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Sex determination in 58 bird species and evaluation of CHD gene as a

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 Zoo Biology 32: 269–276 (2013)

RESEARCH ARTICLE

Sex Determination in 58 Bird Species and Evaluation

of CHD Gene as a Universal Molecular Marker in Bird
Sexing
Milos Vucicevic,1* Marija Stevanov-Pavlovic,1 Jevrosima Stevanovic,1 Jasna Bosnjak,1
Bojan Gajic,2 Nevenka Aleksic,1 and Zoran Stanimirovic1
1
Department of Biology, Faculty of Veterinary Medicine, University of Belgrade, Serbia
2
Department of Parasitology, Faculty of Veterinary Medicine, University of Belgrade, Serbia

The aim of this research was to test the CHD gene (Chromo Helicase DNA-binding gene) as a universal molecular
marker for sexing birds of relatively distant species. The CHD gene corresponds to the aim because of its high degree
of conservation and different lengths in Z and W chromosomes due to different intron sizes. DNA was isolated from
feathers and the amplification of the CHD gene was performed with the following sets of polymerase chain reaction
(PCR) primers: 2550F/2718R and P2/P8. Sex determination was attempted in 284 samples of 58 bird species. It was
successful in 50 bird species; in 16 of those (Alopochen aegyptiacus, Ara severus, Aratinga acuticaudata, Bucorvus
leadbeateri, Cereopsis novaehollandiae, Columba arquatrix, Corvus corax, C. frugilegus, Cyanoliseus patagonus,
Guttera plumifera, Lamprotornis superbus, Milvus milvus, Neophron percnopterus, Ocyphaps lophotes, Podiceps
cristatus, and Poicephalus senegalus), it was carried out for the first time using molecular markers and PCR. It is
reasonable to assume that extensive research is necessary to define the CHD gene as a universal molecular marker
for successful sex determination in all bird species (with exception of ratites). The results of this study may largely
contribute to the aim. Zoo Biol. 32:269–276, 2013. © 2012 Wiley Periodicals, Inc.

Keywords: molecular sexing; CHD gene; 2550F/2718R; bird

INTRODUCTION
More than 50% of bird species are monomorphic
[Griffits et al., 1998], rendering their sexing based on external
morphology impossible. Moreover, even in dimorphic
species, sex determination is problematic in chicks [Kahn
et al., 1998]. Numerous bird protection programs aimed
at the preservation of various species through intensive
bird breeding imply that the sex of individuals is accurate-
ly identified [Ito et al., 2003]. In zoological gardens and
breeding centers, large numbers of birds are bred and traded,
making sex determination extremely important [Vucicevic
et al., 2010]. Gender identification is relevant to veterinary,
medical and ecological sciences, and is helpful in enforcing
legislation and resolving paternity disputes [Lee et al., 2010].
As aviculture is constantly advancing, even individual own-
ers wish to determine bird sex. Accordingly, a number of
recent studies have focused on the development of efficient
molecular methods for sex identification, which are gaining
undivided attention as an aid in research and conservation of
many bird species [Cerit and Avanus, 2007]. Other methods
of avian sex determination are based on the observation of
sex-specific behavior and the comparison of different mor-
phological entities [Baker and Piersma, 1999, Jodice et
al., 2000, Mendenhall et al., 2010, Tella and Torre, 1993].
Surgical methods (laparoscopy and laparotomy), which en-
able the direct observation of gonads, although successful in
most cases, are aggressive [Griffiths and Phil, 2000]. Ultra-
sonography may also be used in sex identification in birds
Grant sponsor: Ministry of Education and Science of Serbia; Grant number:
46002.
*Correspondence to: Milos Vucicevic, Research Assistant, Department of
Biology, Faculty of Veterinary Medicine, University of Belgrade, Blvd.
Oslobodjenja 18, 11000 Belgrade, Serbia.
E-mail: biolog@vet.bg.ac.rs
Received 22 May 2011; Revised 5 December 2011; Accepted 13 December 2011
DOI 10.1002/zoo.21010
Published online 2 May 2012 in Wiley Online Library (wileyonlinelibrary.com).

© 2012 Wiley Periodicals, Inc.

