This study was performed to evaluate whether vitrification method using ethyle glycol and eletron microscopic (EM) grid could be used far the cryopreservation of human oocytes in ART program. Surplus oocytes were obtained from consented IVF patients. These surplus human oocytes were frozen with our vitrification method, Oocytes were exposed to 1.5M ethylene glycol (EG) in DPBS far 2,5 minutes, followed by 5.5M EG plus 1.0M Sucrose in DPBS for 20 seconds. Then oocytes were transferred onto the EM grid and the grid was plunged into LN2 for storage. For thawing, oocytes containing EM grid were sequentially transferred in 1.0M, 0.5M, 0.25M, 0.125M and 0 M sucrose in DPBS solution at the intervals of 2.5 minutes. Thawed and survived oocytes were provided for ICSI. Embryos from vitrified oocytes were transferred to uterus of the patient on 4 to 5 days after ovulation in natural cycles of on 15 to 17 day of hormone replacement cycles. A total of 370 oocytes from 26 patients were thawed and 159 (43.0%) of them survived. One hundred thirty four oocytes (84.3%) were fertilized normally and 126 pre-embryos were transferred to 26 patients, resulting in 5 clinical pregnancies. The pregnancy rate per transfer was 19.2% and implantation rate was 4.0%. Among the five pregnant, 4 patients delivered 4 healthy babies and the one patient was 32-week ongoing pregnancy. From this results, vitrification using ethylene glycol as cryoprotectant and EM grid is a rapid and simple method that can be effectively applied for the cryopreservation of human oocytes in ART program.

Maternal stress was very common symptom that every pregnant women could have experienced during pregnant period. We found that the causative factors of stress for subjects were physical change(50.8%), family relationship(13.6%), change of body image(7.4%), concern baby(6.8%), economic difficulties(6.4%), depress(4.3%), morning sickness(3%), and miscellaneous(3.5%). According to our study, maternal stress during the first trimester negatively correlated with food and nutrients intake of pregnant women and gave low weight gain during pregnancy and low birth weight of infants. It also significantly correlated with monthly income(p〈0.001) and monthly food cost(p〈0.001) during pregnant period. Mean intake of Calcium and Iron were 47~60% of RDA and 35~48 of RDA, respectively. With the Iron supplement the total intake of Iron exceeded 100% of RDA. We also found majority of food items, except seaweeds, fruits and vegetables, were negatively correlated with maternal stress during three trimesters. Additional support system projected by professionals in health-care field could mediate maternal stress and lead to healthy pregnancy outcome.

This study was done to examine the relationship between serum chemical values (urea nitrogen, glucose, total protein and cholesterol) of recipients and pregnancy rate following embryo transfer. Blood samples were taken from 184 Holstein heifers or cows on Day 6 or 7 (Day 0=day of estrus) to analysis for serum urea nitrogen, glucose, total protein and cholesterol concentrations. After selection of recipients, frozen Holstein embryos were thawed and directly transferred to recipients non-surgically. The average serum concentrations of urea nitrogen, glucose, total protein and cholesterol were 13.8 mg/dl, 56.5 mg/dl, 7.2 mg/dl, 124.8 mg/dl, respectively. The average concentrations of serum urea nitrogen and cholesterol were lower (P<0.05) in pregnant recipients (10.7 mg/dl, 99.2 mg/dl) than in non-pregnant recipients (13.0 mg/dl, 122.2 mg/dl), respectively, although the concentrations of glucose and total protein were not different. These results show serum urea nitrogen and cholesterol concentrations can be used important factors for selection of recipients in Holstein.

