• Reproductive and Developmental Biology
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• v.35 no.2
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• pp.137-141
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• 2011

These study was to investigate the in vitro fertilization and viability of fresh and vitrified oocytes. Also, the developmental capacity of IVF and intracytoplasmic sperm injection (ICSI) oocytes were investigated. Then vitrification was performed with the use of 20% ethylene glycol + 20% DMSO + 0.5 M sucrose + 10% FCS + TCM-199 medium. Vitrification immature oocytes are cultured in vitrification solution for 10 min afterwards transferred to expose at room temperature for 5 min. and transferred to the ice water for 5 min. The oocytes were sealed in a 1.0 mm straw and placed in a $LN_2$ container. Frozen oocytes were rapidly thawed in a water bath at $30{\sim}35^{\circ}C$, and then placed in TCM-199 medium containing 0.5 M sucrose for 5 min each, respectively, at $38^{\circ}C$. After being washed for 2~3 times, using fresh medium the oocytes were cultured in TCM-l99 medium supplemented with 5% FCS at $38^{\circ}C$ in 5% $CO_2$ and air. The normal morphology of fresh and vitrified-thawed oocytes were $87.1{\pm}2.1%$ and $54.8{\pm}2.5%$, respectively. The viability rates of fresh and vitrified-thawed oocytes were $70.0{\pm}2.2%$ and $41.9{\pm}2.6%$, respectively. Viability rates of vitrified-thawed oocytes were lower than that of fresh follicular oocytes (p<0.05). The in vitro maturation rates of fresh and vitrified oocytes were $45.1{\pm}3.6%$ and $28.9{\pm}4.4%$, respectively. The IVF rates of fresh follicular and vitrified-thawed oocytes were 34.00.2% and $20.2{\pm}2.6%$, respectively. The in vitro maturation and fertilization rates of vitrified-thawed oocytes were lower than those of the fresh follicular oocytes (p<0.05). A total of 350 oocytes were fixed and stained after co-incubation with spermatozoa, of which 88 had identifiable nuclear material. After IVF for 20 hrs, $25.1{\pm}3.4%$ of the oocytes found to have been penetrated by spermatozoas. Oocytes were fixed and stained after ICSI, and 105 oocytes contained identifiable nuclear material. After IVF and ICSI for 20 hrs, $34.3{\pm}3.4%$ and $59.0{\pm}2.0%$ of the oocytes were found to have been penetrated by spermatozoas. The developmental rates upon ICSI were significantly higher than those of the IVF method (p<0.05).

• Korean Journal of Animal Reproduction
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• v.22 no.2
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• pp.187-194
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• 1998

In order to determine suitable conditions for rapid freezing of porcine embryos, the kind and concentration of cryoprotectants, sucrose concentrations, equilibration time and thawing temperature in freezing medium were examined in relation to the survival of frozen-thawed oocyte and embryos. The results obtained are as follows : 1. The suitable concentrations of cryoprotoctant in the freezing medium which consisted of TCM-199+20% FCS were 1.5M for glycerol, 2.0M for DMSO, 2.5M for ethylene glycol, and 2.0M for propanediol. The sucrose concentration of 0.25M in the medium was found to optimal because the survival rate was markedly higher at this concentration when compared to the others. The survival rate was relatively high when the frozen embryos were thawed at 30$^{\circ}C$ in the freezing medium containing 2.5M cryoprotectants. The equilibration periods of 2.0 and 5.0 minutes revealed the higher survival in the media containing 1.5 or 2.1M glycerol when compared to 10 and 15 minutes. 2. The fertilization rates of frozen-thawed follicular oocytes which matured in vitro for 1, 12, 24 and 48 hours were 6.7~26.7% depending on the maturation time, and the rates were relatively high for those matured for a short period of time. The survival rates of frozen-thawed oocytes which matured in vitro for certain periods and fertilized were 10.0~30.0% depending on the maturation time.

• Korean Journal of Plant Resources
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• v.25 no.2
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• pp.285-292
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• 2012

Self-incompatibility (SI) system is a genetic barrier that prevents self-fertilization and promotes cross-pollination among different S genotypes. In many of these species, SI is controlled by a single genetic locus known as S locus, which prevents the fertilization by pollen with same locus. S RNases are the products of the S-locus expressed in the stylar tissue of Fuji Apple with gametophytic self-incompatibility system. This study investigated the various types of chemicals in order to select more effective inhibitors and activators. The effect on the inhibition of S RNase of Fuji apples was investigated $in$ $vitro$. The result showed that the enzyme activity was reduced 24.3% by Iron(II) Sulfate, significantly. $In$ $vitro$ studies of pollen growth tube showed that pollen tube growth had a higher germination rate (90%) in 10% Sucrose than in 2% sucrose extension medium. Data on the fruit set of apples treated with inhibitor and activator. Double application of $A^+$(Apple Plus, ISTECH Co. Ltd.,)+Vitamin B6 had the highest central fruit set as 86.1%(Andong). One time application of $A^{++}$Vitamin B1 in Yeongju obtained the highest central fruit set (91.9%).

