• Journal of Embryo Transfer
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• v.9 no.3
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• pp.235-241
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• 1994

The study was conducted to determine the optimal hormone and glucose levels during the in vitro culture of bovine oocytes matured and fertilized in vitro for blastocyst development. Oocytes matured in TCM 199 + 10% FCS + hormones and glucose were fertilized in vitro in a TALP medium with swim up separated and heparin-treated epididymal cauda spermatozoa. Oocytes were cultured for 2~5 days in synthetic oviduct fluid medium (SOFM) supplemented with 10% FGS and with different hormone and glucose levels, and further cultured 5 days same medium in SOFM. The results are summarized as follows : The in vitro maturation and penetration rates of porcine oocytes cultured in TCM 199 media containing PMSG, hCG, PMSG + hCG, hCG + $\beta$ estradiol, PMSG + $\beta$ estradiol 0 to20 hours after insemination were 88.0% and 81.8%, 82.6% and 68.4%, 80.0% and 75.0%, 80.0% and 65.0%, 77.3% and 64.7%, respectively. The in vitro maturation and penetration rates of porcine oocytes cultured in TCM 199 media containing PMSG, hCG, PMSG + hCG, hCG + $\beta$ estradiol, PMSG + $\beta$ estradiol 20 to 40 hours after insemination were 92.0% and 87.0%, 92.0% and 82.6%, 91.3% and 81.0%, 85.2% and 73.9%, 87.5% and 81.0%, respectively. The cleavage and in vitro developmental rates to blastocyst of porcine oocytes cultured in TCM 199 media containing 0.05 mM, 0.10 mM, 0.30 mM, 0.50 mM, 1.00 mM, and 3.00 mM glucose lelvels 0~3 days after insemination were 31.5~48.1% and 10.0~16.7%, respectively. The cleavage and in vitro developmental rates to blastocyst of porcine oocytes cultured in TCM 199 media containing 0.05 mM, 0.10 mM, 0.30 mM, 0.50 mM, 1.00 mM, and 3.00 mM glucose levels 4~8 days after insemination were 30.0~53.8% and 8.7~19.2%, respectively. The cleavage and in vitro developmental rates to blastocyst were higher in TCM 199 media containing various glucose levels 0~3 days after insemination than 4~8 days.

• Animal Bioscience
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• v.37 no.6
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• pp.1007-1020
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• 2024

Objective: We increased the nuclear maturation rate of antral follicle derived oocytes by using a pre-in vitro maturation (IVM) culture system and improved the developmental potential of these porcine pathenotes by supplementing with melatonin. Furthermore, we investigated the expression patterns of genes involved in cumulus expansion (HAS2, PTGS2, TNFAIP6, and PTX3) derived from small and medium antral follicles before and after oocyte maturation. Methods: Only the cumulus oocyte-complexes (COCs) derived from small antral follicles were induced with [Pre-SF(+)hCG] or without [Pre-SF(-)hCG] the addition of human chorionic gonadotropin (hCG) during the last 7 h of the pre-IVM period before undergoing the regular culture system. The mature oocytes were investigated on embryonic development after parthenogenetic activation (PA). Melatonin (10^-7 M) was supplemented during in vitro culture (IVC) to improve the developmental potential of these porcine pathenotes. Results: A pre-IVM culture system with hCG added during the last 7 h of the pre-IVM period [Pre-SF(+)hCG] effectively supported small antral follicle-derived oocytes and increased their nuclear maturation rate. The oocytes derived from medium antral follicles exhibited the highest nuclear maturation rate in a regular culture system. Compared with oocytes cultured in a regular culture system, those cultured in the pre-IVM culture system exhibited considerable overexpression of HAS2, PTGS2, and TNFAIP6. Porcine embryos treated with melatonin during IVC exhibited markedly improved quality and developmental competence after PA. Notably, melatonin supplementation during the IVM period can reduce and increase the levels of intracellular reactive oxygen species (ROS) and glutathione (GSH), respectively. Conclusion: Our findings indicate that the Pre-SF(+)hCG culture system increases the nuclear maturation rate of small antral follicle-derived oocytes and the expression of genes involved in cumulus expansion. Melatonin supplementation during IVC may improve the quality and increase the blastocyst formation rate of porcine embryos. In addition, it can reduce and increase the levels of ROS and GSH, respectively, in mature oocytes, thus affecting subsequent embryos.

