• Korean Journal of Animal Reproduction
• /
• v.26 no.4
• /
• pp.385-394
• /
• 2002

This study was constructed the correlations of the embryonic developmental rates and the frequency of chromosome aberration using ear-skin-fibroblast cell in nuclear transfer (NT) derived embryos. Karyoplast-oocyte complexes were fused and activated simultaneously, then cultured for seven days to assess development. The developmental rates of NT and in vitro fertilization (IVF) embryos were 55.4% vs 63.5%, 31.7% vs 33% and 13.4% vs 16.8% in 2 cell, 8 cell and blastocyst, respectively. Firstly, the frequency of chromosome aberrations were evaluated using fluorescent in situ hybridization (FISH) technique with porcine chromosome 1 submetacentric specific probe. Chromosome aberration was detected at day 3 on the embryo culture, the percentages of chromosomal aneuploidy in NT and IVF embryos at 4-cell stage were 40%, 31.3%, respectively. Secondly, embryonic fragmentation was evaluated at 4-cell stage embryo. Frequency of embryonic fragmentations was in 51.3% of NT, 61.3% of IVF, 28.9% of parthenogenetic activation at 4-cell stage. The proportion of fragmentation in NT embryos was higher than activation embryos. This result indicates that chromosomal abnormalities and embryonic fragments are associated with low developmental rate in porcine NT embryo. It is also suggest that abnormal porcine embryos produced by NT related with lower implantation rate, increased abortion rate and production of abnormal fetuses.

• Journal of Embryo Transfer
• /
• v.16 no.3
• /
• pp.173-182
• /
• 2001

The present study was carried out to examine the effect of superoxide dismutase (SOD) on in vitro maturation (IVM) of porcine follicular oocytes and oxygen concentration with SOD 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 and mean numbers of the penetrated sperm were not different in the NCSU-23 maturation media with 0, 100, 500 and 1,000 units/ml SOD. However. the pronucleus formation rates were significantly lower in oocytes matured with addition groups than those of no addition groups of SOD (P>0.05). 2. The rates of blastocyst formation and total cell numbers of blastocyst at day 7 after in vitro fertilization were significantly lower in addition groups than those of the no addition groups of SOD (P>0.05). 3. The rates of blastocyst formation at day 7 after in vitro fertilization were higher in the NCSU-23 culture medium with 100 units/ml SOD than in those cultured with 0, 500 and 1,000 units/ml SOD under the 5% and 20% $O_2$concentrations. However, no differences was found in the total cell numbers of blastocyst among the treatments. In conclusion, these results suggested that the addition of SOD was not adequate for porcine oocyte maturation and further development, also the rates of blastocyst formation and total cell numbers of blastocyst at day 7 of porcine IVM/IVF embryos were not different in the NCSU-23 culture medium under the 5% and 20% $O_2$concentrations.

• Korean Journal of Animal Reproduction
• /
• v.27 no.2
• /
• pp.115-123
• /
• 2003

The present study was carried out to examine the effect of catalase (CAT) on in vitro maturation (IVM) of porcine follicular oocytes and oxygen concentration with CAT on in vitro development (IVD) of porcine IVM/IVF embryos. The results were summarized as follows - 1 . The rates of nuclear maturation, penetrated oocytes, pronucleus formation rates, polyspermic oocytes and mean numbers of the penetrated sperm were significantly lower in oocytes matured with 100, 500 and 1,000 units/ml CAT than those of central groups (P>0.05). 2. The rates of blastocyst formation and total cell numbers of blastocyst at day 7 after in vitro fertilization were significantly lower in CAT treatment groups than those of the central groups (P>0.05). 3. There were not significant difference in the blastocyst development and total cell numbers of blastocyst on in vitro culture of NCSU-23 media with 0, 100, 500 and 1000 units/ml CAT under the 5% and 20% $O_2$ concentrations. These results suggested that the addition of CAT was not helpful for porcine oocyte maturation and further development, also 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 the 5% and 20% $O_2$ concentrations.

• Reproductive and Developmental Biology
• /
• v.28 no.2
• /
• pp.113-117
• /
• 2004

