HAYATI Journal of Biosciences December 2012 Available online at:

Vol. 19 No. 4, p 183-190 http://journal.ipb.ac.id/index.php/hayati

EISSN: 2086-4094 DOI: 10.4308/hjb.19.4.183

Application of Two Molecular Sexing Methods

for Indonesian Bird Species: Implication for Captive
Breeding Programs in Indonesia
SRI SULANDARI∗, MOCH SYAMSUL ARIFIN ZEIN

Research Center for Biology, The Indonesian Institute of Sciences (LIPI), Widyasatwaloka Building,
Cibinong Science Center, Jalan Raya Jakarta Bogor Km. 46, Cibinong 16911, Indonesia

Received September 13, 2012/Accepted December 31, 2012

Visually identifying the sex of a bird can be difficult. It cannot be done in half the world’s species when they
are adults, and virtually none can be sexed as chicks. Despite this, the sex of a bird is vital for captive breeding.
An increased number of birds are being sexed using DNA amplification techniques. In this approach, the CHD-
W and CHD-Z are distinguished by the amplification of an intron present in both genes. PCR products on the gel
electrophoresis vary in size revealing one band in males at the CHD-Z, and two bands in females corresponding
to both the CHD-W and CHD-Z. Two independent sets of primer (P8/P2 and 2550F/2718R) were used to amplify
the CHD gene region from both the Z and W chromosome. One hundred and ten (110) birds were sexed using first
pair of primers: (P8/P2). Sexing results indicated that 81.8% were successfully determined, 12.7% failed to be
amplified and 5.5% were not perfectly determined because the PCR products showed thick band. The thick band
caused misidentified female to male birds. An alternative primer (2550F/2718R) was applied to solve the problem.
Two hundreds and twenty-nine birds were sexed and the results showed 100% successfully determined. From this
study, it is suggested to use a pair of 2550F and 2718R primers for distinguishing a male from a female bird.

Key words: sex identification, Indonesian birds, primer sexing, PCR, captive breeding
___________________________________________________________________________

INTRODUCTION in their captive breeding is that their genetic sexes are

difficult to be identified from their external morphological
Indonesia which is one of rich countries in biodiversity characteristics at the time of pairing. It is believed that
of birds has 1598 species and 372 species of them are bird sexing is one of the important factors for successful
endemic to Indonesia (Sukmantoro et al. 2007). On the ex-situ conservation program. If sex determination in the
other hand, the damaged natural habitat and uncontrolled birds is well established, better conservation program will
exploitation of exotic species lead to Indonesia has the be optimistically achieved.
highest number of threatened birds in the world. A study Sex is one of the most variable to distinguish
reported by Baillie et al. (2004) that Indonesia recorded individuals. Sex identification of birds can provide
118 (7.38%) bird species categorized as endangered researchers with important information regarding the
species in 2004 IUCN Red List of threatened species. ecology and behavior of bird species (Helander et al.
Efforts to protect those birds are conducted through 2007), also provides valuable insights into their breeding
government’s act or through in-situ or ex-situ strategies, conservation and management programs
conservation. (Helander et al. 2007; Garcia et al. 2009; Naim et al. 2011),
Several efforts in ex-situ conservation has been reproduction programs of threatened species (Ellegren &
successfully conducted in Indonesia, such as: ex-situ Sheldon 1997). In birds, the absence of juvenile sexual
conservation in the ZOO; ex-situ conservation in captive dimorphism often makes it difficult or even impossible to
breeding by community, collection and documentation of determine a chick’s sex on the basis of external
fauna specimen (Museum Zoologicum Bogoriense) and morphology. A similar problem exists for fully grown
DNA genome as genetic resources in Division of Zoology, individuals of many birds species where adult sexual
Research Center for Biology, the Indonesian Institute of dimorphism is absent or at least not very pronounced.
Sciences (LIPI). Breeding in captivity can be an important Efforts to determine sex in birds have been done from time
factor as preservation measure for the species. Captive to time. Up to now, there have been various approaches
breeders in several location in Indonesia such as being used for sex identification other than molecular
Indonesian Safari Park or Bird’s park or ZOO or Bird’s techniques for monomorphic birds including avian
association required a technique allowing early sex laparoscopy, biochemical analysis, and cytogenetic
determination of the birds. One of difficulties encountered analysis (Richner 1989; Dubiec & Zagalska-neubauer
_________________
∗
Corresponding author. Phone: +62-21-8765056, 2006). However, these approaches are usually time
Fax: +62-21-8765068, E-mail: ssulanda@yahoo.co.id consuming or invasive to individuals.

