• Clinical and Experimental Reproductive Medicine
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• v.21 no.1
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• pp.63-68
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• 1994

To confirm the overcome of in vitro 2-cell block, ICR mouse I-cell embryos were cultured in CZB media. All embryos in CZB were overcome in vitro 2-cell block and 92% of embryos were developed to the blastocyst at day 4. However, in m-KRB group(control) only 20% of embryos were developed over 2-cell. Any embryos in m-KRB did not develop to the morular stage. Developments and degenerations of ICR mouse I-cell embryos were compared in CZB medium prepared with water of three quality:(l) Milli-Q ultrafiltration water(UF);(2) Milli-Q reverse osmosis water(RO);(3) tap water(TAP). The objective was to evaluate the potential of quality control using ICR mouse 1-cell embryos. The more water was purified, the better embryo developments were supported and the less embryos were degenerated. As a quality control system, the culture of ICR 1-cell mouse embryos in CZB was useful.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.89-96
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• 1998

Anti-phospholipid antihodies (aPL) have important roles in various pregnancy complications such as recurrent miscarrige, growth retardation, placental abruption and stillbirth. However, their biological actions on preimplantation development of oocytes are still unclear. In this study, we investigated whether either aPL containing sera or phospholipids could affect in vitro fertilization and development of mouse oocytes. Sera used in this study were collected from three patients and the presence of aPL in the sera was confirmed by enzymatic-linked immunosorbent assay. When mouse oocytes were cultured in a serum-free, Chatot, Ziomek and Bavister (CZB) medium (Experiment 1), addition of aPL-containing sera (10%) to CZB medium did not. significantly (P>0.05) influence sperm penetration of oocytes. However, development to the blastocyst stage was significantly (P<0.05) inhibited by serum addition, and formation of morulae (16-23% vs. 58%) and blastocysts (0-4% vs. 21%) was markedly reduced compared with no addition (Experiment 2). In Experiment 3, pronuclear stage embryos were cultured for 96 h in GZB medium supplemented with 1 $\mu$g /ml phosphatidyl ethanolamine, 1 $\mu$g/ml phosphatidyl inositol or 1 $\mu$g /ml phosphatidyl choline. No increase in embryo development was found after addition of the phospholipids to CZB medium. These results suggest that 1) aPL have an inhibitory role in preimplantation development of mouse embryos, and that 2) the action of aPL may be related to a specific phospholid (s) rather than the tested phospholipids in the present study.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.333-339
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• 1996

Cumulus cells have possibly influence on fertilization of mouse oocytes and their subsequent development in vitro, because they readily produce lactate and pyruvate and can modify the concentration of substrates in the medium. In vitro fertilization of mouse oocytes with cumulus mass and their developments in five media which were differently composed in concentrations of glucose, lactate and pyruvate were observed. In the absence of glucose (CZ2 medium) decreased (p<0.01) the percentage of fertilization and embryos reaching the blastocyst stage. But, in the same concentration of glucose, lactate and pyruvate as mouse oviductal fluid with (MT1 medium) and without (MT2 medium) cumulus mass and modified CZB medium containing glucose (CZ1 medium) had no effects (p>0.05). These studies indicate that the adjustments of energy substrates concentration to the physiological level did not improve the fertilization of mouse oocytes with cumulus mass and their development in vitro, and the deletion of glucose showed adverse effects.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.275-282
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• 2004

This study was carried out to examine the effects of development media and those change on in vitro development (IVD) of porcine oocytes matured and fertilized in vitro. Putative embryos, which were matured in vitro in modified NCSU-23 (mNCSU-23) supplemented with 10％ porcine follicular fluid (pFF) and were fertilized in mTBM, were developed in vitro as the experimental scheme. The results are as follows. When porcine putative embryos were cultured in vitro in NCSU-23 or CZB/Pig-MEM, the percentage of oocytes cleaved was not different between two systems, but the percentage of blastocysts in NCSU-23 was significantly higher than in CZB/Pig-MEM (P<0.05). And when porcine putative embryos were cultured in vitro in NCSU-23 during 7 days with or without changing media at day 5, which was supplement with or without 10％ fetal bovine serum(FBS), the percentage of oocytes cleaved, blastocysts at day 6, and the cell number of ICM, TE and total of blastocysts at day 7 were not different among three treatments. As a result of this study, it is supposed that NCSU-23 be more favorable, to develop porcine embryos derived from IVM/IVF, than CZB/Pig-MEM, but that demonstration on the effects of changing medium with fresh one stand in need of the more experiments.

