Optimized vapor‑phase cooling and mitochondria‑targeted MitoPBN supplementation enhance cryosurvival of rooster sperm

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.