• Journal of Embryo Transfer
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• v.31 no.3
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• pp.153-159
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• 2016

(-)-Epicatechin gallate (ECG) is a polyphenol compound of green tea exhibiting biological activities, such as antioxidant and anticancer effects. To examine the effect of ECG on porcine oocytes during in vitro maturation (IVM), oocytes were treated with 0-, 5-, 15-, and $25{\mu}M$ ECG. After maturation, we investigated nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels and subsequent embryonic development after parthenogenetic activation (PA) and in vitro fertilization (IVF). After 42 hours of IVM, the $5{\mu}M$ group exhibited significantly increased (p< 0.05) nuclear maturation (89.8%) compared with the control group (86.1%). However, the $25{\mu}M$ group observed significantly decreased (p< 0.05) nuclear maturation (83.5%). In intracellular maturation assessment the 5-, 15-, and $25{\mu}M$ groups had significantly increased (p< 0.05) GSH levels and decreased ROS levels compared with the controls. The 5- and $15{\mu}M$ group showed significantly increased (p< 0.05) embryo formation rates and total cell number of blastocysts after PA (18% and 68.9, 15% and 85.1 vs. 12% and 59.5, respectively) compared with controls. Although the $25{\mu}M$ group observed significantly lower blastocyst formation rates after PA (27.6% vs. 23.2%) than control group, the $5{\mu}M$ group showed significantly increased blastocyst formation rates after PA (37.2% vs. 23.2%) compared to the control group. Furthermore, the $5{\mu}M$ group measured significantly increased blastocyst formation rates (20.7% vs. 8.6%) and total cell number after IVF ($88.3{\pm}1.5$ vs. $58.0{\pm}3.6$) compared to the control group. The treatment of $5{\mu}M$ ECG during IVM affectively improved the porcine embryonic developmental competence by regulating intracellular oxidative stress during IVM.

• Clinical and Experimental Reproductive Medicine
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• v.46 no.4
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• pp.189-196
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• 2019

Objective: We aimed to evaluate the effects of different oxygen conditions (20% [high O₂], 5% [low O₂] and 5% decreased to 2% [dynamic O₂]) on mouse pre- and peri-implantation development using a novel double-channel gas supply (DCGS) incubator (CNC Biotech Inc.) to alter the oxygen concentration during in vitro culture. Methods: The high-O₂ and low-O₂ groups were cultured from the one-cell to the blastocyst stage under 20% and 5% oxygen concentrations, respectively. In the dynamic-O₂ group, mouse embryos were cultured from the one-cell to the morula stage under 5% O₂ for 3 days, followed by culture under 2% O₂ to the blastocyst stage. To evaluate peri-implantation development, the blastocysts from the three groups were individually transferred to a fibronectin-coated dish and cultured to the outgrowth stage in droplets. Results: The blastocyst formation rate was significantly higher in the low-O₂ and dynamic-O₂ groups than in the high-O₂ group. The total cell number was significantly higher in the dynamic-O₂ group than in the low-O₂ and high-O₂ groups. Additionally, the apoptotic index was significantly lower in the low-O₂ and dynamic-O₂ groups than in the high-O₂ group. The trophoblast outgrowth rate and spread area were significantly higher in the low-O₂ and dynamic-O₂ groups than in the high-O₂ group. Conclusion: Our results showed that a dynamic oxygen concentration (decreasing from 5% to 2%) had beneficial effects on mouse pre- and peri-implantation development. Optimized, dynamic changing of oxygen concentrations using the novel DCGS incubator could improve the developmental competence of in vitro cultured embryos in a human in vitro fertilization and embryo transfer program.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.3
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• pp.319-327
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• 2009

