-210 Nottingham - Nottingham eTheses - The University of Nottingham

dissecting
microscope (Leica) and rinsed once in HEPES-buffered-NCSU 23 medium
containing BSA-free NCSU 23 medium (Petters, 1993) supplemented with 1 µM
glutathione, 1 mM cysteine, 5 mg/l insulin, 10% (v/v) porcine follicular fluid (pFF)
and 10 mM HEPES.
Approximately 50 COCs were transferred into 35 mm petri dishes (Nunc) containing
500 gi of maturation medium (BSA-free NCSU 23 medium supplemented with 1 µM
glutathione, 1 mM cysteine, 5 mg/l insulin, 10 ng/ml epidermal growth factor (EGF),
10% pFF, v/v, 1% essential amino acids and 0.5% nonessential amino acids) overlaid
with warm mineral oil or alternatively transferred into 500 µl modified
NCSU 23
medium in four-well dishes (Nunc) both at 39°C in a humidified atmosphere of 5%
CO2 in air. The medium was supplemented with hormones (10 IU/ml PMSGS Intervet
Ireland Ltd, and 10 IU/ml hCG, Organon Laboratories Ltd) during the first 22 h of
culture, the COCs were then moved to fresh modified NCSU 23 medium without
hormones.
2.2 Synchronisation of porcine oocyte maturation
To synchronise meiotic maturation, selected COCs were incubated in maturation
medium (modified NCSU-23 medium) supplemented with
hormones (10 IU/ml
PMSG and 10 IU/ml hCG) and 5 µg/ml CHX for 12 h at 39°C in a humidified
atmosphere of 5% C02 in air. The COCs were then washed three times in maturation
medium with hormones but no CHX and then cultured in maturation medium with
hormones but no CHX for various periods. Or, selected COCs were
incubated in
maturation medium supplemented with hormones (10 IU/ml PMSG and 10 IU/m1
hCG) and 1 mM cAMP for 22 h. After culture for 22 h, COCs were washed three
times in maturation medium without hormones or cAMP and cultured in maturation
medium without hormones or cAMP for various periods at 39°C in a humidified
atmosphere of 5% CO2 in air.
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