This study was carried out to investigate effective condition for producing somatic cell nuclear transfer (SCNT) embryos of Jeju native cattle. As donor cells for SCNT, ear skin cells from Jeju native cattle were used. In experiment 1, the effect of recipient oocyte sources on the development of Jeju native cattle SCNT embryos were examined. Fusion rate of recipient oocyte and donor cell was not different between the Hanwoo and Holstein recipient oocytes (86.0% vs 89.9%). The rate of embryos developing to the blastocyst stage was significantly (p<0.05) higher in Hanwoo recipient oocytes than in Holstein recipient ones (28.2% vs 14.7%). Blastocysts derived from Hanwoo recipient oocytes contained higher numbers of total cells than those derived from Holstein ones ( vs ), although there were no significant difference. The mean proportion of apoptotic cells in blastocyst was not different between the sources of recipient oocytes. In experiment 2, the development of Jeju native cattle and Hanwoo SCNT embryos were compared. Hanwoo oocytes were used as the recipient oocytes. Fusion rate was not different between the Jeju native cattle and Hanwoo SCNT embryos (92.1% vs 92.9%). The blastocyst rate of SCNT embryos was significantly (p<0.05) lower in Jeju native cattle than in Hanwoo (16.9% vs 31.0%). Blastocysts derived from Jeju native cattle SCNT embryos contained smaller numbers of total cells than those derived from Hanwoo ones ( vs ), but there were no significant difference. The mean proportion of apoptotic cells in blastocyst was not different between the Jeju native cattle and Hanwoo SCNT embryos. The present study demonstrated that Hanwoo recipient oocytes were more effective in supporting production of Jeju native cattle SCNT embryos, although Jeju native cattle SCNT embryos showed reduced developmental capacity when compared to Hanwoo SCNT embryos.

발정주기와 상관없이 CIDR을 삽입하는 날에 50 mg progesterone, 2.5 mg estradiol benzoate를 근육주사하였다. CIDR 삽입 후 4, 5일에 28 AU FSH (Antorin R10)을 4일 동안 감량법으로 주사하였다. 6, 7회 FSH 주사 후 25 mg, 15 mg 를 각각 주사한 다음, CIDR는 7회 FSH 주사 후 제거하였다. 1회째 주사 후 48시간에 GnRH를 주사하였다. 공란우는 발정확인 후 12시간 간격

Epigenetic modification including genome-wide DNA demethylation is essential for normal embryonic development. Insufficient demethylation of somatic cell genome may cause various anomalies and prenatal loss in the development of nuclear transfer embryos. Hence, the source of nuclear donor often affects later development of nuclear transfer (NT) embryos. In this study, appropriateness of porcine embryonic germ (EG) cells as karyoplasts for NT with respect to epigenetic modification was investigated. These cells follow methylation status of primordial germ cells from which they originated, so that they may contain less methylated genome than somatic cells. This may be advantageous to the development of NT embryos commonly known to be highly methylated. The rates of blastocyst development were similar among embryos from EG cell nuclear transfer (EGCNT), somatic cell nuclear transfer (SCNT), and intracytoplasmic sperm injection (ICSI) (16/62, 25.8% vs. 56/274, 20.4% vs. 16/74, 21.6%). Genomic DNA samples from EG cells (n=3), fetal fibroblasts (n=4) and blastocysts from EGCNT (n=8), SCNT (n=14) and ICSI (n=6) were isolated and treated with sodium bisulfite. The satellite region (GenBank Z75640) that involves nine selected CpG sites was amplified by PCR, and the rates of DNA methylation in each site were measured by pyrosequencing technique. The average methylation degrees of CpG sites in EG cells, fetal fibroblasts and blastocysts from EGCNT, SCNT and ICSI were 17.9, 37.7, 4.1, 9.8 and 8.9%, respectively. The genome of porcine EG cells were less methylated than that of somatic cells (p<0.05), and DNA demethylation occurred in embryos from both EGCNT (p<0.05) and SCNT (p<0.01). Interestingly, the degree of DNA methylation in EGCNT embryos was approximately one half of SCNT (p<0.01) and ICSI (p<0.05) embryos, while SCNT and ICSI embryos contained demethylated genome with similar degrees. The present study demonstrates that porcine EG cell nuclear transfer resulted in hypomethylation of DNA in cloned embryos yet leading normal preimplantation development. Further studies are needed to investigate whether such modification affects long-term survival of cloned embryos.

