• Journal of Embryo Transfer
• /
• v.10 no.1
• /
• pp.45-51
• /
• 1995

In order to determine the optimum condition and timing for in vitro maturation of oocytes to metaphase of meiosis II (M II), the immatured follicular oocytes were recovered by puncturing the large(1.0~1.5 mm in diameter) and small(<1.0 mm in diameter) follicles in the ovaries of rabbits treated intramuscularly with a single dose of 100 TU PMSG 68 hours previously. The follicular oocytes were classified into three grades by the attachment of cumulus cells. The Grade I and II follicular oocytes from large follicles were cultured in BO-DM medium with 10% FCS, 35 $\mu$g /nl of FSH, 10 $\mu$g /ml of LH and 1 $\mu$g /ml of estradiol-17$\beta$ at 39t in a 5% $CO_2$ incubator for 11 to 23 hours. In 3 hours interval during the culture period, the oocytes were harvested and their cumulus cells were removed with hyaluronidase. The denuded oocytes were stained with Hoechst 33342 dye and their meiotic status and extrusion of the first polar body (PB) were examined under a fluorescence microscope. Also the fragmentation of the first PB and the distance between the first PB and nucleus were examined. The results obtained were as follows: 1. The mean recovery rate of follicular oocytes from the large and small follicles was 59. 9 and 31.3%, respectively. The mean number of oocytes recovered per rabbit and the Grade I percentage were 14.6 and 94.4% in large follicles, but 2.1 and 61.1% in small follicles, respectively. All the parameters examined were different significantly (p<0.05) between both the folliclular size. 2. Most of the follicular oocytes(86.8%) were matured in vitro to M II phase in 14 hours in Grade I oocytes, but the significantly(p<0.05) less oocytes(45.5%) were matured in Grade II oocytes. 3. The first PB was extruded in most of the oocytes(94.7%) in 14 hours of culture with the fragmentation rate of 29.6%, but the fragmentation rate of the first PB increased significantly (p<0.05) as the culture period for maturation was longer to 20 hours(63.5%). 4. The distance between the first PB and nucleus was increased linearly (p<0.05) as the maturation time passed from 14(7.1$\mu$rn) to 23 hours(58.4$\mu$m). 5. From the above results it was concluded that the optimum time for in vitro maturation culture might be 14 hours in the follicular oocytes from rabbit primed with PMSG for 68 hours, expecially when these follicular oocytes were used for recipient cytoplasms in embryo cloning.

• Journal of Embryo Transfer
• /
• v.10 no.1
• /
• pp.65-72
• /
• 1995

The purposes of this study were to produce cloned rabbit embryos and offsprings by nuclear transplantation(NT) using in vitro matured oocytes as nuclear recipient cytoplasm and to determine the effect of frozen nuclei donor embryos on the production efficiency of cloned embryos. The 8cell embryos were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline containing 10% fetal calf serum(FCS) at 40 hours after hGG injection. A portion of collected embryos were preserved at 4$^{\circ}C$ for 24 hours and a portion of them were frozen by vitrification method. The embryos used for donor nuclei were synchronized in the phase of Gi /S transition. The in vitro matured oocytes were used as recipient cytoplasm following removing the nucleus and the first polar body. The synchronized blastomeres from fresh, cooled or frozen embryos were injected into the enucleated oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 $\mu$sec at 1.0 W /cm in 0.28 M mannitol 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 NT embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The results obtained were summarized as follows: 1. The fusion rates of the blastomeres from fresh, cooled and frozen embryos with the in vitro matured and enucleated oocytes were 100, 95.8 and 64, 3%, respectively. 2. Development in vitro to blastocyst was significantly(p<0.05) different between the cloned embryos with the blastomeres from fresh, cooled or frozen embryos as 39.0, 20. 9 and 15.7%, respectively. 3. The mean numbers of cell cycle per day during in vitro culture of cloned embryos blastomeres from fresh, cooled or frozen embryos was 1.31, 1.29 and 1.16, respectively. 4. A total of 77 nuclear transplant embryos were transferred into 6 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer.

