We attempted to control the maturation promoting factor (MPF) activity and investigated the subsequent reprogramming of bovine somatic cell nuclear transfer (SCNT) embryos. Serum‐starved adult skin fibroblasts were fused to enucleated oocytes treated with 2.5 mM caffeine or 150 μM roscovitine. The MPF activity, nuclear remodeling patterns, chromosome constitutions and development of SCNT embryos were evaluated. Methylated DNA of embryos was detected at various developmental stages. The MPF activity was increased by caffeine treatment or reduced by roscovitine treatment (p<0.05). Blastocyst development was higher in the caffeine‐treated groups (27.6%) than that of the roscovitine‐treated group (8.3%, p<0.05). There was no difference in the apoptotic cell index among the three groups. However, the mean cell number of blastocysts was increased in the caffeine‐treated group (p<0.05). Higher methylation levels were observed in the Day 3 embryos of the roscovitine‐treated group (50.8%), whereas lower methylation levels were noted at Day 5 in the caffeine‐treated group (12.5%, p<0.05). These results reveal that the increase in MPF activity via a caffeine‐treatment creates a more suitable condition for nuclear reprogramming after SCNT.

In this study, we aimed to determine whether the evaluated markers of cell death could be found at particular developmental stages of normal porcine in vitro fertilization (IVF) embryos. We investigated the characteristics of spontaneous and induced apoptosis during preimplantation development stages of porcine IVF embryos. In experiment 1, to induce apoptosis of porcine IVF embryos, porcine IVF embryos at 22h post insemination were treated at different concentration of actinomycin D (0, 5, 50 and 500 ng/ml in NCSU medium). Treated embryos were incubated at in 5% , 5% for 8h, and then washed to NCSU medium and incubated until blastocyst (BL) stage. We examined cleavage rate at 2days and BL development rate at 7days after in vitro culture. A significantly lower rate of cleavage was found in the 500 ng/ml group compared to others (500 ng/ml vs. 0, 5, 50 ng/ml; 27.8 % vs. 50.0%, 41.2%, 35.9%), and BL formation rate in 500 ng/ml was lower than that of others (500 ng/ml vs. 0, 5, 50 ng/ml; 8.0% vs. 12.6%, 11.2%, 12.6%). In experiment 2, to evaluate apoptotic cells, we conducted TUNEL assay based on morphological assessment of nuclei and on detection of specific DNA degradation under fluorescence microscope. This result showed that apoptosis is a normal event during preimplantation development in control group (0 ng/ml actinomycin D). A high number of BL derived control group contained at least one apoptotic cell. Actinomycin D treated BLs responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and increase in the incidence of apoptotic cell death. In 500 ng/ml group, the incidence of apoptosis increased at 4-cell stage and later. This result suggested that apoptosis is a process of normal embryonic development and actinomycin D is useful tool for the apoptosis study of porcine preimplantation embryos.

This study was performed to elucidate the effects of addition of and bovine serum albumin (BSA) in vitro maturation (IVM) and in vitro culture (IVC) medium on porcine embryo production. The development rate to the 2 cell () and blastocyst stages () with different BSA concentrations in IVM medium were similar among treatment groups. Blastocyst hatching rate was significantly higher in the control group (0.0mg/ml) than in the group of 1.0mg/ml supplement (20.0% vs. 0.0%; p<0.05). The development rate to the 2 cell () and blastocyst stages () with different concentrations in IVM medium was similar among treatment groups. The development rate to the blastocyst was significantly higher in the group of 1.0mg/ml(15.3%) than in the group of 0.5mg/ml supplement (7.6%, p<0.05). The development rate to the 2 cell and blastocyst stages following the first addition of in IVM medium was significantly higher in the control group (77.0% and 18.9%) and was (77.2% and 16.9%) greater than that observed in other treatment groups (p<0.05). The development rate to the 2 cell stage () and blastocyst stages () with different BSA concentrations in IVC medium was similar among treatment groups. However, blastocyst hatching rate was significantly higher in the group of 3.0mg/ml supplement (30.0%) than in the control group (0.0%; p<0.05). The development rate to the 2 cell stage (), blastocyst () and hatching stages () were not different. The development rate to the 2 cell stage (), blastocyst () and hatching stages () at the different culture periods were similar among treatment groups. This study suggested that if the addition level and periods of addition are adjusted, it is possible to replace BSA in the in vitro porcine embryo production.

