Higher Blastocyst Formation Rate Research Articles

Evaluation of epididymis storage temperature and cryopreservation conditions for improved mitochondrial membrane potential, membrane integrity, sperm motility and in vitro fertilization in bovine epididymal sperm.

The maintaining of the epididymis at lower temperatures during storage and transport improves sperm quality. Our study aimed to test whether epididymis storage temperature (post-mortem) and sperm cryopreservation affect sperm kinetics, membrane integrity, mitochondrial potential and fertility capacity. Thirty-six epididymides were collected from 18 bulls after slaughter and divided into two groups: at 4 or 34°C for 2-3hr. The sperm was collected from the epididymis cauda. The evaluation consisted of computer-assisted sperm analysis (CASA), SYBR14/PI/JC1 to evaluate membrane integrity, mitochondrial membrane potential (MMP) and measurement of lipid peroxidation (TBARS). The sperm was then frozen using an automatic device. After thawing, sperm samples were evaluated by the same variables and further in vitro fertilization rates. Cryopreservation negatively affected sperm motility in samples stored at 4 and 34°C. Nevertheless, the 4°C samples yielded higher rates of blastocyst formation. Pre-freeze sperm motility, progressive motility and velocity were higher in sperm from epididymis stored at 4°C while post-thaw sperm motility, progressive motility and velocity remained the same among samples from epididymis stored at 4 or 34°C. However, with regard to the kinetic patterns, samples collected from epididymis stored at 34°C had lower values when compared to those stored at 4°C prior the cryopreservation process. Our results indicate that epididymis handling conditions after cryopreservation may affect sperm quality after thawing, especially due to compromised MMP in sperm collected from epididymis stored at higher temperatures.

Integrating insulin into single-step culture medium regulates human embryo development in vitro

To evaluate the effect of supplementing single-step embryo culture medium with insulin on human embryo development. Comparative study. Two private centers. The study involved a sibling oocyte split of 5,142 retrieved oocytes from 360 patients. Sibling oocytes split after intracytoplasmic sperm injection for culture from day 0 through day 5 or 6 in insulin-supplemented or control medium. Women were split to receive their embryos from insulin-supplemented or control medium. Clinical pregnancy rate. There were significantly higher rates of clinical, ongoing, and twin pregnancies in the insulin-supplemented arm than in the control arm. On day 3, embryo quality and compaction were higher in insulin-supplemented medium. On day 5, insulin supplementation showed higher rates of blastocyst formation, quality, and cryopreservation. Insulin supplementation of single-step embryo culture medium from day 0 through day 5 or 6 improved clinical pregnancy rate and human embryo development. However, these findings need further confirmation through a multicenter randomized controlled trial that may include other patient populations and different culture media.

The developmental competence of oocytes parthenogenetically activated by an electric pulse and anisomycin treatment.

The aim of this study was to investigate the developmental competence of oocytes parthenogenetically activated by an electric pulse (EP) and treated with anisomycin and to determine whether this method is applicable to somatic cell nuclear transfer (SCNT). Embryos derived from porcine oocytes parthenogenetically activated by an EP and treatment with 0.01µg/mL anisomycin had a significantly improved in vitro developmental capacity. Furthermore, 66.6% of blastocysts derived from these embryos had a diploid karyotype. The blastocyst formation rate of cloned embryos was similar between oocytes activated by an EP and treated with 2mM 6-dimethylaminopurine for 4h and those activated by an EP and treated with 0.01µg/mL anisomycin for 4h. The level of maturation-promoting factor was significantly decreased in oocytes activated by an EP and treated with anisomycin. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR. Our results demonstrate that porcine oocyte activation via an EP in combination with anisomycin treatment can lead to a high blastocyst formation rate in parthenogenetic activation and SCNT experiments.

16 HISTONE DEACETYLASE INHIBITORS PCI-24781 AND QUISINOSTAT IMPROVE THE IN VITRO DEVELOPMENTAL COMPETENCE OF PIG SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

