• Korean Journal of Animal Reproduction
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• v.26 no.4
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• pp.311-320
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• 2002

The objective of this study was to assess the developmental potential in vitro produced embryos frozen-thawed with the various containers, and also examined expression and localization of heat shock protein 70 at these embryos. For the vitrification, 2-cell, 8-cell and blastocyst stage embryos produced by in vitro fertilization (IVF) and nuclear transfer (NT) were exposed the ethylene glycol 5.5 M freezing solution (EC 5.5) for 30 sec, loaded on each containers such EM grid, straw and cryo-loop, and then immediately plunged into liquid nitrogen. Thawed embryos were serially diluted in sucrose solution, each for 1 min. and cultured in CRI-aa medium. Survival rates of the vitrification production were assessed by re-expanded, hatched blastocysts. There were no differences in the survival rates of IVF using EM grid and cryo-loop. However, survival rates by straw were relatively lower than other containers. The use of cryo-loop resulted in only survival of nuclear transferred embryos (43.7%). Also, there embryos after IVF or NT were analysed by semi-quantitive reverse transcription-polymerase chain reaction (RT- PCR) methods for hsp 70 mRNA expression. Results revealed the expression of hsp 70 mRNh were higher thawed embryos than control embryos. Immunocytochemistry used to localize the hsp 70 protein in embryos. Two and 8-cell embryos derived under control condition was evenly distributed in the cytoplasm but appeared as aggregates in some frozen-thawed embryos. However, in the control, blastocysts displayed aggregate signal while Hsp70 in frozen-thawed blastocysts appeared to be more uniform In distribution. Therefore, this result suggests that the exploiting Hsp 70 in the early embryos may be role for protection of stress condition for increase viability of embryos within IVF, NT and there frozen-thawed embryos.

• Journal of Embryo Transfer
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• v.11 no.1
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• pp.15-26
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• 1996

This study was conducted to improve the in vitro maturation(JVM), in vitro fertilization (IVF) and in vitro developmental capacity of oocytes derived from slaughtered Korean native cattle. The recoverd oocytes, obtained from a local slaughter house, were used completely surrounded by at least 3 layers of cumulus cells in combination with a homogeneous cytoplasmic pigmentation. In vitro maturation was induced in TCM-199 or Ham's F-10 supplemented with LH(1O $\mu$g/rnl), FSH(35 $\mu$g/ml), estradiol-17$\beta$(1 $\mu$g/ml) at 39$^{\circ}C$ under 5% $CO_2$ in air for 24 hours. Sperm from caudal epididyrnis and previously matured cumulus-oocytes complexes were cultured for 24 hours in 100 $\mu$l droplets of fertilization media under paraffin oil. The zygotes were cultured with media(TCM-199 with bovine oviductal epithelial cells or CRlaa) for 7 to 10 days. The cleavage rate of IVM-IVF oocytes was significantly (P<0.05) higher following maturation using Ham's F-10 (59.9%) than TCM-199 (51.6%). Development to the blastocysts among cleaved embryos was not signficantly different between maturation media: Ham's F-10 (16.0%) and TCM-199(11.9%). However, the hatching rate was affected significantly (P<0.05) on rnaturation media as 62.9% in Ham's F-10, compared with 41.2% in TCM-199. The cleavage rate of IVM-IVF oocytes was significantly (P<0.05) higher following IVF using m-TALP medium (80.1%) than BO medium (51.6%). The percentage of in vitro developed blastocysts among cleaved embryos was not signficantly different between fertimization media: BO (11.7%) and m-TALP (17.6%). The cleavage and the developmental rate to the blastocysts after IVF in m-TALP or condition medium(CM) with or without oviduct epithelial cell monolayer(OECM) was similar(80.1% and 17.6% in m-TALP, 83.8% and 19.4% in M-TALP with OECM. 82.9% and 18.9% in CM, 87.6% and 16.0% in CM with OECM, respectively). The percentage of in vitro developed blastocysts among cleaved embryos was significantly (P<0.05) higher in TCM-199 medium co-cul tured with bovine oviduatal epithelial cell monolayers(35.2%) than CRlaa medium(1.9%). These results stggest that the most transferable IVF embryos could be produced from Ham's F-10, m-TALP and TCM-199 medium with bovine oviductal epithelial cell monolayers for IVM, IVF and IVC, respectively.

