Transcription activator like effector nucleases (TALENs) are artificial restriction enzymes generated by fusing a TALE DNA binding domain to a DNA cleavage domain which remove and introduce specific genes to produce transgenic animals. To investigate the efficient laboratory techniques for the injection of TALEN mRNA, pEGFP-N1 commercial plasmid were microinjected into porcine parthenogenetic and in vitro fertilization (IVF). In Experiment 1, to investigate injection time, compared 4 different time durations (2 hr, 4 hrs, 6 hrs & 8 hrs) after post activation of parthenogenetic embryos and after 6 hrs of co-incubation with sperms in IVF embryos. There were significant difference (P<0.05) in development to the blastocysts (4.4, 8.9, 3.9, 0.6%), GFP expression in blastocysts (1.3, 5.7, 2.3, 0.0%) which injected after post activation of 4 hrs compared with other 3 groups. IVF embryos after 2 hrs and 4 hrs injected were expressed GFP significantly higher than rest of two groups (P<0.05). In Experiment 2, compared development of 2 different concentrations (20 ng/μl and 50 ng/μl) of EGFP injection. There were significant difference (P<0.05) between two treatments which has higher cleavage (58.8 vs 41.9%), blastocysts development rate (13.0 vs 11.1%) and GFP expressed blastocysts (5.7 vs 0.0%) in 20 ng/μl than the 50 ng/μl in parthenogenetic embryos. In IVF embryos, only 20 ng/μl injected embryos were expressed GFP (4.2%) after 7 days of incubation and 77.3 vs 64.7% of cleavage, 26.4 vs 23.5% development to blastocysts. In Experiment 3, three different volumes (5, 10 and 20 pl) were microinjected into porcine embryos to determine the most appropriate volume. Out of 3 groups, significantly higher development rates of cleavage (68.3, 58.0, 29.3%), blastocysts (11.7, 12.7, 0.5%) and GFP expressed blastocysts (2.9, 7.8, 0.0%) were shown in the 10 pl group (P<0.05). In conclusion, these results imply that 20 ng/μl concentration, 10 pl of volume and injection at 4 hrs after post activation for parthenogenetic and 2∼4 hrs after IVF, 20 ng/μl concentration and 10 pl volume for IVF embryos were more effective microinjection conditions.

Oxygen consumption is a useful parameter for evaluating mammalian embryo quality, since individual bovine embryos was noninvasively quantified by scanning electrochemical microscopy (SECM). Recently, several approaches have been used to measure the oxygen consumption rates of individual embryos, but relationship between oxygen consumption and pregnancy rates of Hanwoo following embryo transfer has not yet been reported. In this study, we measured to investigate the correlation between oxygen consumption rate and pregnancy rates of Hanwoo embryo using a SECM. In addition to, the expression of pluripotent gene and anti-oxidant enzyme was determined using real-time PCR by extracting RNA according to the oxygen consumption of in vivo embryo. First, we found that the oxygen consumption significantly increased in blastocyst-stage embryos (blastocyst) compared to early blastocyst stage embryos, indicating that oxygen consumption reflects the embryo quality (Grade I). Oxygen consumption of blastocyst was measured using a SECM and total cell number of in vitro blastocyst was enumerated by counting cells stained by propidium iodide. The oxygen consumption or GI blastocysts were significantly higher than those of GII blastocysts (10.2 × 1015/mols—1 versus 6.4 × 1015/mols—1, p<0.05). Total cell numbers of in vitro blastocysts were 74.8, 90.7 and 110.2 in the oxygen consumption of below 10.0, 10.0∼12.0 and over 12.0∼1015/mols—1, respectively. Pregnant rate in recipient cow was 0, 60 and 80% in the transplantation of embryo with the oxygen consumption of below 10.0, 10.0∼12.0 and over 12.0 × 1015/mols—1, respectively. GPX1 and SOD1 were significantly increased in over —10.0 group than below 10.0 groups but in catalase gene, there was no significant difference. On the other hand, In OCT-4 and Sox2, pluripotent gene, there was a significant difference (p<0.05) between the below-10.0 (0.98 ± 0.1) and over 10.0 (1.79 ± 0.2). In conclusion, these results suggest that measurement of oxygen consumption maybe help increase the pregnant rate of Hanwoo embryos.

