• Development and Reproduction
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• v.8 no.2
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• pp.119-122
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• 2004

It is well known that unidentified factors in sera, hormones and growth factors promote the proliferation of granulosa cells and nuclear maturation of bovine COCs in vitro. Attemps had been developed the simple composition of culture media and similar system to in vivo conditions has been applied. In the present study, we investigated the effect of TGF-${\beta}$ on in vitro maturation and in vitro development of Hanwoo COCs. When the COCs were matured in TCM 199 containing 0.1, 1 or 10 ng/ml TGF-${\beta}$ for 24 hrs, metaphaseⅡ of COCs were obtained 95.8%, 100% of matured COCs, respectively and there were no differences among the concentrations of TGF-${\beta}$. Matured COCs with TGF-${\beta}$ cultured in maturation medium after in vitro fertilization, developmental rate to blastocyst were 0~0.8%. Matured COCs with TGF-${\beta}$ were cultured in TCM 199+10% FBS, 0.8% BSA, 0.1% PVA, blastocyst formation were showed in 12.4%, 12.8%, 8.5% of those and cultured in IVMD or IVD without serum were 38.4%, 34.8%, respectively. There were significant differences among the media (P<0.05). TGF-${\beta}$ is available for i vitro maturation of bovine COCs, but further investigation would be need for finding the synergistic autocrine/paracrine fashion of other growth factors in early bovine development.

• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.407-417
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• 2000

The present study was to examine effects of various electrical stimulus treatments used for electro-fusion on the preimplantation development of bovine nuclear transfer (NT) embryos with fetal fibroblast cells. Fetal fibroblast cells were isolated from one fetus at day 45 of gestation in Holstein cow, and passaged 3 to 4 times before being transferred into enucleated oocytes. Single fibroblast cells were individually placed into the perivitelline space of enucleated oocytes by using a micromanipulator. At first, the fusion and developmental rates of reconstructed oocytes were compared between different electric stimulation conditions. When fusion of the reconstructed oocyte was induced by different electric pulse periods (15, 30 and 45 $\mu$sec) at a DC pulse of 1.8 kV/cm, 15 (45.5%, 120/264) or 30 $\mu$ sec group (43.9%, 106/241) showed a higher fusion rate than 45 $\mu$sec group (23.2%, 58/250, P＜0.05). However, no difference was detected in the development rate of the fused oocytes to blastocysts between groups. Next experiment was to examine the effects of different electrical field strengths (1.5, 1.8 and 2.1 kV/cm) for 15 $\mu$sec at electrofusion on in vitro development of the NT embryos. As results, there was no difference in the fusion and developmental rates of the NT embryos between electrical strength (P＞0.05). Finally, developmental competence of bovine NT embryos with somatic cells was compared with IVF-derived embryos. Of enucleated oocytes fused with fibroblast cells, 27.4% (75/274) developed to the blastocyst stage, which is similar to that (24.5%, 58/237) of IVF-derived embryos. However, mean nuclei number of NT blastocysts was smaller than that of IVF-derived blastocysts. Thus, we have established an optimal condition (1.8 kV/cm, 15 $\mu$sec) for electric fusion of bovine NT oocytes with somatic cells. The present study indicates that bovine reconstructed embryos with somatic cells normally develop to blastocyst stage in vitro, although having smaller nuclei numbers of blastocysts as compared to IVF-derived embryos.

• Korean Journal of Animal Reproduction
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• v.27 no.1
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• pp.87-93
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• 2003