Follicular fluid meiosis-activating sterol (FF-MAS) has been suggested as a positive factor which could improve the oocyte quality and subsequent embryo development after in vitro fertilization. However, FF-MAS is a highly lipophilic substance and is hard to detect in studying the relationship between MAS and quality of oocyte maturation. The present study focused on the expression of lanosterol 14${\alpha}$-demethylase (LDM), a key enzyme that converts lanosterol to FF-MAS, on mouse oocyte maturation and its potency on development. LDM expression was strong in gonadotropin-primed germinal vesicle stage oocytes, weak after germinal vesicle breakdown (GVBD), and then strong in MII stage oocytes. The LDM-specific inhibitor azalanstat significantly inhibited oocyte fertilization (from 79.4% to 68.3%, p<0.05). Also, azalanstat (5 to 50 ${\mu}M$) decreased the percentage of blastocyst development dosedependently (from 78.7% to 23.4%, p<0.05). The specific inhibition of sterol ${\Delta}14$-reductase and ${\Delta}7$-reductase by AY9944 accumulates FF-MAS and could increase blastocyst development rates. Additionally, in the AY9944 group, the rate of inner cell mass (ICM)/ total cells was similar to that of in vivo development, but the rate was significantly decreased in azalanstat treatment. In conclusion, LDM, the key enzyme of FF-MAS production, may play an important role in fertilization and early development of the mouse embryo, especially in vitro.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.4
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• pp.357-365
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• 2020

The aim of present study was to investigate regulatory mechanism of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation of porcine oocytes. Basically, immature cumulus-oocyte complexes (COCs) were incubated for 22 h in IVM-I to which hormone was added, and then further incubated for 22 h in IVM-II without hormone. As a result, relative cumulus expansion was increased at 22 h after IVM and it was enhanced by treatment of ALA compared with control group (p < 0.05). During IVM process within 22 h, cAMP level in oocytes was decreased at 6 h (p < 0.05) and it was recovered at 12 h in ALA-treated group, while oocytes in control group recovered cAMP level at 22 h. In cumulus cells, it was reduced in all time point (p < 0.05) and ALA did not affect. Treatment of ALA enhanced metaphase-I (MI) and MII population of oocytes compared with oocytes in control group at 22 and 44 h, respectively (p < 0.05). Intracellular GSH levels in ALA group was increased at 22 and 44 h after IVM (p < 0.05), whereas it was increased in control group at 44 h after IVM (p < 0.05). In particular, the GSH in ALA-treated oocytes during 22 h of IVM was higher than control group at 22 h (p < 0.05). Lipid amount in oocytes from ALA group was higher than control group (p < 0.05). Treatment of ALA did not influence to absorption of glucose from medium. Cleavage and blastocyst formation of ALA-treated oocytes were enhanced compared with control group (p < 0.05). These findings suggest that supplementation of ALA could improve oocyte maturation and development competence through increasing GSH synthesis, lipid storage, and regulation of cAMP accumulation during early 22 h of IVM, and these might be mediated by cumulus expansion.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.5
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• pp.648-656
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• 2008

The objective of this study was to establish conditions for transfection of a foreign gene into somatic cells using cationic lipid reagents and to evaluate the effects of transfection on in vitro development of somatic cell nuclear transfer (SCNT) embryos. Green fluorescent protein (GFP) gene was used as a foreign gene and a non-transfected somatic cell was utilized as a control karyoplast. Monolayers of porcine cells were established and subsequently transfected with a GFP-expressing gene (pEGFP-N1) using three types of transfection reagents (LipofectAMINE PLUS, FuGENE 6 or ExGen500). Donor cells used for SCNT included transfected fetal or adult fibroblasts and oviduct epithelial cells, either serum-fed or serum-starved. Oocytes matured in vitro for 42 h were reconstructed with either transfected or non-transfected porcine somatic cells by electric fusion and activation using a single DC pulse of 1.8 kV/cm for $30{\mu}s$ in $Ca^{2+}$ and $Mg^{2+}-containing$ 0.26 M mannitol solution. Reconstructed oocytes were subsequently cultured in NCSU-23 medium for 168 h and the developmental competence and cell number in blastocyst were compared. There were no significant differences (P>0.05) in fusion, cleavage rates or development to the blastocyst stage between non-transfected, transfected, serum-fed and serum-starved cells. However, the rates of GFP-expressing blastocysts were higher in the FuGENE 6 group (71.4%) among transfection reagents and in the fetal fibroblasts group (70.4%) for donor cells. These results indicate that fetal fibroblasts transfected with FuGENE 6 can be used as donor cells for porcine SCNT and that GFP gene can be safely used as a marker of foreign genes in porcine transgenesis.