The purpose of this experiment was to compare the pregnancy rate (PR) according to the state of the ovaries and uterus, according to the number of embryos transferred from cows and heifers and to investigate the method of artificial twin induction with Hanwoo in vitro fertilized (IVF) embryos by embryo transfer (ET). Looking at the PR according to the condition of the ovaries and uterus, the result was not influenced by the condition of the ovaries, but was significantly influenced by the state of the uterus. The PR according to the number of embryos transferred from cows was 36.8%, 53.0%, 50.5% for 1, 2, and 3 embryos, respectively and although there was a higher frequency of twin calves with 3 embryos than 2, the calving rate was the highest with 2 embryos. In case of heifers, the transfer of 1 embryo showed the best pregnancy and calving rate, and although the PR was similar with 2 embryos (67.7 versus 66.4), in case of 2 embryos transferred there was high frequency of embryonic loss (6.1%) occurred when a cow was diagnosed at 28 and 53 d after ET, total loss (21.3%); sum of fetal death, abortion and stillbirth after pregnant diagnosis at 60 day.

Porcine blastocyst’s quality derived from in vitro is inferior to in vivo derived blastocysts. In this study, to improve in vitro derived blastocyst’s quality and then establish porcine ESCs (pESCs), we treated in vitro fertilized (IVF) embryos and parthenogenetic activated (PA) embryos with three chemicals: porcine granulocyte-macrophage colony stimulating factor (pGM-CSF), resveratrol (RES) and β-mercaptoethanol (β-ME). The control group was produced using M199 media in in vitro maturation (IVM) and porcine zygote medium-3 (PZM3) in in vitro culture (IVC). The treatment group is produced using M199 with 2 μM RES in IVM and PZM5 with 10 ng/mL pGM-CSF, 2 μM RES and 10 μM β-ME in IVC. Data were analyzed with SPSS 17.0 using Duncan’s multiple range test. In total, 1210 embryos in PA and 612 embryos in IVF evaluated. As results, we observed overall blastocyst quality was increased. The blastocyst formation rates were significantly higher (p<0.05) in the treatment groups (54.5%) compared to the control group (43.4%) in PA and hatched blastocysts rates in day 6 and 7 were also increased significantly. Total cell numbers of blastocyst were significantly higher (p<0.05) in the treatment group (55.1) compared to the control group (45.6). In IVF, hatched blastocysts rates in day 7 were increased significantly, too. After seeding porcine blastocyst, the attachment rates were higher in the treatment group (36.2% in IVF and 32.2% in PA) than the control group (26.6% in IVF and 19.5% in PA). Also, colonization rates and cell line derivation rates were higher in treatment group than control group. Colonization rates of control group were 10.8% in IVF and 2.4% in PA, but treatment group were 17.75% in IVF, and 13.1% in PA. And we investigated the correlation between state of blastocysts and attachment rate. The highest attachment rate is in hatched blastocyst (78.35±15.74 %). So, the novel system increased quality of porcine blastocysts produced from in vitro, subsequently increased attachment rates. The cell line derivation rates were 4.2% (IVF) and 2.4% (PA) in control group. In treatment group, they were 10.0% (IVF) and 7.2% (PA). We established 3 cell lines from PA blastocysts (1 cell line in control group and 2 cell lines in treatment group). All cell line has alkaline phosphatase activity and express pluri-potent markers. In conclusion, the novel system of IVM and IVC (the treatment of RES during IVM and RES, β-ME, and pGM-CSF during IVC) increased quality of porcine blastocysts produced from in vitro, subsequently increased derivation rates of porcine putative ESCs.

