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.

Direct injection of genome editing tools such as CRISPR/Cas9 system into developing embryos has been widely used to generate genetically engineered pigs. The approach allows us to produce pigs carrying targeted modifications at high efficiency without having to apply somatic cell nuclear transfer. However, the targeted modifications during embryogenesis often result in mosaicism, which causes issues in phenotyping founder animals and establishing a group of pigs carrying intended modifications. This study was aimed to establish a genomic PCR and sequencing system of a single blastomere in the four-cell embryos to detect potential mosaicism. We performed genomic PCR in four individual blastomeres from four-cell embryos. We successfully amplified target genomic region from single blastomeres of 4-cell stage embryo by PCR. Sanger sequencing of the PCR amplicons obtained from the blastomeres suggested that PCR-based genotyping of single blastomere was a feasible method to determine mutation type generated by genome editing technology such as CRISPR/Cas9 in early stage embryos. In conclusion, we successfully genotyped single blastomeres in a single 4-cell stage embryo to detect potential mosaicism in porcine embryos. Our approach offers a simple platform that can be used to screen the prevalence of mosaicism from designed CRISPR/Cas9 systems.

In the early development of parthenogenetic embryo, cytoplasm and nucleic acid fragmentation may be a cause of lower embryo development. The purpose of this study was to evaluate whether embryonic development and apoptosis factors can be reduced by controlling the in-vitro culture environment by the addition of hormones, pregnancy serum and uterine milk. Our study showed that the activity of Casp-3 increased within the cytoplasm when artificially used hormones to induce the incubation environment, and PCNA's manifestation was low. However, the addition of pregnant serum appeared to lower the Casp-3 activity compared to the other groups. In addition, MMP-9 activity was increased and early embryo development and cytoplasmic fidelity were also increased. Therefore, the results of the present study showed that the use of gestational serum in the development of parthenogenetic embryo inhibit apoptosis and increases cytoplasmic reorganization by natural environmental control in in vitro culture.

Imazosulfuron is globally considered as a relatively safe herbicide that controls plant growth by interfering with amino acid synthesis. It is stable, persists in the soil, and has low toxicity; however, studies about the toxic effects of imazosulfuron on non-targeted aquatic vertebrates are scarce. In this study, imazosulfuron was able to induce acute lethality on zebrafish embryos within 48 h. Imazosulfuron also had adverse effects on heartbeats and induced abnormal development with pericardial edema and scoliosis. Moreover, apoptosis and oxidative stress were increased by imazosulfuron in a dose-dependent manner. Thus, all our results showed that imazosulfuron has toxic effects on zebrafish embryogenesis.

This experiment was conducted to analyse the effects of flavone supplementation on the preimplantation development of in-vitro produced porcine embryos. During in-vitro development, immature oocytes and early embryos were exposed to different concentrations of flavone (0, 1μM, 25μM, 50 μM, and 100 μM respectively). Results showed that 100 μM of flavone significantly reduced the intracellular ROS levels of oocytes accompanied with a significant rise in GSH level. In parthenogenesis, no significant change was observed in the cleavage rates whether flavone was supplemented in IVM or IVC media. In IVM supplemented group, the blastocyst development rate was significantly enhanced by 1 μM concentration than other groups (51.5% vs. 41.3%, 44.0%, 36.3%, 31.7%; P<0.05) respectively. However, in IVC group 1 μM concentration significantly improved the blastocysts production than 50 μM and control groups (50.0% vs. 40.5%, 38.0%; P<0.05) respectively. Following nuclear transfer, the cleavage rate of IVM group was significantly more in 1 μM than 50 μM and 100 μM groups (92.9% vs. 89.7%, 87.8%; P<0.05), followed by similar pattern of cloned blastocysts production being significantly higher in 1 μM group than 50 μM, 100 μM and control groups (16.8% vs. 9.0%, 7.1%, 12.8%; P<0.05) respectively. In IVC group, 1 μM concentration resulted in significantly higher cleavage rate than 25 μM and 50 μM groups (91.7% vs. 87.8%, 88.8%; P<0.05) respectively. However, the blastocysts production was significantly higher in 100 μM group than others (26.2% vs. 13.6%, 14.0%, 18.2%; P<0.05) respectively. The optimal concentrations of flavone significantly enhanced the percentages of ICM:TE than control group (43.8% vs. 37.6%; P<0.05) accompanied with significantly higher expression levels of reprogramming related genes. In conclusion, the optimal concentrations of 1 μM during IVM and 100 μM during IVC can significantly improve the production of porcine in-vitro embryos.

