• Journal of Embryo Transfer
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• v.25 no.1
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• pp.1-7
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• 2010

The objective of this study was to examine the effect of vitamin B (pantothenic acid, folic acid, and myo-inositol) that was supplemented to embryo culture medium on in vitro development of parthenogenetically activated (PA) pig embryos. Cumulus-oocyte complexes derived from slaughtered ovaries were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones (hCG and eCG) for the first 22 h and then further cultured in hormone-free medium for an additional 22 h. After maturation culture, metaphase II oocytes that extruded 1st polar body were electrically activated and treated with $5.0\;{\mu}g/ml$ cytochalasin B for 4 h. Then, PA embryos were cultured for 7 days in a modified NCSU-23 that was supplemented with pantothenic acid, myo-inositol, or folic acid at different concentrations ($3{\sim}300\;{\mu}M$) according to the experimental design. Myo-inositol added to culture medium did not show any beneficial or inhibitory effects on embryo cleavage and blastocyst formation. However, $300\;{\mu}M$ pantothenic acid significantly inhibited blastocyst formation compared to control (no addition) (24% vs. 36%, p<0.05). Folic acid ($300\;{\mu}M$) significantly (p<0.05) increased blastocyst formation (56%) compared to control (41%). Our results demonstrated that in vitro development of PA embryos was significantly influenced by vitamin B and addition of $300\;{\mu}M$ folic acid to culture medium improved in vitro development of pig PA embryos.

• Korean Journal of Animal Reproduction
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• v.26 no.4
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• pp.385-394
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• 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
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• v.24 no.2
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• pp.97-104
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• 2009

The objective of this study was to examine the effect of macromolecule in a maturation medium on nuclear maturation, intracellular glutathione (GSH) level of oocytes, and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Immature pig oocytes were cultured in maturation medium that was supplemented with each polyvinyl alcohol (PVA), pig follicular fluid (pFF) or newborn calf serum (NBCS) during the first 22 h and the second 22 h. Oocyte maturation was not influenced by the source of macromolecules during in vitro maturation (IVM). Embryo cleavage and cell number in blastocyst after PA was altered by the source of macromolecule but no difference was observed in blastocyst formation among treatments. Oocytes matured in PVA-PVA medium showed lower rates of oocyte-cell fusion (70.4% vs. 77${\sim}$82%) and embryo cleavage (75% vs. 86${\sim}$90%) after SCNT than those matured in other media but blastocyst formation was not altered (13${\sim}$27%) by different macromolecules. pFF added to IVM medium significantly increased the intracellular GSH level of oocytes compared to PVA and NBCS, particularly when pFF was supplemented during the first 22 h of IVM. Our results demonstrate that source of macromolecule in IVM medium influences developmental competence of oocytes after PA and SCNT, and that pFF supplementation during the early period (first 22 h) of IVM increases intracellular GSH level of oocytes.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.2
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• pp.121-129
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• 2022

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) is an N6-methyladenosine (m⁶A) RNA modification regulator and a key determinant of prem-RNA processing, mRNA metabolism and transportation in cells. Currently, m⁶A reader proteins such as hnRNPA2/B1 and YTHDF2 has functional roles in mice embryo. However, the role of hnRNPA2/B1 in porcine embryogenic development are unclear. Here, we investigated the developmental competence and mRNA expression levels in porcine parthenogenetic embryos after hnRNPA2/B1 knock-down. HhnRNPA2/B1 was localized in the nucleus during subsequent embryonic development since zygote stage. After hnRNPA2/B1 knock-down using double stranded RNA injection, blastocyst formation rate decreased than that in the control group. Moreover, hnRNPA2/B1 knock-down embryos show developmental delay after compaction. In blastocyste stage, total cell number was decreased. Interestingly, gene expression patterns revealed that transcription of Pou5f1, Sox2, TRFP2C, Cdx2 and PARD6B decreased without changing the junction protein, ZO1, OCLN, and CDH1. Thus, hnRNPA2/B1 is necessary for porcine early embryo development by regulating gene expression through epigenetic RNA modification.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2005.10a
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• pp.138-138
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• 2005

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2005.10a
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• pp.39-40
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• 2005

• Journal of Embryo Transfer
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• v.22 no.4
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• pp.265-269
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• 2007

