Researchers have developed a chemically defined cryopreservation solution for rat sperm that eliminates chicken egg yolk and detergent, achieving a 70 percent fertilization rate and successful live pup births through in vitro fertilization.
Standard animal sperm preservation solutions have long depended on additives that carry notable risks, though fertility treatments and reproductive engineering rely heavily on cryopreservation. Conventional methods typically use lactose solutions containing chicken egg yolk and OEP, a detergent formulated with sodium lauryl sulfate. While egg yolk introduces batch variability and the risk of microbial contamination, the detergent can damage oocytes during in vitro fertilization.
Those persistent vulnerabilities prompted a collaborative research team spanning Kyoto University and RIKEN to design an alternative.
The scientists sought a reliable, chemically defined formula that would protect reproductive cells without relying on egg yolk or detergents. Their work details a specific formulation developed to advance laboratory capabilities.
Scientists Build Egg Yolk-Free Solution
First author Kohtaro Morita and colleagues examined sperm motility after thawing samples frozen in a plain lactose solution, establishing an optimal lactose concentration before introducing further protective agents. The research team built their new freezing medium step by step, testing different additives to protect sperm cells during the freeze-thaw cycle.
Next, the investigators added OptiPrep, known to protect sperm during freezing. They then evaluated various cryoprotectants in different combinations and concentrations. The experiments confirmed that combining ethylene glycol and sericin shields sperm plasma and acrosomal membranes from damage, while the nucleotides ATP and dbcAMP enhance post-thaw motility.
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.
Kohtaro Morita, Kyoto University and RIKEN
Entitled Development of a chemically defined cryopreservation solution for rat sperm and production of offspring by in vitro fertilization, the research was released in Biology of Reproduction in 2026 and penned by Kohtaro Morita, Keiji Mochida, Ayumi Hasegawa, Shunya Ihashi, Kento Morita, Saki Wajima, Miho Kohara, Tadashi Sankai, Arata Honda, Kimiko Inoue, Atsuo Ogura, and Masahide Asano.
Yolk-Free Method Produces Viable Embryos and Offspring
To test the efficacy of the new medium, the researchers cryopreserved rat sperm, thawed it, and used it to fertilize fresh oocytes collected from female rats. The resulting two-cell embryos were then transferred into the oviducts of pseudopregnant female rats.
The cryopreserved sperm yielded a fertilization rate of approximately 70 percent when the team compared the performance against freshly prepared sperm, demonstrating that the yolk-free method produces viable embryos and offspring. While lower than the 90 percent rate achieved with fresh sperm, the fertilization performance proved sufficient to obtain fertilized oocytes efficiently.
Subsequent development confirmed that approximately 36 percent of the embryos fertilized using the cryopreserved sperm resulted in live rat pup births. Statistical comparisons revealed no significant difference in offspring production when matched against the fresh sperm control group.
Researchers Enable International Transport
The research team anticipates that eliminating biological contaminants will simplify distribution for researchers worldwide, as reproductive engineering for rats has historically lagged behind that for mice.
Morita pointed out that even though rat reproductive engineering has traditionally trailed behind mouse techniques, this novel freezing medium is anticipated to streamline global shipping of rat sperm and lower microbial infection hazards during offspring generation.
Additional refinements remain necessary before the technique sees widespread laboratory adoption. Conventional egg-containing methods and fresh sperm typically achieve fertilization rates between 80 and 95 percent, leaving a performance gap that the new chemically defined solution must close through further optimization.