In vitro development of reconstructed goat oocytes after somatic cell nuclear transfer with fetal fibroblast cells

Abstract

In the present study the developmental potential of fetal fibroblasts was evaluated using nuclear transfer. Studies were undertaken to workout suitable dc pulse length on electrofusion of fetal goat skin fibroblast cells with enucleated in vitro matured goat oocytes and their capability of forming embryos. Skin fibroblast cells from fetal skin were isolated and cultured in monolayer using RPMI-1640 media with 10% FCS in 5% CO 2 incubator at 38.5±1 °C with 95% humidity. Recipient oocytes recovered from slaughterhouse ovaries were matured in vitro for 22–24 h and enucleated using a Leitz micromanipulator. Serum starved cells, cytochalasin B blocked synchronized cells and growing cells were used for insertion into the perivitelline space of the enucleated oocyte and electrofused with constant ac pulse at 7–10 V, while dc pulses at 250, 300 and 350 V were applied for 5–20 μs followed by culture in a CO 2 incubator to observe proper electrofusion and cleavage. After fusion the embryos were activated with cytochalasin B for 2–4 h, and then were transferred to IVC media (without any supporting cells and growth factors). During the study, it was observed that fetal fibroblast became confluence within 2 days. Proportions of oocytes undergoing zona dissolution did not differ significantly among the three dc pulses during electrofusion after reconstruction of the embryos, and also there was no significant difference for the fusion rates among serum starved cells, cytochalasin B blocked cells and growing cells. It was concluded that around 300 V resulted in better electrofusion, and cytochalasin B blocked synchronized cells and fast growing skin fibroblast cells of goats could be used for nuclear cloning.