High post-thaw survival of ram sperm after partial freeze-drying

Background: Recrystallization damages occur when a frozen sample is held at high subzero temperatures and when the warming process is too slow. Methods: In this work, ram semen diluted in two different concentrations of sugar solutions (Lyo A consisted of 0.4 M sorbitol and 0.25 M trehalose, and the second, Lyo B composed of 0.26 M sorbitol and 0.165 M trehalose) in egg yolk and Tris medium were compared after freezing 10 μL samples to: (1) − 10, − 25, and − 35 °C and thawing. (2) Freezing to − 10 and − 25 °C, holding for 1 h and then thawing, and (3) freezing to − 10 and − 25 °C and drying for 1 h at these temperatures at a vacuum of 80 mTorr, prior thawing. For drying, we used a new freeze-drying apparatus (Darya, FertileSafe, Israel) having a condensation temperature below − 110 °C and a vacuum pressure of 10–100 mTorr that is reached in less than 10s. Results: Results showed that samples in Lyo B solution frozen at − 25 °C had significantly higher sperm motility in partially freeze-dried samples than frozen samples (46.6 ± 2.8% vs 1.2 ± 2.5%, P < 0.001). Moreover, partially dried samples in Lyo B showed higher motility than Lyo A at − 25 °C (46.6 ± 2.8% vs 35 ± 4%). Cryomicroscopy and low-temperature/low-pressure environmental scanning electronic microscope demonstrated that the amount of the ice crystals present in partially dried samples was lower than in the frozen samples. Conclusion: Holding the sperm at high subzero temperatures is necessary for the primary drying of cells during the freeze-drying process. Rapid freeze-drying can be achieved using this new device, which enables to reduce recrystallization damages.