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, CRaa with serum was superior to CRaa 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 CRaa containing long /ml EGF than in the control CRaa(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) CRaa ; b) CRaa + ing /ml PDGF ; CRaa + Sng /ml PDGF ; CRaa + 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 CRaa 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 CRaa with MEF coculture group(50.9%) compared to any other group(CRaa, 22.3%; CRaa+BRL, 32.9%; CRaa+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 (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 (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.

The objective of this study was to develop an effective in vitro production system capable of obtaining more porcine embryos from immature oocytes These experiments were conducted to examine the effect of sperm factor on the IVF and IVD, and the effect of coculture with somatic cells on the IVD of embryos. Although the concentration of epididymal sperm for IVF did not affect on cleavage rate, but 5 x 105 sperm/mi showed the highest cleavage rate(48.7%) and the developmental potential of IVF oocytes from this concentration was also greatly higher (P-stored sperm for l2hrs and the cleavage rate from fresh sperm was significantly higher (P<0.05) than that from frozen sperm, but the developmental potential after IVF was slightly high from the frozen sperm. The cleavage rate of IVF oocytes cocultured with oviductal epithelial cells and cumulus cells was 76.3% and 72.9%, respectively. There was no difference between two coculture systems but this rate was significantly higher(P<0.05) than that of medium alone(42.0%).

The study was conducted to determine the optimal hormone and glucose levels during the in vitro culture of bovine oocytes matured and fertilized in vitro for blastocyst development. Oocytes matured in TCM 199 + 10% FCS + hormones and glucose were fertilized in vitro in a TALP medium with swim up separated and heparin-treated epididymal cauda spermatozoa. Oocytes were cultured for 2~5 days in synthetic oviduct fluid medium (SOFM) supplemented with 10% FGS and with different hormone and glucose levels, and further cultured 5 days same medium in SOFM. The results are summarized as follows : The in vitro maturation and penetration rates of porcine oocytes cultured in TCM 199 media containing PMSG, hCG, PMSG + hCG, hCG + estradiol, PMSG + estradiol 0 to20 hours after insemination were 88.0% and 81.8%, 82.6% and 68.4%, 80.0% and 75.0%, 80.0% and 65.0%, 77.3% and 64.7%, respectively. The in vitro maturation and penetration rates of porcine oocytes cultured in TCM 199 media containing PMSG, hCG, PMSG + hCG, hCG + estradiol, PMSG + estradiol 20 to 40 hours after insemination were 92.0% and 87.0%, 92.0% and 82.6%, 91.3% and 81.0%, 85.2% and 73.9%, 87.5% and 81.0%, respectively. The cleavage and in vitro developmental rates to blastocyst of porcine oocytes cultured in TCM 199 media containing 0.05 mM, 0.10 mM, 0.30 mM, 0.50 mM, 1.00 mM, and 3.00 mM glucose lelvels 0~3 days after insemination were 31.5~48.1% and 10.0~16.7%, respectively. The cleavage and in vitro developmental rates to blastocyst of porcine oocytes cultured in TCM 199 media containing 0.05 mM, 0.10 mM, 0.30 mM, 0.50 mM, 1.00 mM, and 3.00 mM glucose levels 4~8 days after insemination were 30.0~53.8% and 8.7~19.2%, respectively. The cleavage and in vitro developmental rates to blastocyst were higher in TCM 199 media containing various glucose levels 0~3 days after insemination than 4~8 days.

This study was conducted to examine the condition of in vitro culture system and the viability after embryo transfer of in vitro matured-in vitro fertilized (IVM-IVF) bovine embryos. The in vitro development to the blastocyst stage was enhanced by supplying bovine serum albumin(BSA) to co-culture medium with bovine oviduct epithelial tissue(BOET) compared with that in medium supplemented with fetal bovine serum(FBS) (41.2% vs. 26. 3%, P<0.05). After transfer of IVM-IVF blastocysts into the uterine horn of recipient females (Aberdeen Angus), one was pregnant to term and produced a head of male Korean native calf. These results confirm that the in vitro development of IVM-IVF bovine embryos is affected with different protein source in co-culture with BOET, and IVM-IVF embryos can develop to term after in vitro culture and embryo transfer.

