To improve the efficiency of in vitro production of embryos with follicular oocytes in Korean Native cows, the recovery rates, in vitro maturation, fertilization and development, and the time required for collecting and processing oocytes by aspiration with or without slicing were evaluated comparatively. The ovaries were obtained from a local abattoir and placed in physiological saline at 25~28 and brought to the laboratory within 3 hrs. The oocytes were collected by aspiration of follicles(2~6mm) with or without slicing ovaries after aspiration, and classified into Grade I, Grade II, Denuded, Expanded oocytes by the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules. Also the time required for each step of collecting and processing oocytes were measured. The cumulus cells were removed in some Grade I oocytes to measure their size and nuclear configuration before and after in vitro maturation. The Grade I oocytes were matured in vitro(IVM) for 24 hrs. in TGM-199 supplemented with 35g /ml FSH, 10g /ml LH, 1 g /ml at 39 under 5% C02 in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24hrs. and then the zygotes were cocultured in vitro (IVC) with bovine oviductal epithelial cells for 10 days. The results obtained were as follows: The number of oocytes recovered per ovary was averaged 6.6 by aspiration and 11.2 by slicing post aspiration, which summed to 17.8. The number of Grade I oocytes recovered per ovary was averaged 3.1 by aspiration and 3.6 by slicing, which summed to 6.7. The percentage of Grade I to total oocytes recovered was significantly(P<0.05) higher as 48.0 % in aspiration than 31.6% in slicing post aspiration. The time requlred for recovering a Grade I oocyte by aspiration and slicing was 1.1 and 2.5 min, respectively. The mean diameter of Grade I oocytes by aspiration and slicing was similar as 148.7 and 151.5m, respectively. The percentage of Metaphase II stage oocytes after IVM for 24 hours was significantly (P

This study was conducted to improve the production efficiency of in vitro produced (IVP) embryos in Korean Native cows. The optimal conditions and procedures for in vitro maturation(IVM), in vitro fertilization(IVF) and in vitro culture(IVC) of bovine follicular oocytes and IVP embryos were evaluated. Immature follicular oocytes were collected fiom the follicles of bovine ovaries obtained from abattoirs. The oocytes of Grade I and II for IVM were cocultured with monolayered bovine oviductal epithelial cells(BOEG) or granulosa cells in TCM-199 solution supplemented with follicle stimulating hormone, lutenizing hormone, estradiol-17 and heat inactivated fetal calf serum at 39 under 5% in air for 14 to 24 hours. Most of the oocytes(93%) matured to metaphase II in 24 hours. The cocultured IVM oocytes were fertilized in vitro at significantly(P<0.05) higher rate with BOEC(83.8%) and with granulosa cells(84.6%) than the non-cocultured IVM oocytes(73.6%). The IVM-IVF embryos developed to morula and blastocyst at significantly(P<0.05) higher rate in coculture with BOEC(41.2%) than with granulosa cells(23.1%) or conditioned medium(23.4%).

This study was conducted to investigate the effects of co-culture for the development rate to morula /blastocyst stages of early porcine embryos, derived from oocytes matured and fertilized in vitro, with porcine endometrial cell monolayers(PEM) in the two different media, respectively. The rates of embryos developed to 2-, 4-, 8~16-cell and morula /blastocyst stage were 49.6, 40.5, 28.2 and 15.3% in Ham's F-10 with PEM, and 55.3, 45.9, 32.7, and 17.6% in TCM-HEPES with PEM, respectively. The above development rates to morula /blastocyst stages were significantly higher than those of the embryos cultured in the Ham's F-10 and TGM-HEPES without PEM(P<0.05). The in vitro development rates to the morula /blastocyst stage of 1-cell embryos cultured in Ham's F-10 and TCM-HEPES without PEM were 0~1.2%. Especially, most of embryos were observed to arrest the development beyond 4-cell stages. As shown in the above results, the co-culture of in vitro produced porcine embryos with PEM in the two different media enhanced the development of fertilized eggs to morula /blastocyst stages in vitro. However, we didn't find out any differences for the in vitro development to morula /blastocyst stages between Ham's F-10 and TcM-HEPES media.

This study was carried out to investigate the effect of equilibration time, sucrose concentration and age of embryo on survival and developmental rates of bovine IVF expanding blastocysts frozen-thawed by direct transfer method. The bovine oocytes were collected from 2~5mm follicles, matured for 20~24hrs in 5% incubator and then fertilized with frozen-thawed semen. Expanding blastocysts at day 7, 8, 9, 10 and 11 after IVF were frozen in 1.8M ethylene glycol(EG). Survival and hatching rates of frozen-thawed IVF embryos were examined. The results were as follow ; Survival and hatching rate of TVF expanding blastocysts after 10, 20, 3Omin exposure in 1.8M EG were 100,0,90.9, 47.1, 85.0, 75.0 and 62.5% respectively. Survival rates of IVF expanding blastocysts frozen with 1.8M EG and various concentration(0, 0.25, 0.5, 1M) of sucrose were 73.3, 25. 0, 16.7, 9.1% respectively. Survival and hatching rates of IVF expanding blastocysts frozen-thawed according to age of embryo(Day 7, 8, 9,10, 11) were 86.1, 84.8, 79.3, 61.4, 51.3, 74.2, 76.9, 71.7, 63.0 and 65.0% respectively. In conclusion, the age of the embryo(Day 7, 8) is very important for the successful freezing of IVF bovine embryos and 1.8M ethylene glycol not containing sucrose may be effective cryoprotectant for direct transfer method.