270 Vucicevic et al.
[Hildebrandt et al., 1995], but can be difficult due to the
presence of air sacks [Jensen and Durrant, 2006].
Generally, the individual sex is determined genetical-
ly by genes on one of the two sex chromosomes. In mam-
mals, it is the male-specific Y chromosome that promotes the
development of males; contrary to this, in birds, females are
heterogametic (ZW), while males are homogametic (ZZ).
Owing to the difference in the morphology of sex chromo-
somes, cytogenetics may be useful in sex determination of
birds [Kulic et al., 2010]. The W chromosome has lost most
of the genes during the evolution and is, therefore, reduced in
size, while the Z chromosome is highly conserved and, thus,
larger [Djelic and Stanimirovic, 2004]. However, several
setbacks accompany the use of cytogenetics in bird sexing;
it requires aggressive blood sampling and is labor intensive
since bird cells contain large numbers of chromosomes, from
40 to 126, depending on the species [Griffiths and Phil, 2000].
Chromosome examination has developed over time
from the cytogenetic to a molecular level. Griffiths and Ti-
wari (1995) discovered the Chromo Helicase DNA-binding
gene (CHD gene) on the W chromosome. A very closely
related copy of this was afterwards discovered also on the Z
chromosome by Griffiths and Korn (1997). The avian CHD1
genes belong to a family composed of a chromatin organi-
zation modifier (chromo) domain, a SNF2-related helicase/
ATPase domain, and a DNA-binding domain; thus, the acro-
nym CHD stands for these [Fridolfsson and Ellegren, 2000].
Being functional part of DNA and having evolved
very slowly, the CHD gene is highly conserved even among
distant species. Alignment between CHDW sequence in
birds and CHD sequence in mouse is without gaps exclud-
ing two 130 and 175 bp deletions. This fact is extraordinary,
given that the avian and mammalian lineages diverged more
than 200 million years ago [Ellegren, 1996].
Besides the highly conserved CHD1W/CHD1Z genes
[Ellegren and Sheldon, 1997] some others may be used for
molecular sexing such as Wpcki genes [Hori et al., 2000] and
EE0.6 [Itoh et al., 2001]. Undoubtedly, CHD is the most signif-
icant among them, given that it may be used in almost all bird
species, with the exception of ratites [Griffiths et al., 1996]. The
summary of previous data on bird sexing with the CHD gene
in species tested in the current work is on display in Table 1.
Ellegren (1996) suggested that, due to its high degree
of conservation, the CHD gene can serve as an almost uni-
versal tag for the determination of sex in birds. He presented
several molecular methods using this gene for bird sexing:
an restriction fragment length polymorphism (RFLP) ap-
proach and two alternative polymerase chain reaction (PCR)
strategies to exploit possible length differences between the
two copies of the CHD gene (amplifying both with a primer
set 2917F/3088R) or to perform “allele”-specific amplifica-
tion using copy-specific primers (2945F, cfR and 3224R).
To test the universality of the CHD gene for bird
sexing, it is necessary to assess if it is suitable in as
many as possible species. In this study, the molecular sex
identification method was assessed in 58 bird species with
Zoo Biology
two sets of primers: 2550F/2718R [Fridolfsson and Ellergen,
1999] and P2/P8 [Griffiths et al., 1998] in order to verify the
universality of CHD marker for avian gender determination.
METHODS
Sampling and DNA Extraction
A total of 284 samples of 58 species from 15 avian
orders were provided by The Zoological Garden of Bel-
grade (Table 1). One to 10 thoracic feathers were sampled
by plucking from each bird.
DNA was isolated from the feathers using the DNeasy®
Blood & Tissue Kit, Cat. No 69504 (Qiagen, Valencia, CA).
Quills were cut into 2- to 5-mm-long pieces and submerged
into liquid nitrogen in sealed Eppendorf 1.5 ml tubes. Af-
terward, DNA was extracted following a user-developed
protocol (Purification of total DNA from nails, hair, or feath-
ers using the DNeasy® Blood & Tissue Kit). The incuba-
tion step of the protocol at 56°C was done using DTT, buffer
ATL, and proteinase K until the tissue was lysed completely,
which was determined by observation. Ten microlitres of the
obtained DNA isolate were used in the PCR reaction.
PCR Amplification
Two sets of primers were used for the amplification of
the CHD gene: P2 (5´-TCTGCATCGCTAAATCCTTT-3´)
and P8 (5´-CTCCCAAGGATGAGRAAYTG-3´) described
by Griffiths et al. (1998) or 2550F (5´-GTTACTGATTC-
GTCTACGAGA-3´) and 2718R (5´-ATTGAAATGATC-
CAGTGCTTG-3´) by Fridolfsson and Ellergen (1999).
The following six protocols were used.
Protocol 1 (P1)
PCR amplification was performed in 20 μL reactions
containing: 1 x reaction buffer (Kapa Biosystems), 1.5 mM
MgCl2, 100 μM dNTP (Kapa Biosystems), 0.05 U/μL Taq
polymerase (Kapa Biosystems), and 0.5 μM of each prim-
ers P2/P8 (Invitrogen, Carlsbad, CA). The reaction was per-
formed with 10 μL DNA samples.
Protocol 2 (P2)
PCR amplification was performed with the same
concentrations as in Protocol 1, with the exception of the
primers used: here, 1 μM of each primers 2550F/2718R
(Invitrogen, Carlsbad, CA) was used.
For Protocols 1 and 2 we adapted a single thermal profile
to gain the very best results with both sets of primers. Amplifi-
cation was done on PCR thermocycler Mastercycler Personal
(Eppendorf) and MultiGene Gradient (Labnet International
Inc). The thermal protocol consisted of an initial denaturation
step at 95°C for 4 min, followed by 35 cycles of denaturation
(95°C, 30 sec), annealing (55°C, 30 sec) and DNA extension
(72°C, 45 sec), and a final extension step at 72°C for 4 min.
 Sex Determination in 58 Bird Species 271

