• 한국가축번식학회지
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• 제16권1호
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• pp.55-62
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• 1992

Bovine oocytes obtained from ovarian(2 to 5mm in diameter) of slaughtered cows were cultured in TCM 199 with 10~20% estrous-cow-serum(ECS) for 24~25hr at 39$^{\circ}C$ in 5% CO2-95% air. After culture, some oocytes were examined their maturation. The remainder were used to assess the fertilizability with frozen-thawed spermatozoa in a medium containing caffeine and heparin, and subsequent development in media with bovien cumulus cells(BCC) or bovine oviduct epithelial cells(BOEC). The results obtained were summarized as follows ; 1. The maturation rate of the oocytes in TCM199 with 15% ECS group(76.5%) was higher than that of 10% ECS(69.2%) or 20% ECS group(64.8%). 2. The proportions of the oocytes penetrated and the pronuclear oocytes in the presence of caffeine and heparin were 72.1%(62/86) and 93.5%(58/62), respectively. The rate of polyspermy in the fertilized oocytes was 8.1%. 3. When 73 oocytes recovered from fertilization drop were cultured in TC-199 medium with 10% fetal calf serum(FCS), 41 oocytes(56%) cleaved to 2-cell and further stages of embryos. Among these only one embryo developed upto morula stage. 4. The rate of the cleaved oocytes was higher in medium with BCC(80%:59/74) than BOEC(76%:58/76). However, the rate of developed morulae and blastocysts was higher in the medium with BOEC(40%;23/58) than with BCC(34;20/59).

• 한국수정란이식학회지
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• 제12권1호
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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.

• 한국가축번식학회지
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• 제21권4호
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• pp.345-353
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• 1997

Antioxidants and antioxidants with somatic cell co-culture, bovine oviduct epithelial cells(BOEC) and buffalo rat liver cells(BRLC), were studied as a mean of increasing the development and intracellular glutathione(GSH) concnetrations of bovine embryos derived from in vitro matured(IVM) and in vitro fertilized(IVF) oocytes. Cell numbers and intracellular GSH concentrations of blastocysts were also counted. The developmental rate beyond morula stages in CRlaa containing taurine(2.5mM), superoxide dismutase(SOD, 600U) and catalase(250U) were 1%, 75.0%, 64.8% and 62.3%, respectively. The developmental rate in antioxidant groups was significantly higher than in control(P<0.05). The intracellular GSH concentrations of blastocysts cultured in 0, 2.5mM taurine, 600U SOD and 250U catalase were 33.8pM, 39.3pM, 42.3pM and 54.8pM, respectively. This result indicated that the developmental rates and intracellular GSH concentrations of catalase group was significantly higher than any other groups(P<0.05). The developmental capacity in CRlaa plus various antioxidants co-cultured with BOEC were 55.3%(control), 74.1%(2.5mM taurine), 66.7%(600U SOD) and 70.7%(250U catalase) and in CRlaa plus various antioxidants co-cultured with BRLC in control, 2.5mM taurine, 600U SOD and 250U catalase were 63.8%, 75.5%, 71.0% and 73.5%, respectveily, the intracellular GSH concentrations of blastocyst embryos co-cultured with BOEC and BRLC in CRlaa with 0.25mM taurine, 600U SOD and 250U catalase were 73.4pM and 64.4pM, 79.9pM and 67.5pM, 82.3pM and 71.7pM, and 83.0pM and 80.0pM, respectively. Cell numbers of blastocysts were not difference in all experimental groups. These studies indicate that andtioxidants and antioxidant with somatic cell co-culture can increase the proportion of embryo that developed into morula and blastocysts, and the intracellular GSH concentrations of blastocyst embryos.

• 대한수의학회지
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• 제36권4호
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• pp.919-927
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• 1996

