• 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 Embryo Transfer
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• v.31 no.3
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• pp.153-159
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• 2016

(-)-Epicatechin gallate (ECG) is a polyphenol compound of green tea exhibiting biological activities, such as antioxidant and anticancer effects. To examine the effect of ECG on porcine oocytes during in vitro maturation (IVM), oocytes were treated with 0-, 5-, 15-, and $25{\mu}M$ ECG. After maturation, we investigated nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels and subsequent embryonic development after parthenogenetic activation (PA) and in vitro fertilization (IVF). After 42 hours of IVM, the $5{\mu}M$ group exhibited significantly increased (p< 0.05) nuclear maturation (89.8%) compared with the control group (86.1%). However, the $25{\mu}M$ group observed significantly decreased (p< 0.05) nuclear maturation (83.5%). In intracellular maturation assessment the 5-, 15-, and $25{\mu}M$ groups had significantly increased (p< 0.05) GSH levels and decreased ROS levels compared with the controls. The 5- and $15{\mu}M$ group showed significantly increased (p< 0.05) embryo formation rates and total cell number of blastocysts after PA (18% and 68.9, 15% and 85.1 vs. 12% and 59.5, respectively) compared with controls. Although the $25{\mu}M$ group observed significantly lower blastocyst formation rates after PA (27.6% vs. 23.2%) than control group, the $5{\mu}M$ group showed significantly increased blastocyst formation rates after PA (37.2% vs. 23.2%) compared to the control group. Furthermore, the $5{\mu}M$ group measured significantly increased blastocyst formation rates (20.7% vs. 8.6%) and total cell number after IVF ($88.3{\pm}1.5$ vs. $58.0{\pm}3.6$) compared to the control group. The treatment of $5{\mu}M$ ECG during IVM affectively improved the porcine embryonic developmental competence by regulating intracellular oxidative stress during IVM.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.12
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• pp.1844-1853
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• 2019

Objective: We investigated how pituitary adenylate cyclase-activating polypeptide (PACAP) affects embryonic development during pre-in vitro maturation (pre-IVM) using porcine oocytes isolated from small follicles. Methods: We divided the follicles into the experimental groups by size (SF, small follicles; MF, medium follicles) and treated with and without PACAP and cultured for 18 hours (PreSF[-]PACAP; without PACAP, Pre-SF[+]PACAP; with PACAP) before undergoing IVM. The gene expression related to extracellular matrix formation (amphiregulin, epiregulin, and hyaluronan synthase 2 [HAS2]) and apoptosis (Bcl-2-associated X [BAX], B-cell lymphoma 2, and cysteine-aspartic acid protease 3) was investigated after maturation. The impact on developmental competence was assessed by the cleavage and blastocyst rate and total cell number of blastocysts in embryos generated from parthenogenesis (PA) and in vitro fertilization (IVF). Results: Cleavage rates in the Pre-SF(+)PACAP after PA were significantly higher than SF and Pre-SF(-)PACAP (p<0.05). The cleavage rates between MF and Pre- SF(+)PACAP groups yielded no notable differences after IVF. Pre-SF(+)PACAP displayed the higher rate of blastocyst formation and greater total cell number than SF and Pre-SF(-)PACAP (p<0.05). Cumulus cells showed significant upregulation of HAS2 mRNA in the Pre-SF(+)PACAP compared to the SF (p<0.05). In comparison to other groups, the Pre-SF(+)PACAP group displayed a downregulation in mRNA expression of BAX in matured oocytes (p<0.05). Conclusion: The PACAP treatment during pre-IVM improved the developmental potential of porcine oocytes derived from SF by regulating cumulus expansion and apoptosis of oocytes.

