Advances in molecular sexing of monomorphic birds in Indonesia: A scoping-review with potential application on the Bali starling (Leucopsar rothschildi) breeding
DOI:
https://doi.org/10.24193/subbbiol.2026.1.02Keywords:
Molecular sexing, Primer Performance Index, Bali Starling, Conservation geneticsAbstract
Accurate sex determination is essential for ecological research, captive breeding, and conservation of sexually monomorphic birds. In Indonesia, despite exceptional avian diversity and urgent conservation needs, the application of molecular sexing remains uneven across taxa. This scoping review synthesizes evidence from 35 studies published between 2015 and 2025, drawn from 176 records, that employed PCR amplification of chromo-helicase-DNA-binding (CHD) genes using various primer sets and sample types. Blood and feathers were the most commonly used materials (≈80% of records), while swabs, eggshell membranes, and casque tissue were reported infrequently. Primer performance varied markedly among avian families and sample types. CHD1LF/CHD1LR and NP + P2 + MP consistently showed high amplification reliability, whereas P2/P8 and 1237L/1272H yielded inconsistent results. To enable objective comparison, we propose the Primer Performance Index (PPI), a composite metric integrating amplification success with taxonomic breadth. PPI analysis demonstrated that primers with broad family coverage maintained high reliability after normalization, while taxa-restricted primers showed reduced performance. These findings support improved primer selection for conservation breeding, particularly for the critically endangered Bali Starling (Leucopsar rothschildi), and promote more standardized integration of molecular sexing into avian conservation practice in Indonesia.
Article history: Received 20 March 2026; Revised 18 May 2026;
Accepted 18 May 2026; Available online 25 June 2026.
References
Adelia, E. I., Srihanto, E. A., Rustiati, E. L., Master, J., Firwantoni, F., Anggy, F. P., & Neli Pratiwi, D. (2025). Sex determination in racing pigeons (Columba livia) molecularly using young feather samples. Jurnal Agrosci, 2(6), 332–340. https://doi.org/10.62885/agrosci.v2i6.807
Akrom, A. M., Indarjulianto, S., Yanuartono, Y., Susmiati, T., Nururrozi, A., Raharjo, S., Permana, R.G.S., & Sitompul, Y. Y. (2020a). Buccal swabs as sexing material of young nestlings canary bird (Serinus canaria) and pigeon (Columba livia). J. Sain Vet., 38(1), 31-36. https://doi.org/10.22146/jsv.49032
Akrom, A. M., julianto Soedarmanto, I., Nururrozi, A., & Raharjo, S. (2020b). Genotypic sexing in canary (Serinus canaria) bird based on chromodomain helicase DNA-binding 1 (CHD1) gene. J. Bioteknol. Biosains Indones., 7(1), 1-8.
Annisa, F., Fikriyanti, M., & Withaningsih, S. (2021). DNA sexing for gender determination of changeable hawk-eagle (Nisaetus cirrhatus, Gmelin, 1788). E3S Web of Conferences, 249, 03012. https://doi.org/10.1051/e3sconf/202124903012
Argarini, A. D., Ari Nugroho, H., Purwaningrum, M., & Haryanto, A. (2020). Molecular bird sexing on fischeri lovebird (Agapornis fischeri) by using polymerase chain reaction. BIO Web Conf., 20, 04003. https://doi.org/10.1051/bioconf/20202004003
Azzahra, L. S., Srihanto, E. A., Rustiati, E. L., Master, J., Firwantoni, F., Febriyani, D. A., & Pratiwi, D. N. (2025). Sex determination on racing pigeons (Columba livia) molecularly using blood samples. J. Agrosci, 2(6), 323–330. https://doi.org/10.62885/agrosci.v2i6.785
BirdLife International. (2021). Leucopsar rothschildi. The IUCN Red List of Threatened Species 2021: e.T22710912A183006359. https://dx.doi.org/10.2305/IUCN.UK.2021-3.RLTS.T22710912A183006359.en. Accessed on 18 May 2026.
Dharmayanthi, A. B., Muchsinin, A., Pulungan, A., & Zein, M. S. A. (2021). Genetic diversity and sex identification of pelicans in Ragunan Zoo, Jakarta. J. Biol. Indones., 17(2), 105–114. https://doi.org/10.47349/jbi/17022021/105
El Islami, S. I., Purwaningrum, M., & Haryanto, A. (2021). Molecular sex determination of masked lovebird (Agapornis personata) by polymerase chain reaction method. Proc. KOBI 2nd Int. Confer., 1(1), 48-53.
