Earticle

The demand of pharmaceutical proteins and continuously increasing worldwide, hence cost of production of these proteins is a key factor in the competitive market. Recombinant proteins have several benefits over proteins which are isolated from non-recombinant sources. There are several methods developed for expression of recombinant proteins in large scale. Among these mammalian cell culture based expression system is most used world wide followed by expression of recombinant proteins in live animal bioreactor. Although concept of generating therapeutic proteins in the mammalian cell cultures based system existed for more than two decades, it is still less efficient for producing cost effective therapeutics. One of the issues for lack of advancement in this field is exorbitant cost involved in downstream processing of the expressed proteins. This is largely due to lack of a cell culture system which may aid in high level expression of therapeutic protein followed by efficiently processing and secreation of the expressed protein in the culture. To achieve this, development of cheaper and less cumbersome procedures has become necessary. Cultivated mammalian cells have become the dominant system for the production of recombinant proteins for clinical applications because of their capacity for proper protein folding, assembly and post-translational modifications. Thus, the quality and efficacy of a protein can be superior when expressed in mammalian cells versus other hosts such as bacteria, plants and yeast. The mammary gland has generally been considered the organ of choice to express valuable recombinant proteins because milk is easily collected in large volumes and is the best available bioreactor. However, large scale production of recombinant proteins is still an art in spite of increased qualitative and quantitative demand for these proteins. Foreign proteins are commonly reported to be produced in transgenic milk at rates of several grams per liter. Researchers are constantly challenged to improve and optimize the existing expression systems, and also to develop novel approaches to face the demands of producing the complex proteins. Although concept of generating therapeutic proteins in the mammalian cell culture based system existed for more than two decades, it is still less efficient for producing cost effective therapeutics. We have established bovine mammary epithelial stem cells (bMESCs) using various markers such as Nanog, Oct4, Sox9, CK14, CK18, CSN2, CSN3 etc. Antibacterial pharmaceutical proteins were screened using various bio-informatic tools and then selected proteins such human lipocalin-2 (LCN2) and bovine lactoferricin for the transfection in cow bMESCs. We used different mammary specific vector systems as career for the transfer of targeted genes in bMECs. However, transfection in mammary epithelial/ stem cells is little difficult as compare to other cells as these cells have secretory properties. We successfully expressed both pharmaceutical proteins in the cow mammary epithelial stem cells and established the cell lines. Therefore, development of protocols and establishment of cow mammary epithelial/stem cell lines can be used for various studies and as non-GMO bioreactor for the production of pharmaceutical value proteins.

This study firstly investigated whether plasma treatment of fertilized eggs before hatching could affect the growth and reproduction of chickens. Four-day-incubated fertilized eggs exposed to non-thermal dielectric barrier discharge plasma at 11.7 kV for 2 min resulted in the highest growth in chickens. Plasma growth-promoting effect in chickens was regulated by the reactive oxygen species homeostasis and the improvement of energy metabolism via increasing serum hormones and adenosine triphosphate levels which were resulted from the regulation of the demethylation levels of growth and hormone biosynthesis-related genes. Interestingly, plasma-treated male chickens showed more conspicuous growth characteristics than females. Further, aspects of the male reproductive system were improved by the plasma treatment: testosterone levels, sperm quality and fertility increased via the double regulation of DNA methylation and miRNA in the testis. In summary, optimized plasma treatment of fertilized eggs can improve chicken growth and male reproductive capacity; this finding can improve profitability and next-generation potential production in poultry breeding.

Autophagy is an essential cellular mechanism that degrades cytoplasmic proteins and organelles to recycle their components. Moreover, autophagy is essential for preimplantation development in mammals. Here we show that autophagy is also important for reprogramming in somatic cell nuclear transfer (SCNT). Our data indicate that unlike fertilized oocytes, autophagy is not triggered in SCNT embryos during 6 hours of activation. Mechanistically, the inhibited autophagic induction during SCNT activation is due to the cytochalasin B (CB) caused depolymerization of actin filaments. In this study, we induced autophagy during SCNT activation by rapamycin and pp242, which could restore the expected level of autophagy and significantly enhance the development of SCNT embryos to the blastocyst stage when compared with the control (68.5% and 68.7% vs. 41.5%, p<0.05). Furthermore, the treatment of rapamycin and pp242 accelerates active DNA demethylation indicated by the conversion of 5 mC to 5 hmC, and treatment of rapamycin improves degradation of maternal mRNA as well. Thus, our findings reveal that autophagy is important for development of SCNT embryos and inhibited autophagic induction during SCNT activation might be one of the serious causes of low efficiency of SCNT.