The present study was carried out to obtain the pregnancy and delivery rate following transfer of fresh and frozen-thawed Korean native cattle(KNC) blastocysts(1~4 em-bryos / head) produced in vitro to Holstein recipients. The pregnancy rate of fresh and frozen-thawed KNC blastocysts produced in vitro was 50%(7 /14 heads) and 38.5%(5 /13 heads), respectively. The pregnancy rate of frozen-thawed KNC blastocysts produced in vitro frozen using 1.5M ethylene glycol and 1.4M glycerol for cryoprotectant was 33.3%(2 /6 heads) and 42.9 %(3 /7 heads), respectively. Seven calves including 2 sets of twin were born fiom 5 pregnant recipients receiving eleven fresh blastocysts. Three pregnant recipients were aborted among four pregnant recipients receiving twelve frozen-thawed blastocysts and one calf was born from the rest of one pregnant recipient.

The present study was carried out to investigate the effects of cryoprotectants, equilibration step, freezing rate, culture condition following in vitro fertilization, and age and development stage of embryo by freezing with conventional slow freezing and vitrification on survival of frozen-thawed Korean native cattle(KNC) blastocysts produced in vitro. The KNC blastocysts produced in vitro were equilibrated in 1.8M ethylene glycol or 1.4M glycerol and cooled from -6 to -35 at -0.3 or -O.6 /minute. When equilibrated in 1.8M ethylene glycol, survival rate of fiozen4hawed blastocysts was sarne in both -0. 3 /min and -0.6 /min cooling rate(71.4%). With the equilibration in 1.4M glycerol, survival rate was higher in -0.3 /min(63.6%) than in -0.6 /min cooling rate(53.8%). For vitrification of the KNC blastocysts produced in vitro, they were equilibrated in 2-step or 3-step exposure to vitrification solution(25% ethylene glycol + 25% glycerol). Survival rate was sirilar in both 2-step(45.0%) and 3-step exposure(47.4%). According to culture condition following in vitro fertilization, higher survival rate was obtained for blastocysts co-cultured with bovine oviductal epithelial cell(BOEC, 77.3%) than for those cultured with epidermal growth factor(EGF, 65.7%) or for those co-cultured with BOEG + EGF (54.8%). According to embryo age and development stage, higher survival rate was obtained for 7-day ernbryos(70.0%) than 8-day(56.8%) or 9-day(20.0%) for blastocyst stage and obtained for 8-day embryos(74.3%) than 7-day(62.5%) or 9-day(42.9%) for exponded blastocyst. In surnmary, higher survival rate of frozen4hawed KNC blastocysts produced in vitro were obtained by using ethylene glycol for cryoprotectant and -0.3 /min for cooling rate. And higher survival rate were obtained with co-culture with BOEC for culture condition following in vitro fertilization and with 7-day blastocyst or 8-day expanded blasto cyst for embryo age and development stage.

The present study was carried out to investigate the effects of co-culture cells and growth factors on in vitro culture of Korean native cattle(KNC) embryos fertilized in vitro. Two-eight cell embryos were cultured in vitro using 4 types of co-culture cells and 3 growth factors singly or in combination. The results were as follows, In the co-culture of 2~8 cell embryos with bovine oviductal epithelial cell(BOEC), granulosa cell(BGC), uterine epithelial cell(BUEC) and mouse embryonic fibroblast (MEF) monolayers, the developing rate to blastocysts was significantly(P<0.05) higher with BUEC(32.1%) than with MEF(15.3%), BGC(13.2%) and non co-culture control(11.6%). When the morula co-cultured with BOEC for 5 days following in vitro fertilization were co-cultured with BOEC continuously or with BUEC, respectively, the developing rate to blastocysts was higher with BUEC(73.9%) than with BOEC(56.0%). To examine the effects of growth factors on in vitro development of 2~8 cell embryos, epidermal growth factor(EGF), transforming growth factor-l(TGF-l) and insulin-like growth factor-1(IGF-1) were added singly or in combination to TCM 199 maturation medium with respective concentration. In a addition of each 10, 30 and SOng /rnl EGF, the developing rate to blastocysts was the highest in lOng /ml EGF(25.3%). In addition of each 1, 2 and Sng /mi TGF-1, the developing rate to blastocysts was the highest in lng /ml TGF-1(28.8%). In addition of each 50, 100ng/ml JGF-l, the developing rate to blastocysts was higher in 100ng/ml IGF-l(16.5%) than in SOng/mi IGF-1(12.9%). When lOng /ml EGF and lng /ml TGF-l was added singly or in combination, the developing rate to blastocysts was similar in groups added singly or in combination with EGF and TGF-l (23.l~24.6%), although higher than in control(16.7%). In the co-culture of 2~8 cell embryos Wth BOEC + each 10, 30 and 5Ong /rnl EGF, the developing rate to blastocysts was significantly(p<0.05) higher in BOEC + long /ml EGF(32.3%) than in BOEC + 3Ong /ml EGF(18.9%) and BOEC + song /ml EGF(9.7%). In the co-culture of 2~8 cell embryos with BOEC + each 1, 2, Sng /ml TGF-l the developing rate to blastocysts was higher in BOEC + Sng/rnl TGF-l(28.2%) than in BOEC + lng /ml TGF-l(21.7%) and BOEC + 2ng/ml TGF-l(21.4%). In summary, higher developing rate to blastocysts were obtained with co-culture of BUEC for co-culture system, with addition of lOng /ml EGF or lng /ml TGF-l for growth factor culture system, and with co-culture of BOEC + lOng /ml EGF or BOEC + Sng /ml TGF-l for co-culture + growth factor culture system.