The aim of this study was to examine the effects of PCI-24781 and quisinostat, two novel histone deacetylase inhibitors, on the in vitro development of pig somatic cell NT (SCNT) embryos. Pig fetal fibroblasts were used as donor cells for SCNT embryos. In vitro-matured eggs with first polar body were enucleated by aspiration using a 15-μm inner diameter glass pipette. A single donor cell was inserted into the perivitelline space of each egg and electrically fused using 2 direct pulses of 150 V mm–1 for 50 μs in 0.28 M mannitol. After 1 h, embryos were activated by 2 direct pulses of 100 V mm–1 for 20 μs and incubated with 2 mM 6-DMAP for 4 h. Subsequently, the cloned embryos were cultured in medium for 7 days. In Experiment 1, after activation and treatment with 6-DMAP for 4 h, pig SCNT embryos were treated with various concentrations of PCI-24781 or quisinostat for 24 h. In Experiment 2, NT embryos were treated with 0.5 nM PCI-24781 or 10 nM quisinostat for different durations. The rate of blastocyst formation was significantly higher in the 0.5 nM PCI-24781 group than in the control (25.3 v. 10.5%; P < 0.05; Table 1). Moreover, treatment with 10 nM quisinostat dramatically increased the proportion of embryos that reached the blastocyst stage, in comparison with the control group (18.4 v. 10.7%; P < 0.05). Table 1 shows that SCNT embryos treated with 0.5 nM PCI-24781 for 6 h had higher rates of blastocyst formation than control group (25.2 v. 10.2%; P < 0.05). However, the rate of blastocyst formation was significantly higher in the 10 nM quisinostat for 24 h group than in the control (19.8 v. 10.1%; P < 0.05). We determined the treatment conditions of PCI-24781 (0.5 nM for 6 h) and quisinostat (10 nM for 24 h) that significantly improve the in vitro development of pig SCNT embryos. Table 1.Concentration-dependent and time-dependent effects of treatment with histone deacetylase inhibitors (HDACi) PCI-24781 or quisinostat on the in vitro development of pig SCNT embryos

85 CARBOXYETHYLGERMANIUM SESQUIOXIDE CONFERS ANTIOXIDATIVE PROTECTION FOR PORCINE IN VITRO-FERTILIZED EMBRYOS

Reactive oxygen species (ROS) are generated both from the environment as well as from within the embryos during normal embryo development. However, excessive production of ROS creates the defective effect on embryo development. The purpose of the present study was to determine the effect of antioxidant, carboxyethylgermanium sesquioxide (Ge-132), during in vitro culture (IVC) on embryonic development in porcine in vitro-fertilized (IVF) embryos. The different concentrations of Ge-132 (0, 100, 200, and 400 μg mL–1) were treated to the zygotes (n = 233, 234, 233, and 234, respectively). All of the experiments were repeated at least three times. To characterise putative porcine embryonic stem cell (ESC) derived from Ge-132 treated IVF blastocysts, several assays including an assessment of intensive alkaline phosphatase activity, embryoid body formation and expression analysis of pluripotency by immunofluorescence staining were performed. In the result of embryo developmental competence, the 100- and 200-μg mL–1 Ge-132 significantly (P < 0.05) increased cleavage rates (67.4% and 67.1%, respectively; P < 0.05) compared with the control group (61.6%). The 100 μg mL–1 Ge-132 had significantly (P < 0.05) higher blastocyst formation rates (32.6%) than the control group (26.5%). The Ge-132 supplement during IVC significantly (P < 0.05) increased GSH levels in embryos compared with those in the control, which led to the reduced ROS level. Two putative ESC lines were successfully derived from porcine IVF embryos cultured in 100 and 200 μg mL–1 Ge-132 treatment. The established putative porcine ESCs showed alkaline phosphatase activity, pluripotency-associated markers such as OCT4 and NANOG and the capacity for embryoid body formation, which confirm the differentiation potentials. Together, these results suggest that Ge-132 supplementations during IVC enhanced the developmental competence of porcine IVF embryos, most likely by increasing intracellular GSH synthesis, reducing ROS level, and it might help the derivation of putative porcine ESCs. This work was supported, in part, by a grant from the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ011077, PJ011288), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2013R1A2A2A04008751). These are the results of a study by the Leaders INdustry-University Cooperation Project 2015-B-0013–010118, supported by the Ministry of Education, Republic of Korea.

