• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.21-26
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• 2006

This study was examined the effects of concentrations of polyvinylpyrrolidone(PVP) and supplementation of EGF, cysteine and PVP during in vitro maturation on the development of bovine embryos. In experiment 1, 0.1 to 3.0% PVP was supplemented to IVM medium before IVF. The development rates to the blastocyst stage was significantly higher in 0.5% PVP group than 3.0% PVP group (P<0.05). In experiment 2, EGF, rysteine and PVP were supplemented to IVM medium. The hight cleavage rate was obtained from cysteine group, but blastocyst formation rates did not differ among groups. The highest total cell number and inner cell mass (ICM) cell number were observed in cysteine group. In PVP group, ICM cell number was significantly low than those of cysteine and control groups (P<0.05). After embryo transfer, pregnancy rate was significantly low in PVP group compared to other groups (P<0.05). These results indicate that the supplementation of PVP in IVM medium support the embryo development, but has a deteriorate effect on the blastocyst quality.

• Development and Reproduction
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• v.10 no.4
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• pp.239-245
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• 2006

Experiments were conducted to determine the effects of beta-mercaptoethanol(${\beta}-ME$) supplements to the maturation medium on in vitro fertilization(IVF) and intracellular glutathione(GSH) concentration. Bovine cumulus-intact oocytes were matured in TCM-199 medium containing FBS, hormonal supplements, and ${\beta}-ME$(0, 25 and $50\;{\mu}M$) for 12h and 24 h. After culture, cumulus-free matured oocytes were co-incubated with frozen-thawed spermatozoa for 24h. Maturation rate increased(p<0.05) in ${\beta}-ME$ treatment group, but no significant differences among treatment groups. Also, increases(p<0.05) in intracellular GSH concentration before and after fertilization were observed in $50\;{\mu}M\;{\beta}-ME$ supplements to the maturation medium. Male pronuclear formations after IVF was increased(p<0.05) in ${\beta}-ME$ treatment group, but no significant difference among treatment groups. In conclusion, supplementing ${\beta}-ME$ into the maturation medium increased maturation rates, fertilization rates, and intracellular GSH concentrations.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.1
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• pp.17-21
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• 2005

The effect of melatonin on in vitro embryo development and the expression of antioxidant enzyme gene in preimplantation porcine embryos was determined by modified semi-quantitative single cell RT-PCR. Porcine embryos derived from in vitro maturation /in vitro fertilization were cultured in 5% $CO_2$ and 20% $O_2$ at $37^{\circ}C$ in NCSU23 medium. Melatonin was added to medium at concentration of 1nM, 5 nM, and 10 nM. When treated with 1nM (39.0%) of melatonin, the developmental rate of embryos beyond the morula stage were higher than that of control group (31.0%) (p<0.05). Number of inner cell mass and tropectoderm cell in control (23.0${\pm}$0.5 and 17.3${\pm}$0.8), 1 nM (23.6${\pm}$0.6 and 19.0${\pm}$0.5), and 5 nM (23.3${\pm}$1.1 and 16.3${\pm}$0.8) treated with melatonin were higher than in 10 nM (20.0${\pm}$0.5 and 13.3${\pm}$0.8) treated with melatonin (p<0.05). To develop an mRNA phenotypic map for the expression of catalase, bax and caspase-3, single cell RT-PCR analysis were carried out in porcine IVM/IVF embryo. Catalase was detected in 0, 1 and 5 nM supplemented with melatonin, but bax and caspase-3 were detected in 10 nM treated with melatonin.

• Journal of Embryo Transfer
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• v.12 no.3
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• pp.307-313
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• 1997

Experiments were conducted to assess the effect of quality and viability of bovine blastocysts derived from in-vitro culture(IVC) of in vitro matured and fertilized(IVM-IVF) oocytes during their transport 2 hours. Follicular oocytes were collected form ovaries obtained at a slaughterhouse and were cultured for 24 hours in TCM-199. The IVM oocytes were fertilized in vitro with caudal epididymis spermatozoa. Fertilized oocytes were cultured for 7 to 9 days, and embryos that developed to the blastocyst stage were used for the experiment. The blastocysts, packed in straws with storage medium that consisted TCM-199 with HEPES equilibratd in air and supplemented with 10% FCS were transported at 39~(2.0 h). The quality of blastocysts was assessed and ranked as A(excel-lent), B(Good), fair or poor after transportation. The percentages of A and B grade blastocysts after transport duration for < 1 hours(97.7%) were similar to the result from transport duration for 1~2 hours (92.9%) and 2~3 hours(89.6%), but significantly(P<0.05) higher than transpot duration for 3~4 hours(76.3%). The percentages of A and B grade blastocysts after transport duration for two hours from developed blastocyst at 7day(100%) and 8day(85.0%) were higher 9day(96.6%) and >9day (40.0%). And early to expanded blastocyst produced in vitro were transferred to recipient cow by additional embryos at 7 and 8th day after AI. Three of them were pregnant to term and produced four twin calves, and two calves was premature birth. The gestation lengths of male to female and female to female twin were 282 and 281 days, respectively. And birth weight of twin calves were male to female(22.Skg) and female to female twin(20.3Okg), respectively.

