Bird sex determination is fundamental in various ecological and biological studies, although many avian species cannot be sexed visually due to their monomorphic and/or monochromatic appearance. Thus, reliable laboratory methods for sexing are a prerequisite. Most avian nestlings lack sex-related signs, including the Eurasian pygmy owl (Glaucidium passerinum). We performed laboratory sex determination analysis of this species using blood samples of 242 juveniles and nine adults. It relied on the qPCR of the specific intron from the chromo-helicase DNA-binding protein 1 gene. We tested three primer sets, the P2/P8, 2550F/2718R, and CHD1F/CHD1R, commonly used for bird laboratory sexing. The outcomes were displayed on an agarose gel electrophoresis and a plot from melt curve analysis, which had not been previously conducted in Eurasian pygmy owls. We found that only primer set CHD1F/CHD1R proved reliable, as the only one determined sex with one and two band/s and peak/s on the electrophoresis and the melt curve plot for males and females, respectively. The other two primer pairs failed and depicted one band/peak in all specimens regardless of their sex. Therefore, we recommend performing Eurasian pygmy owls' laboratory sexing by qPCR with CHD1F/CHD1R primers only.

Department of Ethology and Companion Animal Science Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague Kamýcká 129 Praha 6 Suchdol 165 00 Czechia

Department of Game Management and Wildlife Biology Faculty of Forestry and Wood Sciences Czech University of Life Sciences Prague Kámycká 129 Praha 6 Suchdol 165 00 Czechia

Laboratory of Anaerobic Microbiology Institute of Animal Physiology and Genetics Czech Academy of Sciences Vídeňská 1083 Praha 4 Krč 142 20 Czechia

Section of Ecology Department of Biology University of Turku Turku 20014 Finland

Zobrazit více v PubMed

Delgado, M. M. & Penteriani, V. Gender determination of Eurasian Eagle-Owls (Bubo bubo) by morphology. J. Raptor Res.38, 375–377 (2004).

Lee, M.-Y. et al. Application of two complementary molecular sexing methods for East Asian bird species. Genes Genom.30, 365–372 (2008).

Ravindran, S. et al. Sex identification comparison of Barn Owls (Tyto alba javanica) using morphological features and molecular-based methods. Slovak Raptor J.12, 47–54. 10.2478/srj-2018-0005 (2018).10.2478/srj-2018-0005 DOI

Newton, I. Population Ecology of Raptors (Poyser, 1979).

Mikkola, H. Owls of Europe (Poyser, 1983).

König, C. & Weick, F. Owls of the World, 2nd edn. (Yale University Press, 2008).

Forsman, D. The Raptors of Europe and the Middle East. A Handbook of Field Identification (Poyser, 1999).

Laaksonen, T. et al. Year- and sex-dependent effects of experimental brood sex ratio manipulation on fledging condition of Eurasian kestrels. J. Anim. Ecol.73, 342–352. 10.1111/j.0021-8790.2004.00811.x (2004).10.1111/j.0021-8790.2004.00811.x DOI

Kouba, M. et al. Low food abundance prior to breeding results in female-biased sex allocation in Tengmalm’s owl (Aegolius funerus). J. Ornithol.161, 159–170. 10.1007/s10336-019-01707-1 (2020).10.1007/s10336-019-01707-1 DOI

Fargallo, J. A., Laaksonen, T., Pöyri, V. & Korpimäki, E. Inter-sexual differences in the immune response of Eurasian kestrel nestlings under food shortage. Ecol. Lett.5, 95–101. 10.1046/j.1461-0248.2002.00290.x (2002).10.1046/j.1461-0248.2002.00290.x DOI

Andersson, M. et al. Adaptive seasonal trend in brood sex ratio: Test in two sister species with contrasting breeding systems. J. Evol. Biol.16, 510–515. 10.1046/j.1420-9101.2003.00533.x (2003). 10.1046/j.1420-9101.2003.00533.x PubMed DOI

Hipkiss, T. & Hörnfeldt, B. High interannual variation in the hatching sex ratio of Tengmalm’s owl broods during a vole cycle. Popul. Ecol.46, 263–268. 10.1007/s10144-004-0195-7 (2004).10.1007/s10144-004-0195-7 DOI

Laaksonen, T., Lyytinen, S. & Korpimäki, E. Sex-specific recruitment and brood sex ratios of Eurasian kestrels in a seasonally and annually fluctuating northern environment. Evol. Ecol.18, 215–230. 10.1023/b:evec.0000035081.91292.17 (2004).10.1023/b:evec.0000035081.91292.17 DOI

