Gel-loading tip vitrification of in vitro-matured bovine oocytes and subsequent embryo production by IVF and nuclear transfer
2005
Tominaga, K.(Hyogo-ken. Technology Center for Agriculture, Forestry and Fisheries, Kasai (Japan)) | Hamada, Y. | Hochi, S.
The purpose of this study was to cryopreserve bovine oocytes for subsequent blastocyst production by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). A vitrification procedure using gel-loading tips as containers was applied to cryopreserve in vitro-matured and denuded oocytes. In Experiment 1, oocytes were vitrified-warmed in vitrification solution (VS) containing 25, 28, 31, or 40% ethylene glycol (EG) and 1.0 M sucrose. The proportions of survived oocytes that appeared to be morphologically normal after warming, and cleaved oocytes after IVF were lower with 25% EG-based VS when compared with 28-40% EG-based VS. Blastocyst yields 8 days after IVF of oocytes vitrified-warmed in 28 and 31% EG-based VS (12 and 17%, respectively) were not significantly different from those of the fresh control group (32%). Day-7 blastocysts derived from vitrified oocytes were composed of a smaller number of inner cell mass (ICM) and trophectoderm cells than the fresh Day-7 blastocysts. In Experiment 2, oocytes vitrifiedwarmed in 31% EG-based VS were subjected to enucleation and SCNT. The proportions of oocytes fused and cleaved in the vitrified group were comparable to those in the fresh control group. Blastocyst yields 6 and 7 days after SCNT of vitrified oocytes were lower than those of control oocytes, but 8 days after the SCNT, the difference became statistically comparable (45 versus 58% in control group). ICM and trophectoderm cell numbers in Day-7 blastocysts derived from vitrified oocytes were smaller than those of control blastocysts due to a slower developmental rate. In conclusion, bovine oocytes cryopreserved by vitrification in gelloading tip were capable of developing into blastocysts after conventional IVF and SCNT, with slightly smaller cell numbers and a slower developmental rate.
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