• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.20-20
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• 2001

It has been reported that cloning cattle is inefficient. One of the problems was placental abnormality, finally resulting in fetal mortality after transfer of nuclear transfer (NT) bovine embryos. This study was focused on the allocations of embryonic cells to the inner cell mass (ICM) or to the trophectoderm(TE) in NT bovine blastocysts. Somatic cells were derived from a Day 45 fetus of gestation, individually transferred into enucleated oocytes and developed to the blastocyst stage in vitro. Differential staining was used to assess the qualify of blastocysts derived from NT, IVF and in vivo. Development rate of NT embryos to blastocysts (25.0%, 41/164) was similar to that of IVF embryos (28.7%, 49/171). The total cell number of NT blastocysts (101.3$\pm$45.9) was not different compared with that of IVF embryos (107.9$\pm$34.2, P>0.05), but was lower than in vivo embryos (122.5$\pm$21.6, P<0.05). Ratio of ICM/total cells was higher in NT embryos (51.6$\pm$ 18.6%) than in IVF and in vivo embryos (42.3$\pm$ 15.3% and 34.9$\pm$8.9%, respectively) (P<0.05). Most IVF (56.8%, 25/44) and in vivo blastocysts(80.8%, 21/26) was distributed in the proportion of ICM/total cells ranging from 20 to 40% group. However, most NT blastocysts was biased in the 40-60%(34.1%, 15/44) and >60% (31.8%, 14/44) groups. Our findings suggest that placental abnormalities or early fetal losses in the present cloning system may be due to aberrant allocation of NT embryos to the ICM cells.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.443-451
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• 1997

These studies were conducted to determine the sex of preimplantation Hanwoo embryos produced in vitro using polymerase chain reaction(PCR). Y chromosome specific and bovine speicific DNA primers were synthesized and tested for embryo sexing. Bovine IVF embryos were produced in TCM 199 and CR1aa medium, and classified by developmental stages on Day 7 to 9. The effects of developmental rates to bovine IVF blastocysts on sex ratio were also investigated using PCR methods. The results obtained in this study were as follows; 1. Developmental rates to blastocyst from IVM/IVF embryos in TCM 199 and CR1aa medium for 9 days were 23.5 and 30.2%, respectively, and there was significant difference between the media(P<0.05). 2. Male to female ratio of early, mid, expanded and hatching balstocyst produced on Day 7 were 0.7:1, 1.4:1, 2.2:1, and 2.5:1, respectively, and male embryos was significantly higher proportion in expanding and hatching blastocysts(P<0.01). 3. On Day 8, male to female ratio of early, mid, expanded and hatching blastocysts were 0.6:1, 1:1, 2.5:1, and 2.7:1, respectively. Both expanded and hatching blastocysts obtained a significantly higher proportion of males(P<0.01). 4. The male : female ratio of early, mid, expanded and hatching blastocyst produced on Day 9 was 0.6:1, 0.8:1, 1:1, and 2.2:1, respectively. Hatching blastocysts had a significantly higher ratio of males(P<0.01). The developmental rate of IVM/IVF embryos to blastocyst for 9 day culture was higher in CR1aa than that in TCM 199 medium. For the sex ratio by developmental stages of IVF embryos, male ratio was higher in expanded blastocyst but female in early blastocysts.

• Reproductive and Developmental Biology
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• v.36 no.4
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• pp.269-273
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• 2012