Copyright © 2012 Institut Pertanian Bogor. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
184 SULANDARI AND ZEIN HAYATI J Biosci

With the development of molecular techniques, MATERIALS AND METHODS

improved sexing techniques have been developed.
Molecular sexing is attractive since it can be potentially Sampling and DNA Extraction. Three hundreds and
provide an accurate and rapid means for sex identification thirty-nine (339) material DNA samples of birds from across
if based on non-invasive techniques (Lessells & Mateman the class aves i.e. 110 samples consisted of 56 species
1996; Ellegren & Sheldon 1997; Sheldon 1998). The and 229 samples consisted of 10 species were used in this
chromosomal sex determination system of bird is different study (Table 1 & 2). Only 8 samples of Macrocephalon
from that of mammals. In birds, female are heterogametic maleo were collected from Sulawesi island, and the
(ZW) while males are homogametic (ZZ), and sexing can remaining samples were collected from bird captivities in
thus be made by the detection of the W chromosome or Java and Bali islands, including Indonesian Safari Park
W chromosome sequences in a sample of unknown sex. (Bali), Indonesian Safari Park (Prigen) and Indonesian
In 1995, Griffiths and Tiwari discovered the first and only Safari Park (Cisarua), Gembiraloka Zoo (Yogyakarta),
avian W chromosome (analogue to Y chromosome in Surabaya Zoo (Surabaya), Taman Margasatwa Ragunan
mammals) “Chromo-helicase-DNA-binding gene” (CHD- (Jakarta), Bali Bird Park (Bali), Bird Traders (West Java),
W). This gene is remarkably conserved and it has been and Pro Animalia. The material DNA samples used in this
shown that a single set of PCR primers can be used to sex study were deposited at the DNA Bank of Indonesian
birds throughout the class aves, with the exception of Fauna, Division of Zoology, Research Center for Biology-
ratites (Griffiths & Tiwari 1996; Griffiths et al. 1996). LIPI. Material DNA (blood and plucked feathers) samples
These sex-specific genetic markers simultaneously which precipitated with ethanol were extracted using
amplify homologous part of CHD-W and the related gene phenol/chloroform procedures (Sambrook et al. 1989).
CHD-Z (referred to as CHD-NW but is actually Z linked DNA Amplification. Molecular technique for sex
(Griffiths & Korn 1997). Because CHD-Z occurs in both identification in birds conducted in this study, based on
sexes it should always be amplified and this ensures that polymerase chain reaction (PCR), in which sex-specific
the PCR reaction has worked. Unfortunately, the two CHD DNA is located by primers and then amplified. The two
products were the same size; therefore Griffiths et al. (1996) CHD-related primer sets (P8/P2 and 2550F/2718R primers)
used a restriction enzyme to selectively cut a fragment used in sex identification were designed to flank the
from the CHD-Z version before gel electrophoresis. Female, fragment of the gene with the intron. This allows
therefore female had two bands and male had one band. discrimination between the products from the Z and W
More recently, Griffith et al. (1998) introduced new chromosomes on a gel. One hundred and ten (110) samples
