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

• Journal of Embryo Transfer
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• v.28 no.2
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• pp.133-139
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• 2013

The objective of this study was to determine the effect of post-activation treatment with cytoskeletal regulators in combination with or without 6-dimethylaminopurine (DMAP) on embryonic development of pig oocytes after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). PA and SCNT oocytes were produced by using in vitro-matured pig oocytes and treated for 4 h after electric activation with $0.5{\mu}M$ latrunculin A (LA), $10.4{\mu}M$ cytochalasins B (CB), and $4.9{\mu}M$ cytochalasins D (CD) together with none or 2 mM DMAP. Post-activation treatment of PA oocytes with LA, CB, and CD did not alter embryo cleavage (85.8~88.6%), blastocyst formation (30.7~ 32.4%), and mean cell number of blastocysts (33.5~33.8 cells/blastocyst). When PA oocytes were treated with LA, CB, and CD in combination with DMAP, blastocyst formation was significantly (P<0.05) improved by CB+DMAP (42.5%) compared to LA+DMAP (28.0%) and CD+DMAP (25.1%), but no significant differences were found in embryo cleavage (77.5~78.0%) and mean blastocyst cell number (33.6~35.0 cells) among the three groups. In SCNT, blastocyst formation was significantly (P<0.05) increased by post-activation treatment with LA+DMAP (32.9%) and CD+DMAP (35.0%) compared to CB+DMAP (22.0%) while embryo cleavage (85.5~85.7%) and blastocyst cell number (41.1~43.8 cells) were not influenced. All three treatments (LA, CB, and CD with DMAP) effectively inhibited pseudo-polar body extrusion in SCNT oocytes. The proportions of oocytes showing single pronucleus formation were 89.6%, 83.9%, and 93.3%, respectively with the increased tendency (P<0.1) by LA+DMAP and CD+ DMAP compared to CB+DMAP. Our results demonstrate that post-activation treatment with LA or CD in combination with DMAP improves pre-implantation development of SCNT embryos and the stimulating effect of cytoskeletal modifiers on embryonic development is differentially shown depending on the origin (PA or SCNT) of embryos in pigs.

• Journal of Embryo Transfer
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• v.31 no.2
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• pp.131-136
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• 2016

Since the first success of animal cloning, somatic cell nuclear transfer presented various ideas in many research areas such as regenerative medicine. However, SCNT embryos has poor survival rate. Therefore, numerous researches carried out to enhance the developmental capability of porcine nuclear transfer embryos. Cytochalasin B, demecolcine, latrunculin A, cycloheximide and 6-dimethylaminopurine are efficient chemicals treated in post-activation procedure to increase the efficiency of SCNT. This review study is aim to investigate the effects of these chemicals applied to post-activation in porcine SCNT. Cytochalasin B, demecolcine, latrunculin A are cytoskeletal manuplators inhibit extrusion of pseudo-polar body. Cytochalasin B and demecolcine showed considerably higher blastocyst formation proportion (26-28%) compared to when they are not treated (16%). And when latrunculin A was treated for postactivation, blastocyst formation proportion was increased in SCNT embryos exposed to LA (38%) than those in control (14%). On the other hand, cycloheximide and 6-dimethylaminopurine are protein synthesis and kinase inhibitors. And they help to maintain $Ca^{2+}$ fluctuation in oocytes. Cleavage and blastocyst rates of NT embryos were increased when they were exposed to CHX (16.9% and 5.4% with no CHX).And 6-DMAP also showed higher blastocyst formation (21.5% compared to 15.7%, control). Although all these chemicals have different mechanisms, they showed developmental competence enhancement in NT embryos. However, there are only few studies comparing each chemical's post-activation effect. Therefore, further research and study should be conducted to find optimal chemical for improving the efficiency of SCNT.