돼지 수정란의 체외 생산 효율성 향상을 위해서는 배발생율과 더불어 고품질의 배를 조기에 선별해야 한다. 체외 배 발생율에 대한 보고는 많지만, 고품질의 배를 선별할 수 있는 기술에 대한 연구는 거의 없었다. 본 연구에서는 돼지 난포란 유래 수정란의 체외배양에 있어서 배반포로의 배 발달과 생존에 미치는 Vitamin K1(vit K1) 첨가 농도, 시기 및 시간의 효과를 검토하였다. 1.0 μM, 3.0 μM 및 6.0 μM vit K1을 배양 1일째 24시간 첨가한 결과, 배반포 발달율이 시험군이 14.5 ± 4.3, 0.0 및 0.0%로써 대조군의 35.5 ± 3.2%에 비하여 유의하게 낮았고(p<0.05), 배반포의 생존율도 대조군이 31.8 ± 2.6%로써 시험군의 22.2 ± 2.9, 0.0 및 0.0%에 비하여 유의하게 높았다(p<0.05). 상기 첨가 농도에서 첨가 시간을 달리한 결과, 1.0 μM 농도에서 6시간 처리군의 배반포 발달율과 생존율이 각각 26.5 ± 2.9% 및 47.2 ± 2.8%로써 가장 높았고 특히, 12시간 처리군보다 유의하게 높았다(p<0.05). 3.0 μM 농도에서는 대조군의 배발달율이 36.4 ± 3.1%로 가장 높았으나, 생존율은 3.0시간 첨가군이 41.7 ± 3.2%로 대조군에 비하여 유의하게 높았다(p<0.05). 6.0 μM 농도에서도 배발달율은 대조군(32.0 ± 2.8%), 생존율은 0.5시간 첨가군(42.9 ± 1.8%)이 가장 높았다. 각각의 vit K1 첨가 농도와 시간을 기준으로 서로 다른 배양 시기에 첨가한 결과, 1.0 μM 6시간 첨가군에서는 배반포 발달율은 배양 4일째 첨가군, 생존율은 배양 2일째 첨가군이 가장 높았다. 한편, 3.0 μM 3.0시간 및 6.0 μM 0.5시간 첨가군에서는 배양 4일째 첨가군의 배반포 발달율(59.5 ± 4.1% 및 50.0 ± 3.6%)과 생존율(72.7 ± 5.4% 및 79.2 ± 4.0%)이 대조군과 다른 시험군에 비하여 유의하게 높았다(p<0.05). 한편, vit K1 첨가에 따른 배반포의 세포 수를 조사한 결과, 첨가군(1.0 μM 6시간 배양 2일째, 3.0 μM 3.0시간 배양 4일째 및 6.0 μM 0.5시간 배양 6일째)이 53.4 ± 5.8, 49.4 ± 3.8 및 51.5 ± 4.5개로써 대조군의 40.2 ± 2.3개에 비하여 유의하게 많았다(p<0.05). 그러나 사멸세포 수는 시험군이 3.2 ± 0.9∼3.7 ± 2.1개로써 대조군의 4.2 ± 1.2개보다 적었으나, 유의차는 없었다. 세포 사멸 유도 유전자인 Bax mRNA 발현은 처리군과 대조군은 비슷하였으나, 세포 사멸 억제 유전자인 Bcl-xL mRNA 발현은 처리군이 대조군보다 높았고 특히, 6.0 μM 0.5시간 배양 4일째 첨가군이 가장 높았다. 이상의 결과로부터 돼지 미성숙 난포란 유래 수정란의 체외 배양에 vit K1의 첨가는 배반포의 생존율과 세포수 증가에 효과적이었다. 그 이유에 대해서는 아직 많은 부분이 밝혀져야 되겠지만, 고품질의 배반포 조기 선발에는 활용이 가능할 것으로 생각된다.

