• 한국수정란이식학회지
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• 제14권3호
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• pp.185-193
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• 1999

To improve the efficiency of in vitro production of embryos with follicular oocytes in pig, the recovery rates, in vitro fertilization and development. The results obtained were as fellows: The number of oocytes recovered 37 ovary was 1,365 by aspiration, 1,884 by slicing and 3,830 aspiration post slicing, per ovary was averaged 103.5 aspiration post slicing than 30.7 by aspiration and 50.8 by slicing (P<0.05). The percentage of grade I and II oocytes recovered was 0.05∼0.2% and 1.7∼2.3% respectively(p<0.05). The fertilization rates of ejaculate and epididymis sperm was 83.0 and 83.1%. And cleavaged rate was 60.8 and 69.0% respectively(P<0.05). However, there were no significant differences between sperm sources. The clevage rates of fertilized oocyte was significantly(P<0.05) higher as B.O(92.8%) than TALP (90.1%) or mTBM (80.1%). And in vitro developed to blastocyst rates of mTBM media used for fertilization was significantly (P<0.05) higher as 12.4%, compared with the results using the media of TALP(1.6%) or B.O (0.0%). The embryos developed 2-cell stage after in vitro fertilization were co-cultured with or without POEC and BOEC in NCSU-23 and TCM-199 media. In vitro developed to blastocyst rates was NCSU-23 with POEC(2.3%) or BOEC(1.2%), but in vitro cultured in TCM-199 medium with POEC or BOEC was not developed to blastocyst. The percentage of embryos that developed to morula stage in 0, 50, 100, 200 and 250uM was 16.6, 22.0, 13.5, 19.0 and 22.0%, respectively.

• 한국가축번식학회지
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• 제23권4호
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• pp.337-345
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• 1999

본 연구는 소 체외수정란의 체외배양 시 공동배양배지 및 첨가되는 단백질원에 따른 소 체외수정란의 체외발육능을 검토하였다. 소의 미성숙 난포란을 체외에서 성숙, 수정시킨 후, BSA 또는 FBS를 첨가한 TCM-199 또는 CR1aa 배양액으로 단순배양 또는 난구세포, 소 난관상피세포 (BOEC) 및 Buffalo rat 간세포 (BRLC)와의 공동배양 후, 체외수정란의 분할율 및 발육능을 검사하였다. 소 성숙 난포란을 체외수정 후, 분할율은 배양액의 종류에 관계없이 BSA를 첨가한 경우에 유의적으로 높았다 (P＜0.01). 분할된 수정란을 BSA 또는 FBS가 첨가된 TCM-l99 또는 CRlaa 배양액 내에서 단순배양한 결과, 배반포 발육율은 단백질원에 관계없이 CR1aa 액에서 배양한 경우가 유의적으로 높았다 (P＜0.05). 분할된 수정란을 난구세포 또는 BOEC와 공동배양 시, TCM-l99 배양액에서는 FBS에 비하여 BSA 첨가구가 높은 배반포 형성율을 보였으나 (P＜0.05), CRlaa 배양액에서는 BSA와 FBS 첨가구 모두 높은 발육율이 얻어졌다. 한편, 분할된 수정란을 BRLC의 단층세포와 공동배양 시에는 배양액의 종류와 관계없이 BSA에 비하여 FBS가 수정란의 발육율을 향상시켰다 (P＜0.05). 본 실혐의 결과는 배양액 중에 BSA 첨가가 소 체외수정란의 분할을 촉진할 수 있으며, 체외수정란을 체세포와 공동배양 시, 수정란의 발육율이 배양액 및 첨가 단백질원의 종류에 따라 영향을 받아, TCM-199 액에서 난구세포 또는 BOEC와 공동 배양하는 경우에는 BSA 첨가가 효과적일 수 있음을 수 있음을 보여준다.

• 한국가축번식학회지
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• 제18권1호
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• pp.39-46
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• 1994

