These experiments were conducted to examine the effects of theophylline, pentoxifylline and heparin on frozen-thawed Hanwoo sperm for enhancing motility and viability of sperm. Frozen-thawed semen collected from one bull was treated in TALP(tyrode-albuminlactate-pyruvate) containing varous concentrations of theophylline and pentoxifylline. After incubated at 5% CO2 in air atmosphere for 6 hours, the motility of sperm after the treatments was characterized by CASA(computer aided semen analysis) system. When monitored notility(MOT) and curvilinear velocity(VCL), theophylline and pentoxifylline exerted their optimal action at the concentration of 30 mM and 3 mM, respectively. No difference of sperm motility was observed when the sperm was treated with both substances compared with a single treatment of each substance. Comparison was then made for evaluating the effect of theophylline and / or pentoxiophylline on the motility and viability of significant treatment effects of each substance, high MOT and VCL values were detected in sperm treated with theophylline. In the case of sperm viability examined by an eosin-nigrosin staining, however, a significant decrease was found after the combined treatment of theophylline+pentoxyphilline than after the treatment with heparin alone or no treatment(P<0.05). In conclusion, theophylline, pentoxiphylline or heparin can be used for enhancing the motional characteristics and viability of frozen thawed Hanwoo semen. Considering characteristics of these substances, theophyline may be useful in the artificial insemination system, which requires vigorous sperm motility. While, heparin supporting sperm viability in vitro can be effectively used for improving in vitro-fertilization system.

The purpose of this study was to develop a breeding technique to increase Hanwoo of superior characteristics. In the present study, reproductive status of Hanwoo such as size of farm, breeding system and gestationi length was investigated. In addition, effect of low dose administration of prostaglandin F2(PGF2) on luteolysis was examined. The size of farm was classified by the total number of cows and the number of breeding stocks, respectively. The distribution of herd size of < 5, 6~10, 11~30, 31~50 and > heads was 31%, 15%, 39%, 4% and 11%, respectively. Furthermore, the distribution of breeding stock size of <5, 6~10, 11~30, 31~50 and > 50 heads was 36%, 28%, 31%, 3% and 3%, respectively. Average parity was 2.1 in breeding stock. In breeding pattern, artificial in semination(A.I), estrus synchronization-A.I and natural mating was 92.7%, 2.4% and 4.9% respectively. Gestational length of Hanwoo was ranged 253~316 days (average length : 285 days) after estrus( estrus=0). To induce luteolysis, PGF2 was injected into ovarian parenchyma by a modified ovarian injector. The effect of administration of 6mg PGF2 on luteolysis and estrus induction was betweer (P<0.01) when PGF2 was administered into ovarian parechyma than when administered intramuscluarly (71 vs. 91%). When PGF2 was injected into ovarian parenchyma, a decreased concentration to 3 mg did not significantly decreaed its luteolytic effect(92%). When AI was performed following PGF2 treatment, the intraovarian injection group yielded a higher pregnancy rate(69 vs. 88%) than the IM injection groups, regardless of the dosage. In conclusion these results suggest that increasing herd size and regular reproductive management are needed to improve reproductive efficiency in Hanwoo industry. Furthermore, intraovarian administration of PGF2 is effective way to induce luteolysis compared with intramuscular injection.

The objective of this study was to determine the cause and prevalence of bovine abortion and stillbirth in Kyungi-do area. Seventy three bovine fetuses were collected from farms and submitted to the College of Veterinary Medicine, Seoul National University. Submitted fetuses were evaluated during a 4-month period (July to November, 1999) for pathological lesion, tissue protozoa, bacteria and viral infection. The average proportion of abortions was decreased with parity in 73 abortion heifers and cows. Monthly incidence rate of bovine abortion was not different in this study. In fetuses from 90 to 282 days gestation, the majority were between 150 and 250 days gestation(58%). The cause of abortion or stillbirth was determined in 51% of the cases examined. In 15(21%) of the fetus, neosporosis were diagnosed by pathological findings. In three (4%) additional fetuses in three additional fetuses, suspected Neosporosis by pathological lesion, and in 3 (4%) fetuses examined Neopsorosis were diagnosed in 15 feturses and in 3 fetuses, Neosporosis was suspected by pathological legions. Neosporosis / viral infection were diagnosed in three additional fetuses). Miscellaneous bacterial infection, BVDV, iatrogenic cause, Neosporosis / IBRV / BVDV, miscellaneous viral, IBRV/BVDV and others were 3(4%), 3(4%), 2(3%), 2(3%), 1(1%), 1(%) and 9(12%) respectively. The cause and incidence of bovine abortion in different area in Kyungi-do was not different in this study.