TABLE 1. List of Birds Included in the Study

Order Species Common name Noa M/Fb Protocol Previous data

Casuariiformes Casuarius Cassowary 1 Not available
casuarius
Dromaius Australian 5 Kahn et al.,
novae- Emu 1998 (different primers)
hollandiae
Rheiformes Rhea Greater Rhea 3 Not available
americana
Podicipediformes Podiceps Great Crested 1 0/1 P3 Not available
cristatus Grebe
Ciconiiformes Ajaia ajaja Roseate 2 1/1 P2 Tomasulo et al.,
Spoonbill 2002 (different primers)
Eudocimus Scarlet Ibis 3 Not available
ruber
Leptoptilus Marabou Stork 6 0/6 P2 Monadjem et al.,
crumeniferus 2010
Platalea Common 4 2/2 P2 Lee et al., 2008 (different
leucorodia Spoonbill primers)
Ciconia ciconia White Stork 3 2/1 P3 Cortes et al., 1999 (different
primers)
Anseriformes Alopochen Egyptian 2 1/1 P2,P3,P4 Not available
aegyptiacus Goose
Anser indicus Bar-headed 2 Not available
Goose
Branta Canada Goose 2 0/2 P2 Boutette et al., 2002
canadensis
Branta Hawaiian 4 Not available
sandvicensis Goose
Cereopsis no- Cape Barren 3 1/2 P2 Not available
vaehollandiae Goose
Cygnus atratus Black swan 9 3/6 P2 Ong and Vellayan, 2008
Cygnus melan- Black-necked 4 1/3 P2,P3,P6 Ong and Vellayan, 2008
coryphus Swan Ong and Vellayan, 2008
Cygnus olor Mute Swan 4 2/2 P2
Falconiformes Gyps fulvus Griffon Vulture 13 9/4 P2,P3 Dolonec and Sinko, 2009
Haliaeetus White-tailed 6 3/3 P2 Lee et al., 2008
albicilla Eagle
Milvus milvus Red Kite 2 1/1 P3 Not available
Neophron Egyptian 6 2/4 P2, P3 Not available
percnopterus Vulture
Falco subbuteo Eurasian 4 1/3 P3 Ito et al., 2003
Hobby
Aquila heliaca Eastern 1 0/1 P3 Cortes et al., 1999 (different
Imperial primers)
Eagle
Galliformes Acryllium Vulturine 9 3/6 P3 Garcia-Moreno and
vulturinum Guineafowl Mindell, 2000
Gallus gallus Chicken 10 5/5 P3 Wang et al., 2007
Guttera Plumed 7 6/1 P3 Not available
plumifera Guineafowl
Gruiformes Balearica Grey Crowned 2 0/2 P2 Duan and Fuerst, 2001
regulorum Crane
Columbiformes Columba African 2 1/1 P2 Not available
arquatrix Olive-pigeon
Columba livia Rock Pigeon 1 0/1 P3 Jensen et al., 2003
Ocyphaps Crested Pigeon 2 0/2 P2 Not available
lophotes

Zoo Biology
272 Vucicevic et al.