• Animal Bioscience
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• v.37 no.6
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• pp.1007-1020
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• 2024

Objective: We increased the nuclear maturation rate of antral follicle derived oocytes by using a pre-in vitro maturation (IVM) culture system and improved the developmental potential of these porcine pathenotes by supplementing with melatonin. Furthermore, we investigated the expression patterns of genes involved in cumulus expansion (HAS2, PTGS2, TNFAIP6, and PTX3) derived from small and medium antral follicles before and after oocyte maturation. Methods: Only the cumulus oocyte-complexes (COCs) derived from small antral follicles were induced with [Pre-SF(+)hCG] or without [Pre-SF(-)hCG] the addition of human chorionic gonadotropin (hCG) during the last 7 h of the pre-IVM period before undergoing the regular culture system. The mature oocytes were investigated on embryonic development after parthenogenetic activation (PA). Melatonin (10^-7 M) was supplemented during in vitro culture (IVC) to improve the developmental potential of these porcine pathenotes. Results: A pre-IVM culture system with hCG added during the last 7 h of the pre-IVM period [Pre-SF(+)hCG] effectively supported small antral follicle-derived oocytes and increased their nuclear maturation rate. The oocytes derived from medium antral follicles exhibited the highest nuclear maturation rate in a regular culture system. Compared with oocytes cultured in a regular culture system, those cultured in the pre-IVM culture system exhibited considerable overexpression of HAS2, PTGS2, and TNFAIP6. Porcine embryos treated with melatonin during IVC exhibited markedly improved quality and developmental competence after PA. Notably, melatonin supplementation during the IVM period can reduce and increase the levels of intracellular reactive oxygen species (ROS) and glutathione (GSH), respectively. Conclusion: Our findings indicate that the Pre-SF(+)hCG culture system increases the nuclear maturation rate of small antral follicle-derived oocytes and the expression of genes involved in cumulus expansion. Melatonin supplementation during IVC may improve the quality and increase the blastocyst formation rate of porcine embryos. In addition, it can reduce and increase the levels of ROS and GSH, respectively, in mature oocytes, thus affecting subsequent embryos.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.3
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• pp.199-207
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• 2001

Objective: The present study was undertaken to examine the effects of magnesium ion in the culture medium on the development of mouse fertilized oocytes either before or after pronuclear formation, and to investigate whether the effect of magnesium ion is related with the redistributional change of mitochondria. Methods : Fertilized oocytes obtained from the oviducts of mice at 15 hr after hCG injection before pronuclear formation (pre-PN) or 21 hr after hCG injection after pronuclear formation (post-PN) were used. The embryos were cultured for 3 days with basic T6 medium-magnesium free and various concentrations of magnesium ion, 0.0, 0.5, 1.0, 2.0, 4.0 or 8.0 mM, respectively. After culture, the developmental stages of embryos and the number of nuclei were evaluated. To observe the effects of magnesium ion on the mitochondrial distribution, fertilized oocytes were collected at 21 hr after hCG injection and cultured for 6 hr with various concentration of magnesium ion. As a control, fertilized oocytes with pronuclei at 27 hr after hCG injection were used. Results: The concentration of magnesium ion to accelerate the in vitro development of mouse fertilized oocytes appeared to be at 2.0 mM for the pre-PN and the post-PN stage embryos. In the mitochondrial redistribution patterns, the embryos cultured in 2.0 mM concentration of magnesium ion showed the highest percentage (22.6%) of distinct perinuclear clustering pattern comparing to other experimental group. Conclusion: The effect of magnesium ion may be related to the cytoplasmic redistribution of mitochondria. This relationship seems to connect the developmental competence of preimplantation mouse embryos in vitro. These results can suggest that higher concentration of magnesium ion (2.0 mM) than those of conventional culture medium ($0.2{\sim}1.2\;mM$) is more suitable for in vitro culture of preimplantation mouse embryos.

