Production of normal offspring from partially zona-incised vitrified mouse oocytes fertilized with cryopreserved spermatozoa using an optimized protocol

The present study was designed to investigate the optimized conditions for cryopreservation of Kunming (KM) mice spermatozoa (Experiment 1) and to compare the developmental potential of IVF embryos produced from fresh oocytes (Group 1), vitrified-warmed oocytes without (Group2) or with partial zona pellucida incised by a piezo manipulator (ZIP) (Group 3) fertilized withfrozen-thawed spermatozoa (Experiment 2). In experiment 1, spermatozoa were cryopreserved with the medium containing raffinose and egg yolk with different concentrations (0∼60%) and then followed by fertilization with fresh oocytes after thawing. The highest cleavage (76.2%) and blastocysts formation rates (63.6%) were obtained when the egg yolk concentration was adjusted to 30%. To optimize the equilibration time, the spermatozoa were equilibrated in the optimized medium for 0, 10, 30, 50, 70, 90 min at 40°C before plunging into liquid nitrogen (LN₂). After thawing, the highest cleavage rate (87.4%) of IVF embryos was observed when equilibrated for 30 min. In experiment 2, the cleavage and blastocyst rates in Group 1 (81.2%, 65.4%) and Group 3 (72.5%, 45.0%) were higher (P < 0.05) than those in Group 2 (22.2% and 13.9%), respectively. When 2-cell embryos obtained in Group 1 and 3 were transferred, 32.1% and 22.7% of embryos in the pregnant receipts developed to term, respectively. In conclusion, the optimized protocol is highly efficient for the cryopreservation of KM mice spermatozoa; the ZIP technique is very useful for improvement of the fertilization efficiency using the cryopreserved gametes and normal offspring can be produced efficiently.