The objective of the present study was to evaluate the effect of disaccharides supplementation in glycerol-free tris (GFT) on dog sperm cryopreservation with respect to pH adjustment of extender and post-thaw incubation. The spermatozoa collected from five dogs were resuspended (5×107 cell/ml) with GFT containing 100 mM of lactose (L), trehalose (T) or sucrose (S) or pH adjusted (6.85) 100 mM of lactose (LP), trehalose (TP) or sucrose (SP). The sperm (500 μl) were loaded in straws, cooled for 50 min at 4℃, frozen using liquid nitrogen (LN2) vapor for 20 min and plunged in LN2. After thawing at 37℃ for 25 s in a water bath, the spermatozoa were incubated at 24℃ for 30 min. The progressive motility, viability, mitochondrial membrane potential (MMP) and mRNA expression of SMCP gene were then assessed. The MMP was evaluated by combined JC-1 plus PI staining. The relative abundance of SMCP was assessed using quantitative real-time polymerase chain reaction (RT-PCR). Adjustment of pH in GFT extender supplemented with disaccharides did not improve sperrm motility and viability. In general, post-thaw incubation increased the progressive motility of spermatozoa. The sperm motility in the group S was significantly (P<0.05) higher than other groups regardless of post-thaw incubation time. Similarly, the sperm viability in the group S was significantly (P<0.05) higher following post-thaw incubation. The higher sperm motility in the group S was also supported with the significantly (P<0.05) higher live sperm having high MMP. There was no significant difference in mRNA expression of SMCP gene among the experimental groups. These results indicate that cryopreservation of dog sperm in GFT supplemented with S and 30 min post-thaw incubation at 24℃ could provide better freezability of dog spermatozoa with improved motility and higher MMP.

In the present study, we evaluated the effect of glucose-fructose and sucrose supplementation in glycerol-free tris (GFT) on sperm motility, viability, ROS level, apoptosis (BAX and BCL2) and motility (SMCP) related gene expression of dog sperm according to different post-thaw incubation time. The spermatozoa collected from five dogs were resuspended (5×107 cell/ml) with GFT containing 86 mM glucose and 86 mM fructose (GF-GFT) or 100 mM sucrose (S-GFT). The sperm (500 μl) were loaded in straws, cooled for 50 min at 4℃, frozen using liquid nitrogen (LN2) vapor for 20 min and plunged in LN2. The progressive motility, viability, ROS (H2O2) level and mRNA expression of spermatozoa were evaluated according to post-thaw incubation time (0 h, 3 h and 6 h) at 24℃. ROS was assessed using H2DCFDA stain by flow cytometry. The relative abundances of BAX, BCL2 and SMCP were assessed using quantitative real-time polymerase chain reaction (RT-PCR). The motility of spermatozoa cryopreserved in GF-GFT was increased throughout the post-thaw incubation time. The motility of spermatozoa cryopreserved in S-GFT was increased at 3 h of post-thaw incubation. Whereas, the sperm ROS level in GF-GFT group was decreased at 6 h of post-thaw incubation. However, the ROS level in the group S-GFT was gradually increased with the progress of post-thaw incubation period. The post-thaw incubation had no substantial effect on mRNA expression of BAX, BCL2 and SMCP genes of dog spermatozoa in both the GF-GFT and S-GFT groups. These results indicate that GF supplementation in GFT improves the progressive sperm motility during the 6 h of post-thaw incubation with maintaining similar sperm viability and is more efficient in reducing ROS after 3 h of post-thaw incubation. The addition of GF in GFT for the cryopreservation of dog spermatozoa and post-thaw incubation would open an option to achieve more functioning spermatozoa for future assisted reproduction practices.

