• Clinical and Experimental Reproductive Medicine
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• v.29 no.1
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• pp.21-28
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• 2002

Objective: To evaluate whether co-culture of oocytes on vero cell monolayers from Day 0 (Day 0 group) after egg retrieval results in an increase in developmental capacity such as fertilization rate, embryo quality, blastulation and clinical pregnancy rate compared with co-culture of oocytes from Day 1 (Day 1 group). Methods: Sperms were treated with Hams F-10 supplemented with 10% human follicular fluid (hFF). Vero cells for co-culture were prepared in TCM-199 with 10% FBS. Oocytes were co-cultured from Day 0 and fertilized oocytes were co-cultured from Day 1 on vero cell monolayers in DMEM with 10% and 20% hFF, respectively after egg retrieval. On day 1, 2 and 5, fertilization rate and grade of embryos and blastocysts were evaluated. Results (fertilization rate, cleavage rate, grade of embryos and blastocysts and pregnancy rate) were considered statistically significant when p value was less than 0.05 using t-test and $x^2$. Results: In sibling oocytes of same cycles, no differences were found in fertilization rate (94.6 vs. 91.4%), cleavage rates (94.6 vs. 91.4%), embryo grade (on day 2 and 3) and blastulation (65.6 vs. 57.0%) and their grade. In different oocytes of different cycles (patients), no differences were found in fertilization (79.8 vs. 78.3%), cleavage rates (77.7 vs. 76.4%) and blastulation (56.0 vs. 45.3%), but pregnancy rate was higher in the Day 0 group than in the Day 1 group (60.0 vs. 42.9%). Conclusions: This study revealed that the embryonic development capacities were not affected by the different co-culture time in the sibling oocytes of same cycles. Although no statistical significance, because of small size of study, there was a trend for higher pregnancy rates in Day 0 group compared to Day 1 group in different oocytes of different cycles.

• Korean Journal of Animal Reproduction
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• v.21 no.1
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• pp.61-69
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• 1997

The present study was carried out to investigate the effects of EGF and anti-EGF on early embryonic development and hatching in mice. Developmental and hatching rates of mouse em-bryos from 2-cell to morular stage which were cultured in Ham's FlO medium supplemented with EGF (1-1,000 ng/ml) or anti-EGF (whole serum diluted from 1:10 to 1:1,000) were compared to those of control When mouse early 2-cell embryos were cultured in the EGF supplemented medium, blastulation was accelerated compared with control. Hatching rate was also significantly (p

• Clinical and Experimental Reproductive Medicine
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• v.31 no.3
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• pp.169-176
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• 2004

Objective: This study was performed to evaluate and compare the embryonic developmental capacity and pregnancy rates in conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) with ejaculated sperm or testicular sperm cycles. Materials and Methods: Fertilization was examined in the following morning after IVF (group I), ICSI (group II) or TESE-ICSI cycles (group III). Fertilized oocytes were co-cultured with Vero cells until embryo transfer (ET). On day 2 and $5{\sim}7$, grades of embryos (<4- or $\geq$4-cell) and blastocysts (BG1, 2, 3 or early) were evaluated. Clinical pregnancy rate was determined by detecting G-sac with transvaginal ultrasonogram. We analyzed the results by $X^2$ and Student's t-test and considered statistically significant when P value was less than 0.05. Results: Fertilization rate was significantly higher (p<0.05) in group I ($79.0{\pm}21.2%$) than in group II and III ($56.8{\pm}21.6%$ and $36.7{\pm}25.3%$). Cleavage and blastulation rate of group I ($95.8{\pm}13.8%$ and $59.5{\pm}25.3%$) were significantly higher (p<0.05) than those of group III ($83.4{\pm}18.6%$ and $40.4{\pm}36.5%$). Clinical pregnancy rate was significantly higher (p<0.05) in group I and II (40.7% and 41.7%) than that in group III (12.5%). No differences were found in the rates of multiple pregnancy and abortion among three groups. Embryonic implantation rate was higher in group I ($15.1{\pm}20.2%$, p<0.05) and II ($14.7{\pm}20.6%$, NS) than that in group III ($5.1{\pm}15.6%$). However, embryonic implantation rate was increased in ET with blastocyst(s) among three groups. Conclusions: Fertilized oocytes obtained from TESE-ICSI were harder to be successfully cultured to blastocyst stage for 5$\sim$7 days than that from IVF cycles. However, all blastocyst(s) ET increased the embryonic implantation rate equally in IVF, ICSI and TESE-ICSI cycles.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.2
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• pp.125-134
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• 2010

