NCSU-23 Medium Research Articles

#77 : A Micro 3D Printed Device for Intracytoplasmic Sperm Injection Removes the Requirement for a Holding Pipette and Reduces Complexity of the Procedure

Background and Aims: One of the most skilled procedures performed by embryologists is intracytoplasmic sperm injection (ICSI), which takes significant experience to master. Conducting the procedure requires a high level of concentration with training a significant burden on clinical laboratory efficiency. Advancement of a micro 3D printed device to hold oocytes has removed the requirement for a holding pipette, reducing the complexity of the ICSI procedure. This study shows that the microICSIÔ device reduces the technical difficulty of ICSI, improving the speed and precision of the procedure. Method: ICSI was performed conventionally (C-ICSI) and within the microICSIÔ device on abattoir derived porcine denuded oocytes matured in vitro for 39-44 h in NCSU-23 medium containing eCG, hCG, porcine follicular fluid, EGF and insulin. Timing data was captured by video recording for pipette setup and injection time per oocyte for two embryologists and compared between each technique. Oocyte injection time was taken from when a sperm loaded injection pipette was in focus with the oocyte until the injection pipette was removed from the oocyte. Results: Results were compared between C-ICSI and microICSIÔ (Mean ± SD). Pipette setup time was reduced from 278 ± 21 seconds to 109 ± 3 seconds by removing the holding pipette. During the procedure, absence of a holding pipette resulted in embryologists adjusting their hands on average only 3 times during oocyte injection compared to 10 times for C-ICSI when moving between microinjector, pipette and microscope controllers. Oocyte traceability was also improved due to the microICSIÔ device’s numbered array. Embryologist’s injection time was reduced from 42 ± 12 seconds for C-ICSI to 14 ± 5 seconds for microICSIÔ per oocyte. Conclusion: The microICSIÔ device reduces pipette set-up time and the complexity of the ICSI procedure, reducing the time required for oocyte injection by half.

Liquid storage of porcine in vitro-produced blastocysts; a practical approach for short storage.

Commercial application of embryo transfer in pig breeding is dependent on the storage of embryos. The aim of this study was to assess the embryo quality of in vitro-produced blastocysts after 3 h liquid storage at 37°C in CO2-free medium by evaluating morphology, in vitro developmental capacity and apoptosis. Blastocysts at days 5 and 6 post-fertilization were randomly allocated to the storage group (HEPES-buffered NCSU-23 medium including bovine serum albumin in a portable embryo transport incubator at 37°C) or a control group (porcine blastocyst medium in a conventional culture incubator). Thereafter, blastocysts were evaluated for morphology and stained to assess apoptosis straight after the 3 h storage period or after a further 24 h conventional incubation. There was no significant difference between the storage and control group after 3 h storage and the further 24 h conventional incubation for any of the parameters, nor for apoptosis straight after the 3 h storage. Embryos that reached the blastocyst stage at day 5 showed less apoptosis (6.6% vs 10.9%, P = 0.01) and a trend for a higher rate of developmental capacity (70.6% vs 51.5%, P = 0.089) than embryos reaching the blastocyst stage on day 6. In conclusion, in vitro-produced porcine blastocysts can be stored for 3 h at physiological temperature in transportable incubators using a CO2-independent medium without compromising quality.

Evaluation of developmental competence of Sus scrofa domesticus (L.) oocyte-cumulus complexes after intra- and extraovarian vitrif ication.

The aim of the present study was to identify the inf luence of extra- (EOV) and intraovarian vitrif ication (IOV) on mitochondrial activity (MA) and chromatin state in porcine oocytes during maturation in vitro. During EOV porcine oocytes were exposed in cryoprotective solutions (CPS): CPS-1 – 0.7 M dimethyl sulfoxide (DMSO) + 0.9 M ethylene glycol (EG); CPS-2 – 1.4 M DMSO + 1.8 M EG; CPS-3 – 2.8 M DMSO + 3.6 M EG + 0.65 M trehalose. At IOV the ovarian fragments were exposed in CPS-1 – 7.5 % EG + 7.5 % DMSO, then in CPS-2 – 15 % EG, 15 % DMSO and 0.5 M sucrose. Straws with oocytes and ovarian fragments were plunged into LN2 and stored. For devitrif ication, the EOV oocytes were washed in solutions of 0.25, 0.19 and 0.125 M of trehalose, the IOV – in 0.5 and 0.25 М trehalose. Oocytes were cultured in NCSU-23 medium with 10 % f luid of follicles, follicular walls, hormones. 0.001 % of highly dispersed silica nanoparticles (ICP named after A.A. Chuyko of the NAS of Ukraine) were added to all media. The methods of fertilization and embryo culture are presented in the guidelines developed by us. MA and chromatin state were measured by MitoTracker Orange CMTMRos and the cytogenetic method. Signif icant differences in the level of oocytes with high-expanded cumulus between control and experimental vitrif ied groups (81 % versus 59 % and 52 %, respectively, p ≤ 0.001) were observed. The percentage of pyknotic cells in native oocytes was 19 %, EOV or IOV oocytes were 39 % and 49 %, respectively. After culture, the level of matured native oocytes was 86 %, 48 % EOV and 33 % IOV cells f inished the maturation ( p ≤ 0.001). Differences were also observed in the level of MA between groups treated by EOV and IOV (89.4 ± 7.5 μA and 149.2 ± 11.3 μA, respectively, p ≤ 0.05). For the f irst time, pre-implantation embryos were obtained from oocytes treated by IOV.

