New egg yolk-free freezing solution enables rat IVF and live births
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Fertility treatments are becoming more advanced, including through 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 egg yolk carries the risk of microbial contamination and high batch variability, while the detergent 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.
The paper is published in the journal Biology of Reproduction.
"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.
Protecting sperm without egg yolk
The team first performed cryopreservation using only the lactose solution, without the egg yolk or OEP, and examined sperm motility after thawing, 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, reduced 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.
Frozen sperm produce live offspring
The researchers used the newly developed solution to cryopreserve rat sperm. After thawing, they used the sperm for IVF with fresh oocytes collected from female rats and transferred the two-cell embryos into the oviducts of pseudopregnant female rats. Finally, the team compared the IVF efficiency and birth rate achieved with the cryopreserved sperm with those achieved with freshly prepared sperm.
Ultimately, the cryopreserved sperm resulted in a fertilization rate of approximately 70%, which was lower than the 90% achieved with fresh sperm but proved sufficient to efficiently obtain fertilized oocytes. Nonetheless, approximately 36% of the embryos that had been fertilized with the cryopreserved sperm resulted in live rat pup births, showing no significant difference in offspring production compared with the fresh sperm group.
Transport gains, with room for improvement
"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 fertilization rate achieved with the newly developed solution is still lower than the 80% to 95% reported with fresh sperm or conventional cryopreservation methods that contain egg yolk, further improvements are necessary. However, this study demonstrates that some of the issues associated with conventional solutions may eventually be overcome.
Publication details
Kohtaro Morita et al, Development of a chemically defined cryopreservation solution for rat sperm and production of offspring by in vitro fertilization, Biology of Reproduction (2026). DOI: 10.1093/biolre/ioag200
Journal information: Biology of Reproduction
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Reproductive healthObstetrics & gynecology Provided by Kyoto University Who's behind this story?
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