Background: Using cryovial for freezing dog spermatozoa provides a practical method to increase extended sperm volume and shorten the time required for equilibration by using a simple freezing techniques. The purpose of this study was to determine the optimal thawing condition for dog sperm cryopreservation using cryovials. Methods: For sperm freezing, cryovials with 200 × 106 sperm/mL were cooled after the addition of tris egg yolk extender (TEY) at 4℃ for 20 min, then TEY with 4% glycerol was added and equilibrated for another 20 min before being aligned over LN2 vapor for another 20 min and plunged directly into LN2. Spermatozoa were thawed in a water bath at 37℃ for varying times (25 sec, 60 sec, 90 sec, and 120 sec) in the first experiment. In the second experiment, spermatozoa were thawed in a water bath at various temperatures and times (37℃ for 1 min, 37℃ for 1 min with gentle stirring, 24℃ for 24 min, and 75℃ for 20 sec). In these experiments, the effect of thawing conditions on motility parameters, viability (SYBR-14/PI), and acrosome integrity (PSA/ FITC) of spermatozoa were investigated. Results: The post-thaw sperm motility parameters, viability, and acrosome integrity were not significantly different across the experimental groups. Conclusions: In this study, the characteristics of spermatozoa frozen using cryovials were not significantly affected by various thawing conditions.

Despite numerous advances in in-vitro embryo production (IVP), many documented factors have been shown to influence the development of mammalian preimplantation embryos and the success of IVP. In this sense, elevated levels of reactive oxygen species (ROS) correlate with poor outcomes in assisted reproductive technologies (ART) due to oxidative stress (OS), which results from an imbalance between ROS production and neutralization. Indeed, excessive production of ROS compromises the structural and functional integrity of gametes and embryos both in vivo and in vitro. In particular, OS damages proteins, lipids, and DNA and accelerates cell apoptosis. Several in-vivo and in-vitro studies report an improvement in qualityrelevant parameters after the use of various antioxidants. In this review, we focus on OS and the source of free radicals and their effects on oocytes, sperm, and the embryo during IVP. In addition, antioxidants and their important role in IVP, supplementation during oocyte in vitro maturation (IVM), in vitro culture (IVC), and semen extenders were discussed. Nevertheless, various methods for determining the level of ROS in germ cells have been briefly described. Still, it is crucial to develop standardized antioxidant supplement systems to improve overall IVP success. Further studies should explore the safety, efficacy, mechanism of action, and combination of different antioxidants to improve IVP outcomes.

Sperm cryopreservation is a fundamental process for the long-term conservation of livestock genetic resources. Yet, the packaging method has been shown, among other factors, to affect the frozen-thawed (FT) sperm quality. This study aimed to develop a new mini-straw for sperm cryopreservation. In addition, the kinematic patterns, viability, acrosome integrity, and mitochondrial membrane potential (MMP) of boar spermatozoa frozen in the developed 0.25 mL straw, 0.25 mL (minitube, Germany), or 0.5 mL (IMV technologies, France) straws were assessed. Postthaw kinematic parameters were not different (experiment 1: total motility (33.89%, 32.42%), progressive motility (19.13%, 19.09%), curvilinear velocity (42.32, 42.86), and average path velocity (33.40, 33.62) for minitube and the developed straws, respectively. Further, the viability (38.56%, 34.03%), acrosome integrity (53.38%, 48.88%), MMP (42.32%, 36.71%) of spermatozoa frozen using both straw were not differ statistically (p > 0.05). In experiment two, the quality parameters for semen frozen in the developed straw were compared with the 0.5 mL IMV straw. The total motility (41.26%, 39.1%), progressive motility (24.62%, 23.25%), curvilinear velocity (46.44, 48.25), and average path velocity (37.98, 39.12), respectively, for IMV and the developed straw, did not differ statistically. Additionally, there was no significant difference in the viability (39.60%, 33.17%), acrosome integrity (46.23%, 43.23%), and MMP (39.66, 32.51) for IMV and the developed straw, respectively. These results validate the safety and efficiency of the developed straw and highlight its great potential for clinical application. Moreover, both 0.25 mL and 0.5 mL straws fit the present protocol for cryopreservation of boar spermatozoa.

