Muhammad Yusuf1*, Sigit Bintara2, Widjiati Widjiati 3, Athhar Manabi Diansyah1, Sahiruddin Sahiruddin1, Masturi Masturi 1, Kurniawan Dwi Prihantoko2, Aeni Nurlatifah2, Suryo Kuncorojakti3, Suhernik Susilowati3, Tulus Maulana4, Syahruddin Said4
1Faculty of Animal Science, Hasanuddin University, Jl. Perintis Kemerdekaan Km. 10 Tamalanrea Makassar, South Sulawesi, Indonesia
2Faculty of Animal Science, Gadjah Mada University, Depok, Sleman Regency, Special Region of Yogyakarta, Indonesia
3Faculty of Veterinary Medicine, Airlangga University, Campus C, Mulyorejo St., Surabaya, East Java, Indonesia
4Research Center for Applied Zoology, National Research and Innovation Agency, Jl. Raya Bogor Km. 46, Cibinong, West Java, Indonesia
*Corresponding author’s email: myusuf@unhas.ac.id
Received: 06 August 2025 / Revised: 11 October 2025 / Accepted: 14 October 2025 / Published Online: 18 November 2025
Abstract
Reproductive efficiency in livestock can be enhanced through sperm sexing technologies; however, conventional methods such as flow cytometry are expensive, technically demanding, and impractical for field use. Simpler, affordable, and biologically validated alternatives are crucial, especially for indigenous breeds like Bali cattle. This study evaluated the effectiveness of a freeze-dried albumin (egg white) gradient (10%–30%) method for separating X- and Y-bearing spermatozoa in Bali bulls, alongside proteomic profiling to confirm the biological distinctiveness of each fraction. Semen from three mature Bali bulls (Bos javanicus) (n = 3; 5 ejaculates per bull; total 15 ejaculates) underwent separation via the freeze-dried albumin gradient. Sperm quality and kinematic parameters were analyzed using Computer-Assisted Sperm Analysis (CASA), while morphometric assessment estimated sexed sperm proportions. Proteomic analysis using liquid chromatography–tandem mass spectrometry (LC-MS/MS) was followed by gene ontology enrichment, hierarchical clustering, and Pearson correlation, with identified proteins validated against literature. The method enriched X-spermatozoa in the upper layer (69.67%) and Y-spermatozoa in the lower layer (73.50%), with significant differences p < 0.05 in motility, viability, and membrane integrity. Proteomics identified 418 proteins, including 45 unique to X-sperm and 159 unique to Y-sperm. GO enrichment linked X-sperm proteins to nuclear and structural roles, while Y-sperm proteins were associated with mitochondrial and motility processes. Clustering distinctly separated the two sperm types, and several proteins correlated strongly with functional traits. Literature validation confirmed sex-specific markers. This method represents a low-cost, biologically validated alternative for practical sperm sexing in Bali cattle, combining sperm quality assessment with proteomics to support sex-preferential breeding
Keywords: Bali bull, Bos javanicus, Sperm sexing, Albumin gradient, Proteomics, Sex-specific proteins
