Researchers developed a new cryopreservation solution that doesn’t use egg yolk or detergent OEP, which carry the risks of microbial contaminations and egg cell damage.
Image Credit: Kohtaro Morita
Scientific Frontline: Extended "At a Glance" Summary: Chemically Defined Rat Sperm Cryopreservation
The Core Concept: A newly developed, chemically defined cryopreservation solution enables the freezing and storage of rat sperm without the use of egg yolk or potentially damaging detergents.
Key Distinction/Mechanism: Unlike conventional freezing solutions that rely on chicken egg yolk and the sodium lauryl sulfate detergent OEP, which carry risks of microbial contamination, high batch variability, and oocyte damage, this synthetic formulation protects sperm cells using a precise blend of OptiPrep, ethylene glycol, sericin, and the nucleotides ATP and dbcAMP.
Major Frameworks/Components:
- Optimization of lactose concentration to support baseline sperm survival.
- Addition of OptiPrep to improve post-thaw sperm motility.
- Integration of ethylene glycol and the cell-protective agent sericin to minimize damage to sperm plasma and acrosomal membranes.
- Supplementation with the nucleotides ATP and dbcAMP to further enhance post-thaw motility.
- Demonstration of a 70 percent in vitro fertilization (IVF) rate and a 36 percent live birth rate, proving statistically comparable to offspring production utilizing fresh sperm.
Branch of Science: Reproductive Biology, Cryobiology, and Veterinary Sciences.
Future Application: This standardized solution will facilitate the safe, efficient international transport of genetically modified rat lines and drastically reduce the risk of microbial contamination in laboratory breeding colonies.
Why It Matters: By eliminating undefined biological components like egg yolk, this formulation standardizes the preservation of valuable animal models, making rat-based biomedical research more reliable, reproducible, and accessible globally.
Fertility treatments are becoming increasingly advanced, including 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 carries the risk of microbial contamination and high batch variability, while the latter has the potential to damage oocytes during in vitro fertilization (IVF).
These issues with current sperm cryopreservation solutions motivated a team of researchers at Kyoto University and RIKEN to 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, and then evaluated 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 egg yolk or OEP, and, after thawing, examined sperm motility to 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 improve 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 pseudopregnant 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%, 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 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 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.
Published in journal: Biology of Reproduction
Authors: 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
Source/Credit: Kyoto University
Edited by: Scientific Frontline
Reference Number: bio100526_01
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