• Journal of Veterinary Clinics
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• v.7 no.2
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• pp.517-519
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• 1990

In vitro maturation and fertilization of oocytes collected from slaughtered bovine ovaries were investigated. Immature bovine extrafollicular oocytes were cultured for 24 hrs. in TCM 199 supplemented with fetal calf serum in a humidified CO$_2$ incubator. Fertilization in vitro was performed using frozen-awed bull semen which was treated by Ca Ionophore A23187. Fourty percentage of oocytes cultured had matured to the metaphase II ; There were-no effects of the concentration of fetal calf serum and of the addition of HEPES on the maturation rate. The mean proportions of in vitro fertilized eggs and of cleaved eggs were 23.1％ and 14.4％, respectively.

• Korean Journal of Animal Reproduction
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• v.23 no.2
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• pp.113-118
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• 1999

This study was carried out to investigate on the improvement of fertilizing ability of in vitro matured oocytes from sperm density and motility by intracytoplasmic sperm injection(ICSI) into the porcine oocytes. 1. The in vitro fertilization and cleavage rates of oocytes from 1.0, 2.0, 3.0, 5.0 ($\times$10$^{6}$ $m\ell$) sperm concentration by IVF and ICSI of porcine oocytes were 46.7%~75.0%, 60.0%~85.7% and 10.6%~25.0%, 20.0%~64.3%, respectively. 2. The in vitro fertilization and cleavage rates of oocytes from 20, 40, 60, 80% of sperm mortilty by IVF and ICSI of porcine oocytes were 46.4%~71.4%, 67.9%~85.7% and 7.1%~21.4%, 28.6%~60.7%, respectively. 3. The in vitro fertilization and developmental rates of oocytes by IVF and ICSI methods were 55.6%~60.0%, 77.8%~80.0% and 17.8%~24.0%, 42.2%~56.0%, respectively. This ICSI method was improved high fertilization rates of porcine oocytes.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.275-282
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• 2004

This study was carried out to examine the effects of development media and those change on in vitro development (IVD) of porcine oocytes matured and fertilized in vitro. Putative embryos, which were matured in vitro in modified NCSU-23 (mNCSU-23) supplemented with 10％ porcine follicular fluid (pFF) and were fertilized in mTBM, were developed in vitro as the experimental scheme. The results are as follows. When porcine putative embryos were cultured in vitro in NCSU-23 or CZB/Pig-MEM, the percentage of oocytes cleaved was not different between two systems, but the percentage of blastocysts in NCSU-23 was significantly higher than in CZB/Pig-MEM (P<0.05). And when porcine putative embryos were cultured in vitro in NCSU-23 during 7 days with or without changing media at day 5, which was supplement with or without 10％ fetal bovine serum(FBS), the percentage of oocytes cleaved, blastocysts at day 6, and the cell number of ICM, TE and total of blastocysts at day 7 were not different among three treatments. As a result of this study, it is supposed that NCSU-23 be more favorable, to develop porcine embryos derived from IVM/IVF, than CZB/Pig-MEM, but that demonstration on the effects of changing medium with fresh one stand in need of the more experiments.

• Korean Journal of Animal Reproduction
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• v.23 no.3
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• pp.221-227
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• 1999

Successful cryopreservation of bovine immature oocytes can increase availably of oocytes for the in vitro fertilization or nuclear transfer. However, it was not reported successful development to the blastocyst stage following in vitro fertilization of cryopreserved bovine immature oocytes. The objective of this study was to determine the incidence of survival, meiotic maturation, fertilization and in vitro development of cryopreserved bovine immature by ultra rapid cooling methods. The oocytes were adversely affected by brief exposure to EFS40 solution in electron microscope grids and plunged directly into liquid nitrogen. After such ultra-rapid cooled immature oocytes were warmed, 78% of oocytes were matured to the metaphase II stage, 50% of oocytes were fertilized after insemination, and 5% of oocytes were developed to the blastocyst stage. Different sodium concentration of sodium ion in the freezing medium did not affect survival, maturation, fertilization and in vitro development of cryopreserved oocytes. These results suggested that immature bovine oocytes can be cryopreserved by ultra-rapid cooling methods.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.13 no.8
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• pp.3532-3536
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• 2012