• Journal of Veterinary Clinics
• /
• v.12 no.1
• /
• pp.877-886
• /
• 1995

The recycling nuclear transplantation(NT) technique has the powerful potential of producing a large number of genetically identical embryos and offsprings from one embryo. Multiple generational cloning by this technique utilizes the NT embryo itself as the donor for the next generation of cloning. In this experiment, we have produced the third generational cloned embryos by recycling NT. Further we examined comparatively the electrofusion rate and in vitro developmental potential in the cloned embryos of the first second and third generations. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulberco's phosphate buffered saline 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 Gl/S transition of 32-cell stage. The first and second generation NT embryos developed to 16-cell were used as donor nuclei for second and third generation. The recipient cytoplasms were utilized the oocytes collected at 14 hours after hCG injection, following revoming the nucleus and the first polar body by micromanipulation. The separated blastomeres were injected into the enucleated recipient oocytes by micromanipulation and were fused by electrical stimulation. The electrofusion rate was seen to be 78.0, 88.0 and 90.3 % in the first second and third generation NT rabbit embryos, respectively. 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. The in vitro developmental potential to blastocyst stage was significantly(P<0.05) decreased in the third(7.2 %) generation NT embryos compared to the first(53.1 %) and second(16.1 %) generation NT embryos. Following in vitro development to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The mean blastomere numbers and cell cycle numbers of NT embryos during the culture period were significantly(p<0.05) decreased in the second(93.9 cells and 6.55 cylces) and third(81.5 cells and 1.35 cylces) generation, compared to the first(189.9 cells and 7.55 cylces) generation.

• Journal of Veterinary Clinics
• /
• v.13 no.2
• /
• pp.149-152
• /
• 1996

This study was carried out to evaluate the efficiency of production of cloned embryos by nuclear translatation (NT) when using 4-cell to compact morula stage embryos as nuclear donor. In micromanitulation and electrofusion of blastomeres from 4-cell to morula stage embryos, the successful injection rate was higher with late stage blastomeres, on the contrary the fusion rate was lower. The in vitro developmental rate of NT embryos was not significantly different between cell-stages of donor blastomeres. Although the overall rate of production of cloned embryos with 4-cell. 8-cell, early and late morula stage embryos was 14.0, 18.0, 15.3 and 14.1%, respectively, the mean number of blastocysts produced with a donor embryo was the most (4.51) with the compact morulae. Therefore, it can be suggested that the embryos at thelate stage is more beneficial for the mulciple production of cloned embryos, If the late stage blastomeres have maintained their totipotency to produce intact offspring.

• Proceedings of the Korean Society of Embryo Transfer Conference
• /
• 2002.06a
• /
• pp.19-25
• /
• 2002

Researches on manipulation pluripotent stem cells derived from blastocysts or promordial germ cells (PGCs) have a great advantages for developing innovative technologies in various fields of life science including medicine, pharmaceutics, and biotechnology. Since the first isolation in the mouse embryos, stem cells or stem cell-like colonies have been continuously established in the mouse of different strains, cattle, pig, rabbit, and human. In the animal species, stem cell biology is important for developing transgenic technology including disease model animal and bioreactor production. ES cell can be isolated from the inner cell mass of blastocysts by either mechanical operation or immunosurgery. So, mass production of blastocyst is a prerequisite factor for successful undertaking ES cell manipulation. In the case of animal ES cell research, various protocol of gamete biotechnology can be applied for improving the efficiency of stem cell research. Somatic cell nuclear transfer technique can be applied to researches on animal ES cells, since it is powerful tool for producing clone embryos containing genes of interest. In this presentation, a brief review was made for explaining how somatic cell nuclear transfer technology could contribute to improving stem cell manipulation technology.

• Proceedings of the Korean Society of Embryo Transfer Conference
• /
• 2001.10a
• /
• pp.25-34
• /
• 2001

The Committee has, for the 9th consecutive year, compiled statistics of embryos collected and transferred worldwide. Geographically, all regions have participated in the survey; however, there are still some places where it has been impossible to retrieve such data (particularly in Asia). The statistics, therefore, are partially underestimated. By contrast, in other areas, the system has proven to be more efficient than before, particularly in North and South America. This has resulted in the present report, which gives a somewhat more satisfactory picture of the current situation of the ET industry. For the second year, it has also been possible to collect data for several species other than cattle. In cattle, the number of in vivo-derived embryos collected and transferred has once again increased with more than half a million embryos trans-ferred in 1999(520,712), a new record. The number of bovine in vitro-produced embryos has remained stable this year as compared with the previous year(approximately 30,000 embryos transferred). However, there are still teams that have not Yet reported their data. More than 10,000 embryos in each of the ovine and caprine species and close to 2,000 cervid embryos were reported transferred in 1999. Some 500 embryos of the equine species and a few thousand in the porcine and rabbit species have also been transferred in 1999. It is concluded that the ET industry continues to be very active, and, in many species, it encompasses a larger segment of the livestock population overall, which is to the farmer s benefit.

