In vitro developmental ability of ovine oocytes following intracytoplasmic injection with freeze-dried spermatozoa

Freeze-drying (FD) is a new and alternative method to preserve spermatozoa in refrigeration or at room temperature. Suitable protection is required to maintain the sperm DNA integrity during the whole process and storage. The aim of this study was to examine the effect of rosmarinic acid and storage temperature on the DNA integrity of freeze-dried ram sperm. In addition, we evaluated the in vitro developmental ability to the blastocyst stage of oocytes injected with freeze-dried sperm. Ram sperm was freeze-dried in basic medium and in this medium supplemented with 105 µM rosmarinic acid. The vials were stored for 1 year at 4 °C and at room temperature. Frozen sperm was used as control. After rehydration, sperm DNA damage was evaluated, observing that the percentage of spermatozoa with DNA damage decreased significantly in the presence of rosmarinic acid, without differences between the two storage temperatures. Moreover, no differences were observed between the freeze-dried group and the frozen-thawed group in terms of blastocyst formation rate. We proved for the first time that ovine spermatozoa can be lyophilized effectively, stored at room temperature for long term, reconstituted and further injected into oocytes with initial embryo development.