TABLE 1. Continued

Order Species Common name Noa M/Fb Protocol Previous data

Psittaciformes Amazona Blue-fronted 2 1/1 P2,P3 Cortes et al., 1999
aestiva Amazon
Amazona Orange-winged 5 1/4 P3,P6 Boutette et al., 2002
amazonica Amazon Miyaki et al., 1998
Amazona Yellow-crowned 2 0/2 P2
ochrocephala Amazon
Aprosmictus Red-winged 4 4/0 P3 Not available
erythropterus Parrot
Ara ararauna Blue-and-gold 32 21/11 P2,P3,P6 Miyaki et al., 1998
Macaw
Ara chloropterus Green-winged 7 3/4 P2,P3,P6 Ong and Vellayan, 2008
Macaw
Ara severus Chestnut-fronted 11 7/4 P3 Not available
Macaw
Aratinga Blue-crowned 8 6/2 P3 Not available
acuticaudata Conure
Aratinga Red-masked 8 6/2 P3 Miyaki et al., 1998
erythrogenys Conure
Aratinga Jandaya 1 0/1 P3 Miyaki et al., 1998
jandaya Parakeet
Aratinga Sun Parakeet 2 2/0 P3 Miyaki et al., 1998
solstitialis
Cacatua alba White 5 1/4 P3 Wang et al., 2007
Cockatoo
Cacatua Salmon-crested 1 1/0 P3 Boutette et al., 2002
moluccensis Cockatoo
Cacatua Yellow-crested 1 1/0 P3 Boutette et al., 2002
sulphurea Cockatoo
Cacatua Sulphur- 2 1/1 P3 Wang et al., 2007
galerita crested
Cockatoo
Cyanoliseus Burrowing 2 0/2 P3 Not available
patagonus Parrot
Pionites Black-headed 2 1/1 P3,P6 Wang et al., 2007
melanocephala Parrot
Psittacus African Grey 35 21/14 P2,P3 Miyaki et al., 1998
erithacus Parrot
Poicephalus Senegal Parrot 1 0/1 P3 Not available
senegalus
Probosciger Palm Cockatoo 3 3/0 P3 Wang et al., 2007
aterrimus
Trichoglossus Rainbow Lorikeet 5 2/3 P3 Wang et al., 2007
haematodus
Cuculiformes Tauraco persa Guinea Turaco 2 2/0 P3 Boutette et al., 2002
Strigiformes Bubo bubo Eurasian 2 Cortes et al., 1999 (different
Eagle-owl primers)
Coraciiformes Bucorvus Southern Ground 9 2/7 P2,P3 Not available
leadbeateri Hornbill
Passeriformes Ramphastos cuvieri Cuvier’s Toucan 1 Not available
Corvus corax Common Raven 3 1/2 P2 Not available
Corvus frugilegus Rook 1 0/1 P2 Not available
Lamprotornis superbus Superb Starling 4 4/0 P3 Not available
a
Number of samples.
b
Male/Female.

