• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.3-7
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• 2002

In vitro embryo culture techniques provide significant contributions not only for a basic research of fertilization and early embryogenesis, but also for a low cost mass production of bovine embryos for transfer, embryo diagnosis, nuclear cloning and the production of transgenic cows. This presentation introduces newly developed serum-free media (IVD101 and IVMD101) that are effective far high yields of transferable embryos of excellent quality from in vitro-matured and fertilized oocytes. Both serum-free media are superior to a conventional serum-containing medium on the increased rates of blastocyst formation, post-thaw embryo viability, and pregnancy after transfer. Furthermore, reduced risks of calf mortality and large calf syndrome are also observed for the serum-free-derived embryos. Serum-derived embryos contain a large number of lipid droplets and immature mitochondria in their cytoplasm that may account for the lower production of transferable embryos and poor embryo quality.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.6
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• pp.966-975
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• 1999

Technologies on preimplantation porcine embryos have been developed quickly and significantly. Successful development of systems for culture of porcine zygotes to the blastocyst stage has made it possible to utilize follicular oocytes for in vitro production of embryos and thus stimulated research on various embryo technologies. Recent technological development of embryo cryopreservation, separation of X- and Y-bearing spermatozoa and non-surgical embryo transfer has also made it easy to utilize in vivo- and in vitro-produced embryos for artificial manipulation to produce clones and transgenic pigs. Further progress in overcoming various problems associated with each embryo technology will result in acceptable efficiency to utilize porcine embryos with a high or increased quality. Combining these technologies will accelerate further expansion of the swine industry not only for meat production but also for the production of therapeutic recombinant proteins and xonografts.

• Journal of Embryo Transfer
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• v.13 no.3
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• pp.235-243
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• 1998

In this study, we investigated the effects of three kinds of culture medium (Charles and Rosenkrans; CRlaa, Tyrode's; TALP, synthetic oviduct fluid: SOF), insulin transferrin + selenium complex (ITS), macromolecules(polyvinyl alcohol: PVA, fetalb-ovine serum: FBS) and NaCl on the development of early bovine embryos. In experiment 1, there were no differences in embryo development among three kinds of embryo culture medium (CR $l_{aa}$ , TALP, SOF). In experiment 2, BSA, FBS and PVA were added each in TALP as macromolecule sources. The developmental rates of embryos in BSA or FBS added TALP were significantly higher than in PVA added one (p〈0.01), but there was no difference between BSA and FBS added groups. In experiment 3, bovine embryos were cultured in TALP with the following supplements: BSA alone(1, 3 or 8 mg/ml, each) or BSA(1, 3 or 8 mg/ml, each)+ITS (10$\mu\textrm{g}$/m1 insulin, 5 $\mu\textrm{g}$/ml transferrin, 5 ng/ml selenium). In higher concentration of BSA and ITS supplemented groups, the developmental rates over compacted morula were higher than others, but there was a significant effect of ITS only in 1 mg/ml of BSA added group (p〈0.05). In experiment 4, the effect of reduced concentration of NaCl was evaluated. The developmental rate over compacted morula in the medium containing 90 mM of NaCl was higher than in 114 mM group (p〈0.05). In conclusion, BSA could be used as a macromolecule source in bovine embryo culture, and ITS, as a serum substitute, could be used for improving of embryonic development. Also, reduction of NaCl concentration from 114 mM to 90 mM may improve the development of bovine embryos.bryos.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.77-86
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• 1998

