-210 Nottingham - Nottingham eTheses - The University of Nottingham

5.2 MATERIALS AND METHODS
5.2.1 Experimental design
In the first experiment, an MPF/MAPK assay system was to be further developed by
modification of methods by Ye et al. (2003). Effects of different media for washing
oocytes prior to storing samples at - 80°C on MPF/MAPK activities in porcine
oocytes were determined. Effects of different media or solution for washing oocytes
prior to storing samples at - 80°C on MPF/MAPK activities in porcine oocytes were
determined. These media or solution included modified NCSU-23 medium, collection
buffer containing 6.4 mM EDTA (PH7.4), 10 mM NaF and 100 mM Na3VO4 in
DPBS and DPBS containing 0.1% PVA.
The next two experiments were to select a suitable caffeine concentration according to
the effects of different concentration on MPF and MAPK activities and survival
development of parthenogenetic embryos. In the second experiment, COCs were
stripped of cumulus cells at 36 hpm. Denuded TI and early MII oocytes were selected
and cultured in maturation medium (modified NCSU-23) until 38 hpm. Groups of
approximately 20 oocytes arrested at TI or early MII stage were cultured in 50 Al
modified NCSU-23 with different concentrations of caffeine (0,5,10 and 20 mM) for
6h until 44 hpm and then MPF and MAPK activities at 44 hpm were measured. As
control, kinase activities were assayed in denuded intact MII oocytes without caffeine
treatment at 44 hpm. Three replicates of batches of oocytes were performed.
In the third experiment, COCs were stripped of cumulus cells at 36 hpm and selected
TI and early MII oocytes were cultured in HEPES-NCSU-23 until 38 hpm. Groups of
approximately 20 oocytes arrested at TI or early MII stage were cultured in 50 . tl
maturation medium (modified NCSU-23) with different concentrations of caffeine (0,
5,10 and 20 mM) for 6h until 44 hpm and then activated. Cleavage, blastocyst
development and total cell numbers of blastocyst stage embryos were evaluated. Four
replicates of batches of oocytes were performed.
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