Freezing-induced uptake of disaccharides for preservation of chromatin in freeze-dried stallion sperm during accelerated aging†

Nonviable freeze-dried sperm have intact chromatin and can be used for fertilization via intracytoplasmic sperm injection. Freeze-dried sperm preferably should be stored at 4°C or lower, because DNA damage accumulates during storage at room temperature. Disaccharides are known to protect biomolecules both during freezing and drying, by forming a glassy state. Their use is challenging because cellular membranes are normally impermeable for disaccharides. In the current study, we demonstrate that membrane impermeable compounds, including lucifer yellow and trehalose, are taken up by stallion sperm when exposed to freezing. Trehalose uptake likely occurs during freezing-induced membrane phase transitions. Stallion sperm was freeze-dried in various formulations consisting of reducing or nonreducing sugars combined with albumin as bulking agent. Chromatin stability was studied during storage at 37°C, using the flow cytometric sperm chromatin structure assay and microscopic assessment of chromatin dispersion and DNA fragmentation after electrophoresis. Freeze-drying did not affect sperm chromatin, irrespective of the formulation that was used. DNA fragmentation index (DFI) values ranged from 5 to 8%. If sperm was freeze-dried without protectants or in a combination of glucose and proteins, DNA damage rapidly accumulated during storage at 37°C, reaching DFI values of respectively 95 ± 4 and 64 ± 42% after 1 month. DFI values of sperm freeze-dried with sucrose or trehalose ranged between 9-11% and 33-52% after 1 and 3 months storage, respectively. In conclusion, freeze-drying sperm with disaccharides results in uptake during freezing, which greatly reduces chromatin degradation during dried storage.