• Korean Journal of Animal Reproduction
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• v.19 no.1
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• pp.1-7
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• 1995

This study was accomplished to illuminate factors of contamination of microbes in the culture medium and effect of antibiotics on prevention of contamination in the medium when bovine follicular oocyte was matured, fertilized and developed in vitro. 1. When washed or unwashed semen diluted with TCM 199 was incubated for 24∼72hr, contamination was come out. 2. When diluted semen with TCM 199 which has penicillin, streptomycin, gentamycin, kanamycin or nystatin was incubated for 24∼72hr, contamination was not come out only in kanamycin. 3. When imported semen which was diluted in TCM 199 with penicillin, streptomycin, gentamycin, kanamycin or nystatin was incubated for 24∼72hr, contamination was not come out in all treatments. 4. When semen which was diluted in BO, CZB, Ham's F10 or TCM 199 was incubated for 24∼72hr, kanamycin showed no contamination in all treatments, but gentamycin showed contamination in CZB, Ham's F10 and TCM 199. 5. When the semen diluted in BO was moved at 24hr after incubation into BO and incubated for 72hr, contamination was not come out, but when it was moved into the TCM 199 and incubated for 72hr, contamination was come out at 48 to 72hr of incubation. 6. When the semen diluted in BO, BO＋BSA or BO＋FBS containing gentamycin, kanamycin or nystatin was incubated for 24∼72hr, the diluted semen in BO or BO＋BSA showed no contamination in all antibiotics but the diluted semen in BO＋FBS showed no contamination only in kanamycin. 7.The Pseudomonas cepacia, Serratia liquefaciens, Klebsiella pneumaniae was respectively isolated in the semen of A, B, and C bull and the microbes are highly affected by amikacin, tobramycin and kanamycin. 8. When bovine folicular oocyte was in vitro matured, fertilized and developed in the simple medium with kanamycin, 26.6% was developed to over 32cell stage embryo.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.11
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• pp.1665-1672
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• 2008

Interspecies somatic cell nuclear transfer (iSCNT) is a useful method to preserve endangered species and to study the reprogramming event of a nuclear donor cell by the oocyte. Although several studies of iSCNT using murine cells and bovine oocytes have been reported, the development of murine-bovine iSCNT embryos beyond the 8-cell stage has not been successful. In this paper, we examined the developmental potential of embryos reconstructed with a murine embryonic fibroblast as the nuclear donor and a bovine oocyte as the cytoplasm recipient. The reconstructed embryos were cultured in CZB (murine medium) or CR1aa (bovine medium). In addition, for the development of a murine-bovine iSCNT blastocyst, the antioxidant ${\beta}$-mercaptoethanol (${\beta}ME$) was supplemented to CR1aa medium. Furthermore, to verify the mouse genome activation in murine-bovine iSCNT embryos, RT-PCR analysis of murine Xist was performed. The development of the murine-bovine iSCNT embryos cultured in CR1aa was significantly higher than that in CZB (p<0.05). With respect to the effect of BME on the development of the murine-bovine iSCNT blastocyst, addition of BME produced a significant increase in blastocyst development (p<0.05). Karyotype analysis confirmed that the reconstructed embryos were derived from murine cells (40XX). The Xist gene was gradually increased from the 8-cell stage to the blastocyst stage. This is the first report of blastocyst development of iSCNT embryos derived from murine somatic cells and bovine oocytes. These results demonstrate that bovine cytoplasm can support the development of later stages of a preimplantation embryo from murine-bovine iSCNT.

• Korean Journal of Animal Reproduction
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• v.21 no.4
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• pp.389-396
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• 1997

Arrest in embryo development during in vitro culture has been reported in various mammals. Although some cause of the arrest have been suggested, little is known of the way that can overcome the arrest using in vitro culture system. The antioxidant, $\beta$-mercaptoethanol($\beta$-ME), has been shown to play an important role in embryo development. This study was designed to examine the effect of $\beta$-ME on the developing boving embryos produced in vitro by IVM and IVF. To select a, pp.opriate concentration of $\beta$-ME during whole culture period (7 days), various concentrations (10, 50 and 100$\mu$M) of $\beta$-ME were added to the CZB medium and their effects was significantly higher in 100$\mu$M of $\beta$-ME. The effects on development of embryos cultured with and without somatic cells to blastocyst stage were greater in FCS treatment (56.6 and 29.3%) than in BSA treatment(25.5 and 12.8%). We also evaluated the effects of $\beta$-ME addition on the blastocyst formation when embryos at different stages were exposed to 100$\mu$M $\beta$-ME. $\beta$-ME promoted increased development of embryo to blastocyst stage and the effect was greater in 6-cell to morula embryos than in embryos fewer than 4-cells at the initiation of treatment. The results suggested that $\beta$-ME can improve bovine embryo development by overcoming the arrest in early development.

