Abstract
Background:
Sperm sexing is an important biotechnology to increase 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, the efficiency of this method may be influenced by separation time and donor bull age.

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–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. Verification of X- and Y-bearing fractions was conducted through molecular amplification of the SRY and GAPDH genes. Semen quality parameters, including motility, viability, abnormality, plasma membrane integrity, and acrosome integrity, were evaluated both before and after cryopreservation.

Results:
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 compared with 45- and 60-minute treatments. Among bull age groups, nine-year-old sires produced the highest quality semen across most parameters, particularly in terms of motility, viability, plasma membrane integrity, 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 nine-year-old bulls. These benchmarks provide a scientific basis for improving the efficiency of sexed semen production and support the sustainable utilization of Bali cattle in tropical breeding systems.

Key words: Bali cattle; Bovine serum albumin; Bull age; Cryopreservation; Sperm.