Improving conservation breeding: a molecular approach to sex determination in Northern bald ibises (G. eremita)

Introduction: Bald ibises (G. eremita) are migratory birds that have been listed as near extinction by the International Union for Conservation of Nature since 1994 due to a dramatic decline in population size. As this species does not exhibit sexual dimorphism, reliable sex determination requires the use of molecular genetic techniques. The aim of this study was to establish an effective and practical molecular method for sex identification in bald ibises. Materials and methods: The study was conducted on blood and feather samples collected from 96 bald ibises housed at the Şanlıurfa Birecik Bald Ibis Breeding Station. The chromodomain-helicase-DNA-binding (CHD) gene region was amplified by polymerase chain reaction (PCR) using the primer pairs 2550 F–2718R, 1237–1272 H, and P2–P8. PCR products were analyzed by agarose gel electrophoresis. Amplicons obtained with the P2–P8 primer pair were further subjected to restriction fragment length polymorphism (RFLP) analysis using the restriction enzyme HaeIII. In addition, high-resolution melting (HRM) analysis was performed using real-time PCR with primers 1237–1272 H. Results: Following HaeIII digestion of PCR products generated with the P2–P8 primer pair, male individuals exhibited a single band of approximately 350 bp, whereas female individuals showed two bands of approximately 350 and 400 bp. HRM analysis also successfully distinguished sexes based on distinct melting profiles. Both blood- and feather-derived DNA samples were suitable for PCR amplification and subsequent analyses. Conclusion: The results demonstrate that PCR–RFLP analysis using the P2–P8 primer pair combined with HaeIII digestion is a reliable method for sex determination in bald ibises. Furthermore, HRM analysis was applied for the first time in G. eremita and proved to be an effective alternative approach. These molecular methods provide practical and accurate tools for sex identification in conservation and breeding programs of this endangered species.