• Journal of Embryo Transfer
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• v.10 no.1
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• pp.73-82
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• 1995

large scale production of cloned embryos requires the technology of multiple generation nuclear transplantation(NT) using NT embryos as the subsequent donor nuclei. The purposes of this study were producing the second generation cloned rabbit embryos, and also to determine the electrofusion rate and in vitro developmental potential comparatively in the cloned embryos of the first and second NT generation. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FCS) at 47 hours after hCG injection In the first generation NT, the nuclear donor embryos were synchronized in the phase of Gi /S transition of 32-cell stage. The first generation NT embryos which were developed to 8-cell were synchronized in Gi /S transition phase of the following 16-cell stage and used as donor nuclei for second generation Synchronization of the cell cycle of blastomeres was induced, first, using an inhibitor of microtuble polymerization, colcemid for 10 hours to arrest blastomeres in M phase, and secondly, using a DNA synthesis inhibitor, aphidicolin for 1.5 to 2 hours to arrest them in Gi /S transition boundary. The recipient cytoplasms were obtained by removing the nucleus and the first polar body from the oocytes collected at 14 hours after hCG injection. The separated donor blastomeres were injected into the enucleated recipient oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 $\mu$sec at 1.25 kV /cm in 0.28 M rnannitol solution The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. Following in vitro culture of the first and second generation cloned embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The results obtained were summarized as follows: 1. The electrofusion rate was found to be similar as 79.4 and 91.5% in the first and second generation NT rabbit embryos, respectively. 2. The in vitro developmental potential to blastocyst stage of the second generation NT embryos (23.3%) was found significantly(p<0.05) lower, compared with that of the first generation NT embryos (56.8%). 3. The mean blastomeres counts of embryos developed to blastosyst stage following in vitro culture for 120 hours and also their daily cell cycles during the culture period were decreased significantly (p<0.05) to 104.3 cells and 1.33 cylces in the second NT generation, compoared with 210.4 cells and 1.54 cycles in the first NT generation, respectively.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.9
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• pp.1354-1360
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• 2007

The aim of this study was to evaluate if oocytes, aspirated from postovulatory ovarian follicles of superovulated rabbits 14 h post-hCG administration, could be efficiently used as ooplasm recipients for somatic cell nuclear transfer (SCNT). Within a common SCNT protocol, a comparison between oocytes recovered by direct aspiration (aspirated) from available ovarian follicles and oocytes flushed out from oviducts (flushed) was carried out. The results showed that maturation and enucleation rates of aspirated oocytes were 70.7% and 69.2%, significantly lower than 95.3% (p<0.01) and 83.6% (p<0.05), respectively, from flushed oocytes. However, following enucleation of matured oocytes as ooplasm recipients for SCNT, no difference was recorded in fusion and cleavage rates, as well as blastocyst development from cleaved embryos or hatching of blastocysts between aspirated and flushed groups. Additionally, some matured aspirated and flushed oocytes were also used for immediate parthenogenetic activation and the resulting embryo development was not significantly different. Results from this study show the following: i) the majority of oocytes aspirated from postovulatory ovarian follicles of superovulated rabbits 14 h post-hCG administration are matured and can be used directly as ooplasm recipients for SCNT; ii) the reconstructed embryos derived from these oocytes have similar in vitro developmental ability to those flushed from the oviducts.

• 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.

• Korean Journal of Animal Reproduction
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• v.14 no.1
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• pp.73-83
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• 1990

These studies were carried out to find the proper conditions for in vitro maturation and fertilization of bovine follicular oocytes and culture methods capable of further developing early embryos. For these objectives, the cleavage rate of oocytes matured and fertilized in vitro was investigated under medium supplemented with hormones and estrous cow serum and season of oocytes collection as well as different cumulus cell stage before insemination. Finally, 2~8 cell embryos were cultured in in vitro and in vitro culture system to investigate developmental capacity into morula. 1. Cleavage rate of oocytes matured in vitro was 27%(20/73) for A(LH＋FSH＋estradil-17$\beta$＋10% FBS), 38%(27/71) for B(LH＋10% ECS) and 27%(15/56) for C(10% ECS), respectively. Supplement B showed more higher rate and 4~8 cell embryos were also obtained much more in this group(67%, 18/27). In vitro maturation rate of follicular oocytes cultured in TCM 199 supplemented withLH and 10% ECS was 88%(75/85). 2. Cleavage rate(15%, 10/65) of oocytes collected in summer was significantly lower than in fall(47%, 42/89). 3. Cleavage rate(15%, 10/65) of oocytes collected in summer was significantly lower than in fall(47%, 42/89). 3. Cleavage rate(15%, 10/65) of oocytes with partially removed cumulus cell before insemination was more higher than that(44%, 27/62) of oocytes with intact cumulus cell. 4. The frequency of development from early cleaved embryos into morula was 6%(4/65), 12%(4/33) for co-culture of cumulus cell monolayer and bovine oviduct epithelial cell monolayer, respectively and 25%(6/25) in ligated rabbit oviduct.