• Clinical and Experimental Reproductive Medicine
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• v.48 no.4
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• pp.352-361
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• 2021

Objective: The study assessed the developmental potential of germinal vesicle (GV) oocytes subjected to in vitro maturation (IVM) after prematuration culture with cilostamide (a phosphodiesterase-3 inhibitor) and the impact of cilostamide exposure on the morphology of meiosis II (MII) oocytes and subsequent embryo quality. Methods: In total, 994 oocytes were collected from 63 patients. Among 307 GV oocytes, 140 oocytes were selected for the experimental group and 130 oocytes for the control group. The denuded GV-stage oocytes were cultured for 6 hours with cilostamide in the experimental group and without cilostamide in the control group. After 6 hours, the oocytes in the experimental group were washed and transferred to fresh IVM medium. The maturational status of the oocytes in both groups was examined at 26, 36, and 48 hours. Fertilization was assessed at 18 hours post-intracytoplasmic sperm injection. Embryo quality was assessed on days 3 and 5. Results: In total, 92.1% of the oocytes remained in the GV stage, while 6.4% converted to the MI stage (p<0.01) after cilostamide exposure. In both groups, more MII oocytes were observed at 36 hours (25.8% vs. 21.5%) than at 26 hours (10.8% vs. 14.6%) and 48 hours (13% vs. 7.9%) (p>0.05). With the advent of cilostamide, blastocyst quality was better in the experimental group than in the control group (p<0.05). Conclusion: Cilostamide effectively blocked nuclear maturation and promoted cytoplasmic growth. Prematuration culture with cilostamide enabled synchronization between cytoplasmic and nuclear maturity, resulting in better blastocyst outcomes.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.161-166
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• 2004

This study was designed to evaluate the effects of nitric oxide scavenger (hemoglobin) and inhibitor (L-nitro-L-arginine methyl ester; L-NAME) with or without cumulus cell on the development of bovine IVM/IVF embryos. When CR/sub 1aa/ medium were supplemented with different dosage (lug/m, 5ug/m and 10ug/ml) of hemoglobin at 48hrs for in vitro culture, the proportion of embryos developing beyond morulae stage in 0, 1ug/ml and 5ug/ml with or without cumulus cell were 23.8%, 33.3 % and 26.8%, and 39.5%, 54.8% and 48.8%, respectively. There was a significantly difference the developmental rate of 1ug/ml hemoglobin intact cumulus cells to any other groups (P<0.05). On the other hand, when added to hemoglobin at 96 hrs, 1 ug/ml hemoglobin with cumulus cell group was significantly increased the percentage of developing into morulae and blastocysts to any other groups (P<0.05), and similar trend that of added at 48hrs. The overall means of the percentage of developing into morulae and blstocysts in 1ug/ml hemoglobin group was significantly increased than those of any other groups (P<0.05) and cumulus co-culture with hemoglobin was increased the in vitro developing rate of IVM/IVF embryos. In CR/sub 1aa/ medium treated with L-NAME 0, 10, 50 and 100mM, the developmental rate of morula plus blastocysts were 55.6%, 64.9%, 58.8% and 66.7%, respectively. The L-NAME did not affect the developmental rate and the cell numbers of blastocysts in all treated groups. These results indicate that hemoglobin and cumulus co-culture can increase the proportion of embryos that developed into morulae and blastocysts, but cell numbers of blastocysts were not affect in all groups.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.77-82
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• 2004

