• Journal of Embryo Transfer
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• v.22 no.4
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• pp.235-243
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• 2007

Our goal was to examine the effects of early denudation on the enucleation efficiency and developmental competence of embryos following somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA). Oocytes were denuded following 30 h of in vitro maturation (IVM) and then cultured with (D+) or without (D-) their detached cumulus cells for additional $10{\sim}14$ h. Control oocytes were denuded after $40{\sim}44$ h of IVM. The size of the perivitelline space was larger at 40 h of IVM ($11.7{\sim}11.8{\mu}m$) than at 30 h ($8.9{\mu}m;$ p<0.01). The distances between the metaphase II (M II) plates and the polar bodies (PBs) were shorter in D+ ($19.4{\mu}m$) and D- oocytes ($18.9{\mu}m$) than in control oocytes ($25.5{\mu}m;$ p<0.01). Enucleation rates following blind aspiration at 40 h of IVM were higher (p<0.01) in D+ (92%) and D- oocytes (93%) compared to controls (82%). Early denudation did not affect oocyte maturation or the in vitro development of SCNT and PA embryos. When SCNT embryos from D+ oocytes were transferred to four gilts, pregnancy was established in two pigs, and one of them farrowed three live piglets. In conclusion, early denudation of oocytes at 30 h of IVM could improve the enucleation efficiency by maintaining the M II plate and the PB within close proximity and support the in vivo development of SCNT embryos to term.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.111-122
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• 2017

The objective of this experiment was to explore the effects of Roscovitine (Rosco) prior to in vitro maturation (IVM) of immature pig oocyte. Brilliant cresyl blue test has been used to select the good quality of oocyte. Specifically, the effects of Rosco exposure on nuclear and cytoplasmic maturation, diameter, intracellular glutathione (GSH) and reactive oxygen species (ROS), and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), and gene expression levels in SCNT embryos have been measured. Cumulus oocyte complexes (COCs) have been exposed in $75{\mu}M$ of Rosco for 22 and 44 h. The COCs that were matured in the IVM for 44 h without Rosco used as control group. Diameter of matured porcine oocytes 44 h culture with Rosco was significantly lower than 22 h culture with Rosco and control groups. GSH was higher in control group than 22 h and 44 h with Rosco but reduction of ROS in 22 h than 44 h with Rosco. In PA, exposure with Rosco 44 h oocytes group has been significantly lower than 22 h and control group in rates of maturation, cleavage and blastocyst formation. Similarly, in SCNT embryos rates of maturation, cleavage and formation of blastocyst have been also significantly lower in 44 h Rosco treated group than other two groups. SCNT embryos treated with Rosco 22 h showed greater expression levels of POU5F1, DPPA2 and NDP52Il mRNA compared with other two groups. Our results demonstrate that Rosco treatment with 22 h prior to IVM improves the development competence of porcine oocyte.

• Journal of Veterinary Science
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• v.23 no.2
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• pp.31.1-31.13
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• 2022

Background: Compared to medium containing 108 mM sodium chloride (NaCl), in vitro maturation (IVM) using a simple medium with reduced (61.6 mM) NaCl increases the cytoplasmic maturation and embryonic development of pig oocytes. Objectives: This study determines the effect of a complex medium containing reduced NaCl on the IVM and embryonic development of pig oocytes. Methods: Pig oocytes were matured in Minimum Essential Medium Eagle-alpha modification (αMEM) supplemented with 61.6 (61αMEM) or 108 (108αMEM) mM NaCl, and containing polyvinyl alcohol (PVA) (αMEMP) or pig follicular fluid (PFF) (αMEMF). Medium-199 (M199) served as the control for conventional IVM. Cumulus cell expansion, nuclear maturation, intra-oocyte glutathione (GSH) contents, size of perivitelline space (PVS), and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) were evaluated after IVM. Results: Regardless of PVA or PFF supplementation, oocytes matured in 61αMEM showed increased intra-oocyte GSH contents and width of PVS (p < 0.05), as well as increased blastocyst formation (p < 0.05) after PA and SCNT, as compared to oocytes matured in 108αMEMP and M199. Under conditions of PFF-enriched αMEM, SCNT oocytes matured in 61αMEMF showed higher blastocyst formation (p < 0.05), compared to maturation in 108αMEMF and M199, whereas PA cultured oocytes showed no significant difference. Conclusions: IVM in αMEM supplemented with reduced NaCl (61.6 mM) enhances the embryonic developmental competence subsequent to PA and SCNT, which attributes toward improved oocyte maturation.