Effects of in vitro maturation media on in vitro fertility of porcine oocytes and early development of embryos

In pigs, embryo productivity is still lower than that in other livestocks. One of the reasons is incomplete maturation of porcine oocytes in in vitro conditions. Therefore in vitro maturation (IVM) plays a crucial role in in vitro production of porcine embryos. It provides prerequisite condition to in fertilization and subsequent development of porcine embryos. In a previous study, effects of NCSU-37-based medium and TCM-199-based media supplemented with porcine follicular fluid (pFF) or Fetal Bovine Serum (FBS) on in vitro maturation of Landrace oocytes collected in Vietnam have been compared, suggesting that NCSU-37 medium supplemented with 10% of porcine follicular fluid (pFF) had the highest rate of oocytes reach to metaphase II stage in comparison to those of the other two TCM-199-based media. In the present study, further experiments were carried out to evaluate the contribution of IVM media on fertilization capability and developmental competence. Porcine oocytes matured in vitro in 3 media: NCSU-37 supplemented with 10% pFF, TCM-199 supplemented with either 10% pFF or 10% FBS were subjected to in vitro fertilization and subsequent in vitro culture to monitor fertility and embryo development. The results showed that penetration and normal fertilization rates in both TCM-199 groups are both higher than that of NCSU-37 group. Moreover, the cleavage and blastocyst rates, and cell numbers of blastocysts which is a criterion for embryo quality were all higher in TCM-199 groups, especially in the group supplemented with pFF. It might be concluded that TCM-199 media supplemented with either pFF or FBS are suitable for effective in vitro maturation of Landrace porcine oocytes collected in Vietnam.

Effect of high hydrostatic pressure on mitochondrial activity, reactive oxygen species level and developmental competence of cultured pig embryos

High hydrostatic pressure (HHP) has been previously used to increase mammalian oocyte and embryo tolerance on subsequent stresses related with different assisted reproductive technologies. Nevertheless, the mechanisms for HHP-induced stress responses in early embryos have not been yet well understood. Previous studies focused mainly on HHP-modified gene expression while possible changes in cellular functions, including modification of energy metabolism and oxidative stress were neglected. Therefore, we aimed to analyze the effect of HHP treatment on the efficiency of subsequent in vitro pig embryos culture in NCSU-23 medium, on mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) level during their pre-implantation development. Porcine embryos were exposed to the hydrostatic pressure of 20 MPa and their quick response to such stress was analyzed 1 h later. In comparison with control embryos, we detected lower ΔΨm by ∼13% only in expanded blastocysts as well as decreased ROS level by ∼30% and ∼42% at the morula and expanded blastocyst stages, respectively. After HHP-treatment at transcriptionally inactive zygote stage and subsequent embryo culture, long-time responses were found: (1) at expanded blastocyst stage manifesting by ΔΨm decrease by ∼16%, (2) at the morula and expanded blastocyst stages in the form of ROS level reduction by ∼38% and ∼33% respectively. Following HHP stress applied at the transcriptionally active morula stage the long-time response in the expanded blastocysts as a decrease of ΔΨm by ∼19% and ROS level by ∼37% was observed. The percentage of obtained expanded blastocysts was higher after culture of HHP-treated zygotes in comparison to the control. Moreover, expanded blastocysts developed in vitro from both HHP-treated zygotes or morulae, exhibited higher total number of cells per blastocyst, higher number of cells in the inner cell mass as well as lower number of TUNEL-positive nuclei per blastocyst and lower TUNEL index, when compared to untreated embryos. Therefore, the HHP stress applied at the zygote stage, enhances developmental potential and quality of in vitro obtained porcine blastocysts due to the both decreased ΔΨm and ROS level. Our findings may contribute to better understanding of the mechanism of HHP-mediated modifications of energy metabolism and oxidative stress during in vitro development of pig embryos.