This study was carried out to study the survival rate of thawed Hanwoo embryos frozen by the slow-rate freezing or the cryotop vitrification method. Hanwoo cumulus-oocyte complexes were recovered from ovaries at a slaughter house, matured for 20~22 hours, fertilized with Hanwoo semen for 5~6 hours, and cultured for 7~9 days in 38.5℃, 5% CO2 incubator. For freezing, Day 7∼9 blastocysts were collected. Embryos for the slow-rate freezing were equilibrated in 1.8 M ethylene glycol (EG) with Dulbecco's phosphate-buffered saline (D-PBS). Programmable cell freezer was precooled down to —7℃, and the straw was seeded during 8 minutes-holding time, and was cooled to —35℃ at the cooling rate of 0.3℃/min, and then was plunged and stored in liquid nitrogen. Embryos for the cryotop vitrification were treated in TCM199 with 0.5 M sucrose, 16% EG, 16% dimethylsulfoxide (DMSO). Embryos were then loaded individually onto cryotop and plunged directly into liquid nitrogen. The survival rates of embryos frozen by these two freezing methods were evaluated at 12 to 24h post-thawing. The survival rates of frozen/thawed Hanwoo embryos by the cryotop vitrification method (56.86 ± 26.53%) were slightly higher than those by the slow-rate freezing method (55.07 ± 26.43%) with no significant difference. Using the cryotop vitrification and the slow-rate freezing of Hanwoo blastocysts on Day 7 following in-vitro fertilization (IVF) treatment, the survival rates of frozen/thawed Hanwoo embryos were 72.65 ± 18.3% and 79.06 ± 17.8%, respectively. The survival rates by the cryotop vitrification were higher than those by the slow-rate freezing on both Day 8 and 9 with significantly higher survival rate on Day 9 (p<0.05). Using the cryotop vitrification and the slow-rate freezing of Hanwoo embryos to compare between three different blastocyst stages, the survival rates of the blastocyst stage embryos were 66.22 ± 18.8% and 45.76 ± 12.8%, respectively with higher survival rate by the vitrification method (p<0.05). And the survival rate of expanded blastocysts was higher than those of early blastocysts and blastocysts in two freezing methods with significantly higher survival rate by the slow-rate freezing method (p<0.05).

In mammal, unfertilized oocytes remain in the oviduct or under in vitro culture, which is called "oocyte aging". This asynchrony negatively affects fertilization in pre- and post-implantation embryo development. Caffeine a phos-phodiesterase inhibitor is known to rescue oocyte aging in several species. The objective of this study is to determine the cytoskeleton distribution in aged oocytes and the embryo developmental ability of aged oocytes in the present or absence of caffeine during maturation. Caffeine treatment increased the incidence of normal spindle assembly of aged oocytes (treatment, 67.57±4.11% aging, 44.61±6.4%) and no significant differences compared to control group. Fluorescence values were compared using ROS (Reactive oxidation species) stain. Fluorescence values appear of con-trol group intensity rate (51.53.±3.80), aging group (68.10±5.54) and treatment of caffeine (45.04±2.98). Aged oocytes that were derived from addition of caffeine to the IVM (in vitro maturation) medium had significantly increased 2-cell that developed to the blastocyst stage compared to the aging group. Blastocysts, derived from caffeine treatment group, significantly increased the total cell number compare aging (90.44±10.18 VS 67.88±7.72). Apoptotic fragments of genomic DNA were measured in individual embryo using TUNEL assay. Blastocyst derived from caffeine treatment group decreased significantly the apoptotic index compared to blastocyst derived from aging group. In conclusion, we inferred that the caffeine treatment during oocyte aging can improve the developmental rate and quality in bovine embryos developing in vitro

