• Asian-Australasian Journal of Animal Sciences
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• v.17 no.8
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• pp.1076-1079
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• 2004

Antibiotics are common additives in culture media during in vitro embryo development, but their effects on oocyte maturation in vitro have not been tested. The effects of penicillin, streptomycin and gentamicin on the maturational competence and subsequent development potential of goat follicular oocytes were examined after parthenogenetic activation in vitro. Maturation rates at 24 h after in vitro maturation, and parthenogenetic development at 48 h after activation, were evaluated by observing the protruding first polar body and the 4 cell stage cleavage, respectively. When streptomycin was present in the maturation medium, the percentages of matured oocytes 24 h after activation were significantly (p<0.01) lower than those from the other groups (42.5-45.7% vs. 69.1-73.8%). Penicillin and gentamicin treatment did not affect the maturation rates or the percentages reaching the 4 cell stage 48 h after activation. There was no significant difference in cleavage rates among the different antibiotic treatments 48 h after activation. Therefore, streptomycin suppresses the in vitro maturation of immature goat oocytes, but does not influence their subsequent development.

• Korean Journal of Agricultural Science
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• v.43 no.1
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• pp.52-60
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• 2016

In animal reproduction, the quality of oocytes and embryos has been evaluated by the expression of specific molecules. Follistatin (FST), which was isolated from follicular fluid, binds and bio-neutralizes the TGF-${\beta}$ superfamily members. Previous studies using the bovine model showed FST could be an important molecular determinant of embryo developmental competence. However, the effect of FST treatment on porcine embryo developmental competence has not been established. In this study, the effect of exogenous FST on porcine embryo developmental competence was investigated during in vitro culture. FST (10 ng/ml) treatment induced a significant decrease in the rate of cell arrest at the 4-cell stage. The expression levels of DNA-methyltransferase 1 (DNMT1), histone deacetylase 1 (HDAC1), and histone deacetylase 2 (HDAC2) were decreased in 4-cell stage embryos. FST treatment also resulted in significant improvements in developmental competence of embryos in terms of blastocyst formation rate and OCT-4 mRNA levels, the latter being related to pluripotency. In conclusion, during in vitro culture, FST treatment significantly ameliorated 4-cell block during embryonic development and improved embryo developmental competence. Therefore, FST treatment may potentially have a functional role in porcine embryogenesis that is broadly applicable to enhance in vitro embryo development.

• Development and Reproduction
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• v.20 no.2
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• pp.127-133
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• 2016

Purine metabolism is known factor for nuclear maturation of oocytes through both follicle cells and oocyte itself. However, it is largely unknown the roles of purine metabolism in the oocyte competence for fertilization and early development. In this study, the effects of adenosine in oocyte competence for development were examined using adenosine and its synthetic inhibitors. Adenosine treatment from GV intact stage for 18 hr (fGV) caused of decrease the fertilization rate but of increase the cleavage rate compared from the other stage treatment groups. Hadacidin did not effect on fertilization rate but increased cleavage rate without stage specificity. Adenosine did not block the effects of hadacidin with the exception of fGV group. Inhibition of purine synthetic pathways the fertilization rate was decreased in the fGV and fGVB groups but increased in the fMII group. Exogenous adenosine caused of decrease fertilization rate in the fGVB group but increase in the fMII group. Cleavage rate was dramatically increased in the adenosine treatment with synthetic inhibitors. It means that the metabolism of purine has stage specific effects on fertilization and cleavage. Exogenous adenosine had only can improve oocyte developmental competence when it treated at GV intact stage. On the other hand, endogenous synthesis in all maturation stage cause of increase the cleavage rate without effects on fertilization. These data suggest that adenosine at GV stage as a paracrine fashion and inhibitions of endogenous adenosine in all stage improve oocyte developmental competence.

• Reproductive and Developmental Biology
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• v.38 no.4
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• pp.143-146
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• 2014

Antioxidants, as reactive oxygen species scavengers, are one of the beneficial additives in serum-free defined culture medium. In this study, three separate experiments were performed to determine the effects of 3-hyroxyflavone added to the culture medium on the developmental competence of follicular bovine oocytes during in vitro maturation (IVM) and/or in vitro culture (IVC). The rate of blastocyst developed from oocytes cultured in IVM medium with 3-hyroxyflavone was significantly higher than that from control oocytes (39.0% vs. 26.3%, p<0.001), respectively. However, oocytes cultured in the medium with addition of 3-hyroxyflavone only at IVC period did not show significance in the blastocyst development when compared with control. When 3-hyroxyflavone was added to both IVM and IVC media, the rate of blastocyst formation was even significantly lower (21.1%) than control (26.5%; p<0.05). The present findings suggested that antioxidative activity of 3-hydroxyflavone added to only IVM medium beneficially affected the developmental competence of follicular bovine.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.4
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• pp.347-356
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• 2020