The purpose of this study was performed to establish the in vitro culture system of porcine in vitro maturation and in vitro fertilization(IVM/IVF) embryo. These studies was to determine the effects of antioxidants(aesculetin, taurine and melatonin) in porcine IVM/IVF embryos. In routine porcine IVM/IVF procedure, oocytes were cultured for 40∼44h incubation in NCSU 23 mediumand matured oocytes were inseminated with frozen semen. Then 2 to 8 cell embryos were removed cumulus cell and were allotted randomly to NCSU 23 containing different concentration of antioxidants in 5% $O_2$ and 5% $CO_2$ at 38.5$^{\circ}C$. Cell numbers of blastocyst were also counted using double fluorescence stain method. Aesculetin were added to NCSU 23 medium at concentration of 1 ug, 5 ug, and 10 ug, when treated with 10 ug(35.7%) of aesucletin at the rate of embryos of the morula plus blatocsyts were higher than those of any other groups (30.2%, 29.5% and 29.2%)(P<0.05). The developmental rates beyond morula stage of porcine embryos in NCSU 23 medium supplemented with taurine 0, 2.5 and 5.0 mM were 26.1%, 26.9% and 31.7%, respectively. The addition of 5.0 mM taurine was higher the developmental rate beyond morula stage than in any other groups. In NCSU 23 medium treated with melatonin 0, 1, 5 and 10 nM, the developmental rate of morula plus blastocysts were 33.3%, 39.1%, 33.3% and 27.9%, respectively. The developmental rate of morula and blascytocys treated with 1nM melatonin was higher than in any other groups(P<0.05). Cell numbers of blastocyst in NCSU 23 treated with melatonin 0, 1, 5 and 10nM were 41.0, 42.6, 39.6 and 33.0, respectively. These results indicate that aesculetin, taurine and melation can increase the developmental rate beyond the morulae and blastocysts in porcine embryos.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.83-87
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• 2004

The objective of this study was performed to establish the in vitro culture system of porcine in vitro maturation and in vitro fertilization(IVM/IVF) embryo. These studies was to determine the effects of melatonin, nitric oxide donor(SNP), and the combination effects of SNP and melatonin in porcine IVM/IVF embryos. In routine porcine IVM/IVF procedure, oocytes were cultured for 40∼44h incubation, and the zygotes were cultured for 40∼44h in NCSU 23 medium. Then 2 to 8 cell embryos were removed cumulus cell and were allotted randomly to NCSU 23 containing different concentration of melatonin, SNP and SNP plus melatonin in 5% $O_2$, 5% $CO_2$ and 90% $N_2$ at 38.5$^{\circ}C$. Cell numbers of blastocyst were also counted using double fluorescence stain method. In NCSU 23 medium treated with melatonin 0, 1, 5 and 10 nM, the developmental rate of morula plus blastocysts were 33.3%, 39.1%, 33.3% and 27.9%, respectivly. This result show that the developmental rate of morula and blascytocys treated with 1 nM melatonin was higher than in any other groups(P<0.05). The developmental rates of morula plus blastocysts were 41.9% in 0 uM SNP, 25.6% in 50 uM and 28.4% in 100 uM, respectively. The developmental rate of morula plus blastocysts were decreased treated with SNP in NCSU 23. In combined effects of SNP plus melatonin (0, SNP 50 uM, SNP 50 uM plus melatonin 1 nM, SNP 50 uM plus melatonin 5 nM and SNP 50 uM plus melatonin 10 nM), the developmental rates beyond morula stage of porcine embryos were 31.3%, 34.1%, 39.5%, 29.4% and 39.5%, respectively. The addition of SNP 50 uM plus maltonin 1 nM, developmental rates of blastocyst was higher rate than in any other groups. Cell numbers of blastocyst in NCSU 23 treated with melatonin 0, 1, 5 and 10 nM were 41.0, 42.6, 39.6 and 33.0, respectively. In combined effects of SNP plus melatonin (0, SNP 50 uM, SNP 50 uM plus melatonin 1 nM , SNP 50 uM plus melatonin 5 nM and SNP 50 uM plus melatonin 10 nM), cell numbers of developed blastocyst were 36.3, 34.6, 39.0, 39.9 and 39.0, respectively. These result show that the cell numbers of blastocyst treated with 0, 1 and 5 nM melatonin were higher than in 10 nM group(P<0.05), but cell numbers of blatocyst produced by SNP plus melatonin were not significantly difference in all experimental groups.

• 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.17 no.4
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• pp.287-294
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• 1994

This study was to investigate the effects of electrofusion, activation and developmental stage of donor embryos on in vitro development of nuclear transplant bovine embryos. A single blastomere nucleus from 8-cell to morula stage embryos produced by in vitro fertilization(IVF) was transferred into a recipient oocyte enucleated at 23∼25 h after in vitro maturation(IVM) or into a recipient oocyte enucleated and cultured for 14∼15 h. In one experiment the nuclear transplant embryos were subjected to additional activation treatments. Fusion rate of nuclear transplant eggs was high at direct current(D.C) voltages of 1.0 and 1.5 kV/cm 991.5 and 93.3%, respectively), but decreased at 2.0kV/cm (81.8%). Additional activation treatments by electric pulases or 7% ethanol did not affect the cleavage and development of nuclear transplant embryos. Development of nuclear transplant embryos slightly increased by delayed nuclear transfer and fusion (42∼43 h after IVM). With this system, blastocysts were obtained from transfer of 8-cell to morula stage donor nuclei (9.6%∼2.4%). The result of this study suggests that nucleo-cytoplasmic interactins, expecially activation of ooplast are very important for the development of nuclear transplant embryos, and donor cell stage does not affect the development of nuclear transplant embryos.