The osmolarity of a medium that is commonly used for in vitro culture (IVC) of oocytes and embryos is lower than that of oviductal fluid in pigs. In vivo oocytes and embryos can resist high osmolarities to some extent due to the presence of organic osmolytes such as glycine and alanine. These amino acids act as a protective shield to maintain the shape and viability in high osmotic environments. The aim of this study was to determine the effects of glycine or/and alanine in medium with two different osmolarities (280 and 320 mOsm) during IVC on embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. To this end, IVC was divided into two stages; the 0-2 and 3-7 days of IVC. In each stage, embryos were cultured in medium with 280, 320, or 360 mOsm and their combinations with or without glycine or/and alanine according to the experimental design. Treatment groups were termed as, for example, "T(osmolarity of a medium used in 0-2 days of IVC)-(osmolarity of a medium used in 3-7 days of IVC)" T280-280 was served as control. When PA embryos were cultured in medium with various osmolarities, T320-280 showed a significantly higher blastocyst formation (29.0%) than control (22.2%) and T360-360 groups (6.9%). Glycine treatment in T320-280 significantly increased blastocyst formation (50.4%) compared to T320-280 only (36.5%) while no synergistic was observed after treatment with glycine and alanine together in T320-280 (45.7%). In contrast to PA embryonic development, the stimulating effect by the culture in T320-280 was not observed in SCNT blastocyst development (27.6% and 23.7% in T280-280 and T320-280, respectively) whereas the number of inner cell mass cells was significantly increased in T320-280 (6.1 cells vs. 9.6 cells). Glycine treatment significantly improved blastocyst formation of SCNT embryos in both T280-280 (27.6% vs. 38.0%) and T320-280 (23.7% vs. 35.3%). Our results demonstrate that IVC in T320-280 and treatment with glycine improves blastocyst formation of PA and SCNT embryos in pigs.

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.

Mitochondrial dysfunction is found in oocytes and transmitted to offspring due to maternal obesity. Treatment of obese mothers with endoplasmic reticulum (ER) stress inhibitors such as salubrinal (SAL) can reverse the mitochondrial dysfunction and result in normal embryonic development. Pig oocytes have also shown ER stress mostly in metaphase II stage. ER stress in oocytes may hinder the in vitro production of pig embryos. This study investigated the effect of ER stress inhibition by SAL treatment during in vitro maturation (IVM) of porcine oocytes at 1, 10, 50 and 100 nM concentrations. Firstly, we tested various concentrations of SAL. SAL at 10 nM showed higher (P < 0.05) developmental competence to the blastocyst stage (55.6%) after parthenogenesis (PA) than control (44.2%) while not different from other concentrations (49.2, 51.6, and 50.8% for 1, 50, and 100 nM, respectively). Secondly, we performed time-dependent treatment at 10 nM of SAL for IVM of oocytes. It revealed that treatment with SAL during 22 to 44 h of IVM significantly improved PA embryonic development to the blastocyst stage compared to control (40.5, 46.3, 51.7 and 60.2% for control, 0 to 22 h, 22 to 44 h and 0 to 44 h of IVM, respectively, P < 0.05). Glutathione (GSH) content is an indicator of cytoplasmic maturation of oocytes. Reactive oxygen species (ROS) have a harmful effect on developmental competence of oocytes. For this, we determined the intraoocyte levels of GSH and ROS after 44 h of IVM. It was found that SAL increased intraoocyte GSH level and also decreased ROS level (P < 0.05). Finally, we performed somatic cell nuclear transfer (SCNT) after treating oocytes with 10 nM SAL during IVM. SAL treatment significantly improved blastocyst formation of SCNT embryos compared to control (39.6% vs. 24.7%, P < 0.05). Our results indicate that treatment of pig oocytes with ER stress inhibitor SAL during IVM improves preimplantation development PA and cloned pig embryos by influencing cytoplasmic maturation in terms of increased GSH content and decreased ROS level in IVM pig oocytes.

