• Korean Journal of Animal Reproduction
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• v.7 no.2
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• pp.41-52
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• 1983

Animal in dustry in Korea urgently needs the domestic introduction and the industrial a, pp.ication of embryo transfer technique. Namely, this technique can be utilized effectively, as means of the improvement of livestocks, as means of the increase of meat production, as means of substitute for the livestock import, and dissemination of new breed. However, as this technique avaliable in our country is remaining initial stage, we can not make use of the technique industrially unless we make much improvement as follows; induction of superovulation, non-surgical recovery of embryos, synchronization between the estrus such cycles of donor and recipient, non-surgical transfer of embryos, etc. Simultaneously, the basic studies such as harvesting oocytes from ovary, in vitro culture of oocytes, in vitro capacitation of spermatozoa, cloning by culture of blastomeres and transfer of nuclei, sexing embryo, etc. should not be neglected in order to make the technique of embryo transfer more simple and convenient. For the success of these studies, universities, national and public institutes, large scale cattle farms, and small scale cattle farms should cooperate each other. For instance, universities undertake basic researches, and the national and public institutes a, pp.y the results of the researches to animal industry along with cooperation by large scale cattle farms. By the help of the cooperative organizations, the technique relevant to our environment and farm condition may be able to be finalized, and to be a, pp.ied to samll scale cattle farm. Consequently, being served to stimulate animal productivity, this technique can be contributed to the development of livestock industry in Korea.

• Journal of Embryo Transfer
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• v.14 no.3
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• pp.219-224
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• 1999

The effect of several potential antioxidants were examined as a means of increasing the in vitro development of in vitro matured and in vitro fertilized oocytes into morulae and blastocysts. Korean native cattle embryos after in vitro fertilization were cultrued for 7 days at 38.5$^{\circ}C$ in CR1aa containing varing concentration of the antioxidants in a gas phases consisting of 5% CO2, 95% humidified air. The results obtained were summarized as follows; The proportion of embryos developed to morulae and blastocysts in CR1aa containing 2.5uM $\alpha$-tocopherol(11.0% and 6.0%) was significantly higher than those of 0, 5.0, and 7.5uM $\alpha$-tocopherol (P<0.05). concentration of 50uM L-ascorbic acid (7.5% blastocysts) did affect the proportion of embryos developing into blastocystes(P>0.05). Addition of 200uM cysteamine was significantly higher than those of 0, 100 and 300uM (P<0.05). When the fertilized oocytes were cultured at 0. 200, 400 and 600uM of selenium for 168 hrs, the morulae rates were 12.2, 5.2, 16.0 and 16.1% respectively, and addition of 200uM selenium was significantly higher than those of 0, 400, 600uM (P<0.05). These results suggested that the addition of $\alpha$-tocopherol, L-ascorbic acid, cysteamine and selenicum can enhanced development to the morulae and blastocysts of in vitro derived fertilized oocytes.

• Korean Journal of Animal Reproduction
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• v.14 no.4
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• pp.245-252
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• 1990

This study was carried out to investigate the factors affecting fertilization in vitro of follicular oocytes with frozen-thawed spermatozoa in Korean Native Cattle. The bovine ovaries were obtained at a slaughter house and the follicular oocytes were recovered by aspirating the follicular fluid from the visible follicles of 3~6mm. The bovine oocytes were matured in vitro for 20~24 hours in TCM-199 containing FCS and hormones. The matured oocytes were fertilized in vitro using Percoll-separated frozen-thawed spermatozoa in BO solution. The effects of dilution and fertilization media, capacitating method, concentration of inseminated sperm and time after insemination of fertilization, were observed. The results obtained are summarized as follows : 1. The fertilization rate of frozen-thawed sperm inseminated in BO solution with caffeine and heparin together(56.4%) was higher than that of sperm inseminated in BO solution with either caffeine(10.5%) or heparin(8.9%) and without both caffeine and heparin(0%)(P<0.05). 2. The fertilization rate(56.3%) of frozen-thawed sperm inseminated in BO solution with both caffeine and heparin without preincubation was higher than that of sperm preincubated(2.9%)(P<0.05). 3. The fertilization with high concentration of frozen-thawed sperm(1.4~1.8$\times$107cells/ml) in BO solution containing caffeine and heparin resulted in higher fertilization rate, 76.7%, than the low concentration of sperm(0.8~1.0$\times$107cells/ml), 32.7%(P<0.01). 4. When the oocytes were inseminated with frozen-thawed sperm in BO solution containing caffeine and heparin without preincubation, fertilization rate increased by time and the rates were 5.9, 46.0 and 59.4% at 8, 16 and 24 hours, respectively.

