The aim of present study was to investigate regulatory mechanism of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation of porcine oocytes. Basically, immature cumulus-oocyte complexes (COCs) were incubated for 22 h in IVM-I to which hormone was added, and then further incubated for 22 h in IVM-II without hormone. As a result, relative cumulus expansion was increased at 22 h after IVM and it was enhanced by treatment of ALA compared with control group (p < 0.05). During IVM process within 22 h, cAMP level in oocytes was decreased at 6 h (p < 0.05) and it was recovered at 12 h in ALA-treated group, while oocytes in control group recovered cAMP level at 22 h. In cumulus cells, it was reduced in all time point (p < 0.05) and ALA did not affect. Treatment of ALA enhanced metaphase-I (MI) and MII population of oocytes compared with oocytes in control group at 22 and 44 h, respectively (p < 0.05). Intracellular GSH levels in ALA group was increased at 22 and 44 h after IVM (p < 0.05), whereas it was increased in control group at 44 h after IVM (p < 0.05). In particular, the GSH in ALA-treated oocytes during 22 h of IVM was higher than control group at 22 h (p < 0.05). Lipid amount in oocytes from ALA group was higher than control group (p < 0.05). Treatment of ALA did not influence to absorption of glucose from medium. Cleavage and blastocyst formation of ALA-treated oocytes were enhanced compared with control group (p < 0.05). These findings suggest that supplementation of ALA could improve oocyte maturation and development competence through increasing GSH synthesis, lipid storage, and regulation of cAMP accumulation during early 22 h of IVM, and these might be mediated by cumulus expansion.

Gangliosides are glycolipids in which oligosaccharide is combined with sialic acids. Our previous studies have suggested an interplay between ganglioside GD1a/GT1b and meiotic maturation capacity in porcine oocyte maturation. Furthermore, ganglioside GD1a and GT1b are known for its antioxidant activity, but it is still unclear whether possible antioxidant role of GD1a and GT1b is involved in porcine embryos development competence during in vitro culture (IVC). Here, the effects of ganglioside GD1a and GT1b on the embryonic developmental competence during in vitro culture of porcine were investigated. The effects of ganglioside GD1a and GT1b on the expression of ST3GAL2 were confirmed during embryos development (2-cell, 4-cell, 8-cell and blastocyst) using immunofluorescent staining (IF). As a result, the fluorescent expression of ST3GAl2 was higher in embryos at 4-8 cells stage than blastocysts. Blastocyst development rate significantly increased in only 0.1 μM GD1a and GT1b treated groups compared with control group. To investigate the cellular apoptosis, we analyzed TUNEL assay. In case of only 0.1 μM GD1a and GT1b treated groups, the total number of cells in blastocyst compared with control group, but there was no significant difference in the rate of apoptotic cells. We identified the intracellular ROS levels using DCF-DA staining. According to the result, ROS production significantly decreased in blastocysts derived from the 0.1 μM GD1a and GT1b treated groups. These results suggest that ganglioside GD1a and GT1b improve the developmental competence of porcine embryos via reduction of intracellular ROS during preimplantation stage.

Embryos produced with serum show the alterations in their ultrastructure, impaired compaction, abnormal blastulation, aberrant mRNA expression profiles and large calf syndrome with greater incidences of stillbirths and deaths after birth. The aim of the present study was to describe in vitro embryo production by analyzing embryo production, fetal production and pregnancy rate in free-serum medium. The OPU-IVP data used in this study from 2016. Approximately, sixteen cows (Hanwoo), which belonged to the Institute of Gyeongsang National University, were used. Two experimental group is used in this study. Serum groups were conducted in March to July and free-serum group was conducted in September to December. The recovered cumulus-oocyte complexes were morphologically classified to four grades based on the compaction of cumulus cells layers and homogeneity of the cytoplasm. The number of oocyte was significantly greater in serum groups than that in free-serum groups (29.61 ± 0.63 vs. 15.6 ± 0.62; p < 0.05). Between serum and free-serum groups indicate that average of 1st and 2nd grade oocytes were no difference (2.38 ± 1.67 vs. 2.38 ± 1.48; p > 0.05), but number of 3rd and 4th grade oocytes were greater in serum groups than that in free-serum groups (7.31 ± 7.64 vs. 5.60 ± 6.29; p < 0.05). Embryo cleaved competence was higher in rate in free-serum groups than that in serum groups (62.1% vs. 58.3; p < 0.05). However, blastocyst developmental rate was no difference between serum and free-serum groups (33.1% vs. 43.5%; p < 0.05). 986 recipients were used for embryo transfer. Pregnancy rate was indicated that between serum and free-serum group was no difference (54.6% vs. 56.3%; p < 0.05). In conclusion, we developed the free-serum system for production of in vitro bovine embryos in order to meet the developmental and qualitative requirements for large scale commercial use.