The present study was to assess the in vitro viability and sexing rate of bovine embryos. Blastocysts were harvested on day 7~9 day after insemination(in vitro and in vivo), and the sex of the embryos was examined using the LAMP method. Embryo cell biopsy was carried out in a $80{\mu}l$ drop $Ca^{2+}$, $Mg^{2+}$ free D-PBS and, biopsied embryos viability were evaluated after more 12 h culture in IVMD culture medium. The formation of recovered embryo to expanded and hatching stages had ensued in higher of sexed embryo in vivo than in vitro (100% vs. 89%, p<0.05), and in vitro, the rates of degeneration after sexing were significantly (p<0.05) higher in vitro than in vivo(11% vs. 0.0%). The rates of the predicted sex were female 61% vs. 56%, and male 39% vs. 44% in vivo and in vitro, respectively. The rates of survival following different biopsy methods were seen between punching and bisection method in vivo and in vitro (100% vs. 89% and 100% vs, 78% respectively). Biopsy method by punching was significantly (p<0.05) higher than bisection between produced embryos in vivo and in vitro. The present data indicate that with microblade after punching for embryo sexing results in high incidence of survivability on development after embryo biopsy. It is also suggested that LAMP-based embryo sexing suitable for field applications.

• Korean Journal of Animal Reproduction
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• v.18 no.1
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• pp.31-37
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• 1994

This study carried out to investigate the effects of cryoprotectants in the medium on the survival rate of rapidly frozen porcine bisected demi-embryos. The porcine bisected demi-embryos following dehydration by cryoprotectants containing sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water bath. Survival rate was defined as development rate on in vitro culture or FDA-test. The results are summarized as follows : 1. The survival rates of without-zona pellucida embryos and 2 blastomeres porcine embryos were 10.0 and 7.1%, respectively. The rate of unfrozen blastomeres (20.00%) was significantly higher than that of non-frozen oocytes. 2. The survival rates of with and without-zona pellucida of bisected porcine embryos by micromanipulator were 20.0 and 14.3%, respectively. 3. The developmental rates of with and without-zona pellucida of bisected poricine embryos by micromanipulator were 13.3 and 7.1%, respectively.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.107-115
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• 1998

The present study was carried out for the comparative study on the collection of bovine follicular oocytes by ultrasound-guided ovum pick-up(OPU) and slaughterhouse-derived (SHD) ovary aspiration and in vitro production of bovine embryos with the follicular oncytes in Korean native cows. Bovine follicular nocytes were observed with a 6.5 MHz convex-array ultrasound transducer designed for intravaginal use and the oocytes were collected with the aspiration equipment attached to the ultrasonograph. Bovine ovaries were collected and transported in phosphate buffered saline from the local slaughterhouse, the follicular oocytes were collected by the aspiration method. The collected follicular oocytes in good quality were matured, fertilized and cultured in the media. The total number of the visible follicles and the recovery rate of follicular oocytes were increased in ultrasonography following follicle stimulating hormone(FSH) treatment in Korean native cows. The mean recovery rate of oocytes was 66.2, 52.8 and 41.7% in the FSH-OPU, non-treatment-OPU and SHD ovaries, respectively. The mean number of recorved oocytes per cow were not significantly(P<0.05) different between the FSH-OPU(14.0$\pm$11.54) and SHD(17.1i6.21) groups, but the numbers in both groups were significantly(P<0.05) higher than the number in the non-treatment-OPU(3.7$\pm$1.57) group. The mean number of usable nocytes in Grade T /11 per ovary was 6.3, 4.8 and 1.3 in the cows of the SHD, FSH-OPU and non-treatment-OPU groups, respectively. The in vitro developmental rate to the blastocyst was not significantly different between the oocytes obtained via OPU(37.1%) and SHD(29.3%). Therefore, the ultrasound-guided OPU technique can be applied to the production of excellent embryos from the high-quality cows, and for the large scale production of in vitro bovine oocytes and embryos, the SHD ovary aspiration method is valuable.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.191-197
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• 1998

