The aim of this study was to examine the effect of acteoside (the cyclin-dependent kinase inhibitor) on the SCNT efficiency with adult fibroblasts in dog. Canine adult fibroblasts were obtained from muscle and cell cycle of fibroblasts was synchronized by culturing to confluency, serum starvation and treating with 30 μM acteoside for 48 h. Cell cycle stages, cell cytotoxicity (apoptosis) and, prduction of reactive oxygen species (ROS) were analyzed using flow cytometry. The canine cells, prepared by confluent-cell culture or treating with 30 μM acteoside for 48 h, were injected into enucleated in vivo matured oocytes, the couplets were electrical fused and activated by calcium ionomycin. SCNT embryos using acteoside-treated fibroblasts were surgically transferred into oviducts of estrus cycle synchronized recipient dogs. In cell cycle synchronization (G0/G1), there was no significant difference between serum starvations (83.9%) and acteoside treated groups (81.3%) that were higher than confluent group (78.5%). In production of apoptosis, confluent and acteoside treated groups (4.3 and 4.5%, respectively) were generated less than serum starvation group (21.8%). In case of ROS, serum starvation group was induced a significantly higher than other groups. After synchronization of the donor cell cycle, either confluent or acteoside treated, cells were placed with enucleated in vivo-matured dog oocytes, fused by electric stimulation, activated, and transferred into naturally estrus-synchronized surrogates. Fusion and cleavage rate of acteoside treated group were 64.1 and 41.5%, which were higher than those of confluent group (53.9 and 20.6%, respectively). The reconstructed embryo development rates to 4-cell and 8-cell in acteoside treated group were 29.5 and 14.8%, respectively, while confluent group showed 11.1 and 3.2%, respectively. Total 54 SCNT embryos using acteoside-treated fibroblasts were transferred into oviducts of 2 recipient dogs and one recipient finally delivered one puppy, whereas din`t detected pregnancy on transfer of cloned embryos reconstructed with confluent cells in 6 surrogate dogs. In conclusion, the results of the current study demonstrated that canine fibroblasts could be successfully arrested at the G0/G1 stage with reduced the formation of ROS and apoptosis after acteoside treatment. This results may contribute to improve the effi-ciency of canine SCNT. * This research was supported by iPET (Grants 110056-3), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

Acteoside acts as an anti-oxidative activity and anti-apoptosis in the cells. But, it has been not studied on maturation and development of porcine oocytes. The aims of the present study were to examine the effects of acteoside on the morphological progress of meiosis, developmental competence, and ROS in porcine oocytes. Oocytes were matured in tissue culture medium-199, supplemented with acteoside at various concentrations: 0 (control), 10, 30 and 50 μM. The oocytes maturation rates of groups supplemented with acteoside were no significantly different (81.13, 85.96, 82.95 and 83.68%, respectively). Level of ROS was significantly decreased in acteoside treated group. Furthermore, the parthenogenetic blastocyst rate was significantly improved in 10 μM acteoside treated group compared with control group (44.83 vs. 27.75%). And we investigated effect of acteoside on the oocytes condition represented by cytoplasmic maturation by homogeneous distribution and formation of cytoplasmic organelles and regulation of apoptosis-related genes. In the results. during IVM, 10 μM acteoside treated oocytes showed that the mitochondria and lipid droplet were smaller and homogeneous distribution in cytoplasm compare with control oocytes. And reverse transcription polymerase chain reaction (RTPCR) of parthenogenetic blstocysts revealed that acteoside increased the anti-apoptotic genes (Mcl-1, Bcl-2 and Bcl-xL), whereas reduced the expression of pro-apoptotic genes (Bax and Bak). In conclusion, based on the results, the effect of acteoside on IVM was not attractive. However, in acteoside treated group, cytoplasmic maturation seemed to be improved with morphologically uniform distribution of cytoplasmic organelles. Furthermore, embryonic development in acteoside treated group was significantly highly increased than that of non-treated group. Our results represents that addition of acteoside to the IVM medium has a beneficial effect in physiology of porcine oocytes, providing a improved method for porcine oocytes in vitro. * This work was supported by a grant (Code# PJ008148) from BioGreen21 Program, Rural Development Administration, Republic of Korea.

