• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.61-69
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• 2007

This study was designed to evaluate effects of BSA, PVA, gonadotropins and follicle shell during IVM of porcine oocytes and subsequent development to the blastocyst stage after IVF. Cumulus oocyte complexes (COCs) were cultured in TCM-199 media containing 4 mg/ml BSA and 1 mg/ml PVA during IVM for 44 hr. To compare the effect of gonadotropins on oocyte maturation, COCs were cultured with FSH+LH, FSH, LH and FSH-LH-free media during IVM. respectively. Also, different number of follicle shells (0, 2, 4 and 6) was used to examine whether the presence of follicle shell in culture medium affects oocyte maturation. The percentages of fertilization and blastocyst formation, respectively, were higher in the medium containing the PVA (49.0 and 17.9%) than those containing the BSA (40.0 and 12.2%). Significantly higher rates of Mil oocytes were in the presence of FSH+LH and FSH (88.6 and 85.1 %) compared to other treatments (64.0 and 53.4% at LH and FSH-LH-free media). Co-culture with inverted follicle shells in 2 ml maturation medium enhanced the developmental competence of porcine oocytes. In conclusion, PVA could be used as a macromolecules instead of BSA, and FSH and follicle shell played important roles in maturation of porcine oocytes.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.2
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• pp.123-129
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• 2019

Many transcription factors are involved in directing the growth of porcine oocytes. The localization and expression level of a given transcription factor often differ at each stage of early embryonic growth, which spans from fertilization to the formation of the blastocyst. A hallmark of the blastocyst stage is the separation of the endodermal and mesodermal ectoderm. The embryo's medium and its effects are known to be crucial during early development compared to the other developmental stages, and thus require a lot of caution. Therefore, in many experiments, early development is divided into the quality of oocyte and cumulus cells and used in experiments. We thought that we were also heavily influenced by genetic reasons. Here, we examined the expression patterns of five key transcription factors (CDX2, OCT4, SOX2, NANOG, and E-CADHERIN) during porcine oocyte development whose expression patterns are controversial in the pig to the literature. Antibodies against these transcription factors were used to determine the expression and localization of them during the early development of pig embryos. These results indicate that the expressions of key transcription factors are generally similar in mouse and pig early developing embryos, but NANOG and SOX2 expression appears to show speciesspecific differences between pig and mouse developing embryos. This work helps us better understand how the expression patterns of transcription factors translate into developmental effects and processes, and how the expression and localization of different transcription factors can crucially impact oocyte growth and downstream developmental processes.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.1
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• pp.33-38
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• 2004

This study was aimed at testing the gene expression of antioxidant enzymes and apoptosis genes for in vitro culture in porcine embryos produced by in vitro maturation/in vitro fertilization (IVM/IVF). Pocine preimplantation embryos obtainted from IVM/IVF can be successfully culture in vitro, but they are delayed or stop to develop at specific developmental stage. Many factors such as reactive oxygen species and apoptosis in an IVM/IVF system followed by in vitro culture influence the rate of production of viable blastocysts. Porcine embryos derived from IVM/IVF were cultured in the atmosphere of 5% $CO_2$ and 20% $O_2$ at $38.5^{\circ}C$ in NCSU23 medium. The patterns of gene expression for antioxidant enzymes and apoptosis genes during in vitro culture in pocine IVM/IVF embryos were examined by the modified semi-quantitative single cell reverse transcriptase-polymerase chain reaction (RT-PCR). Porcine embryos produced by in vitro procedures were expressed mRNAs for CuZn-SOD, GAPDH and GPX, whereas transcripts for Mn-SOD and catalase were not detected at any developmental stages. Expression of caspase-3 mRNA was detected at 2 cell, 8 cell 16 cell and blastocyst, but p53 mRNA was not detected at any stages. The fas transcripts was only detected in blastocyst stage. These results suggest that various antioxidant enzymes and apoptosis genes play crucial roles in vitro culture of porcine IVM/IVF embryos.

