Faculty of Veterinary Medicine, Urmia University Veterinary Research Forum 2008-8140 17 8 2026 08 01 Lack of differential sperm acrosome-associated 1 gene expression between X- and Y-bearing spermatozoa in Bali bulls (Bos javanicus domesticus) 553 558 737804 10.30466/vrf.2026.2077333.4986 EN Widjiati Widjiati Department of Veterinary Reproduction, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia 0000-0002-8376-1176 Suryo Kuncorojakti Department of Veterinary Reproduction, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia 0000-0001-5170-9982 Suherni Susilowati Department of Veterinary Reproduction, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia 0000-0002-2609-088X Muhammad Yusuf Department of Animal Production, Faculty of Animal Science, Hasanuddin University, Makassar, Indonesia 0000-0003-3421-4384 Sahiruddin Sahiruddin Department of Animal Production, Faculty of Animal Science, Hasanuddin University, Makassar, Indonesia 0000-0002-2844-7547 Masturi Masturi Department of Animal Production, Faculty of Animal Science, Hasanuddin University, Makassar, Indonesia 0000-0003-4302-0661 Athhar Manabi Diansyah Department of Animal Production, Faculty of Animal Science, Hasanuddin University, Makassar, Indonesia 0009-0002-2338-8215 Sigit Bintara Department of Animal Breeding and Reproduction, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, Indonesia 0000-0001-8968-9227 Aeni Nurlatifah Department of Animal Breeding and Reproduction, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, Indonesia 0000-0003-3058-1195 Yayuk Kholifah Balai Besar Inseminasi Buatan Singosari (BBIB Singosari), Directorate General of Livestock and Animal Health Services, Ministry of Agriculture of the Republic of Indonesia, Malang, Indonesia 0000-0002-2398-3228 Riski Lesta Mega Department of Veterinary Reproduction, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia 0009-0004-2297-4399 Devia Yoanita Kurniawati Department of Veterinary Reproduction, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia 0000-0001-9046-6741 Viski Fitri Hendrawan Department of Veterinary Reproduction, Faculty of Veterinary Medicine, Universitas Brawijaya, Malang, Indonesia 0000-0003-1089-0070 Intan Firdha Olien Noor Al Ichsan Department of Veterinary Reproduction, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia 0009-0008-4781-8359 Journal Article 2025 11 18 This study aimed to determine whether the <em>sperm acrosome-associated 1 (SPACA1)</em> gene exhibits differential expression between X- and Y-bearing spermatozoa of Bali bulls and assess its potential as an RNA biomarker for sperm sexing. Sexed frozen semen (X or Y fractions) from Bali bulls was obtained from the Singosari National Artificial Insemination Center, Malang, Indonesia. Post-thaw semen quality was evaluated for motility, viability, abnormality, concentration, and plasma membrane integrity. Total RNA was extracted separately from X and Y fractions using easy-BLUE reagent, and <em>SPACA1</em> expression was quantified by RT-qPCR using <em>GAPDH</em> as an internal control. Relative expression was calculated by the 2^−ΔΔCt method, and group differences were analyzed with the Mann-Whitney U test. The <em>SPACA1</em> expression was numerically higher in X-bearing than Y-bearing spermatozoa (1.00 <em>versus</em> 0.51), but the difference was not significant. Thus, <em>SPACA1</em> transcripts do not provide a reliable molecular marker for sperm sex differentiation. Future work should focus on protein-level characterization of <em>SPACA1</em> and exploration of sex-biased surface proteins for immunological sexing. This research supports Sustainable Development Goal 2: Zero Hunger, by contributing to reproductive efficiency in livestock, and Sustainable Development Goal 12: Responsible Consumption and Production, through development of cost-effective, sustainable sperm sexing technologies. https://vrf.iranjournals.ir/article_737804_6385622d6a6839fdeaec6b7ee6e4b85e.pdf