Understanding of the maternal transcriptome increased to elucidate the underlying molecular mechanism of normal oocyte maturation, which depends on a precise sequence of changes in maternal genes expression. Previous reports that the translational potential of a maternal mRNA is generally determined by the length of the poly(A) tail, and deadenylation is usually the first sign of mRNA degradation. However, in vitro cultured system has the underlying molecular mechanisms remain unclear. We determined whether the role of molecular basis, four important maternal genes, C-mos, cyclin-B1 (regulatory subunit of MPF), BMP15 and GDF9, were selected for detection of their precise mRNA expression patterns by real-time PCR and for determination of their polyadenylation status by poly(A) tail PCR during oocyte maturation. In the present study. the abnormal expression of maternal mRNAs prior to zygotic genome activation, which results in suppression of the corresponding protein level, may be responsible for, at least in part, a profound defect in further embryonic development. Reasonable expression of maternal gene is crucial for proper oocyte maturation and further embryonic development.

• Journal of Embryo Transfer
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• v.18 no.2
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• pp.157-161
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• 2003

The study was carried out to investigate the effects of morphology, reproductive cycle, incubation time and activation of oocytes in vitro maturation of cats oocytes and development of IVM/IVF embryos. The results were summarized as follows : 1. The fertilization and developmental rate of fresh and salts-stored oocytes with and whithout cumulus cells were 65.7%, 17.1% and 28.6%, 8.6% and 57.1%, 13.3%, 23.3%, 3.3%, respectively. The rate of oocytes with cumulus cells(13.3%∼65.7%) was higher than that of denuded oocytes(3.3%∼28.6%). 2. The fertilization and developmental rate of oocytes recovered from ovaries collected at different stages of the reproductive cycle were 68.9%, 44.4%, 48.9% and 17.8%, 8.9%, 12.8%, respectively. 3. The fertilization and developmental rate of oocytes in vitro cultured at different time of incubation(24, 36 and 48 h) were 66.7%, 46.7%, 48.9% and 17.8%, 11.1%, 8.5%, respectively. respectively. The rate of oocytes incubated 24 h(66.7%) was higher than that oocytes incubated 36 and 48 h(46.7%∼48.9%). 4. The fertilization and developmental rate of oocytes treated activation and non-activation oocytes were 57.4%, 31.4% and 22.9%, 11.4%, respectively. The rate of oocytes treated activation was higher than that oocyte treat non-activation.

• Journal of Embryo Transfer
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• v.17 no.1
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• pp.55-59
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• 2002

The objective of this study is to determine the developmental competence of in vitro matured bovine oocytes after intracytoplasmic sperm injection(ICSI) with frozen-thawed epididymal spermatozoa. The ovaries were obtained from slaughtered Korean native cows. Oocytes matured in vitro for 24 hrs were fertilized by ICSI with frozen-thawed epididymal spermatozoa. After ICSI, a group of oocytes was activated with 7% ethanol fur 5 min, and the other group was not activated. The oocytes were cultured in TCM-199 medium containing hormones and 10% FCS for 24~30 hrs in a incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$. The percentage of oocytes reaching M II after 24 hrs and 30 hrs of incubation were significantly higher(p<0.05) after culture with TCM-199 media(80.0% and 88.3%) than M I(8.3% and 6.7%). The rate of cleavaged embryos to blastocyst obtained by ICSI treated activation oocytes was significantly higher(p<0.05) than that of nonactivation oocytes(22/46, 47.8% vs 10/39, 25.6%). The rates of embryos development to blastocyst obtained by ICSI treated sperm of flesh, epididymal and frozen-thawed epididymal were 24/45(53.3%), 15/40(37.5%), 11/43(25.6%), respectively and these values of fresh sperm injection were higher than frozen-thawed epididymal sperm. We also concluded that embryos can be produced with ICSI of in vitro matured oocytes by ICSI using frozen-thawed epididymal semen.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.245-255
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• 2004

