• Journal of Embryo Transfer
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• v.20 no.2
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• pp.113-121
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• 2005

This study was conducted to investigate the effects of different hexoses (glucose, mannose, galactose and fructose) on in vitro development of parthenogenetic embryos in the pigs. When the parthnogenetic embryos were cultured in medium with concentrations of 5mM glucose or 1mM galactose, the rates of embyos developed to morula and blastocyst stages were significantly higher than those in another culture conditions (P<0.05). However, high concentration of galactose inhibited development to morula and blastocyst stages. Addition of hexoses at early stage of porcine parthenogenetic embryos were effective for in vitro development. Especially, the embryos cultured in medium with glucose at early stage were effective for development to 2-cell $(72\%)$ and blastocyst $(19\%)$ stages compared with embryo cultured without glucose. From the present results, it is suggested that development of porcine parthenogenetic embryos can improve in medium with 5mM glucose. The concentration of 1mM galactose was also effective for development of porcine parthenogenetic embryos. It also show that parthenogenetic embryos cultured with glucose at early stage can improve in vitro development.

• Journal of Embryo Transfer
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• v.24 no.2
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• pp.105-108
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• 2009

This study was designed to investigate the effect of kinetin on in vitro development of parthenogenetic porcine oocytes exposed to demecolcine prior to activation. In vitro matured metaphase II stage oocytes were incubated in 0 or 2 ${\mu}$g/ml demecolcine supplemented defined culture medium for 3 h and the oocytes were activated electrically. The parthenogenetic porcine embryos were then cultured in 0 or 200 ${\mu}$M kinetin supplemented defined culture medium for 7 days. Regardless of demecolcine treatment, kinetin supplementation increased blastocyst rates significantly (7.0% versus 12.1% and 4.9% versus 8.5%; Control versus Kinetin and Demecolcine versus Kinetin + Demecolcine, respectively, p<0.05). Demecolcine treatment before activation tended to decrease blastocyst rates regardless of kinetin supplementation although it is not statistically significant. Total cell numbers in the blastocysts also tended to be elevated in embryos when supplemented with kinetin, however only the result between Kinetin and Demecolcine groups is statistically significant (37.6 ${\times}$ 7.2 versus 28.1 ${\times}$ 9.5, respectively, p<0.05). In conclusion, the present report shows that kinetin enhances developmental competence of parthenogenetic porcine embryo regardless of demecolcine pre-treatment before parthenogenetic activation when they were developed in defined culture condition.

• Reproductive and Developmental Biology
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• v.37 no.1
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• pp.51-55
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• 2013

The present study was conducted to examine the effect of antioxidant treatment during parthenogenetic activation procedure on the reactive oxygen species (ROS) levels and in vitro development of porcine parthenogenetic embryos. Porcine in vitro matured oocytes were activated by a combination of electric stimulus and 2 mM 6-dimethylaminopurine (6-DAMP) before in vitro culture. During the activation period, oocytes were treated with $50{\mu}M$ ${\beta}$-mercaptoethanol (${\beta}$-ME), $100{\mu}M$ L-ascorbic acid (Vit. C) or $100{\mu}M$ L-glutathione (GSH). To examine the ROS level, porcine parthenogenetic embryos were stained in $10{\mu}M$ dichlorohydrofluorescein diacetate ($H_2DCFDA$) dye 20 h after culture, examined under a fluorescence microscope, and the fluorescence intensity (pixels) were analyzed in each embryo. The parthenogenetic embryos were cultured for 6 days to evaluate the in vitro development. The apoptosis was measured by TUNEL assay. The $H_2O_2$ levels of parthenogenetic embryos were significantly lower in antioxidant treatment groups ($26.9{\pm}1.6{\sim}29.1{\pm}1.3$ pixels/embryo, p<0.05) compared to control ($33.2{\pm}1.7$ pixels/embryo). The development rate to the blastocyst stage was increased in antioxidant treatment groups (32.0~32.5%) compared to control (26.9%, p<0.05), although, there was no difference in apoptosis among groups. The result suggests that antioxidant treatment during parthenogenetic activation procedure can inhibit the ROS generation and enhance the in vitro development of porcine parthenogenetic embryos.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.1
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• pp.94-101
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• 2020

In the early development of parthenogenetic embryo, cytoplasm and nucleic acid fragmentation may be a cause of lower embryo development. The purpose of this study was to evaluate whether embryonic development and apoptosis factors can be reduced by controlling the in-vitro culture environment by the addition of hormones, pregnancy serum and uterine milk. Our study showed that the activity of Casp-3 increased within the cytoplasm when artificially used hormones to induce the incubation environment, and PCNA's manifestation was low. However, the addition of pregnant serum appeared to lower the Casp-3 activity compared to the other groups. In addition, MMP-9 activity was increased and early embryo development and cytoplasmic fidelity were also increased. Therefore, the results of the present study showed that the use of gestational serum in the development of parthenogenetic embryo inhibit apoptosis and increases cytoplasmic reorganization by natural environmental control in in vitro culture.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.10
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• pp.1510-1516
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• 2007

