• 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.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.249-258
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• 1996

To improve the efficiency of in vitro production of embryos with follicular oocytes in Korean Native cows, the recovery rates, in vitro maturation, fertilization and development, and the time required for collecting and processing oocytes by aspiration with or without slicing were evaluated comparatively. The ovaries were obtained from a local abattoir and placed in physiological saline at 25~28$^{\circ}C$ and brought to the laboratory within 3 hrs. The oocytes were collected by aspiration of follicles(2~6mm) with or without slicing ovaries after aspiration, and classified into Grade I, Grade II, Denuded, Expanded oocytes by the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules. Also the time required for each step of collecting and processing oocytes were measured. The cumulus cells were removed in some Grade I oocytes to measure their size and nuclear configuration before and after in vitro maturation. The Grade I oocytes were matured in vitro(IVM) for 24 hrs. in TGM-199 supplemented with 35$\mu$g /ml FSH, 10$\mu$g /ml LH, 1 $\mu$g /ml at 39$^{\circ}C$ under 5% C02 in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24hrs. and then the zygotes were cocultured in vitro (IVC) with bovine oviductal epithelial cells for 10 days. The results obtained were as follows: The number of oocytes recovered per ovary was averaged 6.6 by aspiration and 11.2 by slicing post aspiration, which summed to 17.8. The number of Grade I oocytes recovered per ovary was averaged 3.1 by aspiration and 3.6 by slicing, which summed to 6.7. The percentage of Grade I to total oocytes recovered was significantly(P<0.05) higher as 48.0 % in aspiration than 31.6% in slicing post aspiration. The time requlred for recovering a Grade I oocyte by aspiration and slicing was 1.1 and 2.5 min, respectively. The mean diameter of Grade I oocytes by aspiration and slicing was similar as 148.7 and 151.5$\mu$m, respectively. The percentage of Metaphase II stage oocytes after IVM for 24 hours was significantly (P

• Korean Journal of Animal Reproduction
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• v.24 no.1
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• pp.69-76
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• 2000

This study was carried out to improve of effective culture system on development of IVM/IVF/IVC bovine embryos. The cumulus-oocyte-complexes (COCs) collected from Korean cattle ovaries harvested at a local abattoir were matured in 50 ${mu}ell$ of TCM199 supplemented with 10% fetal bovine serum (FBS) and hormones (35 $\mu\textrm{g}$/$m\ell$ FSH, 10 $\mu\textrm{g}$/$m\ell$ LH, 1$\mu\textrm{g}$/$m\ell$ estradiol 17 $\beta$ under paraffin oil at 39$^{\circ}C$ in a humidified atmosphere of 5% $CO_2$in air. At 24 hrs after culture, matured oocytes were fertilized in vitro for 22~24 hrs with motile semen in which obtained by centrifugation of a frozen thawed semen on Percoll-density gradients (45% vs. 90%) at 500 g for 20 min. The presumptive zygotes were divided into three experimental groups. Single egg (Group 1), 25 (Group 2) or 50 eggs (Group 3) were cultured on cumulus cell in 50 ${mu}ell$ TCM199 supplement with 10% FBS for 6~9 days after fertilization. In vitro developmental rates into the blastocysts in the groups 2 and 3 were significantly (P＜0.05) higher than those of group 1 (37,27 vs. 6%, respectively). Cell number of blastocysts obtained in groups 2 and 3 at day 8 were significantly (P${mu}ell$) resulted in higher developmental competence and cell number of bovine blastocysts produced in vitro than those the culture of single embryos with cumulus cells.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.201-207
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• 2017

To increase the productivity of in vitro development, the antioxidants have been used for culture system of bovine oocytes and embryos. However, comparative studies on these molecules are rare and direct beneficial effects on blastocyst production cannot be discriminated for best results. The study was conducted to determine the influence of N-acetyl-L-cysteine (NAC), N-acetyl-L-cysteine amide (NACA), glutathione (GSH) and cysteamime (CYS) on maturation competence of COCs from GV to MII stage and productivity of blastocyst formation during in vitro fertilization and culture. There was no difference among maturation rates of oocytes to metaphase II with polar body with antioxidants for any of the treatment groups (p>0.05). However, the significant improvement on the rate of blastocysts ($32.3{\pm}5.0%$) was found in 0.1 mM CYS treatment than 0.3 mM NAC, 0.2 mM NACA or 0.5mM GSH (p<0.05). The addition of NAC ($18.8{\pm}3.7%$) or NACA ($21.2{\pm}3.9%$) did not improve development competence to morula and blastocysts than control ($24.4{\pm}4.1%$) and GSH ($26.5{\pm}5.0%$) (p>0.05). Our study showed that medium supplementation with CYS during IVM and IVC improved the rate of bovine embryo development but not with NAC, NACA and GSH addition.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.199-205
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• 2009

