Optimizing sperm sexing in Bali cattle: Effects of separation time and bull age on BSA gradient efficiency.

Abstract

Background: Sperm sexing is an important biotechnology for increasing reproductive efficiency by aligning offspring sex with production goals. For Bali cattle (Bos javanicus), a native Indonesian breed with high genetic and cultural value, sexed semen also contributes to germplasm conservation and sustainable breeding programs. Bovine serum albumin (BSA) gradient separation offers a practical and affordable alternative to flow cytometry; however, separation time and donor bull age may influence the efficiency of this method.

Aim: This study aimed to evaluate the effect of sperm separation time using the BSA method on sperm quality in bulls of different ages, before and after cryopreservation, confirmed by SRY-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amplification.

Methods: Fifty-six ejaculates were collected from Bali bulls aged 8, 9, and 10 years. Samples were subjected to a factorial randomized block design with sperm separation times of 30, 45, and 60 minutes using 5% and 10% BSA gradients. The X- and Y-bearing fractions were verified through molecular amplification of the SRY and GAPDH genes. Semen quality parameters, including motility, viability, abnormality, plasma membrane integrity (IPM), and acrosome integrity, were evaluated before and after cryopreservation.

Results: The separation time significantly influenced both molecular detection and post-thaw semen quality. A 30-minute separation period yielded the clearest discrimination of X and Y sperm and maintained higher post-thaw functional integrity than the 45- and 60-minute treatments. Among bull age groups, 9-year-old sires produced the highest quality semen across most parameters, particularly in terms of motility, viability, IPM, and acrosome integrity.

Conclusion: Optimal conditions for BSA-based sperm sexing in Bali cattle are achieved with a 30-minute separation period using semen from 9-year-old bulls. These benchmarks provide a scientific basis for improving the efficiency of sexed semen production and support the sustainable use of Bali cattle in tropical breeding systems.