Reactive oxygen species (ROS) production and F-actin cytoskeleton dynamics play important roles in the survival rate of blastocysts after the vitrifiedwarming process. However, the protective effects of Mito-TEMPO against cryo-injury and viability through F-actin aggregation and mitochondrial-specific ROS production in vitrificated-warmed bovine embryos have not been investigated. The aims of the present study were to: (1) determine the effects of Mito-TEMPO on embryonic developmental competence and quality by F-actin stabilization during in vitro culturing (IVC), and (2) confirm the effects of Mito-TEMPO through F-actin structure on the cryotolerance of vitrification-warming in Mito-TEMPO exposed in vitro production (IVP) of bovine blastocysts. Bovine zygotes were cultured with 0.1 μM Mito-TEMPO treatment for 2 days of IVC. Mito-TEMPO (0.1 μM) exposed bovine embryos slightly improved in blastocyst developmental rates compared to the non-treated group. Moreover, the viability of vitrified-warmed blastocysts from Mito-TEMPO treated embryos significantly increased (p < 0.05, non-treated group: 66.7 ± 3.2% vs Mito-TEMPO treated group: 79.2 ± 5.9%; re-expanded at 24 hours). Mito-TEMPO exposed embryos strengthened the F-actin structure and arrangement in the blastocyst after vitrification-warming. Furthermore, the addition of Mito-TEMPO into the IVC medium enhanced embryonic survival and quality through F-actin stabilization after the vitrification-warming procedure. Overall, our results suggest that supplementing the culture with 0.1 μM Mito- TEMPO improves the embryonic quality and cryo-survival of IVP bovine blastocysts.

Until recently, there have been many researches about the freezing methods and several methods of cryopreservation. Hypothermic preservation has been used to complement the embryo freezing technology. There is a study to show the successful results for long-term hypothermic preservation. For that reason, FBS and BSA are commonly added to the culture medium to support embryo development. We investigated the effectiveness of hypothermic preservation method at 4℃ according to embryonic developmental stages for Hanwoo embryos and evaluated the effect of FBS and BSA on Hanwoo embryos as a supplemental reagent in hypothermic preservation medium after recovering preserved embryos from hypothermic preservation. The present study reported that survival and hatching rates of embryos at morula stage following storage at 4℃ is Day 7 group was significantly higher (p < 0.05) compared than those of other groups (p < 0.05). As a result, the survival and hatching rates of embryos at the blastocyst stage following storage at 4℃ result is showed that significantly higher (p < 0.05) survival rates than those of other groups an Day 6. The result showed that hatching rate at Day 6 and 7 were significantly lower (p < 0.05) compared with other groups. The result regarding the survival and hatching rates of bovine embryos following storage at 4℃ for 72 h in various concentrations of BSA are shown The results showed that survival rate of 1% BSA group was not significantly different (p < 0.05) compare with control (FBS) group. Also, the results showed that hatching rate of control (FBS) and 1% BSA were significantly different (p < 0.05) compared with other groups. In conclusion, our result demonstrated that the hypothermic preservation did not effect on the survival and hatching rates of embryos after recovering. In addition, the supplementation of BSA in preservation medium showed no difference in the embryo developmental competence after hypothermic preservation compared to FBS treatment. With that, BSA can be an alternative reagent for the hypothermic preservation medium as an energy source and pH buffer.

