This study evaluated the combined effects of different concentrations of the mitochondria‑targeted antioxidant MitoPBN (0, 10, 25, 50, and 100 µM) and vapor‑phase height above liquid nitrogen (4 cm vs. 6 cm) on the post‑thaw quality of rooster sperm. After thawing at 37 °C, sperm motility (CASA), morphology, membrane integrity (HOST), reactive oxygen species, total antioxidant capacity, antioxidant enzyme activities (SOD, GPX), mitochondrial activity (Rhodamine 123), and apoptosis (Annexin V/PI) were evaluated. The data demonstrated that freezing at 4 cm conferred significantly higher total motility (45.3 ± 2.1% vs. 42.5 ± 2.1%) and progressive motility (21.6 ± 1.1% vs. 20.3 ± 1.0%; P < 0.05) compared to 6 cm. MitoPBN supplementation produced a dose-dependent improvement in motility: total motility increased from 40.4 ± 3.0% at 0 µM to 66.1 ± 3.5% at 100 µM, and progressive motility from 18.0 ± 2.1% to 32.2 ± 4.4% (P < 0.001). Membrane integrity and mitochondrial activity also improved with MitoPBN (P < 0.001), while abnormal morphology and apoptotic rates declined markedly (P < 0.01). ROS levels decreased and SOD, GPX, and total antioxidant capacity increased significantly (P < 0.01) with increasing MitoPBN dose. Vapor height alone had a minimal impact on oxidative marker levels. In conclusion, rapid cooling (4 cm vapor height) combined with MitoPBN supplementation synergistically enhanced post-thaw motility, membrane stability, mitochondrial function, and redox balance in rooster sperm.
