The antioxidant capacity and protective ability of astaxanthin in cryopreservation of mouse spermatogonial stem cells

Cryopreservation of spermatogonial stem cells (SSCs) offers several benefits, but it can also cause various forms of damage that may reduce the functionality of these cells. Incorporating antioxidants into the cryopreservation medium can provide protection against the detrimental effects of cryopreservation by reducing levels of reactive oxygen species (ROS). In this study, the protective effect of astaxanthin (AST) was evaluated to establish an optimal cryopreservation method for SSCs obtained from the testes of neonatal male mice. AST was added to the freezing base medium at 1, 10, and 100 μM concentrations, and then compared with the control (freezing medium without any additives) and 100 μM vitamin E as a conventional antioxidant. Viability, oxidative stress status, intracellular ROS generation levels, and the expression of Bax and Bcl2 were measured in frozen-thawed SSCs 3 weeks after culture and purification. The data showed that the presence of antioxidants, especially 10 μM AST, in the freezing medium significantly increases the viability and total antioxidant capacity (TAC) (P < 0.05), and reduces the levels of lipid peroxidation and intracellular ROS accumulation in the frozen–thawed SSCs. Vitamin E, as well as 10 and 100 μM AST reduced apoptosis in mouse SSCs by downregulating Bax and upregulating Bcl2. The results of this study suggest that adding 10 μM of AST to the freezing medium provides protection to SSCs after thawing. Therefore, the inclusion of 10 μM AST in the cryopreservation medium significantly improves the post-thaw viability and antioxidant capacity of SSCs. These findings indicate that AST could serve as a valuable additive for improving the cryopreservation process of SSCs, thereby offering potential benefits for the preservation of male fertility.