Cryopreservation of bovine embryos is used to efficiently implant surrogate mothers. It has been widely accepted that high lipid content in the oocyte interrupts its survival during freeze-thaw cycles. Serum component in the culture medium is thought to increase the embryo`s lipid contents. Conversely, L-carnitine stimulates lipid metabolism by transporting long chain fatty acids into the mitochondria. Objective of this study was to analyze the effect of L-carnitine supplementation in IVM medium and defined IVC medium on the development, lipid contents and the cryosurvival of bovine IVF embryos. 0.0, 1.5, 3.0 and 6.0 mM L-carnitine was supplemented in IVM medium, respectively (IVM-LC 0.0, LC 1.5, LC 3.0 and LC 6.0). Development rate from the 2cell to the morula stages was higher in IVM-LC 3.0 groups than those of IVM-LC 6.0 (p<0.05). But there were no significant differences among the other groups in the blastocyst rates and lipid content results. When 0.0, 1.5, 3.0 and 6.0 mM L-carnitine were supplemented in IVC medium (IVC-LC 0.0, LC 1.5, LC 3.0 and LC 6.0), development competence was not significantly different between those embryos. Lipid contents of embryos treated L-carnitine (IVC-LC 1.5, 3.0 and 6.0) were significantly lower than embryos of non-treated group. L-carnitine was supplemented 0.0, 1.5, 3.0, 6.0 mM during IVM and 3.0 mM during IVC (LC 0.0 - 3.0, LC 1.5 – 3.0, LC 3.0 – 3.0, LC 6.0 – 3.0) and cryosurvival of blastocysts confirmed after freezing-thawing. There were no significant differences on development, but LC 3.0 – 3.0 was significantly lower lipid contents than other groups. And LC 3.0 – 3.0 had better survival rates and hatched rates of blastocysts than LC 0.0 – 0.0. In conclusion, supplementation of L-carnitine in defined IVC medium decreases lipid contents. And L-carnitine supplementation improves cryosurvival and developmental ability of bovine IVF embryos.

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

This study aimed to produce high-quality blastocysts and establish appropriate microinjection conditions for the introduction of target gene. First, we identified embryo development to the blastocyst stage after microinjection using the CRISPR/Cas9 system on the Cas9 protein or mRNA. As a result, we confirmed that blastocyst development in the Cas9 mRNA injected group significantly increased when compared to the Cas9 protein injected group (p<0.05). However, the blastocyst gene targeting rate increased in the Cas9 protein injected group when compared to the Cas9 mRNA injected group (p<0.05). Next, we treated the injection medium with 10 μg/ml of cytochalasin B (CB), and the microinjected embryos were cultured in CR1-aa medium supplemented with 0.1 μM of melatonin (Mela). Consequently, the blastocyst formation rate significantly increased in the CB treated group (p<0.05). After microinjecting embryos with the CB treated injection medium, we investigated blastocyst formation and quality via Mela treatment. Consequently, the Mela treated group demonstrated significantly increased blastocyst formation rates when compared to the non-treated group (p<0.05). Furthermore, immunofluorescence assay using RAD51 (DNA repair detection protein) and H2AX139ph (DNA damage detection protein) showed an increase in RAD51 positive cells in Mela treated embryos. Therefore, we verified the improvement in knock-in efficiency in microinjected bovine embryos using Cas9 protein. These results also demonstrated that the positive effect of the CB and Mela treatments improved the embryonic developmental competence and blastocyst qualities in genetically-edited bovine embryos.

Bovine somatic cell nuclear transfer (bSCNT) embryos can develop to the blastocyst stage at a rate similar to that of embryos produced by in vitro fertilization (IVF). However, the efficiency of somatic cell cloning has remained low, and applications have been limited, irrespective of the nuclear donor species or cell types. One possible explanation is that the reprogramming factors of each oocyte is insufficient or not properly adapted for the receipt of a somatic cell nucleus, because it is naturally prepared only for the receipt of a gamete. Here, we would like to introduce the aggregation method (agSCNT), a new experimental system that enables and increase oocyte volume and examined its subsequent development. Judgement by the blastocyst formation rate or total cell number was significantly higher in the agSCNT group than that in the SCNT group, and was very similar to that in the control IVF group. Moreover, the cleavage formation rate in the agSCNT group (61.5 ± 1.3) was higher than that in the SCNT group (39.7 ± 2.1), while still less than that in the IVF group (75.4 ± 1.3). We also analyzed the epigenetic modifications in bovine IVF, agSCNT, and untreated SCNT embryos. In conclusion, the present study demonstrated that agSCNT improves the in vitro developmental competence and quality of cloned embryos, as evidenced by increased total cell numbers (TC).

