• Korean Journal of Agricultural Science
• /
• v.47 no.3
• /
• pp.657-665
• /
• 2020

The objective of this study was to investigate whether supplementation of pentoxifylline (PTX; phosphodiesterase inhibitor) to thawed boar semen improves the post-thaw motility of sperm and affects the efficiency of artificial insemination (AI) and further development. To determine the concentration of PTX for AI, frozen-thawed semen was incubated with 0, 5, 10, and 20 mM PTX in an extender freezing medium, respectively, after thawing. Kinematic properties of sperm were examined with a computer-assisted semen analysis (CASA) system. In addition, viability and mitochondrial activity were also tested by LIVE/DEAD and a MitoTracker kit. There were no significant differences in the kinetic parameters of thawed sperm between control and treatment groups, but overall assessment parameters such as motility and rapid progressive were higher in the 10 mM PTX group. In the viability and mitochondrial assay, there were no significant differences observed in the PTX treatment, compared to the control. For further analysis, artificial inseminations were performed using frozen semen and 10 mM PTX treated cryopreserved semen, respectively. There were no differences in pregnancy rates and fetus weights among the groups until 30 and 40 days, but litter size was reduced and relatively low-birth weight was observed in the PTX group. In summary, our findings suggest that enhancement of in vitro sperm quality or non-toxicity supplemented by PTX may have detrimental effects on fetus development.

• Proceedings of the KSAR Conference
• /
• 2003.06a
• /
• pp.33-33
• /
• 2003

This study was conduct to compare the efficacy to produce male and female somatic cloned piglets. Maturation of porcine COCs was accomplished by incubation in NCSU-23 medium supplemented with 0.6 mM cysteine, 10% porcine follicular fluid, 1mM dibutyryl cyclic adenosine monophosphate (dbc-AMP, Sigma, USA), and 0.1 IU/ml human menopausal gonadotrophin (hMG, Teikokuzoki, Japan) for 20h and then cultured without dbcAMP and hMG for another 18 to 24 h. Female and male fetal cells were isolated from each fetus, cultured in ES-DMEM medium containing 10% FCS. Enucleated oocytes were fused with fetal fibroblasts (passage 4 to 15). Reconstructed embryos were cultured in NCSU-23 with 4 mg/ml BSA under mineral oil at 39$^{\circ}C$ in 5% $CO_2$ in air. A total of 12,328 nuclear-transferred embryos (1- to 4-cell stage) were surgically transferred into 69 surrogate gilts. Three recipients aborted during the period of conception. Three gilts delivered eleven female piglets, and five recipients gave rise to birth 22 male piglets. The average birth weigh of the cloned piglets was 1.52 kg (1.38~1.83 kg) in female piglets and 0.84 kg (0.45~1.25 kg) in male piglets. Alive cloned pigs was seven in female piglets (63.6%) and four in male piglets (18.2%). The other two recipients is ongoing. This study suggests that female-derived fetal cell as a nuclear donor has more capability on production of cloned piglets than male.

• Proceedings of the KSAR Conference
• /
• 2001.03a
• /
• pp.66-66
• /
• 2001

Since the first birth of pig derived from embryonic cells by nuclear transfer, many researches to produce cloned pig have been carried out. Recently, two reports about the birth of somatic cell cloned pigs using in vivo oocytes and also Betthauser et al. (2000) reported the birth of somatic cell cloned pigs using in vitro oocytes. So here we investigated the effect of activation method and culture medium on in vitro development of porcine nuclear transfer embryo using fetal fibroblast. Oocytes derived from slaughter house obtained ovaries were matured for 42 to 44 h in TCM 199. Matured oocytes were denuded using 0.1% hyaluronidase and then Oocytes with the first polar body were used for enucleation by aspirating the first polar body and adjacent cytoplasm in TCM 199 supplemented with 7.5 $\mu\textrm{g}$ cytochalasin B. Petal fibroblast cells were prepared from 35 days old fetus. To be used as donor cells, fetal fibroblast cells were serum starved for 3 to 5 days and then isolated into single co:1 by trypsinization. Nuclear transfer embryos were fused using 2 times 1.25㎸ for 30$mutextrm{s}$. Fused NT embryos were activated with calcium ionophore (CI) and 6-dimethyl-aminopurine (6-DMAP). Activated oocytes were cultured in NCSU 23 or BECM 3 for 6 days. There was no significant difference between chemical activation and no chemical activation for blastocyst development rate(11.6 vs. 14.8%). However, cell number was significantly higher when NT embryos were activated with CI and 6-DMAP (31.2 vs. 22.6). When NT embryos were cultured in NCSU 23 or BECM 3, blastocyst development rate was 16.4 and 13.2%, respectively, and cell number was 31.5 and 24.1, respectively. These results suggest that chemical activation after fusion and culture in NCSU 23 could increase cell number of porcine NT embryos.