208 EFFECT OF GDF8 AND SB-431542 ON PORCINE OOCYTE DURING IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT

Growth differentiation factor-8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. SB-431542 (SB) is a specific inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors such as ALK4, ALK5, and ALK7. The purpose of this study is investigation of the effects of GDF8 and SB on porcine oocytes during in vitro maturation and subsequent embryonic development. We first performed ELISA to detect GDF8 concentrations in follicular fluid for each size of follicle; sizes were as follows: small (<3 mm), medium (>3 mm and <6 mm), and large (>6 mm) follicle. After detection of the GDF8 concentration in follicular fluid, we investigated the effect of GDF8 and SB treatment during in vitro maturation (IVM) on nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels, and embryonic development after IVF and parthenogenetic activation (PA). Data were analysed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science, IBM, New York, NY, USA) mean ± SEM. The ELISA result showed different concentrations of GDF8 for each grade of follicular fluid: small, 0.479 ng mL–1; medium, 0.668 ng mL–1; and large, 1.318 ng mL–1. During the IVM process, 1.318 ng mL–1 of GDF8 and 5 ng mL–1 of SB were added to the maturation medium as control, SB, SB+GDF8, and GDF8 treatment groups. After 44 h of IVM, GDF8 group (90.4%) showed a significantly higher nuclear maturation rate than control and SB+GDF8 groups (85.4 and 81.7%). The SB group (78.9%) showed significantly reduced nuclear maturation rate compared with control (P < 0.05). The GDF8 treatment group showed a significant decreased intracellular ROS and increased GSH levels compared with other groups (P < 0.05). The SB+GBF8 treatment group showed a significantly better cytoplasmic maturation than the SB treatment group. In the PA embryonic development analysis, the GDF8 treatment group showed a significantly higher blastocyst formation rate compared with other groups (47.9, 37.2, 46.4, and 58.7% respectively; P < 0.05). In the IVF embryonic development analysis, the GDF8 treatment groups showed significantly higher blastocyst formation rate compared with the SB group (28.2 and 42.2%, respectively; P < 0.05). In conclusion, treatment with GDF8 during porcine oocyte IVM improved the embryonic developmental competence via increased cytoplasmic maturation and led to better oocyte maturation from the ALK receptor inhibition by SB.

Linolenic acid improves oocyte developmental competence and decreases apoptosis of in vitro-produced blastocysts in goat.

The effects of α-linolenic acid (ALA) on developmental competence of oocytes in goats were evaluated in this study. Initially, the level of ALA in small and large antral follicles was determined to be in a range of 0.018-0.028 mg/ml (64.6-100.6 μM, respectively). In vitro maturation was performed in the presence of various concentrations (10, 50, 100, or 200 μM) of ALA. Cumulus expansion, meiotic maturation, levels of intracellular glutathione (GSH), embryonic cleavage, blastocyst formation following parthenogenetic activation (PA) and in vitro fertilization (IVF), number of total and apoptotic cells in blastocyst, and expression of Bax, Bcl-2, and p53 genes in blastocyst cells were determined. Compared with the control, no improvement was observed in cumulus expansion in ALA-treated groups. At 50 μM concentration, ALA increased meiotic maturation rate but had no effect on GSH level. When oocytes treated with 50 μM ALA were subsequently used for PA or IVF, a higher rate of blastocyst formation was observed, and these embryos had a higher total cell number and a lower apoptotic cell number. Expression analyses of genes in blastocysts revealed lesser transcript abundances for Bax gene, and higher transcript abundances for Bcl-2 gene in 50 μM ALA group. Expression of p53 gene was also less observed in ALA-treated blastocysts. Our results show that ALA treatment at 50 μM during in vitro maturation (IVM) had a beneficial effect on maturation of goat oocytes and this, in turn, stimulated embryonic development and regulated apoptotic gene expression.

The Extracellular Calcium-Sensing Receptor (CASR) Regulates Gonadotropins-Induced Meiotic Maturation of Porcine Oocytes.

Gonadotropins and epidermal growth factor (EGF) play crucial roles in promoting oocyte maturation. The regulatory network downstream of these key factors is not well understood. The present study was designed to investigate the role of the calcium-sensing receptor (CASR) in porcine oocyte in vitro maturation. CASR expression was up-regulated in oocytes matured in gonadotropin-containing medium. Cortical distribution of CASR was enhanced with gonadotropins but not EGF. Supplementation of a CASR agonist (NPS R-568) in the gonadotropin (FSH and/or LH)-containing maturation medium significantly enhanced oocyte nuclear maturation. Addition of NPS2390, a CASR antagonist, compromised oocyte nuclear maturation. Furthermore, increased cortical distribution and decreased expression of CASR was observed after the NPS R-568 treatment. Oocytes treated with NPS R-568 had higher concentration of CYCLIN B1, decreased reactive oxygen species, and increased glutathione levels, indicative of advanced cytoplasmic maturation. In contrast, NPS2390 treatment compromised oocyte cytoplasmic maturation. A higher blastocyst formation rate after parthenogenetic activation was observed when oocytes were matured in the presence of the CASR agonist, NPS R-568. MAPK3/1 phosphorylation was increased during in vitro maturation and after NPS R-568 treatment, and decreased following CASR antagonist supplementation. Taken together, our data showed that the CASR is a gonadotropin-regulated factor that promotes porcine oocyte maturation in a MAPK-dependent manner.

