• 한국가축번식학회지
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• 제23권2호
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• pp.133-139
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• 1999

본 연구는 생쥐 미성숙란올 초자화 동결-융해하였올 때, 체외/체내 배발달능을 검토하고자 실시하였다. 생쥐 미성숙란은 동해제인 EFS40(40% ethylene glycol, 18% ficoll, 0.5 M sucrose)으로 초자화동결되었으며, 융해 후 16 시간동안 체외성숙을 유도하여, 제 1 극체가 나타난 성숙된 난자를 1~2$\times$$10^{6}$$m\ell$ 농도의 정자로 체외수정시킨 다음, 난할율 ($\geq$ 2- 세포기)과 체외 / 체내 발달율을 조사하였다. 쥐 미성숙란을 초자화 동결 융해하였던 군 (63.1%)의 체외성숙율은 동해제 노출군 (67.5%)과 대조군(66.3%)에 유사하게 나타났으나, 초자화 동결군의 난할율과 배반포형성율 (64.9, 59.0%)은 동해제노출군 (83.7, 74.7%)과 대조군 (90.7, 83.7%) 에 비해 유의하게 감소하였다 (p＜0.05). 그러나, 초자화 동결 융해하였던 생쥐 미성숙란으로부터 얻어진 배반포기배를 가임신 생쥐에 이식하였을 때, 체내발달율인 전체착상율 (31.3%)과 착상된 배로부터 발달된 산자형성율 (66.7%)은 대조군의 결과 (40.8%, 58.1%)와 각각 비교하였을 때 유의차가 인정되지 않았다. 따라서, 생쥐 미성숙란을 초자화 동결-융해하였을 때, 체외발달율은 유의하게 감소하였지만 생성된 배반포기배로부터의 산자발달율은 대조군과 유사하게 나타나, EFS40을 이용한 초자화 동결 방법은 생쥐 미성숙란 동결에 유용하게 이용될 수 있다는 것을 알 수 있었다.

• 한국가축번식학회지
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• 제16권1호
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• pp.15-20
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• 1992

These experiments were undertaken to investigate the rate of in vitro fertilization of bovine follicular oocytes treated with glycosaminoglycans(GAGs). Bovine follicular oocytes were obtained from the ovary of slaughtered animal and matured in media containing the various concentrations of hydluronic acid, chondroitin sulfate or heparin for 26 hours. Epididymal spermatozoa were capacitated and insemination was made by introducing about 10~15 matured oocytes into the suspension of spermatozoa. Six hour after insemination the eggs were transferred to TCM-199 supplemented with FCS(10%) and then examined the embryo development. After in vitro insemination, percentages of ova fertilized were 61.3 or 48.3%, respectively, for the cumulus intact or removed in the percentages of GAGs. However, in case of cumulus-free oocytes treated with GAGs, the fertilization rates were 58.8, 62.1, 58.8, and 61.8%, respectively, showing significant effect compared to 48.3% in cumulus-free oocytes. Our findings suggest that cnondroitin sulfate and heparin are superior to hyaluronic acid in the fertilizatin and pronuclear formation of bovine oocytes.

• 한국가축번식학회지
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• 제14권1호
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• pp.50-56
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• 1990

Bovine oocytes obtained from follicles(2~5mm) of ovaries after slaughter were cultured in TCM 199 medium with 10~20% heat-inactivated estrus cow serum(ECS) for 25~27 hr, at 39$^{\circ}C$ under 5% CO2 in air. At the end of culture period, some oocytes were stained with 1% acetoorcein and examined for the evidence of oocyte maturation. The remainder were used to assess the potential of in vitro fertilization(IVF) with frozen-thawed spermatozoa and subsequent development in media with or without bovine oviduct epithelial cell (BOEC) co-culture. The results obtained were summarized as follows ; 1. The maturation rate of oocyte in vitro in TCM 199 medium with 15% ECS group(76.3) was superior to 10% ECS group(68.3%) and 20% ECS group(64.5%). 2. The IVF rates of oocytes matured in vitro, and formation rate of male and female pronuclei were 63.6%(77/121) and 93.5%(72/77), respectively. The incidence of polyspermy was very low(2.4%). 3. Of 73 oocytes fertilized in vitro and cultured in TCM 199 medium with 10% fetal calf serum for 7 days, 41(56.3%) were cleaved over 2-cell and only 1(2.4%) was developed beyond the 16-cell stage. 4. Of 76 oocytes co-cultured with BOEC, 58(76.3%) were cleavaged and 23(39.7%) were developed to morula and blastocyst stage. The results of this study indicate that co-culture with BOEC deserved a positive effect on the IVF oocyte development through the 16-cell block.

• 한국가축번식학회지
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• 제20권2호
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• pp.179-190
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• 1996

Sexing and developing from splitted embryos which were fertilized in vitro implicate a possibility of production of the superior and sex controlled individuals. This study was carried out to investigate the production of transferable late blastocysts from in vitro fertilized embryos and to analyze sex by chromosome analysis from same embryos. In results, the ratio of cleavage and fertility of bovine follicular oocytes matured in vitro was 90% in co-cultured with granulosa cells. The competence of embryonic development from in vitro matured and fertilized bovine oocytes was 38% in co-cultured with bovine oviductal epithelial cells. To produce a lot of transferable embryos, therefore, the best conditon of culture system was co-cultured with granulosa cells for immature bovine oocytes and then co-cultured with bovine oviductal eptithelial cells for matured and fertilized oocytes. In chromosome analysis, 93% of in vitro fertilized embryos were very important aspect in chromosome preparation from bovine embryos such as duration of colcemid treatment, weakening of zona pellucida, methods of hypotonic treatment and fixation treatment.