• Journal of Embryo Transfer
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• v.10 no.3
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• pp.251-256
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• 1995

This study was experimented that developmental effects of bovine in vitro fertilized embryos by coculture system and supplementation of energy materials into simple media. With the ovaries from slaughter house in vitro maturation by 24h, in vitro fertilization was performed with sperms collected by Percoll gradient method. Fertilized embryos were cocultured in 15% FCS+CZB medium with BOEC(bovine oviductal epithelial cell), GCM (granulosa cell monolayer) and MEFC(mouse embryonic fihrohlast cell). And also in this study, there was trying to improve the early developmental rate of embryos by addition of concentration-controlled Na-pyruvate, D-glucose which were used as energy sources into CZB medium. In vitro developmental rate was confirmed by the cleavage rate of 48h post-IVF and the embryo development rate at 240h culture. In the coculture system BOEC had 20.0% of blastocysts rate, which was higher than that of other coculture systems. To determine the optimum concentration for early embryo developmental rate rapidly, through the gradient of concentrations of Na-pyruvate and D-glucose, we focused on the cleavage rate at 48h and blastocysts rate at 240h. In case of Na-pyruvate, cleavage rate and developmental rate over 3-cell were lower at the concentration of 1.OOrnM than the other treatment concentrations, otherwise the blastocysts rate was higher as 23.2% than the others. That result showed that as like reported group which had higher develop-mental rate over 3-cell was also higher to the blastocysts rate. In case of D-glucose, there was no effects through the concentration changes. It was the result of this study for which the use of BOEC coculture system and 1.OOmM Na-pyruvate as an energy source had an effect upon embryo development.

• Reproductive and Developmental Biology
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• v.29 no.2
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• pp.117-120
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• 2005

This study was conducted to establish the optimal temperature condition before oocyte activation in B6m F1 mouse. In experiment 1, two embryo culture media (CZB vs KSOM) were evaluated for the development of activated mouse oocytes. Parthenogenetic embryos cultured in KSOM showed better blastocyst development than ones cultured in CZB $(56.2\%\;vs\;81.0\%\;p<0.01)$. Two-hour of pre-incubation before activation significantly reduced the number of hatched blastocysts in KSOM $(22.0\%\;versus\;8.8\%\;p<0.05)$. In experiment 2, recovered oocytes were pre-incubated at different temperature conditions before activation. The experimental groups were divided by 5 as follows. Group A: pre-incubation for 120 min at $37^{\circ}C$, Group B: pre-incubation at $37^{\circ}C$ for 90 min then at $25^{\circ}C$ for 30 min, Group C: pre-incubation at $37^{\circ}C$ for 60 min then at $25^{\circ}C$for 60 min, Group D: pre-incubation at $37^{\circ}C$ for 30 min then at $25^{\circ}C$ for 90 min, and Group E: pre-incubation at $25^{\circ}C$ for 120 min before activation. Group A $(67.6\%)$ and B $(66.7\%)$ showed better development to the blastocyst stage than other groups $(Group\;C:\;50.0\%\;Group \;D:\;49.2\%\;Group\;E:\;33.3\%,\;p<0.05)$. The present study indicates that the temperature before activation affects the development of B6D2 F1 mouse parthenogenetic oocytes and exposure to room temperature should be limited to 30-min when the oocytes are left in HEPES-buffered medium for micromanipulation.

• Korean Journal of Animal Reproduction
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• v.20 no.2
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• pp.111-117
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• 1996

This experiment was designed to evaluate the effect of transforming growth factor-$\beta$ (TGF-$\beta$) and insulin-like growth factor-I (IGF-I) in bovine oocyte maturation in the presence or absence of serum on subsequent fertilization and embryo development. In addition, various concent rations of these growth factors were evaluated for the ability to promote development of eight-cell stage embryos to the blastocyst stage. Cumulus-oocyte complexes were recovered from 2 to 6 mm follicles obtained from slaughterhouse ovaries and cultured at 38.5$^{\circ}C$ for 24 hours in TCM-199 (HEPES Modification) with or with out 20 % fetal bovine serum (FBS) to which the following growth factors were added TGF$\beta$ IGF-l or TGF $\beta$ + IGF-I, all at 10 ng/ml each. The matured oocytes were fertilized in IVF-TL medium with frozen-thawed semen at a concentration of 1 ${\times}$ 10$^6$ cells/ml of fertilization medium following Percoll separation. After 24 hours of sperm-egg incubation, the embryos were transferred to CZB medium without glucose for 48 hours and then cultured in TCM-199 with 20% fetal bovine serum (FBS) for 96 hours. The addition of growth factors to IVM medium in the presence of serum had no effect on cleavage and subsequent embryo devlopment to blastocyst. In the absence of serum, TGF- improved cleavage and development to blastocyst compared to control's(p<0.05) and no synergistic effeet of IGF-I + TGF-$\beta$ was observed. In the second experiment, eight-cell embryos obtained by in vitro maturation (IVM) in TCM-199 + 20% FBS without growth facrors and in vitro fertil-ization (IVF) were cultured in the in vitro cuiture (IVC) medium supplemented with 5, 10 ng/ml TGF-$\beta$ or 5, 10, 50, 100 ng/ml IGF-I. Cleavage rate and development to the blastocyst stage was observed during seven days of incubation. The supplementation of 10 ng/ml TGF-$\beta$ to lVC medium for eight-cell embryos improved development to blastocyst (p<0.05) compared to control. In conclusion, these data indicate that the supplementation of growth factors to IVM medium in the presence of serum does not influence cleavage and subsequent embryo development. However, significantly more oocytes matured in serum-free TCM-199 and eight-cell embryos cultured in lVC medium developed to blastocyst with supplementation of 10ng/ml TGF-$\beta$.