In the last few years, methods for in vitro culture of early embryo stages from oocytes matured and fertilized in vitro using suitable cell culture systems have been established. But the factors affecting pregnancy rates following transfer of bovine embryos produced in vitro were not evaluated enough. So this study was performed to investigate the effects of quality and stage of embryos, parity and Corpus Luteum quality of recipients on pregnancy rates following non-surgical transfer of bovine embryos produced in vitro. Oocytes aspirated from small antral follicles of ovaries obtained at a local slaughter house were matured, fertilized with frozen-thawed semen and co-cultured for 6-7 days by utilizing co-culture system with bovine oviduct epithelial cell in vitro. After co-culture, embryos were transfered to recipients on day 7 (estrus=day 0). Recipients were monitored by ultrasonic scanning method or observation for estrus and rectal palpation after 50 days from transfer. The results of this study are follows. 1. Of the 70 recipients, 70%(49 of 70) had not showed estrus sign between day 0 and day 50, but 22.9%(16 of 70) was diagnosed not pregnant. Therefore the overall pregnancy rate of this study was 47.1%(33 of 70). 2. The pregnancy rate of recipients transfered with excellent(66.7%) and good(54.5%) embryos were higher than that of recipients transfered with fair embryos(15.8%) (p<0.05). 3. The pregnancy rate of recipients transfered with morula, compacted morula, blastocyst and expanded blastocysts were 46.2, 55.0, 62.5 and 50.0%, respectively. 4. The pregnancy rates of recipients transfered to heifer and cow were 54.5 and 55.2%, respectively. 5. The pregnancy rates of recipients with CL score I, II(66.7, 63.6%) were higher than those of recipients with CL score III (10%), (p<0.05). Success of transfer of embryos produced in vitro depends on many variables. The important factors identified in this study were the quality of embryos and the CL score of recipient animals after non-surgical transfer of embryos matured, fertilized and cultured in vitro.

• 한국가축번식학회지
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• 제24권4호
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• pp.429-437
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• 2000

본 연구는 retrovirus vector system 에 의해서 EPO와 EGFP 유전자가 전이된 소 태아세포를 이용하여 핵치환된 소 난자에서의 이들 유전자의 성공적인 도입을 조사하였다. Non-starved 소 태아세포는 탈핵된 소 난자의 위란강내로 주입되었다. 소 태아 세포와 난자는 세포간 전기자극에 의해 융합시켰으며, 이후 calcium ionophore와 6-dimethylaminopurine를 이용하여 난활성을 유도하였다. 핵치환에 의해 재구성된 난자는 8일 동안 CRlaa 배양액에서 소 난관상피세포와 함께 공배양하였다. 핵치환에 의해 재구성된 187개와 210(EPO, EGFP)개의 소 난자 중에서, 149개와 158(EPO : 80.0%, EGFP : 75.2%)개의 난자가 분할되었고, 이들 분할된 난자 중 36개와 35(EPO : 24.2%, EGFP : 22.2%)개의 난자가 배반포까지 발달하였다. 이들 배반포에서, EPO 유전자는 PCR에 의해 36개의 모든 난자에서 삽입을 확인하였고, EGFP 유전자의 발현은 형광현미경 하에서 35개의 모든 난자에서 확인하였다. 이 결과는 외래유전자가 삽입된 소 태아 세포를 이용하여 핵치환된 난자는 배반포까지 성공적으로 발달할 수 있다는 것을 나타낸다. 더우기, 이러한 방법은 효율적인 형칠전환 소를 생산하는데 이용될 수 있으리라 사료된다.

• 한국수정란이식학회지
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• 제10권2호
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• pp.121-129
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• 1995

To examine the critical effect of oxygen concentration on embryonic development, in vitro fertilized embryos were cultured in media(TCM199 vs. SOF) supplemented sera(1O% FCS vs. 10% HS) with and without bovine oviduct epithelial cells under two gas atmosphere (5% $CO_2$ in air vs. 5% $CO_2$, 5% $O_2$, 90% $N_2$). Oocytes, obtained from abattoir ovaries, were matured in EGF containing TCM199 medium co-cultured with BOEC for 24 hours, followed by exposure to frozen-thawed, heparin4reated spermatozoa in TALP for 30 hours. And then early embryos(1~2 cell) were cultured in both TCM199 and SOF supplemented with 10% FCS or 10% RS under 5% $CO_2$ in air or 5% COi, 5% $O_2$, 90% $N_2$. Development to morulae and blastocysts was recorded on days 7, after the start of in vitro fertilization. The developmental rates of in vitro fertilized embryos to morulae and blastocysts cultured in SOF with BOEC under 5% $CO_2$, 5% $O_2$, 90% $N_2$(24.4%) were significantly(p<0.05) higher than cultured in SOF with BOEC under 5% $CO_2$ in air(14.1%) at seven days after in vitro fertilization. When early bovine embryos were cultured in TCM 199 and SOF under two different gas atmosphere, there were no significant differences in the developmental rates to morulae and blastocysts between supplements of 10% FCS and 10% HS. The rates of development to morulae and blastocysts were significantly(p<0.01) higher in TCM 199 with BOEC(24.7%) than TCM199 without BOEC(10.9%) under 5% $CO_2$ in air, otherwise SOF without BOEC(36.4%) were significantly (p<0.05) higher than in SOF with BOEC (24.4%) under 5% $CO_2$, 5% $O_2$, 90% $N_2$. In summary, these experiments have proved that the culture system in SOF supplemented 10% ES is effective on in vitro development of early bovine embryos under 5% $CO_2$, 5% $O_2$, 90% $N_2$. In addition, it is effective to development of bovine embryos that TCM 199 should be co-cultured with BOEC and SOF should be cultured without somatic cells under two different gas atmosphere.