Hakkarainen, H. & Korpimäki, E. Reversed sexual size dimorphism in Tengmalm’s owl: Is small male size adaptive?. Oikos61, 337–346. 10.2307/3545241 (1991).10.2307/3545241 DOI

Massemin, S., Korpimäki, E. & Wiehn, J. Reversed sexual size dimorphism in raptors: Evaluation of the hypotheses in kestrels breeding in a temporally changing environment. Oecologia124, 26–32 (2000). 10.1007/s004420050021 PubMed DOI

Krüger, O. The evolution of reversed sexual size dimorphism in hawks, falcons and owls: A comparative study. Evol. Ecol.19, 467–486. 10.1007/s10682-005-0293-9 (2005).10.1007/s10682-005-0293-9 DOI

Miyaki, C. Y. et al. Sex identification of parrots, toucans, and curassows by PCR: Perspectives for wild and captive population studies. Zoo Biol. Publ. Affiliation Am. Zoo Aquar. Assoc.17, 415–423. 10.1002/(SICI)1098-2361(1998)17:5<415::AID-ZOO6>3.0.CO;2-2. (1998).

Jarvi, S. I. & Banko, P. C. Application of a PCR-based approach to identify sex in Hawaiian honeycreepers (Drepanidinae). Pac. Conserv. Biol.6, 14–17. 10.1071/PC000014 (2000).10.1071/PC000014 DOI

Wu, Y. et al. A simple and reliable molecular method for sex identification of the Brown-eared pheasant (Crossoptilon mantchuricum) from non-invasively collected samples. Anim. Biol.61, 163–173. 10.1163/157075511X566498 (2011).10.1163/157075511X566498 DOI

Fitriana, Y. S., Irham, M., Sutrisno, H. & Abinawanto. A molecular genetic approach for sex determination on helmeted hornbill (Rhinoplax vigil) casque: a forensic casework. BIO Web of Conferences 19, 00020. 10.1051/bioconf/20201900020 (2020).

Mazzoleni, S. et al. Long-term stability of sex chromosome gene content allows accurate qPCR-based molecular sexing across birds. Mol. Ecol. Resour.21, 2013–2021. 10.1111/1755-0998.13381 (2021). 10.1111/1755-0998.13381 PubMed DOI

Griffiths, R. & Tiwari, B. Sex of the last wild Spix’s macaw. Nature375, 454. 10.1038/375454a0 (1995). 10.1038/375454a0 PubMed DOI

Ellergren, H. First gene on the avian W chromosome (CHD) provides a tag for universal sexing of non-ratite birds. Proc. R. Soc. Lond. Ser. B Biol. Sci.263, 1635–1641. 10.1098/rspb.1996.0239 (1996).10.1098/rspb.1996.0239 PubMed DOI

Griffiths, R., Daan, S. & Dijkstra, C. Sex identification in birds using two CHD genes. Proc. R. Soc. Lond. Ser. B Biol. Sci.263, 1251–1256. 10.1098/rspb.1996.0184 (1996).10.1098/rspb.1996.0184 PubMed DOI

Takagi, N., Itoh, M. & Sasaki, M. Chromosome studies in four species of Ratitae (Aves). Chromosoma36, 281–291. 10.1007/BF00283247 (1972). 10.1007/BF00283247 PubMed DOI

Smith, C. A., Roeszler, K. N., Hudson, Q. J. & Sinclair, A. H. Avian sex determination: What, when and where?. Cytogenet. Genome Res.117, 165–173. 10.1159/000103177 (2007). 10.1159/000103177 PubMed DOI

Griffiths, R., Double, M. C., Orr, K. & Dawson, R. J. G. A DNA test to sex most birds. Mol. Ecol.7, 1071–1075. 10.1046/j.1365-294x.1998.00389.x (1998). 10.1046/j.1365-294x.1998.00389.x PubMed DOI

Fridolfsson, A.-K. & Ellegren, H. A simple and universal method for molecular sexing of non-ratite birds. J. Avian Biol.30, 116–121. 10.2307/3677252 (1999).10.2307/3677252 DOI

Lee, J.C.-I. et al. A novel strategy for avian species and gender identification using the CHD gene. Mol. Cell. Probes24, 27–31. 10.1016/j.mcp.2009.08.003 (2010). 10.1016/j.mcp.2009.08.003 PubMed DOI

Chang, H.-W. et al. High-throughput avian molecular sexing by SYBR green-based real-time PCR combined with melting curve analysis. BMC Biotechnol.8, 1–8. 10.1186/1472-6750-8-12 (2008). 10.1186/1472-6750-8-12 PubMed DOI PMC

