• Korean Journal of Animal Reproduction
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• v.21 no.4
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• pp.355-362
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• 1997

This study was undertaken in an effort to product embryos through in vitro maturation(IVM), in vitro fertilization(IVF) and in vitro culture(IVC) after cryopreservation of immature and mature porcine oocytes. The experiments were conducted to investigate IVM rate of oocytes frozen with 3 different cryoprotectants and to examine IVF and IVC of frozen-thawed oocytes. The CEI(cumulus cells expansion index) after IVM of frozen-thawed immature oocytes was higher in oocytes frozen with PG＋PEG(propylene glycol plus polyethylene glycol) than those frozen with single cryoprotectant and this index was almost 90% of unfrozen oocyte's index(2.39 vs. 2.66). The IVF rate of all frozen oocytes was very low(68% of unfrozen oocytes) and the IVF rate of frozen immature oocytes was slightly higher than that of frozen mature oocytes(39.0% vs. 34.4%), but polyspermic penetration was higher in frozen immature oocytes(21.9% vs. 19.1%). The cleavage rate after IVF of frozen-thawed oocytes was 9.3% for frozen mature oocytes and 11.3% for frozen immature oocytes and this rate was significantly lower(P<0.05) than that of control(60.7%). The development to 8-cell stage was greatly lower in frozen mature oocytes than in frozen immature oocytes. The results indicate that the use of PG plus PEG as cryoprotectant may be very effective for vitrification of porcine oocytes and the frozen-thawed immature porcine oocytes can be used fro in vitro embryo production based on IVM, IVF and IVC system.

• Journal of Embryo Transfer
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• v.26 no.4
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• pp.251-256
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• 2011

Presently, the effect of 0.5 mM dibutyryl cAMP (dbcAMP)-supplemented maturation medium during different incubation time on meiotic arrest (germinal vesicle) and resumption (metaphase II) of porcine oocytes and embryonic development of porcine oocytes following in vitro fertilization (IVF) or parthenogenetic activation (PA) was determined. Porcine cumulus oocyte complexes (COCs) were cultured in 0.5 mM dbcAMP for 17, 22, 27, or 42 h, and an additional 22 h without 0.5 mM dbcAMP. The nuclear status was examined at each time point. Oocytes cultured from 39~49 h displayed more than 80% meiotic resumption. More than 85 % of meiotic arrest was presented at 17~22 h. Oocytes were cultured for 22 h with 0.5 mM dbcAMP and additional 22 h without dbcAMP to assess developmental potential following IVF or PA. There were no significant differences in blastocyst rates among the dbcAMPIVF, IVF, dbcAMP-PA, and PA groups, although cleavage rate of IVF group was significantly higher than those of dbcAMP-PA, and PA groups. In conclusion, 0.5 mM dbcAMP influenced meiotic maturation of porcine oocytes depending on incubation time of oocyte, although embryonic development was not improved in both IVF and PA.

• 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.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.47-47
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• 2003

The present study was conducted to determine the expression of the antioxidant enzyme(CuZn-SOD, Mn-SOD and GPX and apoptosis gene(caspase-3) for in vitro culture in in vitro maturation and in vitro fertilization(IVM/IVF) embryos in porcine. Porcine embryos derived from IVM/IVF were cultured in NCSU23 medium under 5% $CO_2$ in air at 38.5$^{\circ}C$. The patterns of gene expression for several antioxidant enzyme and apoptosis genes during preimplantion porcine embryo development were examined by the modified semi-quantitative single cell reverse transcriptase- polymerase chain reaction (RT-PCR). Preimplantation porcine embryos produced by IVM/IVF have expressed mRNAs for CuZn-SOD and GPX, whereas transcripts for Mn-SOD have not detected at any developmental stages. Expression of caspase-3 mRNA was detected at 2 cell, 8 cell, 16 cell and morula stages. The fas ligand transcripts were detected in porcine blastocyst. These results suggest that various antioxidant enzymes and apoptosis genes play crucial roles in in vitro culture of porcine IVM/IVF embryos.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.183-187
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• 2009