Uso de oócitos bovinos como citoplasma receptor na produção de embriões por transferência nuclear de célula somática interespécie (NTSCi)

Interspecies embryo clones have been produced by research groups with relative success in some species. Bovine oocytes matured in vitro and enucleated by micromanipulation were used in three experiments as recipeient cytoplasm in nuclear transfer of ovine, caprine and porcine fibroblasts. The fibroblasts were cultivated until the third passage before being frozen and used. The electrofusion was induced by an application of a 20V pulse during 45 ms. The activation was done with 5 mM ionomycin and subsequently 2 mM 6DMAP. NTSC bovine embryos, NTSCi caprine and ovine embryos were cultivated in SOF medium and NTSCi porcine embryos were cultivated in NCSU23 medium. The fusion rates of the reconstructed complexes with bovine cells did not differ from those observed with ovine cells (88.2%), caprine cells (74.1%) and porcine cells (79.4%). The cleavage rates in ovine (60.3%), caprine (68.4%) and porcine (57.1%) NTSCi groups did not differ from the control group NTSC bovine. The blastocyst rate observed in the group of NTSCi ovine embryos (10.3%) was similar to the group of NTSC bovine embryos (12.7%). In NTSCi caprine embryos, 5.3% of the embryos developed up to the blastocyst stage, while in the NTSCi porcine group there was no development up to the blastocyst stage. In conclusion, the bovine cytoplasm was able to support the embryo development in NTSCi up to the blastocyst stage using ovine and caprine fibroblasts as donor cells.

Effects of Antioxidants Supplement in Porcine Sperm Freezing on in vitro Fertilization and the Glutathione and Reactive Oxygen Species Level of Presumptive Zygotes

The present study was aimed to determine the effects of green tea extract (GTE) and beta-mercaptoethanol (β-ME) supplementation in boar sperm freezing extender on in vitro fertilization (IVF) and reactive oxygen species (ROS) and glutathione (GSH) levels of presumptive zygotes (PZs). Experimental groups were allocated into lactose egg yolk (LEY) without antioxidant (control), GTE (1,000 mg/l in LEY) and β-ME (50 μM in LEY). In freezing, spermatozoa extended with LEY were cooled to 5°C for 3 h and then kept at 5°C for 30 min following dilution with LEY containing 9% glycerol and 1.5% Equex STM. The final sperm concentration was 1 × 108/ml. Spermatozoa were loaded into straws and frozen in nitrogen vapor for 20 min. For IVF, oocytes were matured in NCSU-23 medium and co-cultured with spermatozoa following thawing at 37°C for 25 sec. At 12 h following IVF, IVF parameters (sperm penetration and monospermy) were evaluated. In addition, GSH and ROS levels of PZs were determined by Cell Tracker Blue CMF2HC and DCHFDA, respectively. IVF parameters did not show any significant difference among the experimental groups. GSH and ROS levels of PZs were not significantly different between groups. In conclusion, antioxidant supplementation in boar sperm freezing could not influence IVF parameters, ROS and GSH levels of PZs.

Simple storage (CO2-free) of porcine morulae for up to three days maintains the in vitro viability and developmental competence

The advancement of porcine embryo transfer (ET) technology is constrained by regulatory hurdles (liquid nitrogen transportation) or, more importantly, the technical obstacles of using vitrified embryos in combination with nonsurgical deep uterine ET technology. Maintaining embryos in culture during transport and prior ET collides with the need of CO2 gassing and the best choice of culture medium. In this work, we describe storage conditions for short-term embryo CO2-free storage that allowed for a majority of in vivo-derived porcine morulae to survive after 3 days of storage in a liquid state, and to develop to the blastocyst stage unhatched, a sanitary prerequisite for ET. The storage conditions included NCSU-23 medium supplemented with bovine serum albumin, where bicarbonate was partially replaced by HEPES to avoid the need for CO2 gassing, and a temperature of 37 °C. These conditions were able to maintain the functionality of the stored embryos (hatching capacity after exposure to conventional culture conditions) and their developmental competence after ET (normal fetuses by day 38 of pregnancy). Use of this strategy of CO2-free storage should allow the shipment of fresh embryos worldwide in the absence of liquid nitrogen.

56 Sirtinol Treatment Influences Preimplatation Development of Porcine Embryos via Regulation of Autophagy and Apoptosis

Sirtuin (Sirt), nicotinamide adenine dinucleotide dependent class III histone deacetylase, plays an important role in cellular processes including DNA repair, apoptosis, cell cycle, aging, and determining lifespan. In previous studies, levels of Sirt1 to Sirt3 mRNA were detected in porcine embryos for the first time and levels are lower in blastocysts relative to matured oocytes. In addition, mitochondrial dysfunction and hyperglycemia increases LC3 protein levels and apoptosis in porcine parthenotic embryos and modulation of autophagy also influences apoptosis, mitochondrial contents, abnormal autophagosome formation, and maternal mRNA degradation. However, Sirt-mediated mechanisms have not been examined in in vitro-produced embryos of pig. Therefore, we investigated the relationship between Sirt inhibition and autophagy/mitophagy in porcine pre-implantation embryos. After IVF, embryos were cultured in NCSU-23 media in the presence and absence of 100 μM sirtinol (Sirt inhibitor) until the expended blastocyst stage. As a result, there were no significant differences between the rate of cleavage in control (69.22 ± 1.29) and treated groups (72.66 ± 1.08). However, embryos treated with sirtinol had significantly decreased developmental rates to the morula as well as blastocyst stages. Especially, expanded blastocysts (9.90 ± 1.56 v. 2.92 ± 0.94%) were barely observed in sirtinol-treated group. In the levels of Sirt transcripts, Sirt2 mRNA was significantly lower in sirtinol-treated blastocysts compared with controls (P < 0.05), but the levels of Sirt1 and Sirt3 mRNA were similar in both groups. In addition, we found that sirtinol treatment induced autophagy by increasing the expressions of LC3, Beclin1, and ATG5 in blastocysts. Furthermore, we observed that the abundance of mitochondria stained with mitotracker was lower in sirtinol-treated blastocysts than that of control. Finally, we found that sirtinol treatment resulted in a higher total apoptotic index (6.88 ± 0.84) compared with the control (12.84 ± 0.99) in blastocysts. In summary, our findings in this study demonstrated that Sirt inhibition by sirtinol led to lower levels of Sirt2 transcript in blastocysts, reduced developmental capability and embryo quality with regulation of ATGs, LC3 proteins, apoptosis-related genes, and mitochondrial abundance. Therefore, these results suggest that Sirt2 may play an important role in the pre-implantation development of porcine embryos and their quality through the regulation of autophagy/mitophagy and apoptosis pathways. This research was supported by a Grant from the Bio & Medical Technology Development Program (2015M3A9C7030091) of the National Research Foundation (NRF) funded by the Korean government.