The objective of this study was to develop an effective in vitro production system capable of obtaining more porcine embryos from immature oocytes. These experiments were thus conducted to examine the effect of oocytes type and maturation time on the in vitro maturation(IVM) and fertilization(IVF) of oocytes and the in vitro development (IVD)of IVF embryos. 1. The degree of oocyte maturation based on cumulus expansion index(GEI) did not differ for A- and B-typed oocytes but the index of oocyte type C was lower(P<0.05) than that of other oocyte types. 2. When the oocytes of type A and B were matured for 36, 42 and 48hrs, the GEl was not different between the 36- and 42-h maturation but the GEl after 48hrs was greatly lower(P<0.05) than that of other maturation times. 3. The highest cleavage rate(48.6%) of IVF oocytes was obtained from A typed oocytes and 42-h maturation but the developmental potential based on cleavage index was the highest when B-typed oocytes were matured for 42hrs.

The studies on the carried out to investigate the effects of co-culture with uterine fluids and uterine epithelial cells on the in-vitro fertilization and developmental rate of porcine follicular oocytes. The ovaries were obtained from slaughtered swine. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluids from the visible follicles of diameter 3~5 mm. The follicular oocytes were cultured in TCM-199 medium containing hormones and 10% ECS for 46~48 hrs in a incubator with 5% in air at 38.5 and then matured oocytes were again cultured for 12~18 hrs with motile capacitated sperm by preincubation of heparin. The results obtained in these experiments were summarized as follows ; 1.The in-vitro maturation and fertilization rate of porcine oocytes co-cultured with uterine fluids in TCM-199 medium were 68.0% arid 55.7%, the rates were higher than of control, 56.5% arid 38.7%. 2. When the in-vitro fertilized oocytes were co-cultured with porcine uterine epithelial cells, the fertilization rate was 60.3%, the rates were higher than that of control, 35.7%. 3. When the in-vitro fertilized oocytes were co-cultured with porcine uterine epithelial cells, the development rate to be blastocyst was 12.4%, the rates were higher than that of control, 9.2%(p<0.05).

기관분화(器官分化)된 유채의 조직으로부터 재분화되는 식물체의 획득빈도를 높이고자 이에 관여하는 몇가지 요인에 대한 실험을 수행하여 얻어진 결과는 다음과 같다. 유채의 종자, 무균배양(無菌培養)된 자엽(子葉), 하배축(下胚軸) 및 엽절편(葉切片)배양에서 명상태에서 보다는 암상태에서 캘러스의 생장이 왕성하였고, 하배축(下胚軸)과 자엽조직(子葉組織)에서 형성된 캘러스에서 shoot 분화능력이 높은 편이었다. 자엽(子葉) 및 하배축(下胚軸) 조직(組織)의 캘러스형성에는 2, 4-D 단용보다 2, 4-D와 kinetin 또는 BAP를 혼용하는 것이 효과적이었으며, 자엽조직(子葉組織)의 경우는 2, 4-D(1.0 mg/)와 0.1mg/의 kinetin이 첨가된 배지에 형성된 캘러스를 0.1mg/의 NAA와 2.0 mg/의 kinetin이 첨가된 배치에 이식하였을 때 shoot 분화율이 16.7%로 가장 높게 나타났다. 하배축(下胚軸) 조직(組織)에서는 1.0mg/의 2, 4-D와 0.5mg/의 kinetin이 첨가된 배지에서 형성된 캘러스를 0.1mg/의 NAA와 4.0mg/의 kinetin이 혼용된 배지에 이식하였을 때 shoot 분화율(分化率)이 24%로 가장 높게 나타났다. 자엽(子葉) 및 하배축(下胚軸) 조직(組織)의 캘러스 형성과 shoot분화능력의 유채 품종간(品種間) 차이는 매우 뚜렷하게 인정되었고, 자엽(子葉)보다는 하배축(下胚軸) 조직(組織)에서 형성된 캘러스에서 shoot 분화능력이 현저히 높게 나타났다.

The present study was performed to investigate the effects of co-culture with granulosa cells on in vitro fertilization and cleavage of early bovine embryo development. Bovine oocytes were matured for 20-24 hrs in vitro with granulosa cells or without and then fertilized in vitro using frozen-thawed spermatozoa treated with BO-caffeine, BO-BSA(2OmM heparin added). At l8hrs after insemination, oocytes were fixed and examined or further cultured in TCM 199 for 48hrs. The fertilization rates between the control(70.4%) and the groups of co-cultured with granulosa cell(2.5106 cells/ml; 71.6%, 5.0 106/ml; 71.9%, l.0 107/ml; 71.1%) did not differ significantly. The cleavage rates in the groups co-cultured with granulosa cell(2.5 106 cells/mi; 43.6%, 5.0 106/ml; 46.8%. l.0 107/ml; 45.0%)were significantly higher than that of without granulosa cell, respectively(P<0.05). However there were no significant differences between the groups co-cultured with granulosa cells. The result indicated that co-culture with granulosa cell was effective means to cleavage of bovine follicular oocytes but did not affect the in vitro fertilization.