• Reproductive and Developmental Biology
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• v.30 no.2
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• pp.135-141
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• 2006

The present study was conducted to investigate the effects of cumulus cells and porcine follicular fluid (pFF) on plasminogen activator (PA) activity and oocytes maturation in vitro in the pig. The cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) were incubated in NCSU-23 medium with or without 10% pFF for 0, 24, or 48 hr. In the presence of cumulus cells, the proportions of oocytes matured to metaphase-II stage were significantly (P<0.05) higher in medium with pFF than without pFF (69.8 vs. 37.7%, respectively). When COCs and DOs were cultured in the presence of pFF, tissue-type PA (tPA), urokinase-type PA (uPA), and tPA-PA inhibitor (tPA-PAI) were observed in COCs, and PA activities were higher at 48 hr than 24 hr. When COCs and DOs were cultured in the absence of pFF, tPA and tPA-PAI were observed in COCs, and PA activities were increased as duration of culture increased. No PA activities were detected in DOs regardless of pFF supplementation. When porcine oocytes were cultured in the presence of pFF for 24 and 48 hrs, the activities of tPA-PAI, tPA, and uPA were observed in both COCs and DOs. In medium of absence of pFF, PA activities were observed in oocytes with cumulus cells only. On the other hand, three plasminogen-dependent lytic bands (tPA-PAI, tPA, and uPA) were observed in pFF cultures. Particularly uPA activity was higher than the other kinds of PA activity. When oocytes and cumulus cells were separated from porcine COCs at 0 hr of culture, tPA-PAI, tPA, and uPA were detected in cumulus cells at 48 hr of culture, but no PA activities were in DOs. The presence of pFF and cumulus cells in maturation medium stimulated not only nuclear and cytoplasmic maturation in porcine COCs, but also PA production by cumulus cells and COCs. It is possible that PAs produced by cumulus cells migrated through the gap junction between oocyte and cumulus cells. These results suggest that porcine oocytes have no ability to produce PA themselves.

• Korean Journal of Animal Reproduction
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• v.14 no.1
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• pp.23-30
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• 1990

These studies were carried out to investigate the effects of fetal calf serum(FCS), estrous porcine serum(EPS), porcine follicular fluid(PFF), hormone and matured cumulus cell(MCC) on in vitro maturation and fertilization of porcine follicular oocytes. The ovaries and testes were obtianed from slaughtered Landrace sow and boars, respectively. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluid from the visible follicles of diameter 3~5 mm and the semen were prepared from boar's epididymal cauda. The follicular oocytes were cultured in TCM-199 medium containing hormones, FCS, EPS, PFF and MCC for 48hrs. in a incubator with 5% CO2 in air at 36$^{\circ}C$ and then matured oocytes were again cultured for 18~20 hrs. with $1.5\times$106/ml motile capacitated sperm in the modified Tyroide solution containing 100$\mu\textrm{g}$/ml of heparin. The results obtained in these experiments were summarized as follows : 1. The maturation and fertilization rate of the follicular oocytes, cultured in the TCM-199 medium supplemented with 10% FCS and PMSG＋HCG were 55.6~64.5% and 33.3~37.1%, respectively. 2. The maturation and fertilization rate of the follicular oocytes cultured in the TCM-199 medium supplemented with 20% EPS and PMSG＋HCG were 50.0~55.0% and 30.3~33.3%, respectively. 3. The maturation rate(59.0~64.2%) and fertilization rate(34.8~39.3%) of follicular oocytes cultured in TCM-199 medium supplemented 20% FCS and 50% PFF were higher than those of follicular oocytes cultured in TCM-199 medium supplemented with 5%, 10% and 15% FCS and 10% and 50% PFF. 4. The maturation rate(60.0%) and fertilization rate(40.0%) of follicular oocytes cultured in TCM-199 medium supplemented with 20% FCS and granulosa cell (1$\times$106/ml) were significantly higher than those of fiollicular oocytes cultured in TCM-199 medium supplemented with 5%, 10% and 15% FCS and granulosa cell.

• Reproductive and Developmental Biology
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• v.32 no.4
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• pp.249-253
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• 2008

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 1.1kV/cm for $30{\mu}s$ 0.3M 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.

• Journal of Animal Science and Technology
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• v.65 no.4
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• pp.767-778
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• 2023

The aim of the research is to identify that porcine oocytes can function as recipients for interspecies cloning and have the ability to develop to blastocysts. Furthermore each mitochondrial DNA (mtDNA) in interspecises cloned embryos was analyzed. For the study, mouse-porcine and porcine-porcine cloned embryos were produced with mouse fetal fibroblasts (MFF) and porcine fetal fibroblasts (PFF), respectively, introduced as donor cells into enucleated porcine oocytes. The developmental rate and cell numbers of blastocysts between intraspecies porcine-porcine and interspecies mouse-porcine cloned embryos were compared and real-time polymerase chain reaction (PCR) was performed for the estimate of mouse and porcine mtDNA copy number in mouse-porcine cloned embryos at different stages.There was no significant difference in the developmental rate or total blastocyst number between mouse-porcine cloned embryos and porcine-porcine cloned embryos (11.1 ± 0.9%, 25 ± 3.5 vs. 10.1 ± 1.2%, 24 ± 6.3). In mouse-porcine reconstructed embryos, the copy numbers of mouse somatic cell-derived mtDNA decreased between the 1-cell and blastocyst stages, whereas the copy number of porcine oocyte-derived mtDNA significantly increased during this period, as assessed by real-time PCR analysis. In our real-time PCR analysis, we improved the standard curve construction-based method to analyze the level of mtDNA between mouse donor cells and porcine oocytes using the copy number of mouse beta-actin DNA as a standard. Our findings suggest that mouse-porcine cloned embryos have the ability to develop to blastocysts in vitro and exhibit mitochondrial heteroplasmy from the 1-cell to blastocyst stages and the mouse-derived mitochondria can be gradually replaced with those of the porcine oocyte in the early developmental stages of mouse-porcine cloned embryos.

