• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.19-26
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• 1996

In the present study, we investigated devel-opmental ability and transgene expression of IVM/IVF derived porcine embryos following microinjection with SV40-LacZ. A total of 412 IVM/IVF derived embryos were used to examine developmental ability and transgene expression following DNA microinjection. After centrifugation, pronuclei were visible in 60.3% when examined between 18~21h after IVF. Development and transgene expression were assessed after 9 days in culture. The percentages of injected embryos reaching to the morula and blastocyst were significantly lower (P<0.05) than those of non-injected control embryos. However, the percentages of DNA microinjected embryos and non-injected embryos that developed to the blastocyst or hatched blastocyst stage in dual culture systems (NCSU23 and EMEM) were significantly higher (P<0.05) than those in NCSU23 medium alone. As the resuIt of X-gal staining, the proportion of positive embryos was 40~43% in morula and blastocyst stage embryos, however, mosaicism has been observed in the most putative transgenic morulae and blastocysts. In the PCR analysis, the percentages of embryos integrated gGH gene were 45.0 and 44.4% in morula and blastocyst stage, respectively. These results suggest that improved IVM /IVF system and culture condition increased the embryo viability and ex-pression of a microinjected transgene.

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

• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.12
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• pp.1820-1826
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• 2007

In this study, we conducted various experiments in order to develop enhanced cultural conditions for in vitro-produced porcine embryos. All embryos were produced by in vitro maturation (IVM) and fertilization (IVF) of immature oocytes from abattoir-derived ovaries. In experiment 1, we cultured IVF embryos in 4 different groups, namely, 0% bovine serum albumin (BSA), 3% BSA, 0.05% Polyvinyl alcohol (PVA), and 0.5% Polyvinylpyrrolidone (PVP) added to the basal fluid cultural medium, Porcine zygote medium 3 (PZM-3). The rates of embryo development were higher in the group where the PZM-3 media had been supplemented with 3% BSA than the other groups. While not statistically significant, the percent of blastocysts and hatched blastocytes were 6.9% and 25.0% in the 3% BSA group vs. 1.2-6.4% and 0-16.7% in the other groups, respectively. In experiment 2, we added 10% fetal bovine serum (FBS) to PZM-3 on day 0 of culture and observed the development rate of blastocysts per day of culture from days 0 to 5. The development rate of blastocysts was higher at 15.6% on day 4 than on any other day, and was significantly higher than on day 0 or day 1 (p<0.05). The development rate of hatched blastocysts was 26.7% on day 4, and was higher than on any other day. In experiment 3, we cultured IVF embryos with different fluid culture media, grouped as 1) PZM-3+0.3% BSA (day0-day7); 2) PZM-3+0.3% BSA${\rightarrow}$day-4) PZM-3+10% FBS; 3) PZM-3+0.3% BSA${\rightarrow}$PZM-3+0.3% BSA+(day-4) FBS 10%; and 4) PZM-3+0.3% BSA+10% FBS (day0-day7). The development rates of blastocysts and hatched blastocysts were 21.5% and 53.1% in group 3, respectively, which was significantly higher than group 4 with respect to blastocyst development (5.2%, p<0.05) but not hatched blastocysts (14.3%). The total cell number (TCN) of blastocysts in group 3 was higher at $37.8{\pm}16.1$ than the other groups at $16.8{\pm}4.4$ - $30.1{\pm}10.9$; however, this was not significantly different. The results of this study showed that PZM-3 containing 0.3% BSA and supplemented with FBS during the later stage of culture on day 4 resulted in better TCNs and an increased rate of hatched blastocysts.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.85-93
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• 1994

This study was carried out to investigate the effects of gonadotropins added during maturation of porcine oocytes on the in vitro maturation(IVM), in vitro fertilization(IVF) and developmental potential of embryos. The follicular oocytes were cultured in TCM-199 medium containing different combination of gonadotropins(5$\mu$g /ml FSR or 1OIU /ml PMSG and 1O$\mu$g /ml LH or 1OIU /ml hCG), 10% FCS and 10% PFF for 36~48h in a incubator with 5% $CO_2$ in Air at 39$^{\circ}C$ and then matured oocytes were again cultured to 120h after IVF for 6~7h with heparin(100$\mu$g /m')-treated sperm. When the oocytes were matured for 42brs in the medium containing FSH+LH, FSH+hCG, PMSG+LH or PMSG+hCG, the JVF rate of each treatment was 50.0%, 52.9%, 66.7% and 70.0%, respectively. The highest CEI (cumulus cell expansion index) was obtained from PMSG+hCG-added medium and the highest polyspermic penetration resulted from FSH+LH-added medium. The cleavage of IVF oocytes derived from hormone added IVM was significantly(P<0.05) promoted by PMSG+hCG and the cleavage rate after 36-h, 42-h and 48-h maturation aws 53.0%, 56.7% and 45.6%, respectively. The highest developmental potential resulted from the oocytes derived from PMSG+LH -added IVM.