• Reproductive and Developmental Biology
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• v.36 no.1
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• pp.49-53
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• 2012

The present study was conducted to examine the generation of reactive oxygen species (ROS) during micromanipulation procedures in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine enucleated oocytes were electrofused with donor cells, activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. Oocytes and embryos were stained in dichlorodihydrofluorescein diacetate or 3'-(p-hydroxyphenyl) fluorescein dye and the $H_2O_2$ or $^.OH$ radical levels were measured. $In$ $vitro$ fertilization (IVF) was performed for controls. The samples were examined with a fluorescent microscope, and fluorescence intensity was analyzed in each oocyte and embryo. The $H_2O_2$ and $^.OH$ radical levels of reconstituted oocytes were increased during manipulation (37.2~49.7 and 51.0~55.2 pixels, respectively) as compared to those of mature oocytes ($p$<0.05). During early $In$ $vitro$ culture, the ROS levels of SCNT embryos were significantly higher than those of IVF embryos ($p$<0.05). These results suggest that the cellular stress during micromanipulation procedures can generate the ROS in bovine SCNT embryos.

• Journal of Embryo Transfer
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• v.25 no.4
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• pp.215-219
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• 2010

The objective of this study was to examine the reproductive characteristics of cloned miniature piglets produced from surrogate domestic pigs. Somatic cell nuclear transfer (SCNT) miniature pig embryos were transferred into domestic pigs. As controls, domestic pigs of the same breed with surrogates for SCNT embryos and miniature pigs of the same breed with the somatic cell donor were bred by artificial insemination and natural mating, respectively. Surrogate domestic pigs that farrowed cloned miniature piglets had a significantly longer gestation length (118.1 days) than conventionally bred domestic (115.4 days) and miniature (115.5 days) pigs. Furthermore, the birth weight of cloned miniature piglets produced from domestic pigs (743 g) was significantly greater than that of miniature piglets produced by natural breeding (623 g). Also, cloned miniature piglets had a significantly lower weaning rate (49.7%) than conventionally produced domestic (91.5%) and miniature (100%) piglets. No differences were observed between female and male cloned piglets in gestation length, litter size, birth weight, or weaning rate. Our results demonstrate that gestation length is extended in domestic pigs that are transferred with SCNT miniature pig embryos and that cloned miniature piglets have increased birth weight and high pre-weaning mortality.

• Transactions on Electrical and Electronic Materials
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• v.13 no.6
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• pp.267-272
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• 2012

Considering the properties of the carbon nano tubes (CNT), their inclusion into the polymer matrix vastly increases the properties of the resultant composite. However, this is not the case due to the poor interfacial adhesion of the CNT and the polymer matrix. The present approach focuses on increasing the interaction between the polymer matrix and the CNT through the chemical modification of the CNT resulting in allyl ester functionalized carbon nanotubes (ACNT) and silane functionalized carbon nano tubes (SCNT) which are capable of reacting with the polymer matrix during the curing reaction. The addition of ACNT/SCNT into unsaturated polyester resin (UPR) resulted in the improvement of the electrical properties of resulted nanocomposites in comparison to the CNT. The surface resistivity, volume resistivity, dielectric strength, dry arc resistivity, and the comparative tracking index of the nanocomposites were significantly improved in comparison to CNT. The chemical modification of CNT was confirmed via spectroscopy.

• Journal of Embryo Transfer
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• v.30 no.1
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• pp.33-43
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• 2015

Successful somatic cell nuclear transfer (SCNT) has been reported across a range of species using a range of recipient cells including enucleated metaphase II (MII) arrested oocytes, enucleated activated MII oocytes, and mitotic zygotes. However, the frequency of development to term varies significantly, not only between different cytoplast recipients but also within what is thought to be a homogenous population of cytoplasts. One of the major differences between cytoplasts is the activities of the cell cycle regulated protein kinases, maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK). Dependent upon their activity, exposure of the donor nucleus to these kinases can have both positive and negative effects on subsequent development. Co-ordination of cell cycle stage of the donor nucleus with the activities of MPF and MAPK in the cytoplast is essential to avoid DNA damage and maintain correct ploidy. However, recent information suggests that these kinases may also effect reprogramming of the somatic nucleus and preimplantation embryo development by other mechanisms. This article will summarise the differences between cytoplast recipients, their effects on development and discuss the potential role/s of MPF and or MAPK in nuclear reprogramming.