• Korean Journal of Animal Reproduction
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• v.21 no.1
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• pp.53-59
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• 1997

This study was conducted to investigate the effect of hormones on in vitro maturation of porcine follicular oocytes. The results obtained were as follows : 1. The maturation rates of oocytes cultured in medium containing FSH 1, 10, 50 and 100$\mu\textrm{g}$/ml were 44.6, 58.5, 42.6 and 37.9%, respectively. And those were no difference to maturation rates among the medium containing FSH. However, the maturation rate(13.7%) of oocytes cultured without FSH was significantly(P<0.05) lower than those of oocytes cultured with FSH. 2. The maturation rates of oocytes cultured in medium containing hCG 0, 1, 10, 50 and 100IU/ml were 12.2, 11.9, 17.9, 21.9 and 45.6%, respectively. The maturation rate of oocytes cultured with 100IU/ml hCG was significantly(P<0.05) higher than those of oocytes cultured with 0~50IU/ml hCG concentrations. 3. The maturation rates of oocytes cultured in medium containing E2 0, 1, 10, 50 and 100$\mu\textrm{g}$/ml were 13.7, 10.5, 14.9, 11.4 and 5.9%, respectively. There were no differences to maturation rates among the E2 concentrations. 4. The FSH＋hCG treatment was the highest maturation rate in medium containing different combination of FSH, hCG and E2.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.12
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• pp.1703-1712
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• 2015

The in vitro maturation (IVM) efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+) oocytes with low glucose-6-phosphate dehydrogenase (G6PDH) activity have shown superior quality than BCB negative (-) oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG) migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9) and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB- oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.70-70
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• 2002

Sphingosine-1-phosphate(S1P) is one of the sphingolipid metabolites which affect a variety of cellular processes including the proliferation, differentiation, growth, survival, migration and gene expression. The present study was undertaken to investigate the effect of SIP on nuclear maturation of porcine oocytes. In vitro maturation frequency of porcine oocytes were compared in three different media; group Ⅰ: NCSU23＋0.1% PVA, group Ⅱ: NCSU23＋10% PFF(porcine follicular fluid), and group Ⅲ: NCSU23＋10% PFF＋10 ng/㎖ EGF＋2.5 mM β-mercaptoethanol. Each group containing 0.1 ㎎/㎖ cysteine was divided into 4 sub-groups of SIP concentration(0, 50, 500 and 5000nM). Porcine oocytes were incubated in each maturation medium supplemented with hormones(10 IU/㎖ PMSG and 10 IU/㎖ hCG) for 22h and then further cultured in the same medium without the hormones for 22h. After completion of in vitro maturation, the oocytes were fixed and stained to examine nuclear maturation by using a rapid stain method. In the group Ⅰ, the proportions of metaphase Ⅱ stage among oocytes cultured in 0nM(control), 50 nM, 500nM and 5000nM S1P were 45.5%, 66.7%, 56.6% and 48.7%, respectively. (omitted)

• Reproductive and Developmental Biology
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• v.36 no.1
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• pp.71-77
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• 2012

The objective of this study was to examine the effect of thymidine treatment during $in$ $vitro$ maturation (IVM) of porcine follicular oocytes on blastocyst development. Porcine oocytes were treated with thymidine (10 mM, 20 mM and 30 mM) for 2 or 6 hr in the preiods of IVM I and/or II. The survival rates of the blastocysts in the 6 hr treatment groups of 10 mM and 20 mM during IVM I period were significantly higher than those of control group ($p$<0.05). However, the survival rate of the blastocysts in the 2 hr treatment group of 20 mM during IVM II period was significantly higher than control group ($p$<0.05). Furthermore, the survival rate of the blastocysts in the 6 hr treatment group of 30 mM during IVM II period was significantly lower than control group ($p$<0.05). Consistent with the previous result, blastocyst development of both IVM I and II treatment group was also showed as similar pattern. Total and apoptotic cell numbers of blastocysts derived from thymidine treated porcine oocytes were examined by using Tunel assay. The results showed that there was no significant differences in total cell number of blastocysts between thymidine treated and untreated groups. However, apoptosis-positive cells in the thymidine treated group (6 hr IVM I) were significantly lower than those of other groups ($p$<0.05). Taken together, these results indicate that high quality oocytes were selected by DNA synthesis mechanism according to high concentration thymidine treatment during porcine oocyte maturation. Therefore, we concluded that presumptive selected oocytes by thymidine treatment during maturation periods improved the further embryo development and embryonic quality of IVF embryos by decreasing the incidence of apoptosis in preimplantation porcine embryos.