• Journal of Embryo Transfer
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• v.24 no.2
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• pp.105-108
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• 2009

This study was designed to investigate the effect of kinetin on in vitro development of parthenogenetic porcine oocytes exposed to demecolcine prior to activation. In vitro matured metaphase II stage oocytes were incubated in 0 or 2 ${\mu}$g/ml demecolcine supplemented defined culture medium for 3 h and the oocytes were activated electrically. The parthenogenetic porcine embryos were then cultured in 0 or 200 ${\mu}$M kinetin supplemented defined culture medium for 7 days. Regardless of demecolcine treatment, kinetin supplementation increased blastocyst rates significantly (7.0% versus 12.1% and 4.9% versus 8.5%; Control versus Kinetin and Demecolcine versus Kinetin + Demecolcine, respectively, p<0.05). Demecolcine treatment before activation tended to decrease blastocyst rates regardless of kinetin supplementation although it is not statistically significant. Total cell numbers in the blastocysts also tended to be elevated in embryos when supplemented with kinetin, however only the result between Kinetin and Demecolcine groups is statistically significant (37.6 ${\times}$ 7.2 versus 28.1 ${\times}$ 9.5, respectively, p<0.05). In conclusion, the present report shows that kinetin enhances developmental competence of parthenogenetic porcine embryo regardless of demecolcine pre-treatment before parthenogenetic activation when they were developed in defined culture condition.

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.199-205
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• 2016

The aim of this study was to investigate the effect of uterine histotroph on embryo development and the expression of cysteine-rich protein 2 (CRP2), coatomer subunit gamma-2 (G2COP), myoglobin (MYG), vascular endothelial growth factor D (VEGFD), collagen alpha 4 chain (COL4) and galactoside 3-L-fucosyltransferase 4 (FUT4) proteins in porcine embryo during pre-implantation. Uterine histotroph (UH) was collected from uterine horn on corpus albican phase, and embryos were cultured in porcine zygote medium with UH for 168 hours. Cleavage and blastocyst formation of embryo were detected at 168 hours after in vitro fertilization. And CRP2, G2COP, MYG, VEGFD, COL4 and FUT4 proteins were observed using confocal laser microscope. In results, embryo cleavage rate was not significantly changed by UH, but blastocyst rate was significantly (P<0.05) decreased in UH-treated embryos. Moreover, CRP2, G2COP, MYG, VEGFD, COL4 and FUT4 proteins were expressed in blastomere. CRP2 in embryo was significantly overexpressed (P<0.05), but not G2COP, MYG, VEGFD, COL4 and FUT4 proteins. In summary, UH on corpus albican phase was increased CRP2 protein in embryo, and inhibited blastocyst formation in preimplantation porcine embryos, suggesting that CRP2 may play an interrupter on embryo development in pigs.

• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.127-131
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• 2007

Embryonic germ (EG) cells are undifferentiated stern cells isolated from cultured primordial germ cells (PGC). These cells share many characteristics with embryonic stem cells including morphology and pluripotency. Undifferentiated porcine EG cell lines demonstrating capacities of differentiation both in vitro and in vivo have been established. Since EG cells can be cultured indefinitely in an undifferentiated state, whereas somatic cells in primary culture are often unstable and have limited lifespan, EG cells may provide inexhaustible source of karyoplasts in nuclear transfer (NT). In this study the efficiencies of NT using porcine EG and fetal fibroblast cells were compared. Two different techniques were used to perform NT. With conventional NT procedure (Roslin method) involving fusion of donor cells with enucleated oocytes, the rates of development to the blastocyst stage in EG and somatic cell NT were 16.8% (59/351) and 14.5% (98/677), respectively. In piezo-driven microinjection (Honolulu method) of donor nuclei into enucleated oocytes, the rates of blastocyst formation in EG and somatic cell NT were 11.9% (15/126) and 9.4% (9/96), respectively. Regardless of NT methods used in this study, EG cell NT gave rise to comparable rate of blastocyst development to somatic cell NT. Overall, EG cells can be used as karyoplast donor in NT procedure, and embryos can be produced by EG cell NT that may be used as an alternative to conventional somatic cell NT.

