Jae-Gyu Yoo

Neurogenic and cardiomyogenic differentiation of mesenchymal stem cells isolated from minipig bone marrow.

The present study investigated the potential of minipig bone marrow-mesenchymal stem cells (BM-MSCs) to differentiate in vitro into neuron- and cardiomyocyte-like cells. Isolated BM-MSCs exhibited a fibroblast-like morphology, expressed CD29, CD44 and CD90, and differentiated into osteocytes, adipocytes and chondrocytes. Upon induction in two different neuronal specific media, most of BM-MSCs acquired the distinctive morphological features and positively stained for nestin, neurofilament-M (NF-M), neuronal nuclei (NeuN), β-tubulin, galactocerebroside (Gal-C) and glial fibrillary acidic protein (GFAP). Expression of nestin, GFAP and NF-M was further demonstrated by RT-PCR and RT-qPCR. Following cardiomyogenic induction, MSCs exhibited a stick-like morphology with extended cytoplasmic processes, and formed cluster-like structures. The expression of cardiac specific markers α-smooth muscle actin, cardiac troponin T, desmin and α-cardiac actin was positive for immunofluorescence staining, and further confirmed by RT-PCR and RT-qPCR. In conclusion, our results showed the in vitro differentiation ability of porcine BM-MSCs into neuron-like and cardiomyocyte-like cells.

Influence of epidermal growth factor supplementation during in vitro maturation on nuclear status and gene expression of canine oocytes.

This study evaluated the effect of epidermal growth factor (EGF) supplementation during in vitro maturation on the meiotic status and the expression of EGF receptor (EGFr), luteinizing hormone receptor (LHr) and gap junction protein α 5 (GJA5) in canine cumulus-oocyte-complexes (COCs). COCs of ≥110 μm diameter, exhibiting dark pigmentation and completely surrounded by three or more layers of cumulus cells collected from anestrus stage ovaries in natural cycle were matured in TCM-199 supplemented with 10% fetal bovine serum, 0.57 mM cysteine, 10 μg/ml LH and FSH, and different concentrations of EGF (0, 10 and 30 ng/ml). Oocytes cultured for 72 h were fixed to assess the nuclear maturation. Expression of EGFr, LHr and GAJ5 was assessed by immunocytochemistry and real-time PCR. Proportion of metaphase II status of oocytes cultured in in vitro maturation (IVM) medium supplemented with 10 ng/ml EGF for 72 h was significantly (P<0.05) higher than 0 and 30 ng/ml EGF supplemented IVM medium (9.8% vs. 6.5% and 5.2%). In both cumulus cells and oocytes, EGFr protein was undetectable, LHr protein level of expression was low and a strong expression of GJA5 protein was observed. The relative abundance (RA) of EGFr transcript revealed low levels and the LHr expression decreased steadily with addition of EGF. However it did not vary among different concentrations of EGF supplementation. The RA of GJA5 transcript exhibited lower level at 10 ng/ml EGF supplementation. In conclusion, the supplementation of 10 ng/ml EGF in IVM media exerted a positive influence on the progression of maturation to MII phase and the expression level of GJA5 at 72 h, but did not demonstrate any stimulatory role on the expression of EGFr and LHr during the maturation of the canine IVM oocytes.

Differential gene-expression profiles from canine cumulus cells of ovulated versus in vitro-matured oocytes

We compared the nuclear maturation status and gene-expression profiles of canine cumulus cells (CCs) derived from cumulus-oocyte complexes (COCs) that were spontaneously ovulated versus those that were matured in vitro. Cumulus-oocyte complexes were retrieved from uteri by surgical flushing (after spontaneous ovulation) or by ovariectomy follicle aspiration and in vitro maturation. The objective of Experiment 1 was to investigate the nuclear maturation status of in vivo- versus in vitro-matured oocytes. The objective of Experiment 2 was to compare gene-expression profiles of CCs derived from in vivo- versus in vitro-matured COCs. Genes analysed are related to cell maturation, development and apoptosis, including GDF9, MAPK1, PTX3, CX43, Bcl2 and BAX; mRNA expression for all of these genes, except for GDF9, differed (P<0.05) between in vivo- and in vitro-matured CCs. In conclusion, we found that gene-expression profiles are related to the quality of CCs and therefore posit that monitoring gene expression could be a useful strategy to guide attempts to improve in vitro culture systems.

