Molecular bird sexing of sulphur‐crested cockatoo (Cacatua galerita) by polymerase chain reaction method

Sex identification of endangered and protected birds in captivity is very important for conservation programs. Half of the world’s bird species are monomorphic, where male and female are difficult to distinguished morphologically, including cockatoos. Sex identification using molecular bird sexing is more accurate and applicable because it directly targets the sex chromosomes. The purpose of this study was to determine the sex of Sulphur‐crested cockatoo (Cacatua galerita) by detecting differences in the intron size of the chromodomain helicase DNA‐binding 1 (CHD1) gene on the Z and W chromosomes by polymerase chain reaction (PCR) method and to compare of plucked feathers and blood samples as DNA sources for molecular bird sexing. DNA was extracted from feather and blood samples from four C. galerita. Extracted DNA was amplified on the CHD1 gene by PCR method with P2, MP, and NP primers, which were visualized using agarose gel 1.5% under UV transilluminator with a wavelength of 280 nm. The resulting PCR product was detected at 392 bp for the CHD1 Z gene segment and 297 bp for CHD1 W gene segments, where males showed a single DNA band (ZZ) and females showed a double DNA band (ZW). Four C. galeritawere 100% successfully determined, consisting of one female and three males. Elec‐ trophoresis results showed DNA bands from blood samples were thicker and brighter than DNA bands from feather samples.


Introduction
Sulphurcrested cockatoo (Cacatua galerita) is a type of cockatoo found in Eastern Indonesia, Papua New Guinea and Australia (BirdLifeInternational 2019). C. galerita is also known as the yellowcrested big cockatoo because of its similarities with Cacatua sulphurea, but C. galerita has a bigger body with a body length of 380510 mm, a wingspan of more than 260 mm, and bodyweight of ap proximately between 815 to 975 g (Beehler et al. 2001; WorldParrotTrust 2018. This bird has white feathers all over its body, except its yellow crest, the underside of the wings, and tail feathers. It has a black and very compact beak and blue eye circles (Foreshaw 2010; Rowley andKirwan 2016).
Cacatua galerita is one of the curved beak birds com monly kept as a pet by Indonesian since its docile nature and ability to imitate human voices. The rise of illegal cap ture of this bird can be threaten its conservation in nature (Nugroho and Zein 2015). Indonesian government classi fies C. galerita as a protected animal and to maintain its conservation, the Indonesian government has issued sev eral regulations, including Law No. 5 Year 1990 concern ing Conservation of Living Resources and Their Ecosys tems,Government Regulation No. 7 Year 1999 on Pre serving Flora and Fauna Species, and Regulation of the Minister of Environment and Forestry No. 106 Year 2018 regarding Second Amendment to Regulation of the Minis ter of Environment and Forestry No. 20 Year 2018 regard ing Types of Protected Plants and Animals. Conservation status of C. galerita in International Union for Conserva tion of Nature (IUCN) is Least concern (LC) (BirdLifeIn ternational 2019). Although C. galerita is classified as the LC status, the distribution number of its wild population is decreasing. According to the Convention on Interna tional Trade in Endangered Species of Wild Fauna and Flora (CITES), C. galerita is included in the appendix II category (Hay 2008) and not an endangered animal, how ever, the trade of these animals should be regulated to pre vent excessive trading (Beno and Ohee 2009). Preserva tion of cockatoos is essential for conserving the population and prevent extinction.
One of the attemps to preserve cockatoos is the bird conservation activities, including in situ developing natu ral habitats, and ex situ breeding outside of their natural habitat. Ability to distinguish the sex of the cockatoos in captivity is an important aspect that should be considered in breeding for the prospective broodstock and register ing the data of the chicks (Nugroho and Zein 2015). C. galerita are monomorphic birds, where male and female birds have almost the same morphology, size and color, so that distinguishing the sex by using observation alone is rather difficult (Rowley and Kirwan 2016). Various meth ods are developed for bird sexing, such as karyotype, hor mone analysis, laparoscopy, and vent sexing. However, these methods have various disadvantages, such as being timeconsuming, high cost, invasive and difficult to apply in the field, or can only be carried out on a certain period of time. Other methods for bird sexing are the molecular method and morphometric analysis method (Dubiec andZagalskaNeubauer 2006; Cerit andAvanus 2007).
The molecular method, developed by (Fridolfsson and Ellegren 1999), is a method for determining the sex of birds based on deoxyribonucleic acid (DNA), which is based on the differences among the intron size of the Chro modomain Helicase DNAbinding 1 (CHD1) gene in the Z and W chromosomes. Molecular method expedites identi fication of a bird's sex since it can be applied in the bird as young as 57 days; and take for short determination (Pur waningrum et al. 2019). Aves has heterozygous ZW sex chromosomes in females and homozygous ZZ sex chro mosomes in males (Grant 2001). The CHD1 gene was discovered to exist in both Z (CHD1Z) and W (CHD1 W) chromosomes in most ratite bird species with differ ent lengths of introns, thus utilization of CHD1 as a ge netic marker is suitable for sex determination Korn 1997; Ellegren 1996). The CHD1 gene is ampli fied by using the polymerase chain reaction (PCR) method using several primers, such as 2550 F and 0718 R as well as P2 and P8 primer set which successfully performed for sex determination in 58 bird species and evaluation of CHD gene as a universal molecular marker in bird sexing by Vucicevic et al. (2013). The amplicon of DNA fragment from the Z chromosome in male birds and two DNA frag ments in female birds from Z and W chromosomes are generated. There are different sizes length of DNA frag ments due to differences in the length of the amplified in tron (Fridolfsson andEllegren 1999; Liu et al. 2011). Over the past two decades, the study on sex determination using molecular methods is gradually developed since it is con sidered highly accurate, relatively practical, noninvasive, and fast in determining sex in young birds and monomor phic birds (Nugroho and Zein 2015). It has been reported that the application of CHD1 gene for sex determination of avian is a noninvasive technique for sex determination of monomorphic birds (Angat andYusof 2015; Khaerunnisa et al. 2013).