In recent years, an increasing number of studies on pig in vitro maturation(IVM) and in vitro fertilization(IVF) have been separated. the wide range of new technologies, including that in applied molecular genetics, has increased this interest. the production of viable porcine embryos in vitro is a prerequisites for the successful production of transgenic pigs to date. The efficiency of IVM/IVF techniques in the porcine is lower than that obtained in other species such as cattle and mouse. The several problems are generally thought to be the cause of poor results: the low rate of MPN formation derived from inadequate IVM of oocytes, the high incidence of polyspermy after IVF and cell blocking at 4 cell during embryos culture. For there reasons overcoming, many studies have been conducted to improve in vitro embryo-genic competence of oocytes. In the last several years, many maturation culture media have been evaluated and various exogenous factors such as hormones and grows factors have been tested to improve the efficiency of porcine in vitro system. In the study several antioxidants have been examined to improve in vitro fertilization and development of porcine oocytes. In this study, several antioxidants were examined to determine the effects on the development of oocytes to the cleavage, morula and blastocyst stage when added at the maturation(IVM) or in vitro fertilization(IVF) or in vitro culture(IVC) of porcine embryos. Porcine oocytes were matured, fertilized and embryos were cultured in defind conditioned medium in vitro with or without supplementation with the antioxidents of cysteine, catalase and glutathione. 1. Significant improvement of blastocyst rate (27.2％ versus 15.4％, p<0.05) were achieved when catalase(500U/$m\ell$) were added to TCM-199 medium and morula rate(72.0％ versus 53.9％, p<0.05) were significantly higher when glutathione(1.0mM/$m\ell$) were added to TCM-199 medium than those of control. 2. In mTBM medium for oocytes fertilization, the addition of cysteine, catalase and glutathione had no positive effect on embryonic development. glutathione had no positive effect on embryonic development. In conclusion, this study shows that addition of catalase, gluththione during IVM improved the rate of porcine embryo development.

• Journal of Life Science
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• v.13 no.5
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• pp.732-739
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• 2003

The purpose of this study was to determine if the addition of spermatozoa into the culture medium could influence the nuclear maturation of denuded porcine germinal vesicle (GV) oocytes in vitro. Cumulus-oocyte complexes were collected from follicles of 3 to 5 mm in diameter, The cumulus and corona cells were removed from oocytes. Porcine denuded oocytes were cultured in tissue culture medium containing spermatozoa. After 48 h culture, oocytes were examined for the evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II (M II). The proportion of oocytes reaching M II stage was significantly (P<0.01) increased in the oocytes cultured in media containing spermatozoa compared to those in media without spermatozoa $(31.9\pm1.8%\; vs\; 14.9\pm1.0%)$.No differences in the rates of M II were observed among the different period of spermatozoa exposure nor among the spermatozoa from different species. The proportion of oocytes reaching M II stage was significantly different between high and low concentrations of spermatozoa. The present study suggests that mammalian spermatozoa contain a substance(s) that improves nuclear maturation in vitro of GV oocytes. Enhancing effect of spermatozoa for oocytes maturation in vitro is a highly dose-dependent.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.169-174
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• 2007

Despite many efforts to improving canine in vitro maturation(IVM), the efficiency is still low compared to that of other mammalian species. The present study investigated the effects of gonadotropin, epidermal growth factor and cysteine supplementation on in vitro maturation of canine oocytes. Cumulus-oocyte complexes (COCs) were matured in basic medium (TCM-199 containing 10% FBS, 0.11mg/ml sodium pyruvate supplemented with or without $10{\mu}g/ml$ FSH, 10ng/ml EGF and 0.57mM cysteine) for 72 hr at $38.5^{\circ}C$ in humidified atmosphere of 5% $CO_2$ in air. After culture, oocytes were stained with Hoechst 33342 $(10{\mu}g/ml)$ for 30 min at $4^{\circ}C$, and assessed their nuclear status (GV: germinal vesicle, GVBD: germinal vesicle break down, MI: metaphasse I, MII: metaphase II, UK: unknown stage). No differences were observed in GV, MI and MII rate except GVBD rate between with and without gonadotropins addition respectively (6.7%, vs. 17.2%). Supplementation of 10ng/ml of EGF in hormones added IVM medium resulted in significantly (p<0.05) higher MII rate (4.54% vs.7.06%). Although there are no significantly difference, total of MI and MII rates were increased by adding cysteine. In conclusion, the present study indicates that supplementation of $10{\mu}g/ml$ LH and FSH, 10ng/ml EGF and 0.57mM cysteine in canine IVM medium show a positive influence on the progression of maturation to MII at 72 hr.