• 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.32 no.4
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• pp.337-342
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• 2017

The present study was aimed to determine the effects of green tea extract (GTE) and beta-mercaptoethanol (${\beta}-ME$) supplementation in boar sperm freezing extender on in vitro fertilization (IVF) and reactive oxygen species (ROS) and glutathione (GSH) levels of presumptive zygotes (PZs). Experimental groups were allocated into lactose egg yolk (LEY) without antioxidant (control), GTE (1,000 mg/l in LEY) and ${\beta}-ME$ ($50{\mu}M$ in LEY). In freezing, spermatozoa extended with LEY were cooled to $5^{\circ}C$ for 3 h and then kept at $5^{\circ}C$ for 30 min following dilution with LEY containing 9% glycerol and 1.5% Equex STM. The final sperm concentration was $1{\times}10^8/ml$. Spermatozoa were loaded into straws and frozen in nitrogen vapor for 20 min. For IVF, oocytes were matured in NCSU-23 medium and co-cultured with spermatozoa following thawing at $37^{\circ}C$ for 25 sec. At 12 h following IVF, IVF parameters (sperm penetration and monospermy) were evaluated. In addition, GSH and ROS levels of PZs were determined by Cell Tracker Blue CMF2HC and DCHFDA, respectively. IVF parameters did not show any significant difference among the experimental groups. GSH and ROS levels of PZs were not significantly different between groups. In conclusion, antioxidant supplementation in boar sperm freezing could not influence IVF parameters, ROS and GSH levels of PZs.

• Journal of Embryo Transfer
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• v.24 no.1
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• pp.65-69
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• 2009

The aim of the present study was to investigate the cleavage pattern, its developmental ability and apoptosis of porcine embryo in vitro. Morphology data on a total of 919 embryos were analyzed retrospectively. Forty-eight hours after insemination, embryos were classified into five groups based on the cleavage state as follows; 1 cell, 2 cell, 4 cell, 5 to 8 cell and fragmentation. These groups were cultured another 120 hours and then evaluated for blastocyst formation. Blastocyst formation rates were significantly higher in 4 cell (42.5%) and 5 to 8 cell (48.6%) cleaving groups than in other groups (p<0.05). On the other hand, 2 cell and fragmentation groups produced 4.9% and 3,9% blastocysts, respectively. And we could verify that in the event of 2 cell block and fragmentation of embryo. To analyze the apoptotic frequency in preimplantation development of porcine IVF embryos, all cells of each blastocyst were performed by TUNEL assay. There were no significantly differences in the total cell numbers of embryos and apoptotic cell rate in blastocysts among the each classified groups. Data suggest that 4 cell and 5 to 8 cell cleaving embryos at 48 hour after insemination have high developmental competence, and may be an useful parameter to predict the development of preimplantation embryos and to study using preimplanation embryonic research.

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.41-51
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• 2002

The present study was carried out to examine the effect of cysteamine in vitro maturation (IVM) of porcine oocytes and development of porcine IVM/IVF Embryos. The results were summarized as follows : 1. The rates of nuclear maturation, penetrated oocytes, pronuclear formation, polyspermic oocytes and mean numbers of the penetrated sperm were not different in NCSU23 maturation medium with 0, 25, 50 and 100 $\mu$M cysteamine (P〉0.05). 2. The rates of blastocyst formation at day 7 after in vitro fertilization in 0, 25, 50 and 100 $\mu$M cysteamine were 17.9$\pm$6.1, 17.4$\pm$6.3, 24.2$\pm$1.9 and 16.9$\pm$2.0%, respectively. And the total cells were 30.7$\pm$2.4, 34.9$\pm$2.8, 39.6$\pm$2.3 and 36.8$\pm$3.6, respectively. Fifty $\mu$M cystealnine group was significantly higher than those of any other treatment groups (P＜0.05). 3. The ratios of ICM/total cells in 20~40% category were 20.5, 41.6, 19.5 and 31.5%, respectively. Twenty five $\mu$M cysteamine group was higher than those of other groups. 4. The rates of blastocyst formation at day 7 in the NCSU-23 culture medium of porcine IVF-produced embryos with 0, 25, 50, and 100 $\mu$M cysteamine were 16.0$\pm$0.2, 13.6$\pm$1.7, 25.0$\pm$0.8 and 15.7$\pm$4.5%, respectively. And the total cells were 27.0$\pm$3.7, 36.1$\pm$4.8, 34.0$\pm$3.8 and 25.2$\pm$4.4, respectively. Fifty $\mu$M cysteamine group was significantly higher than those of any other treatment groups (P＜0.05). 5. The ratios of ICM/total cells in 20~40% category were 53.8, 30.0, 16.6 and 11.1%, respectively. The addition groups of cysteamine were lower than those of control group. In conclusion, these results suggested that the addition of 50 $\mu$M cysteamine in the IVM medium and 25~50 $\mu$M cysteamine in IVC medium were effective on the blastocyst formation and total cells of blastocysts.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.83-87
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• 2004