This experiment was carried out to develop an in vitro culture system for rabbit embryos. The zygotes or 2-cell embryos were collected from the oviducts of the superovulated and mated does with D-PBS/10% FCS at 24 hours after hCG injection. The in vitro developmental rate of blastocyst formation and the number of nuclei in the embryos were examined under the following treatments; 1) TCM-199 with 10% FCS, 2) EBSS with 10% FCS, 3) rabbit vitreous humor(VH), 4) TCM-199 with 10% FCS＋BOEC, 5) TCM-199 with 10% FCS＋ROEC, 6) EBSS with 10% FCS＋BOEC and 7) EBSS with 10% FCS＋ROEC. For a comparative study of in vivo and in vitro development, the fresh blastocysts, which were developed in vivo for 96 hours after hCG injection, were collected from the uterus and their numbers of nuclei were counted. 1. The zygotes or 2-cell embryos developed to the blastocyst stage in TCM-199, EBSS and VH at the rates of 93, 92 and 89%, respectively. 2. The higher developmental rates 95~98% of blastocyst formation was achieved when the embryos were co-cultured with a monolayer of bovine or rabbit oviductal epithelial cells in TCM-199 or EBSS. No significant difference in developmental rates was shown between bovine and rabbit oviductal epithelial cells. 3. In a comparative study of in vivo and in vitro development, the total numbers of nuclei were significantly less in the in vitro cultured embryos(104~224) than the in vivo developed embryos(1, 0090 at 96 hours after hCG injectin. 4. The mean cell cycle numbers in the embryos cultured for 72 hours in TCM-199 with 10% FCS, EBSS with 10% FCS, TCM-199 with 10% FCS＋BOEC, TCM-199 with 10% FCS＋ROEC, EBSS with 10% FCS＋BOEC and in vivo was 7.38, 6.63, 7.76, 7.69, 7.01 and 9.92, respectively. From these results, it can be suggested the optimal culture system for in vitro culture of rabbit embryos is a co-culture system with bovine or rabbit oviductal epithelial cells in TCM-199 with 10% FCS. Considering the significant reduction in total numbers of nuclei in the in vitro cultured embryos, the advanced research on development of in vitro culture system for rabbit embryos is expected.

• 한국가축번식학회지
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• 제22권1호
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• pp.43-50
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• 1998

The effect of oxygen tension on embryonic development in co-culture was evaluated from the standpoint of the reduction of dissolved oxygen concentration by the oxygen consumption of feeder cells. Three co-culture systems using bovine oviductal epitherial cells (BOEC), African green monkey kidney cells (Vero cells) or buffalo rat liver cells (BRLC) have been compared in terms of development of bovine embryos derived from oocytes matured and fertilized in vitro. Among the co-cultured embryo, Vero cells su, pp.rted the highest developmental rate (29%) and the other two showed the similar rates. When the co-cultures were incubated in three different oxygen tension such as 5, 10, 20% oxygen atmosphere, embryos co-cultured with Vero cells at 10%-O2 resulted in the highest percentage of development. From the measurement of oxygen consumption of feeder cells, BRLC consumed 1.38　10-10 mg-O2/min/cell which was higher than 0.94　10-10 and 0.26　10-10mg-O2/min/cell for Vero cells and BOEC, respectively. Based on the oxygen consumption data, the phenomena of optimum oxygen tension required in embryo development in vitro has been analyzed, and we suggested that gas phase oxygen concentration, oxygen consumption rate of feeder cells and the number of feeder cells should be considered for the design of optimal co-culture system for effective fertilization of embryos in vitro.

• 한국산학기술학회논문지
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• 제21권6호
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• pp.527-535
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• 2020

본 연구의 목적은 가축의 조직에서 유래한 동물세포를 재생산하기 위하여 세포를 동결하는 방법으로 초자화 동결법을 간단하게 이용하는 조건을 확립하고자 하였다. 소난관상피세포를 초자화 동결법에 적용하기 위하여 소 난관상피세포를 0.25ml 스트로에 밀봉하여 액체질소에 직접 노출하였다. 발정 3.5일자의 난관에서 추출된 소 난관상피세포는 polyampholyte가 주성분인 StemCell Keep^TM을 구매하여 초자화 동결을 유도하였고 5, 10, 25, 50, 75 및 100% 농도에서 생존율을 조사하였다. 세포의 생존성은 트립판 블루염색기법과 SYTO-13/PI 핵 염색시약을 이용하여 차별적 생사염색기법을 이용하여 분석하였다. Trypan blue 염색법에서는 각각 5.6±11.8, 12.5±7.2, 53.0±2.7, 85.1±6.9, 79.8±0.6 및 60.7±6.7%의 생존율이 관찰되었고, SYTO-13/PI 염색시약에서는 각각 4.6±2.5, 30.8±12.1, 58.4±2.5, 85.5±1.2, 79.8±0.6 및 71.2±1.2%의 생존율이 관찰되었다. 이러한 결과는 소 난관상피세포는 50% StemCell Keep^TM 농도의 동결배양액을 이용하여 동결 보존하는 것이 가장 우수한 생존율을 얻을 수 있었고 세포 유전자원 은행을 위한 영구보존에 적절할 것으로 판단된다.

