• Journal of Embryo Transfer
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• v.29 no.2
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• pp.119-125
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• 2014

Although the majority of surviving pigs cloned by somatic cell nuclear transfer (SCNT) appear to be physiologically normal, there is a general lack of detailed hemato-physiologic studies for the period of early adulthood to substantiate this claim. In the present study, we investigated variation in blood chemistry and endocrinological parameters between mesenchymal stem cells (MSCs) derived from cloned and normal age-matched female and male miniature pigs. Cloned females and males showed normal ranges for complete blood count assessments. Biochemical assessments showed that ${\gamma}$-GGT, ALT and cholesterol levels of male and female clones were significantly (P<0.05 or P<0.01, respectively) higher than that of age-matched control miniature pigs. Variations in insulin and IGF-1 were higher in female clones than in male clones and controls. Thus, although female and male cloned miniature pigs may be physiologically similar to normal animals, or at least within normal ranges, a greater degree of physiological and endocrinological variation was found in cloned pigs. The above variation must be taken into account before considering cloned female or male miniature pigs for various biomedical applications.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.293-301
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• 1996

In the present study, mouse follicular oocytes and 2-cell embryos(late -zygote stage embryos included) were cultured on the electric pad for electric stimulation in the culture incubator. In addition, follicular oocytes and embryos were tested for maturation and development under higher temperature condition($39^{\circ}C$).Mouse follicular oocyte maturation were not affected by anion electric stimulation and there is no significant difference in GBVD and MI between the control and experiment group after 4hr culture. In the embryo culture, it was found that more morula and blastocyst were found in the electric stimulation group rather than the control(96hr). This may seem to be caused with cytoplasmic $Ca^{2+}$ transient rise by electric stimulation(anion pad). On the other hand higher temperature incubation ($39^{\circ}C$) on the anion pad caused all the embryos degenerated within $12h{\sim}24hr$ culture. This was quite different from large animal embryos(bovine, pig, sheep), in which beneficial effect of high temperature incubation for oocyte maturation and embryo development were found.

• 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.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.271-280
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• 2018

Here, we evaluated the mode of programmed cell death during porcine oocyte maturation by comparing the two major pathways associated with programmed cell death, apoptosis (type I), and autophagy (type II). We investigated the expression and localization of major genes involved in autophagy and apoptosis at mRNA and protein levels. Furthermore, the effect of hormonal stimulation on autophagy and apoptosis was analyzed. We found that the activity of autophagy-associated genes was increased in the cumulus-oocyte complexes (COCs) following follicle-stimulating hormone (FSH) treatment, while the addition of luteinizing hormone (LH) reversed this effect. The expression of proteins associated with autophagy was the highest in FSH-treated COCs. On the other hand, caspase-3 protein level was maximum in COCs cultured with LH. The treatment with rapamycin resulted in the effect similar to that observed with FSH treatment and increased autophagy activity. Thus, hormonal stimulation of pig oocytes resulted in distinct patterns of maturation. The high-quality oocytes majorly relied on the type II pathway (autophagy), while the type I pathway (apoptosis) was more prominent among poor-quality oocytes. Further investigation of this distinction may allow the development of techniques to produce high-quality oocytes in porcine in vitro fertilization.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.337-342
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• 2018

Cryopreservation is mainly used for preservation of boar sperm. However, this method stresses the sperm by reactive oxygen species (ROS), and the conception rate and the litter size are not more efficient than the liquid preservation of spermatozoa. Therefore, we use chitosan which is a natural product derived antioxidant compound. We used GnHA (chitosan+hyaluronic acid) and GnHG (chitosan hydrogel) as chitosan complexes to cryopreserve boar sperm for improve sperm metabolism and function. Sperm parameter (sperm motility, progressive motility, path velocity, straight-line velocity, curvilinear velocity) is measured by computer-assisted sperm analysis (CASA) using frozen sperm with GnHA or GnHG (0, 0.25, 0.5, 1 mg/mL), respectively. Also, lipid peroxidation analysis using malondialdehyde (MDA) is performed to confirm the antioxidative effect of chitosan in frozen spermatozoa. CASA analysis showed GnHA and GnHG are effective against cryopreserved boar sperm. And antioxidant effect is measured by lipid peroxidation analysis. GnHA and GnHG, which is chitosan complex are effective for boar sperm cryopreservation by antioxidant effect.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.4
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• pp.323-328
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• 2020