• Journal of Embryo Transfer
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• v.9 no.2
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• pp.161-166
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• 1994

This experiment was carried out to produce cloned aniraals by nuclear transplantation in rabbits. The ovulated oocytes were collected from the oviducts between 14 and 15 hours after hGG injection. The denuded oocytes were used as nuclear recipient cytoplasm following enucleation by micromanipulation. The blastomeres separated from the 8-cell embryos were used as nuclear donor. The nucleated oocytes receiving a blastomere in the perivitelline space were electrically fused in the 0.28 M mannitol solution at 1.5 kV /cm, 60$\mu$sec for three times. The nuclear transplant embryos which were used and developed to 2- to 4-cell stage in vitro were transferred into the oviducts of synchronized recipient does. A total of 64 nuclear transplant embryos were transferred to 7 recipient does and produced three offspring(4.7%) from a foster mother 31 days after embryo transfer.

• Journal of Embryo Transfer
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• v.4 no.1
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• pp.21-27
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• 1989

Hydrogel chambers made from polymerized 2-hydroxyethyl methacrylate were used for in chamber fertilization. To determine whether sperm motility was preserved in the Hydrogel chamber, chambers which have rabbit sperm or frozen-thawed bovine sperm were transplanted inside of mouse peritoneal cavity and sperm were observed after recovering the chambers in the due time. As a result, it was determined that preservation of sperm motility was good. To determine whether in chamber fertilization was possible, chambers which have rabbit oocytes and sperm were transplanted inside of mouse peritoneal cavity and eggs were observed after recovering the chambers at 84 hr of preservation. As a result, the fact that fertilization and culture was occurred inside of the chamber was determined.

• Journal of Embryo Transfer
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• v.13 no.3
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• pp.219-226
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• 1998

Enucleation of oocytes is an important limiting step for embryo cloning. We propose an enucleation technique based on the removal of chromatin after oocyte activation by aspirating the second polar body containing complemented chromatin. In a preliminary experiment to determine an optimal age of oocytes enucleation in rabbits, oocytes were enucleated at 15~20 hours post hCG. Recently ovulated oocytes were enucleated at a higher rate than aged oocytes. Microsurgical removal of the complemented chromatin in the second polar body was significantly more effective in enucleating than aspiration of a larger cytoplasm volume surrounding the first polar body of metaphase-arrested oocytes(96.8% versus 70.4%; P〈0.05). Moreover, compared with a nuclear transplantation protocol based on enucleation of metaphase-arrested oocytes and preactivated oocytes followed by treatment with 5 $\mu$M ionomycin for 5 min and 2 mM DMAP for 1 hr, there was no significant difference in the rate of blastocyst development. The ease with which modified technique can be performed is likely to render this technique widely useful for research and practice on mammalian cloning.

• Clinical and Experimental Reproductive Medicine
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• v.1 no.1
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• pp.49-54
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• 1974

In the present studies, effect of progesterone on the germinal vesicle break-down of the mouse oocytes cultured in the micro tube was investigated. The results obtained are as follows: As dose of progesterone in the medium rose, accordingly the break-down of the germinal vesicle was suppressed. It was found that $ED_{50}$ was 15.7 ${\mu}g$/ml, and $ED_{90}$ 60.7 ${\mu}g$/ml of progesterone. The dose suppressing the oocyte maturation was apparently higher than that on the rabbit or on the mouse embryonal development. The inhibiting effect of progesterone on the GVBD was reversible. The germinal vesicle of the oocytes were broken down immediately in the medium upon removal of the hormone. Progesterone stops meiosis at any stage upon administration, while dbe AMP or theophylline supresses only the break-down of the nuclear membrane. Recovering of the meiotic division of the oocytes once exposed to progesterone was delayed a little. The inhibiting action of progesterone was not altered by adding more pyruvate or in the presence of higher concentration of the mineral ions in the culture medium.