These experiments were carried out to optimize the parameters of electrical activation, methods of parthenogenetic activation and embryo culture in vitro and meanwhile to isolate embryonic stem cells-like (ESCs) derived from porcine parthenogenetic blastocysts (pPBs). These results showed that, as the electric field strength increased from 1.0 to 2.7 kV/cm, the cleavage rate of parthenogenetic embryos increased gradually but the rate of oocyte lysis was significantly increased when using 2.7 kV/cm field strength. The rate of cleavage in 2.2 and 2.7 kV/cm groups was significantly increased in comparison with that of the 1.0 kV/cm group. A voltage field strength of 2.2 kV/cm DC was used to investigate blastocyst development following activation with a single pulse of 30 or $60-{\mu}sec$ pulse duration. The optimum pulse duration was 30-${\mu}sec$, with a blastocyst rate of 20.7%. Multiple pulses were inferior to a single pulse for blastocyst yield (8.0% vs. 29.9) (p<0.05). For porcine oocyte parthenogenetic activation methods, the rates of cleavage (79.0% vs. 59.8%) and blastocysts (19.4% vs. 3.4%) were significantly increased in electrical activation in contrast to chemical activation with ionomycin/6-DMAP (p<0.05). Rates of cleavage and blastocyst formation in NCSU-23 and PZM-3 embryo media were higher than those of G1.3/G2.3 serial culture media, but there was no significant difference among the three groups. The total cell number of blastocysts in PZM-3 embryo culture media containing $5{\mu}g/ml$ insulin was significantly higher than that of the control (no insulin) ($44.3{\pm}9.1$ vs. $33.9{\pm}11.7$). For isolation of PESCs-like, the rates of porcine blastocysts attached to feeder layers and ICM colony formation in Method B (nude embryo culture) were better than those in Method A (intact embryo culture).

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.145-147
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• 2005

Autocrine or paracrine mediators released by the early embryo are implicated in the support of embryonic development. Their mechanisms and optimal embryo density in the medium, however, are uncertain. This study was conducted to establish the optimal embryo density and culture medium volume in mouse parthenogenetic embryo culture. In experiment 1, culture of parthenogenetirally activated oocytes at a concentration of $2{\~}4$ embryos/${\mu}L$ significantly improved development to the blastoryst stage ($72{\%}{\leq}$) compared with culture at the lower ($0.2{\~}1$e mbryos/${\mu}L,\;0\~37.5\%$) and the higher ($5{\~}6$ embryos/${\mu}L,\;30\~53\%$) concentration for 120 h when the oocytes were cultured in a 5 ${\mu}L$ drop under mineral oil In experiment 2, the embryos cultured at a concentration of $2{\~}4$ embryos/${\mu}L$ in a 10 ${\mu}L$ drop ($81.1{\%}$) showed significantly higher blastocyst rates than those in a 5 ${\mu}L$ drop ($68.5{\%}$). This study optimizes in vitro culture condition by modifying embryo density and the volume of culture medium It may give appropriate level of autocrine and/or paracrine factors to enhance viability and subsequent normal development of mouse parthenogenetic embryos in vitro.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2005.10a
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• pp.108-108
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• 2005

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2000.11a
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• pp.57-57
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• 2000

• Journal of Embryo Transfer
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• v.26 no.2
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• pp.111-115
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• 2011

In general, zona pellucida (ZP) of the blastocyst has to be removed first, then either isolated the inner cell mass (ICM) or ZP-removed whole blastocyst, which is then cultured on the feeder layer to induce ICM outgrowth for the generation of embryonic stem cells (ESC). However, it is unclear whether ICM isolation before seeding on feeder layer is beneficial or not because the interaction between ICM and trophoblasts may affect cellular growth and/or pluripotency during the culture on the feeder. In the present study, two ZP removal methods (mechanically by splitting with a 28-gauge needle versus chemically by the treatment of acid-Tyrode's solution) and two ICM isolation methods (ZP-free whole blastocyst seeding versus mechanical isolation of ICM) were evaluated for the efficient isolation and culture of putative parthenogenetic bovine ESC. The number of maintained outgrown colonies was counted in each experimental group. As the result, mechanical removal of ZP with a needle and followed by whole ZP-free blastocyst seeding on feeder cells tended to attach more on the feeder layer and resulted in more outgrown colonies with its simple and less time-costing benefits. Currently we are generating ESC lines in HanWoo cattle by using this method for initial outgrowth of the parthenogenetic bovine blastocysts.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.237-243
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• 2018

Hedgehog (Hh) pathway plays a key role in development from invertebrate to vertebrate. It is known to be involved in cell differentiation, polarity, proliferation, including the development of vertebrate limb and the establishment of flies' body plan. To investigate how the regulation of Hh pathway affects the development of parthenogenetic murine embryos, the parthenogenetically activated murine embryos were treated with either cyclopamine (Cyc), an antagonist of Hh pathway, or purmorphamine, an agonist of Hh pathway. While Cyc did not affect the blastocyst formation and its total cell number, the chemical reduced the hatching rate of embryos and the expression levels of Fn1 mRNA. The results of the present study show the possibility that Cyc may affect the development of embryos at blastocyst stage by blocking Hh pathway and this may cause detrimental effect to the embryos at peri-, and post-implantation stages.