• Journal of Embryo Transfer
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• v.31 no.4
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• pp.367-374
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• 2016

This study was conducted to evaluate the effect of transfer temperature of epididymis on survival rate of semen and development ability of B6D2F1 mice embryos. No significant differences were noted in the survival rate of semen ($59.0%{\pm}0.1$ vs. $47.6%{\pm}0.1$), in vitro fertilization rate ($90.7%{\pm}0.1$ vs. $90.7%{\pm}0.1$), developmental rate ($90.0%{\pm}0.1$ vs. $90.0%{\pm}0.1$), and blastocysts formation rate ($53.1%{\pm}0.2$ vs. $52.3%{\pm}0.2$) between groups. (NS; P>0.05). However, the zona hatched rate was significantly higher in the $4^{\circ}C$ group compared to those of the $37^{\circ}C$ group ($47.8%{\pm}0.1$ vs. $25.6%{\pm}0.2$; p<0.05). When it comes to cell numbers of blastocysts, the % ICM (/total cells) was significantly higher in the group of $4^{\circ}C$ compared to the $37^{\circ}C$ ($27.0%{\pm}0.1$ vs. $18.3%{\pm}0.1$; p<0.05). However there were no differences in total cell numbers ($72.7{\pm}31.6$ vs. $62.0{\pm}36.6$), ICM cell numbers ($17.0{\pm}7.8$ vs. $14.6{\pm}8.6$), TE cell numbers ($55.8{\pm}29.8$ vs. $64.0{\pm}24.4$), the ratio of ICM:TE ($1:4.2{\pm}4.1$ vs. $1:6.4{\pm}7.2$) between two groups (NS; P>0.05). Taken altogether, it is expected to achieve the best developmental ability of B6D2F1 mice embryos in the transfer temperature of epididymis. Also these results can provide fundamental data to maximize culture condition for in vitro fertilization on B6D2F1 mice. In future, therefore, it is expected that results herein might be applied for in vitro culture of human embryos.

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.53-57
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• 2004

The study was carried out to investigate the effects of morphology, reproductive cycle, incubation time and activation of oocytes on in vitro maturation of cat oocytes and development of IVM/IVF embryos. The results were summarized as follows: 1. When recovered from ovaries collected at different stages of the reproductive cycle (inactive, follicular and luteal stage), the developmental rates of oocytes to GV and MI stage were 72.5% and 27.5%, 57.5% and 7.5%, 62.5% and 17.5%, respectively. 2. The developmental rates of oocytes with cumulus cells to GV and MI stage in different conditions of incubation (5% $CO_2$ , 95% $O_2$ and 10% $CO_2$, 90% $O_2$) were 70.0% and 27.5%, 52.5% and 20.0%, 55.0% and 12.5%, respectively. 3. The developmental rates to GV and MI oocytes when cultured at different time of incubation (17∼20, 21∼24, 25∼28 and 29∼32 h) were 67.5% and 20.0%, 67.5% and 30.0%, 62.5% and 22.5%, 65.0% and 15.0%, respectively. 4. The fertilization and cleavage rates of freshly collected oocytes with and without cumulus cells were 72.5% and 25.0%, 37.5% and 7.5%, respectively. The rates were greater in oocytes with cumulus cells than those without cumulus cells. 5. The fertilization and cleavage rates of oocytes recovered from ovaries collected at different stages of the reproductive cycle (inactive, follicular and luteal stage) were 75.0% and 25.0%, 40.0% and 7.5%, 50.0% and 15.0%, respectively.

• 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.

• Journal of Embryo Transfer
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• v.27 no.1
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• pp.29-35
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• 2012