Zoo Biology

Fig. 1. Ethidium bromide stained agarose gels showing sex de-
termination in different avian species with 2550F/2718R set of
primers.
A. M – Ladder, 1 – Cereopsis novaehollandiae (♀), 2 – C. novae-
hollandiae (♂), 3 – Cygnus atratus (♀), 4 – Ocyphaps lophotes (♀),
5 – O. lophotes (♀), 6 – Columba arquatrix (♀), 7 – C. arquatrix
(♂), 8 – Cygnus atratus (♂), 9 – C. atratus (♀), 10 – C. atratus
(♀), 11 – Amazona aestiva (♂), 12 – A. aestiva (♀), 13 – A. ochro-
cephala (♂), 14 – A. ochrocephala (♂)
B. M – Ladder, 15 – Bucorvus leadbeateri (♀), 16 – B. leadbeateri
(♀), 17 – B. leadbeateri (♂), 18 – B. leadbeateri (♀), 19 – Neoph-
ron percnopterus (♀), 20 – N. percnopterus (♀)
C. M – Ladder, 21 – Ara ararauna (♀), 22 – A. ararauna (♀), 23 –
A. ararauna (♂), 24 – Cygnus melanocoryphus (♀), 25 – C. atratus
(♀), 26 – C. olor (♂), 27 – C. olor (♀), 28 – C. olor (♀), 29 – Pla-
talea leucorodia (♂), 30 – P. leucorodia (♂)
D. M – Ladder, 31 – Milvus milvus (♀)
Protocol 3 (P3)
The amplification was carried out in 25 μL reaction
volume containing 12.5 μL of KAPA2G Robust HotStart
ReadyMix (Kapa Biosystems) and 1.25 μL of each primer
from 2550F/2718R primer set and 10 μL DNA sample.
For samples amplified with KAPA2G Robust Hot-
Start ReadyMix, the recommended thermal protocol was
modified according to troubleshooting recommendations
for low yield, and involved 3 min of initial denaturation
at 95°C, followed by 35 cycles of denaturation (95°C,
15 sec), primer annealing (55°C, 15 sec), extension (72°C, 15
sec), and a final extension step at 72°C, which lasted 8 min.
Protocol 4 (P4)
In samples where amplification with either Protocol 1
or Protocol 2 was unsuccessful or the results were inconclu-
sive, the annealing temperature was varied. Gradient PCR
was done on MultiGene Gradient (Labnet International Inc.)
at annealing temperatures ranging from 50–60ºC (50ºC,
50.5ºC, 51.1ºC, 52.4ºC, 53.9ºC, 55.3ºC, 56ºC, 57.3ºC,
58.4ºC, 59.4ºC, 59.7ºC, and 60ºC).
Protocol 5 (P5)
The reamplification of Alopochen aegyptiacus sample
from previously amplified DNA at annealing temperature
51.1°C with 2550F/2718R primers was done on Multi-Gene
Gradient (Labnet International Inc.). The reaction was per-
formed in 20 μL total volume. The concentration of MgCl2
was reduced to 1 mM and of 2550F/2718R primer set to
0.8 μM. The concentrations of PCR-buffer, dNTP and Taq-
Sex Determination in 58 Bird Species 273
polymerase were unchanged. The thermal profile was re-
duced to 25 cycles, without changes in denaturation, anneal-
ing, and extension temperatures.
Protocol 6 (P6)
The reamplification of previously amplified DNA
samples with Protocol 3 from Ara ararauna, A. chloroptera,
Pionites melanocephala, and Psittacus erithacus, and two
reamplifications of samples from Amazona amazonica, Ara
ararauna, and Cygnus melancoryphus, was performed un-
der the same condition as described in Protocol 3, except
that the thermal profile was reduced to 25 cycles at the same
denaturation, annealing, and extension temperatures.
Visualization of PCR products
The PCR products were visualized with UV light after
staining the 2% agarose gel with ethidium bromide. A commer-
cial O’RangeRulerTM 50bp DNA Ladder or O’RangeRulerTM
200bp DNA Ladder (Fermentas) were used as size markers in
order to judge whether Z- and W-bands were obtained.
RESULTS
Gender determination was attempted in 284 feather
samples originating from 58 bird species (Table 1). It suc-
ceeded in 50 species with primer set 2550F/2718R. With
16 (Alopochen aegyptiacus, Ara severus, Aratinga acuti-
caudata, Bucorvus leadbeateri, Cereopsis novaehollandiae,
Columba arquatrix, Corvus corax, C. frugilegus, Cyanoli-
seus patagonus, Guttera plumifera, Lamprotornis superbus,
Milvus milvus, Neophron percnopterus, Ocyphaps lophotes,
Podiceps cristatus, and Poicephalus senegalus) in those 50
species, there had been no earlier attempts at gender deter-
mination with the protocols described in this study (Table
1, Fig. 1). Amplification with primers P2/P8 resulted in a
single band in all samples where attempted, rendering sex
identification impossible (Fig. 2).
Gradient PCR was done with both sets of primers
when the two bands were not clearly visible and unspecific
products rendered the interpretation of the results difficult.
However, varying the annealing temperature with primer
Fig. 2. Ethidium bromide stained agarose gels showing sex deter-
mination in different avian species with P2/P8 set of primers.
M – Ladder, 1 – Cygnus atratus, 2 – Leptopilos crumeniferus,
3 – Alopochen aegyptiacus, 4 – Corvus corax, 5 – Balearica regu-
