• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.21-26
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• 2006

This study was examined the effects of concentrations of polyvinylpyrrolidone(PVP) and supplementation of EGF, cysteine and PVP during in vitro maturation on the development of bovine embryos. In experiment 1, 0.1 to 3.0% PVP was supplemented to IVM medium before IVF. The development rates to the blastocyst stage was significantly higher in 0.5% PVP group than 3.0% PVP group (P<0.05). In experiment 2, EGF, rysteine and PVP were supplemented to IVM medium. The hight cleavage rate was obtained from cysteine group, but blastocyst formation rates did not differ among groups. The highest total cell number and inner cell mass (ICM) cell number were observed in cysteine group. In PVP group, ICM cell number was significantly low than those of cysteine and control groups (P<0.05). After embryo transfer, pregnancy rate was significantly low in PVP group compared to other groups (P<0.05). These results indicate that the supplementation of PVP in IVM medium support the embryo development, but has a deteriorate effect on the blastocyst quality.

• Korean Journal of Animal Reproduction
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• v.18 no.4
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• pp.299-307
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• 1995

The goal of cell stem cell technology is to produce a viable and genetically normal animal. To achieve this goal various laboratories have followed 2 different pathways beginning with either the culture of 1) single or pooled ICMs grown with or without a feeder layer or 2) single or pooled 16-20 cell stage embryos grown with a feeder layer. Also, thus far embryonic cell cultures or lines have been established by several methods including loose suspension culture for short-term cultures and more commonly murine or bovine fibroblast feeder layers for long-term culture. Pluripotent lines have been derived from 16-cell through blastocyst inner cell mass stages. The efficiency of establishing cell lines and cell proliferation apper to be affected by the number of cells or embryos starting the line. Most attempts to produce offspring from long term STO cell feeder layer cultured ICM or morulae derived ES cells have resulted in pregnancy failure in the first trimester when ES cells were used in cuclear transfer or have failed to retain ES cells in the progeny produced by chimerization. The exception is 1 chimeric fetus from use of morula ES cells in the chimerization with early embryonic cells. There is much to be learned yet about ES cell culture requirements for maintenance of totipotency. If bovine ES cell lines loose imprinting pattern and totipotency with long-term culture and passage as suggested for mouse ES cells, we may be limited to the use of short-term cultures for multiplication of embryos and efficient production of transgenic animals. No bovine ES cell system has yet met all of the criteria indicated for a totipotent ES cell line.

• Reproductive and Developmental Biology
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• v.31 no.1
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• pp.35-41
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• 2007

This study was conducted to examine the effects of energy substrates in different conoentration of carbohydrates in the human oviduct and uterus on the in vitro development of mouse 2-cell embryos. Two-cell embryos were collected from ICR female mice at $46{\sim}50\;hr$ after 5 IU hCG injection and cultured in three different media [control: 0 mM, Guoup A: glucose (G) 0.5 mM + pyruvate (P) 0.32 mM + lactate (L) 10.5 mM, Group B: G 3.15 mM + P 0.1 mM + L 5.83 mM] for 72 hr. Rates of morula formation of group A (72.3%) and B (56.6%) were significantly higher higher (p<0.05) than that of control (34.9%) at 24 hr. However, blastocyst rate was significantly higher (p<0.05) in control (51.8%) than group A (39.8%) and B (28.9%) at hr. At 72 hr, no differences were found in the number of zona-intact, zona-escape and total blastocysts among groups. Mean and ICM cell numbers were significantly higher (p<0.05) in group A (78.0, 13.4) and B (64.4, 11.8) than control (53.1, 5.7), respectively, The percent of ICM were significantly higher (p<0.05) in group A (22.9%) and B (23.7%) than control (14.2%). No differences were round in the TE cell numbers ($34.1{\sim}45.1$). The ICM:TE ratio was significantly higher $34.1{\sim}45.1$ in control (1:6.0) than group A (1:3.4) and B (1:3.4). This study shows that energy substrates added to culture media especially, the oviductal level of carbohydrates increase the developmental capacity of 2-cell mouse embryos.

• Development and Reproduction
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• v.12 no.1
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• pp.77-86
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• 2008

This study was carried out to investigate the effect of leukemia inhibitory factor (LIF) on the derivation of mouse ES cells from isolated blastomeres. Two-cell stage mouse embryos were obtained from superovulated BDF1 female mice. Collected embryos were cultured to blastocyst stage in culture medium supplemented with 0, 1,000, 2,500 or 5,000 U/mL of LIF. Cultured blastocysts were examined by counting the number of cells in the inner cell mass (ICM) and trophectoderm (TE) using differential staining method. When 2-cell embryos were cultured with 2,500 U/ml of LIF, the cell numbers of ICM significantly increased in comparing with those of the control($21.0{\pm}4.0$ vs. $15.9{\pm}5.0$, P<0.01) and 1,000 U/mL of LIF-containing group ($21.0{\pm}4.0$ vs. $16.6{\pm}4.9$, P<0.05). We used an ES cell establishment medium with 20% Knockout Serum Replacement and 0.01 mg/mL ACTH instead of fetal bovine serum. Establishing efficacy of ES cell lines were the highest in 2,500 U/mL of LIF-containing group as 36.7% (11/30). This culture medium was applied to the culture of isolated blastomeres and to derivate ES cell lines. Three ES cell lines (21.4%) from isolated blastomeres of 2-cell stage embryos were established. In further experiments, we could establish one ES cell line (4.0%) from single blastomere of 4-cell stage embryo. The subcultured ES cells and their embryoid bodies were characterized by analyzing gene expression for undifferentiation and differentiation marker gene using immunocytochemistry and RT-PCR. In conclusion, LIF supplementation in culture medium could increase the cell number in ICM of blastocysts and support derivation of ES cell lines from isolated blastomeres.

