• 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.

• Journal of Veterinary Clinics
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• v.9 no.1
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• pp.323-332
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• 1992

The present study was carried out to examine the effect of oviduct epithelium and its conditioned medium on e development of early bovine embryos in vitro. Oocytes obtained from ovarian follicles of slaughtered cows were cultured in TCM199 with 10% fetal calf serum for 22-24hrs and then fertillzed in vitro using frozen-thawed semen treated with BO-caffein, BO-BSA(20mM heparin added). Oviduct epithelium was collected in each stage of the estrus cycle and conditioned medium was the medium in which oviduct epithelium in early luteal stage was cultured. In vitro fertilized bovine embryos of 1～2 cell were co-cultured with oviduct epithelium from different estrus cycles, cultured in conditioned medium, and cultured in rabbit oviduct. The cleavage rates of in vitro fertilized early bovine embryos co-cultured with oviduct epithelial cell from early luteal, luteal and follicular phase of estrus cycle(67.2～70.8%) and cultured in conditioned medium(56.7%) were significantly(p<0.05) higher than that of the control(44.2%) The rate of development to morula or blastocyst stage in oviduct epithelial cell co-culture(15.3～32.5%) from three phase of estrus cycles and conditioned medium(14.5%) were significantly(p<0.05) higher than that of the control(5.2%). The oviduct epithelial cell from early luteal phase gave a significantly( p<0.05) higher rate of development to morula or blastocyst stage than both luteal and follicular phase. The results of in vivo culture in rabbit oviduct of early bovine embryos were 52.1% for the cleavage rate and 26.7% for the rate of development to morula or blastocyst stage.

• Journal of Embryo Transfer
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• v.17 no.2
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• pp.117-121
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• 2002

This study was carried out to investigate the in vitro fertilization rate of canine immature oocytes cryopreserved by vitrification freezing. The vitrification solutions of EPS and EDS were consisted of 40％ ethylene glycol 18％ Ficoll and 0.3M sucrose, and 20％ ethylene glycol, 16.5％ DMSO and 0.5M sucrose in TCM-199 medium supplemented with 10％ FCS, respectively. The oocytes were exposed The developmental rate of in vitro cultured oocytes recovered from ovaries collected at different stages of the reproductive cycle were 3.8％, 10.7％, 46％, respectively. to EFS or EDS at $25^{\circ}C$ and loaded into straw fer 30 sec. The straws was slowly immersed into L$N_2$. Fertilization and survival rate was defined as development rate on in vitro culture or FDA-test. 1. The fertilization rate after vitrification freezing of immature oocytes at 1, 6, 12 and 24 hrs after collection from ovaries was very low(5.3％～31.4％) than the unfrozen oocyte(60.0％). And the fertilization rate after vitrification freezing of immature oocytes was very higher than that of mature oocytes. 2. The survival rate after vitrification freezing of immature oocytes at 1, 6, 12 and 24 hrs after collection from ovaries was 55.0％, 40.0％, 28.6％ and 17.1％, respectively. And the survival rate after vitrification freezing of immature oocytes was slightly higher than that of mature oocytes.

• Reproductive and Developmental Biology
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• v.31 no.4
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• pp.261-265
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• 2007

Insulin, transferrin and selenium (ITS) complex is reported to improve in vitro development of oocytes and embryos. This study was carried out to investigate the effects of ITS during in vitro culture (IVC) of porcine parthenogenetic and nuclear transfer (NT) embryos on subsequent developmental capacity in vitro. The electrically activated oocytes were cultured in Porcine Zygote Medium (PZM-3) with various concentrations (0, 0.1, 0.5, and 1.0%) of ITS for 7 days. Also, the electrically activated reconstructed embryos were cultured in PZM-3 with various concentrations (0, 0.1, 0.5, and 1.0%) of ITS for 6 days. Addition of ITS to culture medium did not affect development of porcine parthenogenetic embryos in vitro. To test the effect of ITS on the in vitro development of porcine NT embryos, factorial experiments were also performed for in vitro maturation (IVM) medium (TCM-199) with or without 1% ITS and culture medium (PZM-3) with or without 0.5% ITS. Addition of 0.5% ITS to culture medium increased (p<0.05) the proportion of NT blastocysts compared with non-treated group. In contrast, addition of 1% ITS to culture medium was ineffective or had a detrimental effect. Also, addition of ITS only to maturation medium increased (p<0.05) the percentage of NT blastocysts formation compared with the control group. In conclusion, addition of ITS to IVM or IVC medium could improve subsequent blastocyst development of porcine NT embryos.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.233-240
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• 1996