• Reproductive and Developmental Biology
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• v.29 no.4
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• pp.207-212
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• 2005

In the present study, we investigated the effects of genotypes on in vitro maturation and fertilization in porcine fresh/frozen-thawed oocytes. The porcine cumulus-oocyte complexes (COCs) were divided into four groups according to whether they were: (1) in vitro matured; (2) cryopreserved and in vitro matured; (3) in vitro fertilized and (4) cryopreserved, and in vitro fertilized. Maturation of porcine COCs was accomplished by incubation in NCSU23 medium. Immature oocytes were cryopreserved by Open Pulled Straws (OPS) method according to Vajta et al., (1998). Oocytes stained by Acetic-Orcein method were observed under the microscope. DNA extracted from the ovaries was analyzed by RAPD (random amplified polymorphic DNA) and SSCP (single strand conformational polymorphisrrt) method. The rates of oocytes maturation and fertilization were significantly high in AA genotype. The results indicated that in vitro maturation and fertilization in porcine fresh/frozen-thawed oocytes may be affected by genotypes in pigs.

• 대한생식의학회:학술대회논문집
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• 1996.11a
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• pp.59-60
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• 1996

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.6
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• pp.966-975
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• 1999

Technologies on preimplantation porcine embryos have been developed quickly and significantly. Successful development of systems for culture of porcine zygotes to the blastocyst stage has made it possible to utilize follicular oocytes for in vitro production of embryos and thus stimulated research on various embryo technologies. Recent technological development of embryo cryopreservation, separation of X- and Y-bearing spermatozoa and non-surgical embryo transfer has also made it easy to utilize in vivo- and in vitro-produced embryos for artificial manipulation to produce clones and transgenic pigs. Further progress in overcoming various problems associated with each embryo technology will result in acceptable efficiency to utilize porcine embryos with a high or increased quality. Combining these technologies will accelerate further expansion of the swine industry not only for meat production but also for the production of therapeutic recombinant proteins and xonografts.

• Reproductive and Developmental Biology
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• v.30 no.2
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• pp.125-133
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• 2006

This study was conducted to detect the apoptosis incidence in blastocysts and to compare the abundance of Bax, Bcl2L1, VEGF and FGFR2 in in vitro fertilized (IVF), parthenogenetic (PAT) and nuclear transfer (NT) embryos. Oocytes matured for 40 hr were enucleated and reconstructed with confluenced fetal fibroblasts (FFs) derived from a ${\sim}45$ day fetus. Reconstructed eggs were then fused with 2 DC pulses (2.0 kV/cm, $30{\mu}sec$) and cultured with $7.5{\mu}g/ml$ cytochalasin B for 3 hr. Parthenotes (PAT) were produced with the same electric strength and culture for NT eggs. The embryos were cultured in NCSU-23 medium at $39^{\circ}C,\;5%\;CO_2,\;5%\l;O_2$ in air. In 3 runs, set of 10 embryos at the 4-cell to blastocyst stages were used to extract total RNA for analyzing the gene expression patterns of pro-apoptotic (Bax), anti-apoptotic (Bcl2L1), vasculogenesis (VEGF), implantation (FGFR2III) using real-time quantitative PCR. Cleavage and blastocyst rates were significantly higher (P<0.05) in IVF and PAT ($79.3{\pm}8.5\;and\;25.5{\pm}6.1,\;and\;85.0{\pm}6.4\;and\;38.6{\pm}5.5$, respectively)than NT counterparts ($65.1{\pm}5.2\;and\;15.6{\pm}3.0$, respectively). Significantly higher (P<0.05) total cells were observed in IVF controls and PAT ($34.7{\pm}5.8\;and\;38.1{\pm}4.1$) than NT embryos ($24.8{\pm}3.2$). Apoptosis index was significantly lower (P<0.05) in IVF than NT embryos. The Relative abundances (RA) of Bax and VEGF were significantly higher (P<0.05) at blastocyst stage in NT than IVF control. The RA of Bcl2L1 and FGFR2III were significantly higher (P<0.05) at blastocyst stage in IVF than NT. The present study observed the abnormal gene expressions in NT embryos at various developmental stages, suggesting certain clues to find out the cause of the low efficiency of NT to term.

