• Journal of Embryo Transfer
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• v.16 no.2
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• pp.73-78
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• 2001

This study was carried out to investigated developmental ability of IVM/IVF derived hanwoo embryos after DNA microinjection. Microinjected hanwoo embryos were cultured fur 7 days. The cleavage rates of DNA injected embrys(36.3%) was significantly lower than those of non-injected embryos(67.4%; p<0.05). The percentage of injected embryos reaching to the morulae and blastocyst was significantly lower than those of non-injected embryos(p<0.05). When injected embryo were cultured contaning L-ascorbic acid and $\alpha$-tocopherol for 168 hrs, the morulae and blastocyst rates were significantly higher than control(p<0.05). These results suggested that the addition of L-ascorbic acid and $\alpha$-tocopherol can enhanced development to the morulae and blastocyst of microinjected embryos and improved culture condition increased the transgenic hanwoo embryos.

• Development and Reproduction
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• v.22 no.4
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• pp.297-307
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• 2018

The objective of this study was to evaluate the effects of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on cumulus expansion, nuclear maturation, fertilization capacity and subsequent development in porcine oocytes. The oocytes were incubated with 0, 25, 50, and $100{\mu}M$ ALA. Cumulus expansion was measured at 22 h, and gene expresison and nuclear maturation were analyzed at 44 h after maturation. Then, mature oocytes with ALA were inseminated, and fertilization parameters and embryo development were evaluated. In results, both of cumulus expansion and nuclear maturation were increased in $50{\mu}M$ ALA groups compared to control groups (p<0.05). However, expression of gap junction protein alpha 1 (GJA1, cumulus expansion-related gene), delta-6 desaturase (FADS1, fatty acid metabolism-related gene), and delta-5 desaturase (FADS2) mRNA in cumulus cells were reduced by $50{\mu}M$ ALA treatment (p<0.05). Cleavage rate was enhanced in 25 and $50{\mu}M$ ALA groups (p<0.05), especially, treatment of $50{\mu}M$ ALA promoted early embryo develop to 4 and 8 cell stages (p<0.05). However, blastocyst formation and number of cells in blastocyst were not differ in 25 and $50{\mu}M$ ALA groups. Our findings show that ALA treatment during maturation could improve nuclear maturation, fertilization, and early embryo development through enhancing of cumulus expansion, however, fatty acid metabolism- and cumulus expansion-related genes were down-regulated. Therefore, addition of ALA during IVM of oocytes could improve fertilization and developmental competence, and further studies regarding with the mechanism of ALA metabolism are needed.

• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.9-17
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• 1996

This study was conducted to investigate freezability of in vitro and in vitro matured rabbit oocytes, possibility of NT using frozen-thawed unfertilized oocytes, and NT efficiency by zona-slit micromanipulation. After freezing of in vitro matured oocytes, 33 to 49% of oocytes appeared normal morphology and 1.0M DMSO and 1.5M glycerol showed slightly high survival rate, but there was no difference in survival between two cryoprotectants. Freezability of in vitro matured oocytes was low in 1.5M glycerol and more sensitive to freezing. Efficiency of enucleation and fusion rate in method B was higher than that in method A and no difference in this efficiency was between 3 groups of oocytes in method B. Cleavage rate and developmental capacity to M＋B stage of fused embryos derived from frozen oocytes was greatly lower than that from fresh oocytes, respectively(39.1% : 79.5% ; 3.1% : 19.3%) and there was no difference in cleavage rate between DC voltages in two group oocytes. Additional incubation in cytochalasin B after electrical stimulation did not affect embryo development. In conclusion, it is suggested that enucleation and nucelar transfer by slitting of zona is more effective method in rabbit and that further study on optimum freezing conditions for in vitro matured oocytes is necessary to use as recipient oocytes.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.8
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• pp.1076-1079
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• 2004

Antibiotics are common additives in culture media during in vitro embryo development, but their effects on oocyte maturation in vitro have not been tested. The effects of penicillin, streptomycin and gentamicin on the maturational competence and subsequent development potential of goat follicular oocytes were examined after parthenogenetic activation in vitro. Maturation rates at 24 h after in vitro maturation, and parthenogenetic development at 48 h after activation, were evaluated by observing the protruding first polar body and the 4 cell stage cleavage, respectively. When streptomycin was present in the maturation medium, the percentages of matured oocytes 24 h after activation were significantly (p<0.01) lower than those from the other groups (42.5-45.7% vs. 69.1-73.8%). Penicillin and gentamicin treatment did not affect the maturation rates or the percentages reaching the 4 cell stage 48 h after activation. There was no significant difference in cleavage rates among the different antibiotic treatments 48 h after activation. Therefore, streptomycin suppresses the in vitro maturation of immature goat oocytes, but does not influence their subsequent development.

• The Korean Journal of Zoology
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• v.20 no.2
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• pp.109-122
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• 1977

Ultraviolet irradiation of the vegetal hemisphere of the fertilized frog egg prior to first cleavage resulted in alterations in neural morphogenesis. The sensitivity to UV irradiation dropped drastically at the point of 2/3 time lapse between fertilization and the appearance of the first cleavage furrow. Scannining electron micrographs revealed a decrease in size of the cells on the surface of the irradiated embryos. However, ectoderm exchange frafts indicated that the ectoderm of the irradiated embryo retains its capaciiy to form a completely normal neural morphology and epidermal surface. These facts were interpreted in terms of a decrease in the capacity for invagination during gastrulation, and subsequent primary embryonic induction.

