31 EFFECT OF ACTIVATION METHOD ON IN VIVO DEVELOPMENT FOLLOWING SOMATIC CELL NUCLEAR TRANSFER IN GOATS

Abstract

The effects of activation method and timing between fusion and activation in goat somatic cell nuclear transfer (NT) were investigated. In vivo-ovulated oocytes were surgically flushed from donors 54 to 62 h after CIDR withdrawal in the breeding season and enucleated after brief ultraviolet exposure. Transfected fibroblasts and epithelial cells from 4 clonal strains were serum-starved for 4 days before NT. Two direct current electric pulses (2 kV cm^–1 each for 10 μs) were used to induce fusion and simultaneous activation. Forty-five minutes after successful fusion, reconstructs received a second activation stimulus delivered either electrically as above (group 1) or by exposure to 2.5 μM ionomycin for 1 min (group 2). Non-fused couplets received another electrical stimulus in a second fusion attempt (group 3). Fused reconstructs from all three groups were cultured in 5 μg mL^–1 of cycloheximide and 5 μg mL^–1 of cytochalasin B for 3 h before culture overnight in AgResearch SOF media. Embryos at the 1- and 2-cell stages were transferred to the oviducts of synchronized recipients 2 days after oestrus. Each recipient received on average 10 to 12 embryos. Pregnancy and fetal development was monitored regularly by ultrasound. Parturition was induced up to 5 days before expected full term. Kids were reared on the recipients until weaning, with supplemental feeding as required. Embryo survival data were analysed by Fisher's exact test. There were no significant differences between groups 1 and 2 in terms of pregnancy and embryo survival rates throughout development. In group 1, 110 embryos were transferred to 11 recipients. Four does (36%) were diagnosed pregnant on Day 30 of gestation, carrying a total of 8 fetuses (7.3%). All 8 were delivered at term; however, one died at birth and another before weaning. In group 2, 202 embryos were transferred to 20 recipients. Thirteen does (65%) were pregnant on Day 30 of gestation, with a total of 23 fetuses (11.4%). One pregnancy was lost by Day 50 and another by Day 100. The remaining 11 pregnancies (55%) were maintained to term, with 18 kids delivered (8.9%). Four died within 1 day of birth, with the other 14 surviving to weaning. In group 3, a total of 63 embryos were transferred to five recipients. However, no fetuses were detected at Day 30; significantly less than for either group 1 (P < 0.05) or 2 (P < 0.003). Overall embryo survival, in terms of live kids at weaning from embryos transferred, was 5.5, 6.9 and 0% for groups 1, 2 and 3, respectively. Using in vivo-ovulated oocytes, activation with either ionomycin or electrical stimulation, combined with cycloheximide and cytochalasin B, are similar in promoting successful development following NT. However, with re-fusion (group 3) the first electrical stimulus delivered 45 min before fusion may cause a partial pre-activation of the oocyte cytoplasm sufficient to preclude development.

Supported by GTC and the NZ Ministry of Science and Innovation.