The objective of the current study was to describe in vitro embryo production in Hanwoo, analyzing oocytes yield and embryo production. The effects of oocytes production and the number of OPU procedures per animal on embryo production were also evaluated. OPU was done every 3～4 days during experimental period and collected oocytes were fertilized in vitro in both OPU and needle puncture groups. First, we compared the recovery rate of oocytes based on OPU session (Experiment 1). The average of collected oocytes was calculated from every 10 session. The average number of total oocytes recovered per animalonsessionwas 5.16 (mean). Second, we compared the recovery rate base on collection period of OPU (Experiment 2). The following results show the difference of the number of recovered oocytes in every month during the procedure between the months of session. Every animal shows the constant number of recovered oocytes for the first 5 months. However, the recovery rate of oocytes was decreased from month 6 to 8. Third, we compared the developmental rate to blastocyst in two groups (Experiment 3). Oocytes by needle puncture were fertilized with frozen-thawing semen; the cleavage rate 24～48 h after in vitro fertilization (IVF) was 75.8% and blastocyst development rate was 18.8% in needle puncture group. Even though there is lower cleavage rate after IVF in OPU group (61.1%), blastocyst development rate was higher compared with needle puncture group (28.4%). In conclusion, Blastocyst developmental rate could be increased by OPU than classical method of needle puncture. Improvement of bio- technique in collecting oocytes could be applied to understand the reproductive physiology in cattle, expecially Hanwoo. Therefore, further investigation should be done to clarify the efficiency and advantage of OPU involved in reproduction in animals and human being.

The objective of this study was to evaluate in vitro production of bovine embryos in Hanwoo. Oocytes were collected by ovum pick up (OPU) from ovaries of genetically high-value Hanwoo or by needle puncture from ovaries of slaughtered cattle. OPU was done every 3 4 days duing experimental period and collected oocytes were fertilized in vitro in both OPU and needle puncture groups. First, We compared the in vitro maturation rate in two groups (Experiment 1). 545 oocytes were recoverd from 4 females by 32 trials of OPU and then 433 oocytes were shown MⅡ stage after in vitro maturation (79.4%). In case of needle puncture group, 1905 oocytes were collected and then 1420 oocytes were matured to MⅡ stage during in vitro culture(74.5%). Second, we compared the developmental rate to blastocyst in two groups (Experiment 2). 1420 oocyte by needle puncture were fertilized with frozen-thawing semen; the cleavage rate 24 48 h after in vitro fertilization (IVF) was 88.6% and blastocyst development rate was 20.5% in needle puncture group. Even though there is lower cleavage rate after IVF in OPU group (84.8%), blastocyst development rate was higher compared with needle puncture group (26.4%). In conclusion, Blastocyst developmental rate could be increased by OPU than classical method of needle puncture. Improvement of bio-technique in collecting oocytes could be applied to understand the reproductive physiology in cattle, expecially Hanwoo. Therefore, further investigation should be done to clarify the efficiency and advantage of OPU involved in reproduction in animals and human being. This research was suppoted by Imsil-gun agricultural technology service center.

A six-year-old male Pekinese dog was referred to the Veterinary Medical Center, Chungbuk National University because of intermittent vomiting, anorexia, alopecia, and pruritus. Generalized alopecia, pigmentation, and papular erythema in the skin were observed on physical examination. Hematological studies indicated a severe pancytopenia and hypoalbuminemia. A hormone analysis indicated a hyperestrogenemia. A circumscribed mass, measuring approximately 3-4 cm in diameter, was observed on abdominal radiographs. Grossly, the cryptorchid testis was enlarged by a firm and white spherical mass, measuring approximately 3 cm in diameter, which, on its cut surface, was creamy white with hemorrhage. The normal scrotal testis was markedly atrophic and soft. Histologically, the intra-abdominal cryptorchid testis contained abundant fibrous tissue stroma and the stromal tissues were arranged in a tubular pattern in which the neoplastic cells tended to palisade. The cells had poor and pale eosinophilic cytoplasm streaming into the center of the tubules. The nuclei were round to oval and tended to be in a basilar location. Regions of hyperplasia and marked squamous metaplasia were observed in many areas of the prostate. Based on the histological findings, we were able to identify these masses as Sertoli cell tumors, and made a final diagnosis as Sertoli cell tumors through immunohistochemistry methods using inhibin-α, vimentin, and neuron-specific enolase.

Temperature signaling can be initiated by members of transient receptor potential (thermo-TRP) channels. Hot and cold substances applied to teeth usually elicit pain sensation. Since odontoblasts constitute a well-defined layer between the pulp and the mineralized dentin, being first to encounter thermal stimulation from oral cavity, they may be involved in sensory transduction process, in addition to their primary function as formation of dentin. We investigated whether thermo-TRP channels are expressed in a odontoblast cell line, MDPC-23. The expressions of thermo-TRP channels were examined using reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry, fluorometric calcium imaging. Analysis of RT-PCR revealed mRNA expression of TRPV1, TRPV2, TRPV4 and TRPM8, but no TRPV3, TRPA1. Immunohistochemical approach failed to detect TRPV1 expression. Whereas the application of 4-phorbol-12,13-didecanoate(10 μM, a TRPV4 agonist), menthol(1 mM, a TRPM8 agonist) and icilin(10 μM, a TRPM8 agonist) produced the enhancement of intracellular calcium concentration, capsaicin(1 μM, a TRPV1 agonist) did not. Our results suggest that subfamily of thermo-TRP channels expressed in odontoblasts may serve as thermal or mechanical transducer in teeth.