This study was conducted to investigate the effects of embryo development by fusion condition on the nuclear transfer with brindle coated cow's ear cells. Ear cells were transferred into an enucleated oocyte and fused with cytoplasm in the fusion condition with 1.9kv/cm, 2.0kv/cm, 2.1kv/cm each 10 and 20ug duration Nuclear transfer embryo were activeted with a combination of 5ug/ml and 1.9mM 6-DMAP (4min, 4h). Fusion rate was 51∼68% range among fusion condition (1.9, 2.0, 2.1kv/cm; 10, 20us). But, cytoplasm lysis rate was increased by higher electric condition (0∼51.8% range). Each parameter's cleavage and blastocyst formation rate were 1.9kv/cm for 10us (75.8 and 19.5%), 20us (69.8 and 48.6%), 2.0kv/cm for 10us (76.9 and 20.0%), 20 us (68.5 and 40.9%), 2.1kv/cm for 10us (70.5 and 44.2%), 20 us (68.5 and 27.0%). We compared the effectiveness of cloning for between brindle coated cow's ear cells and Hanwoo fetal fibroblast cells. There was no significant differences in the fusion rate and developmental rate to the blastocyst stage. After transfer of blastocysts derived from nuclear transfer embryos, pregnancy rates of the Hanwoo fetal fibroblast cells and brindle coated cow's ear cells were checked pregnant on day 60 as assessed by ultrasonography, 40% (2/5) and 15.8% (3/19), respectively. This studies conclude that brindle coated cow's ear cells have the developmental potentiality to term by nuclear transfer. These results demonstrate that the increased the field strength was to be profitable for development of blastocyst or reduce of cytoplasm's damage than increasing the pulse duration.

• Development and Reproduction
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• v.9 no.1
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• pp.49-52
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• 2005

The effects of cytochalasin B was studied for the cleavage and development of in vitro matured porcine follicular oocytes. The follicular oocytes were collected from slaughtered pig ovaries and matured for 65 hours. The matured oocytes were activated by 7% ethanol(v/v) in DPBS and the activated oocytes were subjected to cytochalasin B concentrations of 2.5, 5.0 and $7.5\;{\mu}g/mL$ for 3, 5 and 7 hours, and then the treated oocytes were cultured in NCSU23 with 0.4% BSA for 7 days. The cleavage rates were not different significantly in each treatment. However, the oocytes treated with $5.0\;{\mu}g/mL$ for 5 hours yielded a significantly higher morula rate(19.7%) than oocytes treated with $2.5\;{\mu}g/mL$ for 3 and 5 hours(9.4%). The sum rate of $2.5\;{\mu}g/mL$ concentration(10.5%) by hour was also significantly lower than those of 5.0(18.0%) and $7.5\;{\mu}g/mL$ concentration(14.6%). The blastocyst rate in oocytes treated with $5.0\;{\mu}g/mL$ for 3(9.4%) and 5 hours(9.0%) was significantly higher than the rate in oocytes treated with $2.5\;{\mu}g/mL$ for 3 hours(0%). The sum rate of $5.0\;{\mu}g/mL$ concentration also significantly higher than those of 2.5 and $7.5\;{\mu}g/mL$ concentration. The results demonstrated that the treatment of oocytes with cytochalasin B of $5.0\;{\mu}g/mL$ for $3{\sim}5$ hours was the optimal concentration and duration for parthenogenetic activation and blastocyst formation of in vitro matured porcine oocytes.

• Journal of Embryo Transfer
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• v.22 no.1
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• pp.1-7
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• 2007

This study was conducted to examine the role of intact cumulus cells during in vitro fertilization (IVF) on sperm penetration, male pronuclear (MPN) formation and subsequent embryo development of oocytes matured and fertilized in vitro. Cumulus-oocyte complexes obtained from the slaughtered gilt ovaries were matured for 44 h in TCM199 containing 10% porcine follicular fluid, epidermal growth factor and hormones. After maturation culture, denuded oocytes or oocytes with intact cumulus cells were coincubated with frozen-thawed boar semen for 8h in a modified tris-buffered medium containing 5mM caffeine and 10mM calcium chloride. Putative zygotes were fixed and examined for sperm penetration and MPN formation (Experiments $1{\sim}3$), or cultured in North Carolina State University-23 medium fo. 156 h (Experiment 3). In Experiment 1, sperm penetration was examined after insemination of denuded oocytes and oocytes with intact cumulus cells at the concentration of $7.5{\times}10^5$ sperm/ml. Optimal sperm concentration for IVF of cumulus-intact oocytes was determined in Experiment 2 by inseminating intact oocytes with $2{\sim}5{\times}10^6$ sperm/ml. In Experiment 3, denuded or intact oocytes were inseminated at the concentrations of $7.5{\times}10^5$ and $4.0{\times}10^6$ sperm/ml, respectively, and in vitro embryo development was compared. Sperm penetration was significantly (p<0.01) decreased in cumulus-intact oocytes compared to denuded oocytes (35.2% vs. 77.4%). Based on the rates of sperm penetration and normal fertilization, the concentration of $4.0{\times}10^6$ sperm/ml was optimal for the IVF of intact oocytes compared to other sperm concentrations. The presence of intact cumulus cells during IVF significantly (p<0.05) improved embryo cleavage (48.8% vs. 58.9%), blastocyst (BL) formation (11.0% vs. 22.8%) and embryo cell number $(22{\pm}2\;vs.\;29{\pm}2\;cells)$ compared to denuded oocytes. In conclusion, these results suggest that intact cumulus cells during IVF inhibit sperm penetration but improve embryo cleavage, BL formation and embryo cell number of porcine embryos produced in vitro.