All of ART treatments in Japan are under control of Japan Society of Obstetrics and Gynecology (JSOG). JSOG covers all fields of obstetrics and gynecology in Japan. ART can be performed only in JSOG-certified centers to which at least one ART specialist (M.D.) must belong. ART specialist is certified by Japan Society of Reproductive Medicine (JSRM) which cover the field of reproductive medicine together with JSOG. Each ART center must submit the reports of all of ART treatment cycles every year and the accumulated data is open to public. From the latest data, number of ART treatment cycles in Japan is the largest in the countries registration system is functioned. (Actually, the largest and the second largest countries are China and India, respectively.) However, delivery rate per treatment cycles in Japan is the worst because percentage of cycles with aged women over 40 years old is increasing in recent years. In addition, neither egg donation nor preimplantation genetic screening (PGS) of embryos is permitted from ethical point of view. All of clinical research must be registered to JSOG and get permission in advance. At the end of the research, the report must be submitted. The major characteristics of Japan ART are minimum stimulation and “all freeze”, though these are not working range of embryologist. Needless to describe, minimum stimulation is performed in order to avoid ovarian hyper stimulation syndrome (OHSS). “All freeze” is the strategy to get higher implantation rate by complete synchronization of endometrium cycle and embryo development. In relation to ART laboratory works, very expensive machines (Time-lapse cinematography- equipped dry incubators, automatic vitrification machine, etc.) are developed in recent years. TLC-equipped dry incubator is gradually introduced in many ART centers. Accordingly, single step blastocyst culture medium is also going to be popular in order to reduce laboratory work. As the result of author’s investigation, osmolality of blastocyst medium is significantly increased during 5 days culture in dry incubator when compared with humidified condition (p<0.05) in spite that culture medium is covered by mineral oil. No significant difference in blastulation rate though slightly decreased in dry condition. Rescue ICSI (rICSI), double insemination (rescue conventional insemination: rcIVF), rescue artificial oocyte activation (rAOA) are widely performed in many centers in order to improve fertilization rate. The major unsolved problems in the field of ART are thin endometrial layer cases, aged women and azoospermia. In vitro proliferation of endometrial-derived stem cells, being performed by Prof. S. Kato’s group in Kyushu University, and in vitro germ cells production from iPS cells by two groups, M. Saito in Kyoto University and K. Hayashi in Kyushu University, may be able to solve these problems in the future.

Acute kidney injury (AKI) is one of the most devastating systemic condition that is interrelated with high mortility and morbidity rates. The majority of AKI in clinical practice is caused by ischemia reperfusion or neprotoxicity. The etiology and progression, as well as the mechanism of its resolution and recovery have been continuously studies using rodent models. However, it is of critical importance to evaluate this model in non-murine species to better translate the results from animal studies to clinical studies. Not only for sole AKI, renal Ischemia reperfusion warm injury occurs during donor kidney acquisition in transplantation. Therefore, AKI modeling in large animals, e.g., nonhuman primates, would benefit the understanding its pathophysiology and also maintain the graft function post-transplantation. We established a renal warm ischemia reperfusion model in cynomolgus monkeys, and determined several key evaluation parameters including serum biochemistry and histology works. This presentation will cover the brief story of both how our nonhuman primate research facility was setup, and also several key findings from our monkey experiments.

Mammalian DNA replication occurs only at the fixed place, termed the origin of replication, origin of replication is not recognized by the DNA sequence. It is recognized by the DNA licensing, which indicate that DNA replication is prepared. Furthermore, in the case of mammalian one-cell embryo is separated into paternal and maternal pronuclei, and DNA licensing and DNA replication takes place each in a separate pronuclei. Based on the characteristics of these mammalian one-cell embryo, we tested whether paternal DNA that was destined for degradation was properly licensed, by testing for the presence of MCM7 and ORC2, two licensing proteins, in the paternal pronuclei. We also tested whether the zygote recognized the TOP2B mediated DNA fragmentation in epididymal spermatozoa or the nuclease degradation in vas deferens spermatozoa, by testing for the presence of gamma-H2AX. Sperm DNA fragmentation does not prevent licensing of DNA replication origins. The embryo recognizes DNA that is damaged by nucleases, but not by TOP2B because H2AX is phosphorylated in paternal pronuclei resulting from spermatozoa with fragmented DNA from vas deferens spermatozoa treated with divalent cations, but not from epididymal spermatozoa treated the same way. Our data indicate that the one-cell embryo does harbor a mechanism to prevent the replication of severely damaged DNA from spermatozoa, even though the embryos do not undergo classical apoptosis.

Rheumatoid arthritis (RA) is a common autoimmune disease in the world, and is characterized by synovial hyperplasia, inflammatory cell infiltration, cartilage degradation, bone erosion, joint destruction and an increase in the levels of pro-inflammatory cytokines. Current therapies for RA focus on the inhibition of inflammatory cascade using anti-inflammatory substances to prevent or delay the progression of symptoms, however, the efficacy is varied and limited. Therefore, MSCs are considered as a potential candidate for RA treatment due to their immunomodulation properties in inflammation milieu. Meanwhile, synovial fluid (SF) is a significant source of MSCs in RA therapy; the SF is an easily accessible source of MSCs, which can be obtained during diagnosis or treatment of RA patients without additional harming, in addition, it is well addressed that the population of MSCs derived from SF (SF-MSCs) in arthritis patients is extensively increased in comparison to that in healthy patients. Hence, we have investigated whether the immunomodulation properties of SF-MSCs are capable of improving the symptoms of RA as the new therapeutic approach. For the purpose of the aforementioned objective, we have conducted: establishment of the method for isolation of SF and bone marrow (BM) specimens from minipigs, exploration of the most stable reference genes in porcine MSCs for the further analysis, comparative evaluations between porcine SF- and BM-MSCs with respect to the pluripotent capacities and the immunomodulation properties in RA animal model, comparative characterizations of human SF-MSCs obtained from different disease-graded RA patients, identification for alterations of immunomodulation properties depending on the degree of inflammation milieu in SF-MSCs from human RA patients, and establishment of new large animal RA model in minipig. We hope that these results can contribute to better understand the pathogenesis and etiology of RA as well as develop new therapeutic strategy.