Improved embryo development in Japanese black cattle by in vitro fertilization using ovum pick-up plus intracytoplasmic sperm injection with dithiothreitol.

The purpose of this study was to determine whether dithiothreitol (DTT) treatment of sperm and ethanol activation improve embryo production by intracytoplasmic sperm injection (ICSI). Further, we compared ICSI with standard in vitro fertilization (IVF) in oocytes obtained from cattle. We demonstrated that DTT reduced the disulfide bond in the bovine sperm head. Using oocytes obtained from a slaughterhouse, ICSI-DTT treatment without ethanol showed the highest rate of blastocyst formation. We applied these results to fertilization using ovum pick-up (OPU). Eleven Japanese black cattle served as donors for OPU plus standard IVF (OPU-IVF). Of them, four donors with low embryo development rates were selected to determine whether embryo development was enhanced by OPU plus ICSI (OPU-ICSI). We assessed effects on embryo development following IVF and ICSI in oocytes obtained using OPU. Blastocyst rates were significantly higher for OPU-ICSI than for OPU-IVF. Our results suggest that OPU-ICSI improves the blastocyst development rate in donors with low embryo production compared with the standard OPU-IVF.

Oxygen tension affects histone remodeling of in vitro–produced embryos in a bovine model

In vitro production of bovine embryos is a biotechnology of great economic impact. Epigenetic processes, such as histone remodeling, control gene expression and are essential for proper embryo development. Given the importance of IVP as a reproductive biotechnology, the role of epigenetic processes during embryo development, and the important correlation between culture conditions and epigenetic patterns, the present study was designed as a 2 × 2 factorial to investigate the influence of varying oxygen tensions (O2; 5% and 20%) and concentrations of fetal bovine serum (0% and 2.5%), during IVC, in the epigenetic remodeling of H3K9me2 (repressive) and H3K4me2 (permissive) in bovine embryos. Bovine oocytes were used for IVP of embryos, cleavage and blastocyst rates were evaluated, and expanded blastocysts were used for evaluation of the histone marks H3K9me2 and H3K4me2. Morulae and expanded blastocysts were also used to evaluate the expression of remodeling enzymes, specific to the aforementioned marks, by real-time polymerase chain reaction. Embryos produced in the presence of fetal bovine serum (2.5%) had a 10% higher rate of blastocyst formation. Global staining for the residues H3K9me2 and H3K4me2 was not affected significantly by the presence of serum. Notwithstanding, the main effect of oxygen tension was significant for both histone marks, with both repressive and permissive marks being higher in embryos cultured at the higher oxygen tension; however, expression of the remodeling enzymes did not differ in morulae or blastocysts in response to the varying oxygen tension. These results suggest that the use of serum during IVC of embryos increases blastocyst rate without affecting the evaluated histone marks and that oxygen tension has an important effect on the histone marks H3K9me2 and H3K4me2 in bovine blastocysts.

Improved Bovine Blastocyst Developmental Potential by L-carnitine Supplementation

In this study, the potential role of L-carnitine supplementation in the maturation of oocytes and pre-implantation development of embryos was investigated using bovine as a model. In Experiment 1, bovine oocytes recovered from the abattoir were matured in the absence (control) or presence of L-carnitine, subjected to in vitro fertilization and assessed on their developmental potential up to the blastocyst stage. The nuclear maturation and cleavage rate observed between the control and L-carnitine supplemented group ranged from 83.1% - 87.1% and 57.1% - 67.9%, respectively. Significantly higher blastocyst formation rate and improved total cell count in 0.1- 0.5 mg/ml L-carnitine supplemented groups were observed versus the control (51.0 - 54.2% vs 29.5% and 97.4 - 110.1 ± 2.2 vs 82.5 ± 1.6, respectively (P<0.05). In Experiment 2, zygotes resulting from in vitro fertilization of bovine oocytes were cultured in modified synthetic oviductal fluid medium with or without L-carnitine supplementation. Results showed no significant difference on the blastocyst formation rate among treatment groups, but the total cell count of blastocyst derived from 0.1 - 0.5 mg/ml L-carnitine supplemented groups were higher than the control (98.4 - 115.4 ± 3.1 vs 84.6 ± 3.2, respectively (P<0.05). Overall, the results demonstrated the usefulness of the procedures utilized in the maturation, fertilization and culture of bovine oocytes and early- stage embryos. That, L-carnitine supplementation at the level of 0.1 - 0.5 mg/ml concentration in the maturation and culture media tend to enhance the developmental potential of oocytes and early- stage embryos to the blastocyst stage as indicated by a higher total cell count (improved cell activity).