Direct injection of genome editing tools such as CRISPR/Cas9 system into developing embryos has been widely used to generate genetically engineered pigs. The approach allows us to produce pigs carrying targeted modifications at high efficiency without having to apply somatic cell nuclear transfer. However, the targeted modifications during embryogenesis often result in mosaicism, which causes issues in phenotyping founder animals and establishing a group of pigs carrying intended modifications. This study was aimed to establish a genomic PCR and sequencing system of a single blastomere in the four-cell embryos to detect potential mosaicism. We performed genomic PCR in four individual blastomeres from four-cell embryos. We successfully amplified target genomic region from single blastomeres of 4-cell stage embryo by PCR. Sanger sequencing of the PCR amplicons obtained from the blastomeres suggested that PCR-based genotyping of single blastomere was a feasible method to determine mutation type generated by genome editing technology such as CRISPR/Cas9 in early stage embryos. In conclusion, we successfully genotyped single blastomeres in a single 4-cell stage embryo to detect potential mosaicism in porcine embryos. Our approach offers a simple platform that can be used to screen the prevalence of mosaicism from designed CRISPR/Cas9 systems.

• Journal of Embryo Transfer
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• v.26 no.1
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• pp.79-84
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• 2011

This study was performed to identify the differentially methylated region (DMR) and to examine the mRNA expression of the imprinted H19 gene in day 35 of SCNT pig fetuses. The fetus and placenta at day 35 of gestation fetuses after natural mating (Control) or of cloned pig by somatic cell nuclear transfer (SCNT) were isolated from a uterus. To investigate the mRNA expression and methylation patterns of H19 gene, tissues from fetal liver and placenta including endometrial and extraembryonic tissues were collected. The mRNA expression was evaluated by real-time PCR and methylation pattern was analyzed by bisulfite sequencing method. Bisulfite analyses demonstrated that the differentially methylated region (DMR) was located between -1694 bp to -1338 bp upstream from translation start site of the H19 gene. H19 DMR (-1694 bp to -1338 bp) exhibits a normal mono allelic methylation pattern, and heavily methylated in sperm, but not in oocyte. In contrast to these finding, the analysis of the endometrium and/or extraembryonic tissues from SCNT embryos revealed a complex methylation pattern. The DNA methylation status of DMR Region In porcine H19 gene upstream was hypo methylated in SCNT tissues but hypermethylated in control tissues. Furthermore, the mRNA expression of H19 gene in liver, endometrium, and extraembryonic tissues was significantly higher in SCNT than those of control (p<0.05). These results suggest that the aberrant mRNA expression and the abnormal methylation pattern of imprinted H19 gene might be closely related to the inadequate fetal development of a cloned fetus, contributing to the low efficiency of genomic reprogramming.

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.1-7
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• 2016

Xenotransplantation involves multiple steps of immune rejection. The present study was designed to produce nuclear transfer embryos, prior to the production of transgenic pigs, using fibroblasts carrying transgenes human complement regulatory protein hCD59 and interleukin-18 binding protein (hIL-18BP) to reduce hyperacute rejection (HAR) and cellular rejection in pig-to-human xenotransplantation. In addition to the hCD59-mediated reduction of HAR, hIL-18BP may prevent cellular rejection by inhibiting the activation of natural killer cells, activated T-cell proliferation, and induction of $IFN-{\gamma}$. Transgene construct including hCD59 and ILI-18BP was introduced into miniature pig fetal fibroblasts. After antibiotic selection of double transgenic fibroblasts, integration of the transgene was screened by PCR, and the transgene expression was confirmed by RT-PCR. Treatment of human serum did not affect the survival of double-transgenic fibroblasts, whereas the treatment significantly reduced the survival of non-transgenic fibroblasts (p<0.01), suggesting alleviation of HAR. Among 337 reconstituted oocytes produced by nuclear transfer using the double transgenic fibroblasts, 28 (15.3%) developed to the blastocyst stage. Analysis of individual embryos indicated that 53.6% (15/28) of embryos contained the transgene. The result of the present study demonstrates the resistance of hCD59 and IL-18BP double-transgenic fibroblasts against HAR, and the usefulness of the transgenic approach may be predicted by RT-PCR and cytolytic assessment prior to actual production of transgenic pigs. Further study on the transfer of these embryos to surrogates may produce transgenic clone miniature pigs expressing hCD59 and hIL-18BP for xenotransplantation.