Somatic cell nuclear transfer (SCNT) is the technique which generates embryos by transferring diploid nucleus into an enucleated oocyte, it has produced specific animals successfully in a variety of species. However, the developmental capacity of SCNT embryos is still relatively lower than that of embryos produced in vivo. Oocyte is a kind of lipid rich cells, its quality limits the efficiency of embryo production. L-carnitine is a co-enzyme facilitating the transportation of long chain fatty acids across the inner mitochondria membrane where fatty acids are used for generating adenosine triphosphate (ATP) via beta-oxidation. It also has antioxidant actions which may protect mitochondrial membranes and DNA against damage induced by reactive oxygen species (ROS). Whether L-carnitine is functional in bovine SCNT embryos are unknown. Therefore, the objective of this study was to examine the effects of L-carnitine on oocyte maturation and developmental competence of subsequent SCNT embryos. L-carnitine was supplemented during IVM, then intracellular ROS and GSH levels, mitochondrial activity, gene expression of COCs were analyzed at the end of IVM. SCNT embryos were produced subsequently, apoptosis detection and gene expression evaluation were performed in blastocysts. In the results, treatments with 1.5 mM and 3 mM L-carnitine significantly improved maturation rates (P<0.05). Treatments with 3 mM L-carnitine effectively induced improvement in nuclear maturation, intracellular GSH levels and mitochondrial activity, as well as a reduction in intracellular ROS levels (P<0.05). mRNA levels of CPT1A, ACAA1, ACAA2, AREG, EREG, SOD1, GPX4, GLUT1 and CDC2 transcripts were effectively up-regulated by 3 mM L-carnitine treatments (P < 0.05). Similarly, 3mM L-carnitine induced an increase in blastocyst developmental rates and an improvement in blastocyst quality (P<0.05). Our study indicates that L-carnitine treatment during IVM improves oocyte nuclear maturation and subsequent SCNT embryo development.

Although assisted reproductive technology (ART) has been developed in many mammalian species including cows, the only embryo preservation technology that is available is cryopreservation. In the present study, small molecules were used to preserve embryos at room temperature. The basic medium for embryo preservation consisted of 1% BSA non-cryopreservation medium (BNC) instead of fetal bovine serum (FBS). To maintain survival and prevent damage during embryo storage, three candidate small molecules were selected—CHIR99021, Y-27632 and Thiazovivin—and their concentrations were optimized. Then, the embryos in the small molecule supplemented preservation medium were stored at room temperature. The viability and hatching rate of embryos stored at 10°C were greater for Y-27632-BNC and CHIR99021+Y-27632-BNC compared to BNC. However, the rate was lower for Thiazovivin-BNC compared to BNC. Although there were no surviving embryos after storage at 20°C, the viability and hatching rate of embryos significantly increased in Y-27632-BNC and CHIR99021+Y-27632-BNC compared to BNC. The mechanism by which small molecules enhance survival of embryos during storage was investigated, and expression of heat shock protein 70 was observed to increase. The findings of this work may be useful in improving ART in the agricultural field.

It is well known that the production of transgenic bovine embryos is more difficult than the production of non-transgenic bovine embryos. We performed whether the quality of transgenic bovine embryos expressing the enhanced green fluorescent protein (EGFP) can be improved when cultured for 4 days from day 4 until day 8 after activation with epidermal growth factor (EGF), insulin-like growth factor (IGF), and flavonoid (F) supplements. The EGFP gene was introduced into bovine IVF embryos using microinjector. In experiment, transgenic bovine embryos were cultured in modified CR1aa medium containing 10% FBS at 38.8℃ in an incubator (5% CO2, 5% O2, and 90% N2) for 8 days and embryos were equally divided into four groups [non-treated group (control), 1 μM IGF+EGF (IE), 10 μM F and 1 μM IGF+EGF+10 μ M F (IEF)] at day 4 embryos. In the result, development rate of F treatment group (41.8%) was higher than that of control (33.3%), IE (23.9%) and IEF groups (28.0%). However, hatching rate was significantly high in IE (53.0%) and IEF (65.0%) groups than in control (42.9%) and F group (42.8%) (p<0.05). The EGFP expression rate was not different among all groups (30.0~33.3%) at blastocyst stage. In comparison of total cell number, IE group (145.2±10.4) was significantly higher than control group (101.4±14.3). Apoptotic index of IE group (1.9%) was the lowest compared with that of control (3.3%), F (3.5%) and IEF groups (4.6%). This result demonstrate that the combination of IGF, EGF and flavonoid can be helpful to improve the development potential and the quality of transgenic bovine embryos.

