-210 Nottingham - Nottingham eTheses - The University of Nottingham

were washed three times in embryo culture medium supplemented with 7.5 µg/ml
cytochalasin B and then cultured in embryo culture medium supplemented with 7.5
99/m1 cytochalasin B at 39 °C in 5% CO2.5% 02 and 90% N2 for 6 h.
For SCNT, cytoplast-karyoplast couplets were selected, rinsed once in activation
medium (0.28 M mannitol, 0.05 mM MgC12i and 0.1 mM CaCI2) and activated by
administration of 2 consecutive 60-gsec DC pulses of 1.2 kV/cm. Once activated,
oocytes were washed three times in PZM-3 medium supplemented with 7.5 µg/ml
cytochalasin B and cultured in PZM-3 medium supplemented with 7.5 gg/m1
cytochalasin B and cultured at 39°C in 5% CO2,5% 02 and 90% N2 for 6 h.
2.8 In vitro culture of porcine embryos
Following activation, oocytes or couplets were washed three times in embryo culture
medium (PZM-3) and then cultured in groups of 12-30 in 50 gl drops of embryo
culture medium under mineral oil at 391C in 5% C02,5% 02 and 90% N2 for 7 days.
2.9 Embryo staining and evaluation of development
Blastocysts at day 7 post onset of activation were selected, washed once
in PBS
containing 0.1% PVA and fixed in PBS containing 4% PFA for 10 min. The fixed
blastocysts were washed once again in PBS containing 0.1% PVA and mounted in a
small drop of Vectashield mounting medium containing DAPI (Vector Laboratories,
Inc) on a clean glass slide.
2.10 MPF and MAPK assay
2.10.1 Preparation of oocyte lysate
The preparation of oocyte lysate and analysis of MPF and MAPK activities were
performed as previously described with some modifications (Ye et al, 2003). 50 µl
300 U/ml hyaluronidase was added to each 500 µl modified NCSU-23 medium
containing oocytes and oocytes were denuded by pipetting. Groups of 10 cumulus
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