Cryopreservation is a widely-used efficient means of long-term sperm preservation. However, unlike other types of semen, cryopreserved boar semen has reduced fertility and the efforts continue to optimize post-thawing sperm recovery. In this study, we evaluated the effects of various washing solutions (Hulsen solution, labmade DPBS and commercial DPBS) on post-thawing porcine sperm kinematics (CASA system), viability (SYBR-14/PI) and acrosome integrity (PSA/FITC). We also examined the effect of washing-centrifugation on frozen-thawed semen kinematics. The results indicate that type of washing solution and post-thawing centrifugation alters parameters linked to sperm quality (total motility, progressive motility, viability and acrosome integrity). Significantly higher (p < 0.05) motility and progressive motility were obtained when cryopreserved semen was processed with Hulsen solution. The postthaw percentage of live and intact acrosomal sperm was significantly higher in group 1 (Hulsen solution) as compared to other groups. Following thawing-centrifugation, the results showed significantly higher motility and progressive motility in group 1 than other groups. However, the latter two DPBS groups did not differ statistically. Taken together, Frozen-thawed spermatozoa motility, acrosome integrity and viability can be affected by the type of washing solution used. Moreover, centrifugation of frozenthawed semen has an unfavorable effect on total motility and progressive motility.

The present study was undertaken to evaluate the effect of trisaccharides supplementation in glycerol-free tris (GFT) for the cryopreservation of dog spermatozoa. In the first experiment (E1), dog spermatozoa were resuspended with 50, 75, 100 or 125 mM of raffinose, melezitose or maltotriose and cooled at 4 ℃ for 10 min. To determine the effect of different cooling time, the spermatozoa resuspended with 100 mM of raffinose, melezitose or maltotriose were cooled during 10, 20, 30 or 40 min at 4 ℃ (second experiment; E2). The straws were then aligned horizontally for 10 min on the rack and then plunged into LN2. In the third experiment (E3), to determine the effect of different vapor freezing time, the spermatozoa resuspended with 100 mM raffinose were cooled at 4 ℃ for 20 min and frozen in LN2 for 5, 10, 15 or 20 min and then plunged into LN2. In the fourth experiment (E4), to compare different freezing methods [cooling plus vapor freezing (CV), cooling plus step-down freezing (CS) and direct step-down freezing (SD)], the spermatozoa resuspended with 100 mM raffinose were cooled for 20 min and frozen in LN2 vapor for 5 min in case of CV method. In case of CS method, spermatozoa were cooled for 20 min at 4℃ and then frozen by the step-down freezing method. The straws were then aligned horizontally at 18, 15, 5, and 2 cm respectively from the surface of LN2 for 1, 1, 1.4, and 5 min, respectively in an L shaped straw holder and then plunged into LN2. For SD method, the straws were directly aligned horizontally at the same levels as CS from the surface of LN2 for 1, 1, 1.9, and 5 min, respectively and then plunged into LN2. After thawing at 37℃ for 25 sec, the spermatozoa were then incubated for 30 min in the freezing extender (E1) or in the 50 mM sucrose supplemented GFT (E2, E3, and E4) at 24℃. Following post-thaw incubation, sperm progressive motility and viability were assessed in E1, E2, E3, and E4. In addition, acrosome integrity, and gene expression related to apoptosis (BAX, BCL2, and Caspase10) and sperm motility (SMCP) were evaluated in E4. The results demonstrated that, in E1, using 75 mM trisaccharides resulted in significantly (p<0.05) higher sperm motility in all sugar groups. Using 100 mM melezitose significantly (p<0.05) improved the post-thaw viability than the 100 mM raffinose. The viability in 100 mM maltotriose was similar with 100 mM raffinose and melezitose group. In E2, the different cooling time has no significant effect on post-thaw sperm progressive motility in all the sugar types. In addition, the viability was variable among the different groups. In E3, liquid nitrogen vapor freezing for 5 min resulted in improved motility and viability. The sperm progressive motility was significantly (p<0.05) higher in CV and SD group compared to CS group and the sperm viability was significantly (p<0.05) higher in CV group compared to the other groups in E4. However, the acrosomal integrity of spermatozoa in the group CV was significantly (p<0.05) higher than the group CS and SD. In addition, the expression of SMCP gene was significantly (p<0.05) higher in the CV group than the CS group. In contrast, the expression of Caspase10 significantly (p<0.05) lower in the group CV and SD than the group CS. Furthermore, the ratio of gene expression of BAX and BCL2 was significantly (p<0.05) lower in the group CV than the group CS. Therefore, cryopreservation of dog spermatozoa in 100 mM of raffinose supplemented GFT cooled for 20 min and vapor freezing for 5 min provides better progressive sperm motility, viability, and acrosome integrity with higher expression of SMCP gene and lower expression of caspase10 and BAX/BCL2 ratio following post-thaw incubation in 50 mM sucrose supplemented GFT for 30 min at 24℃.

