A combined technology of transvaginal ovum pick-up(OPU) system with in vitro-oocyte manipulation technique can be used for improving reproductive efficiency in the cattle. The objective of this study was to establish a newly-conceived breeding program using OPU in the pregnant cows. The OPU trial was performed in pregnant cows every 10 days from 40 through 90 days of artificial insemination (Al), and number of follicles in ovary, number of retrieved oocytes and embryo development following in vitro-fertilization, were evaluated. Reduced number of follicles in the ovaries of pregnant cows was firstly detected from 70 days after A' and a significant (P<0.05) decrease in the follicle number (5.4 follicles /donor) was found at 90 days than at 40, 50, 60 and 80 days after Al (8.0~9.2). A similar pattern was also observed in the number of oocytes retrieved by OPU apparatus during experimental period. When retrieved oocytes were matured and inseminated in vitro with frozen bull semen, development of the oocytes to the blastocyst stage was not significantly affected by the retrieval time. Four embryos (morula or blastocyst stage) derived from oocytes retrieved from pregnant cows were nonsurgically transferred to four recipient cows on day 7 of estrus cycle. For the first time in Korea, three of four transferred embryos developed to live calves with normal physiological parameters. In conclusion, an effective breeding program employing pregnant cow can be developed by use of OPU trial and in vitro culture techniques of oocytes ; OPU system could be repeated in pregnant cows with no risk of abortion and viable offsprings were borne after transfer to the recipients.

This study was carried out to establish an effective system for embryo transfer techniques by analyzing several factors affecting in-vivo embryo transfer in Korean cattle Embryos were transferred into a toral of 301 recipients. The results obtained in studies on the factors affecting pregnancy rate after embryo transfer by condition of transfer time were as follows ; 1. The pregnancy rate by the seasons of transferred fresh and frozen embryos were not different, but the pregnancy rate was slightly higher in summer(80.8%). 2. The pregnancy rate by the days of embryo transfer after estrus were not different when fresh embryos were transferred, but the pregnancy rate was highest at 8 days when frozen embryos were transferred(P<0.01, 40.0%). 3. The pregnancy rate at estrus synchronization was remarkably higher with PGF treated than natural (P<0.05, 70.4%, 43.4%). 4. The pragnancy rate by the degree of estrus synchronization was best when the estrus was synchronized in both fresh and frozen embryos (83.3% and 29.7%, respectively), but the pregnancy rate was not different among 2 days. But the pregnancy rate of frozen embryos were slightly higher when the recipients exhibited estrus earlier than donors.

This study was carried out to establish an effective system for embryo transfer techniques by analyzing several factors affecting in-vivo embryo transfer in Korean cattle. Embryos produced in-vivo were transferred into a total of 301 recipients The results obtained in studies on the factors affacting pregnancy rate after embryo transfer by condition of recipients were as follows ; 1. The pregnancy rate by age and parity of recipients showed high in 5~8 and over 12 years old(72.7~73.9%), and 3rd~4th parity(82.1%) for fresh embryos(P<0.05). The pregnancy rate did not differ by age and parity of recipients in frozen embryos. The pregnancy rate of frozen embryos tended to be similar to that of fresh embryos(38.5% and 25.0~36.7%). 2. The number of observation for normal estrus cycles of recipients did not differ In pregnancy rate between one and 2 times in fresh embryos(64.9%, 69.8%). The pregnancy rate by transferred frozen embryos showed significantly higher after 2 times of observation(P<0.05, 16.3%, 37.5%). The pregnancy rate by days open did not differ between fresh and frozen embryos. But the pregnancy rate was slightly higher in 12 months and 6 months of days open for fresh and frozen embryos, respectively(70.1~71.1% and 24.5%, respectively). 3. The pregnancy rate of transferred fresh and frozen embryos into right and left side of uterine horn did not differ(62.1% : 65.9% 25.0% : 24.3%, respectively). The pregnancy rate by the grade of CL was not different in fresh embryos, but the pregnancy rate was significantly higher in the grade A than B for frozen embryos(P<0.01, 43.2%, 16.2%).