The purpose of this study is to evaluate the effects of astaxanthin added to freezing buffer on semen parameters, total sperm oxidation stress after post-thawing of boar sperm and lipid peroxidation (LPO) which is caused by reactive oxygen species (ROS) in sperm membrane. Varying concentrations of astaxanthin (0, 10, 50, 100 and 500 μM) were used in the freezing buffer during cryopreservation to protect the DNA of thawed miniature pig sperm. Semen parameter was measured using computer assisted sperm analysis (CASA) for sperm motility and determine ROS rate, oxidative stress of boar sperm using fluorescence-activated cell sorting (FACS). Sperm motility was higher (p<0.05) in the astaxanthin group than in the control group. Sperm motility and the number of progressive motile sperm was higher (p<0.05) in the astaxanthin 500 μM group (66±1.7%) than in the control group (49.8±4%). In ROS evaluation, the astaxanthin group lowered intracellular O2 and H2O2 in viable sperm. The Yo-Pro-I/HE and PI/H2DCFDA staining as revealed using flow cytometry was lower in astaxanthin groups than in the other groups. As the result, we found that astaxanthin could protect the sperm plasma membrane from free radical and LPO during boar sperm post-thawing.

제2차 말 산업 육성 5개년 계획의 시행 등 말 산업이 전국적으로 확대됨에 따라 승용마 인공수 정을 원하는 농가도 점차적으로 증가하고 있는 추세이다. 말의 경우 신선·냉장정액 사용 시 적기 수정의 어려움이 있어 동결정액의 중요성이 대두되고 있다. 하지만 국내의 말 동결정액 연구는 미 미한 수준이며, 인공수정을 위한 말 동결정액은 전량 수입하여 사용하고 있는 실정이며, 말 동결 정액의 경우 신선정액이나 액상정액에 비해 활력 및 수태율이 현저히 떨어지는 문제점이 있다. 본 연구에서는 제주산마의 동결정액의 성상 개선을 위하여 펜톡시필린 수준(4mM, 8mM, 16mM)을 설 정하여 정자의 운동성, 생존율, 정자막 온전성을 평가하였다. PDE 억제제인 펜톡시필린 첨가는 정 자의 조기 첨체반응을 방지하고 수정률을 높이며 활성산소를 억제하는 효과가 있다고 보고되었 다. 정액의 동결-융해 30분 후에는 펜톡시필린 4mM(T1)처리구와 펜톡시필린 8mM(T2)처리구에서 Progressive Fast Motility(PFM)가 유의적으로 높게 나타났다. 융해 후 1시간에는 T4와 T8에서 대조 구보다 immotil, Motiltiy, Local Motiltiy에서 유의적으로 높은 결과를 보였다. 또한 생존율에서 T8보 다 T4가 유의적으로 높았으며, 펜톡시필린 16mM(T3) 처리구는 생존율은 대조구와 T4보다 높았으 나 Motiltiy가 다른 처리구보다 유의적으로 낮았다. 이상의 결과를 종합해 볼 때 펜톡시필린 4mM 와 8mM를 처리했을 때 비처리보다 정자의 운동성을 유의적으로 증진시키는 효과가 있었으며 펜 톡시필린 16mM 이상의 처리는 정자에 좋지 않은 영향을 미치는 것으로 나타났다.

Little is known to date about neural development of pig and directed differentiation of porcine pluripotent stem cells (PSCs) to neuronal cells remains elusive. To determine whether soluble factors from glioblastoma multiforme (GBM) promoted the neural differentiation from porcine induced PSCs (iPSCs), cells were treated cultured media of GBM cells. First of all, we isolated and established primary GBM cell line (WHO grade IV). The cellular morphology of GBM cancer cell line are dendritic-like with positive expression in NESTIN, SOX2, VIMENTIN and GFAP using immunofluorescence analysis. G-banded karyotype from primary GBM cell line revealed severe numerical chromosomal aberrations. GBM-cultured medium (CM) treated iPSC-NPCs survive well in vitro when supplemented with a combination of growth factors, including EGF and bFGF. The GBM-CM treated differentiated cells showed an increased mRNA expression level of astrocyte marker, GFAP and the dopaminergic neuron marker, tyrosine hydroxylase (TH). However, there was no significant difference in mRNA expression level of oligodendrocyte marker, MBP. The protocol developed in the present study for large animal models might provide an exciting tool to bridge the present gaps in neuroscience studies between rodents and humans.