• Journal of Embryo Transfer
• /
• v.12 no.3
• /
• pp.253-258
• /
• 1997

토끼에서는 수정란 이식과 같은 기본적인 번식공학적 방법의 효율성이 아직 생쥐와 같은 실험동물에 비해 떨어지고 있어 생물공학적인 기술을 응용하는데 큰 어려움이 있다. 특히 유전자 이식에 의한 형질전환 토끼의 생산과 같은 생물공학적인 기술을 실용화하는데 효율이 높은 수정란이식 기술의 개발이 필수적이라고 할 수 있다. 본 연구에서는 토끼에서 수정란 이식 기술의 첫 단계인 과배란 유도를 효율적으로 이용할 수 있는 방법을 정립하기 위해 반복적인 과배란 유도가 배란율 및 수정란의 질적인 면과 양적인 면에 미치는 영향을 조사하였다. 연구방법으로는 FSH와 HCG를 사용하여 과배란을 유도하였고 2.5 개월의 반복처리간격으로 3번의 반복적인 과배란 처리를 한 후 반복처리에 따른 배란율과 배란된 난자의 형태학적 상태, 배양에 의한 발생 능력 상태 등을 감소하였으며 배란수의 변이도 커지는 경향을 나타내었다(첫번째, 32.6(+-)2.5; 두번째 28.7(+-)3.7; 세번째 20.99(+-)3.8). 제 2극체의 돌출, 전핵의 형성, cummulus cell의 존재등에 의한 회수된 난자의 형태학적 관찰에 의한 방법으로 난자를 분류한 결과 과배란의 반복수가 증가함에 따라 다양한 모양의 난자가 회수되어 배란이 광범위한 시간대에 일어나고 있음을 나타내었다. 또한 과배란의 반복적인 유도에 의해 난소의 혈포수는 증가하였으나 채란된 난자의 채외배양에 의한 발육율에는 차이가 없었다. 그러므로 과배란의 반복적인 유도는 공란토의 난소반응에는 영향을 미쳤으나 난자의 질에는 영향을 미치지 않았음을 나타낸다.

• Reproductive and Developmental Biology
• /
• v.28 no.3
• /
• pp.141-145
• /
• 2004

Mucin coat is deposited on the embryos during passage through the oviduct in rabbit. When in vitro cultured blastocysts were transferred to the recipients, the lack of mucin coat might account in part for failure of pregnancy after transfer. The present study were carried out to investigate whether deposition of mucin coat were induced when in vitro cultured blastocysts were transferred to recipients. At 19 ～20 hours post-coitus one-cell embryos were collected by flushing oviducts. These embryos cultured for 72 hours were reached to blastocyst stage. And these blastocysts were transferred to the oviduct of asynchronized (one day later than the donors) and synchronized recipient. To confirm deposition of the mucin coat, blastocysts transferred to the oviduct were recovered at 24 and 48 hours after the transfer. Fifty eight percent of blastocysts recovered from uterus of asynchronous recipient at 24 hours after transfer and 92.9% of blastocysts recovered from uterus of synchronous recipient were 0～10 ㎛ of mucin coat thickness. And 11.8% of blastocysts of asynchronized recipients and 7.1% of blastocysts from asynchronized recipients were in 11～20 ㎛ of mucin coat thickness. When blastocysts were recovered from uterus at 48 hours after transfer, 87.0% of blastocysts from asynchronized recipients and 5.9% of blastocyst from synchronized recipients were in 0～10 ㎛ of mucin coat thickness. And 76.5% of blastocysts of synchronized recipients and 4.4% of blastocysts from asynchronized recipients were in 11～20 ㎛ of mucin coat thickness. From these results it is speculated that the low implantation rate of in vitro cultured rabbit blastocysts transferred to oviduct of recipient was caused by high degeneration of the embryo after transfer and inappropriate deposition of mucin coat.