This study was carried out to determine the effect of cumulus cell attachment and various factors on in vitro maturation of pig foflicular oocytes. Oocytes with various configuration of cumulus cell mass were collected ftom ovaries of mature gilts by asperating with syringe equipped with needles of different gauges, follicle size and with or without cumulus cells. They were cultured in TCM-199 mediun containing FGS(fetal calf serum) for 30~48 hours in incubator with air containing 5% $CO_2$ at 38.5$^{\circ}C$. Mter orcein staining at in vitro maturation condition, GV, GVBD, anaphase, telophase and M II were observed. Results are surumarized as follows: 1. Recovery rates were 55.8, 55.5 and 34.4% when the cumulus-compacted oocytes were collected with 18, 21, 26 gauge needles of syringes, respectively. 2. 79% of oocytes with compacted cumulus cells were at GV stage and most of the oocytes with partially denuded and denuded cumulus cells were from GVBD to M- II stages. 3. Percentage of mature oocytes among those which are follicular diameter of 1~2, 3~6 and over 6 mm was 42.6, 53.2 and 60.8%, respectively. 4. Percentage of mature oocytes among those which are compacted, partially denuded and denuded was 60.5, 46.2 and 35.4% respectively. 5. Percentage of mature oocytes in co-cultured with monolayers of cumulus cells was higher (57.1%) than that found with oocytes cultured alone (53.4%).

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.161-169
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• 1997

This study was conducted to compare the insemination time of bovine oocytes and determine the effects of glucose(1.5 mM) on the development of bovine embryos at early cleavage stage. Oocytes were matured for 24 h, followed by exposure to sperm and cultured in modified Tyrode's media drops or with bovine oviduct epithelial cell monolayer prepared in TCM199(BOECM). Insemination time and culture system were varied in each experiment. In experiment 1, to investigate the developmental capacity of bovine embryos after different time of exposure to sperm, bovine ova and sperm were co-incubated for 18, 30 or 54 h, respectively. The development to blastocysts of 30 and 54 h insemination groups were significantly higher(P<0.05) than 18 h group, and in case of blastocysts of cleaved embryos, 30 h group were significantly higher(P<0.05) than other groups. In experiment 2, we investigated the effect of glucose on early bovine embryos. After 18 h insemination, in vitro fertilized oocytes were separated following 3 groups ; G+0, C+24 and C+48. Oocytes of G+0 group were cultured in glucose added Tyrode's medium after fertilization, oocytes in C+24 and C+48 groups were cultured in glucose free Tyrode's medium after fertilization. After 24 h culture, G+24 group was moved to glucose added medium. All oocytes of 3 groups were moved to BOECM after 48 h culture. The rates of cleavage and development to blastocysts in G+0 group were significantly lower than other groups. In experiment 3, we determined the effects of glucose exposure from 8 to 20 h after insemination on the cleavage and development of oocytes. The oocytes in glucose added group had high capacity of cleavage and further development. This study shows that in bovine oocytes, the optimal exposure to sperm is 30 h and glucose exposure to bovine one-cell embryos is detrimental to their first cleavage and further development in vitro but there has no evidence of detrimental effect of glucose(1.5 mM) exposure to bovine embryos over the two-cell stage in vitro.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.171-179
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• 1997

The objectives of the present study were improvements in the efficiency of developmental rates to morula and blastocyst stages to produce a large number of genetically identical nuclear transplant embryos. The oocytes collected from slaughterhouse ovaries were matured for 24 h and then enucleated and cultured to allow cytoplasmic maturation and gain activation competence. And then the donor embryos were treated for 12 h with 10 $\pi$g /ml nocodazole and 7.5 $\pi$g /ml cytochalasin B to synchronize the cell cycle stage at 26 h after the onset of culture. The blastomeres were transferred into the perivitelline space of the enucleated nocytes and blastomeres and oocytes were fused by electrofusion. The cloned embryos were then cultured in various conditions to allow further development. The age of the recipient(30 vs 40 h) had no significant effect on the fusion rates(82.4 vs 82.1%) and the developmental rates to morula /blastocyst(9.8 vs 11.0%). Effect of Nocodazole treatment on the donor cell cyle synchronization to improve the developmental rates of bovine nuclear transplant embryos was significantly higher than control group(21.4 vs 10.1%, p<0.05). Significant differences were in the percentage of fusion rates(72.9,77.1vs 61.9%) in three types of fusion medium(PBS(+), mannitol and sucrose, p<0.01). The developmental rates of bovine nuclear transplant embryos appeared to be highest in mSOF medium under 5% 0$_2$ condition, but no significant differences were found when compared with TCM199-BOEC and mSOF under two different oxygen ratio(5 and 20%).