• Korean Journal of Animal Reproduction
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• v.24 no.1
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• pp.69-76
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• 2000

This study was carried out to improve of effective culture system on development of IVM/IVF/IVC bovine embryos. The cumulus-oocyte-complexes (COCs) collected from Korean cattle ovaries harvested at a local abattoir were matured in 50 ${mu}ell$ of TCM199 supplemented with 10% fetal bovine serum (FBS) and hormones (35 $\mu\textrm{g}$/$m\ell$ FSH, 10 $\mu\textrm{g}$/$m\ell$ LH, 1$\mu\textrm{g}$/$m\ell$ estradiol 17 $\beta$ under paraffin oil at 39$^{\circ}C$ in a humidified atmosphere of 5% $CO_2$in air. At 24 hrs after culture, matured oocytes were fertilized in vitro for 22~24 hrs with motile semen in which obtained by centrifugation of a frozen thawed semen on Percoll-density gradients (45% vs. 90%) at 500 g for 20 min. The presumptive zygotes were divided into three experimental groups. Single egg (Group 1), 25 (Group 2) or 50 eggs (Group 3) were cultured on cumulus cell in 50 ${mu}ell$ TCM199 supplement with 10% FBS for 6~9 days after fertilization. In vitro developmental rates into the blastocysts in the groups 2 and 3 were significantly (P＜0.05) higher than those of group 1 (37,27 vs. 6%, respectively). Cell number of blastocysts obtained in groups 2 and 3 at day 8 were significantly (P${mu}ell$) resulted in higher developmental competence and cell number of bovine blastocysts produced in vitro than those the culture of single embryos with cumulus cells.

• Journal of Embryo Transfer
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• v.30 no.1
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• pp.7-15
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• 2015

The objective of this study was to investigate the efficiency of nicotinic acid on sperm cryosurvival and fertilization ability in frozen-thawed boar semen. Boar semen was collected by glove-hand method and was frozen using freezing solution treated to 0, 5, 10 and 20 mM of nicotinic acid. The frozen sperm for sperm characteristic analysis was thawed such as viability, acrosome reaction, and mitochondrial integrity. The frozen-thawed sperm was estimated by SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction and Rhodamine123/PI double staining for mitochondrial integrity using a flow cytometry. The embryo was estimated in vitro development and DCFDA staining for reactive oxygen species assessment. As results, frozen-thawed sperm viability was significantly higher in 5 and 10 mM ($61.1{\pm}1.5%$,$64.7{\pm}2.0%$) of nicotinic acid than other groups (0 mM, $52.1{\pm}2.3%$; 20 mM, $47.8{\pm}5.1%$, P<0.05). The live sperm with acrosome reaction was significantly higher in 5 and 10 mM of nicotinic acid ($26.1{\pm}1.8%$, $24.9{\pm}1.5%$) than other groups (0 mM, $35.3{\pm}0.8%$; 20 mM, $36.5{\pm}1.9%$, P<0.05). The live sperm with mitochondrial integrity was significantly higher in 5 and 10 mM ($84.2{\pm}3.6%$, $88.4{\pm}2.3%$) of nicotinic acid than other groups (0 mM, $77.3{\pm}4.4%$; 20 mM, $73.3{\pm}3.6%$, P<0.05). Blastocyst rate of in vitro development was significantly higher in 10 mM ($17.0{\pm}1.3%$) of nicotinic acid than other groups (0 mM, $9.4{\pm}0.5%$; 5mM, $12.6{\pm}0.8%$; 20 mM, $5.0{\pm}1.0%$, P<0.05). Moreover, total cell number was higher in 5 and 10 mM ($53.6{\pm}2.9%$, $57.9{\pm}2.8%$) of nicotinic acid than other groups (0 mM, $41.0{\pm}1.4%$; 20 mM, $23.2{\pm}2.8%$, P<0.05). Hydrogen peroxide in embryos was lower in 5 mM nicotinic acid ($0.7{\pm}0.1%$) than other groups (0 mM, $1.0{\pm}0.1%$; 10mM, $0.9{\pm}0.0%$; 20 mM, $1.4{\pm}1.0%$, P<0.05). In conclusion, nicotinic acid-treated semen improves cryosurvival and quality of spermatozoa. Also, the fertilized oocytes with nicotinic acid improve quality of embryo and blastocyst formation.