• Journal of Embryo Transfer
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• v.28 no.2
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• pp.127-132
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• 2013

The objective of this study was to examine the effect of in vitro maturation (IVM) medium, cytochalasin B (CB) treatment during intracytoplasmic sperm injection (ICSI), and electric activation on in vitro development ICSI-derived embryos in pigs. Immature pig oocytes were matured in vitro in medium 199 (M199) or porcine zygote medium (PZM)-3 that were supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then further cultured in hormone-free medium for an additional 21~22 h. ICSI embryos were produced by injecting single sperm directly into the cytoplasm of IVM oocytes. The oocytes matured in PZM-3 with 61.6 mM NaCl (low-NaCl PZM-3) tended to decrease (0.05

• Reproductive and Developmental Biology
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• v.33 no.1
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• pp.29-33
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• 2009

The brilliant cresyl blue (BCB) has been used to select the developmental competent oocytes in pigs, goats and cows. Growing oocytes have a higher level of active glucose-6-phosphate dehydrogenase(G6PDH) compare to mature oocytes and are rarely stained compared to mature oocytes, because G6PDH converts BCB to colorless. First polar body extrusion regard as a guideline of meoisis completion. Selection of polar body extrude oocyte is more developmental competent to blastocyst than unselected. This study was conducted to compare the BCB test to the polar body extrusion on selection of developmental competent porcine oocytes for the production of blastocyst. Cumulus-Oocytes complex were exposed to 26uM BCB stain diluted in NCSU-23 for 90 min. There was no significant difference embryo development to blastocysts between BCB treated and not treated($19.58{\pm}1.99$ vs $18.75{\pm}2.27%$), which means there was no detrimental effect of BCB exposure to oocytes. Normal fertilization is not differed among treatment groups from 70.0 to 78.4% development to blastocyst, beside polyspermy did not. To compare two different selection methods, BCB test and polar body extrusion, evaluate the developmental competent of IVP embryos. BCB+PB+(blue stained and polar body extruded, $20.71{\pm}0.45%$) and BCB-PB+(colorless and polar body extruded, $20.04{\pm}l.29%$) groups are significantly (p<0.05) higher developed than those of BCB+PB-(blue stained and no polar body, $13.24{\pm}0.73%$) and BCB-PB-(colorless and no poladbody, $7.25{\pm}0.77%$). These results showed that selection of polar body extruded oocytes method is more efficient than that of BCB test.

• Reproductive and Developmental Biology
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• v.37 no.2
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• pp.65-73
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• 2013

The objective of this study was to examine the effects of high concentrations of glucose on porcine parthenotes developing in vitro. Addition of 55 mM glucose to the culture medium of embryos at the four-cell-stage significantly inhibited blastocyst formation, resulting in fewer cells in blastocyst-stage embryos and increased levels of apoptosis and autophagy compared to control. Quantitative reverse transcriptase (RT) PCR analysis revealed that the expression of pro-apoptotic genes (Caspase 3, Bax and Bak) and autophagy genes (Atg6 and Atg8/Lc3) were increased significantly by the addition of 55 mM glucose to the culture medium compared to control. MitoTracker Green fluorescence revealed a decrease in the overall mitochondrial mass compared to control. However, the addition of 55 mM glucose had no effect on mRNA expression of the nuclear DNA-encoded mitochondrial-related genes, cytochrome oxidase (Cox) 5a, Cox5b and Cox6b1. These results suggest that hyperglycemia reduced the mitochondrial content of porcine embryos developing in vitro and that this may hinder embryonic development to the blastocyst stage and embryo quality by increasing apoptosis and autophagy in these embryos.

• Journal of Embryo Transfer
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• v.24 no.1
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• pp.65-69
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• 2009

The aim of the present study was to investigate the cleavage pattern, its developmental ability and apoptosis of porcine embryo in vitro. Morphology data on a total of 919 embryos were analyzed retrospectively. Forty-eight hours after insemination, embryos were classified into five groups based on the cleavage state as follows; 1 cell, 2 cell, 4 cell, 5 to 8 cell and fragmentation. These groups were cultured another 120 hours and then evaluated for blastocyst formation. Blastocyst formation rates were significantly higher in 4 cell (42.5%) and 5 to 8 cell (48.6%) cleaving groups than in other groups (p<0.05). On the other hand, 2 cell and fragmentation groups produced 4.9% and 3,9% blastocysts, respectively. And we could verify that in the event of 2 cell block and fragmentation of embryo. To analyze the apoptotic frequency in preimplantation development of porcine IVF embryos, all cells of each blastocyst were performed by TUNEL assay. There were no significantly differences in the total cell numbers of embryos and apoptotic cell rate in blastocysts among the each classified groups. Data suggest that 4 cell and 5 to 8 cell cleaving embryos at 48 hour after insemination have high developmental competence, and may be an useful parameter to predict the development of preimplantation embryos and to study using preimplanation embryonic research.