The present study was carried out to examine the effects of $O_2$ concentrations and culture media (North Carolina State University (NCSU)-23, porcine zygote medium(PZM)-3 or PZM-4) on in vitro development of porcine IVM/IVF embryos. Porcine oocyte-cumulus complexes were cultured in BSA-free NCSU-23 medium containing porcine follicular fluid (10%), cysteine (0.9 mM), $\beta$-mercaptoethanol (25 $\mu\textrm{g}$/$m\ell$), epidermal growth factor (10 ng/$m\ell$) and hormonal supplements (PMSG and hCG: 10 IU/$m\ell$) for 20∼22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20∼22 h. After culture, cumulus-free oocyte were coincubated with liquid boar spermatozoa for 5∼6h. Putative zygotes were transferred to NCSU-23, PZM-3 and PZM-4 medium under the condition of 5% $O_2$ or 20% $O_2$ concentrations. At 48 h, no mean differences were found in cleavage rates. However, the rates of blastocyst formation at day 7 after in vitro fertilization were significantly higher in PZM-3 medium under the condition of 5% $O_2$ concentration than other treatments (19.9$\pm$2.4 vs. 11.1$\pm$2.0 to 16.0$\pm$2.5%, P<0.05). The total cell numbers of blastocysts were significantly higher in 5% $O_2$ than in 20% O2 (P<0.05). However, no differences was found among the culture media within each $O_2$ concentrations. In conclusion, the use of PZM-3 medium in 5% $O_2$ concentration was effective on in vitro development of porcine IVM/IVF embryos.

• Proceedings of the Korean Society of Embryo Transfer Conference
• /
• 2002.11a
• /
• pp.8-17
• /
• 2002

There have been intensive attempts to establish reliable methods far in vitro production (IVP) methods for of porcine embryos. Although a great deal of progress has been made, our current IVP systems still need to be improved. In this review, we focused on studies about in vitro maturation and fertilization (IVM-IVF) of porcine oocytes and their in vitro culture (IVC), especially on an excellent piglets production system using modified IVP system producing porcine blastocysts with high Quality.

• Journal of Embryo Transfer
• /
• v.29 no.2
• /
• pp.141-148
• /
• 2014

Quercetin and genistein, plentifully present in fruits and vegetables, are flavonoid family members that have antioxidative function and plant-derived phytoestrogen activity. The antioxidative effects of quercetin and genistein on boar sperm characteristics and in vitro development of IVF embryo were investigated. The sperm motility was increased by addition of genistein $50{\mu}M$ for 6 hr incubation compared to control (p<0.05). The sperm viability was increased by addition of quercetin 1 and $50{\mu}M$ and genestein 1 and $50{\mu}M$ for 3 hr incubation. In addition, the sperm viability seemed to be increased dose-dependantly by addition of quercetin or genistein 1 and $50{\mu}M$, respectively (p<0.05). The membrane integrities were not increased by quercetin or genistein treatments for 3 hr or 6 hr incubation period except for quercetin $1{\mu}M$ for 3 hr incubation. In mitochondrial activities, addition of quercetin $50{\mu}M$ for 6 hr incubation increased mitochondrial activity but decreased at $100{\mu}M$ concentration compared with control (p<0.05). When porcine IVF embryos were cultured in PZM-3 medium supplemented with low concentrations of quercetin ($1{\sim}10{\mu}M$), the developmental rates to morula and blastocyst increased but significantly decreased at high concentrations of quercetin ($25{\sim}50{\mu}M$). The highest developmental rate to blastocysts among all concentrations of quercetin was shown at quercetin $10{\mu}M$ (p<0.05). The developmental rates to morula or blastocysts at low ($0.01{\sim}1{\mu}M$) and high ($5{\sim}10{\mu}M$) concentrations of genistein were not significantly different among all treatment group and genistein did not affect on IVF embryo development. These results suggest that quercetin and genistein seem to have positive effects at certain concentrations on sperm characteristics such as motility, viability and mitochondrial activity. In addition, low concentrations of quercetin (1, 5 and $10{\mu}M$) in this experiment, seem to have beneficial effect on porcine IVF embryo development but genistein did not affect on it at all given concentrations ($0.01{\sim}10{\mu}M$).

• Asian-Australasian Journal of Animal Sciences
• /
• v.20 no.5
• /
• pp.711-717
• /
• 2007

In this study, two cell types from porcine females, namely fetal fibroblasts (pFFs) and adult ear fibroblasts (pAEFs) and two passages (3-4 and 7-8) were investigated by evaluating the development rate, blastocyst cell number and the incidence of apoptosis. No significant differences were observed in the cleavage rates of cloned and IVF embryos. The blastocyst rates between the embryos cloned with pFFs ($15.1{\pm}3.2$) and pAEFs ($10.4{\pm}2.6$) did not differ significantly but was significantly (p<0.05) lower in pAEFs than that in IVF ($22.5{\pm}4.5$) embryos. Total cell number in pFFs ($28.4{\pm}4.3$) and pAEFs cloned blastocysts ($24.2{\pm}5.1$) was significantly (p<0.05) lesser than IVF control ($35.4{\pm}3.2$). Apoptosis rates between cloned blastocysts differed significantly (p<0.05) and were significantly (p<0.05) higher than IVF embryos. The blastocyst rates between the cloned embryos cloned with different cell passages did not differ significantly but in embryos cloned with 7-8 cell passage was significantly (p<0.05) lower than the IVF control. Apoptosis signals were detected in IVF and cloned embryos as early as day 3 and the rates of apoptosis increased concurrently with the embryo development. In conclusion, high apoptosis during in vitro preimplantation development resulted in low development rate and total cell number of cloned embryos. Moreover, based on the apoptotic incidence in cloned blastocysts, fetal fibroblasts are more suitable for production of cloned embryos in porcine.

