-210 Nottingham - Nottingham eTheses - The University of Nottingham

4.4 DISCUSSION
For successful porcine SCNT, a reproducible and successful parthenogenetic
activation system is important. Differences between development of porcine
parthenotes following various activation protocols could be due to factors such as
species specificity, in vitro or in vivo origins of the treated oocytes, oocyte age, pulse
number, duration and strength of electric stimulation and the composition of the
electroporation medium, especially the balance of ions in the nonelectrolyte solution
(Prochäzka et al., 1992; Zhu et al., 2002). In the first experiment, a reliable porcine
oocyte parthenogenetic activation method was introduced. MII oocytes obtained by
normal IVM in modified NCSU-23 medium were selected and activated at 44 hpm
and activated oocytes were finally cultured in PZM-3 medium. This protocol was
based on Walker et al. (2002) and Zhu et al. (2002).
As is discussed in Chapter 1, Walker et al. (2002) developed a porcine cloning
protocol reaching an overall cloning efficiency of 5.5% development to piglets in all
recipients, using electrical activation parameters previously reported by Polejaeva et
al. (2000) but with a modified fusion/activation medium. Subsequently both Zhu et al.
(2002) and Lee et al. (2004) developed competitive electrical activation parameters
for porcine oocytes which supported parthenogenetic development to the blastocyst
stage of greater than 50%). However, these best parameters did not lead to a high
frequency of development in SCNT (De Sousa et at, 2002; Lee et al., 2003).
Therefore, in the studies reported here, we adopted the electrical parameters from
Walker et al. (2002) while the concentration of mannitol was changed to 0.28 M so
that the osmolarity of the activation medium was about 295 mOsm.
A range of media have been used for porcine embryo culture. These include modified
Whitten's medium (mWM;
Beckmann and Day, 1993), North Carolina State
University 23 (NCSU-23) medium (Petters and Wells, 1993), Beltsville embryo
culture medium 3 (BECM-3; Dobrinsky et al., 1996) and porcine zygote medium
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