Vol. 9, Issue 12, Part H (2025)

The long-term preservation of mammalian spermatozoa is essential for assisted reproduction, livestock breeding, genetic resource conservation and protection of endangered species. Although cryopreservation in liquid nitrogen (LN₂) is a conventional method, its dependence on continuous LN₂ supply, high maintenance costs and vulnerability to natural disasters highlight the need for robust, low-cost alternatives. Freeze-drying (lyophilization) has emerged as a promising LN₂-independent preservation strategy, enabling stable storage of sperm at room or refrigeration temperatures. Initially used for microorganisms, freeze-drying has been successfully applied to mammalian sperm, with the first live offspring produced via ICSI using freeze-dried mouse sperm in 1998. The technique involves controlled freezing, primary sublimation and secondary desorption to produce a dry, stable product. Protective media containing Tris, EDTA/EGTA or trehalose play critical roles in maintaining DNA and chromosomal integrity. Although freeze-dried sperm lose motility, their genomic and oocyte-activating capacities remain intact, enabling successful fertilization through ICSI. DNA integrity assessed using AO, SCD and TUNEL assays shows no significant increase in fragmentation compared to fresh sperm. Freeze-dried sperm offer major advantages, including simplified storage and transport, reduced risk of pathogen transmission, suitability for remote regions and disaster-prone areas and long-term genetic banking. Future advancements should focus on improving post-rehydration viability, optimizing FD media and expanding the application of freeze-dried sperm for wildlife conservation and global biobanking initiatives.