Objective: The aim of this study was to measure the concentrations of cytokines contained in the hydrosalpingeal fluid and to evaluate the effect on the mouse embryo development with the different cytokine concentration. Methods: The hydrosalpingeal fluids (HSF) were collected during the surgery for hydrosalpinx which was confirmed by hysterosalphingogram. The cytokines in HSF including interleukin (IL)-$1{\alpha}$, IL-$1{\beta}$, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-$\alpha$, Interferon-$\gamma$ (IFN-$\gamma$), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), monocyte chemotactic protein-1 (MCP-1) were measured by ELISA method. HSF were added up to culture media with 5%, 10%, and 30% concentrations. The blastulation rates were compared. Results: IL-$\alpha$, IL-$1{\beta}$, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-$\alpha$, IFN-$\bamma$, VEGF, EGF, and MCP-1 were detected, but the concentrations were different from each sample. IL-6 and IL-10 were increased in HSF-1 group, and IFN-$\gamma$, MCP-1, VEGF were increased in HSF-2 compared with normal serum range. The Th1/Th2 ratio of HSF-2 (IFN-$\gamma$:IL10) was highly elevated to 61.64, compared with that of HSF-1 (3.69). The blastulation rate was significantly decreased in HSF-2 group (27.7%) compared those of the HSF-1 group (74%) and control group (76.7%). It showed the trend that the blastulation rate was decreased depending on the concentration HSF of culture media in HSF-2 group, but it was not statistically significant. Conclusion: The composition and concentration of cytokines in each HSF were different, and increased proinflammatory cytokines in HSF might be associated with poor embryonic development. Further study will be needed about the effect of each cytokines in HSF.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.4
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• pp.237-243
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• 2002

Objective : The aim of this study was to evaluate the influence of different media on blastulation, mean cell number, percentage of inner cell mass (ICM) of total cells and ICM : trophectoderm (TE) ratio in mice. Materials and methods: A total 552 two cell embryos were retrieved from ICR female mice (4 weeks old) at 48 hr after hCG injection (mated just after hCG injection) and cultured in MEM (n=276) or TCM (n=276) supplemented with 20% hFF. The grading of blastocysts from zona-intact (ZiB) to -escape (hatching and hatched, ZeB) was performed at 72 hours after culture. Total, TE and ICM cell numbers were analyzed by differential staining of blastocyst. Statistical analysis was performed using t-test with SigmaPlot-2001. P-values < 0.05 were accepted as statistically significant. Results: The blastulation rate in MEM ($64.9{\pm}4.95%$) was significantly higher (p=0.0031) than that in TCM ($57.2{\pm}5.22%$). No differences were found in the number of ZiB and ZeB between MEM ($31.9{\pm}2.62$, $33.0{\pm}4.58%$), and TCM ($27.2{\pm}4.28$, $30.1{\pm}4.58%$). A total 314 blastocysts (MEM=166; TCM=148) were stained differentially. Mean cell number of blastocysts was significantly higher (p=0.0002) in TCM ($73.1{\pm}3.3$) than in MEM ($61.7{\pm}2.5$). Differential staining was successfully performed in 155 blastocysts (MEM=77; TCM=78). The percentage of ICM was significantly higher in MEM than in TCM ($20.9{\pm}1.3$ vs. $17.1{\pm}1.2%$, p=0.0281). The ICM : TE ratio was higher in TCM than in MEM (1 : $4.85{\pm}0.68$ vs. 1 : $3.78{\pm}0.78$, NS). Conclusion: These results show that MEM increase the blastocyst formation and percentage of ICM of total cells comparing with TCM in mice.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.1
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• pp.25-32
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• 2010