approach in which no restriction enzyme was needed. (Table 1) were sexed using a set of P8/P2 primers (Griffiths
They employ two primers which anneal to conserved et al. 1998). The first set of primer sequences were as
exonic region but then amplify across an intron in both follows: P8: 5'-CTCCCAAGGATGAGRAAYTG-3' and P2:
CHD-W and CHD-Z. Because these introns are noncoding 5'-TCTGCATCGCTAAATCCTTT-3'; 2550F (5’-
they are less conserved and their length usually differ GTTACTGATTCGTCTACGAGA-3’). If a set of P8/P2
between the genes. It leads to the PCR product vary in primers could not differentiate between male to female, an
size. Therefore, the gel electrophoresis immediately reveals alternative primer set (2550F/2718R) was applied to solve
one band the male and two bands in the female. the problem (Fridolfsson & Ellegren 1999). Sum of 229
In 1999 Fridolfsson and Ellegren also developed a samples (Table 2) were sexed using a pair of 2550F/2718R
simple and universal method for molecular sexing of non- primers (Fridolfsson & Ellegren 1999). The second set of
ratite birds, which based on the detection of a constant primer sequences were as follows: 2550F (5’-
size difference between CHD1W and CHD1Z introns. GTTACTGATTCGTCTACGAGA-3’) and 2718R
Using highly conserved primers flanking the intron, PCR (ATTGAAATGATCCAGTGCTTG-3’).
amplification and agarose electrophoresis, females are PCR amplification for both primer pairs were carried
characterized by displaying one (CHD1W) or two out in a total volume of 15 μl. The final reaction condition
fragments (CHD1W and CHD1Z), while males only show were as follows: reaction containing 0.2 mM of each dNTP,
one fragment (CHD1Z) clearly different in size from the 0.3 pmol of each primer, 2.5 mM MgCl2, 0.5 Units of Taq
female-specific CHS1W fragment. DNA polymerase in 1x reaction buffer (10 mM Tris-HCl
It is known that sex identification of birds is essential pH 8.3 and 50 mM KCl), and 0.3 mg/ml of BSA. Reactions
part of ex-situ conservation breeding programmes. of PCR for both primer pairs were made in the tube 0.2 ml
Although the CHD gene has been used successfully in and the reaction process of PCR were carried out on the
many bird species (Griffiths et al. 1998; Miyaki et al. 1998; thermocycler machine Gene Amp*PCR system 9700
Ito et al. 2003; Sacchi et al. 2004; Lee et al. 2007, 2010), but (Applied Biosystem, USA).
we mainly discussed the merits of two such methods for As many as 229 samples from several captivities were
the molecular sexing of captived birds in this study, the identified using the 2550F/2718R primers. Molecular sexing
Griffith et al. (1998; P8/P2) and Fridolfsson and Ellegren was conducted because sex of the 229 birds could not be
(1999; 2550F/2718R). The aim of this work was to test the identified by morphological appearance. It is believed that
2-molecular sexing method on bird species, particularly bird sexing is one of the important factors for successful
for birds kept in captivity in Indonesia. ex-situ conservation program. Female or male must be
Vol. 19, 2012 Molecular Sexing Method 185