Porcine spermatogonial stem cells (SSCs) are an useful model for a successful gene modification of sperm compatible, and various gene delivery systems to transport specific genes into the cytoplasm of porcine SSCs have been introduced for successfully producing transgenic sperms. However, each gene delivery system still suffers from low transfection efficiency and transfected gene expression in porcine SSCs, resulting in making it difficult to produce transgenic offspring. Accordingly, in order to determine type of promoters optimized to porince SSCs, impact of each Cytomegalovirus (CMV), Simian virus 40 (SV40), and chicken b-actin promoter on transfection efficiency of porcine SSCs was investigated using electroporation. For these, the highest transfection efficiency and cell viability were observed in porcine SSCs transfected with 1 μg of the each transgenic vector with a single electric pulse from an electroporator at a voltage of 200 V and a capacitor setting of 500 μF in a 0.4 cm cuvette containing 200μL of standized SSCs medium. Cell viability was measured by trypan blue exclusion assay, and analysis of transfection efficiency and intensity were conducted as counting SSCs expressing enhanced green fluorescent protein (EGFP) and measuring EGFP fluorescent intensity under flow cytometer system. As the results, the highest EGFP expression efficiency and intensity were observed in porcine SSCs introduced with CMV promoter-containing vectors, compared to those with SV40 and chicken b-actin protmoter- containing vectors. However, cell viability showed no significant difference among porcine SSCs experiencing transfection of SV40, CMV and chicken b-actin promotercontaining vector. In conclusion, we found that CMV promoter could effectively stimulate expression of genes transfected to porcine SSCs. This will contribute advances in further research related in SSCs transplantation and production of transgenic animals.

Spermatogonial stem cells (SSC) are promising resources for genetic preservation and restoration of male germ cells in human and animals. However, in our preliminary experiment, it was observed that SSC was fragile when used as nuclei donor for SCNT. This study investigated the potential of porcine SSC as donor nuclei for somatic cell nuclear transfer (SCNT) and developmental competence of SSC-derived cloned embryos. In addition, it was examined whether demecolcine could prevent rupture of SSC during SCNT. SCNT embryos were produced using a standard protocol of our laboratory. After electric activation, SCNT embryos were treated with demecolcine combined with 6-DMAP for 4 h, washed, and then cultured in a porcine zygote medium-3 at 39℃ in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 for 7 days. When SSC was compared with porcine fetal fibroblast (PFF) in the potential to support embryonic development after SCNT, SSC-derived SCNT embryos showed higher (p< 0.05) developmental competence to the blastocyst stage (53.7%) than PFF-derived embryos (28.6%). Treatment of SSC with demecolcine significantly (p<0.01) inhibited the rupture of SSC during SCNT (7.8% vs. 16.4%) and increased fusion of cell-ooplasm couplets compared to no treatment (71.9% vs. 62.8%). In addition, even after demecolcine treatment, SSC-derived SCNT embryos showed a higher blastocyst formation (46.4%) than PFF-derived embryos (27.8%). Our results demonstrate that porcine SSC are a desirable donor cell type for production of SCNT pig embryos and demecolcine increases production efficiency of cloned embryos by inhibiting rupture of nuclei donor SSC during SCNT.

This study was conducted to investigate the endoplasmic reticulum (ER) stress and subsequent apoptosis of porcine somatic cell nuclear transfer (SCNT) embryos induced during SCNT procedures, which compared with IVF embryos. Porcine SCNT embryos were made by micromanipulation and electrofusion/activation. Control porcine embryos were prepared by in vitro fertilization (IVF). Porcine SCNT and IVF embryos were sampled at 3 h-, and 20 h-post SCNT or IVF, and at the blastocyst stage for mRNA extraction. The x-box binding protein 1 (Xbp1) mRNA, the key transcription factor in the ER stress condition was confirmed by RT-PCR analysis. The expressions of ER stress-associated genes, the C/EBP homologous protein (CHOP), binding protein (BiP), activating transcription factor 4 (ATF4), and glucose-regulated protein 94 (GRP94) were confirmed by RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. Prior to SCNT, the splicing of Xbp1 mRNA and increased expressions of ER stress-associated genes were confirmed in the somatic cells treated with tunicamycin (TM), an ER stress inducer. Expression of spliced Xbp1 (Xbp1s) mRNA in the SCNT embryos did not differ with that of IVF embryos, regardless of the embryonic stage. Expression of ER stress-associated genes in SCNT embryos was, however, significantly increased in the all embryonic stages compared to IVF embryos (p<0.05). Apoptotic gene expression was slightly high in the SCNT embryos, but not different from that of IVF embryos in each group. The result of this study indicates that excessive ER stress can be induced by SCNT procedures. Although, ER stress-derived apoptosis was not identified in this study, more research studies are needed on the relationship between SCNT and ER stress-derived apoptosis.