Gangliosides are glycolipids in which oligosaccharide is combined with sialic acids. Our previous studies have suggested an interplay between ganglioside GD1a/GT1b and meiotic maturation capacity in porcine oocyte maturation. Furthermore, ganglioside GD1a and GT1b are known for its antioxidant activity, but it is still unclear whether possible antioxidant role of GD1a and GT1b is involved in porcine embryos development competence during in vitro culture (IVC). Here, the effects of ganglioside GD1a and GT1b on the embryonic developmental competence during in vitro culture of porcine were investigated. The effects of ganglioside GD1a and GT1b on the expression of ST3GAL2 were confirmed during embryos development (2-cell, 4-cell, 8-cell and blastocyst) using immunofluorescent staining (IF). As a result, the fluorescent expression of ST3GAl2 was higher in embryos at 4-8 cells stage than blastocysts. Blastocyst development rate significantly increased in only 0.1 μM GD1a and GT1b treated groups compared with control group. To investigate the cellular apoptosis, we analyzed TUNEL assay. In case of only 0.1 μM GD1a and GT1b treated groups, the total number of cells in blastocyst compared with control group, but there was no significant difference in the rate of apoptotic cells. We identified the intracellular ROS levels using DCF-DA staining. According to the result, ROS production significantly decreased in blastocysts derived from the 0.1 μM GD1a and GT1b treated groups. These results suggest that ganglioside GD1a and GT1b improve the developmental competence of porcine embryos via reduction of intracellular ROS during preimplantation stage.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.147-157
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• 1998

This study was conducted to investigate the effects of in vitro fertilization, culture and embryo development according to in vitro maturation rate, protectant composition and equilibrium time after frozen /thawing of bovine immature oocytes. This results obtained in studies on the effect of different cryoprotectants on the viability, maturation and development of in vitro bovine oocytes were as follow: 1.The post-thawing of immature oocytes matured to metaphase II during culture time for 0 to 26 h, and those group (62~3%) were low than control group (76.7%). The optimal maturation time of frozen-thawed immature oocytes was at 24 h. 2.The viability of cryopreserved immature oocytes was not affected by sort of cryoprotectants. The developmental competence of frozen4hawed oocytes was not affected by cryoprotectants. These results indicate that an optimal maturation time of frozen /thawed immature oocytes was at 24h. Furthermore the viability of cryopreserved immature oocytes was not affected by sort of cryoprotectants and developmental competence of frozen /thawed oocytes.

• Reproductive and Developmental Biology
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• v.38 no.4
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• pp.147-158
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• 2014

Differentiated nuclei can experimentally be returned to an undifferentiated embryonic status after nuclear transfer (NT) to unfertilized metaphase II (MII) oocytes. Nuclear reprogramming is triggered immediately after somatic cell nucleus transfer (SCNT) into recipient cytoplasm and this period is regarded as a key stage for optimizing reprogramming. In a recent study (Dai et al., 2010), use of m-carboxycinnamic acid bishydroxamide (CBHA) as a histone deacetylase inhibitor during the in vitro early culture of murine cloned embryos modifies the acetylation status of somatic nuclei and increases the developmental competence of SCNT embryos. Thus, we examined the effects of CBHA treatment on the in vitro preimplantation development of porcine SCNT embryos and on the acetylated status of histone H3K9 on cloned embryos at the zygote stage. We performed the three groups SCNT: SCNT (NT), CBHA treatment at the porcine fetus fibroblast cells (PFFs) used as donor cells prior to SCNT (CBHA-C) and CBHA treatment at the porcine SCNT embryos during the in vitro early culture after oocyte activation (CBHA-Z). The PFFs were treated with a $15{\mu}M$ of CBHA (8 h) for the early culture and the porcine cloned embryos were treated with a $100{\mu}M$ concentration of CBHA during the in vitro early culture (10 h). Cleavage rates and development to the blastocyst stage were assessed. No significant difference was observed the cleavage rate among the groups (82.6%, 76.4% and 82.2%, respectively). However, the development competence to the blastocyst stage was significantly increased in CBHA-Z embryos (22.7%) as compared to SCNT and CBHA-C embryos (8.6% and 4.1%)(p<0.05). Total cell numbers and viable cell numbers at the blastocyst stage of porcine SCNT embryos were increased in CBHA-Z embryos as compared to those in CBHA-C embryos (p<0.05). Signal level of histone acetylation (H3K9ac) at the zygote stage of SCNT was increased in CBHA-Z embryos as compared to SCNT and CBHA-C embryos. The results of the present study suggested that treatment with CBHA during the in vitro early culture (10 h) had significantly increased the developmental competence and histone acetylation level at the zygote stage.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.6
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• pp.827-834
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• 2018