Table 1. Species and number of samples analyzed using P8 and P2 primers

Family Scientific name Total samples Not amplified Note
Psittacidae Psittaculirostris desmarestii 1 1
Psittaculirostris edwardsii 1 1
Chalcopsitta duivenbodei 2 0
Trichoglossus h. haematodus 3 0
Pseudeos fuscata 4 1
Eos bornea (bornea) 2 1
Trichoglossus h. caeruleiceps 2 1
Trichoglossus euteles 2 0
Loriculus galgulus 3 1
Cacatua goffini 6 0 1-Thick band
Alisterus chloropterus 5 1
Cacatua moluccensis 4 1 1-Thick band
Cacatua sulphurea sulphurea 5 0
Probosciger aterrimus 2 1
Cacatua galerita 2 1
Lorius lory lory 2 0
Lorius garrulus 2 0
Cacatua sulphurea 2 0 2-Thick band
Cacatua s. citrinocristata 4 1
Cacatua alba 5 0
Eclectus roratus 3 0
Chalcopsitta atra atra 1 0
Trichoglossus haematodus 2 0
Psittacula alexandri 1 0
Cacatua galerita triton 1 0
Loriculus pusillus 4 1
Cacatua s. parvula 2 0
Cyclopsitta diopthalma 1 1
Aprosmictus erythropterus 1 0
Charmosyna placentis 1 0
Accipitridae Mivus migrans 1 0
Spilornis cheela 1 0
Accipiter virgatus 1 0
Spizaetus bartelsi 1 0
Cuculidae Centropus bengalensis 1 0
Phaenicophaerus curvirostris 1 0
Falconidae Falco tinnunculus 1 0
Strigidae Strix seloputo 1 0
Otus lempijji 2 0
Ketupa ketupu 2 0
Bubo sumatranus 1 0
Corvidae Corvus macrorhynchos 1 0 1-Thick band
Corvus enca 1 0
Phasianidae Lophura ignita rufa 1 0 1-Thick band
Gallus varius 2 0
Pavo muticus 2 0
Argusianus argus 1 0
Columbidae Goura cristata 1 0
Megapodiidae Macrocephalon maleo 2 0
Bucerotidae Rhyticeros undulates 1 0
Anthracoceros malayanus 1 1
Ardeidae Ardea cinerea 1 0
Irenidae Irena puella 2 0
Paradisaeidae Cicinnurus regius 1 0
Paradisaea minor 1 0
Alcedinidae Pelargopsis capensis (Halcyon capensis) 3 0
Total 56 species 110 identified samples 14 not amplified samples 6 thick band samples

determined correctly in breeding captivity. Before sexing sex of the 5-birds can be determined easily, i.e.: male
of the 229 individual samples, a trial experiment for the Hanging parrot (Loriculus sp.) at the top of his chest
2550F/2718R primers was conducted with small number there is a red circle-shaped, while the female Hanging
samples. We used fiveindividuals of known bird sex as a parrot (Loriculus sp.) yellowish green color. Color
control result for molecular sexing technique. The 5-known difference at the top of the chest is what allows people to
bird sex of Hanging Parrots were used as control in this determine whether it is male or female Hanging parrot.
study (1. Loriculus pusillus ♀ , 2. Loriculus pusillus ♀ , 3. The thermal cycling conditions used for P8/P8 primer
Loriculus pusillus ♀ , 4. Loriculus galgulus ♂ , 5. pairs were initial denaturation at 94 οC for 5 minutes, then
Loriculus pusillus ♀ ). By using morphological characters, 30 cycles of denaturation for 30 seconds at 94 °C,
186 SULANDARI AND ZEIN HAYATI J Biosci

Table 2. List of species birds used for sex identification with 2550F and 2718R primers
Family Scientific name Indonesian names Samples type Total
Megapodiidae Macrocephalon maleo Maleo Blood 8
Sturnidae Leucopsar rothschildi Curik Bali Blood 133
Psittacidae Probosciger aterrimus Kakatua Raja Blood 4
Accipitridae Spizaetus bartelsi Elang Jawa Blood 6
Haliastur Indus Elang Bondol Blood 22
Spizaetus cirrhatus Elang Brontok Blood 7
Haliaeetus leucogaster Elang Laut Blood 2
Spilornis cheela Elang Bido Blood 3
Sturnidae Gracula religiosa Beo Plucked feather 17
Turdidae Zoothera citrina Anis merah Plucked feather 27
Total samples 229

annealing for 45 seconds at 50 °C, and extension for 45 ♂ ♀ ♀ ♂ ♂ ♂♀ ♀ ♀ ♂ ♂ ♂ ♀ ♀