• Journal of Embryo Transfer
• /
• v.17 no.3
• /
• pp.239-249
• /
• 2002

Embryos derived from pig oocytes matured in mSOF are able to develop to blastocysts after IVF. Experiment 1 evaluated the effects of two maturation media (TCM-199 vs mSOF) on maturation rate, fertilization parameters, including penetration, polyspermy, male pronuclear formation, and the mean number of sperm penetrated per oocyte. Experiment 2 and Experiments 3 examined the effects of two maturation media on zona pellucida solubility and cortical granule distribution by transmissible electron microscopy, respectively. Experiment 4 assessed the effects of two maturation media on the in vitro embryo cleavage rate and development to blastocyst. Lastly, experiment 5 examined the cell number of blastocyst. An effect of media (P<0.05) was detected for mSOF on the mean number of sperm per oocyte. In TCM group, zona digestion time (196.5$\pm$15.5 vs 131.6$\pm$20.1 before IVF, 397.5$\pm$30.3s vs 185.3$\pm$16.4s after IVF, p<0.05) was higher in TCM-199 group. No significant effects of media was observed on cortical granule distribution between two groups by TEM. An effect (P<0.05) was observed on embryo development to blastocyst (16% vs 8%) but not on cleavage rates. No significant effects of media was observed on total cell number of blastocyst. We found that the high mean number of sperm penetrated per oocyte and the weaker zona pellucida on the basis of the digestion time was shown in pig oocytes matured in mSOF, however, porcine oocyte maturation with supplemented synthetic oviduct fluid medium (mSOF) resulted in blastocyst cell numbers comparable to those observed with Tissue Culture Medium 199.

• Development and Reproduction
• /
• v.22 no.1
• /
• pp.73-83
• /
• 2018

This study investigates the endoplasmic reticulum (ER) stress and subsequent apoptosis in duced during somatic cell nuclear transfer (SCNT) process of porcine SCNT embryos. Porcine SCNT and in vitro fertilization (IVF) embryos were sampled at 3 h and 20 h after SCNT or IVF and at the blastocyst stage for mRNA extraction. The x-box binding protein 1 (Xbp1) mRNA and the expressions of ER stress-associated genes were confirmed by RT-PCR or RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. Before commencing SCNT, somatic cells treated with tunicamycin (TM), an ER stress inducer, confirmed the splicing of Xbp1 mRNA and increased expressions of ER stress-associated genes. In all the embryonic stages, the SCNT embryos, when compared with the IVF embryos, showed slightly increased expression of spliced Xbp1 (Xbp1s) mRNA and significantly increased expression of ER stress-associated genes (p<0.05). In all stages, apoptotic gene expression was slightly higher in the SCNT embryos, but not significantly different from that of the IVF embryos except for the Bax/Bcl2L1 ratio in the 1-cell stage (p<0.05). The result of this study indicates that excessive ER stress can be induced by the SCNT process, which induce apoptosis of SCNT embryos.

• Korean Journal of Animal Reproduction
• /
• v.25 no.2
• /
• pp.181-189
• /
• 2001

The objective of this study was to evaluate the development of porcine follicular oocytes fertilized by intracytoplasmic sperm injection (ICSI). Cumulus-oocyte-complexes (COCs) were collected by aspiration from follicles of 2~7 mm in diameter from a local slaughterhouse. Oocytes were matured in vitro for 40~44 h, and spermatozoa were prepared by swim-up in the presence or absence of 5 mM dithiothreitol (DTT) and then M II stages of the oocyte were either centrifuged or not centrifuged for the following injection of ooplasm. Injected oocytes were cultured in NCSU 23 medium for 6 to 8 days. The results obtained were as follows: 1. The rates of cleavage and development rates into blastocyst by ICSI were not significantly different between the with (53.0% and 19.7%) or without (48.3% and 23.8%) centrifugation, respectively (P＜0.05). 2. The cleavage and developmental rates to blastocyst after ICSI with or without 5 mM DTT treated-sperm were not significantly different (60.4% vs 16.4% and 45.5% vs 22.2%), respectively (P＜0.05). 3. The cleavage and the developmental rates to btastocyst were not significantly different between the zygotes obtained by IVF (51.8% vs. 22.4%) and ICSI (51.4% vs. 21.6%) (P＜0.05). 4. The number of blastomere in blastocyst stages after IVF or ICSI was not significantly different (46.7$\pm$2.9 and 41.9$\pm$4.6).