Development of a chemically defined cryopreservation solution for rat sperm and production of offspring by in vitro fertilization

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Abstract
In vitro fertilization (IVF) and artificial insemination (AI) using frozen–thawed rat sperm have been established to enable the production of viable offspring. However, the current standard cryopreservation solution, which contains chicken egg yolk and Orvus ES Paste (OEP; sodium lauryl sulfate and other undefined components), poses the risk of microbial contamination and limits the experimental reproducibility of IVF and AI. In this study, we developed a chemically defined cryopreservation solution that does not contain chicken egg yolk or OEP and evaluated its efficacy in IVF. After examining the effects of various cryoprotective agents on sperm motility, we found that OptiPrep (a chemically defined reagent) and ethylene glycol in a lactose solution best preserved sperm motility after freezing and thawing. In addition, sericin, a cell membrane protective agent, effectively reduced damage to the plasma and acrosomal membranes of the sperm head. Although the fertilization rate of the sperm cryopreserved in a chemically defined solution (71.4%) was significantly lower than that of fresh sperm (90.5%), fertilized oocytes were successfully obtained. Approximately 60% of the fertilized oocytes developed to the blastocyst stage in vitro. Furthermore, when two-cell embryos were transferred to the oviducts of pseudopregnant females, 36% developed into offspring. Taken together, our results suggest a new strategy for the cryopreservation of rat strains using a chemically defined sperm-freezing solution, enabling the safe and reproducible production of offspring.

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