seconds at 72 °C. A final run of 48 °C for 1 minute and M100 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
72 °C completed the program for 5 minutes. PCR products
were separated by electrophoresis in 3% agarose gel (FMC
Bioproducts, SeaKem GTG Agarose). While the condition
of PCR used for 2550F/2718R primer pairs were initial 400 bp
denaturation on the temperature of 94 oC for 5 minutes, 300 bp
then 30 cycles of denaturation for for 45 seconds at 94 oC,
annealing for 45 seconds at 46 oC and extension for 90
seconds at 72 oC. At the end of the cycle was followed by
the temperature of 72 oC during 10 minutes. PCR products
Figure 1. Identification of sex in birds with P8/P2 set of primers.
were separated by gel electrophoresis through a 2% The sixteen samples showed in the are: 1. Cacatua
standard agarose gel (Agarose LE, Analytical Grade, moluccensis (Male), 2. Lorius lory lory (Female), 3.
Promega). Lorius garrulus (Female), 4. Chalcopsitta duivenbodei
The gels were run in standard TBE buffer and stained (Male), 5. Pseudeos fuscata (Male), 6. Cacatua suphurea
sulphurea (Male), 7. Probosciger aterrimus (Female),
with ethidium bromide. A commercial O’Range Ruler 100 bp 8. Trichoglossus euteles (Female), 9. Lorius lory lory
DNA Ladder (Fermentas) was used as size marker in order (Female), 10. Cacatua galerita (Male), 11. Spizaetus
to judge whether Z and W-bands were obtained. After bartelsi (Male), 12. Cicinnurus regius (Male), 13. Pavo
electrophoresis at 100V for approximately 45 minutes, gels muticus (Female), 14. Cacatua goffini (Female), 15.
Cacatua goffini (Female), 16. Cacatua alba (Male). ♂
were examined and photographed by digital camera under
=Male and ♀ =Female; M = DNA ladder (100 bp).
UV light. A typical banding pattern was revealed sex of
the birds examined, one band the male and two bands in
the female.
Cyclopsitta diopthalma, Loriculus pusillus, Cacatua
RESULTS galerita, and Cacatua s. citrinocristata. It is also noticed
in this study that not all samples from the same species
Based on the visualization of PCR products, sex of give amplified products. For example, only three out of 4
birds can be determined. The PCR product indicated those samples of Cacatua moluccensis showed amplification.
birds with two bands are females and those with one band Sixteen individual samples of PCR product were
are male. The P8 and P2 sexing results of 30 species of demonstrated molecular sexing technique using P8/P2
Psittacidae (Table 1) showed three species failed to be primers. One hundreds base pairs (bp) of DNA ladder is
identified (Psittaculirostris desmarestii, Psittaculirostris indicated at the first line (Figure 1). The size of PCR
edwardsii, and Cyclopsitta diopthalma) and three species products were between 300 to 400 bp and the size
of psittacidae (Cacatua goffini, Cacatua moluccensis, and differences between the two amplified bands (CHD-W and
Cacatua sulphurea) produced thick single band. CHD-Z) were short. Eight out of sixteen samples (Numbers
We also found thick single band in Corvus 2, 3, 7, 8, 9, 13, 14, 15) were female birds and the remaining
macrorhynchos of corvidae and Lophura ignita rufa of eight samples (Numbers 1, 4, 5, 6, 10, 11, 12, 16) were male
phasianidae. The Anthracoceros malayanus sample of birds (Figure 1). The products of PCR seem different
bucerotidae could also not be determined successfully. appearance between species; for example, the two bands
The results in total (Table 1) showed fourteen samples in the Cacatua goffini (line no.14) are larger than the two
used in this study were failed to be amplified. The 14- in the Pavo muticus (line no.13), also the single band in
unamplified samples were Psittaculirostris desmarestii, Spizaetus bartelsi (line 11) and Cicinnurus regius (line
Psittaculirostris edwardsii, Pseudeos fuscata, Eos bornea 12) showed different length. The different appearance on
(bornea), Trichoglossus h. caeruleiceps, Loriculus agarose gel occurred between Cacatua goffini in line 14
galgulus, Alisterus chloropterus, Cacatua moluccensis, and 15, showing less bright in line 15 than in line 14
Probosciger aterrimus, Anthracoceros malayanus, (Figure 1).
 Vol. 19, 2012 Molecular Sexing Method 187

