Kaempferol-loaded BSA nanoparticles as an alternative cryoprotectant attenuate apoptosis and autophagy in Bovine Spermatogonial Stem Cells (BSSCs) during cryopreservation.

Male infertility has been recognized as a major global health concern, particularly among patients undergoing chemotherapy or radiotherapy. In such cases, preservation of Spermatogonial Stem Cells (SSCs), the only cellular source capable of restoring spermatogenesis, represents a valuable therapeutic strategy. However, oxidative stress is a critical factor contributing to SSC damage during cryopreservation. Osmotic shock and intracellular ice crystal formation promote excessive production of Reactive Oxygen Species (ROS), leading to dysregulation of apoptosis and autophagy pathways and ultimately reducing SSCs survival and functional capacity. Kaempferol (Kae), a naturally occurring flavonoid, possesses potent antioxidant and anti-apoptotic properties, yet its clinical application is limited by poor solubility and stability. Therefore, this study investigated the protective effects of Kae and Kaempferol-Bovine Serum Albumin nanoparticles (Kae-BSA nanoparticles) on Bovine SSCs (BSSCs) during cryopreservation. BSSCs-enriched germ cells were isolated from calf testes and co-cultured with Sertoli cells, while nanoparticles were synthesized using the desolvation method and characterized by FESEM, DLS, and zeta potential analyses. Post-thaw assessments demonstrated that Kae-BSA nanoparticles significantly improved cell viability and was associated with favorable changes in apoptosis- and autophagy-related markers. These findings suggest that nanoparticle-mediated delivery of kaempferol may reduce cryoinjury and improve BSSCs preservation outcomes, although the underlying mechanisms require further investigation.