• Journal of Embryo Transfer
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• v.17 no.1
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• pp.61-65
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• 2002

This study was carried out to study the viability of oocytes when vitrified at various maturation stages. Bovine cumulus-oocyte complexes were recovered from ovaries at a slaughter and then divided into five groups: control group(unvitrified oocytes), 0 hr. group(composed of oocytes vitrified before the onset of maturation) and 10, 14, and 20 hrs groups(vitrified at 10, 14 and 20 hrs after the onset of maturation, respectively). The oocytes remained vitrified for 24 hrs, and then were thawed in 3$0^{\circ}C$. Survival and cleavage rates were investigated by results of in vitro culture and aceto-orcein staining or FDA test. No difference in the incidence of diploid oocytes was observed among the control, non-vitrified group(3.6%) and oocytes vitrified at 14 hrs(6.7%) or 20 hrs(1.7%). However, more diploid oocytes were detected after vitrification at 0 hr.(26.7%) and 10 hrs(21.7%) post maturation. The survival rate of all vitrified immature oocytes(12.0~38.0%) was low, 48.0% of unvitrified oocytes and oocytes vitrified before maturation or 0~ 10 hrs after the onset of maturation were higher than that of other groups. The overall fertilization and cleavage rates of vitrified immature oocytes (32.3 ~ 64.6% and 4.6 ~ 32.3%) were low, and 55.0% of unvitrified oocytes and the rate of immature oocytes were very higher than that of mature oocytes.

• 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.

• Journal of Embryo Transfer
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• v.13 no.3
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• pp.227-233
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• 1998

The aim of these experiments was to investigate the effects of oviduct epithelial cells on bovine in vitro fertilization. Oviduct epithelial cell monolayers (OEC) on the 4-well dish were prepared according to general procedures. Monolayers were formed within 5days. The medium for OEC culture (TCM199 with 10% FBS) was replaced with IVF-TALP 2h before each experiment. Macromolecules/proteins from oviductal conditioned medium (OM) were recovered by ultrafiltration, which desalted and concentrated macromolecules greater than 5kDa, and this OM were added to W medium (experiment 1). The cleavage rate in OM+OEC group was significantly higher than in OM group (p〈0.01). In this experiment 2, oocytes were inseminated on OEC with sperm which had been pre-incubated with OEC for 0 or 4h before insemination. In this experiment, oocytes were exposed to sperm only 8 h for clarifying the effect. After insemination, oocytes were cultured in CRlaa. At 42 h post insemination, oocytes were denuded and examined for evidence of cleavage. The cleavage rates of oocytes which were inseminated with OEC treated sperm for 4 h were significantly higher than those of the other group (p〈0.01). In conclusion, sperm released from OEC have more fertilizing ability than those before attachment.

• Journal of Embryo Transfer
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• v.11 no.1
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• pp.15-26
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• 1996

This study was conducted to improve the in vitro maturation(JVM), in vitro fertilization (IVF) and in vitro developmental capacity of oocytes derived from slaughtered Korean native cattle. The recoverd oocytes, obtained from a local slaughter house, were used completely surrounded by at least 3 layers of cumulus cells in combination with a homogeneous cytoplasmic pigmentation. In vitro maturation was induced in TCM-199 or Ham's F-10 supplemented with LH(1O $\mu$g/rnl), FSH(35 $\mu$g/ml), estradiol-17$\beta$(1 $\mu$g/ml) at 39$^{\circ}C$ under 5% $CO_2$ in air for 24 hours. Sperm from caudal epididyrnis and previously matured cumulus-oocytes complexes were cultured for 24 hours in 100 $\mu$l droplets of fertilization media under paraffin oil. The zygotes were cultured with media(TCM-199 with bovine oviductal epithelial cells or CRlaa) for 7 to 10 days. The cleavage rate of IVM-IVF oocytes was significantly (P<0.05) higher following maturation using Ham's F-10 (59.9%) than TCM-199 (51.6%). Development to the blastocysts among cleaved embryos was not signficantly different between maturation media: Ham's F-10 (16.0%) and TCM-199(11.9%). However, the hatching rate was affected significantly (P<0.05) on rnaturation media as 62.9% in Ham's F-10, compared with 41.2% in TCM-199. The cleavage rate of IVM-IVF oocytes was significantly (P<0.05) higher following IVF using m-TALP medium (80.1%) than BO medium (51.6%). The percentage of in vitro developed blastocysts among cleaved embryos was not signficantly different between fertimization media: BO (11.7%) and m-TALP (17.6%). The cleavage and the developmental rate to the blastocysts after IVF in m-TALP or condition medium(CM) with or without oviduct epithelial cell monolayer(OECM) was similar(80.1% and 17.6% in m-TALP, 83.8% and 19.4% in M-TALP with OECM. 82.9% and 18.9% in CM, 87.6% and 16.0% in CM with OECM, respectively). The percentage of in vitro developed blastocysts among cleaved embryos was significantly (P<0.05) higher in TCM-199 medium co-cul tured with bovine oviduatal epithelial cell monolayers(35.2%) than CRlaa medium(1.9%). These results stggest that the most transferable IVF embryos could be produced from Ham's F-10, m-TALP and TCM-199 medium with bovine oviductal epithelial cell monolayers for IVM, IVF and IVC, respectively.

• 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.