This study was carried out to establish an effective system for embryo transfer techniques by analyzing several factors affecting in-vivo embryo transfer in Korean cattle. Embryos produced in-vivo were transferred into a total of 301 recipients. The results obtained in studies on the factors affecting pregnancy rate after embryo transfer by condition of embryos were as follow ; 1. The pregnancy rate of 301 recipients was 45.2% and higher with fresh embryos than with frozen embryos(63.5% : 21.4%, P<0.01). Embryos superovurated by FSH-P had slightly greater than by SUPER-OV in pragnancy rate, athough these were no difference between two treatments. 2. The pregnancy rates of transferred morulae and blastocysts showed no difference between fresh and frozen embryos(63.5% : 63~6% ; 20.0% : 25.8%). However, the pregnancy rates by quality of flesh and frozen embryos were significantly different(P<0~05). The pregnancy rates were outstandingly high in the grade A, B of fresh embryos(59.0~66.4%), and in the grade A of frozen embryos(43.6%). 3. The number of transferred embryos showed no difference in pregnancy rate, but when frozen embryos transferred, the pregnancy rate was slightly higher with two embryos than that with one embryo.

Experiments were conducted to assess the effect of quality and viability of bovine blastocysts derived from in-vitro culture(IVC) of in vitro matured and fertilized(IVM-IVF) oocytes during their transport 2 hours. Follicular oocytes were collected form ovaries obtained at a slaughterhouse and were cultured for 24 hours in TCM-199. The IVM oocytes were fertilized in vitro with caudal epididymis spermatozoa. Fertilized oocytes were cultured for 7 to 9 days, and embryos that developed to the blastocyst stage were used for the experiment. The blastocysts, packed in straws with storage medium that consisted TCM-199 with HEPES equilibratd in air and supplemented with 10% FCS were transported at 39~(2.0 h). The quality of blastocysts was assessed and ranked as A(excel-lent), B(Good), fair or poor after transportation. The percentages of A and B grade blastocysts after transport duration for < 1 hours(97.7%) were similar to the result from transport duration for 1~2 hours (92.9%) and 2~3 hours(89.6%), but significantly(P<0.05) higher than transpot duration for 3~4 hours(76.3%). The percentages of A and B grade blastocysts after transport duration for two hours from developed blastocyst at 7day(100%) and 8day(85.0%) were higher 9day(96.6%) and >9day (40.0%). And early to expanded blastocyst produced in vitro were transferred to recipient cow by additional embryos at 7 and 8th day after AI. Three of them were pregnant to term and produced four twin calves, and two calves was premature birth. The gestation lengths of male to female and female to female twin were 282 and 281 days, respectively. And birth weight of twin calves were male to female(22.Skg) and female to female twin(20.3Okg), respectively.

In vitro fertilization(IVF) derived morula and blastocyst embryos were bisected by a simple method and cultured in vitro without zona pellucida And also bisected embryos were frozen-thawed and cultured in vitro) to evaluate the survival rate. The results obtained were as follows : The average number of grade I or II immature follicular oocytes recovered by slicing method per ovary was 11.9 from 142 ovaries. Following in vitro fertilization, the rates of cleavage and in vitro development to morula and blatocyst were 61.7 and 32.2% respectively. The successful bisection rate of IVE embryos was 67.51%, and the embryos of blastocyst stage were bisected successfully at significantly(P

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)

Many farmers apply cattle slurry on grassland, often in excess of crop N requirements, resulting in groundwater contamination. Our research objective was the development of grassland management systems that reduce undesirable sideeffects on other compartm

The aim of the study is to describe the fate and transformation of nitrogen in grassland ecosystems. Field experiments were conducted using sandyloam soil under variabling conditions: Zen, fertilization, reduced slurry application(l20kg N ), usual sluny a