• Korean Journal of Animal Reproduction
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• v.25 no.1
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• pp.63-69
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• 2001

This study was carried out to examine the effects of nitric oxide compounds (hemoglobin and L-NAME) on the development of porcine in vitro maturation (IVM) and in vitro fertilization (IVF) oocytes. Cumulus cell free embryos derived from porcine IVM/IVF oocytes were cultured in NCSU23 medium containing 1~5 $\mu\textrm{g}$/$m\ell$ hemoglobin added to 44 and 96hrs in culture times, and in NCSU23 medium containing 0, 10, 50 or 100mM L-NAME. The developmental rates beyond morulae stage in 0, 1 and 5 $\mu\textrm{g}$/$m\ell$ hemoglobin groups add to 44hrs in vitro culture times were 52.4%, 57.6% and 57.4%, respectively. The addition of hemoglobin groups made it slightly higher than the control group. The proportion of embryos developed to morulae and blastocysts in 1 $\mu\textrm{g}$/$m\ell$ hemoglobin add to 96hrs after in vitro culture (70.8%) was a little higher than those of 0 and 5 $\mu\textrm{g}$/$m\ell$ hemoglobin (66.2% and 62.8%). There was no significant difference in all groups (P〉0.05). The developmental rates beyond morulae stage in 0, 10, 50 and 100mM of L-NAME groups add to 96hrs after in vitro culture were 65.2%, 73.5%, 70.1% and 53.3%, respectively 10mM and 50mM L-NAME groups were significantly higher than in 0 and 100mM of L-NAME groups (P＜0.05). In conclusions, these results indicate that L-NAME (10mM, 50mM) can increase the proportion of embryos that develop into morulae and blastocysts but hemoglobin did not affect.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.68-68
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• 2001

Blastocyst formation, consisting of the inner cell mass (ICM) and trophectoderm (TE), is the first differentiation process during embryonic development in mammals. It has been hypothesized that the proportion of ICM to TE in the blastocyst may be crucial for subsequent developmental competence of early embryos, which it may be expressed as a sensitive indicator for evaluating in vitro systems. In this study ICM/total cell ratio of nuclear transfer (NT) embryos was compared with IVF-derived and in vivo embryos. Somatic cell nuclei obtained from a fetus at Day 40 of gestation were transferred into the enucleated oocyte and then cultured in NCSU 23 medium for 6 days as previously described (Koo et al., Biol. Reprod. 2000; 63:986-992). ICM and TE cells of blastocysts were determined by using a differential staining method (Han et al., Biol. Reprod. 1999; 60:1110-1113). Development rate (9.8$\pm$2.5%, 23/225) to the blastocyst stage of NT embryos was lower than IVF embryos (23.8$\pm$2.7%, 53/223). Thus, a difference was detected in the in vitro developmental rate to blastocyst stage between NT and IVF-derived embryos (P<0.05). In the next experiment, we investigated ICM and TE nuclei to assess the quality of blastocysts that produced by NT, IVF and in vivo, respectively. NT blastocysts (27.6$\pm$8.3) showed a smaller total cell number than IVF-derived (42.6$\pm$17.4) and in vivo embryos (283.9$\pm$103.5) (P<0.05). Ratios of ICM/total cells in NT, IVF and in vivo blastocysts were 15.1$\pm$ 18.6% (n=56), 12.3$\pm$9.2% (n=57) and 30.4$\pm$6.8% (n=40), respectively. Individual blastocysts for the ratio of ICM/total cells were assigned to 3 groups (I; <20%, II; 20 to 40% and III;>40%). As the results, most in vivo blastocysts (97.5%, 39/40) were distributed into group II while most NT (78.6%, 44/56) and IVF-derived blastocysts (82.5%, 47/57) were allocated to group I. Thus, our data show that NT or IVF-derived embryos have aberrant morphology during early development in vitro systems, suggesting that these anomalies may result in developmental failures of the NT embryos to term.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.170-170
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• 2004

First of all, in vitro production (IVP) of porcine embryos is an important as initial step to improve bio-technical applications such as transgenesis and cloning for xenotransplantation. In recent years, considerable progress has been achieved in the IVP embryos using advanced methods for in vitro maturation (IVM) and fertilization (IVF). (omitted)

• 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.

• 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.