• Journal of Embryo Transfer
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• v.27 no.4
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• pp.253-258
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• 2012

This study was carried out to effects of ethylene glycol concentration, sucrose and culture day of in vitro production embryo on slow-down freezing in Hanwoo. 6, 7, 8 and 9 day embryos produced in vitro were frozen using 1.8M EG+0.1M sucrose, 1.8M EG+0.5% BSA and 1.5M EG+0.1M sucrose media. Survivability was confirmed after frozen-thawed 24 and 48h and ICM, TE cell number were counted by Hoechst 33342 and PI staining after frozen-thawed 24h. As a result, 1.8M EG+0.1M sucrose group was most significantly (p<0.05) higher compared with the other treatment groups on survivability, TE and total cell number after frozen-thawed 24h ($94.2{\pm}2.6%$, $94.67{\pm}3.4$ and $129.67{\pm}5.5$). ICM number did not found significant (p<0.05) differences between the three treatment groups. in 6, 7, 8 and 9 day of embryos using three types of freezing media, frozen-thawed, 1.8M EG+0.1M sucrose groups with embryos cultured 8 day was significantly (p<0.05) highest survivability to $98.3{\pm}1.7%$ after frozen-thawed 24h. 1.5M EG+0.1 sucrose group with embryos cultured 9 day was significantly higher survivability than group of embryos cultured 8 day after frozen-thawed 24 and 48h. In conclusion, 1.8M EG+0.1M sucrose media is considered to be effective to cryopreservation of embryos cultured 8 and 9 day.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.5
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• pp.693-700
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• 2007

The present study was conducted to examine the effect of different levels of essential and nonessential amino acid in NCSU-23 medium on the in vitro-produced porcine embryo as it develops from the zygote to the blastocyst stage. Four experiments were performed, each with a completely randomized design involving 5 to 8 replications of treatments. In order to know the effect of nonessential amino acids in NCSU-23 medium, 0, 5, 10 and $20{\mu}/ml$ MEM were supplemented there to, (Exp. 1) and the medium was supplemented with same level of essential amino acids (Exp. 2). The combined effect of nonessential (0, 5, 10 and $20{\mu}/ml$ MEM) and essential amino acids (0, 5, 10 and $10{\mu}/ml$ MEM) in NCSU-23 medium (Exp. 3), first 72 h with non-essential amino acids (at 0, 5, 10 and $20{\mu}/ml$ MEM), and last 4 d with essential amino acids with the same level as NEAA (Exp. 4) were examined. The embryo development was monitored and the quality of blastocysts was evaluated by counting the number of total cells and determining the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells. When Eagle's nonessential amino acids (MEM) added to NCSU-23 medium, it significantly increased the likelihood of development to the 2- to 4-cell stage and subsequent blastocyst development. Supplementation of different levels of essential amino acids in the NCSU-23 medium decreased cleavage rate, rate of morula and blastocyst development and the number of ICMs. In the case of the combined effect of essential and nonessential amino acids, better and significant results were found for blastocysts, hatching blastocysts and for ICM numbers which were also dose dependent. With respect to the biphasic effect of nonessential and essential amino acids, nonessential amino acids increased cleavage whereas essential amino acids increased the total cell number. Neither the nonessential nor the essential group of amino acids, on their own, affected blastocyst cell number or the differentiation of cells in the blastocyst. In conclusion, this study determined the role of nonessential and essential amino acids in the culture of the porcine embryo and showed that the embryo requires different levels of amino acids as it develops from the zygote to the blastocyst stage.

• Korean Journal of Animal Reproduction
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• v.20 no.2
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• pp.119-126
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• 1996