Objective: The aim of this study was to compare the effects of $36.5^{\circ}C$ and $38.5^{\circ}C$ incubation temperatures on the maturation of bovine oocytes and developmental competence of embryos. Methods: In experiment 1, oocytes were maturated in bicarbonate-buffered TCM-199 for 22 hours in a humidified atmosphere of 5% $CO_2$ in the air at either $36.5^{\circ}C$ or $38.5^{\circ}C$ and nuclear maturation status were determined. In experiment 2, in vitro fertilized oocytes were allocated randomly into synthetic oviductal fluid medium with or without a mixture of 1 mM L-glutathione reduced and 1,500 IU superoxide dismutase and cultured in a humidified atmosphere of 5% $CO_2$, 5% $O_2$, and 90% $N_2$ in the air at $38.5^{\circ}C$ for 8 days. Results: There were no significant differences between incubation temperatures in terms of oocyte maturation parameters such as cumulus expansion, first polar body extrusion and nuclear maturation. Incubation temperatures during in vitro maturation had no effects on developmental competence of embryos, but supplementation of antioxidants increased (p<0.05) developmental competence of the embryos. Blastocysts from oocytes matured at $38.5^{\circ}C$ had comparatively higher inner cell mass, but low overall and trophectoderm cell numbers (p<0.05). Conclusion: The results of present study showed that maturation of bovine oocytes at $36.5^{\circ}C$ may provide a suitable thermal environment for nuclear maturation and subsequent embryo development.

• Journal of Embryo Transfer
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• v.29 no.2
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• pp.101-109
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• 2014

Matrix Metalloproteinases (MMP)-2 and -9 are participated in embryo development, implantation, remodeling of epithelial cell and ovulation. The objective of this study is to evaluate an impact of MMP2 and MMP9 on embryonic developmental competence as well as gene expression profiles of in vitro-produced bovine embryos. After in vitro fertilization, embryos of all groups were transferred into IVC-2 medium treated with MMP2 and MMP9 to check the optimum concentration on the basis of embryo development competence and cell numbers. The optimum concentrations for MMP2 and 9 were 1,200 ng/ml and 300 ng/ml. The blastocyst development competence was not different among 1,200 ng/ml of MMP2 vs. 300 ng/ml of MMP9 vs. combined MMP2 + 9 vs. control groups ($41.46{\pm}10.66$ vs. $37.73{\pm}8.92$ vs. $45.11{\pm}11.41%$ vs. $41.59{\pm}11.88$, respectively). Furthermore, the developmental competences to hatching and hatched blastocysts were not also different among the same groups ($79.84{\pm}12.63$ vs. $83.3{\pm}17.46$ vs. $78.55{\pm}14.48%$ vs. $72.02{\pm}14.09$). In addition, total cell number was significantly (p<0.05) greater in blastocyst treated with MMP9 300 ng/ml among all treatment groups. On the other hand, there was no significant difference of ICM vs. TE ratio in all groups. The expression of five out of six genes (i.e., MMP2, MMP9, IFNt, SSLP1 and HNRNPA2B1) was different among the groups. The expression of IFNt and HNRNPA2B1 genes was significantly greater in MMP9 (p<0.05), but there was no difference of MMP9 expression between MMP2 and MMP9 group (p>0.05). The normalized expression of MMP2 and SSLP1 was greater in MMP2 than other groups (p<0.05). In conclusion, MMPs treatment during IVC-2 medium was remarkably effected on blastocyst developmental competence and gene expression profiles that are related to embryo quality and implantation.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.4
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• pp.189-194
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• 2018

Objective: The aim of this study was to compare the rate of maturation, fertilization, and embryo development of in vitro-matured human oocytes derived from pregnant and non-pregnant women. Methods: Immature oocytes were obtained by needle aspiration from 49 pregnant women (group 1) who underwent a cesarean section at term and 77 non-pregnant women (group 2) who underwent a gynecological operation during the same period (8 months). Healthy immature oocytes (530 in group 1 and 539 in group 2) were cultured and assessed for maturation 36 hours later. Mature oocytes were inseminated by intracytoplasmic sperm injection and cultured up to 144 hours. Results: The percentage of degenerated oocytes was significantly higher (12.1% vs. 6.3%; p<0.001) in group 1 than in group 2. There was no significant difference in the maturation rate (66.8% vs. 68.1%; p=0.698), fertilization rate (66.7% vs. 67.6%; p=0.857), or the rate of formation of good-quality blastocysts (46.2% vs. 47.2%; p=0.898) in oocytes obtained from pregnant and non-pregnant women. Conclusion: The developmental competence of immature oocytes did not differ between pregnant and non-pregnant women.