302 EFFECT OF GROWTH DIFFERENTIATION FACTOR 8 ON PORCINE OOCYTE IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT AFTER PARTHENOGENETIC ACTIVATION AND IN VITRO FERTILIZATION

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. The purpose of this study was to investigate the effects of GDF8 on in vitro porcine oocytes maturation and subsequent embryonic development after pathenogenetic activation (PA) and in vitro fertilization (IVF). We investigated nuclear maturation, intracellular glutathione (GSH), reactive oxygen species (ROS) levels, sperm penetration (SP) analysis, and subsequent embryonic development after PA and IVF. Each concentration (0, 1, 10, and 100 ng mL–1) of GDF8 was added in maturation medium during process of in vitro maturation. Data were analysed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science) mean ± s.e.m. After 44 h of IVM, no significant difference was observed on nuclear maturation from the different concentration (0, 1, 10, and 100 ng mL–1) of GDF8 treatment groups (85.5, 85.9, 89.4, and 87.6%, respectively) compared with the control (P &gt; 0.05). The 10- and 100-ng mL–1 GDF8-treated groups showed a significant (P &lt; 0.05) decrease in intracellular ROS levels compared with other groups. The embryonic developmental competence after PA was affected with GDF8 treatment during IVM. The 10- and 100-ng mL–1 treatment groups showed significantly (P &lt; 0.05) higher cleavage rates (67.5 and 69.1%, respectively) compared with control group (53.7%). The 10- and 100-ng mL–1 treatment groups also showed significantly (P &lt; 0.05) higher blastocyst formation rates (50.5 and 52.7%, respectively) compared with other groups (34.5 and 35.8%). The IVF embryonic developmental competence also was affected with GDF8 treatment during IVM. The 10-ng mL–1 treatment group showed a significantly (P &lt; 0.05) higher blastocyst formation rates and total cell number compared with control (21.5 and 131.3 v. 15.0 and 92.6%, respectively). Also, in the sperm penetration assessment, the 10- and 100-ng mL–1 treatment groups showed higher mono spermy ratio and fertilization efficiency (32.7 and 27.1, 32.0 and 26.5 v. 22.6 and 19.7%, respectively) than control, which was significant (P &lt; 0.05). In conclusion, the treatment with 10 ng mL–1 of GDF8 during IVM improved the PA and IVF porcine embryo developmental competence by decreasing the intracellular ROS levels.This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2013R1A2A2A04008751), Republic of Korea.

Exogenous expression ofOCT4facilitates oocyte-mediated reprogramming in cloned porcine embryos

OCT4 is a well-established regulator of pluripotency and nuclear reprogramming. To determine if improving OCT4 abundance can facilitate oocyte-mediated reprogramming in cloned porcine embryos, we artificially increased OCT4 levels by co-incubating donor cells with 50 ng/µl OCT4 plasmid. We observed higher rates of blastocyst formation (P < 0.05) and lower levels of blastocyst apoptosis in nuclear-transfer-derived embryos carrying OCT4-incubated donor nuclei (OCT4-SCNT). The beneficial effect caused by exogenous expression of OCT4 involves epigenetic changes, wherein increased histone acetylation (AcH3K9) appeared in OCT4-SCNT embryos at the one-cell and blastocyst stages and reduced histone methylation (H3K9me2) was observed at the one-cell stage (P < 0.05). There was a transient increase in exogenous OCT4 and an up-regulation of endogenous OCT4 level in OCT4-SCNT embryos (P < 0.05), while the expression pattern of epigenetic enzymes was changed. These modifications were accompanied by an up-regulation of CDX2, whose interaction with OCT4 is instrumental for implantation, and a down-regulation of XIST, a negative indicator of reprogramming (P < 0.05). Taken together, our results support a role for exogenous expression of OCT4 in improving the efficiency of nuclear reprogramming while establishing a convenient and timesaving method to improve nuclear-transfer outcomes.

