• Journal of Embryo Transfer
• /
• v.9 no.1
• /
• pp.7-14
• /
• 1994

The present experiments on cryopreservation were designed to examine the effects of solution toxicity, equilibration time and cell stages on the post-thaw survival of bovine IVF embryos. The oocytes were matured in vitro(IVM) for 24 hrs. in TCM-199 supplemented with 35 $\mu$g /ml FSH, 10 $\mu$g /ml LH, 1 $\mu$g /ml estradiol-17$\beta$ and granulosa cells at 39$^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24 hrs., and then the zygotes were co-cultured in vitro(IVC) with bovine oviductal epithelial cells for 7 to 9 days. The bovine IVF embryos were exposed to the EFS solution in one step at room temperature, kept in the EFS solution during different period for toxicity test, vitrified in liquid nitrogen, and thawed rapidly. 1. after the bovine blastocysts were exposed to EFS solution for 2 min. at room temperature and then they were washed in 0.5 M sucrose solution and TCM-199, they were cultured to examined cryoprotectant induced injury during exposure, Most of the embryos(95.0%) developed to reexpanded blastocoels. However, when the exposure time was extended to 5 and 10 min, these development rates dropped dramatically in 5 min. (69.5%) and 10 min. (47.4%), respectively, 2. When the bovine IVF embryos were vitrified in EFS solution after the equilibration for 1 and 2 min. exposure, The embryos to have reexpanded blastocoels following thawing, washing and culture processes were found to he 82.6 and 73.9%, respectively. However, when the exposure time was extended to 3 min, this survival rate dropped to 18.2%. The optimal time for equilibration of bovine IVF blastocysts in EFS solution seemed to he 1~2 min. 3. When the bovine IVF embryos were equilibrated for 1 min. the significantly (P<0. 05) higher post-thaw survival rates were obtained from the embryos of blastocyst stage(81.3%) than morulae stage(5. 1%). The optimal cell stage for viterification with EFS solution proven to he blastocyst stage in bovine IVF embryos. 4. The number of blastomeres of blastocyst stage was examined with nuclear staining with Hoechst 33342 during 7 to 9 days post-insemination. The cell counts of frozen bovine IVF embryos were found significantly(P$\geq$7.5 and those of the fresh embryos 76.6$\geq$7. 1, which were cultured in the sarne period and conditions as frozen embryos.

• Journal of Embryo Transfer
• /
• v.30 no.3
• /
• pp.239-248
• /
• 2015

The nature of molecular mechanisms governing embryonic cell block is largely unknown, but recent reports have demonstrated that proper execution of programmed cell death is crucial for this process. The main objective of this study is to determine effects of programmed cell death on porcine oocytes development in vitro after parthenogenesis. Among the blastocysts matured in 3MA, MAP1LC3A and ATG5 RNA gene expression level increased in the order of Cyst < 3MA < RP. However, Casp-3 and TNF-r RNA gene expression level decreased in the order of RP < 3MA < Cyst. Expression of mTOR within the RP-cultured blastocyst was the most highly to the inner cell mass, while 3MA-cultured blastocyst showed very lowest expression in inner cell mass. The expression of mTOR showed a pattern opposite to that of MAP1LC3A. That is, its expression was the lowest in Cyst group. When the enzymatic activity of MMP-2 and MMP-9 was assessed in culture, the level of active MMP-9 was higher expression in the medium of each RP treatment group, with the level of another treatment group being relatively higher. Analyses of TIMP-2 and TIMP-3 revealed that their expression was higher in groups that did not receive RP treatment. More specifically, the level of TIMP-2 was not affected by Cyst treatment, while the level of TIMP-3 was higher in 3MA and RP treatment group. There was highly cell division activation efficiency of parthenogenesis on cultured system of RP supplement IVC medium. Therefore, these results suggest that embryo development was significantly increased in conditional culture medium with active autophagy as compared to common cultured condition. Further investigation of this distinction may enable the development of innovative improvements for the production of porcine somatic cell nuclear transfer.

• Journal of Chest Surgery
• /
• v.29 no.12
• /
• pp.1342-1346
• /
• 1996

A right thoracotomy was used for the reoperation or mitral valve of 15 patients who had previously undergone a cardiac operation through a median sternotomy. In our experience. this approach provided dn excellent exposure of the nlitral valve and easy cannulations of both cavie with minimal dissection, ilvoiding any damage of cardiac and major vessels during re-sternotomy Arterial cannulation was performed in the ascending aorta in 13 patients And in the femoral artery in 2 patients. In earlier cases, venous cannulation was done in the SVC And IVC through the right atrium and snared. In later cases, this could be done without snaginly of both cavae or by placing a silgle light-angled catheter into the right atrium. Crystalloid cardioplegic solution was infused for myocardial protection. Hypothermia was controlled at 20\ulcorner$25^{\circ}C$. For defibrillation, internal paddles were used In one patient while sterilized external paddles were used in 10 patients. In the remaining four patients. however. the heart beat spontaneously The respirator could be weaned within 48 hours alter the operation and no pulmonary complication was observed. One out of the 15 patients expired due to sudden attack of ventricular tarchycardid developed ten days after the operation, but the rest of the patients were discharged with good condition.