Ethical approval
This study complies with the ethical requirements of the Ethical Clearance Commission of the Faculty of Veteri nary Medicine, Universitas Gadjah Mada (Approval no. 0013/ECFKH/Int./2020), as well as local laws and regu lations.

Specimen collection
The samples used in this study were plucked feathers and a few drops of blood from four Sulphurcrested Cockatoo (C. galerita) from the Yogyakarta Wildlife Rescue Cen ter (WRC) in Wates, Yogyakarta. The blood samples were coded as B1, B2, B3, and B4 and the feathers samples were coded as F1, F2, F3, and F4. The plucked feather sam ples were collected by plucking three to five feathers (sec ondary flight feathers) in the ventral part of the wings. The blood samples were taken by cutting the birds's nails near the blood vessels to reduce stress, and then the blood was collected in microhematocrit containing heparin anticoag ulant. Feather and blood samples were stored in airtight plastic containing silica gel for further processing at the Laboratory of Biochemistry and Molecular Biology, Fac ulty of Veterinary Medicine, Universitas Gadjah Mada.

DNA extraction
About 0.5 to 1 cm from three to five feathers (1 g), includ ing the calamus of each sample, was cut. Theprocedure for DNA extraction from feathers followed the standard procedure on the Geneaid gSYNC™ DNA Extraction Kit (Taiwan) Quick Protocol.

Amplification of CHD1 gene by PCR
The extracted DNA (0.38 µg/µl) was used as a template in the amplification process using the PCR method. DNA fragments were amplified by targeting the CHD1 gene on the sex chromosome using P2 (Griffiths et al. 1998), NP and MP primers (Ito et al. 2003). The sequences of nu cleotides on P2, NP, and MP primers are presented in Ta ble 1.
The amplification process was carried out with a total reaction volume of 25 μl with composition details based on the kit protocol MyTaq TM (Bioline UK). The ampli fication reaction was carried out using a Thermal Cycler (Cleaver Scientific GTC96S) machine with an optimal PCR condition for amplification of DNA fragments, i.e.