• Reproductive and Developmental Biology
• /
• v.33 no.2
• /
• pp.113-117
• /
• 2009

Animals produced by somatic cell nuclear transfer (SCNT) using genetically modified cells are almost always transgenic, implying that this method is more efficient than the traditional pronuclear microinjection method. Most somatic cells for SCNT in animals are fetus-derived primary cells and successful gene integration in somatic cells will depend on transfection condition. The objective of this study is to evaluate the efficiency of electroporation (Microporator) and liposome reagents (F-6, F-HD, W-EX, W-Q, W-M) for tissue-type plasminogen activator (tPA) gene transfection and to estimate the overall efficiency of transfection of Korean native pig fetal fibroblast cells (KNPFF). Electroporation showed significantly higher transfection efficiency than liposome reagents with regard to the transfection of in vitro cultures in the early stages of development (41.7% with Microporator vs. 18.3% with F-6, 20.0% with F-HD 18.5% with W-EX, 5.0% with W-M and 6.3% W-Q,). Colonies identified as tPA-positives were treated once more with G418 for 10 to 14 days and growing colonies were selected again. When the cells of newly selected colonies were subjected to single-cell PCR, reselection of colonies following second round of G418 selection increased the rate of transgene integration per each colony. These results suggest that transfection with electroporation is the most efficient and the second rounds of G418 selection may be an effective method for transfection of porcine fetal fibroblast cells.

• Korean Journal of Veterinary Research
• /
• v.34 no.1
• /
• pp.77-83
• /
• 1994

Three viral strains, causing CPE in porcine alveolar macrophage cell, were isolated from aborted fetus, serum from young pig showing blue-ear sign and lung of suspected pig, respectively. The differential diagnostic results showed no characteristics of Aujeszky's disease virus(ADV), hog cholera virus (HCV), Japanese encephalitis virus(JEV), porcine parvovirus(PPV) and encephalomyocarditis virus (EMCV). However, positive reactions were demonstrated by IFA using monospecific porcine antibodies against Lelystad virus. When the paired sera of experimentally inoculated swine with one of isolate, KPRRSV-l were tested by IPMA, the result indicated that the isolate was related to United States isolate than European LV.

• Korean Journal of Veterinary Research
• /
• v.30 no.4
• /
• pp.441-446
• /
• 1990

Encephalomyocarditis virus infection in pigs, characterized by severe reproductive failure in sows and sudden death in less than 7 day-old-piglets, a period of several months from October to December of 1989, in Korea was occurred. The most outstanding gross lesions at necropsy were found in the heart which were consisted of pale, yellow brown necrotic foci of varying size in myocardium. In some cases, pulmonary edema and liver congestion were observed. Histopathology of the heart revealed the interstitial myocarditis, endocarditis and epicarditis with lymphocytic, plasmacytic and macrophage cell infiltration and, in some cases, calcification of the necrotic myocardial muscle fibers. In some cases of brain, the perivascular cuffing and glial nodules were observed. In the liver and the lacrimal gland varying degrees of multifocal necrosis were seen. The virus was isolated from the heart and the brain in a stillborn piglet and a mummified fetus. This outbreak represents the first case of encephalomyocarditis virus infection of pigs in Korea.

• Reproductive and Developmental Biology
• /
• v.28 no.1
• /
• pp.7-12
• /
• 2004

This study was performed to establish the effective culture condition for the establishment of clonal lines from porcine fetal fibroblast cells. Fibroblasts derived from a pig fetus (Day 50) were cultured and passaged two times before use. A single cell was seeded in 96-well plates, cultured in medium supplemented with different concentrations of FBS, catalase or $\beta$-mercaptoethanol ($\beta$ME), and classified by cell size and morphology. Cells were passaged two times into 4-well dish before freezing. The establishment efficiencies were not different among different concentrations of FBS (0.3 to 5.1%). However, population doubling time (PDT) was significantly decreased by increasing the FBS concentration (P＜0.05). The establishment efficiency of $\beta$ME-added group (10.4%) was significantly higher than those of catalase-added and control groups (3.5%, and 3.5%, respectively, p＜0.05), and PDT was significantly decreased (23.6 vs 28.1, and 25.5 h, respectively, p＜0.05). However, catalase did not show a positive effect on the establishment efficiency. Cell size and morphology did not affect the establishment efficiency and PDT of clonal lines. The result of present study shows that the establishment efficiency of clonal cell lines can be enhanced by the culture in media supplemented with 30% FBS and $\beta$ME.