• 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.27 no.2
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• pp.125-133
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• 2003

The present study was carried out to examine the effect of $\beta$-Mercaptoethanol ($\beta$-ME) on in vitro maturation (IVM) of porcine follicular oocytes and oxygen concentration with $\beta$-ME on in vitro development (IVD) of porcine IVM/IVF embryos. The results were summarized as follows. 1. The rates of nuclear maturation, penetrated oocytes, polyspermic oocytes, pronucleus formation and mean numbers of the penetrated sperms were not significantly different using NCSU-23 maturation media for 0, 25, 50 and 100 $\mu$M $\beta$-ME (P>0.05). 2. The rates of blastocyst formation at day 7 after in vitro fertilization were higher in oocytes matured with 25 $\mu$M $\beta$-ME (25.4$\pm$0.9%) than in those matured with 0 (14.5$\pm$1.6%), 50 (17.3$\pm$1.7%) and 100 $\mu$M (12.4$\pm$1.3%) (P<0.05). However, no differences ware found in total cell numbers of blastocyst among the treatments. 3. The rates of blastocyst formation at day 7 after in vitro fertilization were higher in the NCSU-23 Culture medium With 25 $\mu$M $\beta$-ME (23.6$\pm$2.8%) than in those Cultured With 0 (15.4$\pm$4.4%), 12.5 (17.5$\pm$2.3%) and 50 $\mu$M $\beta$-ME (18.6$\pm$2.1%) Under the 5% and 20% $O_2$ Concentrations (P<0.05). However, no differences was found in total cell numbers of blastocyst among the treatments. These results suggested that the addition of 25 $\mu$M $\beta$-ME in the IVM/IVD media were effective on the porcine embryo production. However, the rates of blastocyst formation and total cell numbers of blastocyst at day 7 of porcine IVM/IVF embryos were not significantly different in the NCSU-23 culture medium under 5% and 20% 02 concentrations.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.77-82
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• 2004

The purpose of this study was performed to establish the in vitro culture system of porcine in vitro maturation and in vitro fertilization(IVM/IVF) embryo. These studies was to determine the effects of antioxidants(aesculetin, taurine and melatonin) in porcine IVM/IVF embryos. In routine porcine IVM/IVF procedure, oocytes were cultured for 40∼44h incubation in NCSU 23 mediumand matured oocytes were inseminated with frozen semen. Then 2 to 8 cell embryos were removed cumulus cell and were allotted randomly to NCSU 23 containing different concentration of antioxidants in 5% $O_2$ and 5% $CO_2$ at 38.5$^{\circ}C$. Cell numbers of blastocyst were also counted using double fluorescence stain method. Aesculetin were added to NCSU 23 medium at concentration of 1 ug, 5 ug, and 10 ug, when treated with 10 ug(35.7%) of aesucletin at the rate of embryos of the morula plus blatocsyts were higher than those of any other groups (30.2%, 29.5% and 29.2%)(P<0.05). The developmental rates beyond morula stage of porcine embryos in NCSU 23 medium supplemented with taurine 0, 2.5 and 5.0 mM were 26.1%, 26.9% and 31.7%, respectively. The addition of 5.0 mM taurine was higher the developmental rate beyond morula stage than in any other groups. In NCSU 23 medium treated with melatonin 0, 1, 5 and 10 nM, the developmental rate of morula plus blastocysts were 33.3%, 39.1%, 33.3% and 27.9%, respectively. The developmental rate of morula and blascytocys treated with 1nM melatonin was higher than in any other groups(P<0.05). Cell numbers of blastocyst in NCSU 23 treated with melatonin 0, 1, 5 and 10nM were 41.0, 42.6, 39.6 and 33.0, respectively. These results indicate that aesculetin, taurine and melation can increase the developmental rate beyond the morulae and blastocysts in porcine embryos.