To investigate the effect of co-transfer of trophoblastic vesicle (TV) with frozen-thawed in vitro Produced (IVP) bovine embryo on pregnancy rate, IVP blastocysts were transferred to synchronized recipients. Elongated blastocysts were recovered at Day 13 to 15, and dissected more than 4 pieces to removed the embryonic disc. Throphoblastic fragments were cultured for 48 hours to make throphoblastic vesicles (TVs). TVs were cryopreserved in ethylene glycol or vitrification solution and frozen-thawed TVs were co-transferred to recipients with frozen-thawed IVP embryos. 1 The recovery rate of elongated blastocyst on Day 13 to 15 was 22.5% (18/80) and the size of recovered elongated blastocysts was 0.2∼5.0mm. 2. Eighteen elongated blastocysts were dissected into 88 pieces and 61.4% of those pieces were formed to TV (54/88) 3. The viability of frozen-thawed TV in ethylene glycol was higher than in vitrified solution (92.8% vs. 68.8%) 4. The pregnancy rate in co-transfer with frozen-thawed TV and IVP blastocyst was better than transfer only IVP blastocysts (50.0% vs. 23.1%).

The aims of these study were to diagnose early pregnancy and reproductive disorders by using progesterone concentration and ultrasonography. The measurement of blood progesterone (P) concentration was conducted to diagnose pregnancy and to detect corpus luteum (CL) or evaluate disorder of CLs. As a result, the incidence rates of reproductive disorders were as follows : SH and EED (41.9%), inacitve ovaries (32.6%), follicullar cyst (9.3%), PCL (7.0%), endometritis (4.7%), pyometra (2.3%) and luteal cyst (2.3%). 61 Cows having Pconcentration 1.0 ng/ml(at the insemination) were increased to 1.0 ng/ml 6day after insemination. 50 cows among 61 cows were diagnosed pregnant. 8 cows among 13 HanWoos having Pconcentration 1.0 ng/ml at the insemination and 1.0 ng/mnl 6 day after insemination had non-ovulatory estrus and the others had Pconcentration 1.0 ng/ml at the insemination and 1.0 ng/ml 6 day after insemination, which was the error of estrus detection. All 13 cows were diagnosed non-pregnant. 47 cows diagnosed pregnant after insemination of Pconcentration 3.0 ng/ml were examined by ultrasonography at 30 day post-insemination. As a result, 41 cows were diagnosed pregnant (87.2%) but 14 cows having Pconcentration 3.0 ng/ml at 21 day after insemination was diagnosed to non-pregnancy. Calving intervals by surveying 100 cows were as follows 11~12 months (20%), 12~13 months (36%), 13~14 months (19%), 14 months (25%), respectively. In conclusion, hormone and ultrasonography help to detect reproductive disorders exactly and diagnose early pregnancy. This study suggest that diagnosis of early pregnancy and reproductive disorder by blood Pconcentration and ultrasonography improve reproduction management of HanWoo.

The ovaries of 178 Holstein heifers or cows (heifer; 41, 1 parity; 72, 2 parity; 65) on Day 6 or 7 (Day 0=day of estrus) were examined by transrectal ultrasonography. Diameter of corpus luteum (CL) and large follicle ( 10 mm), and luteal tissue area were determined by ultrasound system with a 5 MHB rectal probe. Blood samples were taken to progesterone analysis. After selection of recipients, frozen Holstein embryos were thawed and directly transferred to recipients non-surgically. The diameter of CL and luteal tissue area was greater (P<0.01) on Day 7 than on Day 6 in heifers, 1 parity or 2 parity cows, respectively, although progesterone concentrations were not different. The presence of fluid-filled luteal cavitied or multiple CL (2 or more) did not affect serum progesterone concentration. A large follicles were observed in 67.4% of heifers or cows and the average diameter was 14.1 mm. Greater luteal tissue area attributed higher pregnancy in heifers, but not in cows, although there were no difference on pregnancy rate according to progesterone concentration in heifers or cows. The pregnancy rate of recipients contained a large follicle at embryo transfer was lower than that of recipients not contained. These results show ultrasonic assessment of ovaries in Holstein recipients is a reliable tool to determine the follicle and CL for recipient selection.

