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

Abstract

Recrystallization damages occur when a frozen sample is held at high subzero temperatures and when the warming process is too slow. In this work, ram semen diluted in two different concentrations of sugar solutions (Lyo A consisted of 0.4M sorbitol and 0.25M trehalose, and the second, Lyo B composed of 0.26M sorbitol and 0.165M trehalose) in egg yolk and Tris medium were compared after freezing 10μL samples to: (1) - 10, - 25, and - 35°C and thawing. (2)Freezing to - 10 and - 25°C, holding for 1h and then thawing, and (3)freezing to - 10 and - 25°C and drying for 1h at these temperatures at a vacuum of 80mTorr, 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 10-100mTorr that is reached in less than 10s. 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. 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.