The present study was carried out to produce in vitro fertilized embryos with immature follicular oocytes collected by transvaginal aspiration from Holstein cows. A simple aspiration apparatus consists of two stainless steel tubes, an inner tube (needle holder; 1.2cmdiameter, 55cm long) and an outer tube (1.5cm diameter, 4Scm long), and a hand-operated vacuum pump was used. Under epidural anesthesia, the needle guide was passed into the vagina of the cow to a point next to the cervix. An ovary was placed against the wall of the vagina over the end of the aspiration needle by rectal manipulation. As the needlepassed into the ovary, an assistant was asked to apply vacuum(l00mrnHg) and the ovary was manipulated back and forth in all directions over the needle. When all sites of the ovary was aspirated, the needle was withdrawn and the needle guide was moved to the other side of ovary and the procedure was repeated. When the oocyte aspiration procedure was finished, collected fluid was transported to laboratory. Oocytes surrounded with at least 1 layer of cumulus cells were matured, fertilized and cultured in vitro. The results were as follows; Ninety seven oocytes were collected by transvaginal aspiration from seventeen Holstein cows(5.7 /head). The number of oocytes surrounded with at least 1 layer of cumulus cells were 60(61.9%). Following in vitro maturation, fertilization and culture, the cleavage and development rate to morula+blastocyst were 83.3% and 30.0%, respectively. From this study, transferable in vitro fertilized embryos could be produced with imma- ture follicular oocytes collected by transvaginal aspiration from Holstein cows using a simple aspiration apparatus

The present study was carried out to investigate effects of gonadotropin, age of donor, day of estrus cycle gonadotropin injection started and season on embryo production after superovulation in dairy cattles. Embryo collection records were obtained from 177 embryo donor collections from 98 Holstein cows aged from 3 to 9 years during 4 years(1993~1996) at National Livestock Re-search Institute. Superovulation was induced by injections of 3 gonadotropins(FSH-P, FOLLTROPHIN-v* or SUPER-OV*) beginning on days 9 to 14 of the estrus cycle. Em-bryos were collected from donors using a nonsurgical technique on days 6 to 8 after insemi-nation. The results were as follows ;Number of total and freezable embryos per donor cow was affected by gonadotropin(P<0.01). The more number of total and freezable embryos wereobtained by use of FOLLTROPHIN-V (13.2, 7.4) or FSH-P (11.0, 5.7) than SUPER-OV* (5.0, 2.4). Age of donor, the day that gonadotropin was started or season didn't affect total or freezable embryos(P >0.05).

This study was carried out to enhance the efficiency of Korean Native cattle embryos and establish the techniques for producing the twin calves. Bisected embryos without zona pellucida which were divided by simple method not using holding pipette or whole two embryos were transferred to recipients.The pedigrees of monozygotic twin calves produced by transfer of bisected pair embryos were identified. The results obtained were as follows ; The average successful bisection rate was 89.16%. The embryos of blastocyst stage (91.66%) were bisected successfully at significantly (P<0.05) higher rate, compared with the morula stage embryos (86.66%). The average survival rate of bisected embryos following 24 hours culture was 59.02%. The survival rate of morula stage embryos (62.50%) was significantly (P<0.05) higher than that of blastocyst stage embryos (55.5%). For the production of monozygotic twin calves, ten pairs of flesh or frozen demi-em- lymphocytes antigen, the twin calves produced by transfer of bisected pair embryos of Korean Native cattle were identified in pedigrees and confirmed as monozygotes.