The present study aimed to examine the effects of ${\gamma}$-linolenic acid (GLA) supplementation to in vitro culture (IVC) medium on in vitro developmental competence, freezability and morphology of in vitro matured and fertilized bovine embryos. In vitro produced (IVP) bovine zygotes were cultured in IVC medium supplemented with 0 (negative control), 15, 31, 62, 125, 250, 500 or 1,000 ppm GLA, 250 ppm linoleic acid albumin (LAA) and without any supplement as a control. Day 6 blastocysts derived from culture control were cultured in IVC medium containing either 62, 250 GLA or 250 LAA for 24 h, and at Day 7 were subjected to freezing or morphological examination by electron microscope. GLA 15 showed a tendency to have a higher cleavage rate at Day 2 (70.3%) than other groups. The hatching rate at Day 9 in LAA (38.2%) was significantly higher than the control and all treatment groups (p<0.05), while the blastocyst rate in LAA (32.4%) did not differ from those of 15 (30.5%), 31 (27.1%), and 62 GLA (33.1%) or the control (35.1%). GLA in concentrations of 125, 250, 500, and 1,000 ppm had significantly detrimental effect on the blastocyst rate compared to 15, 31 and 62 ppm GLA, LAA, and control groups (p<0.05). In contrast, the highest post-thaw survival rate (100%) was observed in the control group (p<0.01). Large lipid droplets were observed in the cytoplasm of trophoblastic cells, even in the control, but were abundant in GLA groups. Taking the results of the study into consideration, the addition of GLA to the culture medium for IVP bovine embryos at the dose of 15 ppm increased the developmental competence of zygotes and enhanced the cleavage rate up to Day 2. However, blastulation rate and post-thaw survival were not increased when GLA was added to the culture media.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.4
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• pp.193-200
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• 2017

Objective: This study was conducted to investigate the efficacy of laser-assisted hatching (LAH) and various vitrification times for embryonic development and blastocyst cell numbers. Methods: First, 2-cell and 8-cell embryos were collected by flushing out the oviducts. In the control groups, they were vitrified for 8 or 10 minutes without LAH. The LAH groups underwent quarter laser zona thinning-assisted hatching before vitrification (4, 6, and 8 minutes or 4, 7, and 10 minutes, respectively). After incubation, double-immunofluorescence staining was performed. Results: The hatched blastocyst rate 72 hours after the 2-cell embryos were thawed was significantly higher in the 2LAH-ES8 group (33.3%) than in the other groups (p< 0.05). In the control-8 group ($22.1{\pm}4.6$), the cell number of the inner cell mass was higher than in the LAH groups (p< 0.05). The number of trophectoderm cells was higher in the 2LAH-ES6 group ($92.8{\pm}8.9$) than in the others (p< 0.05). The hatched blastocyst rate 48 hours after the 8-cell embryos were thawed was higher in the 8LAH-ES4 group (45.5%) than in the other groups, but not significantly. The inner cell mass cell number was highest in the 8LAH-ES7 group ($19.5{\pm}5.1$, p< 0.05). The number of trophectoderm cells was higher in the 8LAH-ES10 group ($73.2{\pm}12.1$) than in the other groups, but without statistical significance. Conclusion: When LAH was performed, 2-cell embryos with large blastomeres had a lower hatched blastocyst rate when the exposure to vitrification solution was shorter. Conversely, 8-cell embryos with small blastomere had a higher hatched blastocyst rate when the exposure to vitrification solution was shorter.

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.27-32
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• 2016

This study was conducted to evaluate the effects of different volume ($100{\mu}l$ vs. 2 ml) of microdrop culture on B6D2F1 mice oogenesis. In the present study, B6D2F1/CrljOri $F_1$ mice were utilized in order to maximize oogenesis. Also we used TCM-199, Dulbecco's medified Eagle's medium (DMEM), embryo culture medium (Fertilization medium, Cleavage medium, Blastocyst medium), G series medium and One step medium. Blastulation rate was not different between groups ($58.4{\pm}2.9%$ vs. $61.2{\pm}4.8%$). Zona hatched rate ($38{\pm}15.4%$ vs. $27{\pm}3.4%$) and attached rate ($55{\pm}13.9%$ vs. $46{\pm}3.9%$) did not differ by the volume of culture media. Total cell numbers ($59.8{\pm}9.7$ vs. $70.3{\pm}8.7$), ICM cell numbers ($15.8{\pm}0.6$ vs. $16.8{\pm}1.5$), TE cell numbers ($44.0{\pm}9.7$ vs. $53.6{\pm}7.3$), % ICM ($26.4{\pm}2.9%$ vs. $23.8{\pm}3.3%$) and ICM:TE ratio ($1:2.8{\pm}0.4$ vs. $1:3.2{\pm}0.6$) were not different between groups (i.e., $100{\mu}l$ vs. 2 ml). These results show that the capacity of the culture medium did not effect the cell numbers of B6D2F1 mice blastocysts. In summary, these results can provide fundamental data to maximize culture condition for in vitro fertilization on B6D2F1 mice.