Increase of bovine embryos produced by in vitro fertilization (IVF) has been seen. The main reason for producing in vitro fertilized embryos in Korea has been to utilize the genetics of cows with higher carcass grade. Ovaries are collected from the cows in the slaughter house and the information on the carcass grade of the cow can be traced. Embryos produced from cows with higher carcass grade have been favored by the farmers. PCR has been one of the main techniques for sex determination of embryos targeting various genes. Bovine sex determining region Y (SRY) is specific to Y chromosome. However, it requires a control gene for PCR, if the embryo is female. In comparison to SRY, amelogenin can be amplified from male or female embryos with different fragment sizes due to differential splicing in all bovidae. The goal of this study was to determine whether there are any differences in the sex ratio of embryos produced in vitro and to compare the efficiency of sex determination using PCR. Ovaries of Hanwoo were collected and transported to the laboratory in thermal bottles. For in vitro maturation, oocytes were collected from the follicles with less than 8 mm of diameter and placed in either the Brackett & Oliphant media (BO), Tissue culture medium-199 (TCM-199), or IVMD101 media, containing 3% fetal bovine serum (FBS), 0.5 mg/ml FSH, 0.5 mg/ml LH, and 1 mg/ml estradiol-17β. For IVF, frozen sperm from Hanwoo bulls were used. After 22-24h IVF, embryos were transferred and cultured either in BO or TCM-199 with 10% FBS until the embryos were hatched. Hatched blastocysts were stored in PBS frozen, and later thawed and treated with embryo lysis buffer. After isolating genomic DNA, it was used for PCR using primers for casein beta (CSN2), as PCR control, or for male specific SRY primers. Alternatively, primers for amelogenin were used. Sex of embryos was determined and the sex ratio was analyzed. Out of 94 embryos, sex of 83 embryos (88.3%) was determined and there were 40 male embryos (48.2%) and 43 female embryos (51.8%). Sex of 31 embryos was determined using both SRY and amelogenin. Among those, 17 embryos were determined as having identical sex, while 1 embryo was determined as having different sex, and the sex of 11 and 2 embryos were determined only by amelogenin or SRY primers, respectively. In conclusion, the success of determining the sex of embryos by PCR was relatively high. Using amelogenin primer for PCR tends to be more efficient than SRY primer in determining the sex. Slightly higher ratio of female embryos was different from previous years and the cause for the difference may require further investigation.

The study aims to assess the embryo development and survivability of bovine embryos cultured in vitro by addition of cysteine. The rates of metaphase II formation are not significantly different among the three groups(73.8% for TCM199, 76.9% for TCM199 with 0.3mM cysteine and 83.8% for TCM199 with 0.5mM cysteine). No differences on cleavage rate(70.6~74.6%) was observed among three culture medium(70.6% for TCM199, 71.3% for CR1aa, and 74.6% for SOF) with 0.5mM cysteine. However, significantly(P<0.05) higher development rate was obtained in the blastocyst stage by adding 0.5mM cysteine in SOF medium(35.6%) than in TCM199(27.6%) or CR1aa(26.6%). No significant differences in the cleavage rates were observed among the three culture. After freezing the blastocysts cultured with 0.5M cysteine, the re-expansion rates ranged from 61.3% to 86.4% among groups, and hatching rates are from 26.3% to 46.9% among groups. The rates of re-expansion and hatching are significantly(P<0.05) higher in SOF medium(86.4% and 46.9%, respectively) than those in TCM199(61.3% and 26.3%) and CR1aa medium(87.1 and 44.4%). After thawing, the blastocyst re-expansion rate become significantly(P<0.05) higher in in vivo (87.1%) and in vitro (70.3%) embryos. In conclusion, our results demonstrate that supplementation of IVM and IVC media with 0.5mM cysteine improved the quality of in vitro production of embryo and post-thawed embryo. Future studies comparing these culture systems in well-designed trials should be performed.

