Step-wise dilution for removal of glycerol from fresh and cryopreserved equine spermatozoa

Osmotic stress is an important component of the damage to spermatozoa during cryopreservation. Osmotic injury, due to hyperosmolar freezing extenders, changes in relative solute concentration in the extra cellular medium during freezing and differences in the relative permeabilities of penetrating cryoprotectants, such as glycerol, and water occur when cryopreserved spermatozoa are diluted into isosmotic media or when spermatozoa are placed in the female reproductive tract. The purpose of the study reported here was to evaluate the effect of step-wise dilution for the removal of the permeating cryoprotectant, glycerol, from both fresh and cryopreserved equine spermatozoa on their motility and viability. There were significant (P < 0.05) effects of osmolality and dilution method on both total and progressive motility as well as viability in fresh spermatozoa. With the rapid, one-step dilution being significantly more detrimental to viability and motility, compared to the fixed molarity and fixed volume-dilutions. With frozen-thawed spermatozoa there were significant effects of stallion on total and progressive motility of spermatozoa after cryopreservation and thawing; however, treatment (type of dilution after thawing) did not affect post-thaw motility. These data indicate that rapid, single-step removal of glycerol from fresh equine spermatozoa results in a post-hyperosmotic stress that is characterized by a reduction in both motility and membrane integrity. This post-hyperosmotic stress can be reduced by a step-wise dilution for removal of glycerol, which improved the maintenance of both motility and membrane integrity. However a similar benefit for step-wise dilution for removal of glycerol was not observed in cryopreserved equine spermatozoa.