Abstract
Background: Semen preservation is a critical component of artificial insemination (AI) in livestock breeding, with storage conditions playing a vital role in maintaining sperm quality. Pesisir bulls, an indigenous breed from West Sumatra, Indonesia, face reproductive challenges due to limited breeding programs and suboptimal semen preservation techniques. Trehalose, a non-reducing disaccharide, is recognized for its cryoprotective and antioxidant properties, which help preserve sperm motility, viability, and membrane integrity during storage. However, the optimal concentration of trehalose for liquid semen storage in Pesisir bulls remains undetermined.
Aim: This study evaluated the effects of trehalose supplementation in a Tris–egg yolk extender on the motility, viability, plasma membrane integrity, and acrosome integrity of Pesisir bull sperm stored at 5°C for 72 hours.
Methods: Ejaculates from Pesisir bulls with ≥80% individual motility were collected weekly using an artificial vagina. Semen was diluted with a Tris–egg yolk extender containing 0, 15, 30, or 45 mM trehalose to achieve a final concentration of 25 million spermatozoa per mL. Sperm quality parameters were assessed at 0-, 24-, 48-, and 72-hours post-dilution.
Results: Fresh semen had a volume of 3.5 ± 0.2 mL, a milky-white color, thick consistency, a pH of 6.8 ± 0.1, and a characteristic seminal odor. Microscopically, it exhibited +++ mass movement, a concentration of 1,465 ± 50 × 10⁶ sperm/mL, 85 ± 2.1% motility, 88 ± 1.8% viability, 5.0 ± 0.5% abnormalities, and 85.5 ± 2.0% plasma membrane integrity. Trehalose supplementation—particularly at a concentration of 30 mM—significantly (P < 0.05) enhanced sperm motility, viability, plasma membrane integrity, and acrosome integrity compared with the control group. Although storage-induced declines in these parameters were observed across all treatments, 30 mM trehalose supplementation effectively mitigated these reductions, preserving sperm quality over the 72-hour storage period.
Conclusion: Trehalose supplementation in a Tris–egg yolk extender enhanced the preservation of Pesisir bull semen during liquid storage. A concentration of 30 mM trehalose was found to be optimal for maintaining sperm quality at 5°C, offering potential benefits for artificial insemination programs and genetic conservation initiatives.

Key words: Pesisir bull, semen storage, trehalose, sperm quality