• Journal of Embryo Transfer
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• v.29 no.4
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• pp.361-368
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• 2014

This study was designed to evaluate the effect of bovine serum albumin (BSA) in a maturation medium on oocyte maturation and embryonic development in pigs. Immature pig oocytes were matured for 44 h in a medium supplemented with 0.4% (w/v) BSA, 0.1% (w/v) polyvinyl alcohol (PVA), or 10% (v/v) pig follicular fluid (PFF). After IVM, oocytes reached metaphase II stage were activated for parthenogenesis (PA) or used as cytoplasts for somatic cell nuclear transfer (SCNT). Nuclear maturation (89.5%, 90.7% and 91.3% for BSA, PVA and PFF, respectively) and intraoocyte glutathione contents (1.20, 1.16 and 1.00 pixels/oocyte for BSA, PVA and PFF, respectively) were not altered by the macromolecules added to maturation medium. IVM of oocytes in a medium containing BSA (21.4%) and PVA (20.7%) showed significantly lower blastocyst formation after PA than culture in medium with PFF (39.2%). After SCNT, oocytes matured in medium with BSA showed decreased embryonic development to the blastocyst stage (9.2%) compared to those matured in medium with PFF (28.9%), while 23.6% of SCNT oocytes matured in medium with PVA developed to the blastocyst stage. When the effect of BSA in a maturation medium during the first 22 h and the second 22 h of IVM in combination with PFF or PVA was examined, PVA-BSA showed a higher nuclear maturation (94.1%) than BSA-PFF (84.5%). However, there was no significant difference in the blastocyst formation among tested combinations (47.3, 52.2, 50.0, 44.4 and 49.0% for PFF-PFF, PFF-BSA, PVA-BSA, BSA-PVA and BSA-PFF, respectively). Our results demonstrate that BSA and PVA added to maturation medium can support oocyte maturation comparable to PFF-supplemented medium. However, maturation of oocytes in a BSA-containing medium decreases embryonic development after PA and SCNT when compared with the medium supplemented with PFF.

• Journal of Embryo Transfer
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• v.29 no.4
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• pp.385-391
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• 2014

Objective of this study was to investigate the effect of nicotinic acid (NA) on the characteristics in fresh semen of miniature pig. We evaluated viability, acrosome reaction and mitochondrial integrity of sperm on 0, 3, 7 and 10 days during storage period with nicotinic acid. As results, the survival rate of sperm in 15 mM NA (day 3, $87.8{\pm}1.2%$; day 5, $84.0{\pm}2.7%$; day 7, $82.2{\pm}0.9%$) and 30 mM NA (day 3, $87.7{\pm}0.3%$; day 5, $84.4{\pm}2.5%$; day 7, $82.3{\pm}0.7%$) groups were higher than control and 5 mM NA groups in 3, 7 and 10 days of semen storage. The NA-treated sperm on 10 day was used day for observing acrosome integrity. The survival sperm with acrosome reaction was higher in 30 mM NA group (day 3, $2.7{\pm}0.2%$; day 5, $3.3{\pm}0.6%$; day 7, $11.4{\pm}0.3%$) than in the control, significantly (P<0.05). Moreover, the live sperm with mitochondrial integrity was higher in whole treatment groups of NA than control group, significantly (P<0.05). Specially, most mitochondrial integrity on 10 day of semen storage was significantly higher in 30 mM NA group ($90.2{\pm}1.6%$) than other treatment groups (control, $81.8{\pm}3.1%$; 5 mM NA, $83.4{\pm}3.0%$; 15 mM NA, $89.1{\pm}0.7%$, P<0.05). In conclusion, supplement of NA in liquid semen of miniature pig can improve and maintain semen quality, such as viability, acrosome reaction, and mitochondria integrity.