• Journal of Embryo Transfer
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• v.23 no.4
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• pp.245-250
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• 2008

This study was designed to investigate the effect of essential amino acids (EAA) and/or non-essential amino acids (NEAA) on the development of parthenogenetic and somatic cell nuclear transfer (SCNT) porcine embryos in vitro. To evaluate the timing of amino acids supplementation, activated oocytes were cultured in NCSU23-PVA with EAA, NEAA or NEAA+EAA (AAs) during specific periods as below: EAA, NEAA or AAs were supplemented during Day 0 to 6 (whole culture period: ALL), Day 2 to Day 6 (post-maternal embryonic transition period: POST-MET), Day 5 to Day 6 (post-compaction period: POST-CMP), Day 0 to Day 2 (pre-maternal embryonic transition period: PRE-MET), or Day 0 to Day 4 (post-compaction period: PRE-CMP). Supplementation of NEAA decreased cleavage rates in PRE-MET and PRE-CMP and also decreased blastocyst rates in POST-CMP. On the other hand, EAA significantly enhanced blastocyst formation rate in POST-MET and no detrimental effect on embryonic development in other groups. Interestingly, NEAA and EAA had synergistic effect when they were supplemented to the medium during whole culture period. Supplementation of AAs also enhanced SCNT porcine embryo development whereas BSA-free medium without AAs could not supported blastocyst formation of SCNT embryos. In conclusion, existence of EAA and NEAA in defined culture medium variously influences the development of parthenogenetic and SCNT porcine embryos, and their positive effect are only occurred when both EAA and NEAA are supplemented to the medium during whole culture period. Additionally, AAs supplementation enhances the blastocyst formation of SCNT porcine embryos when they are cultured in the defined condition.

• Journal of Environmental Health Sciences
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• v.24 no.2
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• pp.126-133
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• 1998

Effects of B(a)P on preimplantation mouse embryos in vitro were studied. Preimplantation mouse embryos were exposed to a concentration of 0.3, 1, 3 and 10 $\mu$M B(a)P for 72 hrs. The toxicological effects of B(a)P were evaluated by morphological observation of embryos up to the blastocyst stage, and by measuring DNA, RNA and protein synthesis by radioactive precursor incorporation. At 1 $\mu$M B(a)P did not affect preimplantation development but interfered with hatching and ICM formation. Suppressing effect of ICM formation was dose dependent. At the eight cell stage, the developmental rate was decreased at above 3 $\mu$M of B(a)P. At the blastocyst stage, attachment and trophoblast outgrowth were diminished at the 10 $\mu$M of B(a)P and ICM formation was decreased at 1 $\mu$M of B(a)P. Inner cell number of blastocyst was decreased dose dependently. So, number of ICM was one of the most sensitive and toxicological end point. The RNA incorporation rate of 0.1 $\mu ^3$H-uridine was dosedependent and the protein incroporation of 0.5 $\mu Ci ^{35}$S-methionine showed a significant decrease after 48 hrs. But the DNA incorporation rate of methyl-$^3$H thymidine was not affected. Our results suggested that B(a)P did not affect the DNA replication but transcription was inhibited by dose dependent manner. There delay of development during the blastocyst stage was mainly due to the inhibition of RNA synthesis followed by protein synthesis.

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

• Journal of Embryo Transfer
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• v.21 no.3
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• pp.183-190
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• 2006

These studies were conducted to monitor developmental competence of follicular oocytes collected from the carcass of the high meat quality in Korean native cattle using each individual protocol of IVM, IVF and IVC. The follicular oocytes that were collected from the ovaries of the cow yielded 1, $1^+\;and\;1^{++}$ meat quality were matured, fertilized and cultured using each individual protocol of IVM, IVF and IVC. As results, the number of follicular oocytes collected from individual fundamentally-registered cows yielded 1, $1^+\;and\;1^{++}$ meat grade were 28.9, 28.8 and 29.6 per head, respectively. The rates of blastocyst formation after IVM, IVF and IVC were 27.2, 28.7 and 32.9% in the cows yielded 1, $1^+\;and\;1^{++}$ meat quality, respectively. The rate of blastocyst formation was 8.4 per head. The number of follicular oocytes collected from pedigree registered cows yielded 1, $1^+\;and\;1^{++}$ meat quality were 25.8, 27.1 and 27.0 per head, respectively. The rates of blastocyst formation were 23.0, 33.7 and 42.6% in the meat quality of 1, $1^+\;and\;1^{++}$ after in vitro-manipulation, respectively (p<0.05). The rate of blastocyst formation was 8.5 per head. In conclusion, these results suggest that in vitro embryo production system using individual culture system including IVM, IVF and IVC can make good use of the gene from the carcass of the high meat quality in Korean native cattle.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.221-228
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• 2018