Sex determination results of 110 individual samples approximately 650 bp, where a female (♀ ) were represented
using P8/P2 primers, was only 81.8% sex correctly by two amplified bands in the size of 400 and 650 bp
determined. While the remaining, the P2 and P8 primer respectively (Figure 4).
combination used in this study was failed to assign their
sex due to amplification failure (12.7%) and yielding a DISSCUSSION
thick band (5.5%). The primer sets of 2550F/2718R was
applied to solve the problem, particularly for thick band Sex of the parrots (Psittacidae) and Bali starling
problem. They developed a technique that using PCR (Sturnidae) were the most widely identified in this study
primers flanking introns which vary in size between (Tables 1 & 2). This bird is one of the most in demand by
CHD1W and CHD1Z, males being recognized in agarose collectors and bird fancier, because of the beautiful,
electrophoresis as displaying a single PCR product (from elegant appearance and economic value. Sexing molecular
CHD1Z) while females show two different products technique was applied to those birds, because it is difficult
(CHD1W and CHD1Z). to discriminate birds between male and female using
Sex of ten Haliastur indus samples which previously morphological characteristic, on the other hand, early sex
identified using P8 and P2 primers, were retested using a determination for captive breeding of birds must be known.
set of 2550F and 2718R primers. Two set of primers used Besides parrot and Bali starling (Bali mynah) birds, other
to identify 10-Haliastur indus showed different sexing pet birds which are very popular as cage-bird on Java,
results. Sex of the ten samples show numbers 1, 2, 3, 4, 5, including Gracula religiosa (Beo) and Zoothera citrina
6, 7, 9, 10, 11 were all males (Figure 2). The P8/P2 primer set (Orange-headed Thrush or Anis). The species of Gracula
failed to distinguish between male and female in species religiosa birds were preferred because of their versatility
of Haliastur indus. In contrast to a set of 2550F/2718R speaking people and Zoothera citrina birds were preferred
primers in Figure 3, showed that 6 samples (numbers 1, 4, because of having good and melodious voice. Recently,
6, 9, 10, 11) were females and 4 samples (numbers 2, 3, 5, captive birds of Beo and Anis also become a lucrative
7) were males. False negative was obtained in when using business opportunity, captive breeding of the birds
P8/P2 primers. scattered across Indonesia.
Two hundreds and twenty nine samples were Breeding of the 2-species birds need to know the
identified using a pair of 2550F and 2718R primers (Table information of sex correctly. Of the 229 samples tested
2). The results showed that sex of the 229-samples could (Table 2), forty four (44) extracted DNA which consisted
be accurately determined (100%). The band patterns of all of 17 samples of Gracula religiosa and 27 samples of
♂ 229 samples from 10 species were in agreement with Zoothera citrina were derived from plucked feather
patterns expected from known sex of the samples. The samples. DNA is extracted from the cells from the basal tip
results also showed that a male (♂ ) birds was represented of the calamus (Morin et al. 1994) or from the blood clot
by a single band fragment (CHD-Z) visualized at embedded in the shaft (Segelbacher 2002; Horvath et al.
2005). According to Taberlet et al. (1999) a potential
concern with feather-based DNA sampling is that the small
♂ ♂ ♂ ♂ ♂ ♂ ♂ ♂ ♂ ♂ number of cells present on within the feather could result
M 1 2 3 4 5 6 7 9 10 11
in inadequate DNA yields for molecular analysis. The result
of this study approved that bird sex identification of the
44 feather samples were determined successfully.