• 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.33 no.3
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• pp.119-127
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• 2018

The purpose of this study is to examined the electrofusion and activation conditions for the production of porcine somatic cell nuclear transfer (SCNT) embryos. In this study, immature oocytes were cultured in TCM-199 with and without hormones for 22 hours. Skin fibroblasts cells of porcine were transferred into the perivitelline space of enucleated in vitro matured oocytes. Cell fusion was performed with two different pulses that each one pulse (DC) of 1.1 kV/cm or 1.5 kV/cm for $30{\mu}sec$. After fusion subsequent activation were divided into three groups; non-treatment (control) and treatment with 2 mM 6-DMAP or $7.5{\mu}g/ml$ cytochalasin B for 4 hours. Transferred embryos were cultured in PZM-3 (Porcine Zygote Medium-3) in $5%\;CO_2$ and 95% air at $39^{\circ}C$ for 7 day. Apoptosis-related genes (Caspase-3, BCL-2, mTOR, and MMP-2) were analyzed by immunofluorescence staining. There was no significant difference between two different electrofusion stimuli in the cleavage rate; $64.9{\pm}4.8%$ in 1.1 kV/cm and $62.7{\pm}4.0%$ in 1.5 kV/cm. However, blastocyst formation rate (%) was significantly different among three different activation groups (no treatment, 2 mM 6-DMAP or $7.5{\mu}g/ml$ cytochalasin B) combined with electrofusion of 1.1 kV/cm. The blastocyst formation rate was $12.6{\pm}2.5$, $20.0{\pm}5.0$, and $34.9{\pm}4.3%$ in control, 2 mM 6-DMAP, and $7.5{\mu}g/ml$ cytochalasin B, respectively. Immunofluorescence data showed that expression levels of caspase-3 in SCNT embryos undeveloped to blastocyst stage were higher than those in the blastocyst stage embryos. Expression levels of Bcl-2 in blastocyst stage embryos were higher than those in the arrested SCNT embryos. These results showed that the combination of an electric pulse (1.1 kV/cm for $30{\mu}sec$) and $7.5{\mu}g/ml$ cytochalasin B treatment was effective for production of the porcine SCNT embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.1
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• pp.44-51
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• 2012

Calcium ions play an important role in the establishment and maintenance of pregnancy, but molecular and cellular regulatory mechanisms of calcium ion action in the uterine endometrium are not fully understood in pigs. Previously, we have shown that calcium regulatory molecules, transient receptor potential vanilloid type 5 (TRPV6) and calbindin-D9k (S100G), are expressed in the uterine endometrium during the estrous cycle and pregnancy in a pregnancy status- and stage-specific manner, and that estrogen of conceptus origin increases endometrial TRPV6 expression. However, regulation of S100G expression in the uterine endometrium and conceptus expression of S100G has been not determined during early pregnancy. Thus, we investigated regulation of S100G expression by estrogen and interleukin-$1{\beta}$ (IL1B) in the uterine endometrium and conceptus expression of S100G during early pregnancy in pigs. We obtained uterine endometrial tissues from day (D) 12 of the estrous cycle and treated with combinations of steroid hormones, estradiol-$17{\beta}$ ($E_2$) and progesterone ($P_4$), and increasing doses of IL1B. Real-time RT-PCR analysis showed that $E_2$ and IL1B increased S100G mRNA levels in the uterine endometrium, and conceptuses expressed S100G mRNA during early pregnancy, as determined by RT-PCR analysis. To determine if endometrial expression of S100G mRNA during the implantation period was affected by the somatic cell nuclear transfer (SCNT) procedure, we compared S100G mRNA levels in the uterine endometrium from gilts with SCNT-derived conceptuses with those from gilts with conceptuses derived from natural mating on D12 of pregnancy. Real-time RT-PCR analysis showed that levels of S100G mRNA in the uterine endometrium from gilts carrying SCNT-derived conceptuses was significantly lower than those from gilts carrying conceptuses derived from natural mating. These results showed that S100G expression in the uterine endometrium was regulated by estrogen and IL1B of conceptus origin, and affected by the SCNT procedure during early pregnancy. These suggest that conceptus signals regulate S100G, an intracellular calcium transport protein, for the establishment of pregnancy in pigs.