Sirtuin inhibition leads to autophagy and apoptosis in porcine preimplantation blastocysts

Sirtuins are nicotinamide adenine dinucleotide dependent class III histone deacetylase proteins that play a crucial role in several cellular processes, including DNA repair, apoptosis, and lifespan. Previous studies have shown that sirtuin inhibition leads to embryonic developmental arrest and oxidative stress in porcine and murine. However, sirtuin-mediated mechanisms have not been examined in porcine preimplantation blastocysts. We therefore investigated the relationship between sirtuins and autophagy. Embryos were cultured with 100 μM sirtinol (SIRT1/2 inhibitor) in NCSU-23 media after in vitro fertilization. Treatment with sirtinol significantly reduced the rates of morula (21.34 ± 1.84 vs. 11.89 ± 2.01), blastocyst development (17.18 ± 1.81 vs. 9.00 ± 2.02), and total cell number (50.80 ± 1.47 vs. 37.71 ± 1.79), compared to controls, with an associating decrease the levels of Sirt2 transcript. Sirtinol treatment induced autophagy through an increase in LC3 transcript and LC3 protein. BECLIN1 and ATG5 expression showed a slight increase in treated group. Finally, treatment with sirtinol dramatically increased TUNEL indices (6.55 ± 0.84 vs. 11.44 ± 0.81) and fragmentation indices (0.33 ± 0.05 vs. 1.40 ± 0.30). BCL2L1 expression was lower, while Caspase-3 expression was significantly elevated in the sirtinol-treated group. Therefore, these findings suggest that sirtuins may elicit their effects through modifying autophagy and apoptosis, leading to developmental arrest and reducing the quality of porcine preimplantation embryos.

Maternal effect gene expression in porcine metaphase II oocytes and embryos in vitro: effect of epidermal growth factor, interleukin-1β and leukemia inhibitory factor.

Maternal effect genes (MEG) play a crucial role in early embryogenesis. In vitro culture conditions may affect MEG expression in porcine oocytes and embryos. We investigated whether in vitro culture medium supplementation with epidermal growth factor (EGF), IL-1β or LIF (leukemia inhibitory factor) affects the mRNA level of ZAR-1 (zygote arrest 1), NPM2 (nucleoplasmin 2) and DPPA3 (developmental associated protein 3) in porcine MII oocytes and embryos. Cumulus-oocyte complexes (COCs) were matured in NCSU-37 medium (control) or in NCSU-37 with EGF 10 ng/ml, IL-1β 10 ng/ml or LIF 50 ng/ml. After maturation for 44-46 h, MII oocytes were preserved for the analysis of MEG mRNA levels (experiment 1). In experiment 2, COCs were fertilized, and the presumptive zygotes were cultured in the same groups. Then, 2-, 4-, 8-cell embryos, morulae and blastocysts were collected for the analysis of MEG mRNA levels. LIF addition to the maturation medium increased MII oocyte numbers (P < 0.05), while EGF and IL-1β did not affect oocyte maturation. Medium supplementation with EGF resulted in lower DPPA3 mRNA levels in MII oocytes and in 2- and 4-cell embryos versus control embryos (P < 0.05). LIF treatment increased DPPA3 mRNA levels in morulae and blastocysts (P < 0.05). Culture with EGF and IL-1β decreased ZAR-1 and NPM2 mRNA levels in 2-cell embryos (P < 0.05). The inclusion of EGF or IL-1β in the porcine in vitro production system influences ZAR-1, NPM2 and DPPA3 mRNA in MII oocytes and embryos but not beyond the 4-cell stage. LIF stimulates oocyte maturation and affects DPPA3 mRNA in porcine morulae and blastocysts in vitro.

