The ultrasound-guided oocytes cllection (ovum pick-up ; OPU) has become a substitution for superovlation in cattle. The objective of this study was to examine the effect of OPU frequency on the in vitro production of embryos in Hanwoo cattle. Six cycling Hanwoo cows were distributed into two groups for either once or twice weekly OPU sessions. Oocytes were collected by ultrasound-guided follicle aspiration(SA600) using a 6.5HMz transducer and attached with 18 gauge needle, with vacuum pressure of 40 mmHg. The cumulus-oocyte complexes (COCs) collected from each donor were matured in TCM 199 supplemented with 10% fetal bovine serum at 5% CO2 in air at 38.5 for 22h and in vitro matured oocytes were co-incubated with sperm(separated by Percoll gradient) for 6h. The zygotes were co-cultured on cumulus cell monolayer in 10ul droplets in the same culture medium and conditions used for IVM for 7 days. On Day 7 of culture, development to blastocysts was examined. Although the number of oocytes collected was variable depending on individuals, overall embryo production in the twice per week OPU sessions was better that in the once per week sessions(6~21 vs 2~7 blastocysts produced, respectively). Two cows(E, A) were good oocyte donors and embryo production was superior in cow C ; however, cow F was a poor donor as compared to the others. In conclusion, these results suggest that for embryo production, twice weekly OPU sessions were better than once per week for producing embryos in vitro from Hanwoo cattle

This study was conducted to investigate the effects of in vitro fertilization, culture and embryo development according to in vitro maturation rate, protectant composition and equilibrium time after frozen /thawing of bovine immature oocytes. This results obtained in studies on the effect of different cryoprotectants on the viability, maturation and development of in vitro bovine oocytes were as follow: 1.The post-thawing of immature oocytes matured to metaphase II during culture time for 0 to 26 h, and those group (62~3%) were low than control group (76.7%). The optimal maturation time of frozen-thawed immature oocytes was at 24 h. 2.The viability of cryopreserved immature oocytes was not affected by sort of cryoprotectants. The developmental competence of frozen4hawed oocytes was not affected by cryoprotectants. These results indicate that an optimal maturation time of frozen /thawed immature oocytes was at 24h. Furthermore the viability of cryopreserved immature oocytes was not affected by sort of cryoprotectants and developmental competence of frozen /thawed oocytes.

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 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).

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.

The object of this study was to evaluate the effect of uterine epithelial cells on development of Korean native cattle(KNC) oocytes fertilized in vitro. Qocytes were collected from ovaries of slaughtered Korean Native Cows and matured in TCM199 with granulosa cells supplemented with 10% FBS, 5g/ml FSH, 10 JU/ml hCG, and 1g/ml estradiol-17 for 24 hrs. For co-culture of in vitro development of fertilized ova, oviductal epithelial cells (ll0˚cells /ml) obtained from slaughtered cow and uterine epithelial cells(110˚cells /ml) flushed from the superovulated holstein on Day 7 were incubated in 39, 5% , 95% air. Frozen-thawed KNC sperm was capacitated with BO(Brackett & Oliphant, 1975) medium supplemented with 10mM, 5mM-caffein. Matured oocytes were inseminated for 20 hrs. And then fertilized oocytes were washed with culture medium and transferred to oviductal epithelial cells for in vitro development and three days later a portion of embryos were transferred to uterine epithelial cells. Stastical methods of developmental rates on KNC-IVF oocytes was ANOVA-test. Developmental rates of KNC-IVF oocytes was significant higher(P<0.01) when co-cul-tured with uterine epithelial cells(25.2%) than oviductal epithelial cells. Blatocyst cul-tured for 7 to 9 days were frozen by automatic freezer with 1.4M glycerol-PBS. Survival rates of blastocyst was 40.0%. Fourteen frozen-thawed blastocysts were transferred to five holstein heifers on day 7 after natural estrus. Three recipients were observed twin and one recipient was single by ultra-sound systems on days 45 after embryo transfer.

This experiment was carried out to study the determination of survival of vitrified and thawed mammal follicular oocytes by FDA-test. Oocytes were divided into 3 groups according to attachment of cumulus cell. Group A oocytes were tightly surrounded by cumulus cell, group B oocytes were partially surrounded by cumulus cell, and group C oocytes were poorly surrounded by cumulus cell. Vitrification solution developed by our previous study (Kim et al, 1992) which consisted of permeable agent (20 % glycerol + 10 % ethylene glycol) and nonpermeable agent (30 % Ficoll + 10 % sucrose). Oocytes (7~10) loaded into 0.25 ml straw after 10 min equilibration were plunged into liquid nitrogen (- 196) directly. The FDA-score of vitrified and thawed group A oocytes was higher in rat (4.2) than in rabbit (3.9), cow (3.8), mouse (3.4) and porcine (2.4), however that of cumulus cell was higher in rabbit (4.7) than in rat (4.1), cow (2.9), porcine (2.6) and mouse (1.4). The FDA-score of vitrified and thawed group B oocytes were 3.1 (cow), 2.9 (rabbit), 2.9 (mouse), 2.6 (rat) and 2.5 (porcine), respectively. However that of cumulus cell was higher in rabbit (3.7) than in porcine (2.6), rat (2.3), cow (1.7) and mouse (0.3). The FDA-score of vitrified and thawed group C oocytes was higher in mouse (4.1) than in cow (2.9), rabbit (2.6), rat (1.3) and porcine (1.1). As shown in the above results, The survival rates of oocytes were higher in group A than in group B and C except in mouse and cow. These results suggest that the survival of cumulus cell as well as follicular oocytes can be reliably judged by their fluorescence with FDA-test.

본 연구는 체세포 핵이식 복제 수정란의 이식에 의한 고능력 한우를 다량 증식하기 위한 방안을 확립하기 위하여 수행되었다. 본 실험에 공여된 체세포는 육질과 육량 등급이 국내에서 100위 이내의 암소 귀세포를 채취하여 동결 및 계대배양하여 사용하였다. 한편, 핵이식 수정란의 준비를 위하여 도축장에서 채취한 난소에서 난자를 회수하여 22시간 성숙배양 후 난구세포를 제거하고 극체가 존재하는 난자만을 선별하여 recipient cytoplasm으로 이용하였다.