The aim of this study was to develop a chemically defined extender for dog sperm cryopreservation by supplementation of essential and non-essential amino acids solution in EY-free PVA extender. Spermatozoa collected from mature dogs (1 ｘ 108 cell/ml) were frozen with EY-free extender supplemented with 0 (control), 1, 2, 4 % essential amino acids (EAAs) or 1, 2, 4 % non-essential amino acids (NEAAs). Sperm progressive motility, viability and acrosome integrity were evaluated immediately after thawing at 37 ℃ for 25 s and post-thaw incubation at room temperature for 20 min. In addition, to evaluate the synergistic effect of EAAs and NEAAs, spermatozoa were frozen with 0, 0.5, 1 or 2 % EAAs-NEAAs mixture (v:v). Sperm progressive motility, viability and acrosome integrity were evaluated immediately after thawing and post-thaw incubation. Additionally, spermatozoa were frozen using EY-free PVA extender supplemented with 2 % EAAs, 2 % NEAAs or 0.5 % EAAs-NEAAs mixture. The ROS level and phosphatidylserine (PS) translocation (Annexin V-FITC assay) were assessed using flow cytometry. In addition, gene expression level for SMCP (motility-related), apoptosis-related BCL2 and BAX was measured after freezing-thawing. The progressive motility of spermatozoa cryopreserved in EAAs or NEAAs significantly increased (P < 0.05) in all groups compared to the control group regardless of thawing conditions. In addition, 1 % NEAAs significantly protected the acrosome membrane of spermatozoa after freezing-thawing (P < 0.05). However, EAAs has shown no significant effect on viability and acrosome membrane integrity of spermatozoa. On the other hand, addition of EAAs-NEAAs mixture to EY-free PVA extender significantly (P < 0.05) increased sperm progressive motility without any effect on viability. Supplementation of 0.5 % EAAs-NEAAs mixture significantly (P < 0.05) increased the expression level of SMCP, BCL2 and BAX compared to control without significant effect on PS translocation and ROS level. We conclude that essential and non-essential amino acids solution can be effectively used in EY-free extender to improve sperm motility, acrosome integrity and gene expression of SMCP and BCL2 in dog sperm cryopreservation.

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

The objective of this study was to determine the effect of post-activation treatment with cytoskeletal regulators in combination with or without 6-dimethylaminopurine (DMAP) on embryonic development of pig oocytes after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). PA and SCNT oocytes were produced by using in vitromatured pig oocytes and treated for 4 h after electric activation with 0.5 μM latrunculin A (LA), 10.4 μM cytochalasins B (CB), and 4.9 μM cytochalasins D (CD) together with none or 2 mM DMAP. Post-activation treatment of PA oocytes with LA, CB, and CD did not alter embryo cleavage (85.8～88.6%), blastocyst formation (30.7～ 32.4%), and mean cell number of blastocysts (33.5～33.8 cells/blastocyst). When PA oocytes were treated with LA, CB, and CD in combination with DMAP, blastocyst formation was significantly (P<0.05) improved by CB+DMAP (42.5%) compared to LA+DMAP (28.0%) and CD+DMAP (25.1%), but no significant differences were found in embryo cleavage (77.5～78.0%) and mean blastocyst cell number (33.6～35.0 cells) among the three groups. In SCNT, blastocyst formation was significantly (P<0.05) increased by post-activation treatment with LA+DMAP (32.9%) and CD+DMAP (35.0%) compared to CB+DMAP (22.0%) while embryo cleavage (85.5～85.7%) and blastocyst cell number (41.1～43.8 cells) were not influenced. All three treatments (LA, CB, and CD with DMAP) effectively inhibited pseudo-polar body extrusion in SCNT oocytes. The proportions of oocytes showing single pronucleus formation were 89.6%, 83.9%, and 93.3%, respectively with the increased tendency (P<0.1) by LA+DMAP and CD+ DMAP compared to CB+DMAP. Our results demonstrate that post-activation treatment with LA or CD in combination with 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.