In this study, we aimed to determine whether the evaluated markers of cell death could be found at particular developmental stages of normal porcine in vitro fertilization (IVF) embryos. We investigated the characteristics of spontaneous and induced apoptosis during preimplantation development stages of porcine IVF embryos. In experiment 1, to induce apoptosis of porcine IVF embryos, porcine IVF embryos at 22h post insemination were treated at different concentration of actinomycin D (0, 5, 50 and 500 ng/ml in NCSU medium). Treated embryos were incubated at $39^{\circ}C$ in 5% $CO_2$, 5% $O_2$ for 8h, and then washed to NCSU medium and incubated until blastocyst (BL) stage. We examined cleavage rate at 2days and BL development rate at 7days after in vitro culture. A significantly lower rate of cleavage was found in the 500 ng/ml group compared to others (500 ng/ml vs. 0, 5, 50 ng/ml; 27.8 % vs. 50.0%, 41.2%, 35.9%), and BL formation rate in 500 ng/ml was lower than that of others (500 ng/ml vs. 0, 5, 50 ng/ml; 8.0% vs. 12.6%, 11.2%, 12.6%). In experiment 2, to evaluate apoptotic cells, we conducted TUNEL assay based on morphological assessment of nuclei and on detection of specific DNA degradation under fluorescence microscope. This result showed that apoptosis is a normal event during preimplantation development in control group (0 ng/ml actinomycin D). A high number of BL derived control group contained at least one apoptotic cell. Actinomycin D treated BLs responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and increase in the incidence of apoptotic cell death. In 500 ng/ml group, the incidence of apoptosis increased at 4-cell stage and later. This result suggested that apoptosis is a process of normal embryonic development and actinomycin D is useful tool for the apoptosis study of porcine preimplantation embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.1
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• pp.33-38
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• 2004

This study was aimed at testing the gene expression of antioxidant enzymes and apoptosis genes for in vitro culture in porcine embryos produced by in vitro maturation/in vitro fertilization (IVM/IVF). Pocine preimplantation embryos obtainted from IVM/IVF can be successfully culture in vitro, but they are delayed or stop to develop at specific developmental stage. Many factors such as reactive oxygen species and apoptosis in an IVM/IVF system followed by in vitro culture influence the rate of production of viable blastocysts. Porcine embryos derived from IVM/IVF were cultured in the atmosphere of 5% $CO_2$ and 20% $O_2$ at $38.5^{\circ}C$ in NCSU23 medium. The patterns of gene expression for antioxidant enzymes and apoptosis genes during in vitro culture in pocine IVM/IVF embryos were examined by the modified semi-quantitative single cell reverse transcriptase-polymerase chain reaction (RT-PCR). Porcine embryos produced by in vitro procedures were expressed mRNAs for CuZn-SOD, GAPDH and GPX, whereas transcripts for Mn-SOD and catalase were not detected at any developmental stages. Expression of caspase-3 mRNA was detected at 2 cell, 8 cell 16 cell and blastocyst, but p53 mRNA was not detected at any stages. The fas transcripts was only detected in blastocyst stage. These results suggest that various antioxidant enzymes and apoptosis genes play crucial roles in vitro culture of porcine IVM/IVF embryos.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.110-110
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• 2003

The high incidence of polyspermic fertilization is one of the major causes lowering the overall efficiency of porcine IVF. The common procedure for IVF involves the co-culture of both gametes in the medium drop, which increases sperm concentration and incidence of polyspermy. Therefore, the present study was carried out to increase the efficiency of porcine IVF by reducing polyspermy using a modified swim-up method. This method modifies conventional swim-up washing by placing oocytes directly at the time of washing. Sperm pellet was prepared in the tube and mature oocytes were placed on cell strainer with $70 \mu m$ pore size (Falcon 2350) at the top of the tube. After insemination, the oocytes were stained for examination. Also, the developmental potential of fertilized embryos was measured to evaluate for the feasibility of this method. While having similar penetration rates in both methods ($86.67 \pm 2.36% to 83.33 \pm 1.36%$), there was a significant reduction of polyspermy in modified swim-up method ($17.50 \pm 1.60%$) compare to the control ($44.1 \pm 3.70%$ (p<0.05). Subsequent culture showed higher rate of blastocyst formation in modified swim-up method (20.44$\pm$0.99%) than the control ($15.73 \pm 3.26%$) (P<0.05), even though there was no significant difference. These results suggest that, by controlling the number of spermatozoa reaching the oocytes, porcine oocytes might be protected from polyspermy in vitro. Also, the developmental potential of the fertilized embryos using this method could be improved by increasing the pool of spermatozoa with better quality. Further optimization of the procedure required to implicate this method in routine porcine IVF.