Results and Discussion
Electrophoresis from total DNA extraction and DNA am plification from CHD1 gene were analyzed by UV transil luminator. The electrophoresis results in the agarose gel are presented in Figure 1 and Figure 2.
Based on Figures 1 and 2, blood samples and feather samples can be compared. The results of DNA extraction and the results of PCR amplification of the CHD gene were observed. The comparison is presented in Table 2 below.
The interpretation of the electrophoresis of the am plification resultof the CHD1 gene segment in blood and plucked feather samples of C. galerita is presented in Ta ble 3.
The genome DNA was clear and thick in blood sam ples coded B1, B2, B3, and B4. In the plucked feather samples, namely F1, F2, F3, and F4, the genome DNA is clear but thinner than those ofthe blood samples. It shows less genome DNA extracted from the plucked feather sam ple so that it is not visualized maximally. These results are consistent with the previous report by Harvey et al. (2006) that the amount of DNA from feather samples is lower than DNA from blood samples. The extraction re sults were then used as a template to amplify the CHD1 gene on Z and W chromosomes by using the PCR method using P2, NP, and MP primers.
The molecular method for bird sex determination in this study is based on the PCR result, which is a method of in vitro amplification or multiplication of DNA molecules in specific areas bounded by a pair of primers. Amplifica tion of the CHD1 gene was performed on a thermal cycler with annealing temperature of 46°C for 40 cycles. Deter mination of sex in birds with P2, NP, and MP primers can be visualized under UV transilluminator at a wavelength of 280 nm. Male birds will produce one DNA band while female birds will produce two DNA bands. According to Morinha et al. (2012), aves have sex chromosomes that are different from mammals. In birds, the heterogeneous nature is found in the females (ZW) while the males are homogeneous (ZZ). The relationship between the position of the CHD1 gene and sex chromosomes in aves (Z and W chromosomes) causes the CHD1 gene to show differences in Z and W alleles in females (Griffiths and Korn 1997). Ito et al. (2003) reported that the design of the P2, NP, and MP primers was based on differences in sequences between CHD1 W and CHD1 Z genes using 3'terminal mismatch primer on point mutation of the female CHD1 W gene. In contrast to mammals, female birds have het erogametic sex chromosomes, namely W and Z sex chro mosomes, while male birds have homogametic sex chro mosomes, namely Z sex chromosomes. In each species of  bird, molecular sexing universally carried out by detecting the presence of the Chromodomain Helicase DNAbinding (CHD) gene. PCR amplification with the CHD gene tar geted can be used to detect sex differences of birds due to the difference of intron length between the CHD1 Z gene on the Z sex chromosome and the CHD1 W gene on the W chromosome sex chromosome (Angat and Yusof 2015). A set of designed primer pairs will anneal at the posi tion between the introns of the CHD gene. It is possible to differentiate the PCR amplification products based on the length and number of PCR products generated from the Z and W sex chromosomes. In male birds generated PCR products in the form of single band DNA in size of 392 bp. whereas in female birds generated double band DNA in size of 297 bp and 392 bp (Dubiec and Zagalska Neubauer 2006). According to Garofalo et al. (2016), NP is a forward primer while P2 and MP are reverse primers. The P2 primer will attach to the CHD1 Z gene on the Z chromosome, while the MP primer will attach to the CHD1 W gene on the W chromosome and only one fragment (Z) appears in males, while two fragments (Z and W) appear in females because they have different lengths of band due to differences in the length of the amplified introns.
Based on the electrophoresis of results, the CHD1 gene can be used to distinguish the sex of C. galerita ( Figure  2). Previously, CHD1 gene can be used to distinguish the sex of family Cacatuidae, i.e. Cacatua sanguinea, Probosciger aterrimus, and Eolophus roseicapi (Purwan ingrum et al. 2019). The expected size of PCR products was 297 bp for the W chromosome and 392 bp for the Z chromosome. Ito et al. (2003) reported that using P2, MP and NP primers, all of the male bird samples produced 392 bp bands in the CHD1 Z gene segment amplification, while the female birds would produce two 392 bp and 297 bp PCR product bands for CHD1 Z and CHD1 W gene segments.
Blood samples B1, B2, B3, and B4 showed very thick and clear bands, making it easier to distinguish between males and females. Feather samples F1, F2, and F3 pro duced thinner and fairly clear bands. The electrophoresis results in total DNA extraction appeared to be thin, while The comparison table (Table 2) shows that blood sam ples are better sources of DNA for molecular bird sexing compared to plucked feather samples. Birds' blood has nu cleated erythrocytes thus, it contains a lot of nucleus DNA. The source of DNA in feathers is obtained from the basal portion of the feather (calamus) containing a lot of epithe lial cells, but a protein inhibitor named keratin makes the extraction process more difficult and it produces a lower quantity of DNA compared to blood (Hickman et al. 1984). Research conducted by Harvey et al. (2006) and Ravin dran et al. (2019) showed similar comparison results where blood samples were more accurate as a source of DNA than extracted feather samples in molecular bird sexing. As seen in Table 3, the four Sulphurcrested Cockatoo (C. galerita) are identified as one female bird and three male birds.
Molecular sex identification in birds is usually per formed by using plucked feather and blood samples. Other kinds of samples, such as cloaca swabs, buccal swabs, and feces, can also be used, but they require a longer collection time. Blood samples are collected from the jugular vein, brachial vein (wing), or medial metatarsal vein (depending on species and age of the bird) and stored in capillary tubes containing anticoagulants. Feather samples are collected by pulling bird feathers on the ventral part of the wing or chest (Dubiec and ZagalskaNeubauer 2006).
Since the nucleated bird erythrocytes are a source of abundant DNA, only a few blood drops are needed for the DNA extraction process. Harvey et al. (2006) reported that blood samples produce more DNA compared to feath ers, but collecting blood samples from birds requires more energy. Improper collection of blood samples can cause stress to the bird. The blood sample in this study was col lected by cutting the nails close to blood vessels in order to reduce stress in birds, and then the blood was collected in microhematocrit containing heparin anticoagulant.
The use of feathers as samples has the advantage of providing accurate (the accuracy equal to sample from blood), safe and nonlife threatening results. It is also quick, inexpensive, and easy to collect the feathers. DNA samples obtained from feather samples cause less stress on birds and thus more considerate towards the animal wel fare (Cerit and Avanus 2007). According to Cakmak et al. (2017), feather samples produce less DNA compared to blood samples. Hence, DNA produced from feather sam ples is more easily degraded so that the quality and quan tity of DNA is low. However, by considering the speed, ease, and minimal risk, feathers are chosen as samples of molecular bird sexing (Harvey et al. 2006). The feather samples used in this study were collected by plucking the feathers on the ventral part of the wings.

Conclusions
Visualization of the results CHD1 gene segment amplifica tion showed that of four Sulphurcrested Cockatoo (C. ga lerita) tested, there were three males and one female with an intron size of 297 bp on the W chromosome and 392 bp on the Z chromosome. Visualization of DNA extraction results and amplification of blood samples is thicker and clearer than the feather samples, so that blood samples are a better and more accurate source of DNA in molecular bird sexing than feather samples.