• Korean Journal of Animal Reproduction
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• v.19 no.2
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• pp.129-134
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• 1995

This study was carried out to investigate on the survival rate and in vitro developmental rate of bisected porcine embryos and immature oocytes by manipulator and micropipette. Bisected embryos and immature oocytes cultured for 1∼5 days in TCM 199 medium with 20% FCS. Survival rate was defined as development rate on in vitro culture or FDA-test. The results are summarized as follows ; 1. The survival rate of bisected porcine embryos and oocytes were 26.1%, 22.7%, respectively. The survival rate of bisected embryos and oocytes was significantly lower than that of non-bisection embryos(62.5%). 2. The survival rate of bisected porcine embryos in cultured for 12, 24, 48, 72 hrs with 20% FCS＋TCM-199 medium were 26.9%, 19.2%, 19.2% and 11.5%, respectively. 3. The in-vitro developmental rate with and without zona pellucida of bisected porcine embryos by micromanipulator were 30.8%, 25.0%, respectively.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.473-477
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• 1997

This research was performed to investigate the developmental ability of mouse oocytes following cold-culture(4$^{\circ}C$) in vitro. When the oocytes were fertilized after 10 hour cold-culture in D-PBS or Ham's F10 with 0.3% BSA, the cleavage rage of the oocytes was not different in the rate of oocytes fertilized in vitro without cold-culture(126/189, 66.2% vs 88/133, 66.7%) and also the rate of embryos developed to blastocyst did not(123/189, 65.1% vs 82/133). But, when the time of the cold-cultre was extended from 10 to 24 hours, the rate of embryos developed to blastocyst was slightly decreased(73.5% vs 52.2%). However, when the oocytes were cultured for 10 and 24hours at 37$^{\circ}C$, the rate of oocytes developed to blastocyst was significantly decreased than that of oocytes following cold-culture. By the results of this study, it'll be possible to utilize effectively the cold-culture of the oocytes when in vitro fertilization is delayed.

• Journal of Embryo Transfer
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• v.24 no.2
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• pp.121-125
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• 2009

In the present study, effects of concentration of cryoprotectant solutions on the nuclear maturation of vitrifiedthawed bovine oocytes were examined. Also, the developmental capacity of vitrified-thawed immature oocytes following ICSI was investigated. Oocytes were cultured in TCM-199 medium supplemented with 5% FBS at $38^{\circ}$C in 5% $CO_2$ and air. The in vitro maturation rate of vitrified oocytes was 24.5 ${\pm}$ 4.2%. The in vitro maturation rate of vitrified oocytes was lower than that of the control (72.0 ${\pm}$ 3.5%, p<0.05). The in vitro maturation rate of vitrified${\sim}$thawed oocytes incubated in TCM-199 medium supplemented with 1.0${\sim}$5.0 ug CB were 26.7 ${\pm}$ 3.2%, 35.7 ${\pm}$ 3.2%, 54.0 ${\pm}$ 3.0%, 42.5 ${\pm}$ 3.6%, respectively. The in vitro maturation rate (57.0 ${\pm}$ 3.0%) of the vitrified-thawed oocytes treated with 3.0 ${\mu}$g CB for 20 min was the highest of all vitrification groups, although the maturation rate were significantly (p<0.05) lower than those of fresh oocytes. The in vitro maturation rates of the vitrified-thawed (with EDS and EDT) oocytes were 53.8 ${\pm}$ 3.4%, 51.1 ${\pm}$ 3.5%, respectively. This results were lower than the control group (72.0 ${\pm}$ 3.0%). The in vitro developmental rates of the vitrified-thawed oocytes following ICSI were 28.6 ${\pm}$ 4.5%, 25.6 ${\pm}$ 4.3%, respectively. This results were lower than the control group (40.0 ${\pm}$ 4.0%).

• Journal of Embryo Transfer
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• v.24 no.2
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• pp.115-119
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• 2009

In the present study, effects of concentration of cryoprotectant solutions on the nuclear maturation of vitrifiedthawed porcine oocytes were examined. Oocytes were cultured in TCM-199 medium supplemented with 5% FBS at $38^{\circ}$C in 5% $C0_2$ and air. The percentage of monospermy in the toxicity group and vitrification group (22.0 ${\pm}$ 3.0% and 31.5 ${\pm}$ 3.5%) was decreased compared with that of the control group (44.0 ${\pm}$ 4.0%). The percentage of in vitro development to blastocyst in the toxicity group and vitrification group (12.0 ${\pm}$ 2.5% and 14.8 ${\pm}$ 2.8%) was decreased compared with that of the control group (28.0 ${\pm}$ 3.0%, p<0.05). The survival and in vitro developmental rate of oocytes vitrification-thawed with EDS and EDT + TCM-199 medium supplemented with 0.1% PVA were 46.3 ${\pm}$ 3.0%, 54.5 ${\pm}$ 3.8% and 14.8 ${\pm}$ 2.5%, 16.4 ${\pm}$ 2.7%, respectively. This results were lower than the control group (28.0 ${\pm}$ 3.5%). The in vitro developmental rate of embryos vitrified with EDS and EDT supplemented PVA did not have a significant difference. The survival and in vitro developmental rate of vitrified-thawed morula and blastocyst embryos were 44.2 ${\pm}$ 3.5%, 17.3 ${\pm}$ 3.0% and 48.1 ${\pm}$ 4.2%, 18.5 ${\pm}$ 3.5%, respectively. Vitrified morulae and blastcyst embryos had a lower survival and developmental rates than their control counterparts.