154 THE EFFECT OF ZINC ON PORCINE IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT AFTER IN VITRO FERTILIZATION

Zinc (Zn) is one of the abundant transition metals in biology and is an essential component of most cells. However, there are few reports about the effect of Zn in porcine oocytes. The objective was to investigate the effects of supplementary Zn during in vitro maturation (IVM) of porcine oocytes. We investigated nuclear maturation, intracellular glutathione (GSH) levels, reactive oxygen species (ROS) levels, and subsequent embryonic development after IVF. Before the experiment, Zn concentrations in IVM medium and body fluids were measured using inductively coupled plasma spectrophotometer (sensitivity: 1 μM) and treatment concentrations were determined. Zinc concentration was 12.6 μM in porcine plasma and 12.9 μM in porcine follicular fluid. We confirmed that Zn was not detected in IVM medium. A total of 541 cumulus–oocyte complexes (COC) were used for the evaluation of nuclear maturation. The COC were matured in TCM-199 medium supplemented with various concentrations of Zn (0, 6, 12, 18, and 24 μM). After 44 h of IVM, no significant difference was observed in all groups (metaphase II rate: 85.7, 88.7, 90.4, 90.3, and 87.2%, respectively). A total of 100 matured oocytes were examined for the effects of different Zn concentrations (0, 6, 12, 18, and 24 μM) on porcine oocyte intracellular GSH and ROS levels, which were measured through fluorescent staining and image analysis program. The groups of 12, 18, and 24 μM showed a significant (P &lt; 0.05) increase in intracellular GSH levels (1.45, 1.67, and 1.78, respectively) compared with the control and 6 μM group (1.00 and 1.08, respectively). The intracellular ROS level of oocytes matured with 12, 18, and 24 μM (0.82, 0.68, and 0.55) were significantly (P &lt; 0.05) decreased compared with the control and 6 μM groups (1.00 and 1.03, respectively). Finally, the developmental competence of oocytes matured with different concentrations of Zn (0, 6, 12, 18, and 24 μM) was evaluated after IVF. There were no significantly different in cleavage rates. However, cleavage patterns and blastocyst (BL) formation were different. Fragmented embryo ratio of the 12 μM group (14.9%) was significantly lower than that of the other groups (control, 6, 18, and 24 μM: 26.4, 17.8, 18.4, and 18.0%, respectively). Oocytes treated with 12 μM Zn during IVM had a significantly higher BL formation rate (28.2%) after IVF compared with the control (19.8%). In conclusion, these results indicate that Zn treatment as body fluid concentration during IVM improved the developmental potential of IVF in porcine embryos by increasing the intracellular GSH concentration and decreasing the ROS level. This work was supported, in part, by a grant from the Next-Generation Bio Green 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

Significant improvement of pig cloning efficiency by treatment with LBH589 after somatic cell nuclear transfer

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) associates with epigenetic aberrancy, including the abnormal acetylation of histones. Altering the epigenetic status by histone deacetylase inhibitors (HDACi) enhances the developmental potential of SCNT embryos. In the current study, we examined the effects of LBH589 (panobinostat), a novel broad-spectrum HDACi, on the nuclear reprogramming and development of pig SCNT embryos in vitro. In experiment 1, we compared the in vitro developmental competence of nuclear transfer embryos treated with different concentrations of LBH589. Embryos treated with 50 nM LBH589 for 24 hours showed a significant increase in the rate of blastocyst formation compared with the control or embryos treated with 5 or 500 nM LBH589 (32.4% vs. 11.8%, 12.1%, and 10.0%, respectively, P < 0.05). In experiment 2, we examined the in vitro developmental competence of nuclear transfer embryos treated with 50 nM LBH589 for various intervals after activation and 6-dimethylaminopurine. Embryos treated for 24 hours had higher rates of blastocyst formation than the other groups. In experiment 3, when the acetylation of H4K12 was examined in SCNT embryos treated for 6 hours with 50 nM LBH589 by immunohistochemistry, the staining intensities of these proteins in LBH589-treated SCNT embryos were significantly higher than in the control. In experiment 4, LBH589-treated nuclear transfer and control embryos were transferred into surrogate mothers, resulting in three (100%) and two (66.7%) pregnancies, respectively. In conclusion, LBH589 enhances the nuclear reprogramming and developmental potential of SCNT embryos by altering the epigenetic status and expression, and increasing blastocyst quality.

