• Journal of Embryo Transfer
• /
• v.11 no.3
• /
• pp.249-258
• /
• 1996

To improve the efficiency of in vitro production of embryos with follicular oocytes in Korean Native cows, the recovery rates, in vitro maturation, fertilization and development, and the time required for collecting and processing oocytes by aspiration with or without slicing were evaluated comparatively. The ovaries were obtained from a local abattoir and placed in physiological saline at 25~28$^{\circ}C$ and brought to the laboratory within 3 hrs. The oocytes were collected by aspiration of follicles(2~6mm) with or without slicing ovaries after aspiration, and classified into Grade I, Grade II, Denuded, Expanded oocytes by the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules. Also the time required for each step of collecting and processing oocytes were measured. The cumulus cells were removed in some Grade I oocytes to measure their size and nuclear configuration before and after in vitro maturation. The Grade I oocytes were matured in vitro(IVM) for 24 hrs. in TGM-199 supplemented with 35$\mu$g /ml FSH, 10$\mu$g /ml LH, 1 $\mu$g /ml at 39$^{\circ}C$ under 5% C02 in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24hrs. and then the zygotes were cocultured in vitro (IVC) with bovine oviductal epithelial cells for 10 days. The results obtained were as follows: The number of oocytes recovered per ovary was averaged 6.6 by aspiration and 11.2 by slicing post aspiration, which summed to 17.8. The number of Grade I oocytes recovered per ovary was averaged 3.1 by aspiration and 3.6 by slicing, which summed to 6.7. The percentage of Grade I to total oocytes recovered was significantly(P<0.05) higher as 48.0 % in aspiration than 31.6% in slicing post aspiration. The time requlred for recovering a Grade I oocyte by aspiration and slicing was 1.1 and 2.5 min, respectively. The mean diameter of Grade I oocytes by aspiration and slicing was similar as 148.7 and 151.5$\mu$m, respectively. The percentage of Metaphase II stage oocytes after IVM for 24 hours was significantly (P

• Journal of Embryo Transfer
• /
• v.10 no.1
• /
• pp.73-82
• /
• 1995

large scale production of cloned embryos requires the technology of multiple generation nuclear transplantation(NT) using NT embryos as the subsequent donor nuclei. The purposes of this study were producing the second generation cloned rabbit embryos, and also to determine the electrofusion rate and in vitro developmental potential comparatively in the cloned embryos of the first and second NT generation. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FCS) at 47 hours after hCG injection In the first generation NT, the nuclear donor embryos were synchronized in the phase of Gi /S transition of 32-cell stage. The first generation NT embryos which were developed to 8-cell were synchronized in Gi /S transition phase of the following 16-cell stage and used as donor nuclei for second generation Synchronization of the cell cycle of blastomeres was induced, first, using an inhibitor of microtuble polymerization, colcemid for 10 hours to arrest blastomeres in M phase, and secondly, using a DNA synthesis inhibitor, aphidicolin for 1.5 to 2 hours to arrest them in Gi /S transition boundary. The recipient cytoplasms were obtained by removing the nucleus and the first polar body from the oocytes collected at 14 hours after hCG injection. The separated donor blastomeres were injected into the enucleated recipient oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 $\mu$sec at 1.25 kV /cm in 0.28 M rnannitol solution The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. Following in vitro culture of the first and second generation cloned embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The results obtained were summarized as follows: 1. The electrofusion rate was found to be similar as 79.4 and 91.5% in the first and second generation NT rabbit embryos, respectively. 2. The in vitro developmental potential to blastocyst stage of the second generation NT embryos (23.3%) was found significantly(p<0.05) lower, compared with that of the first generation NT embryos (56.8%). 3. The mean blastomeres counts of embryos developed to blastosyst stage following in vitro culture for 120 hours and also their daily cell cycles during the culture period were decreased significantly (p<0.05) to 104.3 cells and 1.33 cylces in the second NT generation, compoared with 210.4 cells and 1.54 cycles in the first NT generation, respectively.

• Journal of Embryo Transfer
• /
• v.25 no.3
• /
• pp.165-169
• /
• 2010

This study was carried out to assess the effect of vitamin E against the reactive oxygen species (ROS) on chemical activation of in vitro matured oocytes. Bovine oocytes were aspirated from slaughtered ovaries and transferred to maturation medium with or without vitamin E ($100\;{\mu}M$). After 22 hours of culture, oocytes with polar bodies were selected and submitted to activation treatments with or without vitamin E. After activation, oocytes were cultured in mSOF medium and rate of development was monitored. For ROS ($H_2O_2$) detection, in vitro matured and activated oocytes were selected and stained with DCFDA and observed under fluorescence microscope. The ROS contents were not significant differences in IVM rate, activation process and embryonic development to blastocysts with or without vitamin E. The cell number of blastocyst showed significant difference (p<0.05) in embryos matured and activated with vitamin E. The results of the present study demonstrated that the exposure of vitamin E in IVM and activation process improved the quality of embryos evaluated by the cell number of blastocysts.

