crumeniferus Marabou Stork 1 Necropsy
Threskiornithidae Platalea minor Black-faced Spoonbill 1 Necropsy
Threskiornis aethiopicus Sacred Ibis 1 1 Necropsy
Phoenicopteridae Phoenicopterus minor Lesser Flamingo 1 1 Necropsy
Phoenicopterus chilensis Chilean Flamingo 1 1 Necropsy
Phoenicopterus ruber Caribbean Flamingo 1 1 Necropsy
Anatidae Aix sponsa Wood Duck 1 Necropsy
Anas platyrhynchos Mallard 1 Necropsy
Anas poecilorhyncha Spot-billed Duck Necropsy
Cygnus atratus Black Swan 1 1 Necropsy
Coscoroba coscoroba Coscoroba Swan 1 Necropsy
Accipitridae Spilornis cheela Crested Serpent-eagle 1 Necropsy
Phasianidae Rollulus rouloul Crested Partridge 1 1 Necropsy
Chrysolophus pictus Golden Pheasant 1 1 Necropsy
Syrmaticus Mikado Mikado Pheasant 1 1 Necropsy
Lophura swinhoii Swinhoe’s Pheasant 1 1 Necropsy
Lophura nycthemera Silver Pheasant 1 1 Necropsy
Phasianus colchicus Common Pheasant 1 Necropsy
Gallus gallus Red Junglefowl 1 1 Appearance and necropsy
Meleagris gallopavo Wild Turkey 1 Necropsy
Cracidae Crax rubra Great Curassow 1 Necropsy
Gruidae Grus virgo Demoiselle Crane 1b 1 Necropsy
Grus antigone Sarus Crane 1b 1 Necropsy
Balearica regulorum Grey Crowned-crane 1b 1 Necropsy
Balearica pavonina Black Crowned-crane 1b 1 Necropsy
Columbidae Caloenas nicobarica Nicobar Pigeon 1 1 Necropsy
Two CHD1 Introns for Avian Sexing 427

Zoo Biology DOI 10.1002/zoo

 TABLE 1. Continued

Intron 1237L/ Intron 2550F/

Familya Genus and speciesa Common namea 1272H 2718R Confirmed method
Columba livia Rock Pigeon 1 1 Necropsy
Streptopelia tranquebarica Red Collared-dove 1 1 Necropsy
428 Wang et al.

Streptopelia decaocto Eurasian Collared-dove 1 1 Necropsy

Caprimulgidae Caprimulgus affinis Savanna Nightjar 1 Necropsy
Musophagidae Tauraco persa Guinea Turaco 1 Necropsy

Zoo Biology DOI 10.1002/zoo

Pycnonotidae Pycnonotus sinensis
Pycnonotus taivanus
Sturnidae Sturnus nigricollis
Muscicapidae Rhyacornis fuliginosus
Corvidae Urocissa caerulea
Timaliidae Yuhina brunneiceps
Heterophasia auricularis
Strigidae Otus spilocephalus
Otus bakkamoena
Otus scops
Ketupa flavipes
Dromaiidae Dromaius novaehollandiae
Struthionidae Struthio camelus
Psittacidae Eos bornea
Lorius garrulus
Trichoglossus chlorolepidotus
Trichoglossus haematodus
Trichoglossus h. massena
Chalcopsitta duivenbodei
Chalcopsitta sintillata
Glossopsitta concinna
Amazona aestiva
Amazona amazonica
Amazona autumnalis
Amazona ochrocephala
Light-vented Bulbul 1b Necropsy
Taiwan Bulbul 1b Necropsy
Black-collared Starling Necropsy
Plumbeous Water-redstart 1 Necropsy
Formosan Magpie 1 1 Necropsy
Formosan Yuhina Necropsy
White-eared Sibia 1 Necropsy
Mountain Scops-owl Necropsy
Collard Scops-owl 1 1 Necropsy
Common Scops-owl 1 1 Necropsy
Tawny Fish-owl 1 1 Necropsy
Emu Necropsy
Ostrich Necropsy
Red Lory 1 1 Necropsy
Chattering Lory 1 Necropsy
Scaly breasted Lorikeet 1 1 Endoscopy
Rainbow Lorikeet 1 1 Necropsy
Massena’s Lorikeet 1 1 Necropsy
Brown Lory 1 1 Necropsy
Yellow-streaked Lory 1 1 Endoscopy
Musk Lorikeet 1 1 Endoscopy
Blue-fronted Amazon 1 1 Necropsy
Orange-winged Amazon 1 1 Endoscopy
Red-lored Amazon 1 Endoscopy
Yellow-crowned Amazon 1 Endoscopy
 Ara militaris Military Macaw 1 1 Necropsy
Ara ararauna Blue-and-Yellow Macaw 1 1 Necropsy
Psittacus erithacus Grey Parrot 1 Necropsy
Pionites leucogaster White-bellied Parrot 1 Endoscopy
Pionites melanocephalus Black-headed Parrot 1 Endoscopy
Pionus maximiliani Scaly headed Parrot 1 1 Endoscopy
Coracopsis vasa Vasa Parrot 1 1 Endoscopy
Poicephalus cryptoxanthus Brown-headed Parrot 1 1 Endoscopy
Poicephalus meyeri Meyer’s Parrot 1 1 Endoscopy
Nandayus nenday Nanday Parakeet 1 1 Endoscopy
Aratinga leucophthalma White-eyed Parakeet 1 1 Endoscopy
Cacatua galerita Sulphur-crested Cockatoo 1 Necropsy
Cacatua moluccensis Salmon-crested Cockatoo 1 Necropsy
Cacatua sulphurea Yellow-crested Cockatoo 1 Necropsy
Cacatua s. citrinocristata Citron-crested Cockatoo 1 1 Endoscopy
Cacatua alba White Cockatoo 1 1 Necropsy
Cacatua ducorpsii Ducorps’s Cockatoo 1 1 Endoscopy
Nymphicus hollandicus Cockatiel Necropsy
Probosciger aterrimus Palm Cockatoo 1 1 Necropsy
Cacatus roseicapillus Galah 1 1 Endoscopy
1The sex could be identified by the presence of one band (male) or two bands (female) on the running gel.
The sex could not be identified.
a
The scientific name (family, genus, and species) and common name are based on 2006 IUCN Red List Categories and Criteria (www.iucnredlist.org).
b
The sex could be identified by the presence or absence of the amplified W chromosome product.
Two CHD1 Introns for Avian Sexing 429

