Post-Thaw Semen Quality of Duroc Boars Supplemented with Trehalose and Glycerol in Beltsville Thawing Solution and TrisYolk Extenders

This study aimed to evaluate the quality of post-thawed semen in Duroc boars following supplementation with trehalose and glycerol as cryoprotectants in different extenders. The extenders used were Beltsville Thawing Solution (BTS) and Tris-Aminomethane Egg Yolk (Tris-Yolk). Semen was collected from a 2.5-year-old Duroc boar using the massage technique. Fresh semen was evaluated macroscopically and microscopically, and only ejaculates with motility >75% and sperm concentration>200 × 10⁶/mL were considered suitable for cryopreservation. Semen was initially diluted using BTS or Tris-Yolk extenders and allocated into four tubes, then maintained at room temperature (20–22°C) for two hours (holding time). Afterward,centrifugation was performed to separate the pellet from the supernatant. The resulting pellet was resuspended with extender supplemented with 100 mM trehalose and 4% glycerol. The semen was equilibrated at 5°C for two hours and packed into 0.5 mL straws before freezing in liquid nitrogen vapor. Post-thaw (PT) evaluation showed that sperm motility in BTS supplemented with trehalose and glycerol was significantly higher (25.88 ± 0.72) compared to BTS without supplementation (21.53 ± 0.76), Tris-Yolk with supplementation (16.56 ± 1.11), and Tris-Yolk without supplementation (15.14 ± 0.88) (P < 0.05). These findings suggest that the combination of 100 mM trehalose and 4% glycerol enhances cryopreservation outcomes by better preserving sperm motility post-thaw, particularly in the BTS extender. In conclusion, trehalose and glycerol supplementation in BTS extender provides a more effective cryoprotective environment for maintaining the post-thaw motility of Duroc boar sperm compared to Tris-Yolk extender or non-supplemented extenders.