The aim of this study was to investigate effects of hyaluronidase during IVM on oocyte maturation, oxidative stress status, expression of cumulus expansion-related (PTX, pentraxin; GJA1, gap junction protein alpha 1; PTGS2, prostaglandin-endoperoxide synthase 2) and fatty acid metabolism-related (FADS1, delta-6 desaturase; FADS2, delta-5 desaturase; PPARα, peroxisome proliferator-activated receptor-alpha) mRNA, and embryonic development of porcine oocytes. The cumulus-oocyte complexes (COCs) were incubated with 0.1 mg/mL hyaluronidase for 44 h. Cumulus expansion was measured at 22 h after maturation. At 44 h after maturation, nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured. Gene expression in cumulus cells was analyzed using real time PCR. The cleavage rate and blastocyst formation were evaluated at Day 2 and 7 after insemination. In results, expansion of cumulus cells was suppressed by treatment of hyaluronidase at 22 h after maturation. Intracellular GSH level was reduced by hyaluronidase treatment (p < 0.05). On the other hand, hyaluronidase increased ROS levels in oocytes (p < 0.05). Only PTGS2 mRNA was enhanced in COCs by hyaluronidase (p < 0.05). Population of oocytes reached at metaphase II stage was higher in control group than hyaluronidase treated group (p < 0.05). Both of cleavage rate and blastocyst formation were higher in control group than hyaluronidase group (p < 0.05). Our present results showed that developmental competence of porcine oocytes could be reduce by hyaluronidase via inducing oxidative stress during maturation process and it might be associated with prostaglandin synthesis. Therefore, we suggest that suppression of cumulus expansion of COCs could induce oxidative stress and decrease nuclear maturation via reduction of GSH synthesis and it caused to decrease developmental competence of mammalian oocytes.

Generally, in vivo, primary oocytes are grown and matured into secondary oocytes in the ovarian follicles. Quality of the oocytes matured in vivo is higher than that of oocytes matured in vitro, indicating the importance of materializing the microenvironment of ovarian follicles for production of high quality oocyte. Therefore, we tried to mimic the stiffness of ovarian follicles using an agarose as a biocompatible natural polymer. Unfortunately, to date, there are no many reports on whether the quality of porcine oocytes can be increased effectively under the soft matrix. Accordingly, we tried to evaluate the effects of IVM using different mechanical properties of agarose substrate on developmental competence of porcine oocytes. Agarose substrate was constructed and cumulus-oocyte-complexes (COCs) retrieved from porcine medium antral follicles were matured on non-coated (control) culture dish or dishes coated with 1% and 2% (w/v) agarose substrate. Then, cumulus expansion, embryonic development after parthenogenetic activation, and gene expression level were analyzed and compared. As the results, significant increase in blastocyst formation and cumulus expansion were detected in COCs matured on 1% (w/v) agarose substrate compared with control. Moreover, oocytes of COCs matured on 1% (w/v) agarose substrate showed significantly higher BMP15 expression level compared with control. Pro-apoptotic gene BAX expression was significantly increased in oocytes of COCs matured on 2% (w/v) agarose substrate compared with control. In the glycolytic enzyme phosphofructokinase (PFKP) gene expression, cumulus cells of COCs matured on agarose substrate showed significantly higher PFKP expression than control while they showed significantly lower BAX expression than control. These results demonstrated that quality of porcine oocytes could be increased efficiently by the IVM of immature oocytes on the soft culture matrix using agarose.