Low efficiency of somatic cell nuclear transfer (SCNT) is attributed to incomplete reprogramming of transfered nu-clei into oocytes. Trichostatin A (TSA), histone deacetylase inhibitor and 5-aza-2’deoxycytidine (5-aza-dC), DNA methy-lation inhibitor has been used to enhance nuclear reprogramming following SCNT. However, it was not known molec-ular mechanism by which TSA and 5-aza-dC improve preimplantation embryo and fetal development following SCNT. The present study investigates embryo viability and gene expression of cloned porcine preimplantation embryos in the presence and absence of TSA and 5-aza-dC as compared to embryos produced by parthenogenetic activation. Our results indicated that TSA treatment significantly improved development. However 5-aza-dC did not improve development. Presence of TSA and 5-aza-dC significantly improved total cell number, and also decreased the apoptot-ic and autophagic index. Three apoptotic-related genes, Bak, Bcl-xL, and Caspase 3 (Casp3), and three autophagic-re-lated genes, ATG6, ATG8, and lysosomal-associated membrane protein 2 (LAMP2), were measured by real time RT-PCR. TSA and 5-aza-dC treatment resulted in high expression of anti-apoptotic gene Bcl-xL and low pro-apoptotic gene Bak expression compared to untreated NT embryos or parthenotes. Furthermore, LC3 protein expression was lower in NT-TSA and NT-5-aza-dC embryos than those of NT and parthenotes. In addition, TSA and 5-aza-dC treated embryos displayed a global acetylated histone H3 at lysine 9 and methylated DNA H3 at lysine 9 profile similar to the parthenogenetic blastocysts. Finally, we determined that several DNA methyltransferase genes Dnmt1, Dnmt3a and Dnmt3b. NT blastocysts showed higher levels Dnmt1 than those of the TSA and 5-aza-dC blastocysts. Dnmt3a is lower in 5-aza-dC than NT, NTTSA and parthenotes. However, Dnmt3b is higher in 5-aza-dC than NT and NTTSA. These results suggest that TSA and 5-aza-dC positively regulates nuclear reprogramming which result in modulation of apoptosis and autophagy related gene expression and then reduce apoptosis and autophagy. In addition, TSA and 5-aza-dC affects the acetylated and methylated status of the H3K9.

Cathepsin B is abundantly expressed peptidase of the papain family in the lysosomes, and closely related to the cell degradation system such as apoptosis, necrosis and autophagy. Abnormal degradation of organelles often occurs due to release of cathepsin B into the cytoplasm. Many studies have been reported that relationship between cathepsin B and intracellular mechanisms in various cell types, but porcine embryos has not yet been reported. Therefore, this study evaluated the effect of cathepsin B inhibitor (E-64) on preimplantation developmental competence and quality of porcine embryos focusing on apoptosis and oxidative stress. The expression of cathepsin B mRNA in porcine em-bryos was gradually decreased in inverse proportion to E-64 concentration by using real-time RT-PCR. When putative zygotes were cultured with E-64 for 24 h, the rates of early cleavage and blastocyst development were decreased by increasing E-64 concentration. However, the rate of blastocyst development in 5 μM treated group was similar to the control. On the other hand, both the index of apoptotic and reactive oxygen species (ROS) of blastocysts were sig-nificantly decreased in the 5 μM E-64 treated group compared with control. We also examined the mRNA expression levels of apoptosis related genes in the blastocysts derived from 5 μM E-64 treated and non-treated groups. Expre-ssion of the pro-apoptotic Bax gene was shown to be decreased in the E-64 treated blastocyst group, whereas expre-ssion of the anti-apoptotic Bcl-xL gene was increased. Taken together, these results suggest that proper inhibition of cathepsin B at early development stage embryos improves the quality of blastocysts, which may be related to not only the apoptosis reduction but also the oxidative stress reduction in porcine embryos.

The present study was performed to investigate the effect of Hh-Ag1.5, a small-molecule chemical agonist of SMOothened receptor, on the in vitro maturation and development of in vitro fertilized (IVF) embryos in pigs. Oocytes or fertilized embryos were cultured in a maturation or embryo culture medium supplemented with 0 (control), 25, 50 or 100 nM of Hh-Ag1.5, respectively. Although the maturation rate were not different among treatment groups, the blastocyst formation rate in the group treated with 25 nM Hh-Ag1.5 was significantly increased compared to other groups (P<0.05). While the highest dose of Hh-Ag1.5 (100 nM) did negatively affect to the embryo development and cell number in blastocysts compared to other groups (P<0.05), the apoptotic cell index in blastocysts was significantly lower in 25 and 50 nM groups than in control and 100 nM groups (P<0.05). The mRNA expression of the proapoptotic gene Bax and the ratio of Bax/Bcl-XL decreased in among treatment groups compared to control (P<0.05). The embryo quality related genes, Tert and Zfp42, were significantly decreased in 50 and 100 nM groups compared with control and 25 nM groups (P<0.05). In conclusion, the addition of 25 nM Hh-Ag1.5 to in vitro maturation and culture medium can enhance the developmental potential as well as quality of IVF embryos in pig.