This study was carried out to determine the optimal condition for successful and efficient c cryopreservation of zygotes, 1-cell embryos, using EFS40 which was 40% (v/v) ethylene glycol diluted in DPBS medium containing 30% Fic-oll (w/v) and 0.3 M sucrose. After mouse zygote produced by IVF was vitrified by two freezing methods, the post-warming survival rates of 1-cell zygotes were assessed as cleavage to the 2-cell stage and development into the hatching blastocysts at 5 day. In the one-step method, when embryos were directly exposed to the vitrification solution at 25$^{\circ}C$ for 1 min., survival and development rates of zygotes were 85.5% and 31.9% In the two-step method, embryos were equilibrated with a dilute 20% EG for 1, 3, 5 min. before 1 min. exposure to EFS40, re-spectively. However, the rates of development (17.7, 3.3, 0%) were lower than that of one-step method. The highest survival rate (95.9%) was obtained by one-step method which exposes embryos in EFS40 for 30 sec. In this condition, 63. 8% of cleaved 2-cell developed into hatching blastocysts. In the cell number of Total and ICM using differential labelling technique, there are no significant differences in the cell number of Total and ICM between blastocysts devel oped in vitrified-thawed embryos (63.2${\pm}$16.9, 1 13.5${\pm}$4.0) and control balstocysts (54.0${\pm}$15.2, 1 12.3${\pm}$4.6). Therefore, these results show that mouse zygotes can be successfully cryopreserved by a simple vitrification method although developmental rates of vitrified embryos were reduced. In conclusion, this proposed vitrifi cation procedures can be useful in the cryopreservation of mouse IVF zygotes.

• The Bulletin of The Korean Astronomical Society
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• v.46 no.1
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• pp.36.2-36.2
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• 2021

In the outskirts of galaxy clusters, weak shocks with M_s < ~3 appear as radio relics where the synchrotron radiation is emitted from cosmic-ray (CR) electrons. To understand the production of CR electrons through the so-called diffusive shock acceleration (DSA), the electron injection into the DSA process at shocks in the hot intracluster medium (ICM) has to be described. However, the injection remains as an unsolved, outstanding problem. To explore this problem, 2D Particle-in-Cell (PIC) simulations were performed. In this talk, we present the electron preacceleration mechanism mediated by multi-scale plasma waves in the shock transition zone. In particular, we find that the electron preacceleration is effective only in the supercritical shocks, which have the sonic Mach number M_s > M_crit ≈ 2.3 in the high-beta (β~100) plasma of the ICM, because the Alfven ion cyclotron instability operates and hence multi-scale plasma waves are induced only in such supercritical shocks. Our findings will help to understand the nature of radio relics in galaxy clusters.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.201-205
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• 2005

The objective of this study was to investigative the effects of amino acids supplementation on maturation, fertilization and embryo development of pig oocytes. Essential amino acids (EA), non-essential amino acids (NA) or both amino acids (EA + NA) were supple-mented to North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (pFF). When the amino acids were supplemented to the maturation medium, the maturation rates were higher (p<0.05) in the NA group than control ($83.3{\pm}0.04\%\;versus\;70.0{\pm}0.05\%$, but the subsequent cleavage rates and development to morula and blstocyst stage between aminoacid supplement groups and control were not different. The developmental rates to morula and blastocysts stage were not significantly different regardless of amino acid supplementation to culture medium. In addition, supplementation of amino acids did not significantly affect the rate of fertilization and polyspermy. When the amino acids were supplement to culture medium, the number of trophectodermal (TE) cells was significantly (p<0.05) higher in amino acid supplement group than that of control ($18.6{\pm}0.5\;versus\;16.1{\pm}0.6$), whereas the numbers of inner cell mass (ICM) cells were not different among the treaonent groups and control ($29.0{\pm}0.9\~31.5{\pm}1.2$). Total cell number was also significantly (p<0.05) higher in EANA group ($50.0{\pm}1.0$) than that of control group ($44.2{\pm}1.1$). These results indicate that the amino acid supplementation to maturation and culture medium may not significantly stimulate early embryo development, but may improve the TE cell number of blastocyst stage in the pig.

• Animal cells and systems
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• v.15 no.4
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• pp.287-294
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• 2011

The accelerated cooling rate associated with vitrification reduces injuries attributed to cryopreservation and improves the post-freezing developmental competence of vitrified embryos. In this study, embryos were vitrified and warmed and morphologically evaluated for their development to blastocysts. Survival rates between the fresh ($96.7%{\pm}3.8%$) and vitrified embryos ($90.7%{\pm}5.1%$) did not differ significantly (P>0.05). The mitochondrial membrane potential of fresh control cells measured by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanide iodide staining was similar to that of cryoprotected and vitrified embryos. Mitochondrial staining with rhodamine 123 did not differ among the fresh, cryoprotected, and vitrified embryos. Moreover, the distribution of $H_2O_2$, assessed by 2',7'-dichlorodihydrofluorescein diacetate staining, did not differ among the groups. The results showed that the developmental rate did not differ significantly among the fresh ($87.8%{\pm}11.3%$), cryoprotected ($83.2%{\pm}7.6%$), and vitrified 2-cell embryos ($75.8%{\pm}14.2%$). The mean number of the inner cell mass (ICM), trophectoderm (TE), and apoptotic cells was counted and statistically compared, and although the number of ICM and TE was decreased in the cryoprotected and vitrified embryos, there were no significant differences among the groups (P>0.05). During the cultivation period, randomly selected blastocysts from each group were stained using either 4',6-diamidino-2-phenylindole and bisbenzimide or the terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling technique. The incidence of apoptosis appeared to be almost identical in all the groups. Droplet vitrification could subsequently lead to high survival and developmental rates of cryopreserved mouse embryos.