The study aimed to evaluate the usability of sterile bag collection (SBC) urinalysis and urine culture for diagnosing urinary tract infections (UTI). Urine culture is key for diagnosing UTI, and transurethral catheterization (TUC) or suprapubic aspiration is recommended for non-toilet-trained children. Although urine testing using SBC is non-invasive and easy, UTI can be diagnosed only if other criteria including clinical symptoms and positive urinalysis results are met. This study included 228 infants who were hospitalized for unexplained fever from October 2015 to June 2016. TUC culture, SBC urinalysis, and urine culture were performed for all patients. UTI was diagnosed when the TUC culture results met the criterion of ≥104 colony-forming units (CFU)/mL. When UTI diagnosis was made based on SBC urine colony counts ≥105 CFU/mL, the false-positive and false-negative rates were 6.3% and 70.0%, respectively. When the criterion was set as ≥104 CFU/mL, they were 23.7% and 30.0%, respectively. When both the criteria of ≥105 CFU/mL and positive urinalysis results were met, the false-positive rate was 2.4%, and the false-negative rate was 80%. Our results suggest that diagnosing UTI using SBC urinalysis and urine culture is not useful in infants with unexplained fever.

Background : Recently, there is a urgent demand for development of new varieties with enhanced resistance to various biotic/abiotic stresses. In order to develop ginseng varieties with such traits, systematic breeding program and comprehensive field studies are prerequisite. Methods and Results : ‘Cheonmyeong' seeds were collected in 2008 from the farmer field of Buyeo. Physiological investigation and propagation were conducted from 2009 to 2011. It was given the name 'Eumseong No. 8 through the observed yield trial from 2012 to 2013 and local adaptability was carried out from 2014 to 2015. All phenotypes including agronomic characteristics, seed yield, and physiological response to biotic/abiotic stresses were investigated according to the ginseng GAP and UPOV guidelines. Yellow-red leaf and pink berry at maturing stage were observed. The time of emergence, flowering and berry maturity of the ‘Cheonmyeong’ were faster than those of ‘Chunpoong’. Stem length of ‘Cheonmyeong’ was shorter than that of ‘Chunpoong’, whereas stem diameter was thicker than that of ‘Chunpoong’. Main root length was shorter but main root diameter is thicker than that of ‘Chunpoong’. Number of seeds of ‘Cheonmyeong’ was fewer than that of ‘Chunpoong’ but 1,000-seeds weight and stratification rate were higher than those of ‘Chunpoong’. The yield performance of this variety was 661 kg/10 a in local adaptability test for two years, which is 22% higher than that of ‘Chunpoong’. ‘Cheonmyeong’ showed strong resistance to phytophthora blight, mulberry mealybug and nematode and moderate resistance to alternaria blight. ‘Cheonmyeong’ did almost not occur yellow spot of aerial part and rusty skin of root, show moderate resistance at high temperatures. Conclusion : Our study demonstrated that ‘Cheonmyeong’ is an ideal variety with heavier root weight and enhanced stress resistance and contribute will enhance biotic/abiotic stress resistance and increase the farmers' income.

Most vacuolar proteins are synthesized on the endoplasmic reticulum (ER) as a proprotein precursor and then transported to vacuoles. In vacuoles, they are converted into the respective mature form. TaVPE1 and TaVPE2 were isolated from the cDNA library that was prepared from the wheat kernels at 16 and 18 days after fertilization (DAF). Additionally, putative TaVPEs (TaVPE3, TaVPE4) were isolated by inverse PCR (IPCR) using GrainGenes database. Each open reading frame (ORF) encodes 495, 497, 495, 456 amino acids, respectively. TaVPEs were closely related in respect to peptide comparisons and their sequence homologies were ranged from 84% to 93%. The TaVPE genes showed various expression patterns in response to exogenous treatment of phytohormones. The transcript of TaVPE1 was detected slightly and steadily after exposure to all phytohormones and the accumulation of TaVPE2 transcript was increased from 24 hours in NaCl treatment. The transcript of TaVPE3 was increased from 48 hours in response to H2O2 and decreased after exogenous application of ABA and salicylic acid. In case of TaVPE4, the transcript of TaVPE4 were weakly detected all time points of each phytohormone treatment.