Mammalian oocytes are sensitive to psychological stress at each period of follicular development. Especially, thermal stress interfere with reproductive condition by inducing formation of reactive oxygen species (ROS) and oxidative stress (OS). ROS lead to oocyte apoptosis, weakening oocyte quality and lowering the fertilization rate. As a result, the pregnancy rate is lowered, leading to infertility. Thermal stress also seems to influence zygotes through physiological changes in the maternal environment surrounding them. Loss of developmental competence suggests hyperthermia-induced oxidative stress in embryos. Interest in organic Lonicera caerulea berries has increased in recent years. They are abundant in various health-improving materials. Berries that found from natural products can be as free as possible from the bioactive toxicity of the active ingredient without side effects, and it can be a big advantage because it can work. Mammalian oocytes are arrested at the first meiotic prophase stage and get their meiotic competence to produce offspring during the development of follicle. A series of nuclear and cytoplasmic maturations are involved in this process and these vary in temperature sensitivity. Our study demonstrated that L. caerulea can relieve the negative effects of maternal hyperthermia by reducing ROS level at the developmental stage.

In this study, we examined sperm penetration and blastocyst developmental rate of oocytes to determine fertilizability of cauda epididymal spermatozoa in Hanwoo bull. One testicle with epididymides were castrated from one Hanwoo bull (14 months of age) and transported to laboratory. Spermatozoa recovered from cauda epididymis by mincing with semen extender (Optixcell, IMV, France) and cryporeserved in liquid nitrogen tank until use. As control, frozen Hanwoo semen was used. Cumulus oocyte complexes (COCs) were collected from follicles (2-8 mm) of slaughtered ovaries and 10 to15 COCs were matured in 50μl droplet with M-199 media supplemented with 10% fetal bovine serum, 10μg/ml FSH, 10μg/ml LH, 10μg/ml EGF for 22 to 24 hours in a humidified atmosphere of 5% CO2 in air. After maturation of COCs, matured COCs were co-incubated with cauda epididymal spermatozoa in 100μl droplet in modified Brackett and Oliphant media supplemented with 2.5 mM theophylline for 12 or 18 hours under 5% CO2 in air. Sperm concentration was adjusted to 5 × 106cells/ml. After IVF for 18 hours, presumptive zygotes were cultured in modified synthetic oviductal fluid with 1mM glutamine, 12 essential amino acids, 10 μg/ml insulin under 5% CO2, 5% O2 in air. In experiment 1, we examined sperm penetration rate at 12 hours of IVF of frozen-thawed epididymal sperm. Total penetration rate among cauda epididymis and control were not significantly different (mean±standard error, cauda epididymis and control vs. 49.7±11.3 and 54.4±12.8%) In experiment 2, cleavage and blastocyst development rate were evaluated at day 2 and day 8 after IVF for 18 hours. Cleavage rate among cauda epididymis and control was similar different (cauda epididymis and control vs. 81.2±3.4 and 82.7±2.5%). However, blastocyst developmental rate of cauda epididymis group was significantly higher than that of control group (cauda epididymis and control vs. 24.4±1.6 and 12.2±2.8%, p<0.05). In conclusion, cauda epididymal spermatozoa in Hanwoo bull has high fertilizability and embryo development. Cauda epididymal sperm can be used as an alternative to ejaculated frozen sperm in vitro.

Poor embryo quality and low blastocyst formation have been major limitations in establishment of cloned embryonic stem cells and production of cloned animals through somatic cell nuclear transfer (SCNT). Aggregation of embryos is a promising method for improving developmental competence of blastocysts. The aim of this study was to improve the blastocyst formation and the quality of parthenogenetic (PA) pig embryos by the aggregation of blastomeres at the 4-cell stage that were cultured in various type of culture dishes with or without phytohemagglutinin (PHA). The PA embryos were produced by the general method of our laboratory. On Day 2 after PA, the zona pellucida of 4 cell-stage embryos were removed by treatment with 0.5% (wt/vol) pronase solution. The 3x zona-free blastomere (ZFB) were randomly distributed in each of the following treatments for aggregation. ZFB were cultured for 5 days at 39℃ in an atmosphere 5% CO2, 5% O2, and 90% N2. In Experiment 1, effect of culture dishes on the aggregation efficiency and developmental competence of PA embryos were investigated. ZFB were cultured on non-coated (control) culture dish or dishes coated with 1% (wt/vol) agarose substrate (AS) or Well of the Well in dishes coated with 1% (wt/vol) agarose substrate (WAS). The ZFB cultured in WAS showed significantly higher (P<0.05) aggregation (81.2%) than AS and control (21.6-45.5%). The mean cell number in blastocysts derived from AS and WAS (81.4-89.3 cells/blastocyst) was significantly higher (P<0.05) than that of control (63.8 cells/blastocyst). In Experiment 2, effects of 150 ug/ml PHA treatment on the aggregation efficiency and developmental competence of embryos were investigated. The ZFB cultured in AS with PHA showed a higher (P<0.05) aggregation rate (90.0%) than that in AS without PHA, control with PHA, and control (39.2%, 57.9% and 17.5%, respectively). In conclusion, aggregation of porcine ZFB treated with PHA and agarose substrate could be a useful technique for producing improving blastocyst development with increased mean cell number of blastocysts in pigs.