This study was undertaken to test the hypothesis the hypothesis that treatment with hCG (5,000 IU) at the time of embryo transfer would enhance pregnancy rates in recipients, and the concentration of plasma urea nitrogen(PUN) in recipients was related to the effect of hCG on the reproductive performance. Blood samples were taken according to experimental condition for the assessment of the endogenous plasma progesterone concentration and plasma urea nitrogen. Concentrations of progesterone in plasma were higher in cows treated with hCG on day 7(estrus=day 0) than in those untreated during 7∼43 days after insemination. The pregnancy rates were 65.5 and 54.6% for the hCG treated and untreated groups, respectively. In recipient group categorized with PUN concentration of <12 mg/이, the pregnancy rates were 68.8 and 46.7% for the hCG treated and untreated groups, respectively. The results suggest that hCG treatment at 7 days after insemination could be used to increase the pregnancy rate of embryo transfer, and transfer, and only the recipients with PUN concentration of <12 mg/dl were influenced by treatment with hCG.

This experiment was undertaken to investigate the effect of administration of Agyoju or Mokhyang on the maintenance of pregnancy, delivery and sex ratio in the mice in different gestation periods. Adult female mice were administered orally at three different periods, from ovulation to implantation (Exp.1), from post-implantation to organogenesis (Exp.2), and from fetal growth to parturition (Exp.3). In Experiment 1, number of fetus implanted and mean body weight were not significantly different. However, the delivery of male offspring was significantly increased in Agyoju and Mokhyang administrated groups than control group. In Experiment 2, the number of fetuses implanted, live offsprins and their body weight at delivery were significantly increased in the Agyoju administered group than Mokhyang and control groups. In Experiment 3, the number of live offspring and sex ratio were not different in both treatments and control group. However, mean body weight at delivery was significantly increased in both treatment groups than that of control group. These results suggest that 1) Agyoju and Mokhyang have beneficial effects in maintenance of pregnacy, and that 2) The action of unknown component(s) in Agyoju may be related to selection of male spermatozoa for fertilization in vivo, and that 3) the administration of Agyju of Mojhyang during mid-and late-pregnance periods were shown the increment of body weight of live offspring without decrease of litter size.

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.

Bovine oocytes surrounded with compact cumulus cells were cultured for 20 to 22 hours(, 5% ) in modified TCM-199 medium supplemented with 5% superovulated cow serum(SCS) and inseminated by in vitro capacitated spermatozoa. Day 7 to 8 embryos were equilibrated for 10 minutes in 1.3M methyl cellosolve(MC) <1.1M diethylene glycol(DEG), 1.8M ethylene glycol(EG), 1.6M propylene glycol(PG) and 1.1 M 1,3-butylene glycol(BG) solutions. They were then loaded into 0.25ml straws, placed into an alcohol bath freezer at , cooled from to at /minute, seeded, held for 10 minutes, and stored in liquid nitrogen. After thawing in water, the embryos wee rehydrated in TCM-199 medium and then cultured for 48 hours in TCM-199 plus 5% SCS. Embryos were considered viable if they progressed to later developmental stages with a good morphology. Some of the embryos frozen in each cryoprotectant were thawed and transferred non-surgically without removing the cryoprotectant. Hatched embryos survived freezing and one-step dilution as follows : EG(50.0%), MC(53.6%), DEG(56.9%), PG(58.0%) and BG(11.5%). The survival rate of embryos cooled at vs. /minute was not significantly different(P<0.05), however, blastocysts hatched most often (P<0.01) in vitro when cooled at a rate of /minute(64.6%), 31/48) than at /minute(22.6%, 12/53). Pregnancy rates resulting from embryos frozen in the different cryoprotectants were as follows : MC(48%, 10/21); DEG(30%, 3/10); EG(74%, 20/27); and PG(40%, 4/10). These results indicate that MC, DEG, EG and PG have utility as cryoprotectants for the freezing and thawing of IVF Bovine embryos.

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)