Brubaker, J. L. et al. A noninvasive, direct real-time PCR method for sex determination in multiple avian species. Mol. Ecol. Resour.11, 415–417. 10.1111/j.1755-0998.2010.02951.x (2011). 10.1111/j.1755-0998.2010.02951.x PubMed DOI

Seidensticker, M. T., Holt, D. W., Detienne, J., Talbot, S. & Gray, K. Sexing young snowy owls. J. Raptor Res.45, 281–289. 10.3356/JRR-11-02.1 (2011).10.3356/JRR-11-02.1 DOI

Wang, P. H. et al. Sex identification in the Collared Scops Owl (Otus bakkamoena) with novel markers generated by random amplified polymorphic DNA. Conserv. Genet. Resour.5, 239–242. 10.1007/s12686-012-9778-3 (2013).10.1007/s12686-012-9778-3 DOI

Eiben, K. et al. Sex determination of the boreal owl (Aegolius funereus) using buccal swabs and improved molecular techniques. J. Raptor Res.51, 68–71. 10.3356/JRR-16-24.1 (2017).10.3356/JRR-16-24.1 DOI

Ravindran, S. et al. Molecular sexing of Southeast Asian barn owl, Tyto alba javanica, using blood and feather. Trop. Life Sci. Res.30, 13–23. 10.21315/tlsr2019.30.2.2 (2019).10.21315/tlsr2019.30.2.2 DOI

Kulibaba, R. O. & Liashenko, Y. V. Analysis of CHD gene polymorphism as a model object for molecular sexing of Eurasian Eagle-Owl (Bubo bubo). Cytol. Genet.55, 324–330. 10.3103/S0095452721040071 (2021).10.3103/S0095452721040071 DOI

Çakmak, E., Pekşen, Ç. A. & Bilgin, C. C. Comparison of three different primer sets for sexing birds. J. Vet. Diagn. Investig.29, 59–63. 10.1177/104063871667 (2017). 10.1177/104063871667 PubMed DOI

Mataragka, A., Balaskas, C., Sotirakoglou, K. & Ikonomopoulos, J. Comparative evaluation of the performance of the PCR assays commonly used for the determination of sex in avian species. J. King Saud Univ. Sci.32, 228–234. 10.1016/j.jksus.2018.04.020 (2020).10.1016/j.jksus.2018.04.020 DOI

Mucci, N., Mengoni, C. & Randi, E. Discrimination of PCR products by colour and size improves the accuracy of sex-typing in avian species. Conserv. Genet. Resour.9, 73–77. 10.1007/s12686-016-0623-y (2017).10.1007/s12686-016-0623-y DOI

Masoero, G., Morosinotto, C., Laaksonen, T. & Korpimäki, E. Food hoarding of an avian predator: Sex- and age-related differences under fluctuating food conditions. Behav. Ecol. Sociobiol.10.1007/s00265-00018-02571-x (2018).10.1007/s00265-00018-02571-x DOI

Korpimäki, E., Hongisto, K., Masoero, G. & Laaksonen, T. The difference between generalist and specialist: The effects of wide fluctuations in main food abundance on numbers and reproduction of two co-existing predators. J. Avian Biol.10.1111/jav.02508 (2020).10.1111/jav.02508 DOI

Vučićević, M. et al. Sex determination in 58 bird species and evaluation of CHD gene as a universal molecular marker in bird sexing. Zoo Biol.32, 269–276. 10.1002/zoo.21010 (2013). 10.1002/zoo.21010 PubMed DOI

Dubiec, A. & Zagalska-Neubauer, M. Molecular techniques for sex identification in birds. Biol. Lett.43, 3–12 (2006).

Wang, L. C. et al. Sex identification of owls (Family Strigidae) using oligonucleotide microarrays. J. Hered.99, 187–192. 10.1093/jhered/esm107 (2008). 10.1093/jhered/esm107 PubMed DOI

Turcu, M.-C., Paștiu, A. I., Bel, L.-V. & Pusta, D. L. Minimally invasive sampling methods for molecular sexing of wild and companion birds. Animals13, 3417. 10.3390/ani13213417 (2023). 10.3390/ani13213417 PubMed DOI PMC

Lois-Milevicich, J., Gómez, R. O., Ursino, C. A., Lois, N. A. & de la Colina, A. Rapid and low-cost molecular sexing of a corvid songbird using a single protocol with two universal primer sets. Ardeola68, 423–432. 10.13157/arla.68.2.2021.sc1 (2021).10.13157/arla.68.2.2021.sc1 DOI