The uptake of glucose for metabolism and growth is essential to most animal cells and is mediated by glucose-transporter (GLUT) proteins. The aim of this study was to determine which class of glucose transporter molecules was responsible for uptake of glucose in the mouse early embryo and at which stage the corresponding genes were expressed. In addition, co-culture system with vero cell was used to investigate the effect of the system on GLUT expression. Two-cell stage embryos were collected from the superovulated ICR female and divided into 3 groups. As a control, embryos were cultured in 0.4% BSA-T6 medium which includes glucose. For the experimental groups, embryos were cultured in either co-culture system with vero cells or glucose-free T6 medium supplemented with 0.4% BSA and pyruvate as an energy substrate. 2-cell to blastocyst stage embryos in those groups were respectively collected into microtubes (50 embryos/tube). Total RNA was extracted and RT-PCR was performed. The products were analysed after staining ethidium bromide by 2% agarose gel electrophoresis. Blastocysts were collected from each group at l20hr after hCG injection. They were fixed in 2.5% glutaraldehyde, stained with hoechst, and mounted for observation. In control, GLUT1 was expressed from 4-cell to blastocyst. GLUT2 and GLUT3 were expressed in morula and blastocyst. GLUT4 was expressed in all stages. When embryos were cultured in glucose-free medium, no significant difference was shown in the expression of GLUT1, 2 and 3, compared to control. However GLUT4 was not expressed until morular stage. When embryos were co-cultured with vero cell, there was no significant difference in the expression of GLUT1, 2, 3 and 4 compared to control. To determine cell growth of embryos, the average cell number of blastocyst was counted. The cell number of co-culture ($93.8{\pm}3.1$, n=35) is significantly higher than that of control and glucose-free group ($76.6{\pm}3.8$, n=35 and $68.2{\pm}4.3$, n=30). This study shows that the GLUT genes are expressed differently according to embryo stage. GLUTs were detectable throughout mouse preimplantation development in control and co-culture groups. However, GLUT4 was not detected from 2- to 8-cell stage but detected from morula stage in glucose-free medium, suggested that GLUT genes are expressed autocrinally in the embryo regardless of the presence of glucose as an energy substrate. In addition, co-culture system can increase the cell count of blastocyst but not improve the expression of GLUT. In conclusion, expression of GLUT is dependent on embryo stage in preimplantation embryo development.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.67-71
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• 2014

In porcine embryo culture, one of reactive oxygen species (ROS) is harmful factors that are made during in vitro culture. To decrease the detrimental effect of ROS on embryo development, superoxide dismutase, catalase and glutathione peroxidase could be used in the embryo culture. Out of these antioxidants, 7,8-dihydroxyflavone (7,8-DHF) was reported its antioxidant effects to prevent the glutamine-triggered apoptosis. Therefore, this study was performed to investigate the most appropriate concentration of 7,8-DHF in porcine embryonic development. For that, 5 different concentration (0, 0.1, 0.5, 1, $2{\mu}m$) of 7,8-DHF was supplemented in the porcine IVM media and then maturation and blastocyst formation rates were compared among 5 groups. In maturation rates of porcine oocytes, significant higher maturation rates was shown in the $1.0{\mu}m$ group compared with another 4 groups ($83.3{\pm}2.1$ vs. $80.7{\pm}1.4$, $79.8{\pm}1.4$, $78.3{\pm}1.2$, $79.4{\pm}1.6$), respectively (P<0.05). In the embryo culture, $1.0{\mu}m$ group also showed the significant higher cleavage rates ($76.8{\pm}3.1$ vs. $62.1{\pm}5.0$, $65.7{\pm}4.0$, $68.6{\pm}3.7$, $64.6{\pm}4.0%$) and blastocyst formation rates - ($39.6{\pm}4.0%$ vs. $28.6{\pm}3.3$, $31.1{\pm}3.9$, $29.3{\pm}2.5$, $39.6{\pm}4.0$, $26.4{\pm}3.2%$), respectively (P<0.05). There was no significant difference among 5 groups in the cell number of blastocyst (P<0.05). In conclusion, supplement of $1.0{\mu}m$ of 7,8-DHF was effective to increase the porcine embryonic development competence as antioxidant to ROS.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.39 no.6
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• pp.571-576
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• 1994

This study was conducted to determine the effects of embryo age, growth regulators, low temperature treatment on regeneration, plant growth, and heading in immature embryo culture of Barley. Shoot and root development was more in older than in younger excised embryos, and more in a medium without kinetin than in one with kinetin. The rate of survival was higher on medium without growth regulators and lower on medium with kinetin 5 mg/L because of retarded plant growth. Embryo age and media did not completely replace low temperature treatment. Twenty- and 14 day-old embryos responded by flowering after 4 weeks of vernalization. Embryo culture at 14- and 20 days after anthesis coupled with 4-week cold treatment shortens generation time of barley. When 20 day immature embryos of barley (cv. Olbori) was treated low temperature treatment for 4 weeks, the rate of heading was better than the other treatment.