Fatona, D. R., Permana, R. G. S., Akrom, A. M., Yanuartono, Y., & Indarjulianto, S. (2025). Sexing of young fischer’s lovebird (Agapornis fischeri) based on chromodomain helicase DNA-binding 1 (CHD1) gene and body weight development. J. Sain Vet., 43(1), 162. https://doi.org/10.22146/jsv.96593
Fitriana, F., Resita, R., Disastra, Y., Alfatik, G. H., Artdita, C. A., Haryanto, A., & Aziz, F. (2022). Comparison of five types of polymerase chain reaction primers for sex identification in columbidae family birds. J. Sain Vet., 40(2), 205-220. https://doi.org/10.22146/jsv.68787
Fitriana, F., Al Qodry, M. F. U. Z., De Lucas, J. C. G., Setyorini, D. R., & Aziz, F. (2023a). Appropriate primer selection improves molecular bird sexing accuracy. Buletin Peternakan, 47(4), 215–219. https://doi.org/10.21059/buletinpeternak.v47i4.83320
Fitriana, F., Resita, R., Disastra, Y., Setyorini, D. R., Haryanto, A., & Aziz, F. (2023b). Evaluation of primers targeting chromo helicase DNA-binding gene (CHD) for molecular sexing identification in four bird families. Livest. Anim. Res., 21(1), 14-20. https://doi.org/10.20961/lar.v21i1.66998
Fitriana, F., Setyorini, D. R., Artdita, C. A., Ummami, R., Haryanto, A., & Aziz, F. (2023c). Comparison of four types molecular sexing primers in different bird families. J. Trop. Anim. Vet. Sci., 13(1), 52-58. https://doi.org/10.46549/jipvet.v13i1.359
Fitriana, Y. S., Irham, M., & Sutrisno, H. (2020). A molecular genetic approach for sex determination on helmeted hornbill (Rhinoplax vigil) casque: a forensic casework. BIO Web Conf., 19, 00020. https://doi.org/10.1051/bioconf/20201900020
Fuadah, K., Rahmawati, A., Fitriana, F., & Aziz, F. (2025). Evaluation of molecular primers for sexing the magpie robin and green cucak via CHD1 gene amplification. J. Trop. Anim. Vet. Sci., 15(2), 64–71. https://doi.org/10.46549/jipvet.v15i2.557
Hall, C. A., Conroy, G. C., & Potvin, D. A. (2025). Ex-situ avian sex skews: determinants and implications for conservation. PeerJ, 13, e19312. https://doi.org/10.7717/peerj.19312
Hidayat, R. F. K., Savitri, D., Putri, I., Nugrahani, W. P., & Haryanto, A. (2021). Molecular bird sexing of tanimbar cockatoos (Cacatua goffiniana) by using polymerase chain reaction method. J. Trop. Biodivers. Biotechnol., 6(2), 59997. https://doi.org/10.22146/jtbb.59997
Marc, S., Boldura, O. M., Paul, C., Tripon, M. R., Otavă, G., & Savici, J. (2025). Cross-species validation of pigeon-specific CHD1 primers for molecular sexing in pet birds. Int. J. Mol. Sci., 26(22), 11142. https://doi.org/10.3390/ijms262211142
Nugraheni, P., Purwaningrum, M., Widayanti, R., & Haryanto, A. (2019). Sex determination of peach-faced lovebird (Agapornis roseicollis) using polymerase chain reaction (PCR) techniques. IOP Conf. Ser.: Earth Environ. Sci., 355(1), 012111. https://doi.org/10.1088/1755-1315/355/1/012111
Nugroho, H. A., & Zein, M. S. A. (2015). Evaluation of sexing methods on black caped lory (Lorius lory, Linnaeus 1758). Zoo Indones., 24(2), 83-93.
Ó Marcaigh, F., Kelly, D. J., Analuddin, K., Karya, A., Lawless, N., & Marples, N. M. (2021). Cryptic sexual dimorphism reveals differing selection pressures on continental islands. Biotropica, 53(1), 121–129. https://doi.org/10.1111/btp.12852
Oktanella, Y. (2017). Sex identification based on molecular marker of CHDZ and CHDW gene in some species of Indonesian birds. Adv. Anim. Vet. Sci., 7(10), 844–847. http://dx.doi.org/10.17582/journal.aavs/2019/7.10.844.847
Pambuko, G., Vanessa, R., & Prastowo, S. (2023, July). Amplification of CHD-1 gene fragment in Z and W sex chromosomes of cemani chicken using a different set of PCR primers. In IOP Conf. Ser.: Earth Environ. Sci., 1208(1), 012058. https://doi.org/10.1088/1755-1315/1208/1/012058
Pamulang, Y. V., & Haryanto, A. (2021). Molecular bird sexing on kutilang (Pycnonotus sp.) based on amplification of CHD-Z and CHD-W genes by using polymerase chain reaction method. Biodiversitas, 22(1), 449-452. https://doi.org/10.13057/biodiv/d220155