• 한국가축번식학회지
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• 제21권2호
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• pp.139-146
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• 1997

소와 돼지의 난관 상피세포가 마우스 초기배의 발달 에 미치는 영향과 체외배양에 있어 최적의 배양조건을 알아보기 위하여 ICR 계통의 마우스에 PMSG 7.5 IU와 hCG 7.5 IU를 각각 복강주사하고 자연교미하여 48시간 경과 후에 난관에서 2-세포 초기배를 D-PBS로 관류하여 회수하였다. 회수된 배아는 소와 돼지의 난관 상피세포와 공배양하여 그 효사를 배반포 발달율과 핵의 수를 조사하였다. 또한 생체내와 실험 관내의 발육상태를 비교하기 위하여 hCG접종후 120 시간동안 생체내에서 발육시킨 신선 배반포배를 자궁 에서 채취하여 그 핵수를 계산하였다. 마우스 초기배는 TCM 199, Ham's F-10, Medicult IVF 배양액에서 소 난관 상피세포 또는 돼지 난관 상피세포와 공배양할 경우 91-97%의 높은 배반포 발달율을 보였으며 난관 상피세포간의 차이는 나타 나지 않았다. 각 배양조건에 따라 배양된 배반포의 핵수는 체내에서 자란 배반포에 비해 체외에서 배양한 배반포에서 유의적으로 적었다. 체외배양중 핵수는 공배양하지 않은 TCM 199, Ham's F-10, Medicult IVF medium 에서 각각 68.1${\pm}$6.00, 67.3${\pm}$4.49, 66.4${\pm}$5.64개로 나타났으며 BOEC와 공배양하였을 경우에는 94.3${\pm}$8.61, 92.5${\pm}$7.60, 92.1${\pm}$6.107B, POEC와 공배양하였을 때는 93.3${\pm}$5.80, 92.9${\pm}$6.53, 92.3${\pm}$7.35개로 체내에서 배양된 배반포의 경우의 107.2${\pm}$7.43개보다 적었다. 이상의 결과로 난관 상피세포인 BOEC와 POEC는 마우스 초기배야와의 체외공배양시 배아의 발달과 분화에 이로운 영향을 주어 발달율과 부화율를 향상시키나 핵수 증가에서는 체내조건보다 미홉한 것으로 사료된다.

• 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.

• 한국수정란이식학회지
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• 제10권1호
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• pp.53-63
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• 1995

The effects of different protein sources (serum vs bovine serum albumin), growth factors (EGF and PDGF) and co-culture with various type of somatic cel1s (BOEC, MEF and BRL) on the in vitro development of in vitro matured / in vitro fertilized bovine oocytes were examined, and the viability of frozen/thawed embryos derived from IVM /IVF was examined. Cell numbers of blastocysts were also counted. In Experiment 1, CR$_1$aa with serum was superior to CR$_1$aa with BSA in producing morulae plus blastocysts from IVM /IVF oocytes(24.4% vs 30.4%, p>0.05). In Experiment 2, more morulae plus blastocysts(42.3%) were produced in CR$_1$aa containing long /ml EGF than in the control CR$_1$aa(33.3%). In Experiment 3, 2- to 8-cell embryos derived from IVM /IVF oocytes were randomly allotted to one of 4 culture groups : a) CR$_1$aa ; b) CR$_1$aa + ing /ml PDGF ; CR$_1$aa + Sng /ml PDGF ; CR$_1$aa + lOng /ml PDGF ; culture resulted in 21.3, 51.2, 41.4 and 45.9%(p<0.05), respectively, developing into morulae and blastocysts. In Experiment 4, 0 and Sng /ml PDGF added to CR$_1$aa coculture with BRL or BOEC yielded 47.5, 42.5, 33.8 and 41.6% morulae and blastocysts, respectively. In Experiment 5, the proportion of embryos into morulae and blastocysts was highest in CR$_1$aa with MEF coculture group(50.9%) compared to any other group(CR$_1$aa, 22.3%; CR$_1$aa+BRL, 32.9%; CR$_1$aa+BOEC, 33.8%, p>0.05). In Experiment 6, survival rate of blastocysts produced by in vitro fertilization when cryoprotectant was removed in 0.7M glycerol+0.7M sucrose and 0.7M sucrose solution for 10 min. after thawing at 2$0^{\circ}C$ (Exp. H, 58.8%) was slightly higher than when cryoprotectant was removed 10%, 6.7% and 3.3% glycerol for 10 min. after thawing at 37$^{\circ}C$ (Exp. I, 54.3%). These study indicate that growth factors and somatic cell co-culture can increase the proportion of embryos that develop into morulae and blastocysts without an increase in the cell number and frozen /thawed method employed this experiment was not different.