This study was conducted to investigate the efficacy of vitrification procedure for the cryopreservation of porcine oocytes and the utilization of vitrified oocytes as recipient cytoplasts for somatic cell nuclear transfer (NT), and observed that porcine oocytes are evaluated by pronuclear formation, and parthenogenetic development. Single fetal donor cells were deposited into the perivitelline space of vitrified enucleation oocytes, followed by electrical fusion and activation. NT embryos were cultured in NCSU-23 medium supplemented with 5% FBS, at $38.5^{\circ}C$ in 5% $CO_2$ and air. 1. When the developmental rates of the oocytes after being culture for $0{\sim}10$ hours vitrified with EDS and ETS were 42.0%, 38.0%, respectively. This results were lower than the control group(62.2%). 2. When the developmental rates of the oocytes after being culture for $0{\sim}10$ hours vitrified-thawed with sucrose and glucose, 5% PVP, NCSU-23 supplemented with 10% FBS were 33.3%, 25.9%, respectively. This results were lower than the control group(55.6%). 3. The fusion and development to the blastocyst stage between the NT embryos constructed with the vitrified and non-vitrified oocytes were significant differences. Developmental rate of oocytes and NT embryos constructed with the vitrified or non-vitrified oocytes were $13.0{\pm}2.4%\;and\;23.2{\pm}2.4%$, respectively.

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.9-12
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• 2016

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-specific growth factors that regulate many critical processes involved in early folliculogenesis and oocyte maturation. In this study, effects of GDF9 and BMP15 treatment during in vitro maturation of porcine oocytes upon development after parthenogenetic activation were investigated. Neither GDF, BMP15 alone nor in combination affects the number and viability of cumulus cells or the rates of oocyte maturation and blastocyst development. However, the treatment of GDF9 on porcine oocytes increased the number of trophectodermal (TE) cells of blastocysts derived from activated oocytes (P<0.05). The treatment of BMP15 increased the cell numbers of both inner cell mass (ICM) and TE cells (P<0.05). The treatment with the combination of GDF9 and BMP15 further increased the numbers of ICM and TE cells, compared with GDF9 or BMP15 treatment alone (P<0.05). In conclusion, the treatment of GDF9 or BMP15 (or both) enhanced the quality of blastocysts via the increased number of ICM and/or TE cells.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.8
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• pp.1095-1101
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• 2016

Ginsenoside Rg1 is a natural compound with various efficacies and functions. It has beneficial effects on aging, diabetes, and immunity, as well as antioxidant and proliferative functions. However, its effect on porcine embryo development remains unknown. We investigated the effect of ginsenoside Rg1 on the in vitro development of preimplantation porcine embryos after parthenogenetic activation in high-oxygen conditions. Ginsenoside treatment did not affect cleavage or blastocyst formation rates, but did increase the total cell number and reduced the rate of apoptosis. In addition, it had no effect on the expression of four apoptosis-related genes (Bcl-2 homologous antagonist/killer, B-cell lymphoma-extra large, Caspase 3, and tumor protein p53) or two metabolism-related genes (mechanistic target of rapamycin, carnitine palmitoyltransferase 1B), but increased the expression of Glucose transporter 1 (GLUT1), indicating that it may increase glucose uptake. In summary, treatment with the appropriate concentration of ginsenoside Rg1 ($20{\mu}g/mL$) can increase glucose uptake, thereby improving the quality of embryos grown in high-oxygen conditions.

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.171-180
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• 2011

In this study I report that in vitro development rates of bovine nuclear transfer embryos activated either with boar sperm cytosolic factor (SCF) or with ionomycin followed by cycloheximide (CHX) and subsequent in vivo developmental rates after embryo transfer are related to blastocyst quality as evaluated by apoptosis analysis. SCF was extracted from porcine semen then purified for post-activation injection after nuclear transfer. The optimal timing for SCF injection was determined to be at least 22 h post-IVM for parthenogenetic activation of bovine oocyies. A total of 364 oocytes were successfully enucleated and 268 (73.6%) fused and were injected with SCF. The survival rate of fused and injected embryos was 109/113 (96.5%) after 2 h. The cleavage rates of nuclear transfer embryos after 3 d of culture in the ionomycin/CHX treated group were significantly higher than those of the SCF-activated group (93.3% vs 81.7%, p<0.01, respectively). However, at 7 d and 9 d there was no significant difference between the total developmental rates to blastocyst for either treatment group. Total blastocyst cell numbers were also not significantly different between the two activation treatments (ionomycin/CHX: 149.5${\pm}$7.7 vs. SCF: 139.3${\pm}$4.4 cells). In contrast, the apoptotic levels in the SCF blastocysts were higher than those produced after the chemical treatment (28.2${\pm}$5.1% vs. 8.8${\pm}$0.6%, respectively). A total of 18 expanded or hatching blastocysts was transferred to nine synchronized recipients in each activation group; 5/9 (55.5%) and 2/9 (22.2%) were pregnant at 40 d in the ionomycin/CHX treatment and SCF activated group, respectively. However, only one went to term in the ionomycin/CHX treatment while none of the pregnancies from the SCF group were maintained by 90 d. In conclusion, these results suggest that SCF derived from different species is a limited activator to be used for activation after bovine nuclear transfer in lieu of a chemical activation protocol.