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 is to investigate the effects of GDF8 on porcine oocytes during in vitro maturation (IVM). We investigated a specific gene transcription levels in oocytes and cumulus cells (CC) after IVM by realtime PCR arry, and specific protein expression and activation levels in matured CCs by western blotting. Each concentration (0, 1, 10, and 100 ng/ml) of GDF8 was added in maturation medium (TCM199) during process of IVM. Data were analyzed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science). Data are presented as the mean and Differences were considered significant at P < 0.05. After 44 h of IVM, oocytes are mechanically denuded from CCs with 0.1% of hyaluronidase, and then the separated oocytes and CCs were sampled following each group. To assess the effect of GDF8 on specific gene transcription level changes as a dose response during IVM, the realtime PCR array was performed. In CCs the 1- and 10 ng/ml of GDF8 supplement group showed the transcription co-factors CBP and SP1, cell metabolic regulator MAPK1, and cumulus expansion related genes Has2, Cox-2, Ptx3 and Areg transcription levels were significantly distinguished with control when hierarchically clustered by Euclidean distance with average linkage method after IVM. In matured oocytes the 10- and 100 ng/ml of GDF8 supplement group showed the maternal factors JMJD3 and Zar1, transcriptional regulator FOXO1, Sirt1 and Sirt2, mitochondrial activity factor Sirt3, ACSL3 and ACADL, anti-apoptosis gene BCL-2, and oocyte secrete factor BMP15 mRNA transcription levels were significantly distinguished compared with control. To determine effect of GDF8 supplement during IVM, the GDF8 down steam canonical regulator SMAD2/3 protein phosphorylation levels analyzed in CCs by western blotting. The 10- and 100 ng/ml supplement groups showed significantly increase phosphorylated (P)-SMAD3 (1.56 and 1.34 times higher than control) protein levels (P < 0.05). In conclusion, supplement of GDF8 during IVM activates FOXO homolog transcription and induced cumulus cells expansion via activation of SMAD3 signaling in CCs. While process of IVM, the transcriptional landscape changes in CCs may consequently result maternal factors accumulation and mitochondrial activation in oocytes.

The use of pigs in neuroscience has increased over the past years because the pigs are closely related to humans in terms of anatomy and physiology. Especially, the blood-brain barrier (BBB) maintains the homeostatic microenvironment in the central nervous system (CNS) and they can provide a valuable tool for studying the neurobiology. However, only a few putative blood-brain barrier (BBB) models have been generated by co-culture of porcine primary cells. The fundamental problem is that they lose some of their phenotypes when maintained in vitro for long-term culture. To establish improved in vitro porcine BBB models, we differentiated novel brain microvascular endothelial cells (BMECs) from porcine induced pluripotent stem cells (iPSCs) using a modified human-based protocol. Briefly, the dissociated single cells from iPSCs were seeded in Geltrex. For differentiation, cells were maintained for 3 days of expansion and then switched to unconditioned medium (UM) lacking bFGF for 6-7 days. Then, we subcultured cells onto collagen/fibronectin coated plates and changed BMEC medium for 2-3 weeks. About two weeks later, we observed a cluster of round cells surrounded by spindle shaped adherent cells termed as colony-forming units (CFU) of putative BMECs. Over time, the cluster of cells disappears and remained adherent spindle-shaped cells showed properties of endothelial cells. Although further studies will be needed, this study would be a great comparative analysis of the porcine and human in vitro BBB model.