37 THE EFFECTS OF A CLASS III HISTONE DEACETYLASE INHIBITOR (SIRTINOL) ON EARLY DEVELOPMENT OF PORCINE CLONED EMBRYOS

Previous studies have demonstrated that treatment of cloned embryos with trichostatin A (TSA) or scriptaid, inhibitors of class I and II histone deacetylases (HDAC), significantly enhanced their developmental competence. In the present study, we investigated the effects of sirtinol, an inhibitor of class III HDAC, on the embryonic development of porcine cloned embryos. Data were analyzed with SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) using Duncan’s multiple range test and all experiments were replicated at least 5 times. In experiment 1, 648 parthenotes were divided into 4 groups (0-, 6-, 12-, and 24-h sirtinol treatment after activation) to investigate optimal treatment time using 100 µM sirtinol. The cleavage rate of the 24-h treatment group (81.3%) was significantly (P &lt; 0.05) decreased compared with the 12-h treatment group (88.4%) but there was no difference compared with the control (86.9%) and 6-h treatment groups (86.9%). The parthenotes treated with sirtinol for 12 h after activation had a significantly higher blastocyst formation rate and total cell number in blastocysts (50.5% and 66.9, respectively) than the control (39.4% and 54.1, respectively). In experiment 2, 806 cloned embryos were divided into 5 groups (0, 50, 100, 150, and 200 µM sirtinol treatment for 12 h after activation) to investigate optimal concentration. There was no significant difference in cleavage rate. The rate of blastocyst formation and total cell number in blastocysts were significantly (P &lt; 0.05) improved by treatment with 150 µM sirtinol for 12 h after activation (28.8% and 51.0, respectively) compared with the control (17.5% and 37.1, respectively). The total cell number in blastocysts was also significantly increased in 50 and 200 µM groups (47.9 and 48.4, respectively) compared with the control (37.1). In experiment 3, we examined the effects of 150 µM sirtinol treatment for 12 h after activation with or without 5 nM TSA on in vitro embryonic development after somatic cell nuclear transfer. The rate of blastocyst formation was significantly improved in sirtinol-treated and TSA-treated groups (30.9 and 31.3%, respectively) but not in the sirtinol with TSA group (27.6%) compared with the control (21.7%). The total cell number in blastocysts was significantly increased by treatment with sirtinol and TSA together (73.9) compared with the control (49.0) but there was no difference in only sirtinol- (59.8) or TSA- (59.2) treated groups. There was no significant difference in cleavage rate among groups. Our results suggest that sirtinol improves the embryonic development of porcine cloned embryos and sirtinol with TSA synergistically increases the blastocyst quality. This work was supported by a grant from the Next-Generation BioGreen 21 program (no. PJ008121012011), Rural Development Administration, Republic of Korea.

Valproic acid improved in vitro development of pig cloning embryos but did not improve survival of cloned pigs to adulthood

The objective was to examine the effects of valproic acid (VPA), a histone deacetylase inhibitor, on in vitro and in vivo development of Wuzhishan miniature pig somatic cell nuclear transfer (SCNT) embryos. Experiment 1 compared in vitro developmental competence of nuclear transfer embryos treated with various concentrations of VPA for 24 h. Embryos treated with 2 mM VPA for 24 h had a greater rate of blastocyst formation compared with control or embryos treated with 4 or 8 mM VPA (21.5% vs. 10.5%, 12.6%, and 17.2%, P < 0.05). Experiment 2 examined the in vitro developmental competence of nuclear transfer embryos treated with 2 mM VPA for various intervals after chemical activation. Embryos treated for 24 h had higher rates of blastocyst formation than the control or those treated for 4 or 48 h (20.7% vs. 9.2%, 12.1%, and 9.1%, P < 0.05). In Experiment 3, an average of 207 (range, 192–216) nuclear transfer embryos from the VPA-treated group were transferred to surrogate mothers, resulting in three pregnancies. Two of the surrogates delivered a total of 11 live piglets. However, for unknown reasons, nine of 11 piglets in the VPA-treated group died within 1 to 5 d after birth. Untreated control embryos (average, 205; range, 179–225) transferred to four surrogate mothers resulted in three pregnancies, two of which delivered a total of 12 live offspring, although four of 12 piglets in the VPA-untreated group died (cause unknown) within 1 to 3 d, whereas eight of the 12 piglets in the VPA-untreated group survived more than 3 or 4 mo. The average birth weight of the two litters from the VPA-treated group tended (P < 0.05) to be lower than that from the control groups (551.6 g vs. 675.2 g). In conclusion, VPA treatment increased the blastocyst formation rate of SCNT porcine embryos; both VPA-treated and the untreated clones developed to term, but offspring from VPA-treated embryos had a lower survival to adulthood than those from control embryos (18.2% vs. 67.0%; P < 0.05).