172 THE APOPTOTIC EFFECTS OF BISPHENOL A-INDUCED MITOCHONDRIAL-DERIVED REACTIVE OXYGEN SPECIES ON MATURATION OF PORCINE OOCYTES IN VITRO

Bisphenol A (BPA) is well known as oestrogen-like chemical and it is widely used in plastic products. Many studies have reported that BPA exposure has a well-known toxicity effect on reproduction function, such as reducing the number of ovulated oocytes, oocyte quality, and maturation rate. Recently, BPA induced mitochondrial-derived reactive oxygen species (mito-ROS) and disrupted mitochondrial homeostasis by increasing of superoxide anions production. In this study, we investigated how the regulation of mito-ROS production may play a critical role in meiotic maturation and expansion of cumulus cells during the in vitro maturation progression of porcine oocytes. Furthermore, we investigated the toxicity effect of BPA exposure on mitochondrial functions and mito-ROS production during porcine oocyte maturation in vitro. All results were analysed using a 1-way ANOVA followed by Bonferroni’s and Tukey’s Multiple Comparison Test and t-tests. First, porcine oocytes were matured in NCSU-23 medium supplemented with BPA (50, 75, and 100 µM) for 44 h. Our results indicated that the rates of matured oocytes were significantly decreased by BPA exposure in a dose-dependent manner (69.4 ± 5.1, 50.9 ± 6.3, and 29.9 ± 5.8% for BPA treatments of 50, 75, and 100 μM) compared with control group (70.2 ± 7.8%; P &lt; 0.05). Next, we confirmed the secretion functions of oocyte and cumulus cell of cumulus-oocyte complex (COC) and ROS production. Cumulus cell secretion factors (has2, tnfaip6, and cx37) mRNA expression in COC were decreased in the BPA-treated (75 µM) group. In addition, mRNA expressions of mitochondrial-specific antioxidant enzymes (sod2, P &lt; 0.001; prdx3, P &lt; 0.01; prdx5, P &lt; 0.001) and mitochondrial apoptosis genes (bax and caspase-3, P &lt; 0.01) were significantly increased in COC of the BPA-treated (75 µM) group. We measured mitochondrial membrane potential and mito-ROS production using JC-1 analysis and Mito-SOX staining, respectively. The BPA treatment caused a rapid decrease of mitochondrial membrane potential maintenance and increase of mito-ROS production in porcine COC. Moreover, mitochondrial-specific ROS scavenger, Mito-Tempo (0.1 µM) treatment was significantly increased the meiotic maturation of porcine oocytes compared with control group (78.5 ± 3.5 v. 65.8 ± 5.0%; P &lt; 0.05). Based on these results, we first confirmed that BPA exposure reduces the meiotic maturation and cumulus cells expansion of COC by increasing mito-ROS production during porcine oocyte maturation in vitro. Therefore, controlling of mito-ROS for mitochondrial function maintenance and apoptosis plays a critical role in improving porcine oocyte maturation in vitro. This work was supported by grants from the Next-Generation BioGreen 21 Program (PJ01117604) and the Bio-industry Technology Development Program (316037–04–1-HD020) through the Rural Development Administration, the Ministry of Agriculture, Food and Rural Affairs, Republic of Korea.

In vitro fertilization of porcine oocytes is affected by spermatic coincubation time

Abstract: The aim was to study the effects of different gamete coincubation times on porcine in vitro fertilization (IVF), and to verify whether efficiency could be improved by reducing oocyte exposure time to spermatozoa during IVF. In groups of 50, a total of 508 immature cumulus-oocyte complexes (COCs) were matured in NCSU-37 medium. The COCs were cultured for 44 hours and then inseminated with in natura semen (2,000 spermatozoa/oocyte). The sperm and oocytes were coincubated according to the following treatments (T): T1 = oocytes exposed to spermatozoa for one hour (173 oocytes), T2 = oocytes exposed to spermatozoa for two hours (170 oocytes), and T3 = oocytes exposed to spermatozoa for three hours (165 oocytes). After these coincubation periods, the oocytes were washed in fertilization medium (TALP medium) to remove spermatozoa not bound to the zona pellucida and cultured in another similar medium (containing no sperm). Eighteen to twenty hours after fertilization, the putative zygotes were stained in Hoechst-33342 to evaluate the IVF results. The penetration rate was higher (P&lt;0.05) after two hours of coincubation time than it was for one or three hours. Furthermore, 68.60% of the ova coincubated with the spermatozoa for two hours were monospermic. The oocytes exposed to spermatozoa for one hour (T1) presented a higher (P&lt;0.01) rate of polyspermy than those in T2 and T3. Fertilization performance (%) did not differ (P&gt;0.05) between oocytes exposed to spermatozoa for one (T1) and three hours (T3). However, optimum (P=0.048) results were obtained after two hours of coincubation, when the rate of fertilization performance was 50.16±8.52%. The number of penetrated sperm per oocyte, as well as male pronucleus formation, did not differ (P&gt;0.05) between the treatments evaluated. Under these assay conditions, especially in relation to the sperm concentration used, gamete coincubation for a period of two hours appears to be optimal for monospermy and fertilization performance. Thus, it is the optimal time period for obtaining a large number of pig embryos capable of normal development.