In most mammals, metaphase II (MII) oocytes having high maturation promoting factor (MPF) activity have been considered as good oocytes and then used for assisted reproductive technologies including somatic cell nuclear transfer (SCNT). Caffeine increases MPF activity in mammalian oocytes by inhibiting p34cdc2 phosphorylation. The objective of this study was to investigate the effects of caffeine treatment during in Vitro maturation (IVM) on oocyte maturation and embryonic development after SCNT in pigs. To this end, morphologically good (MGCOCs) and poor oocytes (MPCOCs) based on the thickness of cumulus cell layer were untreated or treated with 2.5 mM caffeine during 22-42, 34-42, or 38-42 h of IVM according to the experimental design. Caffeine treatment for 20 h during 22-42 h of IVM significantly inhibited nuclear maturation compared to no treatment. Blastocyst formation of SCNT embryos was not influenced by the caffeine treatment during 38-42 h of IVM in MGCOCs (41.1-42.1%) but was significantly improved in MPCOCs compared to no treatment (43.4 vs. 30.1%, P<0.05). No significant effects of caffeine treatment was observed in embryo cleavage (78.7-88.0%) and mean cell number in blastocyst (38.7-43.5 cells). The MPF activity of MII oocytes in terms of p34cdc2 kinase activity was not influenced by the caffeine treatment in MGCOCs (160.4 vs. 194.3 pg/ml) but significantly increased in MPCOCs (133.9 vs. 204.8 pg/ml). Our results demonstrate that caffeine treatment during 38-42 h of IVM improves developmental competence of SCNT embryos derived from MPCOCs by influencing cytoplasmic maturation including increased MPF activity in IVM oocytes in pigs.

The elevated temperature and high humidity has been known as main reason for heat stress on animals and cause detrimental effects on productivity of organisms and physiological conditions of normal bioactivities. The aims of this study were to evaluate the relationship between time of heat shock simulation during in vitro maturation and developmental competence of subsequent embryo after in vitro fertilization. Heat shocked cumulus-oocyte complexes (COCs) of Korean native cattle were subjected to normal conditions for 22, 21, 18 and 12 h respectively and transferred to heat stress inducing condition at 40.5 °C in other incubator for 0 (control), 1 and 4 h. After maturation for 22 h, the oocytes were fertilized and cultured in mSOF media for 8 d and examined the developmental capacity of embryos. There were no differences in maturation and cleavage rates between 0, 1 and 4 h heat socked oocytes, but blastocysts formation were lower in the 4 h heat stressed oocytes. The apoptotic cells of developed blastocysts were also increased in at day 8 with 4 h heat shocked oocytes. These results indicate that heat shock on oocytes during maturation could cause negative effects on the developmental competence of 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.