This study was carried out to investigate the effective genetic resources preservation system using the frozen boar semen. The porcine oocytes were matured for 44 hours in NCSU-23 medium with or without 10% Porcine Follicle Fluid (PFF), 0.5 ${\mu}g/ml$ porcine FSH, 0.5 ${\mu}g/ml$ equine LH, 1.0 ${\mu}g/ml$ 17 $\beta$-estradiol ($E_2$) and 10 ng/ml Epidermal Growth Factor (EGF) under mineral oil at $38.5^{\circ}C$ in humidified atmosphere of 5% $CO_2$ in air. After 44 h of culture, the oocytes were inseminated with frozen-thawed semen and fresh semen prepared with mTBM medium for 6 h. Later, set of 50 presumptive zygotes were transferred into 4-well dish (500 ${\mu}l$) of IVC medium. for embryos freezing, slow-freezing and vitrification methods were used as a cryopreservation. Differences among treatments were analyzed using General Linear Model Procedure by SAS Package (version 6.12) differences were considered significant when p<0.05. Following IVF and IVC, the rates of cleavage and blastocysts formation were significantly higher (p<0.05) in hormone supplemented group than that of hormone-free group (25.7 vs, 12.1). The development rates to cleavage and blastocysts were significantly higher in PZM-5 group than NCSU-23 group (60.3%, 46.6% vs 27.4%, 11.1%). Further improvement was achieved when PZM-5 was supplemented with FBS. Cleavage rates was significantly higher in fresh semen source group than frozen semen (66.7% vs 43.7%). However in blastocysts rates was similar two groups. Post-thaw survival rates of embryos were 1.2% and 2.2% in slow-frezing and vitrification groups, respectively. The results of our study suggest that it is still possible to improve the culture conditions and boar semen cryopreservation for enhance reproductive technology and animal genetic resources conservation.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.4
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• pp.495-499
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• 2006

In our previous studies, we demonstrated that Vascular Endothelial Growth Factor (VEGF) enhances bovine oocyte maturation and early embryonic development in serum supplemented media. In this experiment, to determine the synergistic effect of VEGF with serum components on early embryonic development in vitro in cattle, 1 mg/ml polyvinyl-alcohol (PVA) was replaced with foetal bovine serum (FBS) in maturation and culture media. Bovine oocytes were matured in Synthetic Oviduct Fluid (SOF) supplemented with PVA, PVA+5 ng/ml of VEGF, FBS, or FBS+VEGF. Fertilized oocytes were cultured in the same conditions for 8 days. The development of embryos was examined at 48 h post- insemination and on days 6, 7 and 8. The results were analyzed using repeated measures two- factor ANOVA, in which the effects of VEGF and serum were assigned as two factors. The development rate to 4- to 8-cell embryos at 48 h was significantly higher in the PVA+VEGF group than in the PVA group (44.7% and 31.5%, respectively). However, the highest development rate to 4- to 8-cell embryos was obtained from the FBS+VEGF group (58.8%). On day 8, the blastocyst rates were higher in the PVA+VEGF (22.8%), FBS (32.1%, p<0.05) and FBS+VEGF (42.1%, p<0.05) groups than in the PVA group (17.1%). Two- factor ANOVA of the development rates indicates that VEGF had a significant effect, but had no synergistic effect with serum components on early embryonic development. The results of the present study demonstrate that VEGF improves the in vitro developmental competence of bovine oocytes and/or embryos independent of the effect of serum components.