• Journal of Embryo Transfer
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• v.23 no.4
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• pp.291-298
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• 2008

We separately cultured follicular oocytes collected from individual ovaries of slaughtered Korean native cows and examined both the embryonic development rate and pregnancy rate after embryo transplantation according to the meat yield and quality grades of the source beef carcass. Oocytes from meat yield grade B cows exhibited a higher fertilization rate and embryonic developmental rate to the eight-cell stage than oocytes from grade A or C animals (p<0.05), but there was no significant difference in rate of development to the blastocyst stage among meat yield grades A, Band C. The oocyte cleavage rate and development rate to the eight-cell stage from meat quality grade 3 cattle was higher than grades 1++, 1+, 1 and 2 (p<0.05). Embryos derived from grade animals displayed a development rate to the blastocyst stage of 19.4%, which was also higher than all other meat quality grades (p<0.05). Transplantation of in vitro-cultured oocytes from meat yield grade A ovaries led to a higher pregnancy rate (64.2%) than in vitro-cultured oocytes from meat yield grade B ovaries (56.5%), but there was no significant difference between the two groups in pregnancy or abortion rates. In conclusion, embryonic development rate and pregnancy rate has a close relation to meat quality grades of the source beef carcass, this results is to give information for the Korean native cows improvement of breed.

• Journal of Embryo Transfer
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• v.13 no.1
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• pp.77-86
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• 1998

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.

• Journal of Embryo Transfer
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• v.11 no.1
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• pp.7-14
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• 1996

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, 5$\mu$g/ml FSH, 10 JU/ml hCG, and 1$\mu$g/ml estradiol-17$\beta$ for 24 hrs. For co-culture of in vitro development of fertilized ova, oviductal epithelial cells (l$\times$l0˚cells /ml) obtained from slaughtered cow and uterine epithelial cells(1$\times$10˚cells /ml) flushed from the superovulated holstein on Day 7 were incubated in 39$^{\circ}C$, 5% $CO_2$, 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.

• Korean Journal of Animal Reproduction
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• v.16 no.3
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• pp.277-284
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• 1992

The cattle follicular oocytes matured for 26~28h in culture condition were examined at 4, 5, 6, 7, 8, 10, 12, 14, 16 and 18h after insemination with bull spermatozoa preincubated for 4.5h in the uter isolated from estrous hamsters. After further culture with spermatozoa for 4~18 h, 73~89% of the total oocytes had matured to the second metaphase. None of the follicular oocytes matured in culture, were fertilized 5h after insemination. But when the oocytes were examined at 6, 8, 10, 14 and 18h after insemination, 60, 73, 82, 80 and 87% of oocytes were fertilized, respectively. The majority of the fertilized oocytes had enlarged sperm head at 6h after insemination and a part of the fertilized oocytes begun to develop from enlarged sperm head to male pronuclear stage at 8h after insemination, and most of them developed to male and female pronuclear stage at 10h after insemination. The results suggest that the penetration of spermatozoa into the oocytes may occur earlier than 6h after insemination and development of their pronuclear stage may occur at 8h after insemination.

• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.175-181
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• 2008

This study was conducted to investigate an effective recipient oocyte and culture system for producing of Hanwoo (Korean native cattle) somatic cell nuclear transfer (SCNT) embryos. Hanwoo ear skin fibroblasts were used as donor cells. In vitro matured Hanwoo or Holstein oocytes were enucleated, and single donor cells were transferred into the perivitelline space of the enucleated oocytes. The couplets were subsequently fused and activated. The reconstructed embryos were cultured in a conventional or sequential culture system. In the former, embryos were cultured in CR2aa medium for eight days; in the latter, embryos were cultured in modified CR2aa-A (mCR2-A) for three days and then further cultured in modified CR2aa-B (mCR2-B) for five days. In the experiment with the recipient oocyte, the rate of embryo development to the blastocyst stage was significantly (p<0.05) higher in Hanwoo recipient oocytes than in Holstein ones (48.8% vs 38.9%). BIastocysts derived from Hanwoo recipient oocytes contained significantly (p<0.05) higher numbers of total cells than those derived from Holstein recipient oocytes ($156.0{\pm}68.2$ vs $134.7{\pm}54.8$). There was no difference in the mean proportion of apoptotic cells in blastocysts between the sources of recipient oocytes. In the experiment with the embryo culture system, the blastocyst rate was somewhat higher in sequential system than in conventional system (50.0% vs 43.5%), though there was no significant difference. The numbers of total ($160.0{\pm}69.0$ vs $156.7{\pm}68.4$) and apoptotic cells ($14.0{\pm}10.4$ vs $11.8{\pm}6.4$) were not different between the culture systems. In conclusion, the present study demonstrated that Hanwoo recipient oocytes and the sequential culture system were more effective in supporting the production of Hanwoo SCNT embryos.

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.127-132
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• 1993

Frozen semen obtained from 5 different Korean Native Cattle were used for in vitro fertilization of oocytes. The proportions(3∼11%) of oocytes penetrated in the basic medium with 13.9 mM glucose were very low in different semen. However, the penetration rates of oocytes in the presence of caffeine and heparin increased to 68∼84%. The proportions(10∼52%) of oocytes penetrated in the presence of caffeine alone were low at various glucose concentrations(0∼27.8 mM). In the medium with heparin, significantly(P<0.05 at least) higher penetration rate was obtained in the absence(94%) than in the presence(28∼45%) of 0∼27.8 mM glucose concentrations. The medium with caffeine and heparin, however, produced similar penetration rates(84∼97%) regardless of the various glucose concentrations.

• Journal of Embryo Transfer
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• v.15 no.1
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• pp.39-46
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• 2000

This study was conducted to investigate the effect of hormones, protein sources and anti-oxidants on in vitro maturation (IVM) and in vitro fertilization(IVF) of bovine follicular oocytes. The rates of Holstein follicular oocytes classified as grade A and B(50.2% and 33.2%) were higher than those of Hanwoo cattle(40.3% and 32.0%, P<0.05). The cumulus cell expansion rates of oocytes cultured in TCM-199 and Ham's F-10 medium supplemented with 10% FCS and hormones were higher (81.9~87.6%) than those of non-treated groups (74.5~81.7%). The fertilization rates of oocytes cultured in TCM-199 and Ham's F-10 medim supplemented with 10% FCS, 1% BSA and 10% bFF was 53.8~55.0%, 51.4~52.6%, and 47.0~50.0%, respectively. The polyspermy rates was 13.6~14.2%, 10.0~11.1%, and 10.0%, respectively. When the oocytes were cultured in TCM-199 and Ham's F-10 medium with 50${\mu}{\textrm}{m}$ $\alpha$-tocopherol, the fertilization rates was 62.0 and 60.2%, respectively. In the maturation medium added of 100${\mu}{\textrm}{m}$ cysteamine, the fertilization rates was 64.7 and 66.7%, respectively. The fertilization and polyspermy rates of treated groups were higher than those of non-treated group. The results show that hormones, protein sources and anti-oxidants can provide a benefit for in vitro maturation and fertilization of bovine follicular oocytes.