The reactive oxygen species (ROS) generated during the somatic cell transfer nuclear (SCNT) procedures may cause the mitochondrial dysfunction and DNA damage, which may result in restricts the reprogramming of SCNT embryos and play a key direct role in apoptosis. The present study was conducted to investigate the effect of antioxidant treatment during the SCNT procedures on the inhibition of mitochondria and DNA damages in bovine SCNT embryos. The reconstituted oocytes were treated with antioxidants of 25 μM β-mercaptoethanol (β-ME) or 50 μM vitamin C (Vit. C) during the SCNT procedures. In vitro fertilization (IVF) was performed for controls. Mitochondrial morphology and membrane potential (ΔΨ) were evaluated by staining the embryos with MitoTracker Red or JC-1. Apoptosis was analyzed by Caspase-3 activity assay and TUNEL assay, and DNA fragmentation was measured by comet assay at the zygote stage. Mitochondrial morphology of non-treated SCNT embryos was diffused within cytoplasm without forming clumps, while the IVF embryos and antioxidant treated SCNT embryos were formed clumps. The ΔΨ of β-ME (1.3±0.1, red/green) and Vit. C-treated (1.4±0.2, red/green) SCNT embryos were significantly higher (p<0.05) than that of non-treated SCNT embryos (0.9±0.1, red/ green), which similar to that of IVF embryos (1.3±0.1, red/green). Caspase-3 activity was not difference among the groups. TUNEL assay also revealed that little apoptosis was occurred in SCNT embryos as well as IVF embryos regardless of antioxidant treatment. Comet tail lengths of β-ME and Vit. C-treated SCNT embryos (337.8±23.5 μm and 318.7 ±27.0 μm, respectively) were shorter than that of non-treated SCNT embryos (397.4± 21.4 μm) and similar to IVF embryos (323.3±10.6 μm). These results suggest that antioxidant treatment during SCNT procedures can inhibit the mitochondrial and DNA damages of bovine SCNT embryos.

Autophagy is conserved response to starvation by which cells catabolize their components to create an internal supply of essential nutrients. Ceramide is known to induce autophagy in many cells through down-regulation of amino acid and glucose transporters. The mechanism of starvation induced-autophagy in mouse embryo remains unclear. In order to understand the mechanism by which starvation regulates autophagy, in this study, we investigated nutrient transporters expression and the effect of c2-ceramide on the in vitro development, apoptosis and autophagy via starvation in mouse embryo. Glucose transporters (Glut1 and Glut 3), high levels of transcript were expressed from 1 to 2 cells and gradually decreased through the morula and blastocyst (BL) stages. Amino acid transporters (LAT-1 and 4F2hc) gradually decreased from the zygote to the BL stage. Furthermore, the expression of nutrient transporters (Glut1, 3, LAT-1 and 4F2hc) were significantly reduced at the BL stage after ceramide treatment. Especially, mTOR expression after ceramide treatment of embryos was significantly higher than controls. Ceramide treated embryos exhibited significantly reduced developmental rates and total cell numbers, and increased apoptotic cell death at the BL stage. Consequently, we next evaluated the effect of ceramide treatment on mitochondrial number and morphology. There was a significant decrease in the average mtDNA copy number and the mitochondrial area in ceramide treated BL stage embryos. Both the expression of autophagy-related genes, Lc3, Gabarap, Atg4A and Atg4B, and the synthesis of LC3 were significantly induced at the BL stage. These results suggest that autophagy under starvation condition influences the in vitro development and apoptosis and autophagy, and may play a role in early mouse embryogenesis.

Semen can be divided into two parts. One is cellular part which contains sperms the other is liquid part which is called by seminal plasma. The seminal plasma is a nutritive and protective medium for the sperms. Fructose, which is major energy source, is supplied to sperms swim to female oocyte. Alkalic property protects sperms from hostile environment of female reproductive organ. Also, seminal plasma induces tolerance to preexisted immune cells, and changes intra‐uterine environment to better conditions for fertilized embryos to implant. However, the effects of seminal plasma in in vitro culture of fertilized embryos are unclear. Second fraction of fresh semen was obtained from a normal farm pig. The semen was centrifuged to remove sperms, and then supernatant was filtrated. The filtered seminal plasma was stored in — 30℃. In this study, electrically activated and chemically activated porcine embryos were employed to investigate the developmental rate after 2 hours treatment of none, 0.1%, 0.5%, and 1% seminal plasma in culture media by two days of activation. Both electrically and chemically activated embryos, cleavage rate and cell numbers of blastocysts were not significant difference within four groups. Blastocyst formation rate of electrically activated embryos also did not show significant difference within any groups. However 0.1% seminal plasma treatment group showed significantly increase of blastocyst formation rate in chemically activated group (None; 24.8%, 0.1%; 31.7%, 0.5%; 19.4, and 1%; 16.5%, respectively. p<0.05).

