Researchers
研究者名
Kohtaro Morita
研究者名
Asano, Masahide
Overview
Kyoto, Japan — Fertility treatments are becoming more and more advanced, including with cryopreservation, or the freezing of eggs and sperm. However, studies involving animal sperm often use a lactose solution containing chicken egg yolk and OEP, a detergent containing sodium lauryl sulfate: the former carrying the risk of microbial contamination and high batch variability, while the latter has the potential to damage oocytes during in vitro fertilization.
These issues with current sperm cryopreservation solutions motivated a team of researchers at Kyoto University and RIKEN to see if they could find a better alternative. They set out to develop a more reliable, chemically defined cryopreservation solution for rat sperm that does not contain egg yolk or OEP, which they then evaluated for its IVF efficacy.
“We were particularly interested in whether rat sperm could be cryopreserved without egg yolk, which has been used for many years in conventional sperm preservation solutions,” says first author Kohtaro Morita.
The team first performed cryopreservation using only the lactose solution, without the egg yolk or OEP, and after thawing, examined sperm motility, helping the researchers determine the optimal lactose concentration. They then added OptiPrep, which is known to be effective in protecting sperm during cryopreservation, and found that it improved sperm motility after thawing. The researchers subsequently added various cryoprotectants in different combinations and concentrations to identify the formulation that yielded the highest rat sperm motility.
These experiments helped the scientists confirm that the combination of ethylene glycol and sericin, a cell-protective agent, reduces damage to sperm plasma and acrosomal membranes, and that the nucleotides ATP and dbcAMP improved sperm motility after thawing. The resulting optimal cryopreservation solution consisted of OptiPrep, ethylene glycol, sericin, and the two nucleotides.
The researchers used the newly developed solution to cryopreserve rat sperm, then after thawing used the sperm for IVF with fresh oocytes collected from female rats and transferred the two-cell embryos into the oviducts of psuedopregnant female rats. Finally, the team compared the IVF efficiency and birth rate of the cryopreserved sperm with that of freshly prepared sperm.
Ultimately, the cryopreserved sperm resulted in a fertilization rate of approximately 70 percent, which was lower than the 90 percent achieved with fresh sperm but proved sufficient enough to efficiently obtain fertilized oocytes. Nonetheless, approximately 36 percent of the embryos that had been fertilized with the cryopreserved sperm resulted in live rat pup births, showing no significant difference in offspring production when compared with the fresh sperm group.
“Although reproductive engineering technology for rats has lagged behind that for mice, we expect that this new cryopreservation solution will facilitate the international transport of rat sperm and reduce the risk of microbial contamination when producing offspring,,” says Morita.
As the the fertilization rate achieved with the newly developed solution is still lower than the 80 to 95 percent reported with fresh sperm or conventional cryopreservation methods that contain egg yolk, further improvements are still necessary. However, this study demonstrates that some of the issues associated with conventional solutions may eventually be overcome.