• Reproductive and Developmental Biology
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• v.31 no.1
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• pp.29-33
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• 2007

This study was conducted to examine the development of in vitro fertilized (IVF) and nuclear transfer (NT) embryos following vitrification IVF and NT embryos developed to the blastocyst stage were equilibrated by 3 steps, vitrified and thawed, and their survival and hatching rates were examined. In IVF embryos, higher survival (82.1%, 96/117) and hatching rates (64.1%, 75/117) were obtained respectively after thawing and culture in expanded blastocysts compared to blastocysts (p<0.05). High survival and hatching rates were also obtained by vitrification of NT blastocysts, especially in expanded and hatching blastocysts (81.1 and 78.3%, respectively). The result of this study shows that IVF and NT blastocysts, especially late stage blastocysts, are successfully cryopreserved by vitrification.

• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.307-313
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• 1996

This study was conducted to investigate the effect of S-phase synthronized nuclear transfer on the development of nuclear transplant bovine embryos. A blastomere derived from the 16~32 cell stage bovine embryos was transferred into an enucleated metaphase II(MII) oocytes or activated S-phase eggs. From the MII-phase and S-phase nuclear transfer, 6.3%(4/63) and 13.8%(9/65) of nuclear transplant embryos developed to the blastocyst stage, respectively. In the S-phase nuclear transfer, maximal proportion of embryos developed to the blastocyst stage(16.6%) was obtained after the recipient cell was activated 8 h prior to receving a donor nucleus. MII-phase nuclear transplant embryos showed the PCC state of their nuclear at 1.5~2 h after fusion, whereas, S-phase nuclear transplant embryos did not undergo PCC. The result of this study suggests that if blastomeres of unknown cell-cycle-stage are used, S-phase nuclear transplantation through the activation of enucleated oocytes prior to fusion enhances development of nuclear transplant embryos. This result also suggests that the interval time from oocyte activation to cell fusion may affect the development of nuclear transplant embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.11 no.6
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• pp.655-660
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• 1998

To produce blastocysts more efficiently, it is required to identity accurately the factors involving embryonic cleavage in the chemically defined medium. Effects of pyruvate, lactate, calcium and protein sources on early cleavage of bovine follicular oocytes were investigated. The percentage of IVF embryos cleaved to ${\geq}$ 2-cell or ${\geq}$ 8-cell was higher in pyruvate (+) and lactate (+) (48 or 14%) than in pyruvate (-) and lactate (-) (22% or 4%), than in pyruvate (+) and lactate (-) (28% or 5%) and than in pyruvate (-) and lactate (+) (40% or 10%). Lactate was more effective than pyruvate during early cleavage of bovine embryos in the chemically defined medium. The percentage of IVF embryo cleaved to ${\geq}$ 2-cell and ${\geq}$ 8-cell in calcium (-) (19 and 6%) was significantly (p < 0.05) lower than in calcium (+) (78 and 45%). The percentage of embryos developed to ${\geq}$ 2-cell showed no significant (p < 0.05) difference among BSA, 1 and 20% FBS (57, 57 and 57%). Also the percentage of A grade embryos developed to ${\geq}$ 2-cell showed no significant (p < 0.05) difference among BSA, 1 and 20% FBS (40, 35 and 28%). The percentage of embryos developed to ${\geq}$ 8-cell showed no significant (p < 0.05) difference among BSA, 1 and 20% FBS (33, 23, and 22%). However, the percentage of A grade embryos developed to ${\geq}$ 8-cell in BSA (24%) was significantly (p < 0.05) higher than in 1 and 20% FBS (13 and 8%). The percentage of embryos developed to ${\geq}$ morula showed no significant (p < 0.05) difference among BSA, 1, 10 and 20% FBS (76, 76, 80 and 68%). The percentage of A grade embryos developed to ${\geq}$ morula in 10% FBS (59%) was significantly (p < 0.05) higher than 20% FBS (43%). The percentage of embryos developed to blastocyst showed no significant (p < 0.05) difference among BSA, 1, 10 and 20% FBS (34, 41, 43 and 32%). However, the percentage of A grade embryos developed to ${\geq}$ blastocysts in 10% FBS (25%) was significantly (p < 0.05) higher than in 20% FBS (8%).