Immature nocytes and in VitrO matured Oocytes collected from the slaughtered Korean cattle were frozen slowly with 10% ethylene glycol+5% polyvinyl pyrolidine+0.05M trehalose (l0EPT), 10% ethylene glycol+5% ficoll+0.05M sucrose (1OEFS), or 10% ethylene glycol+5% ficoll+0.05M trehalose (l0EFT) by cell freezer (experiment 1). And also,They were ultra-rapidly frozen with 30% ethylene glycol+10% polyvinyl pyrolidine+0.5M trehalose (3OEPT) or 30% ethylene glycol+18% ficoll+0.5M sucrose (3OEFS) using electron microscope grid (experiment 2). In experiment 1, the cleavage rate was 23.0% when immature oocytes were frozen slowly using various cryoprotectants descrihed above, and 5.1% of cleaved oocytes developed to over morula stage after in Vitro fertilization (IVF). There were no significant differences among these groups. When matured oocytes were frozen slowly, the total cleavage rate was 19.7%, and over morula stage was 3.2%. lOEPT (4.8%) and EFS (4.4%) were slightly more effective than l0EFT (0.0%) for development in vitro. Only in l0EFT treated group, immature oocytes have higher developmental capacity than matured ones, when they were frozen slowly and IVF after thawing. In experiment 2, oocytes were ultra-rapidly frozen using the electron microscope grid with two kind of cryoprotectants described above. In immature oocyte group, the cleavage rate was 13.9% and 5.8% of cleaved oocytes developed to over morula stage after IVF, and in matured group, 25.7 and 7.6%, respectively. There were no significant differences between two kind of cryoprotectants, but in ultra-rapid freezing using electron microscope grid, the efficiency is slightly higher in matured oocyte group.

• Journal of Embryo Transfer
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• v.8 no.2
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• pp.151-157
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• 1993

The present study was undertaken to determine the optimal condition for parthenogenetic activation of rabbit oocytes by electric stimulation in vitro in an attempt to develop nuclear transplantation techniques for cloning mammalian embryos and animals. Freshly ovulated oocytes were collected from superovulated rabbits from 13 to 26 hrs. after hCG injection. The cumulus-free oocytes were activated parthenogetically by repeated stimuli of square direct electric pulses in O.3M mannitol solution. After applying electric stimulations of different voltages, pulse durations and pulse times, all of the oocytes were cultured in TCM-199 with 10% FCS for 96 hours in a 5% $CO_2$ incubator, and their developmental potential in vitro was examined. The higher activation rate (68.9%) was achieved at the voltage of 2.0kv/cm, the pulse duration of 60 $\mu$sec and three pulse times and the activation rate of 100% was achieved at the pulse duration of 100 and 200 $\mu$sec, the voltage of 1.5kv/cm and three pulse times. Although the higher rates of activation of oocytes were achieved at 100 and 200 $\mu$sec, none of them developed to blastocyst in vitro. The oocytes collected 18~20 hours post hCG injection showed the highest rate of activation and development to blastocyst in vitro than the oocytes collected 13~15 or 25~26 hours post hCG injection. Therefore, it can be suggested that the application of electric stimulation of 2.0kv/cm, 60 $\mu$sec and three pulse times to the oocytes collected at 18~20 hours post hCG injection would be more beneficial for the parthenogenetic activation of oocytes in rabbits.

• Reproductive and Developmental Biology
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• v.29 no.2
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• pp.121-125
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• 2005

The present study was performed to determine the ability of canine oocytes to achieve nuclear maturation according to oocyte diameter and different culture environments. All of the collected oocytes were classified by grade 1 to 3 and by their diameters such as $<100{\mu}m,\;<100{\mu}m\;to\;<110{\mu}m,\;<110{\mu}m,\;to\;<120{\mu}m,\;>120{\mu}m,$. Oocytes were cultured in culture medium supplemented with $10\%\;FBS,\;0.4\%\;BSA,\;10\%$ porcine follicular fluid (pFF), $10\%$ canine serum (CS), or $10\%$ canine estrus serum (CES). The mean number of oocytes recovered from estrus status ovaries was significantly higher than that of anestrus status ovaries (p<0.01). The maturation rate of grade 1 oocytes $(>120{\mu}m)$ was significantly higher than that of the other groups (p<0.05). Nuclear maturation to MI to MII in diameter of $>110{\mu}m$ groups was significantly higher than that in $<100{\mu}m$ group (p<0.05). The oocytes cultured in $10\%$ FBS­supplemented group were significantly higher rate of GVBD compared to the other supplemented groups (p<0.05), and oocytes maturation to MI to MII in $10\%$ FBS-, $0.4\%$ BSA-, and $10\%$ pFF-supplemented groups were significantly higher than those in $10\%$ CS-supplemented group (p<0.05). Based on these results, the estrus status and the size of oocyte affect positively to improve nuclear maturation of canine immature oocytes in vitro. Among several protein sources, porcine follicular fluid was the most effective supplementation to culture medium to achieve higher in vitro maturation rate.

