Improvement of Mouse Spermatozoa Freezing at ‐80°C With Ascorbic Acid 2‐Glucoside at Thawing Phase

Background: Sperm cryopreservation is the most common method to maintain a great number of mutant mouse lines. However, the use of liquid nitrogen (LN2) for freezing presents considerable problems in terms of cost, safety, and accessibility. For this reason, the storage of semen samples at -80° in ultra-freezers has been increasingly used in recent years, with evident advantages: it does not require dedicated space, it is cheaper, and it is available even in small laboratories. Objectives: In this work, freezing at -80°C is tested in combination with antioxidant, aimed at reducing membrane damage and oxidative stress. Materials and Methods: Wildtype mouse line sperm were cryopreserved using the CPM method directly at -80°C. The effect of ascorbic acid 2-O-alpha-glucoside (AA2G) spermatozoa treatment, during thawing, was evaluated through a qualitative (motility, vitality, and acrosomal reaction using SCA System) and functional analysis (IVF and embryo development), after 1, 6, and 12 months. Results: Sperm motility and velocity parameters showed no significant differences between treated and control groups across both B6N and B6J strains over 12 months, though overall motility declined with time. Antioxidant treatment (AA2G) significantly improved sperm viability and in vitro fertilization (IVF) outcomes in both strains, particularly B6N, without affecting acrosome reaction rates or blastocyst development. Discussion: Sperm cryopreservation at -80°C, while cost-effective, induces strain-dependent damage affecting sperm viability and fertility, particularly in B6J mice. Supplementation with the antioxidant AA2G during capacitation significantly improved post-thaw sperm survival and in vitro fertilization outcomes in both B6N and B6J strains, supporting its use in optimizing cryopreservation protocols without liquid nitrogen. Conclusion: These results may help to design new protocols or optimize those already validated by promoting the use of antioxidants in freezing at -80° as they have a significant effect on reducing oxidative stress, making spermatozoa qualitatively comparable to freezing in LN2.