• Korean Journal of Animal Reproduction
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• v.21 no.3
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• pp.293-302
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• 1997

Follicular oocytes of Grade I and II were collected from 2~6 mm ovarian follicles and matured in vitro (IVM) for 24 hrs in TCM-199 su, pp.emented with 35$\mu\textrm{g}$/ml FSH, 10$\mu\textrm{g}$/ml LH, and 1$\mu\textrm{g}$/ml estradiol-17$\beta$ at 39$^{\circ}C$ under 5% CO2 in air. They were fretilized in vitro (IVF) by epididymal spermatozoa capacitated with heparin for 12 hrs. The zygotes were then co-cultured in vitro with bovine oviducted epithelial cells (BOEC) for 7 to 9 days. The optimal time for IVM, the successful enucleation of IVM oocytes by micromanipulation at different oocyte ages after IVM, and the ideal culture system for IVM for effective IVF and in vitro development of IVM-IVF embryos was examined for in vitro production of nuclear recipient oocytes and nuclear donor embryos. To improve the efficiency of nuclear transplantation (NT) of IVF embryo into IVM follicular oocytes, this study evaluated the optimal electric condition and oocytes age for activation of IVM oocytes and in vitro development of NT embryos. In vitro development of NT embryos with preactivation or non-preactivation in enucleation oocytes, cell number of IVN-IVF embryos, and NT embryos wre also examined. The results obtained were as follows; 1. The most suitable enucleation time was at 24 hpm (83.3%) rather than that of 28 hpm(69.6%) and 32 hpm(50.0%). 2. There was no difference among the fusion rates of NT embryos at the voltages of 0.75, 1.0 and 1.5 kV/cm, but the in vitro development rates to morule and blastocyst were significantly (P<0.05) higher at the voltage of 0.75(12.5%) and 1.0kV/cm (12.6%) compared to 1.5kV/cm(0%). 3. No significant difference in activation rates were seen in NT embryos stimulated for 30, 60 and 120 $\mu$sec (71.7, 85.2 and 71.9%, respectively), but the in vitro development rates to morulae and blastocyst were significantly (P<0.05) higher in the oocytes stimulated for 30 $\mu$sec (11.6%) and 60 $\mu$sec(10.7%) than 120 $\mu$sec(0.0%). 4. The fusion rates (71.0 and 87.3%) and the in vitro development rates (9.1 and 12.7%) to morula and blastocyst were seen in the NT embryos stimulated at 28 and 32 hpm under the condition of 1.0 kV/ml, 60 $\mu$sec. However, at 24 hpm the fusion rates were 64.8% and the in vitro development to morula and blastocyst were not seen. 5. The fusion rates between the 8~12, 13~17 and 18~22-cell stage of IVM-IVF embryos were not significantly different. The in vitro development rates of the fused embryos to morula and blastocyst which were received from a blastomere of 8~12, 13~17 and 18~22-cell stages of IVM-IVF embryos were 14.9, 8.3 and 6.5%, respectively. 6. The in vitro development rate of the enucleated recipient oocytes with preactivation (24.2%) to morula and blastocyst was significantly (P<0.05) higher than that of non-preactivation (12.8%). 7. The cell numbers of NT blastocyst and IVM-IVF blastocyst cultured during 7~9 days were 63$\pm$11 and 119$\pm$23, and then their the mean cell cycle number were 5.98 and 6.89, respectively.

• Journal of Embryo Transfer
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• v.25 no.2
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• pp.117-125
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• 2010

Several cloned animals have been produced using somatic cell nuclear transfer (SCNT) and have interested in producing the transgenic cloned animals to date. But still its efficiency was low due to a number of reasons, such as sub-optimal culture condition, aberrant gene expression and nuclear reprogramming. The purpose of this study was to analyze gene expression pattern in in vitro fertilized (IVF) or SCNT pre-implantation embryos. IVF- or SCNT-embryos were cultured in media supplemented with different proteins (FBS and BSA) or energy sources (glucose or fructose). Blastocysts from IVF or SCNT were analyzed using semi-quantitative RT-PCR in terms of developmentor metabolic-related genes. Culture medium supplemented different proteins or energy sources had affected on the expression of developmental or metabolic genes in the SCNT blastocysts.

