Review on advances in sperm preservation: Freeze-drying as an alternative to cryopreservation

Abstract

Sperm preservation is a vital component of assisted reproductive technologies (ART), livestock breeding programmes, and conservation efforts for endangered species. Although cryopreservation in liquid nitrogen (LN₂) remains the gold standard, it poses significant challenges, including prohibitive costs, contamination risks, and logistical complexities. In response, freeze-drying, or lyophilization, has emerged as a promising alternative, enabling long-term sperm storage at ambient temperatures while maintaining DNA integrity. This review provides an in-depth examination of the principles, benefits, and limitations of freeze-drying in comparison to conventional cryopreservation. We discuss key factors influencing the efficiency of freeze-drying, including the role of protective additives such as trehalose, EDTA (Ethylenediaminetetraacetic Acid), and EGTA (Ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid), optimal storage conditions, and precise rehydration techniques. Furthermore, we highlight successful applications of freeze-dried sperm across various species and outline future directions for optimising this technology in both veterinary and human reproductive medicine. By exploring the potential of freeze-drying, this review aims to contribute to the advancement of sperm preservation techniques and their applications in biomedicine and conservation.