Petrou, E. L., Scott, L. C., McKeeman, C. M., & Ramey, A. M. (2024). Molecular sexing of birds using quantitative PCR (qPCR) of sex‐linked genes and logistic regression models. Mol. Ecol. Resour., 24(4), e13946. https://doi.org/10.1111/1755-0998.13946
Pinayungan, P. P. W. T., Lovela, A. R., Aksono, E. B., Ismudiono, Setiawan, B., Rachmawati, K., & Rahmahani, J. (2024). Sex identification of the sun conure (Aratinga solstitialis) using calamus based on polymerase chain reaction. Media Kedokt. Hewan, 35(1), 21–27. https://doi.org/10.20473/mkh.v35i1.2024.21-27
Purwaningrum, M., Nugroho, H. A., Asvan, M., Karyanti, K., Alviyanto, B., Kusuma, R., & Haryanto, A. (2019). Molecular techniques for sex identification of captive birds. Vet. World, 12(9), 1506–1513. https://doi.org/10.14202/vetworld.2019.1506-1513
Ratri, I. N. D., Rahmawati, I. P., Nugrahani, W. P., & Haryanto, A. (2022). Molecular bird sexing of small yellow-crested cockatoo (Cacatua sulphurea, Gmelin 1788) using polymerase chain reaction method. J. Trop. Biodivers. Biotechnol. 7, 1-12. https://doi.org/10.22146/jtbb.76463
Samad, A., Solihin, D. D., Sumantri, C., & Purwantara, B. (2023). Phylogeography of the maleo senkawor (Macrocephalon maleo Sal. Muller 1846) based on cytochrome B gene in Sulawesi and their sex determination using molecular sexing. Indones. J. Anim. Vet. Sci., 28(1). https://doi.org/10.14334/jitv.v28i1.3107
Savitri, D., Putri, I., Nugrahani, W. P., Purwaningrum, M., & Haryanto, A. (2021). Molecular bird sexing of sulphur-crested cockatoo (Cacatua galerita) by polymerase chain reaction method. Indones. J. Biotechnol., 26(1), 1–6. https://doi.org/10.22146/ijbiotech.54611
Sitohang, L. R. (2017). Molecular sexing of Javan hawk-eagle and changeable hawk-eagle confiscated by BKSDA in Yogyakarta. Thesis. Atma Jaya University, Yogyakarta.
Sutomo, Yuni, L. P. E. K., Iryadi, R., & Etten, E. V. (2023). Habitat suitability modeling for Jalak Bali (Leucopsar rothschildi) in East Java, Bali, and Lombok: A potential sites for its ex-situ conservation. AIP Conf. Proc., 6(1), 050005. https://doi.org/10.1063/5.0118658
Tricco, A. C., Lillie, E., Zarin, W., O'Brien, K. K., Colquhoun, H., Levac, D.,... & Straus, S. E. (2018). PRISMA extension for scoping reviews (PRISMA-ScR): checklist and explanation. Ann. Intern. Med., 169(7), 467-473. https://doi.org/10.7326/M18-0850
Turcu, M. C., Paștiu, A. I., Bel, L. V., & Pusta, D. L. (2023). Minimally invasive sampling methods for molecular sexing of wild and companion birds. Animals, 13(21), 3417. https://doi.org/10.3390/ani13213417
Vera, F., Wajjwalku, W., Yuda, P., & Daryono, B. S. (2021). A new primer set in CHD1 gene for bird sex identification. Biodiversitas, 22(11), 4977-4982. https://doi.org/10.13057/biodiv/d221133
Wirastika, P. I. P., Yuda, I. P., & Zahida, F. (2015). Sex determination of Bali starling (Leucopsar rothschildi) using molecular sexing. KnE Life Sci., 2(1), 114. http://dx.doi.org/10.18502/kls.v2i1.128
Withaningsih, S., Ilham, M. F., & Rosdianto, A. M. (2024). Morphometric and DNA sexing accurately in male Javan hawk-eagle (Nisaetus bartelsi) determination at Kamojang Eagle Conservation Center, West Java, Indonesia. Biodiversitas, 25(3). https://doi.org/10.13057/biodiv/d250332
Yuda, P., & Saputra, A. W. (2021). Eggshell membrane for DNA sexing of the endangered maleo (Macrocephalon maleo). F1000Research, 9, 599. https://doi.org/10.12688/f1000research.23712.4
Yuda, P., & Wajjwalku, W. (2022). Using feathers for molecular sexing of straw-headed bulbul (Pycnonotus zeylanicus) offsprings. J. Trop. Biodivers. Biotechnol., 7(1), 67129. https://doi.org/10.22146/jtbb.67129
Yuda, P., Kinanti, R. A., & Wijaya, A. (2020). Use of swab for DNA sampling from confiscated raptors for molecular sexing. IOP Conf. Ser.: Earth and Environ. Sci., 590(1), 012011. https://doi.org/10.1088/1755-1315/590/1/012011
Zein, M. S. A., Haryoko, T., Sulistya Fitriana, Y., Sulistyadi, E., & Prawiradilaga, D. M. (2017). Molecular DNA and phenotypic DNA study application in cockatoo release program. J. Biol. Indones., 13(1), 157–169. https://doi.org/10.47349/jbi/13012017/157
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