This experiment was carried out to investigate the ovarian responses of the ovulation point, ovarian weight and size, the number of ovarian follicles and collected embryos, and to study the effects of the developmental stages (oocytes, 2-4 cell. 8-16 cell and morulae), additional levels of Ficoll (0, 15, 30%) on the survival rate (FDA-test) of rat embryos frozen in vitrification solution (20% glycerol + 10% ethylene glycol + 10% sucrose). Sunanarized results was as follows; 1. The mean ovulation point per head was 7, and the weight of ovaries was 0.03g. The size of ovary was 5.9 mm(L) and 4.6 mm(W), and the number of ovarian follicles over and below 2 mm was 4.7 and 8.7, respectively. The number of the collected embryos per head was 5.5 (79%). 2. 2. The FDA score of embryos frozen in 20 G 10 E 10 S without Ficoll was 2.8 (oocyte), 2.6 (2-4 cell), 3.9 (8-16 cell) and 3.6 (morula), respectively. However, there were no significant differences among treatments. 3. The FDA score of embryos frozen in 20 G 10 E 10 S with 15% Ficoll was 3.4 (oocyte), 4.0 (2-4 cell), 4.7 (8-16 cell) and 4.8 (morulae), respectively (P>0.05). 4. The FDA score of embryos frozen in 20 G 10 E 10 S with 30 % Ficoll was 3.7 (oocyte), 3.2 (2-4 cell), 4.4 (8-16 cell) and 4.4 (morulae), respectively (P>0.05). 5. As shown in the above results, the higher survival rate was obtained in the treatment of 15% Ficoll than that of 30%. And the survival rate (FDA-test)of the oocytes and 2-4 cell stages of the rat embryos was lower than that of 8~16 cell and morulae stages. It was considered that 8-16 cell and morulae could be available for the successful freezing by vitrification of rat embryos with 15% Ficoll except for oocytes.

This study was carried out to select the best cryoprotectant and to establish optimal concentration of the cryoprotectant in ultrarapid freezing of mouse 4-cell embryos and morulae, respectively. We investigated survival of ultrarapid frozen embryos according to various cryoprotectants such as glycerol, ethylene glycol, propylene glycol and dimethyl sulfoxide (DMSO). Suvival of the embryos frozen at different concentrations (3.0, 4.0 and 5.0 M) of indivisual cryoprotectant was also tested. Preimplantation developmental rate (96.3%, 83/86) of 4-cell mouse embryos treated with 4.0 M ethylene glycol after ultrarapid freezing and thawing was higher than those of other cryoprotectants (glycerol, propylene glycol and DMSO). In the ultrarapid freezing of mouse morulae, the highest developmental rate (98.8%, 89 /90) of the embryos to blastocysts was shown in the group of 5.0 M glycerol. Thus, these results demonstrate that 4.0 M ethylene glycol and 5.0 M glycerol are optimal for ultrarapid freezing of 4-cell mouse embryos and morulae, respectively.

The efficient cryopreservation of embryos requires optimal times of dehydration and rehydration This study was carried out to investigate the effect of various times of dehydration and rehydration The effects were evaluated through testing morphological normality and developmental ability of 1 cell mouse embryos which were ultrarapidly frozen and thawed. The 1 cell embryos were dehydrated for 1.5, 3, 5, and 10 minutes using mPBS-BSA containing 3.SM DMSO and 0.25M sucrose on cooling chamber or on ice. After ultrarapidly frozen and thawed, they were rehydrated for 0, 0.5 and 5 minutes with mPBS-BSA containing 0.25M sucrose at room temperature. The results obtained were as follows: The embryos that were rehydrated for 0.5 minutes showed higher normality than the embryos for 0 and 5 minutes did. The embryos that were dehydrated for 10 minutes showed higher normality than the embryos for 1.5, 3, and 5 minutes did. The developmental ability of normal thawed-embryos was high in 10 minute dehydration treatment compared to other treatments. However, it was not affected by cooling methods (on ice and on cooling chamber) for embryo dehydration.