Towards increasing stallion sperm longevity by storage at subzero temperatures in the absence of ice

The aim of cell preservation technologies is to slow down damaging reactions by lowering the storage temperature. Upon dilution in a stabilizing extender, stallion sperm can be stored at refrigerator temperatures for several days. Cryopreservation allows storage for decades, but freezing and thawing cause damage and viability losses. It is assumed that by storing cells at subzero temperatures in a non-frozen supercooled state, the damaging effects of ice formation can be avoided. In this study, we have investigated if stallion sperm can be stored at -10°C in the absence of ice, and compared viability during supercooled storage with that during storage at 5°C. We found that addition of 2% Ficoll-400 to buffered saline and covering with mineral oil depressed the sample freezing point and inhibited surface-catalyzed nucleation. This allowed storage in a supercooled state at -10°C for up to 7 days. Supplementing specimens with sperm, however, increased the incidence of sample freezing. Nonetheless, with 50×10⁶ sperm mL^-1, about 40% of the samples turned out to be non-frozen. Adding 100 mM sucrose was found to preserve sperm membrane intactness during supercooled storage, although this resulted in lower percentages as found with refrigerated storage. Sperm motility appeared to be lost during supercooled storage but could be partly restored by substituting buffered saline with a milk-based extender as base medium. Percentages of membrane intact sperm, however, were found to be lower. Supercooled storage holds promise for semen preservation, but further optimization of the storage solution is required to preserve sperm motility.