The present study examined the histological characteristics of adult testis in the long-beaked common dolphin (Delphinus capensis) from Korean waters and the localization of DEAD-box polypeptide 4 (DDX4; a germ cell marker) and vimentin (a Sertoli cell marker) expression in the dolphin testis compared with that in terrestrial mammals, including dogs and rats. The seminiferous tubules of dolphin testis have very small or completely closed lumens, and spermatogenic cells and Sertoli cells within the tubules cannot be differentiated. Immunohistochemical analysis showed that, in the dolphin testis, DDX4- and vimentin-positive cells were scattered extensively within the tubule, whereas in the dog and rat testis, DDX4 immunoreactivity was localized in spermatogenic cells of the adluminal compartment, and vimentin immunoreactivity was localized in Sertoli cells of the basal compartment in the seminiferous epithelium. These results suggest that the histological characteristics of the seminiferous tubules in the dolphin testis differ from those of terrestrial species.

A lot of works have been dedicated to clarify the reasons why the establishment of embryonic stem cells (ESCs) from pig is more difficult than that from mouse and human. Several concomitant factors such as culture condition including feeder layer, sensitivity of cell to cell contact, definitive markers of pluripotency for evaluation of the validity and optimal timing of derivation have been suggested as the disturbing factors in the establishment of porcine ESCs. Traditionally, attempts to derive stem cells from porcine embryos have depend on protocols established for mouse ESCs using inner cell mass (ICM) for the isolation and culture. And more recently, protocols used for primate ESCs were also applied. However, there is no report for the establishment of porcine ESCs. Indeed, ungulate species including pigs have crucial developmental differences unlike rodents and primates. Here we will review recent studies about issues for establishment of porcine ESCs and discuss the promise and strategies focusing on the timing for derivation and pluripotent state of porcine ESCs.

The influenza viruses can be spread from birds to people. In this process, the pig is the intermediate host, and this virus is amplified and produces many mutations in pigs. Therefore, we attempted to develop the influenza-resistant pigs for the study of the virulence test and the transgenic (TG) animal model for translational research. At interferon- α, γ treated cells, the porcine Mx2 protein has been observed near the nuclear envelope and inhibits influenza virus proliferation, but not in common cells. So, we tried to produce the Mx2 gene over-expressed pig by somatic cell nuclear transfer(SCNT).First, we establish the Mx2 gene over expressed cells for the preparation of the TG donor cells. Porcine fetal fibroblasts were transfected with cytomegalo virus vector which include the porcine Mx2 gene. The established transgenic cell was injected into the enucleated ooplasm for the production of the Mx2-TG cloned embryos. Total, 511 female TG porcine SCNT embryos (TG-SCNTembryos) were made. The 511 female TG-SCNT embryos were transferred to five surrogates. On 25 days after embryo transfer, two of female embryos’ surrogates were diagnosed as pregnant (pregnancy rate, 40%). On day 114, we obtained six cloned piglets and four mummies from two of female embryos’ surrogate. Being analyzed by PCR, all female piglets were not integrated with Mx2 gene. Hereby, we again established newly male MX-TG cell line for donor cell of SCNT. 427 male TG-SCNT embryos were made. From these, 38 of male TG-SCNT embryos were cultured in in vitro to confirm the developmental capacity of TG-SCNT embryos. Among these porcine SCNT-TG embryos, 26 embryos (68.4%) were cleaved. Finally, 5 transgenic porcine SCNT embryos (13.2%) developed to the blastocyst stage. All male TGSCNT blastocysts were proved to be integrated with Mx2 gene as PCR analysis. Therefore, we expect that newly birth male piglets will be targeted with MX2 gene. The remaining 389 male embryos were transferred to four surrogates. On 25 days after embryo transfer, one of male embryos’ surrogates was diagnosed as pregnant (pregnancy rate, 25%). Now, pregnant surrogate have maintained at 88 days after embryo transfer and shown more than eight embryonic sacs. This study has presented new possibilities of production of influenza virus resistant pig by SCNT for translational research. * This work was supported by a grant from Next-Generation BioGreen 21 program (# PJ008121), Rural Development Administration, Republic of Korea.

