Inhibition of extracellular ice crystals growth for testing the cryodamaging effect of intracellular ice in a model of ram sperm ultra-rapid freezing

We aimed to test the common belief, that as the sperm cell is the smallest cell in an organism and, therefore, it contains a small amount of intracellular water, the intracellular ice should not strongly affect the sperm cell during the cryopreservation. To investigate the effect of intracellular ice, we developed a novel approach of ram spermatozoa ultra-rapid freezing with the inhibition of the growth of extracellular ice crystals during freezing. In our approach, ram sperm was supplemented with cell-impermeable synthetic ice blocker 1,4-Cyclohexanediol, leaving a sample unsupplemented as a control. Sperm in cryostraws were ultra-rapidly frozen, and then thawed. We hypothesized, that only in the case when the intracellular ice plays the role in the cryodamage, the post-freezing intactness of spermatozoa frozen in the presence of extracellular ice inhibitors should be equal (or even less) to the intactness of spermatozoa frozen without extracellular inhibitors. No statistically significant difference (p = 0.98) was observed between the post-freezing intactness of sperm ultra-rapidly frozen in the presence of 1,4-Cyclohexanediol and the intactness of sperm frozen without 1,4-Cyclohexanediol. We concluded that the intracellular ice plays the role in sperm cryodamage, at least in the model of ram sperm ultra-rapid freezing.HighlightsHere, we investigated the cryodamaging factors during ram sperm ultra-rapid freezing;Optimized ultra-rapid freezing will provide positive economic implications for the sheep industry worldwide.