• Journal of Embryo Transfer
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• v.25 no.1
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• pp.15-20
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• 2010

This study was performed to identify the optimal timing for oocyte donor replacement during OPU procedure. OPU was carried out to collect oocytes from every donor at an interval of $3{\sim}4$ days (2 times a week). The collected oocytes were matured in vitro in TCM-199 supplemented with 10% FBS, 10 mg/ml of FSH and 1 mg/ml of estradiol for 24 h. After 24 h of exposure to sperm, the presumptive zygotes were cultured in CR1aa medium supplemented with 4 mg/ml of BSA for 3 days before being changed to CR1aa medium with 10% of FBS for another $3{\sim}4$ days. The mean numbers of retrieved oocytes were remained constantly up to 3 months ($6.0{\pm}0.5$, $6.2{\pm}0.7$, $5.2{\pm}0.6$), but significantly decreased at over 4 to 6 months ($3.7{\pm}0.5$, $2.8{\pm}0.4$, $1.2{\pm}0.2$) (p<0.05). The blastocyst development potential was also very similar rate from 1 to 3 months (37.2%, 40.4% and 44.6%), but significantly decreased from 4 to 6 months (24.8%, 29.3% and 28.6%, respectively) (p<0.05). The production of OPU derived embryos in periods of 1 to 3 months ($2.2{\pm}0.3$, $2.5{\pm}0.3$ and $2.3{\pm}0.4$) were significantly higher than those in 4 to 6 months ($0.9{\pm}0.2$, $0.8{\pm}0.2$ and $0.3{\pm}0.2$, respectively) (p<0.05). In conclusion, the efficient periods for the production of OPU derived embryos was until 4 months, twice per week to produce over 64 transferable embryos and then replace new donor after 3 months use. The best replacement time is 3 months and could be maximized production of OPU derived embryos.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.31 no.4
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• pp.599-607
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• 1998