Changes of Ganglioside GM3 Expression in Porcine Oocyte Maturation and Early Embryonic Development in vitro

Gangliosides exist in glycosphingolipid-enriched domains on the cell membrane and regulate various functions such as adhesion, differentiation, and receptor signaling. Ganglioside GM3 by ST3GAL5 enzyme provides an essential function in the biosynthesis of more complex ganglio-series gangliosides. However, the role of gangliosides GM3 in porcine oocytes during in vitro maturation and early embryo development stage has not yet understood clear. Therefore, we examined ganglioside GM3 expression patterns under apoptosis stress during maturation and preimplantation development of porcine oocytes and embryos. First, porcine oocytes cultured in the NCSU-23 medium for 44 h after H2O2 treated groups (0.01, 0.1, 1 mM). After completion of meiotic maturation, the proportion MII (44 h) was significantly different among control and the H2O2 treated groups (76.8±0.3 vs 69.1±0.4; 0.01 mM, 55.7±1.0; 0.1 mM, 38.2±1.6%; 1 mM, P<0.05). The expressions of ST3GAL5 in H2O2 treated groups were gradually decreased compared with control group. Next, changes of ST3GAL5 expression patterns were detected by using immunofluorescene (IF) staining during preimplantation development until blastocyst. As a result, we confirmed that the expressions of ST3GAL5 in cleaving embryos were gradually decreased (P<0.05) according to the early embryo development progress. Based on these results, we suggest that the ganglioside GM3 was used to the marker as pro-apoptotic factor in porcine oocyte of maturation and early embryo production in vitro, respectively. Furthermore, our findings will be helpful for better understanding the basic mechanism of gangliosides GM3 regulating in oocyte maturation and early embryonic development of porcine in vitro.

136 COMPARISON OF NCSU-23 AND ALPHA-MINIMAL ESSENTIAL MEDIA IN THE DEVELOPMENT OF ISOLATED PORCINE PREANTRAL FOLLICLES IN VITRO

To develop a preantral follicular culture system that will support follicular growth and result in fertilizable oocytes, we conducted an experiment designed to determine the best medium for culture. In our preliminary experiment, we compared 2 common base media used for porcine oocytes: α-minimal essential medium and NCSU-23. Ovaries were collected from prepubertal gilts at a local abattoir and transported to the laboratory in saline solution (0.9% NaCl) maintained at 30–35°C. The ovaries were cut into small pieces (1–3 mm), and preantral follicles were isolated mechanically. Preantral follicles from 280 to 300 μm in diameter were collected into a small dish containing medium TCM199 (Lonza 12–117F) supplemented with 5% fetal bovine serum. The follicles were transferred from the collecting medium to the culture medium that consisted of base medium (NCSU23 or α-minimal essential medium) supplemented with 3.5 μg mL–1 of insulin, 10 μg mL–1 of transferrin, 100 μg mL–1 of l-ascorbic acid, 7.5% porcine serum, and 1.5 ng mL–1 of FSH. The follicles were randomly distributed to the different experimental treatments and cultured for 6 days in 24-well cell culture plates, with 3 follicles per well in 280 μL of culture medium. The culture was carried out at 38.5°C in 5% CO2 in air. Culture medium was changed every 2 days with freshly prepared medium. The diameters of follicles were measured every 2 days, and each follicle was photographed and evaluated at 20× magnification. Forty-two follicles per group were analysed and collected in 4 replicates. Data were statistically analysed with ANOVA using the Generalized Linear Model (GLM) procedure (SPSS, version 18, SPSS Inc., Chicago, IL, USA), where the independent variable was the sample (group and day of culture). Tukey's post hoc test was used to perform multiple comparisons; the α level was set at 0.05. All data were expressed as quadratic means with standard error of the means. Only the antrum formation was evaluated by chi-square test. The results, reported in Table 1, show that there was no statistical differences between follicle size between NCSU23 or α-minimal essential media, but at Day 6 there was a positive trend (P = 0.08). Otherwise, when we compared the size inside the groups, we observed that the preantral follicles grew more in α-minimal essential media than in NCSU23. The percentages of antrum formation were 65 v. 76% (NCSU23 and α-minimal essential media, respectively). These results support the use of α-minimal essential media because it had a positive effect on the antrum formation, and that after Day 4 some follicles could undergo a regression phase. Future studies will be necessary to evaluate the molecular status and the hormone production. Table 1.Follicle size (μm) from Day 0 to Day 6

81 ALTERED PROTEIN COMPOSITION OF PORCINE FOLLICULAR FLUID DUE TO A HIGH-FIBRE DIET AND THE POTENTIAL FOR OPTIMISATION OF IN VITRO CULTURE MEDIA