Cathepsin B, a lysosomal cystein protease that plays an important role in the degradation of intracellular proteins in lysosomes, is detected in a wide variety of cells including bovine oocytes and embryos. Although the mode of action of cathepsin B is not fully understood, a strong relationship was observed between cathepsin B and apoptosis in many types of cells. Cathepsin B was found to induce the apoptotic pathway through activating initiator caspases rather than executioner caspases. Thus, the aim of this study was evaluated the effect of capthesin B inhibitor, E-64, on blastocyst developmental competence and subsequent preimplantation quality of the IVF and SCNT bovine embryos. After IVF and SCNT procedures, presumptive bovine embryos were cultured in CR1aa medium supplemented with E-64 for 24 h. Then, samples were additionally cultured in CR1aa medium without E-64 for 5 days. In our results, the frequency of blastocyst formation was higher when treated with E-64 compared with the control group (p<0.05). Furthermore, the blastocyst cell number was enhanced and apoptosis reduced (TUNELpositive nuclei number) by E-64 treatment in both IVF and SCNT bovine embryos (p<0.05). In the real-time quantitative RT-PCR, the expression of anti-apoptotic Bcl-xL gene was shown to be increased in the blastocyst stage, whereas expression of proapoptotic Bax was decreased. In conclusion, our results indicate that E-64 improves the developmental competence and embryonic qualities of bovine IVF and SCNT embryos by modulating cathepsin B induced apoptosis during the preimplantation stage.

Pig embryonic stem cells (ESC) has been suggested to become important animal model for therapeutic cloning using embryonic stem cells derived by somatic cell nuclear transfer (SCNT). However, the quality of cloned embryo and derivation rate of cloned blastocyst has been presented limits for derivation of cloned embryonic stem cell. In this study, we have tried to overcome these problems by aggregating porcine embryos. Zonafree reconstructed SCNT Embryos were cultured in micro-wells singularly (non-aggregated group) or as aggregates of three (aggregated groups) at the four cell stage. Embryo quality of the cloned embryos and attachment on feeder layer rate significantly increased in the aggregates. The aggregation of pig SCNT embryos at the four-cell stage can be a useful technique for improving the quality of pig cloned blastocyst and improvement in the percentage of attachment on the feeder layer of cloned embryos. * This work was supported by the BioGreen 21 Program (PJ0081382011), Rural Development Administration, Republic of Korea.

The coupling of autophagy and endoplasmic reticulum (ER) stress has been implicated in a variety of biological processes. However, little is known regarding the involvement of the autophagy/ER stress pathway in early embryogenesis or the underlying mechanism (s). Here, we showed that the developmental competence of in vitro-produced (IVP) bovine embryos was highly dependent on the autophagy/ER stress balance. Although relative abundances of autophagy-associated gene transcripts, including LC3, Atg5, and Atg7 transcripts, were high in oocytes and throughout the early stages of preattachment development, extensive autophagosome formation was only detected in fertilized embryos. Using inducer and inhibitor of autophagy, we showed that transient elevation of autophagic activity during early preattachment development greatly increased the blastocyst development rate, trophectoderm cell numbers, and blastomere survival; these same parameters were reduced by both inhibition and prolonged induction of autophagy. Interestingly, the induction of autophagy reduced ER stress and associated damage, while the developmental defects in autophagy-inhibited embryos were significantly alleviated by ER stress inhibitor treatment, indicating that autophagy is a negative regulator of ER stress inearly embryos. Collectively, these results suggest that early embryo genesis of IVP bovine embryos depends on an appropriate balance between autophagy and ER stress. These findings may increase our understanding of important early developmental events by providing compelling evidence concerning the tight association between autophagy and ER stress, and may contribute to the development of strategies for the production of IVP bovine blastocysts with high developmental competence.