Lysophosphatidic acid (LPA) is an important signaling molecule. Here, the effect and mechanism of LPA on the preimplantation development of porcine embryos during in vitro culture (IVC) was examined. Porcine embryos were cultured in porcine zygote medium (PZM-3) supplemented with 30 μM LPA during different days. There was a significantly higher cleavage rate in Day 1-7 and significantly higher total cell number of blastocysts in Day 1-3 and Day 4-7. It was also found that messenger RNA (mRNA) expression level of PCNA, BCL2 and BAX in blastocysts obtained from D1-7 group were significantly higher and BCL2/BAX mRNA ratio in D1-3 group was significantly lower than control group but Day 4-7 and Day 1-7 groups were comparable with control group. Treatment with 20 μM PLC inhibitor significantly decreased the embryo cleavage rate and blastocyst formation rate. Moreover, LPA as an activator of PLCs, enhanced the 30 μM LPA + 20 μM U73122 group embryo cleavage rate which similar with control group. In conclusion, the results suggest that treatment with LPA during IVC improves the porcine early embryo cleavage by activation of PLC signaling pathway and regulate the mRNA expression that contribute to total cell number of blastocysts during blastocyst formation.

Lysophosphatidic acid (LPA) is an important signaling molecule which mediates many different cellular responses. The purpose of this study was to investigate the effect of in vitro culture (IVC) medium supplemented with LPA on the preimplantation embryonic development of porcine embryos derived from in vitro fertilization (IVF). Embryos derived from IVF were cultured in PZM-3 medium supplemented with 30 μM LPA on Day 1 to Day 7, Day 1 to Day 3 (early stage), or Day 4 to Day 7 (late stage), or without LPA. Moreover, the messenger RNA (mRNA) expression of obtaining blastocysts from each group were analyzed. Data were analyzed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science) mean ± SEM. There was a significantly higher cleavage rate in Day 1 to Day 7 than control (71.25% and 57.46%, respectively) and significantly higher total cell number of blastocysts in Day 1 to Day 3 and Day 4 to Day 7 than control (56.07,56.53 and 45.19, respectively). The results also showed that the mRNA expression level of PCNA, Bcl-2 and Bax in Day 1 to Day 7 group blastocysts were significantly higher than control and the expression level of Bax in Day 1 to Day 3 was also significantly higher than control. Moreover, it also showed that Bcl-2/Bax mRNA ratio in D1-3 group was significantly lower than control but D4-7 and D1-7 groups were comparable to control group. In conclusion, our results suggest that treatment with 30 μM LPA during IVC improves the porcine early embryo cleavage and the blastocyst total cell number after IVF and regulating the mRNA expression of blastocysts during blastocyst formation.

Post-ejaculation of sperms into the female reproductive tract, acquisition of sperm capacitation is an essential step in the fertilization process. Accordingly, during in-vitro fertilization, the successful fertilization requires necessarily induction of capacitation in the retrieved sperms. To date, many candidate substances have been considered as capacitation inducers. However, there were no reports about the comparison of efficiency inducing sperm capacitation among diverse capacitation inducers. Therefore, we tried to determine an inducer showing the best capacitation performance in mouse sperms by comparing the preimplantation development of in-vitro-fertilized embryos using sperms experiencing capacitation by a variety of capacitation inducers. For these, calcium, progesterone, bovine serum albumin (BSA), heparin, lysophosphaticylcholine (Lyso-PC) were used as candidate capacitation inducers. Optimized concentration of each inducer were determined by accessing ratio of sperms experiencing acrosome reaction using coomassie G-250 blue staining. Subsequently, in vitro fertilization was performed using sperms incubated in each optimized concentration inducer. The ratio of fertilized oocytes was observed. As the results, Calcium at 2.7 mM and 0.3% (w/v) BSA showed the highest fertilization rates compared to 15 μM progesterone, 50 mM heparin, and 100 μM Lyso-PC. From these results, we found that 2.7 mM calcium and 0.3% (w/v) BSA were the most effective sperm capacitation inducers of mouse sperm for in vitro fertilization. From these results, we could identify that, among diverse sperm capacitation inducers, 2.7mM calcium and 0.3% (w/v) BSA were the most effective inducers for in vitro fertilization.