Positive effects of treatment of donor cells with aphidicolin on the preimplantation development of somatic cell nuclear transfer embryos in Chinese Bama mini‐pig (Sus Scrofa)

To optimize somatic cell nuclear transfer (SCNT) procedures in mini-pigs, the present study was designed to examine the effects of donor cell types and aphidicolin (APC) treatment on in vitro development of reconstructed embryos. Oviduct epithelial cells (OEC), ear fibroblast cells (EFC) and cumulus cells (CC) derived from mini-pigs were treated with serum starvation only or serum starvation followed by treatment of 0.1 µg/mL APC. The reconstructed embryos were cultured for 7 days to evaluate their developmental competency. Cleavage and blastocyst formation rates of reconstructed embryos derived from the OEC by APC treatment were significantly higher than the serum starvation (61.82% vs. 56.25%, 24.55% vs. 17.86%; P < 0.05). The cleavage rate from the EFC was significantly increased by APC treatment compared to serum starvation only (63.36% vs. 57.01%; P < 0.05). In the ooctyes with the CC, the reconstructed embryos could yield high blastocyst formation rate by APC treatment (29.63%; P < 0.05). In the presence of APC, CC gave rise to the highest cleavage and blastocyst formation rates among the three cell types. Therefore, our results suggest that treatment of CC with serum starvation plus APC prior to nuclear transfer is more suitable in SCNT of mini-pigs.

52 THE EFFECTS OF DONOR CELL CYCLE AND THE TIMING OF OOCYTE ACTIVATION ON DEVELOPMENT OF BOVINE NUCLEAR TRANSFERRED EMBRYOS IN VIVO

The cell cycle of donor cells and recipient cytoplasts are important factors affecting development of nuclear transferred (NT) embryos. We previously showed that bovine NT embryos using pre-activated cytoplasts and early G1 cells had a high in vitro developmental rate (SSR, 2008, 41st Annual Meeting). The objective of the present study was to evaluate the effects of donor cell cycle (early G1 or G0 phase) and the timing of oocyte activation on fetal development of bovine NT embryos. Adult fibroblasts from ear skin tissue of Japanese black cattle were used as donor cells. The G0 phase cells were synchronized by serum-starvation, and the G1 phase cells were prepared from actively dividing M phase cells. NT embryo production was performed by 2 kinds of protocols as follows: 1) recipient oocytes were activated by Ca ionophore (CaI), followed with cycloheximide (CH) for 2 h, and fused with synchronized donor cells followed with cytochalasin D (CD) and CH for 1 h, then CH for 4 h (pre-activated), 2) unactivated oocytes were fused with synchronized donor cells and activation was performed by CaI 1 h after fusion, followed by with CD and CH 1 h, then CH for 4 h (post-activated). After activation treatments, NT embryos were cultured in IVD101 medium for 7 days. Then, blastocysts were transferred to recipient cows. Diagnosis of pregnancy was made by ultrasonography at days 30, 60, and 90 (Day 0 = the day of embryo transfer). As shown in Table 1, the blastocyst formation rate of the NT embryos derived from early G1 cells in the pre-activated group was higher than that from G0 cells in the post-activated group (36% v. 23%, P &lt; 0.05). After embryo transfer, 29, 67, and 50% of recipient cows were pregnant at Day 30 in G0 post-, G1 post-, and G1 pre-activated groups, respectively. However, only 1 embryo (14%) of G0 post-activated group developed to term. In conclusion, bovine NT embryos using early G1 cells and pre-activated cytoplasts showed a high blastocyst formation rate, but the full-term development of bovine NT embryos could not be improved by using early G1 cells and pre-activated cytoplasts. Table 1.Effect of the timing of oocyte activation on developmental ability of bovine NT embryos derived from early G1 or G0 phase cells

Development of bovine embryos cultured in CR1aa and IVD101 media using different oxygen tensions and culture systems

The aim of the present study was to optimise the culture conditions for the in vitro production of bovine embryos. The development of in vitro fertilised bovine oocytes in CR1aa supplemented with 5% calf serum and IVD101 culture media were compared using traditional microdrops and Well of the Well (WOW) culture systems either under 5% or 20% oxygen tension. After 7 days of culture, a significantly higher blastocyst formation rate was obtained for embryos cultured in CR1aa medium compared to those cultured in IVD101, irrespective of O2 tensions and culture systems. The blastocyst formation in IVD101 was suppressed under 20% O2 compared to 5% O2 . Despite their similar total cell numbers, higher rates of inner cell mass (ICM) cells were observed in blastocysts developed in IVD101 medium than in those developed in CR1aa, irrespective of O2 tensions. There was no significant difference in blastocyst formation, total, ICM and trophectoderm (TE) cell numbers between embryos obtained by microdrop and WOW culture systems irrespective of the culture media and O2 tensions used. In conclusion, CR1aa resulted in higher blastocyst formation rates irrespective of O2 tension, whereas IVD101 supported blastocyst formation only under low O2 levels but enhanced the proliferation of ICM cells.