| Summary: | 博士 === 國立屏東科技大學 === 熱帶農業暨國際合作系所 === 98 === The Contents of Abstract in This Thesis:
The objective of this study was to investigate the effects of nuclear transfer of porcine oocytes matured in vitro, and to establish the optimal nuclear transfer condition for porcine oocyte. In addition, the investigation of preservation conditions on the porcine oocytes was also studied.
Results of the study indicated that the different in vitro maturation media (M199 and NCSU23) significantly affected the activation and development of reconstructed porcine oocytes with fresh cumulus cells (F0) as donor cells. The reconstructed oocytes derived from the NCSU23 matured oocytes gave significantly higher activation rate, one (pro) nuclear (1N) formation rate, and embryo development rate than the M199 group. Thus, NCSU23 was a better maturation medium than M199 for reconstructed porcine oocytes.
Cultured cumulus cells as donor cells significantly affected the activation and development of reconstructed porcine oocytes. The activation rate, one (pro) nuclear formation (1N) and embryo development rate of F4 group were significantly higher then the F0 group. In addition, the cultured cumulus cells as the donor cell favored the reconstructed oocytes to proceed to more advanced development such as morula stage. These effects were described as the cause of more synchronization of cell stage of donor and recipient cells for F4 group. The cultured cumulus cells were injected into the enucleated oocytes to obtain the reconstructed oocytes. The chosen injection positions were the perivitelline space, ooplasm edge and ooplasm center. The activation rate, (pro) nuclear formation rate and embryo development rate of the ooplasm edge group were obviously greater the perivitelline space and ooplasm center groups. Furthermore, the injected position at the ooplasm edge favored the more advanced development. The ooplasm edge would be an optimum injected position.
The in vitro maturation time of porcine oocytes significantly affected the activation and development of reconstructed porcine oocytes. A maturation time of 48 h was found to be an optimum condition.
The cultured donor somatic cells [cumulus cells (F4) and porcine ear cells (F10)] did not affect the activation rate, (pro) nuclear formation rate and embryo development rate of the reconstructed oocytes significantly. However, the ear cell group exhibited higher percentage of morula than the cumulus cell group after embryo development, indicating that the ear cell group favored more advanced embryo development.
The activation method affected the activation rate, (pro) nuclear formation rate and embryo development rate of the reconstructed porcine oocytes significantly. The activation method in which the reconstructed oocytes were activated simultaneously with a single DC pulse of 2.0 kV/cm and then placed in the embryo culture medium for 4 h followed by activated at 1.2 kV/cm was found to be the best one among the four activation methods.
Preservation condition of porcine ovaries significantly affected the in vitro maturation, activation and development of porcine oocytes. The preservation temperature significantly affected the maturation of porcine oocytes. The effect was described as a compromise of suppressions of autolysis around physiological temperature and frostbite due to low temperature. A preservation temperature at about 25℃ showed maximum maturation rate for a preservation time of 8 h. The preservation temperature for collected ovaries also significantly affected the activation and embryo development of porcine oocytes. Base on the maturation rate, the activation rate and cleavage rate, a preservation temperature at about 25℃ would be an optimum temperature for a preservation time of 8 h.
Different preservation media for ovaries also significantly affected the in vitro maturation, activation and embryo development of the porcine oocytes. Preservation temperature significantly affected the maturation of porcine oocytes. A preservation temperature at about 25℃ showed optimum maturation rate for a preservation time of 8 h for the PBS, 0.9% NaCl, and BCS preservation media.
The maturation rates and activation rates of oocytes of ovaries preserved in PBS or BCS were significantly higher than those in 0.9% NaCl solution. Among these three preservation media, PBS exhibited highest cleavage rate indicating that PBS should be as better preservation medium for porcine ovaries at 25℃ for 8 h or longer period.
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