• Reproductive and Developmental Biology
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• v.36 no.4
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• pp.255-260
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• 2012

The purpose of this study was to assess follicular viability and competence through in vitro culture of preantral follicles isolated from vitrified mouse whole ovaries. Mouse preantral follicles were enzymatically isolated from vitrified- warmed and fresh ovaries and cultured for 10 days followed by in vitro oocyte maturation. In vitro matured oocytes were fertilized and cultured to the blastocyst stage. Five minutes pre-exposure to vitrification solution of whole ovaries had significantly higher (p<0.05) oocyte survival and maturation rates than between 10 min exposure groups. Oocyte diameter was significantly smaller (p<0.05) in the 5 and 10 min exposure groups ($69.4{\pm}2.8$ and $67.8{\pm}3.1$) when compared to that of control group ($71.7{\pm}2.1$). There was no statistical significant difference in blastocyst development rates between vitrification group (8.6%) and the fresh control group (12.0%). The mean number of cells per blastocyst was significantly lower (p<0.05) in the vitrification group ($41.9{\pm}20.2$) than in the fresh control group ($55.1{\pm}22.5$). The results show that mouse oocytes within preantral follicles isolated from the vitrified whole ovaries can achieve full maturation, normal fertilization and embryo development.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.123-130
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• 2017

This study was conducted to investigate the effects of alpha-lipoic acid (aLA) as an antioxidant that decrease the reactive oxygen species (ROS) in bovine embryonic development. Slaughterhouse derived bovine immature oocytes were collected and 4 different concentrations (0, 5, 10 and 20 mM) of aLA was supplemented in bovine in vitro maturation (IVM) medium. After 20 hrs of IVM, maturation rates, levels of ROS and glutathione (GSH), and further embryonic development after parthenogenetic activation (PA) and in vitro fertilization (IVF) was investigated according to aLA concentrations. Maturation rate was significantly higher in 10 mM group than other groups (80.5% vs. 62.9, 73.9, 64.2%; P<0.05). In the levels of ROS and GSH in matured oocytes as an indicator of oocyte quality, significantly better results were shown in 5 and 10 mM groups compared with other 2 groups. After IVM, significantly higher rates of blastocyst formation were shown in 10 mM groups in both of PA (27.9% vs. 18.8, 22.3, 14.2%; P<0.05) and IVF (32.6% vs. 23.9, 27.3, 16.2%; P<0.05) embryos. In addition, significantly more cell total cell number and higher inner cell mass ratio in 10 mM PA and IVP blastocysts showed developmental competence in 10 uM groups. Therefore, based on the entire data from this study, using $10{\mu}M$ of aLA confirmed to be the optimal concentration for bovine oocyte maturation and embryonic development.

• Journal of Embryo Transfer
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• v.29 no.3
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• pp.265-271
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• 2014

Implementation of smart embryo technologies in cattle e.g. ovum pick-up followed by in vitro embryo production (OPU-IVP). Seasonal variation is important factor for follicular growth, oocytes quality, quantity and developmental competence. Therefore the aim of present study was carried out to investigated whether the seasons (hot and cool) effect on follicular development, oocyte recovery and subsequent embryo development. Follicular oocytes were aspirated from Korean native cows (Hanwoo) by the ovum pick-up (OPU) method, which was performed 24 times during two different seasons, the hot (July to September) and cool (October to December), from OPU donors. The recovered oocytes were classified according to morphological categories and used for in vitro embryo production (IVEP). The mean number of total follicles was significantly higher (p<0.05) during the hot season ($18.32{\pm}2.26$) compared to cool season ($15.41{\pm}3.34$). Furthermore, seasons did not significantly effect on the number of oocytes recovered (hot season: 41.16% vs. cool season: 46.14%). However, the average number of Grade A oocytes was significantly greater during hot ($1.75{\pm}1.86$) season compared to the cool season ($1.00{\pm}1.46$), but there was no significant difference of other grades oocytes. The cleavage rate (hot: 66.67% vs. cool: 63.3%) and embryo development (hot: 58.95% vs. cool: 56.97%) did not differ significantly between the seasons. In conclusion, the results of present study suggest that the season (hot and cool) does not have effects on the oocyte recovery and embryo developmental competence of in vitro cultured embryos.