• Journal of the Korean Society of Radiology
• /
• v.13 no.6
• /
• pp.895-900
• /
• 2019

Ultrasound is known to be harmless to the human body and is widely used in obstetrics and gynecology to confirm the diagnosis and development status of fetus. Diagnosis Although long - term use of ultrasound may cause changes in body temperature, studies on the uterine temperature changes due to ultrasound have been lacking. The purpose of this study was to investigate the change of temperature according to ultrasonic scanning time using a self - produced uterine model phantom. Ultrasound equipment and a 4MHz convex probe were used to construct the uterine model phantom similar to the human uterus using acrylic and pig uterus, which are tissue equivalents. Three probe type thermometers were installed to measure the inside of the acrylic water tank, the uterus, and the atmospheric temperature. The temperature of the uterine phantom was ascertained by measuring the temperature of the subject for 6 hours, 361 times. In this study, the possibility of human body temperature elevation due to ultrasound could be confirmed and this study will be used as the basic data of ultrasonic heat absorption study.

• Reproductive and Developmental Biology
• /
• v.30 no.2
• /
• pp.119-124
• /
• 2006

The present study compared the developmental potential of cloned porcine embryos with mesenchymal stem cells (MSCs), fetal fibroblasts (FFs) and cumulus cells (CCs) by assessing the cleavage and blastocyst rate, total cell number, inner cell mass (ICM) ratio and apoptosis. MSCs were isolated by ficoll gradients from femur of -6 month old female pig, and maintained for primary cultures. FFs from a female fetus at ${\sim}30$ day of gestation were established, and CCs were obtained from cumulus oocyte complexes (COCs) aspirated from $3{\sim}6$ mm follicles in diameter. Donor cells at $3{\sim}4$ passage were employed for nuclear transfer (NT). COCs were matured and fertilized in vitro(IVF) as control. Cleavage rate was significantly (P<0.05) higher in IVF than in NT embryos with MSCs, FFs and CCs ($82.7{\pm}8.9%\;vs\;70.6{\pm}5.4,\;68.7{\pm}5.1\;and\;63.4{\pm}5.6%$, respectively). However, blastocyst rates in IVF and NT embryos derived from MSCs ($24.5{\pm}2.8\;and\;20.4{\pm}8.3%$) did not differ, but were significantly (P<0.05) higher than NT derived from FFs and CCs ($10.6{\pm}2.7\;and\;9.8{\pm}2.1%$). Total cell number and the ratio of ICM to total cells among blastocysts cloned from MSCs ($35.4{\pm}5.2\;and\;0.40{\pm}0.09%$, respectively) were significantly (P<0.05) higher than those from FFs and CCs ($24.9{\pm}6.2%\;vs\;0.19{\pm}0.16,\;23.6{\pm}5.5\;and\;0.17{\pm}0.16%$, respectively). Proportions of TUNEL positive cells in NT embryos from FFs and CCs ($6.9{\pm}1.5\;and\;7.4{\pm}1.7%$, respectively) were significantly (P<0.05) higher than in MSCs ($4.8{\pm}1.4%$) and IVF ($2.3{\pm}0.9%$). The results demonstrate that MSCs have a greater potential as donor cells than FFs and CCs in achieving enhanced production of cloned porcine embryos.

• Asian-Australasian Journal of Animal Sciences
• /
• v.25 no.8
• /
• pp.1102-1116
• /
• 2012

During embryo implantation in pigs, the uterine endometrium undergoes dramatic morphological and functional changes accompanied with dynamic gene expression. Since the greatest amount of embryonic losses occur during this period, it is essential to understand the expression and function of genes in the uterine endometrium. Although many reports have studied gene expression in the uterine endometrium during the estrous cycle and pregnancy, the pattern of global gene expression in the uterine endometrium in response to the presence of a conceptus (embryo/fetus and associated extraembryonic membranes) has not been completely determined. To better understand the expression of pregnancy-specific genes in the endometrium during the implantation period, we analyzed global gene expression in the endometrium on day (D) 12 and D15 of pregnancy and the estrous cycle using a microarray technique in order to identify differentially expressed endometrial genes between D12 of pregnancy and D12 of the estrous cycle and between D15 of pregnancy and D15 of the estrous cycle. Results showed that the global pattern of gene expression varied with pregnancy status. Among 23,937 genes analyzed, 99 and 213 up-regulated genes and 92 and 231 down-regulated genes were identified as differentially expressed genes (DEGs) in the uterine endometrium on D12 and D15 of pregnancy compared to D12 and D15 of the estrous cycle, respectively. Functional annotation clustering analysis showed that those DEGs included genes involved in immunity, steroidogenesis, cell-to-cell interaction, and tissue remodeling. These findings suggest that the implantation process regulates differential endometrial gene expression to support the establishment of pregnancy in pigs. Further analysis of the genes identified in this study will provide insight into the cellular and molecular bases of the implantation process in pigs.