The mesenchymal stem cells (MSC) has been investigated as a source of stem cell therapy to replace and treat damaged cells. Human endometrial epithelial and stromal cells was isolated from hysterectomy tissue and the direct evidence of stem/progenitor cells in the human endometrium was identified. Endometrium derived stem cells (EnMSCs) are known to have a high proliferative ability, genetic stability, lack of tumorigenicity and low immnunogenicity during long-term cultivation. Here, we aimed to identify MSC in canine endometrium and characterize its potential to differentiate into decidua cells. EnMSCs were isolated from thrown-away spayed uterus of adult canine depending on their estrus cycle, and identified by flow cytometry, immunocytochemistry and flow cytometry with MSC specific markers. We then characterized the ability of EnMSCs by the doubling-time analysis, colony-forming units and MSC differentiation assays. Isolated EnMSCs expressed stem cell specific genes (Sox2, Oct4, Nanog, MCAM, Endoglin, Susd2 and IGTB) and MSC surface markers (CD90, CD44 and CD117). EnMSCs are also differentiated into adipogenic, osteogenic and chondrogenic cells morphologically under modified conditions with the expression of lineage specific genetic markers. EnMSCs showed higher proliferation ability than canine amniotic fluid derived MSCs which were used as a positive control. EnMSCs were cultured at low density (10, 20, cells/cm2) and initiated to form small colonies of loosely-arranged cells and gradually formed large colonies of densely-packed cells which underwent self-renewal with high proliferative potential which is similar to the clonogenicity feature of human endometrium-derived stem cells. EnMSCs were then induced to differentiate into decidua cells with 0.5 mM dbcAMP. After 14 days, EnMSCs changed their morphology into the elongated and rounded shape. The induced decidual cells expressed PRL and IGFBP1 which are typically expressed in decidua cells. In conclusion, we successfully isolated and characterized MSC in the canine endometrium which differentiated into decidua cells. These results showed that endometrium may be a promising source of stem cells, and furthermore raise the possibility of canine EnMSCs as a novel hypothetical decidualisation model of infertility associated with decidualisation insufficiency and implantation failure.

The production of feline induced pluripotent stem cells (iPSCs) can solve the problems that are related with existing unstable supply and demand of eggs as well as ethical aspects about embryonic stem cell at the same time. On the basis of excellent proliferation, it is to facilitate the researches about human disease like FIV and Allergen at the level of cells, not experimental animals. But, a lot of advanced researches are lean too much towards on the transduction using DNA type virus that have the risk of tumorigenesis during reprogramming and on the mLIF-dependent culture condition for the production of feline iPSCs. This being so, this study shows the reprogramming results using Sendai virus vector that is RNA type virus and have no the footprint after transduction. In addition, the feline iPSCs were stably cultured in bFGF-dependent culture condition during the reprogramming step and culture step. In conclusion, we found the bFGF-dependent culture condition in feline iPSCs and suggested the approach using Sendai virus vector as an alternative for reprogramming without concern about tumorigenesis. These methods can be universally applicable to not only the researches about reconstruction and conservation of feline species, but also to a lot of deep studies related with iPSCs or LIF, bFGF to find new approaches.

Growth differentiation factor8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. Overall of the current studies, the GDF8 is detected in oviduct fluid and uterus which led us to suggest that the GDF8 may effect on preimplantation embryonic development and act paracrine role to correlate with successful late-blastocyst implantation in in vivo. The purpose of this study is the effects of GDF8 on porcine parthenogenesis (PA) embryo development during in vitro culture (IVC). We were investigated the effect of GDF8 supplement during PA embryo IVC by cleavage and blastocyst formation rate and patterning analysis. Data were analyzed by on way ANOVA, followed by Tukey’s range test. Respectively 0.2, 2 and 20 ng/mL of GDF8 were added during IVC followed experiment design as control, 0.2, 2, and 20 GDF8 supplement groups. After 48h of embryo culture time, no significant difference was observed on cleavage rate from the different concentration (0, 0.2, 2, and 20 ng/ml) of GDF8 supplement groups (65.7%, 66.0%, 66.3%, and 65.8%, respectively). After 120h of embryo culture time, the 0.2 and 2 group showed significantly (p<0.05) higher blastocyst formation rate than control (40.4% and 36.4% VS 40.4%, respectively). In embryo developmental pattern analysis, the 0.2 ng/ml GDF8 supplement groups showed significantly higher (p<0.05) 2-3 cell cleavage- and early blastocyst pattern compared with control (12.0% and 10.4% VS 6.6% and 6.2%, respectively). However there are no significantly different pattern was observed in other groups. In conclusion, the 0.2 ng/ml of GDF8 supplementation during porcine PA embryo IVC significantly changed embryonic developmental patterns. However there are further studies are required such as analysis of blastocyst total number, specific gene transcription pattern, and ICM/TE rate to make clarify and support the conclusion.