Interspecies somatic cell nuclear transfer (iSCNT) is a valuable tool for studying the interactions between an oocyte and somatic nucleus. The object of this study was to investigate the developmental competence of in vitro‐matured porcine oocytes after transfer of the somatic cell nuclei of 2 different species (goat and rabbit). Porcine cumulus oocytes were obtained from the follicles of ovaries and matured in TCM‐199. The reconstructed embryos were electrically fused with 2 DC pulses of 1.1 kV/cm for 30 μs in 0.3 M mannitol medium. The activated cloned embryos were cultured in porcine zygote medium‐3 (PZM‐3), mSOF or RDH medium for 7 days. The blastocyst formation rate of the embryos reconstructed from goat or rabbit fetal fibroblasts was significantly lower than that of the embryos reconstructed from porcine fetal fibroblast cells. However, a significantly higher number of embryos reconstructed from goat or rabbit fetal fibroblasts cultured in mSOF or RDH, respectively, developed to the morular stage than those cultured in PZM‐3. These results suggest that goat and bovine fetal fibroblasts were less efficacious than porcine‐porcine cloned embryos and that culture condition could be an important factor in iSCNT. The lower developmental potential of goat‐porcine and porcine‐bovine cloned embryos may be due to incompatibility between the porcine oocyte cytoplasm and goat and bovine somatic nuclei.

This study was designed to investigate the effect of essential amino acids (EAA) and/or non-essential amino acids (NEAA) on the development of parthenogenetic and somatic cell nuclear transfer (SCNT) porcine embryos in vitro. To evaluate the timing of amino acids supplementation, activated oocytes were cultured in NCSU23-PVA with EAA, NEAA or NEAA+EAA (AAs) during specific periods as below: EAA, NEAA or AAs were supplemented during Day 0 to 6 (whole culture period: ALL), Day 2 to Day 6 (post-maternal embryonic transition period: POST-MET), Day 5 to Day 6 (post-compaction period: POST-CMP), Day 0 to Day 2 (pre-maternal embryonic transition period: PRE-MET), or Day 0 to Day 4 (post-compaction period: PRE-CMP). Supplementation of NEAA decreased cleavage rates in PRE-MET and PRE-CMP and also decreased blastocyst rates in POST-CMP. On the other hand, EAA significantly enhanced blastocyst formation rate in POST-MET and no detrimental effect on embryonic development in other groups. Interestingly, NEAA and EAA had synergistic effect when they were supplemented to the medium during whole culture period. Supplementation of AAs also enhanced SCNT porcine embryo development whereas BSA-free medium without AAs could not supported blastocyst formation of SCNT embryos. In conclusion, existence of EAA and NEAA in defined culture medium variously influences the development of parthenogenetic and SCNT porcine embryos, and their positive effect are only occurred when both EAA and NEAA are supplemented to the medium during whole culture period. Additionally, AAs supplementation enhances the blastocyst formation of SCNT porcine embryos when they are cultured in the defined condition.

Somatic cells such as oviduct epithelial cell, uterine epithelial cell, cumulus-granulosa cell and buffalo rat river cell has been used to establish an effective culture system for bovine embryos produced in in vitro. But nitric oxide (NO) metabolites secreted from somatic cells were largely arrested the development of bovine in vitro matured/ in vitro fertilized (IVM/IVF) embryos, suggesting that NO was induced the embryonic toxic substance into culture medium. The objective of this study was to investigate whether BOEC co-culture system can ameliorate the NO-mediated oxidative stress in the culture of bovine IVM/IVF embryos. Therefore, we evaluated the developmental rate of bovine IVM/IVF embryos under BOEC co-culture system in the presence or absence of sodium nitroprusside (SNP), as a NO donor, and also detected the expression of growth factor (TGF-p , EGF and IGFBP) and apoptosis (Caspase-3, Bax and Bcl-2) genes. The supplement of SNP over 5 uM was strongly inhibited blastocyst development of bovine IVM/IVF embryos than in control and 1 uM SNP group (Table 2). The developmental rates beyond morulae stages of bovine IVM/IVF embryos co-cultured with BOEC regardless of SNP supplement (40.4% in 5 uM SNP+ BOEC group and 65.1% in BOEC group) were significantly increased than those of control (35.0%) and SNP single treatment group (23.3%, p<0.05: Table 3). The transcripts of Bax and Caspase-3 genes were detected in all experiment groups (1:Isolated fresh cell (IFC), 2:Primary culture cell (PCC), 3:PCC after using the embryo culture, 4: PCC containing 5 uM SNP and 5: PCC containing 5 uM SNP after using the embryo culture), but Bcl-2 gene was not detected in IFC and PCC (Fig. 1). In the expression of growth factor genes, TGF-p gene was found in all experimental groups, and EGF and IGFBP genes were not found in IFC and PCC (Fig. 2). These results indicate that BOEC co-culture system can increase the development beyond morula stages of bovine IVM/IVF embryos, possibly suggesting the alleviation of embryonic toxic substance like nitric oxide.