M 1 2 3 4 5 6 7 8 9 10 1112 1314 15 16

Figure 2. Sex identification in ten individuals of Haliastur indus

with P8/P2 set of primers. =Male, M=DNA Ladder
600 bp
(100 bp).
400 bp

♀ ♂ ♂ ♀ ♂ ♀ ♂ ♀ ♀ ♀
M 1 2 3 4 5 6 7 9 10 11 ♀ Figure 4. Identification of sex in birds with 2550F/2718R set of
primers. 1. Control , 2. Control ♀ , 3. Probosciger
aterrimus ( ♀ ), 4. Probosciger aterrimus ( ♂ ),
5.Leucopsar rothschildi (♀ ), 6. Leucopsar rothschildi
(♂ ), 7. Macrocephalon maleo (♀ ), 8. Macrocephalon
maleo (♂ ), 9. Haliastur indus (♂ ), 10.Haliastur indus
(♀ ), 11. Spizaetus cirrhatus (♀ ), 12. Spizaetus cirrhatus
( ♀ ), 13. Spizaetus bartelsi ( ♀ ), 14. Spilornis cheela
Figure 3. Sex identification in ten individuals of Haliastur indus ( ♂ ), 15. Haliaeetus leucogaster ( ♂ ), 16. Spizaetus
with 2550F/2718R set of primers. ♂ = Male, ♀ = Female, cirrhatus (♂ ). ♂ = Male, ♀ = Female, M = DNA Ladder
M = DNA Ladder (100 bp). (100 bp).
188 SULANDARI AND ZEIN HAYATI J Biosci