• Journal of Embryo Transfer
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• v.22 no.4
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• pp.251-255
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• 2007

This study was carried out to examine on developmental competence of Hanwoo embryos cultured in vitro according to culture conditions and freezing methods. The in vitro developmental competence to blastocyst stage at Day 8 of culture in SOF was significantly (p<0.05) higher than that in CR1aa (30.3% vs. 18.4%). The in vitro developmental rate of morula and blastocysts cultured in group culture was significantly (p<0.05) higher than that in individual culture (41.4% and 36.0% vs. 21.1% and 10.5%, respectively). The cell number of Day 8 blastocysts in group culture was significantly (p<0.05) higher than that in the individual culture ($120.1{\pm}12.8\;vs.\;94.1{\pm}12.1$, respectively). The survival rates of frozen-thawed balstocysts that were exposed in 1.5 M ethylene glycol or 1.5 M ethylene glycol containing 0.1 M sucrose were 77.5% and 78.7%, respectively. The survival rates of blastocysts cultured for 48 h in slow freezing and vitrification was not significantly different (73.3 and 74.0%). In conclusion, in vitro developmental competence of bovine embryos was influenced on the culture medium (SOF) and culture method (Group culture). Survival rate of frozen-thawed of bovine embryos was not influenced on freezing solutions and freezing methods.

• Journal of Embryo Transfer
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• v.8 no.2
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• pp.91-95
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• 1993

The studies on the carried out to investigate the effects of co-culture with uterine fluids and uterine epithelial cells on the in-vitro fertilization and developmental rate of porcine follicular oocytes. The ovaries were obtained from slaughtered swine. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluids from the visible follicles of diameter 3~5 mm. The follicular oocytes were cultured in TCM-199 medium containing hormones and 10% ECS for 46~48 hrs in a incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 12~18 hrs with motile capacitated sperm by preincubation of heparin. The results obtained in these experiments were summarized as follows ; 1.The in-vitro maturation and fertilization rate of porcine oocytes co-cultured with uterine fluids in TCM-199 medium were 68.0% arid 55.7%, the rates were higher than of control, 56.5% arid 38.7%. 2. When the in-vitro fertilized oocytes were co-cultured with porcine uterine epithelial cells, the fertilization rate was 60.3%, the rates were higher than that of control, 35.7%. 3. When the in-vitro fertilized oocytes were co-cultured with porcine uterine epithelial cells, the development rate to be blastocyst was 12.4%, the rates were higher than that of control, 9.2%(p<0.05).

• Reproductive and Developmental Biology
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• v.38 no.4
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• pp.143-146
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• 2014

Antioxidants, as reactive oxygen species scavengers, are one of the beneficial additives in serum-free defined culture medium. In this study, three separate experiments were performed to determine the effects of 3-hyroxyflavone added to the culture medium on the developmental competence of follicular bovine oocytes during in vitro maturation (IVM) and/or in vitro culture (IVC). The rate of blastocyst developed from oocytes cultured in IVM medium with 3-hyroxyflavone was significantly higher than that from control oocytes (39.0% vs. 26.3%, p<0.001), respectively. However, oocytes cultured in the medium with addition of 3-hyroxyflavone only at IVC period did not show significance in the blastocyst development when compared with control. When 3-hyroxyflavone was added to both IVM and IVC media, the rate of blastocyst formation was even significantly lower (21.1%) than control (26.5%; p<0.05). The present findings suggested that antioxidative activity of 3-hydroxyflavone added to only IVM medium beneficially affected the developmental competence of follicular bovine.

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.13-20
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• 2004