This study investigated the effect of Charcoal:Dextran Stripped fetal bovine serum (CDS FBS) and heat-inactivated FBS (HI FBS) in embryo culture medium on their ability to support in vitro development of bovine embryos. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, and cryo-tolerance. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly (P<0.05) higher in medium containing CDS FBS than in medium containing HI FBS (42.84 ± 0.78% vs. 36.85 ± 0.89%, respectively). Furthermore, the beneficial effects of CDS FBS on embryos were associated with a significantly reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. The post-thaw survival rate of blastocysts was significantly (P<0.05) higher in the CDS FBS than in the HI FBS group (85.33 ± 4.84% vs. 68.67 ± 1.20%). Quantitative real-time PCR showed that the mRNA levels of acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain, hydroxymethylglutaryl-CoA reductase, and insulin-like growth factor 2 receptor were significantly increased upon culture with CDS FBS. Moreover, the mRNA levels of sirtuin 1, superoxide dismutase 2, and anti-apoptotic associated gene B-cell lymphoma 2 in frozen-thawed blastocysts were significantly (P<0.05) higher in the CDS FBS group than in the HI FBS group, however, the mRNA level of the pro-apoptotic gene BCL2-associated X protein was significantly reduced. Taken together, these data suggest that supplementation of medium with CDS FBS improves in vitro bovine embryo developmental competence and cryo-tolerance.

처녀생식은 관심 개체의 생식세포에서 반수체 유전물질을 이배체로 복원시킴으로써, 요구되는 특정형질을 다음세대에 거의 소실 없이 전달할 수 있다. 쥐의 처녀생식을 통한 난모세포의 활성화 는, 앞선 논문 들에서 알려진 바, strontium에 가장 유의적으로 그 비율이 높다. 그러나 기존의 strontium의 유의성을 살펴보고자 했던 실험의 경우는, 배양될 배아들을 단일 단계만의 배지에서 실험을 진행한 사례가 다수이다. 따라서, 본 실험은 활성화된 쥐의 parthenotes의 배반포까지의 발 달률 및 생존률을 높이기 위해, R1ECM배지에서 KSOM배지로의 두 단계 배양 체계를 기반으로 진행하였다. 또한, 이미 보고된 glucose의 농도에 따른 배아의 발달률 변화와 상피세포 성장인자의 첨가로 인한 발달률 증가가, 단일 단계 배양이 아닌 두 단계 배양 체계에서도 유의적 결과를 보이 는가를 살펴보았다. R1ECM배지에서 One cell embryo 까지의 배양을 진행했으며, 이후 3그룹으로 나누어, 각각 KSOM만을, modified KSOM (high glucose)을, KSOM+EGF을 사용하여 각각 2, 4, 8-cell과 배반포까지의 발달률을 비교하였다. 각 그룹은 통제집단 (KSOM으로의 배지 교체를 하지 않음.)과 비교했을 때, 모두 유의적 발달률을 보였다. 특히, KSOM+EGF 집단이 가장 특기할 만한 결과치를 보였다. 다만, glucose의 농도를 달리한 KSOM 집단은, KSOM만을 사용한 집단과 큰 차 이를 보이지는 못하였다. KSOM 배지 배양에서 glucose의 농도 변화는 오히려 배지의 전체 농도의 지나친 증가를 야기한 것처럼 보였는데, 이는 좀 더 세밀한 조절을 해야 할 필요가 있을 것이다. 실험에 이용된 암컷 쥐의 난모세포는 hCG 주입 후 16시간이 지난 시점에서 채취되었는데, 본 실 험과 hCG 주입 이후의 경과된 시간과의 연관성은 향후의 추가적 연구가 필요해 보인다. 쥐 난 모 세포의 처녀생식 활성화를 위해 cytochalasin B와 cycloheximide가 첨가된 1.25 mM의 strontium을 사 용했는데, 해당 시약에서 cytochalasin B의 작용을 통해 세포 분열의 진행 단계에서 arrest 현상을 피할 수 있었던 것으로 사료된다.