The results showed different amplification such as that the gender of some avian species cannot be identified
one band was less bright than the other bands on an by the P2/P8 PCR-based protocol (Griffiths et al. 1998).
agarose gel (Figure 1; Line 14 & 15). DNA sample of By using P8/P2 set of primers a negative outcome of test
Cacatua goffini (Line 14) was extracted from liver, while was obtained from this study. Furthermore, the results
DNA sample of Cacatua goffini (Line 15) was extracted were confirmed that apparently among those of 10
from feather. DNA quantity of plucked feather is lower individuals produce offspring. This result gave an approval
concentration than DNA quantity of liver. That result likely that the PCR method using a primer set of 2550F/2718R
occurred because prior to amplification, the DNA was able to correctly identify the sex of 10 individual
concentration of each sample was not equated in this samples of Haliastur indus. Based on Griffiths et al. (1998),
study. One reason from Griffiths et al. (1998) stated that this problem is always associated with systems based on
primer competition occurred during the amplification the sole detection of W-linked sequences: a negative
process, i.e. the primers may match one CHD gene slightly outcome of a test can result both from the sample being
less well that the other. Harvey et al. (2006) supported our male and a technical failure.
result give evidence that feathers can provide sufficient Position of the amplified bands, CHD-W and CHD-Z
DNA for molecular sexing reactions. This low cost, speed, was in between 300-400 bp (Figures 1 & 2). The P8/P2
and ease of collection, storage, and transport of feather primers amplified two alternative PCR fragments, but the
samples are the major advantages (Duan & Fuerst 2001). size difference between two fragments were too short.
The results indicated that only one out of 4 samples Because the length in P8/P2-amplified Z- and W-fragments,
of Cacatua moluccensis was failed to be identified (Table which are extremely short, making it hard to resolve them
1). This problem of not all samples succeed to be amplified on agarose gels (Chang et al. 2008). According to
was due to unknown reasons. It might be caused by Fridolfsson and Ellegren (1999), the difference in size
different quality of DNA sample. There may be too little between Z-and W-spesific fragments amplified with the
DNA present in the isolated DNA sample to amplify. DNA P8/P2 primers, ranging 10-80 bp. Furthermore Ito et al.
concentration varied among samples. As described above (2001) discovered that the size difference between CHD1Z
that yield of DNA was dependent on sources of DNA and CHD1W differ between species 2-8 bp in Accipitridae.
material. In general, Sambrook et al. (1989) stated that That is why several PCR products tried using P8/P2 primers
isolation DNA aim to separate DNA present in the nucleus in Accipitridae (Figure 2) and visualized either using
of the cell from other cellular components. In birds, standard agarose or poly acrylamide gel agarose, showed
because the nucleated erythrocytes of birds make avian female and male are still often indistinguishable. Dawson
blood an unusually rich source of nuclear DNA. DNA in et al. (2001) reported that the fragments amplified with the
plucked feather samples is typically present in much lower P8/P2 primers cannot be distinguished on agarose gel in
copy number than DNA from blood or tissue samples. the auklets. Moreover, the assignment of sex on the basis
The results obtained six-amplified samples were shown of the P8/P2 primers may be in some species difficult
thick single band using P8/P2 primers (Table 1). We found because of a polymorphism in the Z chromosome (Dawson
thick band in this study because agarose gel et al. 2001; Dubeic & Zagalska-Neubauer 2006).
electrophoresis of P8/P2 products, however this bands Things to consider for this sexing study, if high
can not be used for the differentiation of the sexes. Griffiths concentration DNA in the isolated sample was directly
et al. (1998) explained that the PCR did produce CHD-W used to amplify, amplification always failed. The high
and CHD-Z bands but the introns were so similar in size concentration DNA will inhibit the PCR reaction. In that
that they could not be distinguished on a 3% agarose gel, case, the samples might be amplified, but both sexes
and they also suggested to use an 8% denaturing yielding a single amplification product of identical size.
acrylamide gel whose resolution is easily sufficient to They failed to produce a double band for female birds,
discriminate the two products. Thus, sexing primers therefore female bird was identified being male. To test
designed by Griffiths et al. (1998) seem not always possible this theory, information of DNA concentration on each
to separate by standard agarose electrophoresis. Ellegren sample was needed. The isolated DNA samples were
(1996) further suggested to use of single-strand measured using spectrophotometer. DNA concentration
conformation polymorphism (SSCP) analysis or Griffiths of several materials varied. From measurement of DNA
et al. (1996) suggested to differentially cut the PCR concentration, the results indicated that blood has higher
products with enzymes such as HaeIII or MaeII to allow DNA concentration than feather. After DNA concentration
their separation on agarose gel. Other methods were was known, several dilutions of the isolated DNA samples
developed to overcome the limited difference in the length were generated in this study. We found the best PCR
of intron for CHD-Z and CHD-W by using PCR-RFLP product in gel electrophoresis derived from DNA
(Sacchi et al. 2004; Reddy et al. 2007), RAPD (Wu et al. concentration between 50-100 ng/μl, and finally this was
2007), and AFLP (Huang et al. 2007) fingerprintings. used as a default DNA template in the PCR reaction.
Naim et al. (2011) also reported that the P2 and P8 Even though we identified sex of a limited number of
primer combination failed to assign sex for all individuals representative species (Table 1 & 2), it is likely that
of white-bellied sea eagle. Increasing evidence molecular determination of a male from a female using
(Fridolfsson & Ellegren 1999; Sacchi et al. 2004; De volo 2550F/2718R was more effective way than using P8/P2. A
et al. 2005; Huynen et al. 2006; Reddy et al. 2007) shows problem of thick band was never found with 2550F/2718R
Vol. 19, 2012 Molecular Sexing Method 189

primers, so incorrect identification of female to male bird laboratory works. Thanks also to members of ornithology
was avoided. The results of amplified bands (CHD-W and laboratory, Division of Zoology, RCB-LIPI who helped in
CHD-Z) produced in this study were the same as previous species identification during sampling.
results obtained by Ong and Vellayan (2008); Vucicevic et
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