Polo-like kinase 1 (plk1) shows multiple events of somatic cell and mammalian oocyte division. In mice, Plk1 distributes to the centromeres from prophase to anaphase and compose spindle apparatus at different stages of mitosis in spindle organization. Somatic cell nuclear transfer (SCNT) has a number of advantages however it is difficult to apply to basic or translational researches due to its low cloning efficiency. The causes of this low cloning efficiency are unclear. However, they are attributed to the cumulative results of several biological and technical factors. In this study, a biological factor plk1 was investigated. B6D2F1 mice (7–8 weeks old) were superovulated with 10 IU of pregnant mare’s serum gonadotropin and 9 U of human chorionic gonadotropin (HCG) 48 hr later. The oocytes were then collected 14 hr after HCG injection and cultured on potassium simplex optimized medium (KSOM). The plk1-specific inhibitor BI2536 was used to understand the influence of plk1. The 2-cell stage embryos were assessed by fluorescence immunoassay. In consequence, all BI2536-treated embryos failed in the first mitotic division which showed plk1 have critical role in the first mitotic division of the mouse embryo. SCNT requires enucleation of oocyte and injecting a donor cell into the enucleated cytoplast. In this process, a respectable amount of plk1 that co-localize with nucleus may be removed together. Fluorescence immunoassay and qPCR were used to monitor the change of plk1 level during SCNT. There was significant difference between the control and enucleated embryos in the level of plk1. In all division-failure 2-cell embryos, incorrect positioning of plk1 was found. Taken together, this results demonstrate that plk1 is critical for successful mitotic division of mouse SCNT 1-cell embryos.

The nature of molecular mechanisms governing embryo development is largely unknown, but recent reports have demonstrated that proper execution of programmed cell death is crucial for this process. The main objective of this study is to examine the mode of programmed cell death during nuclear transfer embryos development in porcine. In particular, the relative employment of two major pathways in programmed cell death; e.g. apoptosis (type I) and autophagy (type II) was compared. Oocytes use in the study was matured in vitro in the presence of 10% FBS maturation medium. After nuclear transfer embryos were cultured for each programmed cell death control factor [Cysteamine(Cyst : 0.4mM), 3-methyladenine(3MA : 2.5mM) and Rapamycin(RP : 100nM)] in TCM-199 medium supplemented with 0.1% BSA. In this study results of among the blastocysts development in 3MA; PCNA, MAP1LC3A and ATG5 RNA gene expression level increased in the order of IVF<Cyst < 3MA < RP. However Casp-3 and TNF-r RNA gene expression level decreased in the order of IVF < 3MA and RP< Cyst. The expression of mTOR showed a pattern opposite to that of MAP1LC3A. That is, its expression was the lowest in Cyst group. And next experiments analysis of MMP expression patterns. Analysed this MMPs enzyme activation to evaluate the effectiveness of high quality brastocyst culture in porcine. In this results of the enzymatic activity of MMP-2 and MMP-9 was assessed in culture, the level of active MMP-9 was higher expression in the medium of each 3MA and RP treatment group, with the level of another treatment group being relatively higher. These results suggest that the autophagy activation culture medium is more effective for stable and innovative nuclear transfer embryos development.

Abnormal epigenetic reprogramming of donor nuclei is supposed to be one of the factors that causes low development efficiency of mammalian somatic cell nuclear transfer (SCNT). Trichostatin A (TSA) is an inhibitor of histone acetylase, and so development of SCNT embryos could be increased by treatment with TSA. In the present study, we examined the effect of TSA on in vitro development of porcine embryos derived from NT (nuclear transfer) by investigating the status of histone acetylation in TSA-treated and control NT embryos and the expression of developmental related genes. In this study, we found that incubating　NT embryos with 40nM TSA for 24h after activation could improved the blastocyst formation rate from 13.7% to 32.5%. Thechange in histone acetylation level as a reslut of TSA treatment were validated using immunofluorescence and confocal microscopy. Immunofluorescence results indicated that the level of aetylation at histone 3 lysine 18 (AcH3K18) was increased at early embryo development stage after TSA treatment. furthermore, we compared the expression patterns of several genes (developmental related genes; Oct4, Sox2, Nanog, Cdx2, the imprinting genes; igf2r). TSA treatment improved the expression of development related genes such as Oct4, Cdx2, Nanog as well as the imprinted genes like igf2r. In conclusion, our results demonstrated that TSA treatment improves the in vitro development of porcine NT embryos, increased the global histone acetylation (AcH3K18) and enhances the expression of some developmentally important genes (Oct4, Cdx2, Nanog) at blastocyst stages.

