74 PREGNANCY ESTABLISHMENT OF NUCLEAR TRANSFERRED EMBRYOS AFTER VITRIFICATION AND EMBRYO TRANSFER IN CATTLE

The success of cryopreservation of bovine cloned embryos is important to commercial application of somatic nuclear transfer (NT) in the dairy and beef industry, especially for solving the problem of embryo transfer (ET) between fresh NT embryos and recipients. This experiment was conducted to test the possibility of establishing pregnancy by embryo transfer of vitrified bovine NT embryos. Oocytes were aspirated from antral follicles of slaughterhouse ovaries, and subsequently cultured in maturation medium for 20 h. Cumulus cells were then denuded from the oocytes by vigorous vortexing for later enucleation and donor nuclear transfer. Skin fibroblast cells used for NT were derived from cultured ear explants taken from an elite dairy cow, and cumulus cells were cultured from the cumulus-oocytes complexes collected by ultrasound-guided transvaginal retrieval. Fibroblasts and cumulus cells were cultured at passage 5 or 6 in 10% FBS DMEM at 37°C in 5% CO2 humidified air, and used as nuclear donors. After donor cell transfer, somatic cell-cytoplast pairs were then fused by applying two direct current pulses at 2.0 kV cm-1 for a duration of 10 µs/pulse. Fused embryos were activated in 10 mg mL-1 cycloheximide in M199 + 7.5% FBS for 4 h. Embryos were cultured in CR1aa plus 3 mg mL-1 BSA for 2 days (initiation of activation = Day 0) at 39°C, 5% CO2, 5% O2 and 90% N2, and then cultured on bovine cumulus monolayers in CR1aa medium supplemented with 7.5% FBS for 5 successive days. Expanding and hatching blastocysts on Day 7 were selected for cyropreservation via liquid nitrogen surface vitrification (LNSV). Vitrification solution contained HEPES-buffered TCM199 supplemented with 20% FBS, ethylene glycol and dimethylsulphoxide. A droplet of 1–2 µL vitrification solution containing two blastocysts was directly dropped onto a cooled surface within 30 s after 3 min incubation in equilibration solution. Vitrified NT embryos were kept at -150°C vapor phase until recipients were synchronized to Day 7 for ET. Embryos are warmed and subsequently washed several times in rehydration solutions and M199 + 7.5% FBS medium. The warmed embryos from initial trials were cultured for 2 h to evaluate their viability after cryopreservation. Non-surgical transfer was carried out to transfer two embryos to a synchronous recipient, and pregnancy was determined by palpation via rectum around Day 70 of transfer. After warming of vitrified embryos, similar high survival rates were achieved in NT embryos derived from either cumulus (93.8%, n = 16), or fibroblast cells (95.8%, n = 48) as nuclear donors, respectively (P > 0.05). The pregnancy data (Table 1) on Day 70 of gestation indicated that there were no significant differences among ET groups with fresh NT blastocysts, vitrified cumulus NT and fibroblast NT embryos (P > 0.05). This study demonstrates that cryopreserved bovine NT embryos via LNSV vitrification can maintain an in vivo developmental viability comparable to freshly produced NT counterparts. Further developmental potential of vitrified NT embryos to term is under investigation. Table 1 Pregnancy outcomes of vitrifield bovine NT embryos after embryo transfer

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