lorum, 6 – Psittacus erithacus. 7 – Neophron percnopterus, 8 –
bucorvus leadbeateri, 9 – B. leadbeateri, 10 – Columba arquatrix,
11 – Amazona aestiva, 12 – Cereopsis novaehollandiae, 13 – Ara
ararauna, 14 – A. ararauna, M – Ladder
Zoo Biology
274 Vucicevic et al.
set 2550F/2718R at 51.1°C while processing two samples
from A. aegyptiacus, the visualization of the bands was
enabled (Fig. 3A). In one of these samples, we determined
female sex and in the other, since the band was faint (Fig.
3B), we proceeded with reamplification, which led to con-
clusion that the second individual was a male (Fig. 3C).
KAPA2G Robust HotStart ReadyMix was used with
2550F/2718R primer set for some samples from species
listed in Table 1 with P3 (Fig. 4A). These samples were
successfully amplified with KAPA2G Robust HotStart
ReadyMix, which enabled sex determination.
In some samples when KAPA2G Robust HotStart
ReadyMix (Kapa Biosystems) was used, it was necessary,
in order to achieve clear results, to perform one (Fig. 4B) or
two successive reamplifications (Fig. 4C).
DISCUSSION
Because there is a high degree of conservation even
among distant species, the CHD gene is an interesting mark-
er that provides an opportunity for developing a universal
method for molecular bird sexing. PCR amplification of the
CHD gene with both sets of primers used in this study pro-
duces a single Z-band in male and two bands (Z and W) in
female birds.
Having in mind that the aim of this study was to test
the CHD genes in terms of their universality as molecular
markers for bird sex determination, feathers were sampled
from species with a relatively wide distribution through-
out the avian phylogeny in order to create a sample as
representative as possible.
Generally, the use of 2550F/2718R gave good results
in the 50 species, while sex determination was not successful
Fig. 3. Amplification of Alopochen aegyptiacus samples using Gra-
dient PCR.
A. M – Ladder, 1 – 50.5°C, 2 – 51.1°C, 3 – 52.4°C, 4 – 53.9°C, 5 –
55.3°C, 6 – 56°C, 7 – 57.3°C, 8 – 58.4°C, 9 – 59.4°C, 10 – 59.7°C
B. M – Ladder, 11 – Alopochen aegyptiacus (51.1°C) (♀), 12 – A.
aegyptiacus (51.1°C) (♂)
C. M – Ladder, 13 – Alopochen aegyptiacus (♂)
Zoo Biology
with the P2/P8 primers (Table 1). Both sets of primers flank
an intron while annealing at exon sites, and the difference in
the intron length between the two chromosomes allows the
visualization of two bands. The difference between Z- and
W-bands resulting from the amplification with 2550F/2718R
primers is expected to be in the range of 150 to 250 bp [Fri-
dolfsson and Ellegren, 1999], thus enabling the separation and
visualization on agarose gel. The products amplified with P2/
P8 primer set generally do not resolve well in a standard aga-
rose gel electrophoresis in the case of females and may often
be misinterpreted as a single band, which is a characteristic of
a male [Ong and Vellayan, 2008]. The expected difference in
intron size in phylogenetically close species (Ara ambiqua, A.
ararauna, A. cyanoptera, A. macao, Branta sandvicensis, Ca-
loenas nicobarica, Coracias indicus, Dendrocygna viduata,
Electus roratus roratus, and Psitacula cyanocephala) is 30–50
bp, but ranges from 10 to 80 bp [Jensen et al., 2003]. There-
fore, these PCR products are more difficult to clearly resolve
on agarose gel and, in most cases, require the use of polyacryl-
amide gels or other high-resolution methods such as capillary
electrophoresis [Lee et al., 2010]. The deployment of high-
resolution techniques would probably produce good results
with our samples with which it was impossible with P2/P8
primers on agarose gel, but it is not economically justified.
The design of primers 2550F/2718R is such that W-
fragment is the smaller one, enabling bird sexing even if only
one fragment is visualized due to the size differences between
the bands [Dawson et al., 2001]. In our samples, this occurred
in Ajaia ajaja and Neophron percnopterus and had been
Fig. 4. Sex determination in different avian species with KAPA2G
Robust HotStart ReadyMix.
A. M – Ladder, 1 – Psittacus erithacus (♀), 2 – Aratinga erythrog-
enys (♀), 3 – Psittacus erithacus (♀)
B. M – Ladder, 4 – Pionites melanocephala (♂), 5 – P. melano-
cephala (♀), 6 – Psittacus erithacus (♀), 7 – P. erithacus (♂), 8 – P.
erithacus (♂), 9 – P. erithacus (♂), 10 – P. erithacus (♂), 11 – Ara
chloroptera (♀), 12 – A. chloroptera (♂), 13 – A. ararauna (♂),
14 – A. ararauna (♂)
C. M – Ladder, 15 – Amazona amazonica (♂), 16 – Ara ararauna
(♀), 17 – A. ararauna (♀), 18 – A. ararauna (♂)