This study was carried out to establish the techniques for producing the calves of genetically superior Korean Native cattle by transfer of frozen-thawed embryos. The effects of some factors related to embryo recovery following superovulation and pregnancy rate following transfer of frozen-thawed embryos were evaluated. Also calving state was investigated. The results obtained were as follows ; The mean number of total and transferrable embryos recovered per superovulated cow was 8.72 and 4.90, respectively, from a total of 72 superovulations using 34 donor cows. There were no significant differences in the number of total or transferrable embryos recovered per superovulated cow between products of follicle stimulating hormone (FSH), years, seasons, and collection numbers. The pregnancy rate was found 44.44% following transfer of frozen-thawed embryos of Korean Native cattle to a total of 180 recipient cows including 82 Angus, 27 Charolais, 62 Hereford and 9 Korean Native cows. The pregnancy rate was significantly (P<0.05) higher in the transfer of excellent (42.99) and good embryos (40.17%), compared with fair (5.90%) grade embryos. And the pregnancy rate was significantly (P<0.05) higher in the transfer of embryos of morula stage (43.86%) than blastocyst stage (15.51%). But there were no significant differences in pregnancy rates between natural and induced estrus estrus asynchrony of 1 days, breeds, and parities of recipient cows. The normal calving rate of 80 pregnant cows following transfer of frozen4hawed em-bryos was 87.5% and the other 10 pregnant cows showed abortion during the period from pregnancy diagnosis at 50~60 days to calving. The average gestation length of normally delivered recipients was 288.50 days and the average birth weight of 70 calves born was 24.22 kg. The gestation length was significantly (P<0.05) shorter in the recipients delivering female calves (286.70 days) than males (289.39 days). But there were no significant differences in gestation tength and birth weight of calves born between the recipient breeds.

This study was carried out to increase the viability of bovine frozen4hawed in vitro produced (IVP) embryos and pregnancy rate by direct transfer method. Cumulus-oocyte complexes were aspirated from excised Hanwoo ovaries and matured in TGM 199 for 20~22 hours at 38.5 in 2% in air. Matured oocytes were fertilized with capacitated sperm for 6 hours and then co-cultured with cumulus cells for 9 days. 63% of the oocytes cultured was deaved and 29% out of them developed into blastocysts. Good or excellent grade of blastocysts on D 7 or 8 were frozen with 1.8M ethylene glycol as a cryoprotectant for direct transfer. Frozen embryos were thawed at 2 water for 10 sec following 4~5 second in air. For the survival assay of frozen4hawed lVP blastocysts, they were cultured in TCM 199 supplemented with 100M -mercaptoethanol and 20% FCS for 72 hours. The percentage of embryos developed to re-expanded or hatched after 72 hours culture was 95. 5 and 77.3%, respectively. When frozen-thawed Ivp embryos were transferred to 43 synchronized recipients by direct transfer method, eighteen recipients (41.8%) was pregnant. The highest pregnant was in naturafly synchronized recipients (71.4%), but induced estrus by using PRID(29.2%) and PGF(20.0%) was showed lower pregnancy rate. The pregnancy rate was higher in day 7 blastocysts(56.0%) than day 8 blastocysts(22.2%). (Key words: in vitro produced, blastocyst, frozen-thawed, direct transfer)

This study was carried out to establish an effective and practical system for commercialization of embryo production techniques by analyzing several factors influencing in vivo embryo production on days and seasons of flushing in Korean native cattle. In vivo embryos were flushed 226 times from 128 donors. The results obtained for the factors influencing in vivo embryo production on days and seasons of flushing were as follows :1.The percentages of fertilized, transferable and freezable embryos by seasons were significantly different in both FSR-P and SUPER-OV(P<0.01). The percentages of them were highest in sunrrner with FS H-P and highest in autumn with SUPER -OV.2. The production of transferable and freezable embryos by flushing days was highest in 8 days with FSH-P, and there was no difference between 7 and 8 days for SUP ER-OV. 3. The failure rates of recovery were 17.0% in SUPER-OV and 21.2% in FSH-P, respectively. The donors superovulated but failed recovery were 8.5% in SUPER-OV and 12.9% in FSH-P, respectively. Nonsuperovulated donors was 8.4% and donors giving less than 2 eggs at recovery was 8.4% in both FSH -P and SUPER-OV 4. The donors returned to normal estrus after superovulation were 34.1% after 1 cycle,39.4% after 2 cycles, and 16.7% after 3 cycles by FSH-P, respectively. For SUPER-OV, they were 55.3, 33.0 and 9.6%, respectively. Generally, normal estrus after the treatment of superovulation was earlier and the occurrence of ovarian cyst was also lower in SUP ER-OV than in FSH-P.5.The percentages of blastocyst in embryos flushed at 7~8 days after estrus were 21. 9% and 54.3% in FSH -P and SUPER-OV, respectively. The development of embryos was faster in SUPER-OV than in FSH-P.(Key words : in vivo embryo, flushing days, superovulation, FSH-P, SUPER-OV)