Mitochondrial dysfunction is found in oocytes and transmitted to the offspring due to maternal obesity. This is curable by endoplasmic reticulum (ER) stress inhibitors such as salubrinal (SAL). Recently pigs are considered as a model animal for biomedical research due to its physiological similarity with human. Pig oocytes have shown ER stress mostly in metaphase II stage. ER stress is hindering the in vitro embryo production (IVP). This study investigated the effect of ER stress inhibition by using SAL during 44 h of in vitro maturation (IVM) of oocytes at 1, 10, 50 and 100 nM concentrations. Firstly, we defined the concentration of SAL during IVM of pig oocytes. SAL at 10 nM showed higher (44.2 to 55.6%, P<P0.05) development competence to the blastocyst state than control and other concentrations after parthenogenetic activation (PA). Secondly, we sorted out the time-dependent treatment at 10 nM of SAL for IVM of oocytes. It revealed that treatment with SAL during 22 to 44 h and 0 to 44 h of IVM improved PA embryonic development significantly (40.5, 51.7 and 60.2% for control, 22 to 44 h and 0 to 44 h of IVM, respectively, P<0.05). Glutathione (GSH) level is an indicator of cytoplasmic maturation of oocytes. Reactive oxygen species (ROS) have a harmful effect on development competence of oocytes. For this, we determined the intraoocyte levels of GSH and ROS after 44 h of IVM. It was found that SAL increased intraoocyte GSH level and also decrease ROS level (P<0.05). Finally, we performed somatic cell nuclear transfer (SCNT) after treating oocytes with 10 nM SAL during IVM. SAL treatment significantly improved blastocyst formation of SCNT embryos compared to control (24.7 vs. 39.6%, P<0.05). Our results indicate that treatment of pig oocytes with ER stress inhibitor SAL during IVM improves preimplantation development cloned pig embryos by influencing cytoplasmic maturation in terms of increased GSH content and decreased ROS level in IVM pig oocytes.

Lysophosphatidic acid (LPA) is a member of the phospholipid autacoid family and has growth factor and hormone-like activities on various animal cells. In this study, we investigated the effect of LPA on porcine embryo development. Porcine parthenogenetic embryos were treated into various concentrations of 0 (control), 0.1, 1 and 10 μM LPA (0 LPA, 0.1 LPA, 1 LPA and 10 LPA) during in vitro culture for 7 days or cultured in basic culture medium until day 4 and treated LPA from day 4 to day 7. In the LPA treatment for culturing from day 0 to day 7, there was no significant difference on cleavage and blastocyst formation rate. In addition, the blastocyst development proportion which was classified as expanded, hatching, or hatched blastocystshas was no significant difference among all groups. In the LPA treatment for culturing from day 4 to day 7, 0.1 and 1 LPA groups were presented increased blastocyst formation compared to other groups, but cleavage rate and over-expanded blastocyst formation rate were not significantly different among all LPA treated groups. The total cell number was not different but apoptosis was reduced when 1 LPA treated from day 4 to day 7. The relative mRNA expression level of anti-apoptosis gene, BCL2L1 was higher and pro-apoptosis gene, BAK was lower in the 1 LPA treated group than the control. In comparison with the control and the 1 LPA treated group using time-lapse monitoring system, 1 LPA treated embryo was accelerated developmental speed via morula compaction and expanded blastocyst. The 1 LPA treated group significantly increased the relative expression levels of gap junction and tight junction related genes, GJD1, CDH1 and ZO-1 compared to the control. These results indicated that 1 μM LPA supplementation for culturing from day 4 to day 7 post activation is efficient in blastocyst formation and LPA may be helpful for embryo developmental capacity.