Among laboratory animals, pigs are anatomically and physiologically closer to human. Transgenic (TG) pigs can be widely applied as models of human diseases. Many researchers created TG pigs which have specific modified genome under a constitutively active promoter. A constitutively active promoter is effective to express a target gene, but the uncontrollable expression often results in unwanted outcomes. In this study, as a way to solve these problems, we tried to regulate the expression of target genes by tetracycline (Tet) on/off system. We tested the operation of Tet on/off system in TG donor cells. Miniature porcine fetal fibroblasts were transfected with universal doxycycline- inducible vector and an enhanced green fluorescent protein (eGFP) was used as the target gene. The induced transgene expression by doxycycline was detected on fluorescence microscopy. On one day after 1 μg/ml doxycycline treatment, the fluorescence intensity for TG cells was increased. And we then performed Somatic Cell Nuclear Transfer (SCNT) to confirm the working of Tet on/off system in the porcine SCNT-TG embryos. Total 649 transgenic porcine SCNT embryos were made. From these, 64 of SCNT embryos used in invitroculturewith1 μg/mldoxycycline. Among these porcine SCNT-TG embryos, 39 embryos (60.9%) were cleaved. Finally, 15 transgenic porcine SCNT embryos developed blastocyst. Induced transgene expression was observed all of cleaved embryos and blastocysts. The remaining 585 embryos were transferred to 6 surrogates. On 25 days after embryo transfer, two surrogates were diagnosed as pregnant (pregnancy rate =33.3%). On day 113 (one day prior to delivery), we obtained six cloned TG piglets from first pregnant surrogate. Unfortunately, all TG piglets died because their surrogate died suddenly at delivery time. However, we could obtain the TG cell lines from the cloned TG piglets. Being analyzed by PCR, all piglets were found to be eGFP gene targeted. Now, second pregnant surrogate have maintained at 80 days after embryo transfer and shown more than three embryonic sacs. This data suggested that, Tet on/off system can control target gene expression in transgenic porcine SCNT embryos. This result has presented new possibilities of regulation of target gene expression in cloned TG pigs by Tet on/off system. * This work was supported by a grant from Next-Generation BioGreen 21 program (# PJ008121), Rural Development Administration, Republic of Korea.

Porcine blastocyst’s quality derived from in vitro is inferior to in vivo derived blastocysts. In this study, to improve in vitro derived blastocyst’s quality and then establish porcine ESCs (pESCs), we treated in vitro fertilized (IVF) embryos and parthenogenetic activated (PA) embryos with three chemicals: porcine granulocyte-macrophage colony stimulating factor (pGM-CSF), resveratrol (RES) and β-mercaptoethanol (β-ME). The control group was produced using M199 media in in vitro maturation (IVM) and porcine zygote medium-3 (PZM3) in in vitro culture (IVC). The treatment group is produced using M199 with 2 μM RES in IVM and PZM5 with 10 ng/mL pGM-CSF, 2 μM RES and 10 μM β-ME in IVC. Data were analyzed with SPSS 17.0 using Duncan’s multiple range test. In total, 1210 embryos in PA and 612 embryos in IVF evaluated. As results, we observed overall blastocyst quality was increased. The blastocyst formation rates were significantly higher (p<0.05) in the treatment groups (54.5%) compared to the control group (43.4%) in PA and hatched blastocysts rates in day 6 and 7 were also increased significantly. Total cell numbers of blastocyst were significantly higher (p<0.05) in the treatment group (55.1) compared to the control group (45.6). In IVF, hatched blastocysts rates in day 7 were increased significantly, too. After seeding porcine blastocyst, the attachment rates were higher in the treatment group (36.2% in IVF and 32.2% in PA) than the control group (26.6% in IVF and 19.5% in PA). Also, colonization rates and cell line derivation rates were higher in treatment group than control group. Colonization rates of control group were 10.8% in IVF and 2.4% in PA, but treatment group were 17.75% in IVF, and 13.1% in PA. And we investigated the correlation between state of blastocysts and attachment rate. The highest attachment rate is in hatched blastocyst (78.35±15.74 %). So, the novel system increased quality of porcine blastocysts produced from in vitro, subsequently increased attachment rates. The cell line derivation rates were 4.2% (IVF) and 2.4% (PA) in control group. In treatment group, they were 10.0% (IVF) and 7.2% (PA). We established 3 cell lines from PA blastocysts (1 cell line in control group and 2 cell lines in treatment group). All cell line has alkaline phosphatase activity and express pluri-potent markers. In conclusion, the novel system of IVM and IVC (the treatment of RES during IVM and RES, β-ME, and pGM-CSF during IVC) increased quality of porcine blastocysts produced from in vitro, subsequently increased derivation rates of porcine putative ESCs.