In this stuyd, the effect of the dominant follicle aspiration for the superovulatory response in HanWoo was investigated. The criterion for the presence or absence of a dominant follicle based on their morphological examination. The dominant follicle was aspirated 48hr before the onset of superovulation treatment by 6.5MHz convex probe connected with a carrier and superovulation induced by FSH (Super-Ov Tyrer, Texas, U.S.A) adminstered twic a day s.c. over 4 day in a decreasing regimen. From 13 HanWoo scanned daily to determine the presence and growth of the dominant follicle, its an average diameter of 15.4mm was measured and an average diameter of corpora lutea was 18.7mm on day of follicular aspiration. In the experiment, a follicular remove by ultrasound-guided aspiration, the ovarian response was significantly enhanced when animals were superovulated in the aspiation of a dominant follicle compare with animals superovulated non-aspiration of a dominat follicle. In the aspiration of a dominant follicle donors yieleded more corpora lutea(14.44.7 vs 8.63.4) and transferable embryos(8.94.2 vs 5.42.7) than control. In cows in which the dominant follicle had been aspirated under sonographical control 2 days before superovuation, the number of corpus lutea and transferable embryos were significantly enhanced compared with animals superovulated in the presence of a dominant follicle (14.44.7 vs 6.92.7, ; 8.94.2 vs 3.31.6). After 7 days of artificial insemination, the embryos at 7 days were cllected by uterine flushing after dominant follicle insemination, the embryos at 7 days were collected by uterine flushing after dominant follicle aspiration and superovulation treatment, and evaluated their quality by morphological criteria. Sixteen embryos with excellent and good grade were transferred into 8 recipient cows. Six pregnancies were identified at 60 and 120 days of gestation by rectal palpations. In conclusion, the present study showed that 1) the presence or absence of a dominant follicle signficicnatly affects superovulatory responses, and 2) ultrasound-guided follicular aspiration of the dominant follicle and superovuation treatment provides an accurate and procedure to increase ovarian responses in HanWoo.

The present study was performed to evaluate the best electric fusion condition in nuclear transfer, Korean Native Cattle fibroblasts were used as nucleic donors. Oocytes from slaughterhouse were matured in vitro for 22 h and enucleated. Each individual cells were transferred into enucleated ocytes and reconstructed embryo were placed into the fusion chamber. In experiment 1, pulse were performed by altering pulse duration at 1. 75kv/cm, 1 time. When pulse duration is 30, fusion and development rates is higher than other conditions. In experiment 2, the effect of different pulse number were studied at the pulse duration 30 and the same pulse intensity. When pulse number was one, fusion rates were higher than other conditions. The fused embryos were moved to culture medium and assessed their development to blastocyst. These results showed that best fusion condition was 30 and one time. And the fibroblasts derived from Han Woo can be reprogrammed by nuclear transplantation and develop subsequently in vitro.

핵 이식에 공여되는 수핵난자의 효과적인 동결보존을 위하여 한우 성숙난자를 1.0 M dimethylsulfoxide(DMSO) 또는 1.0 M glycerol이 함유된 동결보호제를 이용하여 처리하거나, 동결보호제 처리 후 완만동결법을 이용한 동결융핼르 시행하여 상기 실험처리로 야기되는 투명대 경화현상을 관찰하였다. 도축장 유래의 난소에서 미성숙난자를 채취한 후 10% 소 태아혈청을 함유한 TCM-199을 이용하여 22∼24 시간 동안 체외성숙배양을 이해

The development potential of bovine somatic cells was evaluated using nuclear transfer. A single donor cell derived from fetus of HanWoo(Korean Native Cattle) was selected and deposited into perivitelline space of each enucleated oocyte before electrical fusion and activation. Nuclei of donor cells starved for 7 days (37%) tended to support the development of reconstitute embryo the blastocyst stage better than those of donor cells starved 3, 14 and 30 days. The cleavage rate was significantly lower(P<0.05) in reconstitute embryos derived from large size donor cells(51.2%), than those from small medium size donor cells(76.6 and 73.5, respectively). The developmental rate to blastocyst of reconstructed embryos from medium size donor cells was higher than those from small and medium size donor cells. This study demonstrates that an appropriate culture period for induction into quiescent stage and the size of donor cells effect on the efficiency of nuclear transfer using cultured bovine cells.