The objective of this study was to establish the effect of post-activation treatment with cytoskeletal regulators of CB, CB+CHX, CB+DC, CB+6’DMAP on embryonic development of pig oocytes after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). PA and SCNT embryos were produced by using in vitro matured pig oocytes and treated for 4 h after electric activation with cytochalasins B (7.5 μg/ml), CB+cycloheximide (10 μg/ml), CB+demecolcine (0.4 μg/ml), and CB+2mM 6-Dimethylaminopurine. Post-activation treatment of PA oocytes with CB, CB+CHX, CB+DC and CB+6’DMAP no significant differences were found in embryo cleavage (83.2～91.5%), mean cell number of blastocysts (40.6～ 42.3% cells/blastocyst) but significantly (P<0.05) differences blastocyst formation (28.6～36.4%). When PA oocytes were treated with CB, CB+CHX, CB+DC, CB+6’DMAP, blastocyst formation was significantly (P<0.05) improved by CB (36.6%) compared to CB+CHX (30.9%), CB+DC (28.6%) and CB+6’DMAP (35.2%). In SCNT, was not significantly (P<0.05) increased by post-activation treatment with CB+CHX (81.3%), CB+DC (83.9%) and CB+6’DMAP (90.0%) compared to CB (84.5%) on embryo cleavage, blastocyst formation (19.1%~23.6%) and blastocyst cell number (39.6～41.4% cells/blastocyst) also were not influenced. But increased tendency in CB+6’DMAP. In addition, we investigated survivin expression in porcine SCNT embryos during the early developmental stages. The levels of survivin mRNA in 2-4 cell stage SCNT embryos were significantly higher 6’DMAP treated group than other treatment groups of SCNT embryos. These observations suggested that 2-4 cell cleaving embryos at have high developmental competence, and which may be influenced by survivin expression in porcine SCNT embryos. Our results demonstrate that post-activation treatment with CB, CB+CHX, CB+DC, CB+6’DMAP improves pre-implantation development of SCNT embryos and the stimulating effect of cytoskeletal modifiers on embryonic development is differentially shown depending on the origin (PA or SCNT) of embryos in pigs.

Ganglioside GT1b, glycosphigolipids with three sialic acid, is known to play an important role in signal transduction such as epidermal growth factor receptor (EGFR). EGF is also known to induce resumption of meiosis and cumulus cells expansion during porcine oocyte maturation. Therefore, this study was conducted to evaluate the effects of ganglioside GT1b on resumption of meiosis and cumulus cells expansion in porcine oocyte maturation. First, porcine cumulus-oocyte complexes were cultured in NCSU-23 medium supplemented with GT1b (0, 1, 2 and 4 μM) at 44 h. We observed that the proportion of the metaphase II (M II) stage was significantly increased in the 2 μM GT1b (78.0 ± 2.3) treated group than in the other groups. Furthermore, expression of cumulus cells expansion factor genes (Has2, TNFAIP6, Ptx3) were significantly increased in the 2 μM GT1b treated group than in the other groups. Next, we investigated the meiotic maturation and the expressions of cumulus cells expansion factor genes after GT1b and/or EGF treatment. The proportion of the M II stage was significantly higher in the GT1b+EGF (90.1 ± 2.3) treated group than in the other groups. Moreover, expressions of cumulus cells expansion factor genes were significantly increased in the GT1b+EGF treated group than in the control group. After in vitro fertilization, fertilization rate, preimplantation development competence and quality of blastocyst were improved in oocytes derived from GT1b+EGF treated group. Taken together, these results suggest that exogenous ganglioside GT1b improving the developmental competence of porcine embryos via increase of resumption of meiosis and cumulus cells expansion during in vitro maturation of porcine oocytes.

The clustered regularly interspaced short plalindromic repeats(CRISPR)/CRISPR associated protein (Cas9) system can be applied to produce transgenic pigs. We applied CRISPR/Cas9 system to generate hG-CSF targeted pig parthenogenetic embryos. Using sigle guided RNA targeted to pig hG-CSF genes was injected into cytoplasm of in vitro matured oocyte before electrical activation. The CRISPR/Cas9 vector were diluted in Tris-EDTA buffer (TE buffer) and injected with different concentration of 0 (sham injection), 2.5 and 25 ng/ul. In results, regardless of the concentrations of vector, the cleavage and blastocyst rate were not significantly different among three groups. Since plasmid DNA was used for microinjection, we investigated whether DNA vectors were integrated into the genome. Genomic PCR of the coding sequence of Cas9 variants and hG-CSF was performed to detect genomic integrants. Each blastocysts were collected into a microtube, and then PCR was performed. Overall 32 embryos are not expressed targeted gene.