This study was carried out to establish an effective and practical system for commercialization of embryo production techniques by analyzing several factors influencing in vivo embryo production on condition of donors treated FSH-P and SUPER-OV in Korean native cattle. In vivo embryos were flushed 226 times from 128 donors. The results obtained for the factors influencing in vivo embryo production by conditionof donors treated FSH-P and SUPER-OV were as follows :1. Age and parity of donors did not affect the transferable and freezable embryos among the treatments in FSH-P but the transferable and freezable embryos were decreased after 6 years old and 4th parity in SUPER-OV(P<0.01). 2. The production of embryos on the weight of donors was higher in over 400kg of body weight in FSH-P(P<0.01) and was higher in over 450kg than 400~450kg of body weight in SUPER-OV(P<0.05). For FSH-P embryo production was better responded in 350~450kg of body weight with 30~32mg doses, and showed a better result in over 450kg body weight with 32~34mg doses.(Key Words : in vivo embryo, donors, FSH-P SUPER-OV)

I. The Factors Influencing In Vivo Embryo Production by Condition of Superovulation Treatment These studies were carried out to establish an effective and practical system for comrnercialization of embryo production techniques by analyzing several factors influencing in vivo embryo production on superovulation treatment in Korean native cattle. In vivo embryos were flushed 226 times from 128 donors.The results obtained from the studies on the factors influencing in vivo embryo production by superovulation treatment were as follows : FSH-P had a significiant advantage(83.0%) over SUPER-OV in the percentage of fertilized embryos(P<0.01). No difference was found loetween FSH-P and SUPER-OV in the percentage of transferable and freezable embryos.2. The response of superovulation by SUPER-OV was greater than that of FSH-P The donors having 8~9 and more than 10 of corpora lutea(CL) derived by FSH-P were 40.0%(most frequent) and 33%, respectively. The donors having more than 12 and 10 CL derived by SUPER -OV were 33.3% (most frequent) and 56.6%, respectively.3. Embryo production after treatment of repeated superovulation was remarkablely decreased at 3rd time by FSH-P but did not differ among 1, 2 and 3rd times by SUPER-OV. Embryo production on intervals of repeated superovulation was significantly different for the number and percentage of fertilized, transferable and free-zable' embryos in FSH-P (P<0.01) and rernarkablely decreased in repeated superovulation of 81~120 interval days. The SUPER-OV showed no differences in interval days of repeated superovulation and was found better than FSH-P in the response of repeated superovulation. (Key words : in Vivo embryo, superovulation, FSH -P, SUPER-OV)