This study reports a proteomic analyses on porcine follicular fluid (FF) obtained from a previous nutritional trial, where oocytes from gilts fed a high-fibre (HF) diet for the first 19 days of their third oestrous cycle produced blastocysts with more cells following in vitro maturation (IVM) and IVF compared with oocytes from control-fed (CON) pigs. Oocytes were matured in TCM-199 supplemented with LH and FSH at 0.5 μg mL–1 and 10% of the animals’ own pooled FF. Following IVF, resultant embryos were cultured in NCSU-23 medium for 6 to 7 days. We hypothesize that FF protein composition is altered by the HF diet and that this confers the reproductive benefits previously observed. The FF had previously been stored at –80°C after the IVF trials and was thawed for the current study, which compared the protein composition of pooled Day 19 FF from 12 CON pigs and 12 HF pigs. These gilts were a subset of the pigs described above with the largest FF volumes. The protein composition of pooled FF from 6 CON pigs whose oocytes produced blastocysts was compared with FF from 6 CON pigs whose oocytes did not produce blastocysts. The same analysis was carried out with the 6 HF pigs that produced blastocysts and the 6 HF pigs that did not produce blastocysts. Equal numbers of samples from animals were selected for experimental balance. The proteomic study was carried out in duplicate. Abundant proteins were depleted from FF by Proteominer enrichment. Samples were labelled by isotopic di-methylation, where in each analysis, one sample was labelled with a heavy methyl group, the other with a light methyl group. Proteins were detected by liquid chromatography tandem mass spectrometry. Protein identifications were filtered using a 1% false discovery threshold and a requirement for two or more peptides detected for each protein. Differentially expressed proteins (DEPs) were identified as having heavy/light ratios greater than 1.2 or less than 0.8, which are recognised cut-off points for differential expression in proteomics. Over 140 DEPs were detected between CON and HF samples, indicating a nutritional influence on FF protein composition. Over one-third (37%) of these DEPs were also differentially expressed in the blastocyst versus no blastocyst analyses, suggesting that the altered FF protein composition may affect IVF outcome. DEPs were submitted into Ingenuity Pathway Analysis to highlight associated canonical pathways and upstream regulators. Top ranking canonical pathways detected included coagulation system, acute phase response, and LXR/RXR activation pathways. Potential upstream regulators detected by IPA included transforming growth factor beta, tumour protein P53, and beta-oestradiol. These pathways and upstream regulators could serve as potential avenues for elucidating the mechanism(s) by which the HF diet results in the reproductive benefits and could lead to the refinement of IVM and IVF culture conditions. This study was funded by AHDB Pork and BBSRC.

80 REACTIVE OXYGEN SPECIES LEVEL IN PIG EMBRYOS CULTURED IN PRESENCE OF HYALURONAN

It has been reported that during in vitro embryo culture reactive oxygen species (ROS) are generated and are detrimental to embryo development. A recent study (Smorag et al., Proc. 9th ICPR, 2013, 110) demonstrated that an addition of 1 mg mL–1 of hyaluronan (HA) to porcine embryo culture medium improves the development of zygote to blastocyst stage and the quality of produced embryos. Moreover, the embryos cultured with HA showed lower inner mitochondrial membrane potential (Romek et al., 2015 Proc. Symp. Progress in Cell Biology: Mitochondria and Chloroplast, Krakow, 31). Based on the beneficial effect provided by supplementation of HA during embryo culture, we investigated the ROS level in porcine embryos cultured with HA. Porcine zygotes were obtained surgically after flushing the oviducts of superovulated and inseminated gilts. In the experimental group, zygotes were cultured up to the blastocyst stage in NCSU-23 medium supplemented with 1 mg mL–1 of HA (CROMA, Pharma GmbH, Leobendorf, Austria), in an atmosphere containing 5% CO2 in air, at 39°C. In the control group, HA supplementation was omitted. To measure ROS level, embryos at the stages 2–4 and 8–16 cell, morula, and blastocyst (experimental group) and zygote, 2–4 and 8–16 cell, morula, and blastocyst (control group) were labelled with 5 μM CM-H2DCFDA dye (Molecular Probes Inc., OR, USA) for 30 min at 39°C. Labelled embryos were then examined under a Nikon Eclipse microscope with a CCD camera. The total amount of fluorescence emitted from each individual embryos and proportional to the ROS level was measured in arbitrary units. The data were analysed using one-way ANOVA and post-hoc Tukey test. ROS level (mean ± standard error of the mean) in the experimental group was 8.21 ± 2.65 (n = 25), 10.31 ± 3.13 (n = 18), 9.08 ± 2.89 (n = 21), and 20.45 ± 2.38 (n = 31) for 2–4 cell, 8–16 cell, morula, and blastocyst, respectively, whereas in the control group was 9.15 ± 3.43 (n = 15), 7.11 ± 3.13 (n = 18), 8.67 ± 3.04 (n = 19), 11.47 ± 2.46 (n = 29), and 54.74 ± 2.89 (n = 21) for zygote, 2–4 cell, 8–16 cell, morula, and blastocyst, respectively. For experimental and control groups, ROS levels remained unchanged up to morula. On the contrary, at the blastocyst stage from the experimental group ROS level decreased significantly (P ≤ 0.05) in comparison with blastocysts from the control group. In conclusion, porcine blastocysts derived from zygotes cultured with supplementation of 1 mg of HA possess a significantly lower ROS level than blastocysts cultured without HA. This suggests that HA supplementation in culture medium can reduce the ROS level in porcine cultured blastocysts. The project was funded by the National Science Center based on decision number DEC-2012/07/B/NZ9/01326.