The osmolarity of a medium that is commonly used for in vitro culture (IVC) of oocytes and embryos is lower than that of oviductal fluid in pigs. In vivo oocytes and embryos can resist high osmolarities to some extent due to the presence of organic osmolytes such as glycine and alanine. These amino acids act as a protective shield to maintain the shape and viability in high osmotic environments. The aim of this study was to determine the effects of glycine or/and alanine in medium with two different osmolarities (280 and 320 mOsm) during IVC on embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. To this end, IVC was divided into two stages; the 0-2 and 3-7 days of IVC. In each stage, embryos were cultured in medium with 280, 320, or 360 mOsm and their combinations with or without glycine or/and alanine according to the experimental design. Treatment groups were termed as, for example, "T(osmolarity of a medium used in 0-2 days of IVC)-(osmolarity of a medium used in 3-7 days of IVC)" T280-280 was served as control. When PA embryos were cultured in medium with various osmolarities, T320-280 showed a significantly higher blastocyst formation (29.0%) than control (22.2%) and T360-360 groups (6.9%). Glycine treatment in T320-280 significantly increased blastocyst formation (50.4%) compared to T320-280 only (36.5%) while no synergistic was observed after treatment with glycine and alanine together in T320-280 (45.7%). In contrast to PA embryonic development, the stimulating effect by the culture in T320-280 was not observed in SCNT blastocyst development (27.6% and 23.7% in T280-280 and T320-280, respectively) whereas the number of inner cell mass cells was significantly increased in T320-280 (6.1 cells vs. 9.6 cells). Glycine treatment significantly improved blastocyst formation of SCNT embryos in both T280-280 (27.6% vs. 38.0%) and T320-280 (23.7% vs. 35.3%). Our results demonstrate that IVC in T320-280 and treatment with glycine improves blastocyst formation of PA and SCNT embryos in pigs.

• Journal of Embryo Transfer
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• v.25 no.3
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• pp.179-187
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• 2010

Embryonic compaction is essential for normal preimplantation development in mammals. The present study was to investigate the effects of compaction patterns on developmental competence of pig embryos. The proportion of blastocyst formation derived from compacted morula was higher than those of compacting and pre-compacting morula (P<0.01). Nuclei numbers of inner cell mass (ICM), trophectoderm (TE), and total of blastocysts derived from compacted group were also superior to those of compacting and pre-compacting groups (P<0.05). Then, compaction patterns, developmental ability and structural integrity were compared between mono- and poly-spermic embryos. The rate of compacted morula in mono-spermic embryos was higher than that of poly-spermic embryos (P<0.05). Especially, the rate of blastocyst formation derived from compacted embryos in mono-spermic embryo group was higher than that of poly-spermic embryo group (P<0.05), although no difference was detected between the two groups in the structural integrity. Finally, we confirmed that beta-catenin was differentially expressed according to compaction patterns in morula and blastocyst stage embryos. In conclusion, our results suggest that the compaction patterns during preimplantation development play a direct role in developmetal competence and quality of pig embryos.

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.315-317
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• 2015

In vitro culture of murine embryos is an important step for in vitro production systems including in vitro fertilization and generations of genetically engineered mice. M16 is widely used commercialized culture media for the murine embryos. Compared to other media such as potassium simplex optimization medium, commercial M16 (Sigma) media lacks of amino acid, glutamine and antibiotics. In the present study, we optimized M16 based embryo culture system using commercialized antibiotics-glutamine or amino acids supplements. In vivo derived murine zygote were M16 media were supplemented with commercial Penicillin-Streptomycin-Glutamine solution (PSG; Gibco) or MEM Non-Essential Amino Acids solution (NEAA; Gibco) as experimental design. Addition of PSG did not improved cleavage and blastocyst rates. On the other hand, cleavage rate is not different between control and NEAA treated group, however, blastocyst formation is significantly (P<0.05) improved in NEAA treated group. Developmental competence between PSG and NEAA treated groups were also compared. Between two groups, cleavage rate was similar. However, blastocyst formation rate is significantly improved in NEAA treated group. Taken together, beneficial effect of NEAA on murine embryos development was confirmed. Effect of antibiotics and glutamine addition to M16 media is still not clear in the study.