• Journal of Embryo Transfer
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• v.14 no.2
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• pp.121-129
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• 1999

The objective of this study was to produce calves by transfer of embryos derived from slaughter house(SHD) and ultrasound-guided ovum pick-up (OPU). At 60 hrs after injection of 400 mg FSH dissolved in 25% polyvinylpyrrolidone(PVP) by single dose, ultrasound-guided follicular oocyte aspiration was ferformed. Day-7 and day-8 blastocysts produced by in vitro maturation (IVM), fertilization (IVF) and culture(IVC) of the oocytes derived from SHD and OPU were nonsurgically transferred into recipients. The results obtained were as follows. The cleavage rate and the development rate to blastocysts were not significantly (P<0.05) different between the oocytes obtained by SHD (72.9% vs. 34.1%) and OPU (75.9% vs. 38.4%). The oocyte recovery rate from the number of follicles by ultrasound-guided aspiration were not significantly (P<0.05) different between Holstein (61.7%) and Hanwoo (60.1%), but the rate of oocytes useful for IVF was significantly (P<0.05) higher in Hanwoo (69.3%) than Holstein (59.6%). The cleavage rate and the development rate to blastocysts was not significantly (P<0.05) different between Holstein (74.9% vs. 39.2%) and recipients on day 8 of estrus cycle resulted in 13 pregnancies (34.2%). One of them was sacrificed during gestation period due to mastitis and another was aborted spontaneous. The resulting 14 calves were morphologically normal at birth. Seventy eight fresh OPU-IVF embryos were transferred into 21 recipients on day 8 of estrus cycles, resulting in pregnancy of 12 recipients (41.4%). Two of them were sacrificed during gestation period due to mastitis and the other two were aborted. Nevertheless, the 11 OPU-calves have been born normally.

• Korean Journal of Animal Reproduction
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• v.21 no.4
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• pp.345-353
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• 1997

Antioxidants and antioxidants with somatic cell co-culture, bovine oviduct epithelial cells(BOEC) and buffalo rat liver cells(BRLC), were studied as a mean of increasing the development and intracellular glutathione(GSH) concnetrations of bovine embryos derived from in vitro matured(IVM) and in vitro fertilized(IVF) oocytes. Cell numbers and intracellular GSH concentrations of blastocysts were also counted. The developmental rate beyond morula stages in CRlaa containing taurine(2.5mM), superoxide dismutase(SOD, 600U) and catalase(250U) were 1%, 75.0%, 64.8% and 62.3%, respectively. The developmental rate in antioxidant groups was significantly higher than in control(P<0.05). The intracellular GSH concentrations of blastocysts cultured in 0, 2.5mM taurine, 600U SOD and 250U catalase were 33.8pM, 39.3pM, 42.3pM and 54.8pM, respectively. This result indicated that the developmental rates and intracellular GSH concentrations of catalase group was significantly higher than any other groups(P<0.05). The developmental capacity in CRlaa plus various antioxidants co-cultured with BOEC were 55.3%(control), 74.1%(2.5mM taurine), 66.7%(600U SOD) and 70.7%(250U catalase) and in CRlaa plus various antioxidants co-cultured with BRLC in control, 2.5mM taurine, 600U SOD and 250U catalase were 63.8%, 75.5%, 71.0% and 73.5%, respectveily, the intracellular GSH concentrations of blastocyst embryos co-cultured with BOEC and BRLC in CRlaa with 0.25mM taurine, 600U SOD and 250U catalase were 73.4pM and 64.4pM, 79.9pM and 67.5pM, 82.3pM and 71.7pM, and 83.0pM and 80.0pM, respectively. Cell numbers of blastocysts were not difference in all experimental groups. These studies indicate that andtioxidants and antioxidant with somatic cell co-culture can increase the proportion of embryo that developed into morula and blastocysts, and the intracellular GSH concentrations of blastocyst embryos.

• Journal of Embryo Transfer
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• v.23 no.3
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• pp.133-139
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• 2008

DNA methylation is one of the reasons for poor survival of clone animals. The OCT-4 gene is essential for maintaining pluripotency of embryonic stem (ES) cells and early embryos. We previously reported that the 5'-promoter region of Oct-4 gene was a target of DNA methylation and the methylation status was changed variously during embryonic development in bovine. The study conducted to examine the expression and methylation pattern of tissue-dependent differentially methylated region (T-DMR) of Oct-4 gene in bovine somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF) blastocysts. The Oct-4 gene expression was evaluated by RT-PCR and fluorescence immunocytochemistry. The methylation pattern of T-DMR was analyzed using restriction mapping and bisulfite sequencing methods. The Oct-4 transcripts were highly expressed in IVF, while they were not expressed in SCNT. The Oct-4 protein was not detected or expressed at very low level in SCNT, the intensity of Oct-4 protein, however, was strong in IVF. On the other hand, the T-DMR of Oct-4 gene was hypermethylated in SCNT compared to that of IVF. These results suggested that expression and the failure of demethylation of Oct-4 gene was closely associated with incomplete development of SCNT embryos.