The objective of this experiment was to explore the effects of Roscovitine (Rosco) prior to in vitro maturation (IVM) of immature pig oocyte. Brilliant cresyl blue test has been used to select the good quality of oocyte. Specifically, the effects of Rosco exposure on nuclear and cytoplasmic maturation, diameter, intracellular glutathione (GSH) and reactive oxygen species (ROS), and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), and gene expression levels in SCNT embryos have been measured. Cumulus oocyte complexes (COCs) have been exposed in 75 μM of Rosco for 22 and 44 h. The COCs that were matured in the IVM for 44 h without Rosco used as control group. Diameter of matured porcine oocytes 44 h culture with Rosco was significantly lower than 22 h culture with Rosco and control groups. GSH was higher in control group than 22 h and 44 h with Rosco but reduction of ROS in 22 h than 44 h with Rosco. In PA, exposure with Rosco 44 h oocytes group has been significantly lower than 22 h and control group in rates of maturation, cleavage and blastocyst formation. Similarly, in SCNT embryos rates of maturation, cleavage and formation of blastocyst have been also significantly lower in 44 h Rosco treated group than other two groups. SCNT embryos treated with Rosco 22 h showed greater expression levels of POU5F1, DPPA2 and NDP52Il mRNA compared with other two groups. Our results demonstrate that Rosco treatment with 22 h prior to IVM improves the development competence of porcine oocyte.

This study was conducted to establish the optimal chemical post-activation conditions in porcine embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) using 4 different chemical compositions (cytochalasin B (CB), cyclohexamide (CHX), demecolcine (DC), 6-dimethylaminopurine (DMAP). Porcine embryos were produced by PA and SCNT and then, cultured for post-activation with CB (7.5 μg/mL), CB (7.5 μg/mL) + CHX (10 μg/mL), CB (7.5 μg/mL) +DC (0.4 μg/mL), and CB (7.5 μg/mL) + DMAP (2 mM). In PA embryonic development, cleavage rates have been significantly higher in CB group (94.7%) and CB+DMAP group (94.1%) than that of CB+CHX and CB+DC group (88.1 and 84.3%, respectively). There have been no significant differences in blastocyst formation rates among the four groups. In cell number of blastocyst was shown in CB group (42.3%) significantly higher than CB+CHX and CB+DC group (40.6 and 40.6%, respectively). In SCNT embryonic development, CB+DMAP group (89.7%) significant differences were found on embryo cleavage rates when compared with other three groups. Blastocyst formation rates in CB+DMAP group (26.9%) were significantly higher when compared with CB, CB+CHX, and CB+DC groups (25.5, 20.2, and 22.1%, respectively). In blastocyst cell number, CB+DMAP group (41.4%) was found higher significant difference compared with other three groups. Additionally, we have investigated survivin expression in early development stages of porcine SCNT embryos for more confirmation. Our results establish that CB group and CB+DMAP group for 4 h during post-activation improves pre-implantation improvement of PA and SCNT embryos.

The objective of this study was to establish the effect of post-activation treatment with cytoskeletal regulators of CB, CB+CHX, CB+DC, CB+6’DMAP on embryonic development of pig oocytes after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). PA and SCNT embryos were produced by using in vitro matured pig oocytes and treated for 4 h after electric activation with cytochalasins B (7.5 μg/ml), CB+cycloheximide (10 μg/ml), CB+demecolcine (0.4 μg/ml), and CB+2mM 6-Dimethylaminopurine. Post-activation treatment of PA oocytes with CB, CB+CHX, CB+DC and CB+6’DMAP no significant differences were found in embryo cleavage (83.2～91.5%), mean cell number of blastocysts (40.6～ 42.3% cells/blastocyst) but significantly (P<0.05) differences blastocyst formation (28.6～36.4%). When PA oocytes were treated with CB, CB+CHX, CB+DC, CB+6’DMAP, blastocyst formation was significantly (P<0.05) improved by CB (36.6%) compared to CB+CHX (30.9%), CB+DC (28.6%) and CB+6’DMAP (35.2%). In SCNT, was not significantly (P<0.05) increased by post-activation treatment with CB+CHX (81.3%), CB+DC (83.9%) and CB+6’DMAP (90.0%) compared to CB (84.5%) on embryo cleavage, blastocyst formation (19.1%~23.6%) and blastocyst cell number (39.6～41.4% cells/blastocyst) also were not influenced. But increased tendency in CB+6’DMAP. In addition, we investigated survivin expression in porcine SCNT embryos during the early developmental stages. The levels of survivin mRNA in 2-4 cell stage SCNT embryos were significantly higher 6’DMAP treated group than other treatment groups of SCNT embryos. These observations suggested that 2-4 cell cleaving embryos at have high developmental competence, and which may be influenced by survivin expression in porcine SCNT embryos. Our results demonstrate that post-activation treatment with CB, CB+CHX, CB+DC, CB+6’DMAP improves pre-implantation development of SCNT embryos and the stimulating effect of cytoskeletal modifiers on embryonic development is differentially shown depending on the origin (PA or SCNT) of embryos in pigs.