• Journal of Embryo Transfer
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• v.15 no.3
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• pp.219-230
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• 2000

This study was carried out to determine the factors on achieving good viability of embryos biopsied fur sexing, to investigate pregnancy rate following embryo transfer(ET) with sexed embryos, and to confirm the accuracy for the calves bort following ET with sexed embryos by polymerase chain reaction(PCR). To investigate viability of Hanwoo embryos after biopsy for sexing, fresh and frozen/thawed embryos were biopsied according to different developmental day of blastocysts, different stage of blastocysts, and different biopsy grade and the embryos themselves were incubated for 2 hours in TCM199 after microsection to be evaluated morphologically for recovery as blastocyst. The results obtained were as follows : 1. The rate of oocytes cleaved in vitro and the rate of blastocyst of the cleaved oocytes were 52.5% and 21.6%, respectively. The rate of blastocyst on day 8 was 11.2%, denoting the highest rate during whole culture period posterior to in vitro fertilization(IVF) 2. After biopsy for sexing, the viability rate of blastocyst on day 7, 8 and 9 was 75.0%, 88.4%, and 100.0%, respectively and the viability of early, mid, and expanded blastocyst after biopsy was 75.0%, 88.9%, and 91.1%, respectively The viability rate of fresh and frozen/thawed embryos was 89.9%, 71.4%, respectively. And the viability of expanded, hatching, and hatched blastocyst of frozen/thawed embryos was : 75.0%, 75.0%, and 50.0%, respectively. The viability of embryos according to biopsy grade of 10∼20%, 21∼30%, and 31∼40% was 85.7%, 91.5%, and 71.4%, respectively. 3. Pregnancy rate after transfer with biopsied embryo between flesh and frozen/thawed embryos was 22.6% and 20.0%, respectively. 4. In comparison between sex by PCR method and sex of calves born after embryo transfer, the accuracy of sex deterimination was 92.3% (12/13).

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.189-194
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• 1997

The ovaries of Korean native cows or heifers were obtained from a slaughter house and kept on 28~3O˚C and transported to laboratory within 2 hrs. The follicular oocytes were collected follicles. The oocytes were matured in vitro for 24 hrs. In TCM-199 supplemented with 35 $\pi$g /ml FSH, 10 $\pi$g /ml LH, 1 $\pi$g /ml estradiol-17 and granulosa cells at 39˚C under 5% $CO_2$ in air. The caudal epididymis of Korean native bulls were obtained from a slaughter house and transported to laboratory within 30 minutes. Swim-up of collected spermatozoa and freezing sperm was layered under 2ml fertilization B. 0. medium in two tissue culture tubes and held at a 45˚C angle for 0~2 hrs. They wrer fertilized in vitro by freezing sperm treated with heparin for 24 hrs, and then the zygotes were co-cultured in vitro with bovine oviductal epithelial cells for 7 to 9 days. The follicular oocytes recovered were classified into 41.7% as grade I, 51.5% as grade II and 6.8% as graed III. The number of oocytes recovered per ovary was averaged 8.3 and they were classifed into 2.3 as grade I, 2.5 as grade II and 2.3 as grade III. The cleavage rate of matured oocytes was significantly(P

• Journal of Embryo Transfer
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• v.15 no.3
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• pp.271-277
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• 2000

This study was conducted to establish the optimal culture conditions for in vitro production of bovine embryos derived from slaughter house ovaries. Cumulus-oocyte- complexes (COCs) collected by aspiration from follicles of 2~7 mm in diameter were matured in Ham's F-10 medium supplemented with 0.01 $\mu\textrm{g}$/m1 epidermal growth factor (EGF) at 39$^{\circ}C$ in a humidified atmosphere of 5% $CO_2$in air. After 24 hrs of culture, the oocytes were co-cultured with epididymal sperm selected off by Percoll-density gradient in TALP medium for 24 hrs. The presumptive zygotes were cultured in HECM-6 medium for 3 d post-insemination, and followed by cultured in TCM199 medium until 7 to 10d post-insemination. The cultures were compared of their cleavage and development into later stage in culture medium by additions of different protein sources (PVA, BSA and BCS) and by different embryo density. The rates of cleavage and development rates into blastocyst were not significantly (P<0.05) different among the culture media containing with BSA (75.0% and 40.5%), BCS (76.7% and 38.0%) and PVA (72.5% and 42.2%), respectively. Significantly (P<0.05) higher blastocysts rates were obtained in culturing of 30 and 40 embryos in each 50$\mu$l droplets of culture medium than in 5, 10 and 20 embryos. These results indicate that the optimal density of embryos is 30~40 embryos in a 50$\mu$l droplet of culture medium. Furthermore there is no effect of different protein sources on early embryonic development.