The motility of frozen-thawed sperm is very different among not only species but also among individuals within the same species. Since the optimal protocol needs to be adjusted for each species produce good quality frozen sperm, we investigated the pretreatment, cooling, freezing and thawing methods of sperm cryopreservation. In the first part of sperm cryopreservation, we carried out either the isolation of sperm from human semen or the addition of a neutralizer to boar semen to protect the sperm from bacteria-released endotoxins. During the cooling and freezing processes, we determined the optimal combination of hyper-osmolality solution and low concentration of glycerol. Although the motility of the sperm immediately after thawing was very high after this novel freezing technique, the motility decreased in a time dependent manner due to the increase of intracellular Ca2 in sperm. To suppress Ca2 uptake just after thawing, we added the Ca2 chelator, EGTA to the sperm thawing media, which improved the post-thawed sperm motility and sperm membrane integrity, and decreased the rate of sperm DNA fragmentation. Using the frozen-thawed boar sperm, the conception rate and the number of pups per delivery were significantly greater than those of the conventional method. This method should contribute to human infertility care.
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