적정 배양액의 선정, ITS의 첨가와 BSA의 농도조절 및 NaCl 농도의 조절을 통해 소 수정란의 무혈청, 체세포배제 배양체계를 확립하기 위하여 수행한 실험에서 다음과 같은 결론을 얻었다. 1. 배양액으로 CRlaa, TALP 및 SOF를 사용하여 발육률을 검토한 결과, 발육률의 유의적인 차이는 나타나지 않았다. 2. 배양액내의 고분자물질원으로 BSA, FBS 및 PVA를 첨가하여 사용한 결과 BSA 및 FBS 첨가군이 PVA 첨가군보다 유의적으로 높

난관상피세포와 그 배양액에서 분비된 고분자 분획이 소정자의 수정능력에 어떠한 영향을 미치는지 알아보고자 실시한 실험에서 다음과 같은 결론을 얻었다. 1. 난관상피세포배양액으로부터 MW 5 kDa cut-off bucket을 사용하여 탈염 및 농축을 실시, 단백질/고분자분획을 회수하고 이를 체외수정용 배양액에 첨가하고 난관상피세포 monolayer와 공배양을 통해 체외수정을 실시한 결과 고분자분획첨가 및 난관상피세포 공배양(OM+OEC)군이 고분자분획첨가

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.

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.

The embryogenesis stimulating activity(ESA) had been shown in co-culture of embryos with bovine oviduct epithelial cell(BOEC) and culture in BOEG-conditioned medium. The present study was undertaken to purify and quantify the embryotropic proteins and to determine the optimum concentration of the embryotropic protein for the proper development of embryos. In BOEC-conditioned medium, five major bands of proteins were detected(66, 53, 40, 32 and 24 kDa) by SDS-PAGE. From these proteins, 288pg of protein that had a 32kDa molecular weight was purified by gel filtration column and perfusion chromatography ion-exchange column. When purified protein was supplemented to the in vitro culture media at various concentrations in protein-free media, 2.5g /ml supplement group showed significantly higher rates of embryo development into morula /blastocyst stages than other groups(p<0.05). In conclusion, we purified 32kDa protein from BOEC-conditioned medium and this protein showed optimum embryogenesis stimulating effect at 2.5g /ml.

From September 1993 to August 1997, we treated ovarian disorders in 1,782 repeat breeder cows after diagnosis by ultrasound on 35 farms in Kyeong-ki do. The rates of ovarian appearance were 59.8% of CL group, 16.7% of ovarian atrophy or hypofunction, 15.4% of luteal cyst, 4.3% of follicular cyst and 3.7% of follicle group in diagnosis with rectal palpation and ultrasound. The results of treatment for ovarian disorders were 1,316 cows(73.8%) in estrus, 348 cows(19.5%) in non-detected and 118 cows(6.6%) in unidentified. The rates of PGF, GnRH and mineral vitamin complex treatment to estrus were 79.6, 69.2 and 50.3%. Two groups were treated with 5 ml PGF intramuscular injection(I.M.) and 1.5 ml PGF intraovarian injection(I.O.), and the results of 1.5ml PGF I.0. were significantly higher than that of 5ml PGF I.M. in inductiom estrus(p<0.05). The pregnant rates were 29.8% in total repeat breeder cows with ovarian disorders following diagnosis and treatment. In summary, rectal palpation and ultrasonography were proven to be useful tools of diagnosis and treatment in ovarian disorders, and it was also suggested that the response to treatment with PGF I.0. was better than PGF I.M.

Supplementation of maturation medium with additional granulosa cells has beneficial effect on in vitro maturation of bovine follicular oocytes and their subsequent cleavage and development in vitro. However, maturation system using granulosa cells have some disadvantages that collection of granulosa cells is cumbersome and metabolic activity of the cells is variable according to ovarian cycle or follicular size. We hypothesized that bovine immsture oocytes matured without granulosa cell coculture can fertilize and develop normally if the medium volume per oocyte is reduced during in vitro maturation. Immature oocytes were matured for 24 hours in a TCM199 containing 10% fetal calf serum, anterior pitultary hormone (0.02 AU /ml Antrinⓡ) and estradiol with or without granulosa cells in vitro. In Group 1, 35 to 40 oocytes were matured in a well of 4-well plastic dish containing 500 l of maturation medium and granulosa cells, and 9 to 10 oocytes were matured in a 50-l drop of maturation medium without granulosa cells in Group 2. After maturation, oocytes were coincubated with sperm for 30 hours in a modified Tyrode's medium (IVF). Inseminated oocytes were cultured in a microdrop (30 l) of a synthetic oviduct fluld medium (SOFM) containing BSA, Minimum Essential Medium essential and non-essential amino acids for 9 days. As a preliminary experiment, we investigated the beneficial effect of granulosa cells during maturation on subsequent cleavage and development using the same type of culturedishes (4-well dish). Granulosa cells could not increase embryo cleavage after fertilization but significantly improved (p<0.05) embryo development to expanding blastocyst (Table1 and 2). In Group 1, 68 and 80% of inseminated oocytes have cleaved at 30 hours and 2 days after IVF, respectively, which is similar (p>0.05) to the result of Group 2 (69% at 30 hours and 78% at 2 days after IVF). The oocytes in Group 2 showed 21 and 11% of developmental rates to expanding and hatching blastocysts, respectively, which was not significantly different (p>0.05) from those (20 and 10%, respectively) of oocytes in Group 1. In conclusion, it has been clarified that a microdrop culture system without granulosa cells for in vitro maturation can support bovine embryonic development to blastocyst in vitro as readily as a granulosa cell coculture system.

