Optimizing Semen Cryopreservation in Inner Mongolia Cashmere Goats: Combined Effects of Centrifugation Parameters and L-Proline Supplementation

This study optimized the cryopreservation protocol for cashmere goat semen by testing centrifugation speeds (750, 1000, 1250, 1500 rpm) for seminal plasma removal and L-proline concentrations (10, 30, 50 mmol/L) in a freezing extender. Semen from six 3-year-old breeding bucks of Inner Mongolia cashmere goats was evaluated post-thaw in terms of motility, membrane integrity, antioxidant capacity, and artificial insemination (AI) outcomes (<i>n</i> = 130 does). The results demonstrated that the group that underwent centrifugation at 1250 rpm saw significantly improved sperm motility (<i>p</i> < 0.05), curvilinear velocity (VCL, <i>p</i> < 0.05), and straight-line velocity (VSL, <i>p</i> < 0.05) compared to the other groups. The addition of 30 mmol/L L-proline further enhanced post-thaw sperm motility (<i>p</i> < 0.05), plasma membrane integrity (<i>p</i> < 0.05), and acrosome integrity (<i>p</i> < 0.05), while significantly reducing reactive oxygen species (ROS, <i>p</i> < 0.05) and malondialdehyde (MDA, <i>p</i> < 0.05) levels. This group also exhibited the highest antioxidant capacity, as indicated by elevated levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) (<i>p</i> < 0.05). AI trials revealed that semen treated with 1250 rpm centrifugation and 30 mmol/L L-proline achieved the highest kidding rate (56.82%), significantly outperforming the control group (37.21%, <i>p</i> < 0.05). Meanwhile, no significant differences were observed in prolificacy or offspring sex ratio (<i>p</i> > 0.05). In conclusion, this study demonstrates that combining 1250 rpm centrifugation for seminal plasma removal with the addition of 30 mmol/L L-proline to the freezing extender significantly improves the quality of cryopreserved cashmere goat semen and enhances AI outcomes.