Introduction Porcine embryonic stem cells (pESCs) derived from cloned embryos might be a useful animal model in biomedical research, however, establishment of cloned pESCs is difficult by its incomplete nuclear reprogramming. Here, we report the improved development competence of porcine cloned embryos by vitamin C (VC) supplement to establish the pESCs. Materials and Methods Slaughterhouse-derived oocytes were in vitro matured for 44h and parthenogenetic and cloned embryos were produced using matured oocytes. Both of embryos were cultured for 6 days in PZM-5 media and development rates were examined. Four different concentration of VC (0, 25, 50, 100, and 200 μg/ml) was supplemented in IVM and IVC media and preimplantation developments in the 5 groups were compared in both of embryos Results and Discussion In the cleavage rates of IVM group, significantly higher rate was shown in 50 mg/ml group than other groups (84.5 ± 0.6% vs. 69.8 ± 5.5, 75.7 ± 1.8, 80.4 ± 0.2, 72.4 ± 0.1%; P<0.05), respectively. Significantly higher rates of blastocyst development also were shown in 50 mg/ml group than other groups (27.0 ± 2.0% vs. 20.4 ± 1.4, 22.1 ± 1.3, 23.7 ± 1.2, 19.6 ± 1.3%; P<0.05), respectively. In the cleavage rate of IVC group, non-significantly different with each group (84.0 ± 1.3, 86.7 ± 1.0, 88.4 ± 1.4, 76.7 ± 3.0, 64.6 ± 4.4; P<0.05). In the blastocyst rate of IVC group, significantly higher rate was shown in 25mg/ml and 50 mg/ml group than other groups (22.3 ± 1.7, 23.8 ± 1.7% vs. 19.1 ± 1.3, 15.9 ± 1.0, 5.8 ± 1.5%; P<0.05) In conclusion, supplement of 50μg/ml of VC in IVM and IVC media enhanced the development of porcine parthenogenetic embryos and these results will be a helpful information in the development of porcine cloned embryos and derivation of its embryonic stem cells.

Introduction The objective of this study was to investigate the effects of staining of porcine cumulus-oocytes complexes (COCs) by brilliant cresyl blue (BCB) test prior to in vitro maturation may be used to select developmentally competent oocytes. Furthermore, milrinone can be used to promote developmental competence of porcine embryos produced during parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). Materials and Methods Slaughterhouse-derived porcine cumulus-oocyte-complexes (COCs) were exposed to BCB and treated oocytes divided into BCB+ (colored cytoplasm), BCB- (colorless cytoplasm) groups. After division into 2 groups, intracellular glutathione (GSH) and reactive oxygen species (ROS) of matured oocytes were compared. And, preimplantation development of PA and SCNT embryos were also compared between 2 groups. BCB- oocytes were exposed to milrinone with different concentrations (0, 50, 75, and 100μM) for 6 h prior to IVM for further development of embryos. Results and Discussion GSH was higher in BCB+ group than BCB- group whereas ROS was lower in BCB+ than BCBgroup. In parthenogenetic embryos, BCB+ oocytes group was significantly higher on maturation (87.5 vs 80.6, 71.3%), cleavage (88.6 vs 82.9, 76.3%), and blastocyst formation rates (34.3 vs 27.8, 25.3%) than control and BCB- oocytes groups, respectively. Moreover, ratio of ICM:TE cells were higher in BCB+ oocytes group (30.3% vs. 28.6, 26.4%, respectively) than other groups. In cloned embryos, the significant higher blastocyst formation rates were shown BCB+ groups (30.6% vs. 26.0, 20.1%) than BCB- groups. To improve the cytoplasmic maturation in BCB- oocytes, 4 different concentrations of milrinone (0, 50, 75, and 100μM) were supplemented in the IVM media for 6 h. BCB- oocytes supplemented with 75μM milrinone showed the significantly higher rates of blastocyst formation than other groups. Our results demonstrate that staining of porcine oocytes with BCB before IVM may be used for selection of good quality oocytes and milrinone supplementation can be used to improve embryo developmental competence of porcine embryos.