The studies were carried out to investigate the effects of co-culture with cumulus cells and oviduct epithelial cells on the in vitro fertilization and cleavage rate of bovine follicular cocytes and to determine the optimum thawing temperature and equilibration time on in vitro developmental rate of frozen bovine embryos. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes were cultured in TGM-199 medium containing 10 IU /ml의 PM SG, 10 IU /ml의 hCG, ip g/ml의 -estradiol and 10% FCS for 24~48 hrs in incubator with 5% in air at 38.5. The bovine embryos following dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquld nitrogen and thawed in 3 water. Survival rate was defined as developmental rate on in vitro culture or FDA-test. The results are sunanarized as followes :1. The in vitro fertilization and in vitro developmental rates of bovine oocytes co-cultured with cumulus cells in TCM499 medium were 75.0~76.8% and 17.3~27.6%, respect-ively. And in-vitro fertilization rates of cumulus-enclosed oocytes(55.4%)were significantly(p<0.05) higher than cumulus-denuded oocytes (23.1%). 2. The in vitro fertilization and in vitro developmental rates of bovine oocytes co-cultured with l l04cells /ml, 1 x l06cells /ml, lx l08cells /ml and 1 x l015cells /ml oviduct epithelial cells in TCM-199 medium were 74.5~77.8% and 15.7~21.20 respectively.3. The in-vitro fertilization and in vitro developmental rates of bovine oocytes cocultured in '1CM-199 media containing PMSG, hCG, PMSG+hCG. PMSG+-estradiol, hCG+-estradiol 0 to 40 hrs after insemination were 74.0~77.4% and l8.9~23.l%, re-spectiv ely.4.The survival rates of bovine embryos thawed after rapid freezing in the freezing medium containing a various concentration of sucrose added 1.5M and 2.OM glycerol,DMSO and propanediol were 23.5~31.4% and 20.6~34.l%, respectively. 5. The temperature thawed at 3 after rapid freezing of bovine embryos resulted in a significantly higher embryos survival rate than did at 2 and 35.6. The equilibration time on the survival rates of bovine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time (10~20min.). (Key words : bovine embryos, co-culture, freezing, in vitro development)

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 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 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 Gphase, respectivley. The in vitro developmental rate to blastocyst stage with the S and Gphase 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 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 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.

The present study was conducted to investigate the influence of embryo cell stage at 18h post-fusion and oocyte preactivation on sebsequent in vitro developmental potential in the nuclear transplant rabbit embryos. The embryos of 16-cell stage were collected and synchronized to G phase of 32-cell stage. The recipient cytoplasms were obtained by removing the first polar body and chromosome rnass from the oocytes collected by non-dis-ruptive microsurgery procedure. The separated G phase blastomeres of 32-cell stage were injected into non-preactivated recipient cytoplasms. Otherwise, the enucleated recipient cytoplasms were preactivated by electrical stimulation at 18h post-hCG injection and the separated G phase blastomeres of 32-cell stage were injected. Mter culture until 20h post-hOG injection, the nuclear transplant oocytes were electrofused by electrical stimulation. The fused nuclear transplant embryos were classified into 3~4-cell, 2-cell and 1-cell stage at 18 hrs post-fusion and cultured until the embryos reached blastocyst stage. The developmental rate to blastocyst stage was significantly (P <0.05) higher in all the reconstituted embryos of 3~4-cell stage(58.0%) than in 2 and icell stage. The developmental rate to blastocyst stage in the embryos of 3~4-cell stage at 18 hrs post-fusion was significantly (P<0.05) higher in the reconstituted without oocyte preactivation(77.8%) than in the oocyte-preactivated embryos (33.3%). These results indicated that the higher rate of in the in vitro development to blastocyst stage might be obtained form the embryos which were reconstituted with nuclear donor of G phase and non-preactivated oocyte, and developed more rapidly for 18 hrs post-fusion.

This study was carried out to produce twin calves by embryo transfer in Hanwoo and investigate the pregnancy and twin rate by recipient's conditions. All recipients were bred at estrus by artificial insemination with Hanwoo semen and then transfered an additional embryo produced in vivo or in vitro to tbe uterine horn contralateral to the corpus luteum on Day 7. The results obtained were as follows ; 1. The pregnancy rate was higher in young recipients of 3 years (68.8%) than in old ones of 10 years and greater(36.4%). And for CL size pregnancy rate was 57.9, 45.4 and 60.1% in large, medium and small size of CL of recipients, respectively. 2. 447recipients were transferred an additional embryos at 7th day after Al and average pregnancy rate was 57.5% and twin production rate was 22.2%. 3. Average pregnancy and twin production rate by direct transfer methods of frozenthawed IVF embryos was 56.0 and 16.7%. 4. The ratio of male to female twin in a total of 55 twin pairs was 54.6%, and average gestation lengths of male to female and female to female twin were 280.65.4 and 279.715.4 days, respectively. Average birth weight of twins was beavior in male and male twin(23.2i5.8kg) than in male and female twin(20.52.6kg).