Activation is a crucial step in mammalian somatic cell nuclear transfer (SCNT). Recently we described the application of the handmade cloning technique for porcine SCNT that uses oocytes without zonaa pellucidae (zona-free) in a micromanipulation-independent procedure (Kragh et al. 2004 Reprod. Fertil. Dev. 16, 315–18). The purpose of the present study was to investigate the effect of a combined electrical and chemical activation of zona-free porcine oocytes. Cumulus-oocyte complexes were aspirated from ovaries of sows and matured for 41 h. Subsequently, the cumulus cells were removed by the addition of 1 mg/mL hyaluronidase in HEPES-buffered TCM-199. For zonae pellucidae removal, oocytes were incubated in 8 mg/mL pronase in HEPES-buffered TCM-199 supplemented with 20% cattle serum for 10 s. Three independent experiments with four treatments were conducted, and oocytes were activated by a combined electrical and chemical activation. Oocytes were washed once in activation medium (0.3 M mannitol, 0.1 mM MgSO4, 0.1 mM CaCl2, and 0.01% polyvinyl alcohol) and transferred to a chamber with two wires (0.5-mm separation) covered with activation medium. After the electrical pulse, oocytes were incubated in culture medium (NCSU-37 containing 4 mg/mL BSA) supplemented with 5 μg/mL cytochalasin B and 10 μg/mL cycloheximide for 6 h. Activated oocytes were cultured in culture medium using the wells of wells system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–64) in the submarine incubation system (Vajta et al. 1997 Theriogenology 48, 1379–85). The rate of development into blastocysts was checked on Day 7 of culture. In treatment 1, zona pellucida-intact oocytes were first activated by a single DC pulse of 1.25 kV/cm for 80 μs, and subsequently cultured in cytochalasin B and cycloheximide for 6 h. In treatments 2 and 3, oocytes without zonae pellucidae were activated by a single DC pulse of 1.25 and 0.85 kV/cm for 80 μs, respectively, and subsequently cultured in cytochalasin B and cycloheximide for 6 h. In treatment 4, oocytes without zonae pellucidae were bisected by hand under a stereomicroscope using a microblade in 5 μg/mL cytochalasin B in TCM-199 supplemented with 15 mg/mL BSA, re-fused/activated by a single DC pulse of 1.25 kV/cm for 80 μs in activation medium, and cultured in cytochalasin B and cycloheximide for 6 h. The rates of blastocyst formation from activated oocytes (mean ± SEM) in treatments 1, 2, 3, and 4 were 55 ± 4%, 40 ± 2%, 49 ± 1%, and 41 ± 8%, respectively. In conclusion, the combined electrical and chemical activation efficiently induced parthenogenetic development of zona-free oocytes. Also, a more authentic control model for activation during SCNT was established by activating and producing blasctocysts from re-fused bisected oocytes.
Application of the modified handmade cloning technique to
pigs
