• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.75-83
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• 1993

Purine has been identified in the preparation of follicular fluid and shown an activity in maintaining oocyte meiotic arrest. Therefore this study was performed to examine the inhibitory effect of purine on germinal vesicle break down(GVBD) in the presence and absence of human fetal cord serum(HFCS) or human mature follicular fluid(HMFF), as a protein source, in vitro culture. Immature oocytes(GV stage) were collected from ovaries of 21∼28 days old ICR mice by puncturing the antral follicles with a fine needle, at 48 hrs after PMSG injection. Some of the oocytes were denuded by drawing the cumulus-enclosed(complex) oocytes in and out of a pasteur pipet. Complex oocytes and denuded oocytes were cultured 3 hrs. in T6 media containing 0.75mM adenosine or/and 4mM hypoxanthine, with HFCS or HMFF. Their GVBD rates were observed at every 1 hr. during the culture time. Both adenosine and hypoxanthine have shown a time-dependent inhibitory effect on GVBD in complex and denuded oocytes and the inhibitory effect was maximized in culture medium containing hypoxanthine and adenosine. HFCS and HMFF increased the GVBD rates in the presence of the purines, thus HFCS and HMFF may contain a factor that could reverse the inhibitory effect of purines. Also complex oocytes were more sensitive to not only the inhibitory effect of purines but the promoting action of HMFF on GVBD than denuded oocytes. Therefore it was reconfirmed that granulosa cells play an important part in meiotic arrest and resumption.

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.113-120
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• 1993

To provide the optimal culture conditions for the developm,ent of in vit개 produced embryos, we have been investigated various culture media as well as co-cultrue systems using porcine cumulus cells or mouse fetal fibroblast cells. Porcine ovaries were brought to the laboratory from local slaughter house within 1 hour after slaughtering and cumulus oocytes complexes were recovered from antral follicles(3~5mm) with 23 gauge needle. To maturate follicular oocytes, cumulus oocytes complexes were washed three times with TCM-199 containing 25mM HEPES and incubated(39$^{\circ}C$, 5% CO2 in air) in various maturation media for 42 hrs. Ejaculated and liquid storaged boar spermatozoa capacitated with different sperm capacitation methods and media were rpepared for fertilizing of matured follicular oocytes in vitro. Fertilization was performed by adding 5~10${\mu}\ell$ fo capacitated spermatozoa containing 1~5$\times$105 sperm/ml to droplets. Eighteen to twenty-eight hours after sperm insemination, fertilized eggs were washed three times with culture media and transferred to the various culture media, to the culture media with a monolayer of somatic cells. The in vitro development rates of 1-cell embryos cultured with three times with culture media and transferred to the various culture media, to the culture media with a monolayer of somatic cells. The in vitro development rates of 1-cell embryos cultured with three different media, m-KRB, BECM and TCM-HEPES were 0~1.0%, showing extremely lower rates. Especially, most of embryos were observed to arrest the development beyond 4-cell stages. The rates of embryos developed to 2-, 4-, 8-, 16-, 32-cell and morula or blastocyst stage in co-culture with porcine cumulus cells and mouse fetal fibroblast cells were 61.1~67.0%, 59.0~58.0%, 42.5~43.1%, 28.4~30.2% and 20.4~21.0%, respectively. These development rates upto morula or blastocyst stages were significantly higher than those of the embryos cultured in the basic culture medium(P<0.01). These findings suggest that co-culture of in vitro fertilized eggs with porcine cumulus cells or mouse fetal fibroblast cells enhance the development of fertilized eggs to morula or blastocyst stage in vitro.

• Korean Journal of Veterinary Research
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• v.36 no.4
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• pp.919-927
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• 1996

In the last few years, methods for in vitro culture of early embryo stages from oocytes matured and fertilized in vitro using suitable cell culture systems have been established. But the factors affecting pregnancy rates following transfer of bovine embryos produced in vitro were not evaluated enough. So this study was performed to investigate the effects of quality and stage of embryos, parity and Corpus Luteum quality of recipients on pregnancy rates following non-surgical transfer of bovine embryos produced in vitro. Oocytes aspirated from small antral follicles of ovaries obtained at a local slaughter house were matured, fertilized with frozen-thawed semen and co-cultured for 6-7 days by utilizing co-culture system with bovine oviduct epithelial cell in vitro. After co-culture, embryos were transfered to recipients on day 7 (estrus=day 0). Recipients were monitored by ultrasonic scanning method or observation for estrus and rectal palpation after 50 days from transfer. The results of this study are follows. 1. Of the 70 recipients, 70%(49 of 70) had not showed estrus sign between day 0 and day 50, but 22.9%(16 of 70) was diagnosed not pregnant. Therefore the overall pregnancy rate of this study was 47.1%(33 of 70). 2. The pregnancy rate of recipients transfered with excellent(66.7%) and good(54.5%) embryos were higher than that of recipients transfered with fair embryos(15.8%) (p<0.05). 3. The pregnancy rate of recipients transfered with morula, compacted morula, blastocyst and expanded blastocysts were 46.2, 55.0, 62.5 and 50.0%, respectively. 4. The pregnancy rates of recipients transfered to heifer and cow were 54.5 and 55.2%, respectively. 5. The pregnancy rates of recipients with CL score I, II(66.7, 63.6%) were higher than those of recipients with CL score III (10%), (p<0.05). Success of transfer of embryos produced in vitro depends on many variables. The important factors identified in this study were the quality of embryos and the CL score of recipient animals after non-surgical transfer of embryos matured, fertilized and cultured in vitro.