| Two experiments were conducted to investigate the cryopreservation of porcine early stage embryos (2-cell to 8-cell embryos). Vitrification and warming of embryos were carried out in the same manner for different solutions.The first experiment was conducted to prepare matured oocytes for constructing somatic cell nuclear transplantation (SCNT) embryos to be later used in vitrification, also to compare the effect of the maturation time in oocyte maturation. Oocytes were in vitro matured for 44-48 h in TCM 199 supplemented with 10% newborn calf serum (NCS),10 IU/ml pregnant mare serum gonadotropin (PMSG),10 IU/ml human chorionic gonadotropin (hCG), 1μg/ml 17β-E2 (estradiol),10% porcine follicular fluid (pFF),69μg/ml L-Cysteine. Maturation duration adopted in this study was able to provide considerable number of high quality oocytes with first polar body (pbI) for production of SCNT embryos.The second part of the first experiment was carried out to compare the developmental rates of embryos constructed by porcine cytoplast nuclei reprogrammed from porcine cumulus cells and mouse cumulus cells with cleavage rates 75.5% vs 73.6% respectively in the 2-cell stage and 56.8% vs 28.1% respectively in the 8-cell stage. It was also found that pSCNT and mouse-pig interspecies somatic cell nuclear transplantation (iSCNT) embryos progressed through a few mitotic divisions indicated that the factor contained in the porcine ooplasm were capable of reprogramming the foreign nucleus and driving cell division. In addition, no significant differences were found in the development of iSCNT and pSCNT embryos (p>0.05). However, under our experimental conditions an important finding of our SCNT study was that both porcine-porcine SCNT and mouse-porcine iSCNT embryos progress arrested consistently at the 8-cell stage in vitro.The objective of the second experiment of this study was to investigate the effect of three different vitrification solutions VS-1:ES40 (40% Ethylene Glycol + 0.5M sucrose); VS-2:EFS40 (40% EG+60% FS) and EDFS40 (40% EG+20% DMSO+20% FS) on the survival rate of early stage porcine embryos (2-,4- and 8-cell stage) by using open-pulled straws (OPS) method. All vitrification solutions used in this study were prepared in TCM199 supplemented with 10% NCS and held at 39℃, in addition all embryo handling was performed in the laboratory at ambient temperature (22-24℃). All embryos were divided into three groups and distinguished each other by the vitrification solution used in OPS. Each group of embryos was first equilibrated in a culture medium TCM-NCS supplemented with 20% v/v of Ethylene Glycol (EG20) for 5 minutes. Then placed in a 100μl drop of vitrification solutions (VS) for 30 sec and immediately embryos were loaded onto OPS and plunged directly into liquid nitrogen (LN2) at-196℃. After storage in liquid nitrogen, embryos were warmed for in vitro culture by the direct warming method where straws containing the embryos were removed from the liquid nitrogen and immersed vertically in the first well of a Petri dish containing droplets of 100μl of TCM-NCS with 0.5M sucrose for 5 min. Embryos were then transferred to 100μl TCM-NCS with 0.25 M sucrose for another 5 min. All warming media were held at 39℃. Finally embryos were washed three times in PBS and maintained at 39℃.FDA stain was used to evaluate the survival rate of the vitrified embryos after thawing. The results showed that ES40 gave the highest survival rate of the embryos following vitrification-thawing procedure among the others cryoprotectant solutions used in this experiment. Furthermore, it has also been demonstrated that there is no statistical difference between ES40 and EDFS40 at survival rates (p>0.05), but significant differences were found between ES40 and EDFS40 compared to EFS40.The results demonstrate that pSCNT and iSCNT embryos can be successfully vitrified and warmed. Additional research is needed to verify that vitrify 2-to 8-cell stage embryos by OPS method can also develop into later stages. |
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