The purpose of this study is to improve production efficiency of vitrified-thawed transgenic bovine embryos. Transgenic bovine embryos were produced by injection of FIV-GFP lentiviral vector into perivitelline space of in vitro matured MІІ stage oocytes, and then in vitro fertilization. EGFP-expressing transgenic bovine blastocysts were cultured in serum-containing and serum–free medium. These blsatocysts were vitrified by pull and cut (PNC) container made with 0.25 cm plastic straw. Results indicate that total developmental rates of normal IVF embryo cultured in serumcontaining and–free medium into blastocyst were not significantly different (22.3 vs 21.5%) and those of GFPexpressing transgenic bovine embryo into blastocyst showed no significant difference between serum-containing (13.9%) and–free medium (13.1%). However, developmental rate of GFP transgenic embryo was significantly (P<0.05) lower than its of normal IVF embryos. In additional study, we vitrified GFP transgenic normal bovine blastocysts using PNC vitrification method. Survival rate of vitrified-thawed GFP transgenic blastocyst (23.1%) was significantly (P<0.05) lower than its of normal blastocysts (68.9%). Although, survival rate of vitrified-thawed GFP transgenic blastocyst was lower than its of normal blastocyst, our result may suggested that PNC vitrification method is feasible to cryopreserve transgenic embryos. Our next plan will be the production of GFP express transgenic bovine derived from vitrified-thawed embryos using PNC method.

Developmental potential of cloned embryos is related closely to epigenetic modification of somatic cell genome. The present study was to investigate the effects of applying histone deacetylation inhibitor, trichostatin A (TSA) to activated porcine embryos on subsequent development of porcine parthenogenetic and nuclear transfer embryos. Electrically activated oocytes were treated with 5 nM TSA for different exposure times (0, 1, 2 and 4 hr) and then the activated embryos were cultured for 7 days. The reconstructed embryos were treated with different concentrations of 0, 5, 10 and 25 nM TSA for 1 hr. Also 5 nM TSA was tested with different exposure times of 0, 0.5, 1, 2 and 4 hr. And fetal fibroblast cells were treated with 50 nM TSA for 1, 2 or 4 hr and with 5 nM TSA for 1 hr. Cumulus-free oocytes were enucleated and reconstructed by TSA-treated donor cells and electrically fused and cultured for 6 days. In parthenogenetic activation experiments, 5 nM TSA treatment for 1 hr significantly improved the percentage of blastocyst developmental rates than the other groups. Total cell number of blastocysts in 1 hr group was significantly higher than other groups or control. Similarly, blastocyst developmental rates of porcine NT embryos following 5 nM TSA treatment for 1 hr were highest. And the reconstructed embryos from donor cells treated by 50 nM TSA for 1 hr improved the percentage of blastocyst developmental rates than the control group. In conclusion, TSA treatment could improve the subsequent blastocyst development of porcine parthenogenetic and nuclear transfer embryos.

