-210 Nottingham - Nottingham eTheses - The University of Nottingham

protocol was a further development based on previous protocol (Ye et al., 2003). The
images obtained were close to those in Wehrend and Meinecke (2001) and Kubelka et
al. (2002) with 2 clear bands representing MPF and MAPK activities individually.
The MPF and MAPK double kinase assay allows the simultaneous measurement of
MPF and MAPK activities, minimising the inevitable errors by individual assays.
This method has also been successfully applied in assessment of ovine oocyte
MPF/MAPK activities recently (data not shown).
Next, which caffeine concentration was better to treat enucleated oocytes was
determined and toxicity experiments for caffeine treatment were introduced. Caffeine
was shown to have no significant effects on either MPF or MAPK activities of
porcine oocytes selected at TI and early MII stage after 6 hours incubation with
caffeine. Parthenogenetic developmental potential of TI and early MII oocytes treated
by different concentration of caffeine was compared and 5 mM caffeine was chosen
for future experiments (Chapter 5). Interestingly, by selection of cAMP treated
oocytes at relatively earlier maturation stage (TI and early MII), the frequency of
blastocyst formation can reach about 50%, which was different from the number (32.8
± 5.3%) using cAMP treated MII oocytes, indicating selection of oocytes at earlier
stage could be beneficial for parthenotes. Also, 5 mM caffeine did not effectively raise
either kinase activity in TI enucleated oocytes peaking at 44 hpm during the
prolonged culture. Based on this, cell transfer at 42 hpm and activation at 44 hpm
were decided.
Finally, the development of SCNT embryos using TI enucleated oocytes treated with 5
mM caffeine was examined. Caffeine has been individually reported to result in
improved remodeling of the donor genome in porcine and ovine SCNT embryos
(Kawahara et al., 2005; Iwamoto et al., 2005; Kwon et al., 2008; Lee and Campbell,
2006). In particular, Lee and Campbell (2006) presented a detailed investigation into
the effects of caffeine on ovine oocytes and SCNT produced embryos. They
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