Alzheimer’s disease (AD) is a progressive neurodegenerative disease associated with memory loss and cognitive impairments. An AD transgenic (Tg) pig model would be useful for preclinical testing of therapeutic agents. In this study, we report the use of somatic cell nuclear transfer (SCNT) to produce transgenic pigs over-expressing the human AD related genes (APP, APPswedish, Presenilin 1, and Tau). Transgenic embryos were generated by SCNT of from ear fibroblasts expressing AD genes. A total of 1808 (average 258) SCNT embryos were transferred into 7 recipients. Pregnancy was successfully maintained in one recipient. We obtained 1 cloned male piglet from a surrogate gilts and the weight of piglet was 935 g. But, the male piglet died two days after birth. The piglet expressed AD related genes by PCR and western blot analysis. Transgenes were expressed in multiple tissues, and at especially high levels in brain. AD Tg pig might be very useful for studying the disease and for testing new therapeutics in preclinical studies of human AD.

The various functions of mitochondria are essential for the transition from oocyte to embryo and for early embryonic development up to the blastocyst stage. Mitofusin 1 (MFN1) is the main mediator of mitochondrial fusion and homeostasis. Mfn1 proteins are located in the outer mitochondrial membrane. The mitochondrial membrane potential is a central indicator of cellular viability that reflects indicators of metabolic activity such as oxidative phosphorylation and the electron transport process. We investigated the MFN1 expression level in porcine SCNT blastocysts. The results showed that the MFN1, mitochondrial membrane potential and ATP levels were significantly decreased in the SCNT blastocysts. Whereas Bad, Bax, Caspase-3 and Caspase-9 levels were significantly increased compared with the parthenogenetic blastocysts. This study suggests that downregulation of MFN1 could affect the blastocyst development of porcine SCNT embryos through mitochondrial function and cellular apoptosis.

C-Phycocyanin (C-PC), a protein from green microalgae, has been suggested to possess various biological activities, including antioxidant and free radical scavenging properties. The aim of the current study was to explore the effects of C-PC on the maturation of porcine oocytes and subsequent developmental competence after parthenogenetic activation and somatic cell nuclear transfer (SCNT) as well as the underlying mechanisms. There was no significant improvement in nuclear maturation rates between the control and C-PC supplementation groups (1, 3, 5, 10 μg/mL). However, supplementation of 5 μg/mL C-PC in the maturation medium significantly increased blastocyst formation and hatching rates after parthenogenetic activation (59.6±3.6% and 33.0±2.6% vs. 49.8±3.5% and 27.4±2.4%, respectively). In addition, the presence of CPC during the maturation period significantly improved blastocyst formation rates and total cell numbers after SCNT (24.8±1.9% and 42.2±3.3 vs. 21.6±2.2% and 39.5±3.4, respectively) compared to the control group. Furthermore, cellular proliferation and the expression of pluripotency-related genes (SOX2 and NANOG) were increased in cloned blastocysts derived from the C-PC supplemented group. Importantly, C-PC supplementation during maturation not only improved cumulus expansion and increased the expression of cumulus expansion-related genes (HAS2, PTX3, and PTGS2), but also enhanced antioxidant capacity, improved mitochondria function, and decreased cathepsin B activity in porcine oocytes. These results demonstrate that C-PC may be useful for improving porcine oocyte quality and subsequent developmental competence in embryos.

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 μ M) 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/ H2DCFD a 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.

Vitrification is a common technique of cryopreservation in animal genetic resources of oocytes or embryos and replacing slow freezing methods. This study examined the factors of preparing open pulled straw (OPU) that could be used for vitrification with digital heating gun (DHG). Thermal performance of heat transfer of DHG is more stable and adjustable than traditional way of heating method like hot-plate or alcohol lamp. Thermal setting value of DHG was adjusted to 250, 350, 400 and 450℃ and production rate of wanted OPS was examined. The thickness of wall and outer diameter of OPU were measured by microscopy. OSP production efficiency of 0.5 mL straw was observed at 0, 66.1, 90.5 and 85.7% for 1~2 sec and 9.5, 33.3, 47.6 and 23.8% for 2~3 sec of heating time. OPS with inner diameter in the range of 200~650 μm was produced with 0.5 mL straw, and OPS with inner diameter in the range of 150~300 μm was obtained with 0.25 mL straw. OPS at the desired length and size could be obtained easily when straw was heated for 1.6~2.3 seconds at 350~400℃. Production rates of 0.25 mL straw were higher than those of 0.5 mL straw, so it was needed more attention to control time and power that were required for optimal heat transfer with 0.5 mL straw. Based on the data obtained in this study, hand-made OPSs could be produced for vitrification devices to freeze COCs, oocytes and embryos of with various size of outer diameter.