• Journal of Embryo Transfer
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• v.9 no.3
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• pp.269-277
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• 1994

The objective of this study was to develop an effective in vitro production system capable of obtaining more porcine embryos from immature oocytes These experiments were conducted to examine the effect of sperm factor on the IVF and IVD, and the effect of coculture with somatic cells on the IVD of embryos. Although the concentration of epididymal sperm for IVF did not affect on cleavage rate, but 5 x 105 sperm/mi showed the highest cleavage rate(48.7%) and the developmental potential of IVF oocytes from this concentration was also greatly higher (P$^{\circ}C$-stored sperm for l2hrs and the cleavage rate from fresh sperm was significantly higher (P<0.05) than that from frozen sperm, but the developmental potential after IVF was slightly high from the frozen sperm. The cleavage rate of IVF oocytes cocultured with oviductal epithelial cells and cumulus cells was 76.3% and 72.9%, respectively. There was no difference between two coculture systems but this rate was significantly higher(P<0.05) than that of medium alone(42.0%).

• Reproductive and Developmental Biology
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• v.33 no.1
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• pp.7-11
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• 2009

This study was performed to confirm the microtubule assemblies and methylation patterns of porcine IVF and parthenogenetic embryos. Cumulus-oocyte complexes were collected and matured in vitro for 42 hr. Oocytes were fertilized by prepared fresh sperm or activated parthenogenetically by exposure to electric stimulation and 6-dimethylaminopurine. Porcine IVF and parthenogenetic embryos were cultured in vitro for 6 days. Embryos were stained by immunofluorescence staining method to observe the dynamic of nucleus and microtubules in the first mitotic phase and the methylation patterns in different developmental stages. After then, samples were confirmed and analyzed through a laser-scanning confocal microscope. IVF embryos had a centrosome originated from sperms, which was shown a $\gamma$-tubulin spot. However, $\gamma$-tubulin spot was not observed in parthenogenetic embryos. A lower methylation level was observed in IVF embryos compared to parthenogenetic ones at the morula and blastocyst stages. In conclusion, it is considered that microtubule assemblies and genetic regulation mechanism differ between parthenogenetic and IVF embryos.

• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.289-297
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• 2000

This study was conducted to investigate whether various protein sources and co-culture affect in vitro development of porcine zygotes derived from In vitro maturation/fertilization (IVM/IVF). These results obtained in these experiments are summarized as follows 1. When porcine oocytes matured and fertilized In vitro were cultured in NCSU 23 medium supplemented with various BSA concentrations (0.4, 0.8 and 3.2%), In vitro developmental rates of porcine zygotes to blastocyst stage were 22.9, 18.4 and 14.6%, respectively. High concentration of BSA (3.2%) showed a smaller nuclei number (36.1$\pm$11.8) of blastocysts than 0.4 and 0.8% BSA groups (53.2$\pm$27.4 and 61.2$\pm$22.5, respectively) (P<0.05). This result indicates that high concentration of BSA is detrimental on preimplantation development of IVF-derived porcine embryos. 2. No differences were detected in the developmental rate and mean nuclei number of porcine embryos between 10 and 20% FBS concentrations in culture medium. 3. IVF-derived porcine embryos co-cultured with mouse or porcine embryonic fibroblast cells showed a lower development to the blastocyst stage than those without co-culture system. Consequently, the present study suggests that high concentration of BSA as a protein source in culture medium suppresses development potential of porcine embryos produced In vitro. In addition, co-culture with somatic cells is not effective on in vitro development of IVF-derived porcine embryos to blastocyst stage.