To improve the developmental potential of bovine somatic cell nuclear transfer (SCNT) embryos, this study compared the developmental rates to blastocyst stage in the SCNT embryos using donor fibroblasts treated with 5-azacytidine (5AC) and S-adenosylhomocysteine (SAH) at different concentrations. Their reprogramming efficiency level was investigated with level of telomerase activity. Donor fibroblasts isolated from adult ear skin of a cow were exposed to 5AC and SAH at different concentrations during 2 passages. After nuclear transfer into enucleated recipient oocytes, the cleavage and developmental rates were significantly (p<0.05) decreased in the SCNT embryos using 5AC-treated fibroblasts (5AC-SCNT embryos), compared with those of non-treated control (control-SCNT embryos) and SAH-treated fibroblasts (SAH-SCNT embryos). The developmental rates to blastocyst stage tended to be slightly increased in the SAH-SCNT embryos at each of the concentrations, and especially, the developmental rates in the SCNT embryos using 1.0 mM SAH-treated fibroblasts were significantly (p<0.05) higher than that of control SCNT embryos. The mean numbers of total and ICM cell in blastocysts were also significantly (p<0.05) decreased in the 5AC-SCNT embryos, compared with those of other SCNT blastocysts. Further, the level of telomerase activity was also significantly (p< 0.05) decreased in the 5AC-SCNT embryos than those of control and SAH-SCNT embryos. Whereas, a significantly (p<0.05) up-regulated telomerase activity was observed in SAH-SCNT embryos, compare with that of control-SCNT embryos. In conclusion, SCNT embryos using hypomethylated donor cells with SAH, not 5AC, may improve the developmental potential and reprogramming efficiency.

We investigate the effect of L-glutathione (GSH), an antioxidant, treatment during the somatic cell nuclear transfer (SCNT) procedures on the in vitro development and DNA methylation status of bovine SCNT embryos. Bovine in vitro matured (IVM) oocytes were enucleated and electrofused with a donor cell, then activated by a combination of Ca-ionophore and 6-dimethylaminopurine. The recipient oocytes or reconstituted oocytes were treated with 50 μM GSH during these SCNT procedures from enucleation to activation treatment. The SCNT embryos were cultured for 7 days to evaluate the in vitro development, apoptosis and DNA methylation in blastocysts. The apoptosis was measured by TUNEL assay and caspase-3 activity assay. Methylated DNA of SCNT embryos at the blastocyst stages was detected using a 5-methylcytidine (5-MeC) antibody. The developmental rate to the blastocyst stage was significantly higher (P<0.05) in GSH treatment group (32.5±1.2%, 78/235) than that of non-treated control SCNT embryos (22.3±1.8%, 50/224). TUNEL assay revealed that the numbers of apoptotic cells in GSH treatment group (2.3±0.4%) were significantly lower (P<0.05) than that of control (3.8±0.6%). Relative caspase-3 activity of GSH treated group was 0.8±0.06 fold compared to that of control. DNA methylation status of blastocysts in GSH treatment group (13.1±0.5, pixels/ embryo) was significantly lower (P<0.05) than that of control (17.4±0.9, pixels/embryo). These results suggest that antioxidant GSH treatment during SCNT procedures can improve the embryonic development and reduce the apoptosis and DNA methylation level of bovine SCNT embryos, which may enhance the nuclear reprogramming of bovine SCNT embryos.