Detection of Tick-Borne Pathogens in the Korean Water Deer (Hydropotes inermis argyropus) from Jeonbuk Province, Korea.

The objective of this study was to investigate the prevalence of tick-borne pathogens in the Korean water deer (Hydropotes inermis argyropus). Pathogens were identified using PCR which included Anaplasma, Ehrlichia, Rickettsia, and Theileria. Rickettsia was not detected, whereas Anaplasma, Ehrlichia, and Theileria infections were detected in 4, 2, and 8 animals, respectively. The most prevalent pathogen was Theileria. Of the 8 Theileria-positive animals, 2 were mixed-infected with 3 pathogens (Anaplasma, Ehrlichia, and Theileria) and another 2 animals showed mixed-infection with 2 pathogens (Anaplasma and Theileria). Sequencing analysis was used to verify the PCR results. The pathogens found in this study were identified as Anaplasma phagocytophilum, Ehrlichia canis, and Theileria sp. To the best of our knowledge, this is the first report identifying these 3 pathogens in the Korean water deer. Our results suggest that the Korean water deer may serve as a major reservoir for these tick-borne pathogens, leading to spread of tick-borne diseases to domestic animals, livestock, and humans. Further studies are needed to investigate their roles in this respect.

Detection of Anaplasma sp. in Korean Native Goats (Capra aegagrus hircus) on Jeju Island, Korea

Anaplasma species are obligate intracellular pathogens that can cause tick-borne diseases in mammalian hosts. To date, very few studies of their occurrence in Korean native goats (Capra aegagrus hircus) have been reported. In the present study, we investigated Anaplasma infection of Korean native goats on Jeju Island, Republic of Korea, and performed phylogenetic analysis based on the 16S rRNA gene sequences. Our results showed that Anaplasma infection was found mostly in adult female goats. The phylogenetic tree revealed that the 7 sequences identified in Korean native goats could belong to Anaplasma sp. and were distinct from A. marginale, A. centrale, and A. ovis. The results indicated that the sequences identified to belong to Anaplasma were closely related to sequences isolated from goats in China and were clustered within the same group. To our knowledge, this is the first study to detect Anaplasma sp. infection in Korean native goats.

The effects of artificial activation timing on the development of SCNT-derived embryos and newborn piglets

This study investigated the effects of two different activation regimens on the developmental potential of somatic cell nuclear transfer (SCNT) embryos and postnatal survivability of the cloned piglets. In vitro matured oocytes were enucleated and reconstructed with porcine fetal fibroblasts. On the basis of the activation regimen used, the reconstructed porcine embryos were allocated into two groups: Group 1-simultaneous electrical pulses and activation group (SFA group); and Group 2-electrical fusion without calcium followed by electrical pulses with calcium after colcemid and cytochalasin B treatment for 5h (DA group). Embryonic development in both SFA and DA groups was determined at day 6 of culture in NSCU-23 medium. To investigate the post-implantation development after the two activation methods, embryos were cultured for 1 day and then transferred into the oviducts of estrus-synchronized recipients. DA group had significantly (p<0.05) higher cleavage rates than SFA group. However, the developmental rate to the blastocyst stage and the mean cell number of blastocysts did not differ (p>0.05) between SFA and DA groups. Moreover, the pregnancy rate of SFA group was not significantly different compared to DA group. A total of 20 cloned piglets (SFA group-8 live piglets, DA group-11 live piglets and one stillborn) were obtained in the present study. The birth weight of the cloned piglets (live births) did not differ (p>0.05) between the two groups. Furthermore, no difference was observed in the postnatal survival rates of the cloned piglets obtained using two different activation regimens. These results suggest that the timing of artificial activation and additional chemical treatments do not affect the developmental rate of porcine SCNT embryos. Remarkably, the pregnancy rate and postnatal survivability of the cloned piglets did not vary between SFA and DA groups.

Identification of bovine viral diarrhea virus infection in Saanen goats in the Republic of Korea

Bovine viral diarrhea virus (BVDV) is one of the most important viral pathogens of livestock and causes substantial economic losses to the livestock industry worldwide. BVDV is not necessarily species specific and is known to infect domesticated and wild ruminants. In the present study, BVDV infection was identified in two Saanen goats from one farm, and two different viral subtypes were found, BVDV-1a and BVDV-2a. Each isolate was closely related to cattle isolates identified in the Republic of Korea. The two sequences obtained in this study were not consistent with border disease virus (BDV). The incidence of BVDV in this farm apparently occurred in the absence of contact with cattle and may be associated with grazing. This study demonstrates that BVDV infection may be possible to transmit among goats without exposure to cattle. Therefore, this result indicates that Saanen goats may act as natural reservoirs for BVDV. This is the first report of BVDV-1a infection in a Saanen goat.