This study was conducted to investigate the developmental ability of caprine embryos after somatic cell interspecies nuclear transfer. Donor cells were obtained from an ear-skin biopsy of a caprine, digested with 0.25% trypsin-EDTA in PBS, and primary fibroblast cultures were established in TCM-199 with 10% FBS. After maturation, expanded cumulus cells were removed by vigorous pipetting in the presence of 0.3% hyaluronidase. The matured oocytes were dipped in D-PBS plus 10% FBS＋7.5 $\mu\textrm{g}$/ml cytochalasin B and 0.05 M sucrose. The reconstructed oocytes were electrically fused with donor cells in 0.3 M mannitol fusion medium. After the electofusion, embryos were activated by electric stimulation. Interspecies nuclear transfer embryos with bovine cytoplasts were cultured in TCM-199 medium supplemented with 10% FBS including bovine oviduct epithelial cells for 7∼9 day. On the other hand, the NT embryos with porcine cytoplasts were cultured in NCSU-23 medium supplemented with 10% FBS for 6∼8 day at $39^{\circ}C, 5% CO_2$ in air. In caprine-bovine NT embryos, the cleavage(2-cell) rate was 36.8% in confluence and 43.8% in serum starvation. The developmental rate of morula- and blastocyst-stage embryos was 0.0% in confluence and 18.8% in serum starvation. In caprine-porcine NT embryos, the cleavage(2-cell) rate was 76.7% in confluence and 66.7% in serum starvation. The developmental rate of morula and blastocyst stage embryos was 3.3% in confluence and 3.0% in serum starvation, and no significant difference was observed in synchronization treatment between donor cells. In caprine-bovine NT embryos, the cleavage(2-cell) rate of cultured donor cells was 30.8% and 17.6% in 5∼9 and 10∼14 passage(P＜0.05). The developmental rate of morula and blastocyst stage embryos were significantly higher(P＜0.05) in 5∼9 passage(23.1%) than in 10∼14 passage(0.0%) of cultured donor cells. In caprine-porcine NT embryos, the cleavage rate was significantly higher(P＜0.05) in 5∼9 passage(86.7%) than in 10∼14 passage(50.0%) of cultured donor cells. The developmental rate of morula and blastocyst stage embryos were 3.3 and 0.0% in 5∼9 and 10∼14와 passage of cultured donor cells. In caprine-bovine NT embryos, the developmental rate of morula and blastocyst stage embryos were 22.6% in interspecies nuclear transfer, 33.9% in in vitro fertilization and 28.1% in parthenotes, which was no significant differed. The developmental rate of morula and blastocyst stage embryos with caprine-porcine NT embryos were lower(P＜0.05) in interspecies nuclear transfer(5.1%) than in vitro fertiltzation(26.9%) and parthenotes(37.4%).

• Journal of Embryo Transfer
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• v.33 no.3
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• pp.169-175
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• 2018

It has been claimed that artificial insemination (AI) of cows with frozen-thawed semen treated with commercially produced kits, Wholemom (in favour of female gender) increases the birth chance of calves with desired sex ratio by approximately 85% without decrease of pregnancy rates. Hence, this study was conducted to investigate the efficacy of wholemom kits as combined with frozen-thawed bovine semen during in vitro fertilization on the in vitro fertilization and developmental efficiency and sex ratios such as some reproductive parameters in bovine. For this, 1,737 oocytes were in vitro fertilized and developed. Agglutination effects on bovine after treatment of Wholemom kit were observed by time passage and dose respectively. To determine sex of embryos, Bovine embryo Y-specific gene primers(ConEY) and Bovine specific universal primer(ConBV) were used as multiple PCR method. Fertilization rate of wholemom-treated group was significantly lower than its of control group[66.9% (1,156/1,737) in Wholemom-treated group; 75.0% (610/813) in control group]. However, developmental rate after fertilization of both wholemom-treated and control groups were not significantly different [26.1% (404/1,156) in Wholemom-treated group; 27.4% (224/610) in control group]. Sex ratio of in vitro fertilized embryo with frozen-thawed semen treated with wholemom kit was determined by multi PCR. Female ratio in wholemom-treated group [85.4% (173/201)] was significantly higher than its of control group [47.2% (66/141)]. In conclusion, wholemom treatments of semen used in the in vitro fertilization and development of bovine oocytes provided increase in female ratio with decrease of fertilization rate.

• Reproductive and Developmental Biology
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• v.35 no.2
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• pp.131-136
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• 2011

The in vitro maturation rate of vitrified-thawed canine oocytes was $30.8{\pm}3.4%$. The in vitro maturation rate of vitrified oocytes was lower than that of the control ($52.0{\pm}2.5%$, p<0.05). The in vitro maturation rate of vitrified-thawed oocytes were significantly (p<0.05) lower than those of fresh oocytes. The in vitro maturation and developmental rates of the vitrified-thawed oocytes were $17.5{\pm}2.5%$ and $8.8{\pm}3.4%$, respectively. This results were lower than the control group ($43.6{\pm}3.2%$ vs $20.0{\pm}3.0%$). SOD1 gene expression of 1~2 mm of follicle size were higher than those of above 6 mm follicle size. SOD2 gene expression of 1~2 mm of follicle size were significantly higher than those of above 6 mm follicle size (p<0.01). The expression pattern of SOD1, 2 was constantly expressed in both groups but strongly expressed in follicles (1~2 mm) group when compared to the above 6 mm follicles. SOD gene expression between groups the fresh and vitrified oocytes groups were significant differences in rates. However, RGS gene expression between groups the fresh and vitrified oocytes groups were no significant differences in rates.