The objectives of the present study were improvements in the efficiency of developmental rates to morula and blastocyst stages to produce a large number of genetically identical nuclear transplant embryos. The oocytes collected from slaughterhouse ovaries were matured for 24 h and then enucleated and cultured to allow cytoplasmic maturation and gain activation competence. And then the donor embryos were treated for 12 h with 10 g /ml nocodazole and 7.5 g /ml cytochalasin B to synchronize the cell cycle stage at 26 h after the onset of culture. The blastomeres were transferred into the perivitelline space of the enucleated nocytes and blastomeres and oocytes were fused by electrofusion. The cloned embryos were then cultured in various conditions to allow further development. The age of the recipient(30 vs 40 h) had no significant effect on the fusion rates(82.4 vs 82.1%) and the developmental rates to morula /blastocyst(9.8 vs 11.0%). Effect of Nocodazole treatment on the donor cell cyle synchronization to improve the developmental rates of bovine nuclear transplant embryos was significantly higher than control group(21.4 vs 10.1%, p<0.05). Significant differences were in the percentage of fusion rates(72.9,77.1vs 61.9%) in three types of fusion medium(PBS(+), mannitol and sucrose, p<0.01). The developmental rates of bovine nuclear transplant embryos appeared to be highest in mSOF medium under 5% 0 condition, but no significant differences were found when compared with TCM199-BOEC and mSOF under two different oxygen ratio(5 and 20%).

This study was conducted to compare the insemination time of bovine oocytes and determine the effects of glucose(1.5 mM) on the development of bovine embryos at early cleavage stage. Oocytes were matured for 24 h, followed by exposure to sperm and cultured in modified Tyrode's media drops or with bovine oviduct epithelial cell monolayer prepared in TCM199(BOECM). Insemination time and culture system were varied in each experiment. In experiment 1, to investigate the developmental capacity of bovine embryos after different time of exposure to sperm, bovine ova and sperm were co-incubated for 18, 30 or 54 h, respectively. The development to blastocysts of 30 and 54 h insemination groups were significantly higher(P<0.05) than 18 h group, and in case of blastocysts of cleaved embryos, 30 h group were significantly higher(P<0.05) than other groups. In experiment 2, we investigated the effect of glucose on early bovine embryos. After 18 h insemination, in vitro fertilized oocytes were separated following 3 groups ; G+0, C+24 and C+48. Oocytes of G+0 group were cultured in glucose added Tyrode's medium after fertilization, oocytes in C+24 and C+48 groups were cultured in glucose free Tyrode's medium after fertilization. After 24 h culture, G+24 group was moved to glucose added medium. All oocytes of 3 groups were moved to BOECM after 48 h culture. The rates of cleavage and development to blastocysts in G+0 group were significantly lower than other groups. In experiment 3, we determined the effects of glucose exposure from 8 to 20 h after insemination on the cleavage and development of oocytes. The oocytes in glucose added group had high capacity of cleavage and further development. This study shows that in bovine oocytes, the optimal exposure to sperm is 30 h and glucose exposure to bovine one-cell embryos is detrimental to their first cleavage and further development in vitro but there has no evidence of detrimental effect of glucose(1.5 mM) exposure to bovine embryos over the two-cell stage in vitro.

