| dc.description.abstract | Porcine parthenogenetic embryonic stem cells (ppESCs) have been successfully
established but have not been maintained for large passage number. There are
tremendous factors that affect the establishment of ppESCs, in which, blastocyst
quality is one of them. Therefore, our main purpose of this study was to improve the
blastocyst quality by adding the combination of fetal bovine serum (FBS), amino acid
(AA, including 1% essential amino acid, 0.5% non-essential amino acid) and insulintransferrin-selenium (ITS) to the in vitro development (IVD) duration; and then use
the high-quality blastocyst from the former experiment to improve the ppESCs
establishment. In the first experiment, the oocytes were collected from 4-6mm in
diameter of follicles and cultured to the morula stage. The morulae were then
continuously cultured in 5 groups: FBS, FBS+ITS, FBS+AA, FBS+AA+ITS and control
group on day 4 of IVD. In the next experiment, the inner cell mass (ICM) isolation
from the high-quality blastocysts was performed by 2 methods: whole-blastocyst
culture and ICM dissection method. The result showed that FBS+AA+ITS had the best
performance with the significant high percentage in the hatched blastocyst day 8
(58.02%) and carrying significant high cell number. This result indicated that the
combination of FBS+AA+ITS on day 4 had significant effect on the formation and
hatching of blastocyst. With high quality, the hatched blastocysts day 8 from
FBS+AA+ITS were kept on to the inner cell mass (ICM) isolation for ppESCs
establishment. In the second experiment, the ICM dissection method had significant
attachment rate, comparing to that of whole-blastocyst culture (44.98% vs 27.92%,
p<0.05, respectively). Interestingly, the ICM colony from whole-blastocyst culture
and ICM dissection method was able to be maintained over 5 passages with no
significant differences (1.17% and 3.04%, respectively). This result suggested that
both whole-blastocyst culture and ICM dissection did not effect on the ICM quality but
only the attachment rate of ICM. In conclusion, the combination effect of
FBS+AA+ITS significantly improved the quality and quantity of blastocyst.
Additionally, the high quality of blastocyst together with the ICM dissection method
were considered suitable for the establishment of ppESC. In the future, the porcine
ICM colony should be continued to maintain for further culture to increase the passage
number. | en_US |