• 한국수정란이식학회지
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• 제15권1호
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• pp.67-75
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• 2000

This study was conducted to establish the optimal conditions for in vitro embryo production using oocytes derived from follicles of slaughter-house ovaries. The ovaries of Hanwoo were obtained from a local slaughter-house. The oocytes were aspirated from visible follicles of 2~7mm in diameter. The recovered oocytes which were completely surrounded by at least 2 layers of cumulus cells and a homogeneous cytoplasmic pigmentation were used. The selected oocytes were washed 3 or 4 times with D-PBS containing 10% bovine calf serum (BCS) and matured in vitor (IVM) in Ham's F-10 supplemented with 10% BCS or 0.01 $\mu\textrm{g}$/ml epidermal growth factor(EGF) at 39$^{\circ}C$ under 5% CO2 in air for 24 hours. They were fertilizqed in vitro (IVF) with fresh sperm separated by Percoll density gradient or swim-up in TALP media. The zygotes were cultrued with or without bovine oviductal epitherial cells(BOEC) in media(HECM-6 supplemented with 11 amino acid and / or TCM-199 supplemented with 10% BCS) for 7 to 10 days. The results obtained were as follow: The cleavage rate and developmental rate to blastocyst after maturation and IVF were not significantly different between Ham's F-10 with EGF(76.0% vs. 44.0%) and BCS(75.9% vs. 43.6%)(P<0.05). The cleavage rate and development rate to blastocyst after fertilizing by swim-up or Percoll method were not signifciantly(P<0.05) different between swim-up (80.2% vs. 29.2%) and Percoll(81.9% vs. 26.5%) (P<0.05). The cleavage rate in TCM 199(80.5) was signficiantly higher than that in HECM-6 (72.0%) (P<0.05). However, developmental rate to blastocyst using TCM 199 following HECM-6 for 3 or 4 days (42.2%) was significantly higher than that in TCM-199 alone(26.7%)(P<0.05). The cleavage rate and development rate of embryos produced in vitro by exchange timing for HECM-6 media were not significantly different between in day 3(78.6% vs. 45.5%) and day 4(75.0% vs. 43.2%)(P<0.05). The cleavage rate and developmental rate to blastocysts according to co-culture system were not significantly different between with (74.2% vs. 41.4%) and without BOEC(73.95 vs. 43.5%) (P<0.05). The number of blastomere in blastocyst stage after co-culture with or without BOEC was not significantly different (106.7$\pm$5.1 and 96.6$\pm$4.0). In conclusion, the most transferable IVP embryos could be produced from Ham's F-10 medium for IVM, Percoll density gradient method for IVF sperm separation and in vitro culture in HECM-6 until day 3 or day 4, and then transferred into TCM-199 until day 9 within adequate embryo density in culture droplets after insemination.

• 한국가축번식학회지
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• 제18권2호
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• pp.113-119
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• 1994

To improve nuclear transplantation(NT) efficiency and to produce a large scale genetically identical cloned calves, examined the in vitro development capacity after co-culture of bovine oviductal epithelial cells (BOEC) and granulosa cells in TCM-199 supplemented with 10% fetal calf serum (FCS) with early bovine embryos derived from in vitro matured fertilized(IVM-IVF) oocyte. In addition, the age dependence of IVM oocyte on electro-stimulation and the effective electric voltage on in ivtro development of bovine NT embryos were examined. The results obtained were summerized as follows; 1. The cleavage rates of IVM-IVF bovine embryos in co-culture with bovine oviductal epithelial cells and granulosa cells were not significantly different(P<0.05), but the developmental rate into morula and blastocyst stage were different showing 38.3 and 20.2%, respectively. 2. The activation (82.5%) and development in vitro(8.6%) into later embryo stages of the aging oocytes of 32 hours post-maturation (hpm) were significantly higher than those of 24 hpm at direct current (DC) voltage of 1.5kV/cm, 60$\mu$sec pulse duration and 1 pulse time. 3. The fusion rates of NT eggs of 32 hpm following to different DC voltages from range 0.75 to 1.5kV/cm were not differ, but the developmental rate into morula and blastocyst stages at DC voltages of 0.75 and 1.0kV/cm were higher(11.4 and 12.6%, respectively) than those of 1.5kV/cm(0%). From these results, it can be suggested the optimal culture system for in vitro culture of IVM-IVF bovine embryos is a co-culture system with BOEC in TCM-199 supplemented 10% FCS. The effective time and the DC voltage for activation, electrofusion and in vitro development of NT embryos derived from IVM-IVF bovine embryo are 32hpm and 0.75~1.0kV/cm. But to improve NT efficiency, the advanced research (cell cycle synchronization, micromanipulation, culture system, etc.) is needed.