• 한국수정란이식학회지
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• 제9권3호
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• pp.261-268
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• 1994

Immatured bovine follicular oocytes added with serum, hormones, granulosa cells and bovine oviduct epithelium cells were fertilized in vitro after in vitro maturation. In vitro maturation and early development capacity were examined and IVF-derived embryos were transferred and to recipients and effects of sperm treatment on in vitro capacitation were investigated. The rate of in vitro maturation was improved when they were co-culutred with granulosa cells in the TCM199 medium added with 10% FCS and hormones. The percentage of acrosome reaction was not differed between sperm treatments and sperm of above 25% under-went AR during 30 min preincubation with caffeine and heparin. The cleavage rate of oocytes in vitro fertilized in TCM199 medium added with 10% FCS and hormones, GC or BOEG higher than that in medium with 10% FCS and GC. But the rate was not significantly different between GC and BOEG The cleavage of rate oocytes cultured in medium containing serum, hormones and BOEG was 80.2% and more embryos were developed to Blastocyst (17.3%). The selected embryos were transferred to 9 recipients by surgical or nonsurgical method but did not result in pregnancy.

• 한국가축번식학회지
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• 제21권3호
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• pp.281-285
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• 1997

The study was conducted to investigate on in vitro developmental rates of bisected bovine embryos co-culture in 10% FCS＋TCM-199 media containing hormones, oviductal epithelial cells and cumulus cells 0 to 7 days after bisection. In vitro developmental rates was defined as development rates on in vitro culture or FDA-test. The results are summarized as follows : 1. In vitro developmental rates of bisected bovine embryos co-cultured in 10% FCS＋TCM-199 media containing PMSG＋hCG, PMSG＋$\beta$-estradiol, hCG＋$\beta$-estradiol, PMSG, hCG 0 to 3 days and 4 to 7 days were 16.7~30.0% and 11.1~25.0%, respectively. In vitro developmental rates of bisected embryos co-cultured in 10% FCS＋TCM-199 media containing hormones significantly higher than that of non co-culture. 2. In vitro developmental rates of bisected bovine embryos co-cultured 10% FCS＋TCM-199 media containing oviductal epithelial cells 0 to 3 days and 4 to 7 days were 25.0% and 22.2%, respectively.

• 한국수정란이식학회지
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• 제11권1호
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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.

• Clinical and Experimental Reproductive Medicine
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• 제48권4호
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• pp.362-367
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• 2021

Objective: The impact of early mechanical removal of cumulus cells on fertilization and embryonic development is not yet precisely known. This study aimed to investigate the effects of early and late cumulus cell removal on fertilization, polyspermy, embryonic development potential, blastocyst development, and clinical outcomes. Methods: A prospective study was conducted of patients who underwent in vitro fertilization between September 2019 and October 2020. Sibling oocytes were randomly allocated after insemination to early cumulus cell removal at 6 hours (group I) and late cumulus cell removal at 16-18 hours (group II). If total fertilization failure (TFF) was determined to have occurred at early cumulus cell removal, rescue intracytoplasmic sperm injection (ICSI) was performed. Fertilization, embryonic development, and pregnancy outcomes were compared. Results: A total of 912 oocytes were assigned to group I (458 oocytes) and group II (454 oocytes). Fertilization, polyspermy, embryo quality, and pregnancy outcomes were not significantly different between both groups. Rescue ICSI enabled fertilization of 79.2% of the TFF oocytes. Conclusion: Early cumulus cell removal at 6 hours had no significant difference in fertilization, polyspermy, embryo development, or obstetric and perinatal outcomes compared to late removal. Early cumulus cell removal combined with early rescue ICSI may have the potential to help couples with TFF.

• 한국수정란이식학회지
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• 제23권2호
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• pp.93-100
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• 2008

This study was conducted to establish an in vitro maturation (IVM) system by selection of efficient macromolecule in the porcine in vitro production (IVP) technology. To choose the efficient macromolecules in the development of porcine embryos, the effects of 3 kinds of macromolecules (porcine serum; PS, porcine follicular fluid; pFF, and polyvinyl alcohol; PVA) supplemented in IVM media on the maturation, cleavage, and development rates to blastocyst of parthenogenetic activation (PA) and in vitro fertilization (IVF) embryos were examined. The maturation rates of porcine oocytes in media supplemented with PS were significantly higher than those with pFF and PVA (92.4% vs. 85.4%, 77.1%; p<0.05). In the cleavage and development to blastocyst rates, supplement with PS or pFF in the IVM media was more effective than PA. However, there were no significant differences in cleavage and development to blastocyst between PS and pFF group. From the results of this study, it was demonstrated that PS was optimal macromolecule in the porcine IVM media.

• 한국수정란이식학회지
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• 제15권1호
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• pp.95-102
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• 2000

Techniques for manipulation of spermatozoa and oocytes have been widely used for in vitro production(IVP) of Hanwoo. This study was conducted to examine the effects of theophylline and heparin on frozen-thawed Hanwoo sperm for enhancing the efficiency of IVP technique. Oocytes were inseminated with forzen bull semen treated with either theophylline or heparin for examining the effect of each substance on fertilization and subsequent development. More (P<0.05) oocytes formed pronucleus and develop to the morula and blastocyst stages after inseminated with sperm treated with heparin than after inseminated with sperm treated with theophylline. The pregnancy rate after embryo transfer was higher after heparin treatment than after theophylline treatment, but did not differ significantly. There was no significant difference of offspring delivery between two groups. In conculsion, theophylline and heparin can be used for enhancing the efficiency of IVP system for Hanwoo. Considering characteristics of these substance, theophylline 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.