In mature oocytes, maturation promoting factor (MPF) activity is playing important roles in arrest at M-phase and its continuous phenomenon, oocyte aging. In most mammals, metaphase II oocytes show high MPF activity and have been used as ooplasts in somatic cell nuclear transfer (SCNT). Caffeine has been found to regulate MPF activity in mammalian oocytes. Caffeine inhibits p34cdc2 phosphorylation and increases MPF activity. The present study investigated the effects of caffeine treatment during last 4 hours of in vitro maturation (IVM) on oocyte maturation and embryonic development after parthenogenesis (PA) and SCNT. The IVM medium was medium-199, 10% (v/v) PFF, cysteine, pyruvate, epidermal growth factor, kanamycin, insulin, and hormones. Immature oocytes were matured in IVM medium without or with 2.5 mM caffeine during the last 4 hours of IVM. The in vitro culture medium for embryonic development was porcine zygote medium-3 containing 0.3% (w/v) bovine serum albumin. Nuclear maturation (83.6–87.2%) and intraoocyte glutathione contents (0.9–1.0 pixels/oocyte) of oocytes were not influenced by the caffeine treatment. The membrane fusion of cell-cytoplast couplets (75.5–76.5%) and cleavage (85.4–86.2%) were also not altered by the caffeine treatment. However, caffeine-treated oocytes showed higher (P<0.05) blastocyst formation after SCNT (47.5 vs. 34.3%) than untreated oocytes. Our results demonstrate that caffeine treatment during last 4 hour of IVM improves the developmental competence of SCNT embryos probably by influencing MPF activity.

The periods of elevated temperature and high humidity has been longer since last ten years and cause problems in program of artificial insemination or at the efficiency of in vitro production of transferable embryos. The aims of this study were to evaluate the relationship between time of heat shock (0, 1, 2 and 4), during in vitro maturation and developmental competence of subsequent embryo after in vitro fertilization. The develpmentat rate and percetage of apoptotic cells were evaluated on matured oocyte and day 8. 41℃ Heat treatment after IVM culture significantly decreased the developmental capacity of IVF embryos. Also the number of apoptotic cell in COCs, morula and blatostcysts was started to increase at 2 hr heat treatment but did not affect on the rate of maturation. These results indicate that heat treatment for 2 to 4 hr at 41℃ have negative effects on maturation rate of COCs and lower the developmental competence of heat shocked oocyte derived embryos.

The cloning efficiency is extremely low despite successful somatic cell nuclear transfer (SCNT) method producing cloned animals in several mammals. In general, faulty epigenetic modifications underlying the incomplete reprogramming of donor cell nuclei after SCNT mainly results in low cloning efficiency. The nuclear reprogramming process involves epigenetic modifications, such as DNA demethylation and histone acetylation, which may be an important factor in enhancing the cloning efficiency. Recently, the histone deacetylase inhibitors (HDACi), such as trichosatin A (TSA) and m-carboxycinnamic acid bishydroxamide (CBHA), to increase histone acetylation have been used to improve the developmental competence of SCNT embryos. Therefore, we compared the effects of TSA with CBHA on the in vitro developmental competence and pluripotency-related gene expression (Oct4, Nanog and Sox2) in porcine cloned blastocysts and histone acetylation pattern (H3K9ac). The porcine cloned embryos were treated with a 50nM concentration of TSA and 100μM concentration of CBHA during the in vitro early culture (10h) after cell fusion and then were assessed to cleavage rate, development to the blastocyst stage and pluripotency-related gene expression in NT blastocyst also, level of histone acetylation in zygote, 2cell, 4cell stage. As results, Although NT, TSA and CBHA treated NT embryos were not different between all groups for cleavage rates, the developmental competence to the blastocyst stage was significantly increased in CBHA treated embryos (22.7%) compared to that of normal NT and TSA treated NT embryos (8.1% and 15.4%)(p<0.05). In addition, all of pluripotent transcription factors (Oct4, Nanog and Sox2) were expressed in the CBHA treated NT embryos, however, Sox2 and Oct4 were expressed in TSA treated NT embryos and expression pattern of CBHA treated NT embryos is particularly similar to that of IVF embryos. Also, CBHA treated NT embryos were increased in level of histone acetylation (H3K9ac) at the zygote, 2-cell, 4-cell stage compared to those of NT and TSA treated NT embryos. In conclusion, the treatment of CBHA as a histone deacetylase inhibitor significantly increased the developmental competence of porcine NT embryos and pluripotency-related gene expressions(Oct4, Nanog and Sox2) in NT blastocysts and level of histone acetylation (H3K9ac).

