Chitosan-coated nano-green-tea (Nano-GTE) in a tris-soybean lecithin extender improves post-thaw motility, antioxidant status, DNA integrity, and fertility of Ossimi Ram Semen

Cryopreservation reduces ram sperm viability and fertilizing ability by inducing oxidative stress, membrane damage, and DNA fragmentation. This study evaluated whether adding chitosan-stabilized nano green tea extract (Nano-GTE) to a Tris-soybean lecithin extender improves post-thaw semen quality and fertility of Ossimi rams. Ejaculates (pooled from five mature rams) were extended with Tris-soybean lecithin containing 0 (control), 50, 100, 150, or 200 µg/mL Nano-GTE, equilibrated at 5 °C for 4 h, frozen in 0.25 mL straws, stored in liquid nitrogen and thawed at 37 °C for 30 s. Post-thaw progressive motility, viability and membrane integrity were significantly higher in the 100 µg/mL group (PrM: 46.3 ± 0.21% vs. control 41.5 ± 0.22%; Viability: 47.1 ± 0.27% vs. control 42.8 ± 0.24%; Membrane integrity: 47.6 ± 0.26% vs. control 43.9 ± 0.27%; P < 0.001). DNA fragmentation (Comet assay) was lowest in the 100-150 µg/mL groups (DNA damage: 5.0 ± 0.21% at 100 µg/mL vs. control 8.3 ± 0.21%; P < 0.001). Antioxidant capacity (TAC) in the post-thaw medium increased at 100 µg/mL, while markers of membrane damage (MDA, AST, ALT, LDH) were reduced compared with control (P < 0.05). Fertility (pregnancy rate after cervical AI) was higher with semen from the 100 and 150 µg/mL groups, whereas the 200 µg/mL dose impaired some parameters. Together, these results indicate that supplementing a Tris-soybean lecithin extender with Nano-GTE at 100 µg/mL markedly improves the post-thaw quality and fertility of ram sperm; higher doses (≥ 200 µg/mL) may be detrimental.