The coat color of mammals is determined by the melanogenesis pathway, which is responsible for maintaining the balance between black-brown eumelanin and yellow-reddish pheomelanin. The melanogenesis-associated genes controlling pigmentation act as a complex and interact with each other to cause phenotypic and genotypic variations in cattle. That the MC1R genotype of Korean native cattle with dark muzzle was e/e or E+/e, while the genotype of Korean native cattle with light muzzle was E+/E+, which is a variant of the MC1R genotype in the Korean native cattle. Especially, the MC1R expression type is shows how much pigmentation, important factor in deciding its status in the coat and nose colours. However, information regarding the coat or nose colours-associated gene regulation of korean cattle is not yet unknown. Therefore, in this study was to investigate the expression patterns of melanogenesis-associated genes in black dot nose(korea brindle cattle) and normal nose(korea native cattle). Using microarray clustering and real-time polymerase chain reaction techniques, we analysed that the expression of genes involved in the mitogen-activated protein kinase (MAPK) and Wnt signaling pathways is distinctively regulated in the dark and light muzzle tissues. Differential expression of tyrosinase was also noticed, although the difference was not as distinct as those of MAPK and Wnt. We hypothesize that emphasis on the MAPK pathway in the Korea brindle cattle induces eumelanin synthesis through the activation of cAMP response elementbinding protein and tyrosinase, while activation of Wnt signaling counteracts this process and raises the amount of pheomelanin in the native cattle. Regarding the increasing interest in the genetic diversity of cattle stocks, genes we identified for differential expression in the brindle cattle vs. native cattle may serve as novel markers for genetic diversity among cows based on the coat and muzzle color phenotype.

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

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

To obtain in vivo matured oocytes for dog cloning, serum progesterone (P4) level were employed for ovulate determination. Radioactive immunoassay (RIA) is a traditional serum hormone assay method with highly radioactivity. The aim of this study was to evaluate the reliability of RIA and to compare its canine serum P4 concentration determination accuracy to that of the electric chemiluminescence immunoassay (ECLI). To obtain in vivo matured oocytes for canine somatic cell nuclear transfer, serum P4 levels were accurately measured with both methods of RIA and ECLI. Although both methods detected similar P4 level before ovulation, the mean P4 concentration using ECLI was significantly higher than that using RIA from 3days before ovulation. Following ovulation, oocytes were collected by surgery, and a lower percentage of mature oocytes were observed using ECLI (39%) as compared to RIA (67%) if 4-8ng/ml of P4 were criteria for determination of ovulation. On other hand, high percentage of mature oocytes was observed using ECLI when 6–15 ng/mL of progesterone was criteria for ovulation determination. To determine whether in vivo oocytes obtained by ECLI method could be used for canine cloning, six canines were selected as oocyte donors and two puppies were produced after SCNT and embryo transfer. In conclusion, compared to the traditional RIA method, the ECLI method is a safe and reliable method for canine cloning.

Canine cloning have been succeeded for a decade. To obtain in vivo matured dog oocytes, Serum progesterone (P4) level were employed for ovulate determination. However, accuracy of P4 methods is not satisfied. The aim of this study was to compare both methods of serum estradiol (E2) and P4 on the accuracy of canine ovulation determination. Canine serum P4 and E2 concentration during both proestrus and estrus were detected. Correlation between accuracy of each method and environment temperature were analyzed. Following ovulation, oocytes were collected by surgery. As a result, higher percentage of mature oocytes was obtained when using E2 (56.43%) as compared to P4 (39.60%). Accuracy of P4 increased from spring (30.76%) to summer (47.92%) and decreased in autumn (37.50%) and winter (29.16%) gradually. Especially, E2 maintained about 50% to 65% whatever the season and temperature. Correlation analyze showed that dynamic of P4 accuracy highly correlated with environment temperate (Rp4=0.862) but E2 could not be affected by the temperature (RE2=0.199). To determine whether obtained oocytes by E2 method could be used for canine cloning, twenty canines were selected as oocyte donors, and two puppies were produced after somatic cell nuclear transfer(SCNT) and embryo transfer(ET) with the oocytes by E2 method. In conclusion, comparing to the P4 method, the E2 is an accuracy and reliable method for canine cloning.