Seasonal changes in hemograms and Theileria orientalis infection rates among Holstein cattle pastured in the mountains in the Republic of Korea.

In the current study, we compared seasonal changes in complete blood counts (CBCs) and rates of infection with a tick-borne pathogen between Holstein cattle housed indoors and those maintained outside on pasture. There were differences in white blood cell (WBC) parameters, but the changes were not associated with seasons or the housing type. Analysis of red blood cell (RBC) parameters showed lower values in August and November versus March, and in the cattle maintained on pasture versus the housed cattle. In comparison with the RBC count of the housed cattle in March (10.1M/μL), the RBC counts of the pastured cattle were significantly lower in August (7.8M/μL; p<0.01) and November (7.5M/μL; p<0.01). The hematocrit (HCT) also showed a decrease in March (33.5%), August (30.0%, p<0.01) and November (28.5%, p<0.01). According to PCR analysis, the Theileria infection rate among the pastured cattle in March was only 11%, but this rate increased to 22% and 60% in August and November, respectively. The RBC count (7.4M/μL) and HCT (27.7%) values in Theileria-positive pastured cattle in November showed a dramatic decrease compared to those of cattle examined in March. Phylogenetic analysis revealed that these Theileria isolates correspond to T. orientalis. These results suggest that a remarkable increase in tick infestation in mountainous areas in the summer may cause increased rates of infection with T. orientalis, leading to significant changes in the RBC profile after grazing. Therefore, these hematological changes may be associated with T. orientalis infection caused by tick-biting; thus, additional studies on the pathogenicity of T. orientalis are needed.

Influences of somatic donor cell sex on in vitro and in vivo embryo development following somatic cell nuclear transfer in pigs.

Objective The present study investigates pre- and post-implantation developmental competence of nuclear-transferred porcine embryos derived from male and female fetal fibroblasts. Methods Male and female fetal fibroblasts were transferred to in vitro-matured enucleated oocytes and in vitro and in vivo developmental competence of reconstructed embryos was investigated. And, a total of 6,789 female fibroblast nuclear-transferred embryos were surgically transferred into 41 surrogate gilts and 4,746 male fibroblast nuclear-transferred embryos were surgically transferred into 25 surrogate gilts. Results The competence to develop into blastocysts was not significantly different between the sexes. The mean cell number of female and male cloned blastocysts obtained by in vivo culture (143.8±10.5 to 159.2±14.8) was higher than that of in vitro culture of somatic cell nuclear transfer (SCNT) groups (31.4±8.3 to 33.4±11.1). After embryo transfer, 5 pregnant gilts from each treatment delivered 15 female and 22 male piglets. The average birth weight of the cloned piglets, gestation length, and the postnatal survival rates were not significantly different (p<0.05) between sexes. Conclusion The present study found that the sex difference of the nuclear donor does not affect the developmental rate of porcine SCNT embryos. Furthermore, postnatal survivability of the cloned piglets was not affected by the sex of the donor cell.

Detection and Identification of Sarcocystis cruzi (Protozoa: Apicomplexa) by Molecular and Ultrastructural Studies in Naturally Infected Korean Cattle ( Bos taurus coreanae ) from Daejeon, Korea

To survey the prevalence of Sarcocystis infections, 210 heart samples were collected from Korean native cattle (Bos taurus coreanae) at an abattoir in Daejeon Metropolitan City, Republic of Korea. Sarcocysts were detected form 31 specimens (14.8%) and identified as Sarcocystis cruzi via transmission electron microscopy. The wall of S. cruzi has flattened protrusions that did not contain fibrils or microfilaments. The protrusions arose irregularly from the base, contained a fine granular substance, lacked internal microfilaments, and measured approximately 0.21–1.25 μm in length and 0.05–0.07 μm in width. Sequence analysis revealed 99.5% homology to S. cruzi. This is the first report on the prevalence of S. cruzi in native cattle from the Republic of Korea.