The aim of the present study was to compare two different serum-free media, modified synthetic oviduct fluid (mSOF) and modified potassium simplex optimization medium (mKSOM) containing 20% RD (RPMI1640 + DMEM, 1:1 v/v) (RD-mKSOM), for in vitro culture (IVC) of bovine embryos. After in vitro maturation and fertilization, the presumptive zygotes were cultured in two different serum-free conditions for 7 days and 9 days to evaluate blastocyst formation and hatching, respectively. Serum supplemented conventional CR2 medium was used as control. After 7 day of culture, there was no significant difference in cleavage and blastocyst formation rates among three groups (mSOF, 59.3 and 30.1%; RD-mKSOM, 65.0 and 41.5%; control, 51.6 and 38.0%, respectively). Hatching rate was significantly higher in control (69.0%) than other experimental groups (mSOF, 22.0%; RD-mKSOM, 39.5%) (P＜0.0001 and P＜0.001, respectively). Although both serum-free conditions showed lower hatching rates than serum-added control, in serum-free groups, RD-mKSOM showed significantly higher hatching rate than mSOF (P＜0.001). In addition, one-step using RD-mKSOM may facilitate IVC procedure than two-step culture system. In conclusion, the results indicate that one-step RD-mKSOM is more suitable defined culture system for IVC of bovine embryos than two-step mSOF.

Programmed cell death or apoptosis is associated with changes in K+ concentration in many cell types. Recent studies have demonstrated that two-pore domain K+ (K2P) channels are involved in mouse embryonic development and apoptotic volume decrease of mammalian cells. In cerebellar granule neurons that normally undergo apoptosis during the early developmental stage, TASK-1 and TASK-3, members of K2P channels, were found to be critical for cell death. This study was performed to identify the role of K+ channels in the H2O2-induced or cryo-induced cell death of mouse and bovine embryos. Mouse and bovine two-cell stage embryos (2-cells) exposed to H2O2 for 4 h suffered from apoptosis. The 2-cells showed positive TUNEL staining. Treatment with high concentration of KCl (25mM) inhibited H2O2-induced apoptosis of 2-cells by 19%. Cryo-induced death in bovine blastocysts showed positive TUNEL staining only in the cells near the plasma membrane. Cryoprotectant supplemented with 25 mM KCl reduced apoptosis slightly compared to cryoprotectant supplemented with 5 mM KCl. However, the combination of antioxidants (β-mercaptoethanol) with 25 mM KCl significantly decreased the rate of H2O2-induced and cryo-induced apoptosis compared to treatments with only antioxidants or 25 mM KCl. These results show that blockage of K+ channel efflux for a short-time reduces H2O2- and cryo-induced apoptosis in mouse and bovine embryos. Our findings suggest that apoptosis in mouse and bovine embryos might be controlled by modulation of K+ channels which are highly expressed in a given cell type.

This study was carried out to investigate the effects of tissue inhibitor of matalloproteinase-1 (TIMP-1), Activin A and Heparin binding epidermal growth factor (HB-EGF) on in vitro production of bovine embryos. In experiment 1, presumptive zygotes were cultured in the medium supplemented with TIMP-1 (0.5 μg/ml), Activin A (100 ng/ml), or HB-EGF (100 ng/ml) at 39 ℃ in a humidified atmosphere of 5% (v/v) CO2, 5% (v/v) O2 and 90% (v/v) N2. In experiment 2, TIMP-1 + HB-EGF or Activin A + HB-EGF combinations were supplemented in the culture medium. The developmental rate to blastocysts, hatching rate and total cell numbers of the blastocysts were evaluated in both experiments. The embryos cultured in medium without growth factor supplementation was used as control group. In experiment 1, the embryos cultured in medium supplemented with TIMP-1 and Activin A showed significantly higher developmental rate to blastocysts than those cultured with HB-EGF and control (36.9%, 34.1%, 21.2% and 23.1%, respectively) (P<0.0001). However, the hatching rate of blastocyst was significantly higher in embryos with HB-EGF than those with TIMP-1, Actvin A and Control groups (84.4%, 58.8%, 51.4% and 49.3%, respectively) (P<0.001). Total cell number per blastocyst was also significantly higher in embryos with HB-EGF group (174.3±2.5) than those with TIMP-1, Activin A (149.7 and 150.0, respectively) (P<0.05) and Control (119.0) (P<0.001). In experiment 2, embryos cultured with combined treatment of Activin A and HB-EGF resulted in significantly higher rates of blastocysts formation (48.0%), hatching rate (89.7%) and total cell number in blastocyst (182.3±2.1) than those with TIMP-1 and HB-EGF combination group (32.0%, P<0.001; 76.6%, P<0.05; 165.7±4.2, P<0.001, respectively). Our data demonstrate that in vitro production of bovine embryos could be improved by combined supplementation of Activin A and HB-EGF in culture medium.

