-210 Nottingham - Nottingham eTheses - The University of Nottingham

The selection of cumulus oocyte complexes (or COCs) is also important to porcine
IVM. Cumulus cells were suggested to play an important role not only in acquiring
the developmental competence associated with oocyte cytoplasmic maturation but
also in protecting oocytes against cell damage by oxidative stress during oocyte
maturation (Tatemoto et al., 2000). In the experiment, oocytes with at least four layers
of cumulus cells were selected for a better developmental competence.
Porcine oocyte IVM system is basically composed of maturation medium, additives
(hormones, growth factors and follicular fluid, etc. ) and culture system (the culture
temperature, humidity and gas phase, etc. ). In the experiment, NCSU-23 was chosen
as basic medium because it is most widely used for IVM in porcine cloning and has
been reported to improve developmental competence (Wang et al., 1997a).
Ozawa et al. (2006) reported the proportion of embryos that developed to the
blastocyst stage was significantly increased with supplement of 0.5 or 1.0 µM
glutathione. Yoshida et al. (1992) observed that media containing a high concentration
of cysteine could remarkably promote further development of embryos after in vitro
fertilisation. Insulin increased the developmental potential of porcine oocytes and
embryos (Lee et al., 2005c). EGF is usually added in the porcine oocyte maturation
medium to improve either nuclear or cytoplasmic maturation. 10 ng/ml
EGF during
IVM enhanced the subsequent postcleavage development to the blastocyst stage
(Abeydeera et al., 1998b). Vatzias and Hagen (1999) showed that pFF improved IVM,
reduced polyspermy, and increased normal fertilisation rates in vitro of porcine
oocytes. Addition of essential and non-essential amino acids also benefited for nuclear
maturation and male pronuclear formation, an indicator of cytoplasmic maturation (Ka
et al., 1997). In this study, oocytes were cultured in BSA-free NCSU 23 medium
(Petters, 1993) supplemented with 1 µM glutathione, 1 mM cysteine, 5 mg/l insulin,
10 ng/ml EGF, 10% pFF (v/v), 1% essential amino acids and 0.5% nonessential
amino acids. The frequency of gilt-derived MII oocytes (91.0 ± 1.4%) was achieved at
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