-210 Nottingham - Nottingham eTheses - The University of Nottingham

5.2.2 In vitro maturation of porcine oocytes
COCs were collected and oocytes were matured as described in Chapter 2 (section
2.1).
5.2.3 Synchronisation of porcine oocyte maturation
COCs were synchronised as described in Chapter 2 (section 2.2), section 4.2.6 by
preincubation in modified NCSU-23 medium at 39°C in a humidified atmosphere of
5% CO2 in air, which was supplemented with hormones (10 IU/ml PMSG and 10
IU/ml hCG) and 1 mM cAMP for 22 h and without hormones and cAMP
for further
various periods.
5.2.4 MPF and MAPK assay
MPF and MAPK assay was performed as described in Chapter 2 (section 2.10).
Groups of 10 cumulus stripped oocytes were washed once in DPBS containing 0.1%
PVA, collection buffer containing 6.4 mM EDTA (PH7.4), 10 mM NaF and 100 mM
NaVO4 in DPBS or modified NCSU-23 medium at 39°C before placed into 0.5 ml
tubes with 5 µl of ice-cold lysis buffer.
5.2.5 Selection of TI and early MII oocytes
Approximately 50 COCs in 400 Al modified NCSU-23 were transferred into a 15 ml
conical polystyrene tube at 36 hpm, as described in Chapter 2 (section 2.4).
5.2.6 TI oocyte enucleation
A portion of cytoplasm containing the extruding TI spindle was aspirated from the
selected oocytes at 36-38 hpm as described in Chapter 2 (section 2.5.2). Enucleated
oocytes were cultured in maturation medium with or without 5 mM caffeine.
5.2.7 Caffeine treatment
Caffeine treatment for selected TI and early MII oocytes or TI enucleated oocytes was
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