• Asian-Australasian Journal of Animal Sciences
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• v.8 no.6
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• pp.641-645
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• 1995

This study was conducted to develop a method for production of nuclear transplant bovine embryos using in vitro-matured (IVM) oocytes and to examine the effect of different conditions of electrofusion on fusion rate and developmental capacity of donor nucleus transplanted to enucleated oocytes. Eight- to sixteen-cell embryos derived from oocytes matured and fertilized in vitro used as donor blastomeres and IVM oocytes were used as recipient oocytes. Oocytes were enucleated immediately after 23-24 h IVM and then reconstituted with a donor blastomere in two different micromanipulation media. Fusion rate and subsequent development of the reconstituted oocytes was compared under the different electric stimuli and recipient oocyte ages. Success rate of enucleation was significantly higher in TCM-199 medium containing FCS than in DPBS. The high fusion rate(75-94%) and development (6.4-14.8%) to morulae and blastocyst (M + B) were obtained from 0.6-0.75 kV/cm DC voltage, although total cleavage was not different among the electric pulses. Most optimal condition of electric stimulation for fusion and development was 1 DC voltage of 0.75 kV/cm, in which 80.5% of oocytes were fused, 80.0% and 31.7% of which was cleaved and developed to M + B, respectively. No M + B was obtained from 1.2 kV/cm DC voltage regardless of pulse frequency. Recipint oocyte age at electrofusion greatly affected the cleavage and subsequent development to M + B, showing high rate at 40-41 h oocyte maturation. These results suggest that a suitable condition of electrofusion for donor nuclei derived from IVF may be 1-2 DC pulses of 0.7 kV/cm for $70{\mu}sec$ and that processing of a transplanted nucleus in IVM oocytes may be affected by maturation age of recipient oocytes.

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.59-63
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• 2004

1. The concentrations of Ang. II were 7.20.91 ${\times}$ $10^3$ , 3.80.34 ${\times}$ $10^3$, 3.50.30 ${\times}$ $10^3$, 2.80.22 ${\times}$ $10^3$ pg/ml in bovine follicular fluids from 1∼3 mm, 3∼5 mm, 5∼7 mm and 8∼10 m follicles, respectively. The concentrations of Ang. II decreased in follicular fluids from large follicles. 2. When oocytes were cultured in media containing various concentrations of Ang. II, a higher proportion of oocytes developed to MII stage in medium with 100 ng/ml (79.5%) Ang II compare to that without Ang. II (58.8%). When oocytes from different sizes of follicles were separately cultured in media containing 100 ng/ml Ang. II, maturation rates were higher in oocytes from small and medium follicles those from controls. 3. GSH content in oocytes cultured for 24 hrs in TCM-199 medium containing 10 and 100 ng/ml of Ang. II was also higher than that of oocytes cultured in medium containing 0 or 10 ng/ml Ang. II. When oocytes were cultured in media containing 0, 10, 100, 1,000 ng/ml of Ang. II, the concentrations of GSH were 5.1M, 5.5M, 7.2M, 8.7M, respectively. 4. When oocytes were cultured in media containing various concentrations of 10, 100, 1,000 ng/ml Ang. II, in vitro maturation and developmental rates were 84.0%, 90.0%, 78.0% and 28.0%, 36.0%, 20.0%, respectively. When oocytes were cultured with an addition of Ang. II in media, in vitro maturation rates higher than that of their controls (76.0%).