Real time B-mode ultrasound was used to detect the early conceptus in 187 Korean native cattles between days 10 and 60 after last insemination. The ultrasound diagnostic findings were systemically confirmed by palpation per rectum after the 60th day of last insemination. The embryonic vesicle and the embryo proper within the veside were first visible on mean day fl and 23, respectively. The heartbeat of the embryo proper could be detected on day 26, and the limb buds, placentomes, amnion, fetal movement, umbilical cord, optic area and split hooves were first visible on day 33, 34, 34, 44.5, 45, 32 and 48, respectively. The mean length of embryo proper was 3.8mm on day 23 which later increased to 56. 6rnrn on day 60. When ultrasound was used to detect the conceptus between days 20 and 30 after insemination and palpation per rectum after the 60th day of insemination, the accuracy rates of pregnancy detection by ultrasound scanning at days 20, 22, 24, 26, 28, 30 were 44.4, 69.2, 78.6, 87.5, 90.0, 93.3%. In summary, the early pregnancy diagnosis of Korean native cattle with ultrasound appears high accuracy rates. It is considered that ultrasound can be used in veterinary practice well.

The present study was undertaken to examine the critical effect of + concentration on electrostimulation and post-electrostimulation media for electric activation of in vitro matured oocytes of Korean Native Cattle. Oocytes collected from slaughterhouse ovaries were matured in TCM 199 containing FSH, estradiol-17 and FBS with granulosa cell monolayer for 24 hours and denuded with hyaluronidase. And then cumulus-free oocytes were submitted to a DC field of 1.0 kV/cm for 60 sec in electroporation media(0.28 M mannito' and PBS) with different + concentations (0.00, 0.05, 0.10 and 0.15 mM). Stimulated oocytes were stained and examined for pronuclear formation after incuhation in SOF for 12 hours. The rates of pronuclear formation in hovine oocytes electrically stimulated in 0.28 M mannitol with 0.05, 0.10 and 0.15 mM +(60.3, 82.2 and 75.0%) were significantly higher than without +(6.3%) at 12 hours after an electric pulse(p<0.005). The activation rates of Korean Native Cattle oocytes stimulated in PBS supplemented with 0.05, 0.10 and 0.15 mM +(71.0, 75.8 and 75.4%) were significantly higher than without +(23.5%) after post-stimulation incubation(p<0.005). After incubation of oocytes in SOF with and without + following electric stimulation in 0.28 M mannitol with 0.10 mM +, the rates of pronuclear formation of bovine oocytes in +-free SOF(85.7%) was significantly higher than in SOF with 1.71 mM +(62.5%, p<0.05). When oocytes were stimulated in two electrostimulation media supplemented with + and incubated in +-free SOF, there were no significant differences in the rates of pronuclear formation hetween 0.28 M mannitol and PBS. These results indicate that a single electric pulse could induce activation of Korea Native Cattle oocytes in 0.28 M mannitol and PBS supplemented with +. Furthermore, to improve the activation rates, it was hetter that stimulated oocytes were incubated in +-free SOF after electric stimulation than in SOF with +.

The aim of this study was to evaluate the effect of recombinant bovine somatotropin (bST; Boostin-S, LG Chem) treatment with FSH (Super OV) or PMSG on superovulatory response for transvaginal ultrasound-guided oocyte retrieval (TVR) in calves. Eight Korean Native Cattle(KNC) heifer calves; 150 to 240 days old; were randomly assigned to four treatment groups: 1) FSH(75 mg); 2) FSH (75 mg) + bST(500mg) 3) PMSG(1;000 IU); 4) PMSG(1, 000 IU) + bST(500 mg). Experimental calves in group 1 (n=2) and 2(n=2) were weekly superovulated for 4 consecutive weeks with daily injection of FSH for 3days and the next day subjected to TVR session. Animals in group 3 (n=2) and 4(n=2) were weekly stimulated for 4 consecutive weeks with a single dose of 1, 000 IU PMSG. TVR was performed on 72 hours after PMSG injection. Calves in group 2 and 4 was received injection of 500 mg of bST every 10 days. At each TVR session, follicle number and size were recorded; the oocytes collected and graded according to cumulus and cytoplasm investment. Collected oocyte were determined viable oocyte according to morphological quality with granulation of oocyte and number and status of cumlulus cells. IVM and IVF were performed and assessed cleavage rate on day 3 after fertilization. A Sonovet 600(Medison, Co., Ltd) realtime ultrasound scanner with a 6.5 MHz convex transducer, fixed at the tip of 500 mm estended handle equipped with a needle guide was used in collecting oocyte. Differences between groups were analysed by chi-square test. The population of large follicle (5 nun) and aspiration rate were not significant different among the 4 groups. But, the number of small follicles (<5 mm) and aspirated oocyte in the KNC calves treated with bST were 1.3~1.6 times higher than in KNC calves treat with FSH or PMSG alone. In conclusion, the administration of bST with FSH or PMSG at superovulation for TVR in calves was increase the nurnber of small follicle which was influenced the number of aspiratable follicle.