Matrix Metalloproteinases (both MMP2 and -9) play a pivotal role of the embryos hatching and implantation. Therefore, the objective of this study was carried out to investigate the influence of MMP2 and MMP9 on embryo development potential and subsequent effect at molecular level. There was no significant difference of cleavage rate among the groups. The development competence of blastocyst was significantly higher (P<0.05) in MMP9 treatment (39.81±16.61) than that to the combined treatment of MMP2 and –9 (23.68±0.27), but there was no significant difference among the control vs. MMP2 vs. MMP9 (35.05±2.74 vs. 32.71±6.18 vs. 39.81±16.61, respectively). On the other hand, the hatching rate of blastocysts was significantly lower (P<0.05) in combined group of MMP2 and –9 (12.55±0.09) (Table1). The expression level of MMP2 and MMP9 was significantly lower (P<0.05) in the entire treatment groups than that in the control group. But the expression of MMP9 was significantly higher (P<0.05) when compared in the entire treatment groups. The relative expression embryonic developmental gene, IFNt expression level significantly lower (P < 0.05) in the MMP9 embryos. The placenta establishment genes, PLAC8 and SSLP1, expression were significantly higher (P < 0.05) in the MMP2 embryos compared to other groups. Transcription regulation gene, HNRNPA2B1, was higher (P < 0.05) in the combined group of MMP2+MMP9 than that in the other groups. In conclusion, our results suggest that MMPs to culture medium improves the blastocyst development rate and further impact on target gene expression analysis.

Matrix Metalloproteinases (MMP)-2 and -9 are participated in embryo development, implantation, remodeling of epithelial cell and ovulation. The objective of this study is to evaluate an impact of MMP2 and MMP9 on embryonic developmental competence as well as gene expression profiles of in vitro-produced bovine embryos. After in vitro fertilization, embryos of all groups were transferred into IVC-2 medium treated with MMP2 and MMP9 to check the optimum concentration on the basis of embryo development competence and cell numbers. The optimum concentrations for MMP2 and 9 were 1,200 ng/ml and 300 ng/ml. The blastocyst development competence was not different among 1,200 ng/ml of MMP2 vs. 300 ng/ml of MMP9 vs. combined MMP2 + 9 vs. control groups (41.46 ± 10.66 vs. 37.73 ± 8.92 vs. 45.11 ± 11.41% vs. 41.59 ± 11.88, respectively). Furthermore, the developmental competences to hatching and hatched blastocysts were not also different among the same groups (79.84 ± 12.63 vs. 83.3 ± 17.46 vs. 78.55 ± 14.48% vs. 72.02 ± 14.09). In addition, total cell number was significantly (p<0.05) greater in blastocyst treated with MMP9 300 ng/ml among all treatment groups. On the other hand, there was no significant difference of ICM vs. TE ratio in all groups. The expression of five out of six genes (i.e., MMP2, MMP9, IFNt, SSLP1 and HNRNPA2B1) was different among the groups. The expression of IFNt and HNRNPA2B1 genes was significantly greater in MMP9 (p<0.05), but there was no difference of MMP9 expression between MMP2 and MMP9 group (p>0.05). The normalized expression of MMP2 and SSLP1 was greater in MMP2 than other groups (p<0.05). In conclusion, MMPs treatment during IVC-2 medium was remarkably effected on blastocyst developmental competence and gene expression profiles that are related to embryo quality and implantation.

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.

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