• Journal of Embryo Transfer
• /
• v.32 no.3
• /
• pp.201-207
• /
• 2017

To increase the productivity of in vitro development, the antioxidants have been used for culture system of bovine oocytes and embryos. However, comparative studies on these molecules are rare and direct beneficial effects on blastocyst production cannot be discriminated for best results. The study was conducted to determine the influence of N-acetyl-L-cysteine (NAC), N-acetyl-L-cysteine amide (NACA), glutathione (GSH) and cysteamime (CYS) on maturation competence of COCs from GV to MII stage and productivity of blastocyst formation during in vitro fertilization and culture. There was no difference among maturation rates of oocytes to metaphase II with polar body with antioxidants for any of the treatment groups (p>0.05). However, the significant improvement on the rate of blastocysts ($32.3{\pm}5.0%$) was found in 0.1 mM CYS treatment than 0.3 mM NAC, 0.2 mM NACA or 0.5mM GSH (p<0.05). The addition of NAC ($18.8{\pm}3.7%$) or NACA ($21.2{\pm}3.9%$) did not improve development competence to morula and blastocysts than control ($24.4{\pm}4.1%$) and GSH ($26.5{\pm}5.0%$) (p>0.05). Our study showed that medium supplementation with CYS during IVM and IVC improved the rate of bovine embryo development but not with NAC, NACA and GSH addition.

• Korean Journal of Heritage: History & Science
• /
• v.47 no.3
• /
• pp.60-85
• /
• 2014

The study is for tracing the mutational process, considering the transformational process of wooden chamber tombs in Gyoungju region during 2~6 century. In addition, class features and the meaning of tombs were studied. First of all, the wooden chamber tombs were classified into 10 different styles(1A~IVC), based on the filling materials, the ways of structure, and the structure of burial chamber. There were only one or two tombs that belongs to the highest classes 'Ga'~'Da' in each period, which has the numeral features. Furthermore, the styles of the central wooden chamber tombs according to the period and the region have distinctive features and have changed so far. It shows that there are the rank among the tombs in the regions. In other words, regarding the ranks of the class 'Ga'~'Cha', there were strict rules and the limitations on the size of the tombs and the number and the quality of the burial relics. It means that the inner-outer size or the structure of the tombs and the number, quality of the burial goods depended on the buried person's birth or social classes, or achievements. Based on Shila social classes, the class 'Ga' tombs were for the highest classes when considered the size, burial relics, and the quality. The class 'Na' and 'Da' tombs were for the higher classes, 'Ma' for middle classes, 'Ba', 'Sa' for lower classes. In addition, 'Ah', 'Ja' tombs were for higher commoner, 'Cha' for lower commoner. Thus, class 'Ga'~'Sa' is the leading class of Shila.

• Clinical and Experimental Reproductive Medicine
• /
• v.23 no.1
• /
• pp.25-32
• /
• 1996

This work has been carried out to examine the number of Total, ICM and TE cells of F1 mouse blastcysts at day 4 after IVF by differential labelling of the nuclei with polynucleotide-specific fluorochromes and to obtain a fundamental information of preimplantation mouse embryo development. Blastocysts produced by superovulated B6CBA F1(C57BL/${\times}$CBA) eggs were inseminated with $1{\times}10^6$spermatozoa/ml and cultured in M16 medium at $37^{\circ}C$, 5% $CO_2$ incubator for 95hrs. Blastocysts were classified as early, middle, expanded and hatching stage according to the developmental morphology; blastocoel expansion and zona thickness. The results obtained in these experiments were summarized as follows; 1) The development rate of blastocysts at 95hrs after IVF was 86.7% and classified blastocysts to early, middle, expanded and hatching were 16.3%, 18.9%, 10.5% and 40.9%, respectively. 2) The numbers of total blastomere using bisbenzimide in the classified blastocysts to early, middle, expanded and hatching were 35.6${\pm}$1O.4, 49.4${\pm}$8.6, 60.8${\pm}$1O.7 and 62.7${\pm}$13.9, respectively. 3) In ICM and TE cell number by using differential labelling with polynucleotide-specific fluorochrome in the classified blastocysts to early, middle, expanded and hatching; ICM numbers were 9.6${\pm}$3.0, 13.6${\pm}$3.9, 16.0${\pm}$3.3 and 19.5${\pm}$4.6, respectively and TE cell numbers were 30.6${\pm}$5.1, 39.9${\pm}$5.8, 42.2${\pm}$8.1 and 43.7${\pm}$11.1, respectively. These results showed the same increase pattern according to development advance level. Also, when compared with the results of total count were obtained between bisbenzimide only and differential labelling, both of them showed the same increase pattern according to development level and at the same time their cell numbers were almost the same. So, rapid and simple cell count method using differential labelling can be used for the examination of later preimplantation development or as an indicator of embryo quality according to the variables of culture conditions.