The present study examined the expression of porcine sirtuin 1–3 (Sirt1–3) genes in immature (germinal vesicle; GV stage), mature (metaphase II; MII stage) oocytes, preimplantation embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). We also investigated the role of sirtuins in oocyte nuclear and cytoplasmic maturation, and embryonic development of PA and IVF embryos using sirtuin inhibitor [5 mM nicotinamide (NAM) and 100 μM sirtinol]. The expression of Sirt1–3 mRNA was significantly (p<0.05) up-regulated during IVM. The expression patterns of Sirt1–3 mRNA in preimplantation embryos of PA, IVF and SCNT were gradually (p<0.05) decreased from MII stage of oocyte to blastocyst stage. Especially, the expressions of Sirt1 and Sirt3 in SCNT blastocysts were significantly lower than IVF blastocysts. Treatment with nicotinamide (NAM) during IVM resulted in significantly decreased nuclear maturation but it was restored when NAM treated with 2 μM resveratrol (RES; known as antioxidant and sirtuin activator) compared to the control (control: 88.9%, NAM: 67.9% and NAM+RES: 86.4% respectively). Intracellular reactive oxygen species (ROS) level of oocytes matured with NAM was significantly increased and with NAM+RES was significantly decreased compared to the control. Treatment with sirtuin inhibitors during IVC resulted in significantly decreased blastocyst formation and total cell number of blastocyst derived from PA (NAM: 29.4% and 29.6, sirtinol: 31.0% and 30.3, and control: 40.9% and 41.7, respectively) and IVF embryos (NAM: 10.4% and 30.9, sirtinol: 6.3% and 30.5, and control: 16.7% and 42.8, respectively). There was no significant difference in cleavage rate both PA and IVF embryos. Oocytes treated with NAM during IVM showed significantly lower expression of PCNA, Bax, Bcl-2, POU5F1 and Sirt1–3 compared to the control. Oocytes treated with NAM+RES during IVM restored gene expression except POU5F1. Similarly, PA derived blastocysts treated with NAM during IVM showed down-regulation of PCNA, Bax, Bcl–2, POU5F1 and Sirt1–2. The blastocysts derived from PA embryos treated with sirtuin inhibitors during IVC showed lower (p<0.05) expressions of POU5F1 and Cdx2 genes. Also, Sirt2 mRNA expression was significantly decreased in sirtinol treated group and Sirt3 mRNA expression was also significantly de -creased in both NAM and sirtinol treated groups compared to the control. These findings indicate that Sirt1–3 which are transcribed and stored during oocyte maturation may have physiological and important roles in porcine oocyte maturation and preimplantation embryonic development by regulating gene expressions. * This work was supported by a grant from Next-Generation BioGreen 21 program (# PJ008121), Rural Development Administration, Republic of Korea.

The study investigated the effects of trans-ε-viniferin on in vitro maturation (IVM) and gene expression. Three experiments were conducted. Firstly the trans-ε-viniferin was purified from the leaves and stems of the Vitis amurensis , a common wild grape found in Korea, Japan, and China. In the first experiment, a total of 594 cumulus oocytes complexes (COCs) were used for the evaluation of the nuclear maturation. COCs were matured with various concentrations of trans-ε-viniferin (0, 0.1, 0.5, 1.0 and 5.0 μM). After IVM 42 44 h, the nuclear maturation was evaluated. In the second experiment, a total of 300 matured oocytes were used to examine the effects of different trans-ε-viniferin concentrations (0, 0.5 and 5.0 μM) on porcine oocyte intracellular GSH and ROS levels. In the third experiment, the gene expression of oocytes matured with trans-ε-viniferin (0.5 μM) and the untreated group were evaluated after IVM. As results, we observed that trans-ε- viniferin treatment during IVM did not improve the nuclear maturation. But significantly increased (p<0.05) intracellular GSH levels in 0.5 μM group (0 μM vs. 0.5 μM; 14.6 vs. 16.8 pmol/oocyte) and reduced ROS levels (0 μM vs. 0.5 μM and 50 μM; 174.6 vs. 25.7 and 23.8 pixel/oocyte). The trans-ε-viniferin treatment during IVM of recipient oocytes promoted higher (p<0.05) expression of Dnmt1 mRNA in 0.5 μM treatment group than in the control group. But, the other gene expressions (PCNA, OCT4, caspase3, BAK, BAX and sit1) did not significantly differ from the control. In conclusion, these results indicated that the trans-ε-viniferin treatment during porcine IVM increased the cytoplasmic maturation through increasing the intracellular GSH synthesis, reducing ROS levels and increasing the Dnmt1 gene expression.