Embryo transfer (ET) is the final procedure for getting pregnancy through assisted reproductive technology such as IVF (in vitro fertilization), SCNT (somatic cell nuclear transfer). In our laboratory, the porcine cloned embryos loaded in ET medium are carried for 3 hours by portable incubator because of the great distance from the laboratory to the experimental farm. Thus, before transferring into recipient, porcine cloned embryos are exposed in vitro condition for long time. Medium which is used in this process is the TALP (Tyrode’s medium supplemented with 10 mM HEPES), but it includes little nutrients for embryo. Thus, the aim of this study is to determine whether ET media containing nutrients affect the in vitro development of embryos compared to TALP. For the experiment, porcine zygote medium (PZM)-5 which has amino acids for developing embryo was chosen as ET medium containing nutrients, added 10 mM Hepes as PZM-5 does not contain buffering system. For experiment, we carried out parthenogenesis through a chemical method using Thi/DTT. Parthenogenetic embryos were cultured in PZM-5 for 2 days, and then they were randomly divided into two group; loaded in a straw with TALP or PZM-5-Hepes, respectively. They were stored in a portable incubator for 3 hours to simulate the time consumed in ET, thereafter embryos in both TALP and PZM-5-Hepes groups were respectively cultured in PZM-5 for additional 5 days. All experiments were repeated 5 times. In result, blastocyst formation rate were 22.46%±1.47 and 23.17%± 2.13, respectively and total cell number were 32.9±2.22 and 37.09±2.18, respectively. There is no significant difference between TALP and PZM-5-Hepes groups. * Further study will investigate effect of PZM-5-Hepes on in vivo development of porcine cloned embryo. This study was supported by IPET (#311011-05-1-SB010), RNL Bio (#550-20120006), Institute for Veterinary Science, the BK21 program and TS Corporation.

Limited success of somatic cell nuclear transfer(SCNT) is attributed to incomplete reprogramming of transferred donor cell. Several approachs, such as histone deacetylase inhibitors and DNA methyltransferase inhibitors have been used to improve the efficiency of somatic cell nuclear transfer. Recently, it is reported that pre-treatment of somatic cells with undifferentiated cell extract, such as embryonic stem cell and mammalian oocytes is an attractive alternative ways to reprogramming control. The aim of this study was to evaluate the early development of porcine cloned embryos produced with porcine ear skin fibroblasts pre-treated with extract from porcine induced pluripotent stem cell (iPSC). For transport of porcine iPSC extract into cultured porcine ear skin fibroblasts, the ChariotTM reagent system was used. Treated cells were cultured for 3 days, and used for the analysis of histone H3K9 acetylation and SCNT The acetylation status of H3K9 was increased in cells treated with iPSC extract and cultured for 3 days compared with control. But, no significant difference was observed between the extract treated and control groups. After SCNT. no difference was observed in the rate of fusion (86.6% vs 86.2%) and embryo cleavage (86.6% vs 87.1%) between the extract treated and control groups. Also, no significant difference was noted in blastocyst rates (23.4% vs 28.4%) as well as cell numbers (43.8±10.8 vs 41.2±11.6) with extract treated group compared with control group. Overall apoptosis rate in blastocyst was not differences between the extract treated and control groups (4.6±3.5% vs 6.0± 5.8%). However, blastocyst rate with high apoptotic cells(>10% appototic cells) was significantly lower in extract treated group when compared with control group (7.1% vs 21.8%).. Our results demonstrated that pre-treatment of porcine ear skin fibroblasts using porcine iPSc extract had beneficial effect on the decreasing apoptosis in the blastocyst cultured in vitro, although there was no effect on the embryonic development.