• Journal of Animal Reproduction and Biotechnology
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• v.39 no.1
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• pp.48-57
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• 2024

Background: Cadmium (Cd) is toxic heavy metal that accumulates in organisms after passing through their respiratory and digestive tracts. Although several studies have reported the toxic effects of Cd exposure on human health, its role in embryonic development during preimplantation stage remains unclear. We investigated the effects of Cd on porcine embryonic development and elucidated the mechanism. Methods: We cultured parthenogenetic embryos in media treated with 0, 20, 40, or 60 µM Cd for 6 days and evaluated the rates of cleavage and blastocyst formation. To investigate the mechanism of Cd toxicity, we examined intracellular reactive oxygen species (ROS) and glutathione (GSH) levels. Moreover, we examined mitochondrial content, membrane potential, and ROS. Results: Cleavage and blastocyst formation rates began to decrease significantly in the 40 µM Cd group compared with the control. During post-blastulation, development was significantly delayed in the Cd group. Cd exposure significantly decreased cell number and increased apoptosis rate compared with the control. Embryos exposed to Cd had significantly higher ROS and lower GSH levels, as well as lower expression of antioxidant enzymes, compared with the control. Moreover, embryos exposed to Cd exhibited a significant decrease in mitochondrial content, mitochondrial membrane potential, and expression of mitochondrial genes and an increase in mitochondrial ROS compared to the control. Conclusions: We demonstrated that Cd exposure impairs porcine embryonic development by inducing oxidative stress and mitochondrial dysfunction. Our findings provide insights into the toxicity of Cd exposure on mammalian embryonic development and highlight the importance of preventing Cd pollution.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.4
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• pp.305-310
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• 2019

Embryos produced with serum show the alterations in their ultrastructure, impaired compaction, abnormal blastulation, aberrant mRNA expression profiles and large calf syndrome with greater incidences of stillbirths and deaths after birth. The aim of the present study was to describe in vitro embryo production by analyzing embryo production, fetal production and pregnancy rate in free-serum medium. The OPU-IVP data used in this study from 2016. Approximately, sixteen cows (Hanwoo), which belonged to the Institute of Gyeongsang National University, were used. Two experimental group is used in this study. Serum groups were conducted in March to July and free-serum group was conducted in September to December. The recovered cumulus-oocyte complexes were morphologically classified to four grades based on the compaction of cumulus cells layers and homogeneity of the cytoplasm. The number of oocyte was significantly greater in serum groups than that in free-serum groups (29.61 ± 0.63 vs. 15.6 ± 0.62; p < 0.05). Between serum and free-serum groups indicate that average of 1st and 2nd grade oocytes were no difference (2.38 ± 1.67 vs. 2.38 ± 1.48; p > 0.05), but number of 3rd and 4th grade oocytes were greater in serum groups than that in free-serum groups (7.31 ± 7.64 vs. 5.60 ± 6.29; p < 0.05). Embryo cleaved competence was higher in rate in free-serum groups than that in serum groups (62.1% vs. 58.3; p < 0.05). However, blastocyst developmental rate was no difference between serum and free-serum groups (33.1% vs. 43.5%; p < 0.05). 986 recipients were used for embryo transfer. Pregnancy rate was indicated that between serum and free-serum group was no difference (54.6% vs. 56.3%; p < 0.05). In conclusion, we developed the free-serum system for production of in vitro bovine embryos in order to meet the developmental and qualitative requirements for large scale commercial use.