previously described in Accipitridae, Anatidae, Falconidae,
Gruidae, and Scolopacidae [Fridolfsson and Ellergen, 1999].
In species where sex could not be unambiguously de-
termined (Anser indicus, Branta sandvicensis, Bubo bubo,
Ramphastos cuvieri, Eudocimus ruber, Casuarius casuarius,
Dromaius novae-hollandiae, and Rhea americana), a single
band of same size was present in all samples of a certain spe-
cies. Our endeavors to determine sex with the CHD marker
in ratites resulted in failure, which is concordant with the
findings of other authors [Kahn et al., 1998; Fridolfsson and
Ellegren, 1999] because in those birds, there seems to be a
lack of heteromorphic sex chromosomes [Wang et al., 2007].
For the remaining five nonratite species in which the deter-
mination of gender failed, there are no data on molecular
sexing performed previously, which renders any comparison
impossible.
Modifying the protocol in terms of annealing tempera-
ture, reamplification, or the use of specialized master mix
(KAPA2G Robust HotStart ReadyMix) was necessary to sex
several bird species. This could be expected given that sev-
eral PCR inhibitors are present in feathers, such as pigments
[Baignet et al., 2005], dead cells, RNA [Nielsen et al., 2000],
or various microorganisms. Molecular sexing could also be
influenced by the interspecies intron variations within the
CHD gene [Kahn et al. 1998] and a polymorphism on the W
chromosome [Dawson et al., 2001].
Given that the design of 2550F/2718R primer set is
such that they span an intron, the difference in product size
is expected not only between Z and W chromosome, but
also between the bands from the same chromosome but be-
tween different species, and even intraspecies differences
have been described in Gallus gallus subspecies [Lee et al.,
2010]. These differences could be correlated to difference
in intron size between species, or can be due to mutations.
In the samples of this study, most of Z bands and W bands
were in the range as expected based on data previously pub-
lished [Fridolffson and Ellegren, 1999]. Relatively small
variations between different species were observed, which
requires further research with the use of more resolute
methods.
CONCLUSIONS
Given that both the bird species, which were
successfully sexed and those which were not are widely
distributed across the phylogenetic tree, it may be concluded
that the failure of sex determination in this study does not
correlate to evolutionary relations between the bird spe-
cies. Overall, the protocols used in this study gave good
results with samples taken from phylogenetically relatively
distant avian species. Successful sexing of 50 bird species
with a single pair of primers and only slight modifications
of PCR conditions and thermal protocols suggest that by
improving this methodological approach, a universal mo-
lecular method for sexing various species of nonratite birds
could be accomplished. This is in accordance with the as-
Sex Determination in 58 Bird Species 275
sumption made by Ellegren (1996) that the CHD gene can
serve as an almost universal tag for the determination of
sex in birds.
ACKNOWLEDGEMENT
We declare that the experiment complies with the cur-
rent laws of Serbia, where it was performed.
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