Localization and quantitative analysis of Cx43 in porcine oocytes during in vitro maturation

Many studies of the main gap junction protein, Cx43, have been conducted in porcine oocyte research, but they have been limited to investigations of cumulus-oocyte complexes (COCs). In this study, we verified Cx43 not in COCs, but in porcine oocytes during maturation, and conducted a quantitative time course analysis. The location and dynamics of Cx43 were examined by immunocytochemistry and western blotting, respectively. COCs were cultured in NCSU23 medium and processed for immunocytochemistry and western blotting at 0, 14, 28, and 42 h after denuding. A Cx43 signal was detected on oolemmas, transzonal projections and the surface of zona pellucidae. Western blotting showed that Cx43 band density increased from 0 to 14 h, and gradually decreased thereafter. Our results clarified that Cx43 is localized in the ooplasmic membrane through zona pellucidae and its level changes over time during culture in porcine oocytes.

Generation of large pig and bovine blastocysts by culturing in human induced pluripotent stem cell medium.

We investigated the effect of human induced pluripotent stem cell (hiPS) medium on porcine somatic cell nuclear transfer and bovine in vitro fertilized early blastocysts, in comparison with North Carolina State University (NCSU)-37 medium and in vitro culture (IVC)-II medium. After 2 days of culture, the diameter of the portion of the blastocyst that was extruded from the zona pellucid dramatically differed between porcine blastocysts cultured in hiPS medium and those cultured in NCSU-37 medium (221.47 ± 38.94 μm versus 481.87 ± 40.61 μm, P < 0.01). Moreover, the diameter of the portion of the blastocyst significantly differed between bovine blastocysts cultured in hiPS medium and those cultured in IVC-II medium (150.30 ± 29.49 μm versus 195.58 ± 41.59 μm, P < 0.01). Furthermore, the total number of cells per porcine and bovine blastocyst was more than two-fold higher in blastocysts cultured in hiPS medium than in those cultured in NCSU-37 medium (44.33 ± 5.28 and 143.33 ± 16.05, P < 0.01) or IVC-II medium (172.12 ± 45.08 and 604.83 ± 242.64, P < 0.01), respectively. These results indicate that hiPS medium markedly improves the quality of porcine and bovine blastocysts.

300 GANGLIOSIDE IMPROVES MEIOTIC MATURATION AND PREIMPLANTATION DEVELOPMENT OF PORCINE OOCYTES

Gangliosides are sialic acid-conjugated glycosphingolipids that are believed to regulate cell differentiation as well as the signals of several signal molecules, including epidermal growth factor receptor (EGFR). These compounds are localised in a glycosphingolipid-enriched microdomain on the cell surface and regulated by the glycosphingolipid composition. However, the role of gangliosides in porcine embryos is not yet clearly understood. Therefore, in this study, the relationship between ganglioside (GD1a) and EGFR activation was investigated during meiotic maturation and further development in porcine oocytes. Porcine oocytes were cultured in the NCSU-23 medium supplemented with or without EGF and GD1a for 44 h. Assessment of meiotic maturation was performed by using aceto-orcein staining method. Also, protein levels in matured oocytes were evaluated by Western blot analysis. After fertilization, presumptive zygotes were cultured in the PZM-3 medium for 6 days. All experiments were repeated more than 3 times. Data were analysed by using Student's t-test and Duncan's multiple range test. The proportion of GV arrested oocytes at 22 h was significantly different between the EGF + GD1a-treated and untreated group (41.6 ± 1.5 v. 25.0 ± 0.0%; P &lt; 0.05). After completion of meiotic maturation (44 h), the proportion of MII was significantly different between the EGF + GD1a-treated and untreated group (89.9 ± 3.6 v. 57.4 ± 5.3%; P &lt; 0.05). Also, expression of EGFR protein in matured porcine oocytes was increased in the presence of EGF and GD1a. After IVF, the percentage of penetrated oocytes was significantly higher in the EGF + GD1a-treated group (89.1 ± 2.3%), resulting in higher than normal pronucleus formation (2PN of 43.1 ± 5.2%). Finally, in result, differences in preimplantation developmental potential were detected between the oocytes that were matured with or without EGF and GD1a (50.4 ± 6.1 v. 27.2 ± 2.7%; P &lt; 0.05). These results suggest that GD1a improves the developmental competence of embryos via enhanced meiotic maturation of porcine oocytes by EGFR activation.