Post-thaw N-acetylcysteine treatment promotes the recovery of cryopreserved spermatogonial stem cells

Spermatogonial stem cells (SSCs) are essential for male fertility but are highly vulnerable to oxidative damage during cryopreservation. This study investigated whether short-term antioxidant treatment during early post-thaw recovery improves SSC viability and function. Murine germ cells enriched for SSCs were cryopreserved and treated with <italic>N</italic>-acetylcysteine for 12 h immediately after thawing, followed by 6.5 days of culture without supplementation. Treatment with 0.4 mM <italic>N</italic>-acetylcysteine significantly enhanced post-thaw proliferation (128.2 ± 6.9%, <italic>p</italic> < 0.05) compared with vehicle control. Comparable to α-tocopherol, <italic>N</italic>-acetylcysteine markedly reduced intracellular reactive oxygen species concentrations to near-baseline levels and attenuated the activation of apoptosis-related proteins, including FAS, caspase-8, cytochrome c, and caspase-3/7. Importantly, <italic>N</italic>-acetylcysteine treatment preserved undifferentiated spermatogonia marker expression and significantly increased functional recovery in vivo, as demonstrated by a higher total number of SSC-derived colonies following transplantation (889.7 ± 686.4 vs. 1903.5 ± 1616.9 colonies, <italic>p</italic> < 0.05). Collectively, these findings identify early post-thaw redox modulation as a critical determinant of SSC recovery and establish <italic>N</italic>-acetylcysteine treatment as a practical adjunct to cryopreservation protocols for fertility preservation in animal reproduction.