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

Abstract

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.

Keywords: Freeze-drying; Freezing; Motility; Preservation; Sperm.

Fig. 1

A schematic workflow of the freeze-thawing and freeze-drying experiments performed

Fig. 2

Mini freeze-drying apparatus (Darya, FertileSafe Ltd., Nes-Ziona, Israel). This figure depicts a schematic sketch of the Darya freeze-drying device

Fig. 3

Cryomicroscopy images of samples frozen (a, b) and freeze-dried (c, d) showing differences in unfrozen fraction amounts. Samples were cooled to − 10 °C for 10 min in Lyo A (Fig. 2b, d) and Lyo B (Fig. 2a, c). Magnification of ×10. The black and white arrows point to the ice crystals and the unfrozen fraction

Fig. 4

Low-temperature scanning electron microscope images. Sample frozen in Lyo A solution, magnification of ×500 (a). Sample frozen with Lyo A solution and dried for 20 min, magnification of ×100K (b)