The objective of this study was to investigate the influence of the addition of cryoprotectants to freezing extenders on post thawing boar sperm quality as well as investigate their optimum concentrations. To evaluate the effect of trehalose (0, 80, 100, 120 mM) added in egg yolk extender freezing medium on motion characteristics, motility, viability and DNA integrity of spermatozoa at post thawing of cryopreservation. Semen were collected from 10 boars and frozen on the same day. Qualified semen samples (motility >80%) from each boar were subdivided four groups, 0 (control), 80 mM (T1), 100 mM (T2) and 12 0 mM (T3). Motility was assessed for % motile cell characteristics using computer-assisted semen analysis (CASA; SAIS SI-100, Medical supply, Korea). Frozen boar sperms were thawed in Beltsville Thawing Solution (BTS) then incubated at 38 celsius degree for 20 minutes. Computer progressive motility, average path velocity, straight line velocity, curvilinear velocity and total motility were higher (p<0.05) in adding 120 mM treatment group (T3) as compared with control. Moreover, trehalose addition did not damage DNA integrity of spermatozoa. Nevertheless, trehalose concentration treatment did not affect the viability of the spermatozoa as the concentration increased. The data showed that trehalose as an additive cryoprotectant was able to improve sperm quality without on sperm DNA integrity in cryopreservation.

Although assisted reproductive technology (ART) currently exists, the only embryo preservation technology that is available is cryopreservation. In the present study, small molecules were used to hold embryos at room temperature. The basic medium for embryo holding for a short period of time at 4℃, 10℃ and 20℃ consisted of 1% BSA non-cryopreservation medium (BNC) instead of fetal bovine serum. To maintain survival and prevent damage during embryo incubation, three candidate small molecules were selected-CHIR99021, Y-27632 and Thiazovivin- and their concentrations were optimized. The viability and hatching rate of embryos incubated at 10℃ were greater for Y-27632-BNC and CHIR99021+Y-27632-BNC compared to BNC. However, the rate was lower for Thiazovivin-BNC compared to BNC. Although there were no surviving embryos after incubation at 20℃, the viability and hatching rate of embryos significantly increased in Y-27632-BNC and CHIR99021+Y-27632-BNC compared to BNC. The pregnancy rate of embryos incubated at 20℃ was also greater in the CHIR99021+Y-27632- BNC group compared to that in the frozen group. The mechanism by which small molecules enhance survival of embryos during incubation was investigated, and expression of heat shock protein 70 was observed to increase. The findings of this work may be useful in improving ART in the agricultural field.

Egg yolk (EY) has been widely used in semen extender as a cryoprotectant. However it contains a biologically hazardous compound. Therefore, the aim of this study was to investigate the effect of Polyvinyl Alcohol (PVA) as a chemically defined substitute for EY in dog sperm cryopreservation. Spermatozoa collected from five dogs were resuspended (1×108 cell/mL) with Tris extender 1 supplemented with 0 (control), 0.025, 0.05, 0.1, 0.2 or 0.3 g PVA. The extended spermatozoa were cooled to 4℃ for 1 h. The cooled spermatozoa were diluted (1:1) with Tris extender 2 containing 1 M glycerol and PVA concentrations used in the 1st dilution and then were loaded in 0.5 mL straws. The straws were kept at 4℃ for additional 30 min and were frozen using LN2 vapor. Sperm progressive motility was assessed immediately after thawing and post-thaw incubation at 24℃ for 20 min, respectively. Viability, acrosome integrity and reactive oxygen species(ROS) level were assessed following post-thaw incubation. As results, supplementation with PVA significantly(p<0.05) increased progressive motility compared to control group both immediately after thawing and post-thaw incubation. Moreover, the sperm progressive motility in 0.1g PVA group was significantly(p<0.05) higher than the rest of the PVA and control groups at post-thaw incubation. Although there were no significant differences in ROS level and viability among the groups, the acrosome integrity of spermatozoa in all PVA groups was significantly higher compared to the control group. We suggest that supplementation of Tris extender with PVA is effective for protecting dog spermatozoa during freezing with higher progressive motility and acrosome integrity. Therefore, PVA can be used as asubstitute for EY in the cryopreservation of dog spermatozoa.

