Effects of Timing of Fusion and Culture Medium on the Development of Bovine Eggs Receiving Blastomeres from 8 to 32-cell Embryos In Vitro

Effects of timing of fusion and culture medium on the development of bovine eggs receiving blastomeres from 8-to 32-cell embryos were examined in vitro. Donor embryos were produced from immature oocytes obtained from ovaries following in-vitro maturation, fertilization and culture for 3 to 4 days. Recipient oocytes were also used after in-vitro maturation for 22 hours in media supplemented with various agents. Electrofusion (750V/cm for 50μsec, DC pulses 2 times) was performed at 30 or 40 hours from maturational culture of recipient oocytes. Reconstituted eggs were co-cultured with follicular cells in TCM-199 medium supplemented with hormones (LH, FSH, estradiol) and/or SOD + thioredoxin, or in M16 medium replaced glucose with gultamine. The results obtained were as follows. Overall fusion rate was 31 to 57%. Reconstituted eggs fused at 40 hours developed to 8-cell stage onwards at the higher rate than those of fused at 30 hours (7 to 23% VS 7 to 32%). However, supplements of hormones, SOD + thioredoxin or gultamine did not improve the developmental ability of nuclear transfer eggs.