This study is performed to evaluate the effect of insulin in the porcine parthenogenetic embryo development. In porcine embryo culture, insulin is helpful factor in the process of embryo development. To identify this, insulin is used in pig embryos development. Therefore, this study was performed to investigate the effect of insulin on early embryonic development in pigs. For that, insulin positive or negative (0, 10 ug/mL) was supplemented in the porcine IVM media and then compared two groups divided by the cytoplasm of the black groups and white ring groups based on the distribution of lipid material of the cell cytoplasm in microscope. In maturation rates of porcine oocytes, significant higher black group rates were shown in the insulin positive groups compared with other groups (56.0±2.1 vs 46.2±0.3). In the embryo culture, black groups were showed the significant higher cleavage rates (82.1±0.8, 78.3±0.1 vs 63.2±0.3, 63.4±0.0), and blastocyst formation rates (15.5±3.6, 16.6±0.4 vs 11.7±1.3, 7.4±0.2) regardless of whether the addition of insulin. Also, black groups were showed higher cell number of blastocyst (33.2±2.5, 35.5±2.6 vs 31.2±2.1, 31.3±2.2). In conclusion, supplement of insulin producing black group in vitro maturation, it was effective in vitro maturation and embryonic development of pig embryos.

Edaravone (Eda) is a potent scavenger of inhibiting free radicals including hydroxyl radicals (H2O2). Reactive oxygen species (ROS) such as H2O2 can alter most kinds of cellular molecules such as lipids, proteins and nucleic acids, cellular apoptosis. In addition, oxidative stress from over-production of ROS is involved in the defective embryo development of porcine. Previous study reported that Eda has protective effects against oxidative stress-like cellular damage. However, the effect of Eda on the preimplantation porcine embryos development under oxidative stress is unclear. Therefore, in this study, the effects of Eda on blastocyst development, expression levels of ROS, and apoptotic index were first investigated in preimplantation porcine embryos. After in vitro fertilization, porcine embryos were cultured for 6 days in PZM medium with Eda (10 μM), H2O2 (200 μM), and Eda+H2O2 treated group, respectively. Rate of blastocyst development was significantly increased (P<0.05) in the Eda treated group compared with only H2O2 treated group. And, we measured intracellular levels of ROS by DCF-DA staining methods and investigated numbers of apoptotic nuclei by TUNEL assay analysis is in porcine blastocyst, respectively. Both intracellular ROS levels and the numbers of apoptotic nucleic were significantly decreased (P<0.05) in porcine blastocysts cultured with Eda (10 μM). More over, the total cell number of blastocysts were significantly increased (P<0.05) in the Eda-treated group compared with untreated group and the only H2O2 treated group. Based on the results, Eda was related to regulate as antioxidant-like function according to the reducing ROS levels during preimplantation periods. Also, Eda is beneficial for developmental competence and preimplantation quality of porcine embryos. Therefore, we concluded that Eda has protective effect to ROS derived apoptotic stress in preimplantation porcine embryos.

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.

체외 배양액에 성장호르몬 및 사이토카인의 첨가는 초기배 발육 및 생산된 배반포의 질에 영향을 미칠 수 있다. 본 연구는 돼지 유도만능줄기세포(porcine induced pluripotent stem cell, piPSC)의 조정배지(conditioned medium, CM)가 돼지 난자의 체외성숙 및 단위발생 후 초기배 발육에 미치는 영향을 검토하기 위하여 수행하였다. 난자-난구세포 복합체(cumulus-oocyte complex, COC)는 0(control), 25, or 50%의 줄기세포 배양액(stem cell medium, SM) 또는 CM이 첨가된 체외성숙 배양액으로 배양하였으며, 성숙된 난자는 활성화 유도 후 같은 농도의 SM 또는 CM을 첨가한 체외배양액에서 배양하였다. 체외 성숙율은 CM-25% 그룹에서 대조구보다 유의적으로 높았으나(p<0.05), 다른 SM 또는 CM 처리구와는 차이가 없었다. 배반포 형성율은 CM-25% 그룹(29.2%)에서 대조구(20.7%), SM-50%(19.6%) 및 CM-50%(23.66%) 처리구보다 유의적으로 높았다(p<0.05). 배반포에서의 세포수 및 세포사 비율은 SM-25% 그룹이 대조구에 비하여 유의적인 차이가 나타났다(p<0.05). 난자의 질과 연관되어 있는 유전자들(Oct4, Klf4, Tert 및 Zfp42)의 발현은 CM-25% 그룹에서 대조구보다 유의적으로 증가되었다(p<0.05). 따라서 본 실험의 결과 체외성숙(IVM) 및 체외발달(IVC) 배양액에 25% 수준의 CM의 첨가는 돼지 단위발생 난자의 배발달과 난자의 질적 향상에 기여하는 것으로 사료된다.