We determined the optimal polyvinyl Alcohol(PVA) concentration in previous study. However, the sperm progressive motility was lower compared to viability. To improve the motility, we determined the effect of pH adjustment in PVA-extender and post-thaw incubation time on dog sperm viability, motility, reactive oxygen species(ROS) level and gene expression levels related to motility(SMCP) and apoptosis(Bcl-2). Spermatozoa collected from five dogs were resuspended (1×108 cell/mL) with Tris extender 1 containing 0.1, 0.2, 0.3 g PVA with non-adjustment of pH (6.45) and adjustment of pH (6.85), respectively. The extended spermatozoa were cooled to 4℃ for 1 h. The cooled spermatozoa were diluted (1:1) with Tris extender 2 containing 1 M glycerol and PVA concentrations used in the 1st dilution and then loaded in 0.5 mL straws. The straws were kept at 4℃ for additional 30 min and were frozen using LN2 vapor. Sperm viability was assessed after cooling and post-thaw incubation at 24°C for 20 min. Sperm progressive motility was assessed after cooling, post-cooling incubation at 24°C for 20 min, immediately after thawing and post-thaw incubation at 24°C for 20 min. In addition, ROS level and the relative abundance of Bcl-2 and SMCP mRNA(qRT-PCR) were measured following postthaw incubation. As results, adjustment of pH to 6.85 in PVA-Tris extender significantly (p<0.05) improved sperm progressive motility compared to non-adjustment groups with no significant effect on sperm viability, ROS and mRNA expression level of Bcl-2 and SMCP. Post-thaw incubation at 24°C for 20 min significantly improved the progressive motility in all pH adjusted groups. We conclude that adjustment of pH to 6.85 and post-thaw incubation in PVA-Tris extender could considerably improve motility in dog sperm cryopreservation.

Mastitis in dairy animals is a costliest disease worldwide and caused by more than 150 micro-organisms. Mastitis is defined as inflammation of mammary gland which is characterized by pathological changes in mammary gland and physical and chemical changes in milk. Cow heifers are the future milk producers of every dairy herd. Heifer mastitis is a disease that potentially threatens production and udder health in the first and subsequent lactation leading to economic losses for dairy farms. Unfortunately, most producers regard young heifers as uninfected, and the presence of mastitis is not observed until calving or until first signs of clinical mastitis in early lactation. Therefore, present study was focused to understand the distribution pattern of mastitis etiological agents in cow heifer’s with their molecular characterization. We found Coagulase Negative Staphylococci (CNS) as chief etiological agents of heifers mastitis followed by Staphylococcus aureus, Streptococcus spp., E. coli etc. Moelcular characterization of most prevalent bacteria was done using bacterial specific genes such as nuc gene, E. coli gene, S agl gene etc. Effective treatment of mastitis is major challenge to field veterinarians, researchers and farmers, use of traditional antibiotics have only upto 60% care rate due to various factors. Hence, we also focused to find out the alternative ways to fighting against low cure rate by developing nano-particles based approaches to improve the treatment of mastitis. We used chitosan nano-particles as vehicles for the Ciprofloxacin. Chitosan-Ciprofloxacin nano-particles were synthesized and characterized for in vitro use as antibacterial agent against mastitis origin bacteria. Present study found that Chitosan-Ciprofloxacin nano-particles had good antibacterial activity with the slow release properties.

황체는 난소 내 존재하는 일시적인 내분비 조직으로서, 임신과 관련된 성 호르몬을 분 비하며, 발정주기에 따라 형성과 퇴행을 반복한다. 황체의 다이내믹한 생리적 현상을 연 구하기 위해서는 황체세포의 종류와 역할을 알아야 하는데, 일반적으로 황체는 progesterone을 분비하는 luteal steroidogenic cell과 혈관을 형성하는 luteal endothelial cell로 구성 되어 있다. 따라서 본 연구는 황체를 구성하는 세포의 기능을 알아보기 위하여 실시하였 다. 황체 내의 혈액은 0.75% Na4EDTA를 처리하여 제거하였고, DNase I, collagenase A 및 trypsin-EDTA를 처리하여 세포를 분리하였다. 원심분리를 통하여 분리된 세포는 38°C, 5% CO₂조건에서 16시간동안 배양하였다. 16시간 후, 부착된 세포는 luteal steroidogenic cells (LSCs)로 판단하였으며, 군집으로 성장하는 세포는 luteal endothelial cells (LECs)과 luteal steroidogenic cell-like cells (LSC-like cells)로 판단하고 passage를 실시하였다. 분 리된 세포는 PCR을 이용하여 세포의 종류를 검증하였다. 3beta-hydroxysteroid dehydrogenase (3β-HSD), platelet endothelial cell adhesion molecule (PECAM-1), vascular endothelial cardherin (VE-cadherin), epithelial cardherin (E-cadherin), endothelin-1 (ET- 1), endothelial nitric oxide synthase (eNOS) 및 fibroblast growth factor 7 (FGF7) mRNA 발 현 결과, 우리는 황체로부터 LSCs, LECs, LSC-like cells의 세포를 순수하게 분리된 것을 확인하였다. 그리고 LSC-like cells의 유래를 명확하게 하여 황체에서 일어나는 다이내믹 한 생리현상을 이해하는데 중요한 역할을 할 것이다.