• Reproductive and Developmental Biology
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• 제28권1호
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• pp.21-27
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• 2004

본 연구는 이종간 핵이식란의 생산성 향상에 기여하기 위한 기초연구로서 핵이식 수정란의 융합과 활성화 과정에 있어서 수핵난자 및 전기적 융합조건이 핵이식 수정란의 융합 및 체외 발달에 미치는 요인들을 조사하기 위하여 실시하였다. 도축되어지는 소 및 돼지의 난소에서 난포란을 채취하여 TCM-199 및 NCSU-23에 혈청 및 호르몬을 첨가하여 39$^{\circ}C$, 5% CO$_2$ 배양기내에서 24 및 48시간 체외성숙을 실시하여 수핵난자를 준비하고, 공여세포의 준비는 산양의 귀세포를 채취하여 0.25% Trypsin-EDTA의 처리로 세포를 분리, 배양하여 사용하였으며, 계대배양과 함께 세포는 TCM-199 ＋ 10% FBS ＋ 10% DMSO로 동결을 실시하였다. 핵이식은 성숙된 난자의 극체 및 전핵을 laser system으로 투명대를 drilling 하여 제거하고 준비된 공여세포를 핵이 제거된 난자에 주입하여 전기적 자극으로 융합을 실시하여 융합된 난자는 전기적 자극으로 활성화를 유도하였다. 활성화가 이루어진 복제 수정란은 수핵란이 소난자의 경우 monolayer가 형성된 10% FBS가 첨가된 TCM199 배양액에서 7∼9일 동안 체외배양하였으며, 수핵란이 돼지의 경우 10% FBS가 첨가된 NCSU-3 배양액으로 6∼8일 동안 체외배양을 실시하여 배반포기로 유도하였다. 본 연구에서 얻은 결과를 요약하면 다음과 같다. 전기자극의 세기를 1.95 kv/cm와 2.10 kv/cm로 주었을 때 수핵란이 소 난자의 경우 융합율은 47.7및 44.6%였으며, 분할율도 41.9 및 54.5%로써 차이가 없었다. 수핵란이 돼지 난자인 경우는 융합율은 51.3 및 46.1%로써 차이가 없었으며, 분할율도 75.0및 84.9%로써 차이가 없었다. 전기자극 시간을 30 또는 60${\mu}$sec, 횟수는 1 또는 2회 주었을 때 수핵란이 소 난자의 경우 융합율은 30 ${\mu}$sec 1회(50.8%) 와 2회(31.0%) 간에 차이가 없었으나, 60${\mu}$sec 1회(19.3%)가 가장 낮았다(P＜0.05). 융합란의 분할율은 30${\mu}$sec 1회(53.3%)와 2회(50.0%) 간에 차이가 없었으나, 60${\mu}$sec 1회(18.2%)보다 유의적(P＜0.05)으로 높게 나타났다. 돼지 난자의 경우 융합율은 30${\mu}$sec 1회(48.1%), 2회(45.2%)및 60${\mu}$sec 1회(48.6%)간에 차이가 없었으며, 분할율은 30${\mu}$sec 1회(78.4%)와 60${\mu}$sec 1회 (79.4%)간에 차이가 없었으나, 30${\mu}$sec 2회(53.6%)보다 유의적(P＜0.05)으로 높게 나타났다. 이종간 핵이식란의 체외발달에 있어서 수핵란이 소 난자의 경우 상실배와 배반포기로의 발달율이 22.6%로써 단위발생란 30.6%와 차이가 없었으며, 돼지 난자의 경우는 이종간 핵이식란이 5.1%로써 단위발생란 37.4%보다 유의적(P＜0.05)으로 낮게 나타났다. 이상의 실험결과로 보아 산양의 체세포를 이용한 이종간 핵이식 복제수정란의 생산을 위하여 수핵란으로 소와 돼지를 사용하여 복제수정란의 발달을 확인할 수 있었으며, 이종간 핵이식에 있어서 수핵란, 공여세포, 융합, 활성화 및 배양조건 등 아직 초보 수준에 있으며, 앞으로 보다 많은 연구를 통하여 이러한 문제들이 해결되면 멸종위기 상태에 있는 동물들의 종 보존에도 활용이 가능할 것으로 생각된다.