• Proceedings of the KSAR Conference
• /
• 2001.10a
• /
• pp.55-55
• /
• 2001

Monosaccaride hexose, may have different role in embryo development of different species. Glucose, fructose and galactose are glycolysible substrates but their effect on embryo development is not identical. Glucose has negative effect to early embryonic stage in several species whereas it is inevitable after compaction. For fructose, it can support blastocyst formation in hamster, mouse and bovine embryo. Effect of galactose is known as detrimental even at a low concentration while glucose has adverse effect only at high concentration in hamster. (omitted)

• Korean Journal of Animal Reproduction
• /
• v.18 no.1
• /
• pp.39-46
• /
• 1994

This experiment was carried out to develop an in vitro culture system for rabbit embryos. The zygotes or 2-cell embryos were collected from the oviducts of the superovulated and mated does with D-PBS/10% FCS at 24 hours after hCG injection. The in vitro developmental rate of blastocyst formation and the number of nuclei in the embryos were examined under the following treatments; 1) TCM-199 with 10% FCS, 2) EBSS with 10% FCS, 3) rabbit vitreous humor(VH), 4) TCM-199 with 10% FCS＋BOEC, 5) TCM-199 with 10% FCS＋ROEC, 6) EBSS with 10% FCS＋BOEC and 7) EBSS with 10% FCS＋ROEC. For a comparative study of in vivo and in vitro development, the fresh blastocysts, which were developed in vivo for 96 hours after hCG injection, were collected from the uterus and their numbers of nuclei were counted. 1. The zygotes or 2-cell embryos developed to the blastocyst stage in TCM-199, EBSS and VH at the rates of 93, 92 and 89%, respectively. 2. The higher developmental rates 95~98% of blastocyst formation was achieved when the embryos were co-cultured with a monolayer of bovine or rabbit oviductal epithelial cells in TCM-199 or EBSS. No significant difference in developmental rates was shown between bovine and rabbit oviductal epithelial cells. 3. In a comparative study of in vivo and in vitro development, the total numbers of nuclei were significantly less in the in vitro cultured embryos(104~224) than the in vivo developed embryos(1, 0090 at 96 hours after hCG injectin. 4. The mean cell cycle numbers in the embryos cultured for 72 hours in TCM-199 with 10% FCS, EBSS with 10% FCS, TCM-199 with 10% FCS＋BOEC, TCM-199 with 10% FCS＋ROEC, EBSS with 10% FCS＋BOEC and in vivo was 7.38, 6.63, 7.76, 7.69, 7.01 and 9.92, respectively. From these results, it can be suggested the optimal culture system for in vitro culture of rabbit embryos is a co-culture system with bovine or rabbit oviductal epithelial cells in TCM-199 with 10% FCS. Considering the significant reduction in total numbers of nuclei in the in vitro cultured embryos, the advanced research on development of in vitro culture system for rabbit embryos is expected.

• Korean Journal of Plant Tissue Culture
• /
• v.27 no.3
• /
• pp.233-238
• /
• 2000

To obtain a basic information of the development of Genus Viola, morphological and histological observation of in vitro calli and cells in Viola culture cells were investigated. There were two callus types obtained by long term subculture of wild viola (Viola partrinii DC. ) petiole callus. One was friable callus - soft and pale green in color and small cells in size, and the other was compact callus - compact and deep bluish green in color, large cells in size. In scanning electron microscopic observation, friable callus was composed of voculated cell around small. cell clump, while compact callus was composed of cells filled with protoplasm Somatic embryogenesis was observed from suspension culture of the compact callus.