Regulation of bovine interferon-tau (bIFNT) production, a conceptus secretory protein implicated in the process of maternal recognition of pregnancy, However, its expression in vivo and its transcriptional regulation are not yet well characterized. Among more than bIFNT gene sequences characterized, approximately 90% of bIFNT transcripts expressed in utero is derived from bIFNT1 gene and amounts of transcripts from other bIFNT genes such as new type I bIFNTcl. It was hypothesized that the variation in expression levels exhibited by the bIFNT1 and bIFNTc1 genes was due to identical in the proximal promoter regions of these bIFNT genes. In the present inverstigation, the 5’-upstream (positions ‒1,000 to +51) region of bovine IFNT1 and IFNTc1 genes were analysed using bovine ear-derived fibroblast (EF) cell line, which would provide a more suitable system for studies of the bovine trophoblast transcription factor ETS2. Luciferase assay, the 5’-upstream region (positiond ‒1,000 to +51) of bIFNT1 and bIFNTc1 was then truncated at positions ‒637, ‒389, ‒262, ‒222, or ‒157, or point-mutated at potential ETS-binding site. As compared to the wild-type constructs, those with deletion and mutation constructs were reduced bIFNT transcription similarly. Also, these findings were confirmed by mutated examining idented ETS2 binding site on the bIFNT1 and bIFNTc1 genes 5’-upstream region, ETS2 transcription factor expression constructs revealed the importance of a ETS2 binding site at (between -79 to -70) was idented in the bIFNT1 and bIFNTc1 genes. These results demonstrate these the short promoter region including the ETS2 binding site, gene display identical by transcription factor ETS2, tissue-specific expression and developmental regulation during pregnancy, in determining bovine IFNT genes transcription by the trophectoderm.

For evaluating the boar semen quality, sperm motility (MOT) is an important parameter because the movement of spermatozoa indicates active metabolism, membrane integrity and fertilizing capacity. Zygote arrest 1 (ZAR1) is oocyte-specific protein involved in the initiation of embryo development. but their functions have not been confirmed in pig until now. Therefore, this study was conducted to analyze their association with sperm motility and kinematic characteristics. DNA samples from 105 Duroc pigs with records of semen motility and kinematic characteristics〔Total motile spermatozoa (MOT), Curvilinear velocity(VCL), Straight-line velocity(VSL), the ratio between VSL and VCL(LIN), Amplitude of Lateral Head displacement(ALH)〕 were analyzed. A SNP in coding region of ZAR1 g.4095 C>A in intron 4 was associated with MOT in Duroc population. Therefore, we suggest that the porcine ZAR1 gene may be used as a molecular marker for Duroc boar semen quality, although its functional effects were not defined yet. These results might shed new light on the roles of ZAR1 in spermatogenesis as candidate gene for boar fertility, but still the lack of association across populations should be considered.

GRP78 is typically expressed in mammalian cells and plays a prominent role in the maintenance of cell homeostasis and development. Additionally, chicken serum-mediated proliferation has regulated the chGRP78 to control prevented apoptosis in chicken cells, thus allowing the required chGRP78-mediated anti-apoptosis. However, the precise molecular mechanisms underlying the chGRP78-mediated apoptosis process remained to be defined. Previously, we reported the effects of chGRP78 on chicken embryo fibroblast (CEF) and DF-1 cell proliferation. Here we identify the AKT1 as a key target of chGRP78 in apoptosis mechanism. Using immunoprecipitation and peptide sequencing, chGRP78 binding sites were detected in AKT1-mediated protein. chGRP78 causes activation of AKT1, and chGRP78 knockdown cells show a decreased level of AKT1. Importantly, activation of AKT1 depletion reciprocally inhibits the apoptosis and promotes proliferation in chicken cells. Taken together, AKT1-mediated signaling pathway plays a critical role in chGRP78-stimulated fibroblast survival and anti-apoptosis, which, suggests an important implications of our findings for the maintenance of chicken fibroblast cells through the inhibition of apoptosis.

돼지 생식기 호흡기 증후군은 Porcine respiratory and reproductive syndrome virus (PRRSV)에 의해 자돈이나 육성돈에서 발병하는 전염병으로 이를 제어할 수 있는 치료제 의 개발은 전무한 상황이다. 이전의 연구에서 재조합 가수분해 항체인 3D8 scFv 단백질 은 nuclease 활성으로 바이러스의 유전자를 무작위적으로 제어할 수 있다고 보고하였고, 이를 바탕으로 형질전환 돼지를 개발하였다. 이에 본 연구는 3D8 scFv 형질전환 돼지에 서 PRRSV에 대한 항바이러스 효과를 알아보고자, 3D8 scFv 형질전환 돼지와 일반돼지 의 교배를 통해 실험군 3D8 scFv 형질전환 돼지와 대조군 일반돼지를 생산하여 본 연구 에 공시하였다. 약 1개월 령의 돼지를 돼지의 비강에 바이러스를 주입한 직접 감염 군과 동일한 방에 같이 사육한 접촉 감염 군으로 나누어 실험을 수행하였으며, 바이러스의 감 염률은 혈중바이러스 농도 분석, 항체가 형성 ELISA 분석 그리고 평균일당증체율(Average Daily Weight Gain, ADWG) 분석을 통해 확인하였다. 접촉 감염 군의 경우 3D8 scFv 형질전환돼지에서 대조군에 비해 혈중바이러스 농도 및 항체가 형성이 저하되고 평 균일당증체율이 증가하여 바이러스에 대한 저항성을 보였으나 유의적인 차이는 확인할 수 없었다. 반면에, 직접 감염 군의 경우 혈중에서 PRRS 바이러스 농도는 유의적(p< 0.01)으로 낮았고, 평균일당증체율에서도 유의적인 차이(p<0.05) 를 보여주었다. 본 실험 을 통해 형질전환 돼지에 도입된 3D8 scFv 유전자로부터 발현된 핵산 가수분해 효소 3D8 scFv에 의한 PRRSV 저항성을 확인할 수 있었으며, 이 연구를 통해 향후 3D8 scFv 형질전환 돼지는 PRRSV 감염 및 전이 연구에 활용할 수 있을 것으로 기대된다.