• Korean Journal of Animal Reproduction
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• v.10 no.2
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• pp.211-217
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• 1986

This experiment was conducted to determine the effect of FSH on in vitro maturation and in vitro fertilizing ability of oocytes recovered from normal follicles of different sizes in superovulated rabbits. Follicular oocytes recovered were cultured in modified Ham's F12 medium containing 0, 0.1, 1.0 and 10$\mu\textrm{g}$ FSH/ml for 18 hours and investigated the degree of cumulus cells expansion and nuclear maturation, which were fertilized with in vivo capacitated rabbit sperm. 1. The number of normal follicles<1.5mm, 1.6 to 2.5mm and> 2.5mm in diameter at 16 to 18hrs after HCG administration was 4.8 (38.8%), 5.5(45.4%) and 3.3(15.8%), respectively. Average percent of oocytes recovered was 69.7% and larger follicles tended to have a higher percent, recovery rate than smaller follicles. 2. The degree of cumulus expansion in medium containing 0.1$\mu\textrm{g}$ FSH/ml was similar to that of control, but markedly decreased under the level of above 1$\mu\textrm{g}$ FSH/ml. The proportions of oocytes which reached the second meiotic metaphase were 57.1, 61.5, 43.8 and 45.0% in medium containing 0, 0.1, 1.0 and 10$\mu\textrm{g}$ FSH/ml, respectively. Oocytes from larger follicles showed a higher nuclear maturation than that from smaller follicles. 3. In vitro fertilization rate of oocytes matured under 1$\mu\textrm{g}$ FSH/ml was slightly, not significant, higher than that of others. 4. Progesterone level in follicular fluid was about 67 to 71ng/ml with no difference in follicular sizes and estradiol-17$\beta$ level was under 25pg/ml.

• Journal of Embryo Transfer
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• v.12 no.1
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• pp.11-20
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• 1997

The influence of cryopreservation of donor embryos on the in vitro developmental potential in the nuclear transplant rabbit embryos was evaluated. The embryos of 16-cell stage were collected and cryopreserved with EFS solution by vitrification method. The frozen embryos were thawed and synchronized to S and G$_1$ phase of 32-cell stage. The recipient/ cytoplasms were obtained by removing the first polar body and chromosome mass from the oocytes collected by non-disruptive microsurgery procedure. The separated S and G$_1$ phase blastomeres of 32-cell stage were injected into enucleated recipient cytoplasms by micromanipulation. After culture until 20 hrs post-hCG injection, the nuclear transplant oocytes were electrofused and activated by electrical stimulation. The fused nuclear transplant embryos were co-cultured with rabbit oviduct epithelial cells. After in vitro culture for 120 hrs, the nuclear transplant embryos developed to blastocyst stage were stained with Hoechst 33342 dye and their blastomeres were counted. The electrofusion rate was significantly (P<0.05) reduced in the frozen nuclear donor,compared with fresh donor nuclei as 80.0 vs 62.8% in S phase and 81.7 vs 64.8% in G$_1$phase, respectivley. The in vitro developmental rate to blastocyst stage with the S and G$_1$phase of fresh embryos(26.3 and 61.1%, respectively) was found significantly (P<0.05) higher, compared to the S and G]phase of frozen embryos(11.9 and 34.6%, respectively). When frozen as well as fresh donor embryos were synchronized to G$_1$ phase, the in vitro developmental rate to blastocyst stage was significantly (P<0.05) higher, compared with S phase donor nuclei. The cell counts of nuclear transplant embryos developed to blastosyst stage were significantly (P<0.05) more in G$_1$ phase of fresh or frozen embryos (180.1 and 125.7 cells, respectively), compared with S phase nuclear donor (145.1 and 103.7 cells, respectively). From the above results it was concluded that the rabbit embryos cryo- preserved by vitrification might be available as nuclear donor, though the developmentalpotential and cell counts of nuclear transplant rabbit embryos were decreased significantly.