The objective 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 melatonin, nitric oxide donor(SNP), and the combination effects of SNP and melatonin in porcine IVM/IVF embryos. In routine porcine IVM/IVF procedure, oocytes were cultured for 40∼44h incubation, and the zygotes were cultured for 40∼44h in NCSU 23 medium. Then 2 to 8 cell embryos were removed cumulus cell and were allotted randomly to NCSU 23 containing different concentration of melatonin, SNP and SNP plus melatonin in 5% $O_2$, 5% $CO_2$ and 90% $N_2$ at 38.5$^{\circ}C$. Cell numbers of blastocyst were also counted using double fluorescence stain method. 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%, respectivly. This result show that the developmental rate of morula and blascytocys treated with 1 nM melatonin was higher than in any other groups(P<0.05). The developmental rates of morula plus blastocysts were 41.9% in 0 uM SNP, 25.6% in 50 uM and 28.4% in 100 uM, respectively. The developmental rate of morula plus blastocysts were decreased treated with SNP in NCSU 23. In combined effects of SNP plus melatonin (0, SNP 50 uM, SNP 50 uM plus melatonin 1 nM, SNP 50 uM plus melatonin 5 nM and SNP 50 uM plus melatonin 10 nM), the developmental rates beyond morula stage of porcine embryos were 31.3%, 34.1%, 39.5%, 29.4% and 39.5%, respectively. The addition of SNP 50 uM plus maltonin 1 nM, developmental rates of blastocyst was higher rate than in any other groups. Cell numbers of blastocyst in NCSU 23 treated with melatonin 0, 1, 5 and 10 nM were 41.0, 42.6, 39.6 and 33.0, respectively. In combined effects of SNP plus melatonin (0, SNP 50 uM, SNP 50 uM plus melatonin 1 nM , SNP 50 uM plus melatonin 5 nM and SNP 50 uM plus melatonin 10 nM), cell numbers of developed blastocyst were 36.3, 34.6, 39.0, 39.9 and 39.0, respectively. These result show that the cell numbers of blastocyst treated with 0, 1 and 5 nM melatonin were higher than in 10 nM group(P<0.05), but cell numbers of blatocyst produced by SNP plus melatonin were not significantly difference in all experimental groups.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.4
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• pp.485-493
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• 2002

Different factors may affect the sensitivity of porcine oocytes during cryopreservation. The effect of two methods (cooling and vitrification), four cryoprotectants [glycerol (GLY), 1, 2-propanediol (PROH), dimethyl sulfoxide (DMSO) or ethylene glycol (EG)] and two vitrification media (1 M sucrose (SUC)+8 M EG; 8 M EG) on the developmental capacity of porcine oocytes at the germinal vesicle (GV) stage or after IVM at the metaphase II (M II) stage were examined. Survival was assessed by FDA staining, maturation and cleavage following IVF and IVC. A toxicity test for different cryoprotectants (GLY, PROH, DMSO, EG) was conducted at room temperature before cooling. GV and M II-oocytes were equilibrated stepwise in 1.5 M cryoprotectant and diluted out in sucrose. The survival rate of GV-oocytes in the GLY group was significantly lower (82%, p<0.01) than that of the other group (92 to 95%). The EG group achieved a significantly higher maturation rate (84%, p<0.05) but a lower cleavage rate (34%, p<0.01) than the DMSO group and the controls. For M II-oocytes, the survival rates for all groups were 95 to 99% and the cleavage rate of the GLY group was lower than the PROH-group (21 vs 43%, p<0.01). After cooling to $10^{\circ}C$, the survival rates of GV-oocytes in the cryoprotectant groups were 34 to 51%, however, the maturation rates of these oocytes were low (1%) and none developed after IVF. For M II-oocytes, the EG group showed a significantly higher survival rate than those of the other cryoprotectant groups (40% vs 23-26%, p<0.05) and the cleavage rates of PROH, DMSO and EG group reached only 1 to 2%. For a toxicity test of different vitrification media, GV and M II-oocytes were equilibrated stepwise in 100% 8 M EG (group 1) and 1 M SUC + 8 M EG (group 2) or equilibrated in sucrose and then in 8 M EG (SUC+8 M EG, group 3). For GV-oocytes, the survival, maturation and cleavage rates of Group 1 were significantly lower than those in group 2, 3 and control group (p<0.05). For M II-oocytes, there were no differences in survival, maturation and cleavage rates between groups. After vitrification, the survival rates of GV and M II-oocytes in group 2 and 3 were similarly low (4-9%) and none of them matured nor cleaved after in vitro maturation, fertilization and culture. In conclusion, porcine GV and M II-oocytes do not seem to be damaged by a variety of cryoprotectants tested, but will succumb to a temperature decrease to $10^{\circ}C$ or to the process of vitrification, regardless of the cryoprotectant used.