There are diverse methods of cryopreservation of mammalian embryos with variable degrees of success. Although cryopreservation technique of mammalian embryos has been advanced, freezing stress affect to cellular event such as apoptosis and autophage in embryos. The objective of the study is to investigate the affection of to survival, development, live offspring, apoptosis and autophagy on embryo. Mouse embryos were vitrified and thawed using normal straw and modified cut standard straw (M-CSS), then in vitro cultured until blastocyst stage and transferred to recipient. Recovery rates (100 vs 99.2%), survival rates (99.2 vs 78.6%), developmental rates (18.4 vs 10.7%), total cell numbers (45 vs 37), preganacy rates (34.5 vs 25%) and offspring numbers (10.1 vs 4.9 %) of M-CSS group are significantly higher than those of normal straw vitrified group. Also, rate of apoptosis in blastocysts developed using M-CSS (1.9%) was significantly lower than using normal straw vitrification (2.7%). Apoptosis-related gene, caspase 3, was expressed at the highest level in blastocysts derived from normal straw group. However, no differences of autophagy related gene, Atg6 and expression of LC3 between normal straw and M-CSS groups were observed. In conclusion, the standard vitrification procedure induces mitochondrial apoptosis in zygotes in an autophagy-independent manner, whereas the novel M-CSS procedure may improve embryo vitrification.

The purpose of this experiment is to compare the pregnancy rate (PR) according to the state of the ovaries and uterus, according to the number of embryos transferred from cows and heifers and to investigate the method of artificial twin induction with Hanwoo in vitro fertilized (IVF) embryos by embryo transfer (ET). Looking at the PR according to the condition of the ovaries and uterus, the result was not influenced by the condition of the ovaries, but was significantly influenced by the state of the uterus. The PR according to the number of embryos transferred from cows was 36.8%, 53.0%, 50.5% for 1, 2, and 3 embryos respectively, and although there was a higher frequency of twin calves with 3 embryos than with 2, the calving rate was the highest with 2 embryos. In case of heifers, the transfer of 1 embryo showed the best pregnancy and calving rate, and although the PR was similar with 2 embryos (67.7 versus 66.4), in case of 2 embryos transferred there was high frequency of embryonic loss( 6.1%) occurred when a cow was diagnosed at 28 and 53 d after ET, total loss (21.3%; sum of fetal death, abortion and stillbirth after pregnant diagnosis at 60 day).

The present study was conducted to examine the generation of reactive oxygen species (ROS) during micromanipulation procedures in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine enucleated oocytes were electrofused with donor cells, activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. Oocytes and embryos were stained in dichlorodihydrofluorescein diacetate or 3'-(p-hydroxyphenyl) fluorescein dye and the H2O2 or ˙OH radical levels were measured. In vitro fertilization (IVF) was performed for controls. The samples were examined with a fluorescent microscope, and fluorescence intensity was analyzed in each oocyte and embryo. The H2O2 and ˙OH radical levels of reconstituted oocytes were increased during manipulation (37.2~49.7 and 51.0~55.2 pixels, respectively) as compared to those of mature oocytes (p<0.05). During early in vitro culture, the ROS levels of SCNT embryos were significantly higher than those of IVF embryos (p<0.05). These results suggest that the cellular stress during micromanipulation procedures can generate the ROS in bovine SCNT embryos.

The objective of this study was to investigate the effectiveness of cryopreservation methods for the effect of various vitrification containers, such as EM-grid, OPS, or cryo-loop on the survival and developmental rate of vitrified mouse pronuclear embryos, and mouse cleavage embryo, at 21, 24, 27 and 30 hr after hCG injection. Post-thaw cleavage was similar among treatments, while the developmental rates of mouse blastocyst and hatched blastocyst were higher ( <0.05) in 27 hr and 30 hr than 21 hr. The developmental rate of hatched blastocyst at vitrified cleavage mouse embryos in cryo-loop was significantly higher than vitrified pronuclear embryos of control group as well as EM-grid and OPS ( <0.05). The developmental rate using cryo-loop was higher than EM-grid, but in case of OPS at vitrified cleavage and mouse pronuclear embryos, no significant difference was noticed. These results of our study show that the developmental rates of mouse embryos were unaffected by various vitrification containers, but in case of mouse embryos and hatched blastocysts at late vitrified pronuclear embryos the developmental rates were higher than early vitrified pronuclear embryos. Moreover, the developmental rate of hatched blastocyst at vitrified cleavage mouse embryos was significantly higher than vitrified pronuclear embryos. For better execution of this study, it will be mandatory to include improvement of vitrification containers, cryopreservation methods and conditions, higher survival rate, safe preservation, contamination and embryo loss.