To clarify annual reproductive cycle of Korean dark sleeper, Odontobutis platycephala (Iwata et Jeon), we examined the seasonal changes of gonadosomatic index (GSI), the proportional frequency of oocyte development stages in the ovary and the changes of sex steroid hormone levels in blood from December 1995 to November 1997. In July and August, GSI was 0.35 to 0.72 and most oocytes in the ovary were chromatin-nucleolus stage and perinucleolar stage (proportional frequency: $87\%\~96\%$). In September, GSI was 1.20 $\pm$ 0.12, some oocytes in the ovary were yolk vesifle stage (proportional frequency: $22.8\%$) and vitellogenic stage which appeared very rarely(proportional frequency: $2.2\%$). GSI increased gradually from October and reached 4.59± 0.61 to December. During this period, oocytes of vitellogenic stage increased slightly (proportional frequency in December: $22.1\%$). In January, GSI was 4.32 $\pm$ 0.72 but the proportional frequency of oocytes in vitellogenic stage increased (proportional frequency: $51.2\%$). from February, GSI was increased sharply and reached to 10.51 $\pm$ 1.04 in March, the highest value throughout the year and the proportional frequency of oocytes in vitellogenic stage also reached the highest levels (proportional frequency: $60\%$). From April, GSI was gradually decreased and fell down to 1.11 $\pm$ 0.35 in June. During this period, the proportional frequency of mature oocytes was the highest in April (proportional frequency of mature oocyte stage: $40\%$ in April, $12\%$ May, $5\%$ June) throughout the year, and atretic ovarian follicles were appeared. The blood level of estradiol-17$\beta$ ($E_2$), which stimulates the hepatic synthesis and secretion of vitellogenin, was $0.84{\pm}0.20\;ng/m{\ell}$ in August, and thereafter was not changed until December. from January, it increased sharply and reached the highest level of $ 2.85{\pm}0.35\;ng/m{\ell}$ in March throughout the year, but fell to $0.14{\pm}0.02\;ng/m{\ell}$ in July(P<0.05), 17$\alpha$-hydroxprogesterone(17$\alpha$-OHP) was the peak $13.37{\pm}0.52ng/m{\ell}$ in March, but no significant changes in other period(below $3ng/m{\ell}$, P<0.05). 17$\alpha$, 20$\beta$-dihydroxy-4-pregnen-3-one(17$\alpha$, 20$\beta$-P), which was known as the final maturation inducing hormone in teleost, was $0.74{\pm}0.09ng/m{\ell}$ in April and $0.54{\pm}0.07ng/m{\ell}$ in May, but no significant changes in other period (below $0.26\;ng/m{\ell}$, p<0.05). Taken together these results, the annual reproductive cycle of O. platycephala divided into 4 periods as follows: 1) ripe and spawning period from April to June, main spawning period was from April to May, 2) Resting period from July to August, 3) Growing period from September to December, 4) Maturing period from January to March. Moreover, It was showed that the changes of sex steroid hormone in blood played a important roles in the annual reproductive cycle of O. platycephala.

• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.425-435
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• 1998

To improve the efficiency of nuclear transplantation in bovine, in this study the development in vitro of nuclear transferred (NT) embryos was compared by different activation regimens of the enucleated oocytes. The effect of developmental stage and culture system of donor nuclei on fusion and development in vitro of NT embryos were also evaluated. Oocytes were collected from Hanwoo ovaries obtained from slaughterhouse and matured in Ham's F-10 supplemented with hormones. After 20~22 h maturation, the oocytes were vortexed to be free from cumulus cells and subsequently their nucleus and the first polar body were removed. Enucleated oocytes were divided into 3 groups for activation; the oocytes of group I were activated with ionomycin for 5 min and subsequently incubated in 6-dimetylarninopurine (DMAP) for 4 h, Those of group II were treated with DMAP for 4 h at 39 h after onset of in vitro maturation (IVM) and those of group III were kept in room temperature ($25^{\circ}C$) for 3 h at 39 h after onset of IVM. After in vitro fertilization (IVF) the embryos for muclear donor were cultured either by group culture (20 embryos /50 ${mu}ell$ drop) or individually (1 embryo /50 ${mu}ell$ drop) for 4 day and 5 day. At day 4 and 5 after IVF, blastomeres were separated in calcium-magnesium free medium, and then classified into small (day 5: $\leq$ 38 ${\mu}{\textrm}{m}$, day 4: $\leq$ 46 ${\mu}{\textrm}{m}$) and large (day 5 : $\geq$ 38 ${\mu}{\textrm}{m}$, day 4 ; $\geq$ 46 ${\mu}{\textrm}{m}$). The separated blastomeres were replaced into enucleated and activated recipient cytoplasm. The blastomere-oocyte complexes were fused by electrically. The NT embryos were cultured in TCM-199 containing 10% FCS in 39$^{\circ}C$, 5% $CO_2$ incubator for 7 day. The results obtained were summarized as follows; There were no differences in fusion and development to blastocyst between groups as group I (68%, 10%), group II (75%, 14%) and group III (73%, 9%), respectively. However, the cell number in blastocyst of NT embryos in group III were significantly fewer than in the other groups (P＜0.05). No differences in fusion and development to blastocyst were found between individual or group cultured and between small or large blastomeres of day 4 and day 5 donor embryos. From these results, it was concluded that the combination of ionomycin and DMAP, or treatment of DMAP at 39 h after onset of IVM were useful for the efficient of production of NT bovine embryos, and the individual cultured embryos could be simply used as donor nuclei for NT bovine embryo.