• Korean Journal of Animal Reproduction
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• v.21 no.3
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• pp.247-253
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• 1997

The objective of this study was to examine the fertilization and embryonic development rates of porcine oocytes matured in vitro according to the morphological normality of epididymal sperm in porcine IVF. The results obtained in this experiment were summarized as follows : 1. When the ratio of morphological normality of epididymal sperm was divided into the three groups with $\leq$ 10%, 10-30% and $\geq$ 50%, the fertilization and embryonic devel-opment rates of 능50% group (64%. 26%) w were significantly higher than those of other two groups ($\leq$10%: 27%, 6% and 10-30%: 36%. 5%) (p$\leq$50% group of morphologically normal epididymal sperm was adjusted to 100% (5 x 10$^5$ cells/ml), the fertilization a and development rates (63%, 27%) of epidymal sperm were similar to those of frozen-thawed ejaculated sperm (56%, 35%). 3. Also, when the fertilization and development rates of epididymal sperm were analyzed according to the oocyte : sperm ratio (1:6000, 1: 6650, 1: 7700, 1: 10000), the fertilization and development rates indicated high, in 1:6000 (68%, 32%) and 1:6650 (89%, 31%), the ratio of oocyte: sperm. Therefore, when the percentage of morphological normality of epididymal sperm is more than 50, the embryonic development a can be obtained similar to that of frozen thawed ejaculated sperm. Also, these result suggested that the morphological evaluation of normal sperm in porcine IVF using epididymal sperm sho비d be prerequisite for the more effective embryonic developm ment.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.7
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• pp.956-965
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• 2019

Objective: The main goal of this study was to provide a morphological indicator that could be used to select high-quality oocytes of appropriate meiotic and developmental capabilities in pig. The higher quality of immature oocytes, the higher success rates of in vitro maturation (IVM) and in vitro fertilization (IVF). Thus, prior to the IVM culture, it is important to characterize oocytes morphologically and biochemically in order to assess their quality. Two of the largest indicators of oocyte quality are the presence of cumulus cells and status of chromatin. To investigate the effects of porcine oocyte chromatin configurations on the developmental capacity of blastocysts, we assessed oocyte chromatin status according to follicle size and measured the developmental potency of blastocysts. Methods: To sort by follicle size, we divided the oocytes into three groups (less than 1 mm, 1 to 3 mm, and more than 3 mm in diameter). To assess chromatin configuration, the oocytes were assessed for their stages (surrounded nucleolus [SN] germinal vesicle [GV], non-surrounded nucleolus [NSN] GV, GV breakdown, metaphase I [MI], pro-metaphase II [proMII], and metaphase II [MII]) at different maturation times (22, 44, and 66 h). To assess the development rate, oocytes of each follicle size were subjected to parthenogenetic activation for further development. Finally, GV oocytes were grouped by their chromatin configuration (SN, SN/NSN, and NSN) and their global transcriptional levels were measured. Results: SN GV oocytes were more suitable for IVF than NSN GV oocytes. Moreover, oocytes collected from the larger follicles had a greater distribution of SN GV oocytes and a higher developmental capacity during IVM, reaching MII more quickly and developing more often to blastocysts. Conclusion: Porcine oocytes with high-level meiotic and developmental capacity were identified by analyzing the relationship between follicle size and chromatin configuration. The porcine oocytes from large follicles had a significantly higher SN status in which the transcription level was low and could be better in the degree of meiotic progression and developmental capacity.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.129-135
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• 2006

This study was conducted to investigate the effect of in vitro maturation (IVM) duration of porcine follicular oocytes on maturation rate, polyspermic rate, and subsequent embryo development. The nuclear maturation rates of oocytes matured for 36, 38, 40, 42 and 44 hr were similar between 68.0, 78.0, 79.5, 73.8 and 81.8% respectively. There was no significant difference in the rates of polyspermy after in vitro feritilization (IVF). The cleavage rate in the group of 36 hr was significantly higher in than that of 40, 44 hr (p<0.05) but not to 38 and 42 hr. The development rate to blastocyst stage was significantly higher in the group of 38 hr (23.1%) than that in the group of 44 hr (15.6%) (p<0.05) but not to 36, 40 and 42 hr. These results suggest that the aged oocytes for 44 hr is not required for the production of bias to cysts derived from porcine IVF embryos.