The present study was conducted to examine the effect of antioxidant treatment during parthenogenetic activation procedure on the reactive oxygen species (ROS) levels and in vitro development of porcine parthenogenetic embryos. Porcine in vitro matured oocytes were activated by a combination of electric stimulus and 2 mM 6- dimethylaminopurine (6-DAMP) before in vitro culture. During the activation period, oocytes were treated with 50 μM β-mercaptoethanol (β-ME), 100 μM L-ascorbic acid (Vit. C) or 100 μM L-glutathione (GSH). To examine the ROS level, porcine parthenogenetic embryos were stained in 10 μM dichlorohydrofluorescein diacetate (H2DCFDA) dye 20 h after culture, examined under a fluorescence microscope, and the fluorescence intensity (pixels) were analyzed in each embryo. The parthenogenetic embryos were cultured for 6 days to evaluate the in vitro development. The apoptosis was measured by TUNEL assay. The H2O2 levels of parthenogenetic embryos were significantly lower in antioxidant treatment groups (26.9±1.6～29.1±1.3 pixels/embryo, p<0.05) compared to control (33.2±1.7 pixels/embryo). The development rate to the blastocyst stage was increased in antioxidant treatment groups (32.0～32.5%) compared to control (26.9%, p<0.05), although, there was no difference in apoptosis among groups. The result suggests that antioxidant treatment during parthenogenetic activation procedure can inhibit the ROS generation and enhance the in vitro development of porcine parthenogenetic embryos.

Embryo reconstruction by somatic cell nuclear transfer (SCNT) has been used to demonstrate that mammalian somatic cells can dedifferentiate into a totipotent nucleus when introduced into an oocyte. This process of dedifferentiation of somatic cells after nuclear transplantation is defined as nuclear reprogramming, although this terminology gives little information on the molecular events that characterize this process. When planning on strategies for nuclear reprogramming by nuclear transfer (NT) one may suggest that converting the somatic nuclear configuration into an embryonic state is highly desirable, so this may promote a succession of events similar to those occurring during early embryo development. In the following part of this overview we will discuss the results of many studies that have investigated different aspects of nuclear remodelling after SCNT.

본 연구는 돼지의 난포란을 체외성숙하여 세포질내정자주입(ICSI)에 의해 생산된 체외수 정란의 체외발달율을 평가하기 위하여 실시하였다. 세포질내정자주입에 의한 체외수정란의 발달율은 서로 다른 보존상태의 정자인 신선정자, 액상정자 및 동결-융해된 정자를 이용하 더라도 수정율과 배발달율에는 영향을 미치지 않는 것으로 나타났다. 세포질내정자주입 후 난자의 전기적 활성화를 처리한 실험군이 활성화를 처리하지 않은 실험군에 비해 수정율과 배반포기배로의 발달율에 있어서는 높은 경향을 나타내었으나, 체외수정 실험군 및 전기적 활성화를 처리한 실험군과 전기적 활성화를 처리하지 않은 실험군간 배반포기배의 할구수는 유의적인 차이를 나타내지 않았다. 또한 각각의 실험군에서 얻은 배반포기배의 염색체를 분 석한 결과, 정상 이배체 염색체상의 비율에 있어서도 유의성을 나타내지 않았다.

The present study was to assess the in vitro viability and sexing rate of bovine embryos. Blastocysts were harvested on day 7～9 day after insemination(in vitro and in vivo), and the sex of the embryos was examined using the LAMP method. Embryo cell biopsy was carried out in a 80 μl drop Ca2+, Mg2+ free D-PBS and, biopsied embryos viability were evaluated after more 12 h culture in IVMD culture medium. The formation of recovered embryo to expanded and hatching stages had ensued in higher of sexed embryo in vivo than in vitro (100% vs. 89%, p<0.05), and in vitro, the rates of degeneration after sexing were significantly (p<0.05) higher in vitro than in vivo(11% vs. 0.0%). The rates of the predicted sex were female 61% vs. 56%, and male 39% vs. 44% in vivo and in vitro, respectively. The rates of survival following different biopsy methods were seen between punching and bisection method in vivo and in vitro (100% vs. 89% and 100% vs, 78% respectively). Biopsy method by punching was significantly (p<0.05) higher than bisection between produced embryos in vivo and in vitro. The present data indicate that with microblade after punching for embryo sexing results in high incidence of survivability on development after embryo biopsy. It is also suggested that LAMP-based embryo sexing suitable for field applications.