Zoo Biology DOI 10.1002/zoo

430 Wang et al.

(78.75%) were successfully identified using intron 1237L/1272H; the remaining 17

(21.25%) were not because of very weak or no amplified products from both
genders.
The failure in sex determination of some avian species using intron 1237L/
1272H implies the existence of genetic diversity. We therefore investigated another
genetic segment, intron 2550F/2718R, to identify the sex. Fifty-nine (73.75%) species
were successfully sexed using intron 2550F/2718R. However, the remaining 21
(26.25%) species were not because of faint or indistinct bands on the gels.
Intron 1237L/1272H worked well in most birds in this study, but it failed in
Ciconiidae, Muscicapidae, Timaliidae, and some species of Psittacidae. Their genders
could be determined using 2550F/2718R. In contrast, sex could not be identified
using intron 2550F/2718R for Accipitridae, Cracidae, Caprimulgidae, Musophagidae,
Pycnonotidae, and some species of Threskiornithidae, Anatidae, Phasianidae, and
Psittacidae; however, intron 1237L/1272H was successful in these species.
Seventy-three avian species covering 19 families were successfully sexed
(91.25%) with introns 1237L/1272H and 2550F/2718R operating complementarily.
Both introns also worked as mutual confirmation. This method was easily carried
out with a unified reaction condition for all species. The two-intron sexing approach
can be applied to more avian species, as proven using necropsy, endoscopy, or
behavior evidence.

DISCUSSION
A bird’s sex could be identified based on the size difference of a given intron
between CHD1W and CHD1Z. However, the identification results from a single
intron tended to be ambiguous for certain species [Griffiths et al., 1998; Fridolfsson
and Ellegren, 1999; Ito et al., 2003], limiting its universal application. For instance,
where only one gel band appears for both genders, both amplified CHD1Z and
CHD1W were too weak to identify, or the CHD1W band was too faint compared to
CHD1Z. The first condition may be because of similar CHD1Z and CHD1W intron
lengths in the tested species, with potential improvement in resolution by substituting
polyacrylamide gel [Griffiths et al., 1998; Kahn et al., 1998] or primer redesigning
[Ito et al., 2003]. However, these alternatives are considerably troublesome. The
latter two ambiguous results may arise from the nucleotide diversity. Jensen et al.
[2003] tried using two primer pairs on a given intron. However, varied PCR
conditions and gel concentrations were used to suit different species. Sex
identification using a given intron with a simple and unified manner thus seems
impossible in these species. Those indistinct results were denoted negative symbols in
our study to exclude any ambiguity. The sex of a wider range of avian species was
successfully determined here with two CHD1 introns operating complementarily.
The gender of certain species could still be identified according to the presence or
absence of CHD1W product (Table 1).
Seven species in this study, spot-billed duck, black-collared starling, Formosan
Yuhina, mountain scops-owl, cockatiel, emu, and ostrich, could not be sexed using
either intron 1237L/1272H or 2550F/2718R. The emu and ostrich seem to lack
heteromorphic sex chromosomes, making sex unidentifiable using genetic markers
on CHD1 [Kahn et al., 1998; Fridolfsson and Ellegren, 1999], EE 0.6 [Ogawa et al.,
1997; Itoh et al., 2001], or Wpkci [Hori et al., 2000; O’Neill et al., 2000]. However, a

Zoo Biology DOI 10.1002/zoo

 Two CHD1 Introns for Avian Sexing 431

new ratite sex-linked marker has been recovered using random amplification of
polymorphic DNA assay [Huynen et al., 2002]. Repetitive G/C-rich sequences on
CHD1W have also made sexing possible on polyacrylamide gels in kiwi [Huynen
et al., 2006]. The failure of sex determination in other species may reflect greater
nucleotide diversity in the tested segments. Further candidate genetic markers should
be investigated for these species in future studies.

CONCLUSIONS
Avian sexing is difficult to accomplish using one CHD1 intron for all species
because of the genetic diversity, and by which multiple modifications are needed.
Genders of 73 avian species covering 19 families were successfully identified using
two CHD1 introns operating complementarily in a unified reaction condition. Sexing
a wider range of avian species is therefore simple to achieve based on this two-intron
approach.

ACKNOWLEDGMENTS
We would like to thank Chun-Yu Huang, for providing feather samples from
some parrot species.

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Zoo Biology DOI 10.1002/zoo