• Journal of Embryo Transfer
• /
• v.32 no.3
• /
• pp.201-207
• /
• 2017

To increase the productivity of in vitro development, the antioxidants have been used for culture system of bovine oocytes and embryos. However, comparative studies on these molecules are rare and direct beneficial effects on blastocyst production cannot be discriminated for best results. The study was conducted to determine the influence of N-acetyl-L-cysteine (NAC), N-acetyl-L-cysteine amide (NACA), glutathione (GSH) and cysteamime (CYS) on maturation competence of COCs from GV to MII stage and productivity of blastocyst formation during in vitro fertilization and culture. There was no difference among maturation rates of oocytes to metaphase II with polar body with antioxidants for any of the treatment groups (p>0.05). However, the significant improvement on the rate of blastocysts ($32.3{\pm}5.0%$) was found in 0.1 mM CYS treatment than 0.3 mM NAC, 0.2 mM NACA or 0.5mM GSH (p<0.05). The addition of NAC ($18.8{\pm}3.7%$) or NACA ($21.2{\pm}3.9%$) did not improve development competence to morula and blastocysts than control ($24.4{\pm}4.1%$) and GSH ($26.5{\pm}5.0%$) (p>0.05). Our study showed that medium supplementation with CYS during IVM and IVC improved the rate of bovine embryo development but not with NAC, NACA and GSH addition.

• Asian-Australasian Journal of Animal Sciences
• /
• v.13 no.7
• /
• pp.894-896
• /
• 2000

In vitro fertilization can be used for salvaging superior buffalo germplasm which otherwise goes waste after the slaughter of animals. This technology has also increased our basic understanding of growth of germ cells and embryos. The requirement of growing embryos is peculiar and stage specific. In the present study the cleavage and development of buffalo embryos were studied with homologous (buffalo) and heterologous (goat) oviductal cell co-culture systems. The cleavage rate improved significantly (p<0.01) in both homologous and heterologous co-culture as compared to control (55.3, 46.8 and 11.4%). The morula formation using homologous and heterologous oviductal cells also increased significantly as compared to control group (43.6, 21.9 & 1.9%). There was no blastula formation in control group, but addition of oviductal cells either from homologous or heterologous species significantly increased the blastula formation (9.5, 12.5%). The cleavage rate and embryo development was slightly better (non significant) in homologous as compared to heterologous oviductal cell culture. It was concluded that the use of oviductal cell co-culture (homologous and heterologous species) have significantly improved cleavage and development of buffalo embryos in vitro.

• Journal of Animal Reproduction and Biotechnology
• /
• v.39 no.3
• /
• pp.169-178
• /
• 2024

Background: Typical difficulties encountered during in vitro fertilization (IVF) to produce embryos in pigs include poor pronucleus formation and poor-quality fertilized embryos because of high polysperm invasion. In this study, we evaluated the effects of supplementation with apple seed extract (ASE) and coculture systems on porcine in vitro-fertilized embryo culture. Methods: Slaughterhouse-derived ovaries were used to obtain cumulus-oocyte complexes (COCs). COCs were conventionally used to perform IVF. We examined the differences in apoptosis and metabolism during development following addition of ASE to normal culture and coculture systems. Matrix metalloproteinases (MMPs), cell development-related factors, and apoptotic proteins were compared in porcine embryos produced under different conditions. Results: The expression of genes related to insulin-like growth factor (IGF) signaling was increased in the coculture system. In the ASE group, early apoptosis and necrosis were reduced in fertilized embryos and the late survival rate increased. Supplementation of the coculture system with ASE led to increased expression of BCL-2 and decreased expression of Casp-3 in the cytoplasm, thereby lowering the apoptosis rate and inducing MMP expression. In addition, compared with the extract-supplemented group in normal culture, the activity of MMP-2 decreased in the coculture system supplemented with ASE, activity of MMP-9 increased, and the expression of dynactin p62 and BrdU in the cytoplasm was higher than that in the other groups. Conclusions: The coculture system increased the activity of the embryonic cytoplasm compared with the non-coculture system. Supplementation with ASE may induce cell activity and inhibit the expression of apoptotic factors.

• Journal of Embryo Transfer
• /
• v.33 no.1
• /
• pp.41-48
• /
• 2018

In the last several decades, cell therapy research has increased worldwide. Many studies have been conducted on cell therapy, and have revealed that transplanted cells did not survive for long, and implanted cells remained inactive causing immune rejection depending on the patient's condition. Therefore, studies on cell-free therapy need to be conducted. To overcome these limitations, an alternative is the use of supernatant from cells, called "conditioned media (CM)." During in vitro cell culture, culture media supply nutrients to maintain cell characteristics and viability. In the culture, cells not only consume nutrients but also release beneficial proteins and substances, which are called "secretome." CM from cells can be stored for a long time and is easy to handle. Moreover, secretome in CM can also be measured; exact amount of secretome is important to set the standard value for disease treatment. Here, we reviewed studies on CM and confirmed that various secretomes from CM were identified in these studies. Moreover, these findings could benefit cell and animal studies in future. In conclusion, CM could be a potential candidate for an alternative to cell therapy.