Differentiated nuclei can experimentally be returned to an undifferentiated embryonic status after nuclear transfer (NT) to unfertilized metaphase II (MII) oocytes. Nuclear reprogramming is triggered immediately after somatic cell nucleus transfer (SCNT) into recipient cytoplasm and this period is regarded as a key stage for optimizing reprogramming. In a recent study (Dai et al., 2010), use of m-carboxycinnamic acid bishydroxamide (CBHA) as a histone deacetylase inhibitor during the in vitro early culture of murine cloned embryos modifies the acetylation status of somatic nuclei and increases the developmental competence of SCNT embryos. Thus, we examined the effects of CBHA treatment on the in vitro preimplantation development of porcine SCNT embryos and on the acetylated status of histone H3K9 on cloned embryos at the zygote stage. We performed the three groups SCNT: SCNT (NT), CBHA treatment at the porcine fetus fibroblast cells (PFFs) used as donor cells prior to SCNT (CBHA-C) and CBHA treatment at the porcine SCNT embryos during the in vitro early culture after oocyte activation (CBHA-Z). The PFFs were treated with a 15 μM of CBHA (8 h) for the early culture and the porcine cloned embryos were treated with a 100 μM concentration of CBHA during the in vitro early culture (10 h). Cleavage rates and development to the blastocyst stage were assessed. No significant difference was observed the cleavage rate among the groups (82.6%, 76.4% and 82.2%, respectively). However, the development competence to the blastocyst stage was significantly increased in CBHA-Z embryos (22.7%) as compared to SCNT and CBHA-C embryos (8.6% and 4.1%)(p<0.05). Total cell numbers and viable cell numbers at the blastocyst stage of porcine SCNT embryos were increased in CBHA-Z embryos as compared to those in CBHA-C embryos (p<0.05). Signal level of histone acetylation (H3K9ac) at the zygote stage of SCNT was increased in CBHA-Z embryos as compared to SCNT and CBHA-C embryos. The results of the present study suggested that treatment with CBHA during the in vitro early culture (10 h) had significantly increased the developmental competence and histone acetylation level at the zygote stage.

본 연구에서 한우를 공란우로 사용하여 OPU 방법으로 가장 더운 계절의 hot season과 선선한 cool season의 두 계절의 차이에 따른 생성된 난포의 수, 난자 회수율, 난자 등급율, 수정율 및 배반포 발달 능력을 분석하여, 두 계절이 공란우의 번식 능력에 미치는 영향에 관하여 조사하였다. 1. 계절의 영향이 OPU 공란우의 난포 생성 수에 미치는 결과는 난포 생성 개수는 1154개(18.32±2.26), 971개(15.41±3.34)로 hot season 그룹이 유의적으로 높은 것을 알 수 있었다(p＜0.05). 2. 계절에 따른 난자 수 및 난자 회수율은 hot season 그룹의 475개(7.54±3.14), 41.16%로 cool season 그룹 448개(7.11±3.42), 46.14%와 비교하여 유의적인 차이가 없었다(p＜0.05). 3. OPU를 통하여 회수된 두 계절별 난자 등급은 Grade A는 Hot season 그룹 110개(1.75±1.86), Cool season 그룹 63개(1.00±1.46)로 hot season 그룹이 cool season 그룹과 비교하여 유의적으로 높았다(p＜0.05). 하지만 다른 등급인 Grade B는 87개(1.38±1.60) vs. 97개(1.54±1.39), Grade C는 166개(2.63±2.43) vs. 170개(2.70±2.04), Grade D는 112개(1.78±2.65) vs. 118개(1.87±1.86)로 hot season과 cool season 간의 유의적인 차이를 보이지 않았다(p＜0.05). 4. 계절에 따른 체외 수정 후의 수정률은 hot season과 cool season 각각 242(66.67%)와 209(63.3%), 배반포 발달율 214(58.95%) vs. 188(56.97%)로 수정률과 배반포 발달율은 유의적인 차이가 없었다(p＜0.05). 본 연구의 결과로 계절에 따른 영향에 의해 공란우의 난포생성수와 A등급의 난자 출현율에서 유의적인 차이를 보였다. 하지만 나머지 등급의 난자 출현율, 수정률 및 배반포 발달율은 차이가 없는 것으로 보아, 계절의 차이로 인한 한우 공란우의 번식 능력에 미치는 영향은 미비하다고 판단된다.