• Journal of Embryo Transfer
• /
• v.22 no.3
• /
• pp.155-160
• /
• 2007

During in vitro culture of mammalian oocytes and embryos, the cells are exposed to the risks that cause cell injury or death. Numerous studies have been reported that the cell injury may be induced by the action of free radicals generated by auto-oxidation. This study was undertaken to investigate the optimal culture condition system for in vitro culture of porcine embryos. We first evaluated the effect of culture media on the porcine embryo development. NCSU-23 and PZM-5, culture medium tested, were failed to produce significant difference on the rate of blastocyst formation. In NCSU-23, the developmental rate was slightly higher than that in PZM-5. During in vitro maturation (IVM), fertilizaton (IVF), and culture (IVC) under 5 or 20% oxygen ($O_2$), the rates of cleavage and development were insignificantly different from each other under our culture condition (20% $O_2$, in NCSU-23), the mean cell number per blastocyst was $40{\pm}10$. These results showed that medium and $O_2$ concentration had no significant effect on the development of porcine embryos.

• Journal of Chest Surgery
• /
• v.41 no.5
• /
• pp.591-597
• /
• 2008

Background: Pulmonary endarterectomy is widely accepted as a treatment for chronic thromboembolic pulmonary hypertension. Based on our experiences, we sought to find ways to reduce perioperative complications and to improve surgical outcomes in patients undergoing pulmonary endarterectomy. Material and Method: This study was designed as a retrospective analysis of 20 patients with pulmonary hypertension who underwent pulmonary endarterectomy between January 1998 and March 2008. All patients presented with chronic dyspnea. Deep vein thrombosis (DVT) was the major cause of chronic pulmonary thromboembolism (55%). Seventeen patients (85%) underwent inferior vena cava (IVC) filter placement. Thirteen patients underwent surgery under total circulatory arrest, while the others underwent surgery while on low flow cardiopulmonary bypass. Concomitant tricuspid annuloplasty was done in 6 patients (66%) whose tricuspid regurgitation was as severe as grade IV/IV. The mean follow-up duration was $45{\pm}32$ months. Result: Using of University of California, San Diego (UCSD), thromboembolism classification, 4 patients (20%) were type 1, 8 patients (40%) were type II, and 8 patients (40%) were type III. Right ventricular systolic pressure was reduced significantly from $77{\pm}29$ mmHg to $37{\pm}19$ mmHg after pulmonary endarterectomy (p<0.001). The degree of tricuspid regurgitation and the NYHA functional class were all improved postoperatively. Reperfusion edema occurred in 7 cases (35%). The incidence of reperfusion edema was higher in the UCSD type III group than in the other group (25% vs 50%, p=0.25) and the length of postoperative intensive care unit stay was longer in type III group ($5{\pm}2$ days vs $9{\pm}7$ days, p=0.07). The early mortality rate was 10%, and the late mortality rate was 15% (n=3); one death was due to progression of underlying non-Hodgkin's lymphoma, and the other deaths were related to recurrent thromboembolism and persistent pulmonary hypertension, respectively. Conclusion: Pulmonary endarterectomy, as a curative surgical method for treating chronic thromboernbolic pulmonary hypertension, should be performed aggressively in patients diagnosed with chronic thromboembolic pulmonary hypertension, and an effort should be made to reduce the frequency of perioperative complications and to improve surgical outcomes.

• Korean Journal of Animal Reproduction
• /
• v.20 no.1
• /
• pp.35-42
• /
• 1996

This study was carried out to investigate the effect of straw loading method and thawing protocol on the in vitro development of in vitro produced mouse blastocysts cryopreserved by vitrification. Three loading types of straw I, Il and III on loading and sealing method were made for vitrification. The ability of the solution on straw loading methods to remain vitreous during warming was tested by exposed in air for 1 to 10 s sec. and then plunged the vitrified straws into water bath at 25°C. Embryos to be vitrified were equilibrated to the 20% EG for 5min. and exposed in EFS 40 for 1min. The plug ends of Straw I and Straw II were sealed with straw powder and Straw III was treated straw powder, followed by heat sealing and then plunged into LN$_2$. The results obtained in these experiments were summarized as follows; 1) Straw I embryo column mostly changed from transparent to opaque upon thawing without exposure in air for 3-6 sec. Straw II embryo column was I improved partially but was not remained completely vitreous during warming. However, when Straw lll loading method was used, the embryo column was remained vitreous completely. 2) High survival rates and development rates of each groups (middle blastocysts and hatching blastocysts) of vitrified embryos were obtained by using Straw III loading method than Straw I method (P<0.05). And the range of s standard error was low in Straw lll method. 3) When the embryos vitrified-frozen were placed in air for 3, 5 and l0sec. and then warmed rapidly in water bath at 25$^{\circ}C$, the survival rates after 24h of culture were 72.7-87.1% and the development rates to hatching stage after 48h of culture were 34.0-48.4%. There were no significantly differences according to exposure time in air during warming. In conclusion, the present results showed that highly survival and low standard error of vitrified-frozen mouse bIastocysts were obtained by using straw lll loading, double sealing and appropriate 2 step warming method.

• Korean Journal of Animal Reproduction
• /
• v.20 no.2
• /
• pp.111-117
• /
• 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-$\beta$.