• Korean Journal of Veterinary Research
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• v.57 no.2
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• pp.89-95
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• 2017

This study was conducted to determine the effects of biophoton treatment during in vitro maturation (IVM) and/or in vitro culture (IVC) on oocyte maturation and embryonic development in pigs. An apparatus capable of generating homogeneous biophoton energy emissions was placed in an incubator. Initially, immature pig oocytes were matured in the biophoton-equipped incubator in medium 199 supplemented with cysteine, epidermal growth factor, insulin, and gonadotrophic hormones for 22 h, after which they were matured in hormone-free medium for an additional 22 hr. Next, IVM oocytes were induced for parthenogenesis (PA) or provided as cytoplasts for somatic cell nuclear transfer (SCNT). Treatment of oocytes with biophoton energy during IVM did not improve cumulus cell expansion, nuclear maturation, intraoocyte glutathione content, or mitochondrial distribution of oocytes. However, biophoton-treated oocytes showed higher (p < 0.05) blastocyst formation after PA than that in untreated oocytes (50.7% vs. 42.7%). In an additional experiment, SCNT embryos produced from biophoton-treated oocytes showed a greater (p < 0.05) number of cells in blastocysts (52.6 vs. 43.9) than that in untreated oocytes. Taken together, our results demonstrate that biophoton treatment during IVM improves developmental competence of PA- and SCNT-derived embryos.

• 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.

• Journal of Embryo Transfer
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• v.22 no.3
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• pp.155-160
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• 2007

During in vitro culture of mammalian oocytes and embryos, the cells are exposed to the risks that cause cell injury or death. Numerous studies have been reported that the cell injury may be induced by the action of free radicals generated by auto-oxidation. This study was undertaken to investigate the optimal culture condition system for in vitro culture of porcine embryos. We first evaluated the effect of culture media on the porcine embryo development. NCSU-23 and PZM-5, culture medium tested, were failed to produce significant difference on the rate of blastocyst formation. In NCSU-23, the developmental rate was slightly higher than that in PZM-5. During in vitro maturation (IVM), fertilizaton (IVF), and culture (IVC) under 5 or 20% oxygen ($O_2$), the rates of cleavage and development were insignificantly different from each other under our culture condition (20% $O_2$, in NCSU-23), the mean cell number per blastocyst was $40{\pm}10$. These results showed that medium and $O_2$ concentration had no significant effect on the development of porcine embryos.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.7-14
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• 1994

The present experiments on cryopreservation were designed to examine the effects of solution toxicity, equilibration time and cell stages on the post-thaw survival of bovine IVF embryos. The oocytes were matured in vitro(IVM) for 24 hrs. in TCM-199 supplemented with 35 $\mu$g /ml FSH, 10 $\mu$g /ml LH, 1 $\mu$g /ml estradiol-17$\beta$ and granulosa cells at 39$^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24 hrs., and then the zygotes were co-cultured in vitro(IVC) with bovine oviductal epithelial cells for 7 to 9 days. The bovine IVF embryos were exposed to the EFS solution in one step at room temperature, kept in the EFS solution during different period for toxicity test, vitrified in liquid nitrogen, and thawed rapidly. 1. after the bovine blastocysts were exposed to EFS solution for 2 min. at room temperature and then they were washed in 0.5 M sucrose solution and TCM-199, they were cultured to examined cryoprotectant induced injury during exposure, Most of the embryos(95.0%) developed to reexpanded blastocoels. However, when the exposure time was extended to 5 and 10 min, these development rates dropped dramatically in 5 min. (69.5%) and 10 min. (47.4%), respectively, 2. When the bovine IVF embryos were vitrified in EFS solution after the equilibration for 1 and 2 min. exposure, The embryos to have reexpanded blastocoels following thawing, washing and culture processes were found to he 82.6 and 73.9%, respectively. However, when the exposure time was extended to 3 min, this survival rate dropped to 18.2%. The optimal time for equilibration of bovine IVF blastocysts in EFS solution seemed to he 1~2 min. 3. When the bovine IVF embryos were equilibrated for 1 min. the significantly (P<0. 05) higher post-thaw survival rates were obtained from the embryos of blastocyst stage(81.3%) than morulae stage(5. 1%). The optimal cell stage for viterification with EFS solution proven to he blastocyst stage in bovine IVF embryos. 4. The number of blastomeres of blastocyst stage was examined with nuclear staining with Hoechst 33342 during 7 to 9 days post-insemination. The cell counts of frozen bovine IVF embryos were found significantly(P$\geq$7.5 and those of the fresh embryos 76.6$\geq$7. 1, which were cultured in the sarne period and conditions as frozen embryos.