• Asian-Australasian Journal of Animal Sciences
• /
• v.16 no.1
• /
• pp.15-22
• /
• 2003

Experiments were conducted to evaluate the effect of blastomere diameters and cell cycle stages on the subsequent development of nuclear transplant rabbit embryos (NT-embryos) using nuclei derived from the 16- or 32-cell stage embryos. All blastomeres and NT-embryos were cultured individually in modified Ham's F-10 medium supplemented with 10% rabbit serum (RS) at $38^{\circ}C$ and 5% $CO_2$ in air. The diameter of blastomeres from 16-cell stage embryos was found twice of those from 32-cell stage (51 vs 27 ${\mu}m$). Significant differences were observed in cleavage rates ($\geq$3 divisions) in the isolated single blastomeres (54 vs 48 for 16-cell; 28 vs 14 for 32-cell, p<0.05), but the fusion rates of oocytes with transferred nuclei were similar between small and large single blastomeres derived from either 16-cell or 32-cell stage embryos. When 16-cell stage blastomeres were used as nuclear donors, cleavage rates ($\geq$3 divisions) of the NT-embryos were greater in the small nuclear donors than in the large donors (73 vs 55%, p<0.05). On the contrary, significantly higher cleavage (43 vs 6%, p<0.05) and developmental rates (14 vs 0%, p<0.05) were observed in the large blastomere nuclear donor group of the 32-cell stage embryos. When the cell cycle stages were controlled by a microtubule polymerization inhibitor (Demicolcine, DEM) or the combined treatment of DEM and Aphidicolin (APH), a DNA polymerase inhibitor, fusion rates were 88-96% for the 16-cell donor group (without DEM treatment), which were greater than the 32-cell donor group (54-58%). Cleavage rates were also greater in the transplants derived from G1 nuclear donor group (93-95%) than those from the DEM and APH combined treatment (73%) for the 16-cell donor group (p<0.05). No significant difference was detected in the morula/blastocyst rates in either donor cell stage (p>0.05). In conclusion, it appeared that no difference in the developmental competence between large and small isolated blastomeres was observed. When smaller 16-cell stage blastomeres were used as nuclear donor, the cleavage rate or development of NT-embryos was improved and was compromised when 32-cell stage blastomeres were used. Therefore, control nuclear stage of the donor cell at $G_1$ phase in preactivated nuclear recipients seemed to be beneficial for the cleavage rate of the reconstructed embryo in the 16-cell transplant, but not for subsequent morula or blastocyst development.

• The Korean Journal of Zoology
• /
• v.32 no.4
• /
• pp.314-321
• /
• 1989

배의 발생과 분화에 따른 혈청 alpha-fetoprotein (AFP)의 생리적 기능 규명을 위한 기초실험의 일환으로, 모체와는 완전히 독립된 상태에서 발생과 분화가 이루어지는 계배의 혈청을 재료로 하여 CM-Sephadex C-50, hydroxyapatite 및 DEAE-Sepharose A-25등의 크로마토그라피 방법에 의해 닭 혈청 AFP를 분리하였다. 계 AFP는 분자량이 약 60 Kd로 사람 및 다른 포유동물 AFP의 분자량 (64-74 Kd)보다 작았다. 닭 AFP, 계배혈청 및 닭 혈청 등을 토끼에 면역하여 항체를 제조하였고, 이를 이용하여 닭의 배발생 단계에 따른 혈청 AFP 함량의 변화를 전기영동 및 면역학적 방법으로 정성 및 정량 분석을 실시하였다. 혈청 AFP 함량은 발생 7일-배 (AFP 농도 : 0.81 mg/ml)부터 점차 증가하여 13일-배에서 최고치인 2.46 mgfml로 나타났으며 그 이후 급격히 감소되어 부화 직후에는 매우 낮은 농도 (0.22mg/ml)였고 부화 4일에 거의 없어 졌으며, 이와 같은 AFP 함량의 변화는 혈청 알부민 함량의 변화와 거의 반비례 함을 알았다. For the preliminary step to investigate the site of AFP synthesis, the ontogenic characteristics and the physiological function of AFP, alpha-fetoprotein was isolated from chick embryo serum through the procedures of CM-Sephadex C-50, hvdroxvapatite and DEAE-Sephadex A-50 chro-matography. The isolated AFP was proved to be pure and its molecular weight was found to be about 60 Kd. With this purified AFP, rabbit anti-chick hor was produced. The serum AFP level in chick embryo has been investagated by using polyacrylamide gel electrophoresis, immunoelec-trophoresis and single radial immunodiffusion from 7 days of incubation until 4 days after hatch-ing. It was found that the AFP level is increased from 0.81 mg/ml at daw-7 embryo to maximum value of 2.46 mg/ml at day-13 embryo, followed by rapid decreases until hatching.