The technique of SCNT is now well established but still remains inefficient. The in vitro development of SCNT embryos is dependent upon numerous factors including the recipient cytoplast and karyoplast. Above all, the metaphase of the second meiotic division (MII)　oocytes have typically become the recipient of choice. Generally high level of MPF present in MII oocytes induces the transferred nucleus to enter mitotic division precociously and causes NEBD and PCC, which may be the critical role for nuclear reprogramming. In the present study we investigated the in vitro development and pregnancy of White-Hanwoo SCNT embryos treated with caffeine (a protein kinase phosphatase inhibitor). As results, the treatment of 10 mM caffeine for 6 h significantly increased MPF activity in bovine oocytes but does not affect the developmental competence to the blastocyst stage in bovine SCNT embryos. However, a significant increase in the mean cell number of blastocysts and the frequency of pregnant on 150 days of White-Hanwoo SCNT embryos produced using caffeine treated cytoplasts was observed. These results indicated that the recipient cytoplast treated with caffeine for a short period prior to reconstruction of SCNT embryos is able to increase the frequency of pregnancy in cow.

Solution of glycerol, ethylene glycol, sucrose, dextrose (GESD) and cryotop methods were carried out to investigate the survivability on vitrification of embryos. Embryos cultured in vitro were vitrified by GESD of 10 or 8 step and cryotop methods of 6 step, from cryopreservation step to frozen-thawed and culture step. Survival rate and ICM, TE cells of embryos were investigated after frozen-thawed 24 h. As a results, cryotop method was significantly (p<0.05) higher ( vs. , ) than GESD 10 or 8 step methods on survivability. Also, In ICM cell number, cryotop method was significantly (p<0.05) higher to cells than GESD 8 step method. TE cell number was significantly (p<0.05) highest to cells in cryotop method. On the other hand, survival rate, TE and total cell number were all the significantly (p<0.05) high, except ICM in GESD 10 step method between GESD 10 step method and GESD 8 step method. In conclusion cryotop method was to be most effective, but it is considered necessary to study vitrification method for step-by-step freezing and thawing process.

This research was investigated the relationship between the number of the transferable embryos and estrus expression rate, BCS (Body Condition Score), which affect the nutritional state of the cow, in Holstein donor cows. CIDRs were inserted into the vaginas of twenty two head of Holstein cows, regardless of estrous cycle. Superovulation was induced using folliclar stimulating hormone (FSH). For artificial insemination, donor cows were injected with and estrus was checked about 48 hours after the injection. Then they were treated with 4 straws of semen 3 times, with 12-hour intervals. Embryos were collected by a non-surgical method 7 days after the first artificial insemination. When BCS was 2.5, the total number of collected ova was 7.3 + 1.9, which is significantly lower (p<0.05) than the numbers 15.4 + 2.8 and 15.4 + 2.1 that were obtained when BCSs were 2.75 and 3.0, respectively. Whereas the numbers of transferable embryos were 5.2 + 1.4 when BCS was 2.5, which was smaller than the numbers 6.0 + 2.1 and 8.5 + 1.8 that were obtained when BCSs were 2.75 and 3.0, respectively; however, the differences were not significant. As for estrus induction rate, the cow groups whose BCSs were 2.75 and 3.0 showed 100.0% and 95.0%, respectively. Whereas the cow group whose BCS was 2.5 showed 57.1%, and the differences were significant (p< 0.05). As for estrous expression rate, the cow groups whose BCSs were 2.5, 2.75 and 3.0 showed 100.0%, 100.0% and 85.7%, respectively; however, the differences were not significant. According to the result of this research, it is considered that the total number of collected ova and the number of transferable embryos will be affected by the nutritional state before and after in vivo embryo production and superovulation treatment, and that although the mechanism is not clear, poor stockbreeding management and nutritional level would cause the decrease of ovum recovery rate and the number of transferable embryos in high-producing cows. On the other hand, diverse researches on the superovulation treatment method that is suitable for high-producing Holstein donor cows would contribute to preventing ovarian cyclicity disorder, as well as to the early multiplication of cows with superior genes by increasing the utilization value of donor cows.