Introduction The objective of this study was to investigate the effects of staining of porcine cumulus-oocytes complexes (COCs) by brilliant cresyl blue (BCB) test prior to in vitro maturation may be used to select developmentally competent oocytes. Furthermore, milrinone can be used to promote developmental competence of porcine embryos produced during parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). Materials and Methods Slaughterhouse-derived porcine cumulus-oocyte-complexes (COCs) were exposed to BCB and treated oocytes divided into BCB+ (colored cytoplasm), BCB- (colorless cytoplasm) groups. After division into 2 groups, intracellular glutathione (GSH) and reactive oxygen species (ROS) of matured oocytes were compared. And, preimplantation development of PA and SCNT embryos were also compared between 2 groups. BCB- oocytes were exposed to milrinone with different concentrations (0, 50, 75, and 100μM) for 6 h prior to IVM for further development of embryos. Results and Discussion GSH was higher in BCB+ group than BCB- group whereas ROS was lower in BCB+ than BCBgroup. In parthenogenetic embryos, BCB+ oocytes group was significantly higher on maturation (87.5 vs 80.6, 71.3%), cleavage (88.6 vs 82.9, 76.3%), and blastocyst formation rates (34.3 vs 27.8, 25.3%) than control and BCB- oocytes groups, respectively. Moreover, ratio of ICM:TE cells were higher in BCB+ oocytes group (30.3% vs. 28.6, 26.4%, respectively) than other groups. In cloned embryos, the significant higher blastocyst formation rates were shown BCB+ groups (30.6% vs. 26.0, 20.1%) than BCB- groups. To improve the cytoplasmic maturation in BCB- oocytes, 4 different concentrations of milrinone (0, 50, 75, and 100μM) were supplemented in the IVM media for 6 h. BCB- oocytes supplemented with 75μM milrinone showed the significantly higher rates of blastocyst formation than other groups. Our results demonstrate that staining of porcine oocytes with BCB before IVM may be used for selection of good quality oocytes and milrinone supplementation can be used to improve embryo developmental competence of porcine embryos.

Introduction Porcine embryonic stem cells (pESCs) derived from cloned embryos might be a useful animal model in biomedical research, however, establishment of cloned pESCs is difficult by its incomplete nuclear reprogramming. Here, we report the improved development competence of porcine cloned embryos by vitamin C (VC) supplement to establish the pESCs. Materials and Methods Slaughterhouse-derived oocytes were in vitro matured for 44h and parthenogenetic and cloned embryos were produced using matured oocytes. Both of embryos were cultured for 6 days in PZM-5 media and development rates were examined. Four different concentration of VC (0, 25, 50, 100, and 200 μg/ml) was supplemented in IVM and IVC media and preimplantation developments in the 5 groups were compared in both of embryos Results and Discussion In the cleavage rates of IVM group, significantly higher rate was shown in 50 mg/ml group than other groups (84.5 ± 0.6% vs. 69.8 ± 5.5, 75.7 ± 1.8, 80.4 ± 0.2, 72.4 ± 0.1%; P<0.05), respectively. Significantly higher rates of blastocyst development also were shown in 50 mg/ml group than other groups (27.0 ± 2.0% vs. 20.4 ± 1.4, 22.1 ± 1.3, 23.7 ± 1.2, 19.6 ± 1.3%; P<0.05), respectively. In the cleavage rate of IVC group, non-significantly different with each group (84.0 ± 1.3, 86.7 ± 1.0, 88.4 ± 1.4, 76.7 ± 3.0, 64.6 ± 4.4; P<0.05). In the blastocyst rate of IVC group, significantly higher rate was shown in 25mg/ml and 50 mg/ml group than other groups (22.3 ± 1.7, 23.8 ± 1.7% vs. 19.1 ± 1.3, 15.9 ± 1.0, 5.8 ± 1.5%; P<0.05) In conclusion, supplement of 50μg/ml of VC in IVM and IVC media enhanced the development of porcine parthenogenetic embryos and these results will be a helpful information in the development of porcine cloned embryos and derivation of its embryonic stem cells.

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