The objective of this study was to investigate the relationship between body weight, body condition score (BCS), blood urea nitrogen (BUN), glucose, cholesterol and number of transferable embryos for the purpose of improving reproductive performance in Hanwoo donors. Seventy five cows, at random stages of the estrous cycle, received a CIDR together with injection of 1mg estradiol benzoate and 50 mg progesterone, and gonadotropin treatment begann. Four days later, the animals were superovulated with a total of 28AU FSH (Antorin, 2AU = 1 ml) administered twice daily in constant doses over 4 days. On the 3rd administration of FSH, CIDR was withdrawn and 25 mg was administered. Cows were artificially inseminated twice after estrous detection at 12 hr intervals. The cows received GnRH at the time of 1st insemination. Embryos were recovered 7 days after the 1st insemination. In conclusion, cows with body weight < 400, 400~450 and > 450kg had number of transferable embryos of , and , cows with BCS <2.25, 2.25~2.75 and had number of transferable embryos of , and respectively. These data indicate that a body weight and BCS for superovulation of CIDR-treated Korean native cows does not affect the embryo yield.

In the present study, the effect of cysteine and NT or bisphenol A (BP) on in vitro aturation (IVM) of porcine oocytes were examined. COCs was cultured in NCSU-23 medium supplement with 10% FCS which had previously been covered with mineral oil and equilibrated in a humidified atmosphere of 5% CO2 and 95% air at 38℃. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 medium supplement with 0.5～10.0 mM cysteine were 34.0±3.2%, 36.0±3.5%, 48.0±3.8%, 22.0±3.2%, respectively. The IVM rate of oocytes cultured in NCSU-23 medium supplement with 0.5～5.0 mM NT for 48 hrs were 24.0±4.2%, 18.0±4.9%, 8.0±2.2%, respectively. NT affects oocyte in vitro maturation rate in a dose-dependent. This result were significantly lower than the control group. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 medium supplement with 1.0 mM NT+5.0 mM cysteine (38.0±4.3%) were significantly higher than that of NT treatment. The IVM rate of oocytes cultured in NCSU-23 medium supplement with 0.05～5.0 mM BP for 48 hrs were 20.0±4.7%, 10.0±5.3%, 6.0±3.2%, respectively. The IVM rate of oocytes cultured in NCSU-23 medium supplement with BP was significantly lower cultured non supplement of BP (44.0±3.5%). BP affects porcine oocyte maturation rate in a dose-dependent manner. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 medium supplement with 1.0 mM BP+5.0 mM cysteine (32.0±3.2%) were increased than that of BP treatment.

Somatic cell nuclear transfer (SCNT) and induced pluripotent stem cell (iPS) experiments have generally demonstrated that a differentiated cell directly converts into a undifferentiated or pluripotent state. In SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor cell nuclei to the recipient cytoplasm of matured oocytes. Although nuclear reprogramming of cells by the ex-ovo methods is not always consistent or efficient, it has been suggested that a combination of nuclear reprogramming technique may improve the efficiency or frequency of normal development of SCNT embryos. Here we hypothesized that treatment of somatic cells with extracts prepared from GV stage sturgeon's oocytes prior to their use as nuclear donors for SCNT will improve subsequent development. We reported a reversible permeabilization protocol with digitonin to deliver sturgeon oocyte exteact (SOE) to porcine fetal fibroblast cell nuclei ex ovo. Porcine fibroblasts were permeabilized by 4 μg/ml of digitonin for 2 min at 4℃ and then incubated in SOE for 7h at 15 18℃ followed by resealing of cell membrane. As results, no difference was observed in the number of fused couplets or the number of fused couplets that cleaved between the extract treated or control group. However, there was a significantly decrease in the percentage of fused couplets that developed to the blastocyst stage in the SOE treated group (p<0.05). Histone acetylation status was determined using an antibody to acetylation at lysine 9 on histone 3 (H3K9Ac). The intensity of H3K9Ac staining in 1-cell stage NT embryos was significantly increased when treated with the SOE (p<0.05), similar to that in 1-cell stage IVF embryos. In addition, porcine NT embryos reconstructed by using donor cell exposed to SOE prior to cell fusion significantly decreased developmental competence to the blastocyst stage but increased pluripotent gene expressions (Sox2, Nanog and Oct3/4) when compared with those in normal NT embryos (p<0.05).