Embryonic genome activation (EGA) is the first major transition that occurs after fertilization, and entails a dramatic reprogramming of gene expression that is essential for continued development. Although it has been suggested that EGA in porcine embryos starts at the four-cell stage, recent evidence indicates that EGA may commence even earlier; however, the molecular details of EGA remain incompletely understood. The RNA polymerase II of eukaryotes transcribes mRNAs and most small nuclear RNAs. The largest subunit of RNA polymerase II can become phosphorylated in the C-terminal domain. The unphosphorylated form of the RNA polymerase II largest subunit C-terminal domain (IIa) plays a role in initiation of transcription, and the phosphorylated form (IIo) is required for transcriptional elongation and mRNA splicing. In the present study, we explored the nuclear translocation, nuclear localization, and phosphorylation dynamics of the RNA polymerase II C-terminal domain in immature pig oocytes, mature oocytes, two-, four-, and eight-cell embryos, and the morula and blastocyst. To this end, we used antibodies specific for the IIa and IIo forms of RNA polymerase II to stain the proteins. Unphosphorylated RNA polymerase II stained strongly in the nuclei of germinal vesicle oocytes, whereas the phosphorylated form of the enzyme was confined to the chromatin of prophase I oocytes. After fertilization, both unphosphorylated and phosphorylated RNA polymerase II began to accumulate in the nuclei of early stage one-cell embryos, and this pattern was maintained through to the blastocyst stage. The results suggest that both porcine oocytes and early embryos are transcriptionally competent, and that transcription of embryonic genes during the first three cell cycles parallels expression of phosphorylated RNA polymerase II.

This research was investigated the relationship, in high-producing Holstein donor cows, between the number of the transferable embryos and the blood serum concentrations of Blood Urea Nitrogen (BUN), glucose and cholesterol, which affect the nutritional state of cows. CIDRs were inserted into the vaginas of twenty two heads of Holstein cows, regardless of estrous cycle. Superovulation was induced using folliclar stimulating hormone (FSH). For artificial insemination, donor cows were injected with and estrus was checked about 48 hours after the injection. Then they were treated with 4 straws of semen 3 times, with 12-hour intervals. Embryos were collected by a non-surgical method 7 days after the first artificial insemination. The total numbers of ova collected from 3 experimental groups whose blood BUN concentrations were <10 mg/dl, 11~18 mg/dl and mg/dl were 8.9, 12.5 and 19.0, respectively; whereas the numbers of transferable embryos were 5.8 + 1.9, 7.9 + 2.8 and 5.2 + 1.4, respectively. When glucose concentration was <60 mg/dl, the total number of collected ova was 9.9, which was smaller than when the concentration was 60~70 mg/dl or mg/dl. When glucose concentration was 60~70 mg/dl, the number of transferable embryos was 7.1 + 2.4, which was slightly larger than the numbers 6.4 + 2.1 and 6.1 + 1.7 that were obtained when the concentrations were <60 mg/dl and mg/dl, respectively ; however, the differences were not significant (p>0.05). When cholesterol concentrations were <150 mg/dl, 150~200 mg/dl and mg/dl, the total numbers of collected ova were 11.2, 11.3 and 8.6, respectively. Whereas the numbers of transferable embryos were 7.1 + 2.1, 7.3 + 1.9 and 5.6 + 1.3, respectively ; however, the differences were again not significant (p>0.05). The result of this research showed no significant difference in ovum recovery rate and the number of transferable embryos according to major metabolite concentrations in high-producing Holstein donor cows. However, it is considered that the failure of maintaining proper nutritional status would cause the fall in in vivo embryo productivity.

The development of embryos reconstructed by nuclear transfer is dependent upon numerous factors including the type of recipient cell, method of enucleation, the type of donor cell, method of reconstruction, activation, the cell cycle stage of both the donor nucleus and the recipient cytoplasm and the method of culture of the reconstructed embryos. Many of these points which have been reviewed extensively elsewhere (Sun and Moor, 1995; Colman, 1999; Oback and Wells, 2002; Renard et al., 2002; Galli et al., 2003b), here we will concentrate on main area, the production of suitable cytoplast and nuclear donor, nuclear-cytoplasmic coordination, oocyte activation, culture of reconstructed embryos, and the effects that this may have on development.