The purpose of this study was to examine the effect of growing stages of the Korean Native Striped Bull (KNSB) on the freezability and fertility of frozen-thawed semen. First, we investigated the total motility (TM) and progressive motility (PM) according to the diluent used for semen freezing. Second, we examined the effect of the age of KNSB on semen volume, TM and PM of fresh and frozen-thawed semen. Third, we examined the effect of frozen semen from the different age of KNSB on the $in-vitro$ fertilization rate, and the artificial insemination pregnancy rate. The diluents used in this experiment were Triladyl$^{(R)}$ and Tris-egg yolk extender (EYE). Semen was collected from 5 KNSB in the growing stage (15 months) and 5 adult KNSB (36 months). When Triladyl or Tris-EYE extender was used for semen freezing, there was no difference of the mean TM and the mean PM. However, the mean TM was significantly higher in Bull No. 1885 than Bull No. 4283 ($p$ <0.05). The mean volume of semen collected from the 15-month-old bulls (2.3 ml) was significantly lower ($p$ <0.05) than that from the 36-month-old bulls (5.0 ml). The mean semen concentration was similar for the 15-month-old ($2.1{\times}10^9$ spermatozoa/ml) and 36-month-old ($1.8{\times}10^9$ spermatozoa/ml) bulls. For the 15-month-old and 36-month-old bulls, the mean TM of fresh semen were 93.7% and 88.3%, respectively, and the mean PM were 97.0% and 88.3%, respectively; the 15-month-old bulls showed a particularly high PM ($p$ <0.05). For the 15-month-old and 36-month-old bulls, the mean TM (56.0% and 58.0%, respectively) and the mean PM (64.0% and 70.7%, respectively) of frozen-thawed semen did not differ. The development rates of embryos after $in-vitro$ fertilization and the pregnancy rate after artificial insemination using frozen-thawed semen did not differ according to the bull's age. In summary, semen volume differed according to the bull's age, but semen concentration and survival rate, the $in-vitro$ fertilization rate, and the pregnancy rate did not differ according to the stripe bull's age. Accordingly, semen from bulls in the growing stage can be collected and frozen for the preservation and multiplication of rare livestock.

• The Journal of Korean Obstetrics and Gynecology
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• v.31 no.2
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• pp.103-112
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• 2018

Objectives: The aim of this paper is to report that herb medicine effects on four infertility patients with ovarian cysts and the menstrual irregularity as a result of ovarian hyperstimulation syndrome. Methods: Four infertility patients with ovarian cysts had the same symptom of menstrual irregularity for preparing In Vitro Fertilization (IVF). They were treated by twice a day herb medication Changbudodam-tang. Then we observed the effects of treatments by regularity of menstruation, checking with ultrasonography of ovarian cyst size. Results: After herb treatments, the sizes of ovarian cysts decreased and one of them disappeared. Above all, menstrual regularity was recovered for preparing IVF. So they could have a positive progress of IVF. Conclusions: These cases show that herb medicine has its effective treatments for the implantational surroundings on infertility patients with ovarian cyst and menstrual irregularity because of ovarian hyperstimulation syndrome.

• Proceedings of the KSAR Conference
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• 2001.10a
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• pp.48-49
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• 2001

While transgenic manipulation in mice have been very successful the same is not true for cattle and pigs. The inability to isolate ES cells from the bovine and porcine has precluded the utilization of the gene targeting technology in these species. Fortunately new advances in cloning by nuclear transfer have opened up a unique opportunity to undertake precise genetic modification in cattle and pigs. The ability of a number of different laboratory groups to successfully clone cattle is due to numerous research programs focused on nuclear transfer in cattle, and the enormous base of knowledge developed over the last 20 years involving the application of assisted reproductive techniques in cattle. Successful and repeatable procedures for in vitro oocyte maturation, in vitro fertilization, and in vitro embryo culture are now well established for cattle. In our laboratory we have utilized nuclear transfer to reproduce the genotypes of several animals, selected for cloning based on their inherent genetic value. Results that we have obtained to date are similar to those reported by other laboratories. (omitted)

• Clinical and Experimental Reproductive Medicine
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• v.26 no.3
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• pp.447-456
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• 1999

Objective: The purpose of this study was to evaluate outcome of intracytoplasmic sperm injection (ICSI) using epididymal and testicular sperm in patients with azoospermia. Methods: From March, 1993 to May, 1999, a retrospective clinical analysis was done of a total of 140 cycles in 112 patients who underwent ICSI. Subjects were divided into three groups: ejaculated-ICSI group included 42 cycles in 34 patients with ejaculated sperm who underwent ICSI due to severe oligospermia and past history of failed or poor fertilization in the previous in vitro fertilization and embryo tranfer (IVF-ET) cycles, microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection (MESA-ICSI) group included 50 cycles in 42 patients with congenital absence of the vas deferens (CAVD) or unreconstructable obstructive azoospermia and testicular sperm extraction and intracytoplasmic sperm injection (TESE-ICSI) group included 48 cycles in 36 patients with no spermatozoa which can be retrieved from epididymis or non-obstructive azoospermia. Results: Normal two-pronuclear fertilization rates were similar in three groups: 64.4% for ejaculated-ICSI group, 59.4% for MESA-ICSI group and 60.4% for TESE-ICSI group. The pregnancy rates were 26.2%, 26.0% and 25.0% respectively. There were no significant differences in the fertilization, cleavage, and clinical pregnancy rates among ICSI cycles using ejaculated, epididymal and testicular sperm. Conclusion: Epididymal and testicular sperm obtained in azoospermic patients can fertilize oocyte successfully and may lead to be similar fertilization rates and clinical pregnancy rates to ejaculated sperm.