Background: Freeze-drying sperm has been developed as a new preservation method where liquid nitrogen is no longer necessary. An advantage of freeze-drying sperm is that it can be stored at 4 °C and transported at room temperature. Although the successful freeze-drying of sperm has been reported in a number of animals, the possibility of long-term preservation using this method has not yet been studied.
Methodology/principal findings: Offspring were obtained from oocytes fertilized with rat epididymal sperm freeze-dried using a solution containing 10 mM Tris and 1 mM EDTA adjusted to pH 8.0. Tolerance of testicular sperm to freeze-drying was increased by pre-treatment with diamide. Offspring with normal fertility were obtained from oocytes fertilized with freeze-dried epididymal sperm stored at 4 °C for 5 years.
Conclusions and significance: Sperm with -SS- cross-linking in the thiol-disulfide of their protamine were highly tolerant to freeze-drying, and the fertility of freeze-dried sperm was maintained for 5 years without deterioration. This is the first report to demonstrate the successful freeze-drying of sperm using a new and simple method for long-term preservation.