• Clinical and Experimental Reproductive Medicine
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• v.19 no.2
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• pp.125-131
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• 1992

The possible effect of human follicular fluid(hFF) on the growth and development of fertilized oocytes and embryos is important because the fallopian tubes are exposed to FF after follicular rupture and the processes of fertilization and embryo cleavage occur inside the fallopian tubes. Previously, it was suggested that human FF might adversely affect on the development of early mouse embryos. In order to investigate the effect of hFF on the development of embryos, early mouse embryos were cultured in media containing various protein sources as bovine serum albumin(BSA), fetal cord serum(FCS) and FF. And we evaluated the development of early mouse embryos in terms of the morphology, cleavage rate, and cell count of blastcysts. There were no significant differences in the morula and blstocyst formation rates of 2-cell mouse embryos cultured in the media containg three different protein sources and three different concentrations of FF. The blastocyst formation rate of 1-cell mouse embryo cultured in FF group was significantly higher than that cultured in BSA group(P<0.05). The morula and blastocyst formation rates of 2-cell mouse embryos of the group cultured in the media containing FF were comparable with those of other two groups, in addition, the cell count of blastocysts of FF group in the 2-cell embryo culture was higher than those of BSA group and HCS group(P<0.01), and this finding was also noted in 1-cell embryo culture. There was no difference in the morula and blastocyst formation rates of the 2-cell mouse embryos cultured in the media containing different concentrations of FF. These results suggest that mature human follicular fluid has no inhibitory activity on the development of early mouse embryos even in high concentration and may be a good protein source which is positively associated with the development of mouse embryos in vitro especially in 1 cell embryo culture.

• Development and Reproduction
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• v.18 no.3
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• pp.153-160
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• 2014

Adaptive development of early stage embryo is well established and recently it is explored that the mammalian embryos also have adaptive ability to the stressful environment. However, the mechanisms are largely unknown. In this study, to evaluate the possible role of aquaporin in early embryo developmental adaptation, the expression of aquaporin (AQP) 5 gene which is detected during early development were examined by the environmental condition. To compare expression patterns between in vivo and in vitro, we conducted quantitative RT-PCR and analyzed localization of the AQP5 by whole mount immunofluorescence. At in vivo condition, Aqp5 expressed in oocyte and in all the stages of preimplantation embryo. It showed peak at 2-cell stage and decreased continuously until morula stage. At in vitro condition, Aqp5 expression pattern was similar with in vivo embryos. It expressed both at embryonic genome activation phase and second mid-preimplantation gene activation phase, but the fold changes were modified between in vivo embryos and in vitro embryos. During in vivo development, AQP5 was mainly localized in apical membrane of blastomeres of 4-cell and 8-cell stage embryos, and then it was localized in cytoplasm. However, the main localization area of AQP5 was dramatically shifted after 8-cell stage from cytoplasm to nucleus by in vitro development. Those results explore the modification of Aqp5 expression levels and location of its final products by in vitro culture. It suggests that expression of Aqp5 and the roles of AQP5 in homeostasis can be modulated by in vitro culture, and that early stage embryos can develop successfully by themselves adapting to their condition through modulation of the specific gene expression and localization.

• 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.

• Journal of Embryo Transfer
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• v.14 no.3
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• pp.163-169
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• 1999

This study was carried out to investigate the effect of co-culture system(bovine oviduct epithelial cells; BOEC) and defined culture system(modified TALP ; mTALP) on the development of IVM-IVF embryos, and survival of in vitro produced blastocysts after freezing and thawing. Occytes from the slaugheterhous ovaries were matured and fertilized using general protocol. The results obtained were as the following: 1. Survival rates of frozen-thawed blastocysts using 10% glycerol as cryoprotectant was higher in day 7 blastocysts than in Day 8 and 9 blastocysts from co-cultrue system, but survival rate of frozen-thawed blastocysts was higher in Day 10 blastocysts than in day 8 and 9 blastocysts from defined culture system. Regardless of their age, survival rate of frozen-thawed blastocysts was significantly higher (p<0.05) in co-culture system than in defined culture system. 2. The cell number of blastocysts was significanlty higher (p<0.05) in Day 7 blasotcysts than in Day 8 and 9 blastocysts from co-cultures, but the cell number of blsstocysts was significantly higher (p<0.05) in Day 10 blastocysts than in Day 8 and 9 blastocysts from defined culture system. Regardless of the culture system, blastocysts with higher cell number showed higher survival rates after freezing and thawing.