To date, CRISPR/CRISPR-associated protein 9 (Cas9)-mediated genome editing technology is expected to become the most efficient tool for economic trait improvement in livestock. However, one of the critical issues for genetic modification by CRISPR/Cas9 is non-specific DNA double strand breaks which is called as off-target effect. In this study, we examined the mutated Cas9, nickase to modulate the specific target gene in chicken primordial germ cells (PGCs) as well as DF1 cells. Chicken myostatin which is famous for inhibitor of muscle cell growth and differentiation during myogenesis was targeted to be mutated with the Cas9-D10A nickase. After co-transfection of the Cas9- D10A nickase expression vector with eGFP gene and two targeted guide RNAs (g- RNAs), the GFP-positive cells were sorted out by fluorescence-activated cell sorting (FACS). In DF1 cells which is chicken embryonic fibroblast cell line, the mutant induction efficiency was 100% in the targeted myostatin site by the genotyping analysis of the knockout DF1 cells. In DF1 cells, number of the deleted nucleotides ranged from 2 to 39 nucleotide deletion. In addition, 6 expected off-target sites were predicted and analyzed but any non-specific mutation in the off-target sites was not observed. We also verified myostatin gene knockout with Cas9-D10A nickase in chicken primordial germ cells (PGCs). In conclusion, the knockout technical platform with the Cas9- D10A nickase can be efficiently applied to functional genomics study in poultry and finally adapted to generate the knockout poultry.

본 연구는 칡소의 모색과 MC1R 유전자형 및 MC1R 유전자의 SNP와의 상관관계를 분 석하고자 실시하였다. 또한 황모와 흑모를 가진 칡소 암소를 모(dam)로 사용함으로써, 칡 소 유전자원의 다양성 확보와 종축으로서의 활용가능성을 검증하고자 수행하였다. 칡소 55두의 모색을 황모, 흑모, 호반모로 구분하고 이들과 한우 10두의 DNA를 추출한 후 PCRRFLP 방법으로 MC1R의 유전자형을 확인하였으며, Taqman SNP genotyping assay를 이용하여 c.866C>T(L 195F) SNP 유형을 결정하였다. 한우의 MC1R 유전자형은 모두 ee 였고, SNP 유형은 C/C였다. 칡소의 MC1R 유전자형은 E+E+, E+e, ee였고, SNP 유형은 C/C와 C/T 였다. 칡소 중 황모 시험군(E+E+, E+e, ee)과 호반모 시험군(E+E+, E+e)에서의 SNP 유형은 C/C가 C/T보다 빈도가 높았다. 그러나 칡소 중 흑모 시험군(E+E+, E+e)에서 는 C/T가 C/C보다 빈도가 더 높았으며, C/T의 비율이 0.357로 황모나 호반모 시험군보다 높아 흑모색 발현에 영향을 줄 수도 있다고 사료된다. 칡소 55두의 성(sex)별로 SNP를 분석한 결과, 암소에서는 C/C가 19두, C/T는 11두였고, 수소에서는 C/C가 21두, C/T가 4 두로서 모두 C/C빈도가 높았다. 칡소 중 황모와 흑모 시험군에서 가계의 MC1R 유전자 형과 SNP 유형을 분석하였다. 모의 모색이 황모이고, MC1R 유전자형이 ee, SNP가 C/C 인 개체와 부(sire)의 모색이 황모이고, E+E+와 C/T인 개체를 교배한 경우, 자손 1두가 (E+e, C/T) 호반모색을 발현하였다. 모가 황모이고, E+E+, C/C인 개체와 부가 호반모이며, E+e, C/C인 개체를 교배한 경우, 자손 2두는 호반모색을 발현하였다. 모가 황모이고, E+e, C/C인 개체와 부가 호반모이며, E+E+, C/C인 개체를 교배했을 때, 자손 5두 중 2두 는(C/C) 호반모색을, 3두는 황모색을 발현하였다. 모가 황모이고, E+e와 C/T인 개체와 부 가 호반모이며, E+E+, C/C인 개체를 교배했을 때, 자손 2두 중 1두(E+E+, C/C)는 호반모 색을 발현하였다. 모가 E+e와 C/T인 개체와 부가 호반모이며, E+e, C/C인 개체를 교배했 을 때, 자손 2두 중 1두가(E+E+, C/T) 호반모색을 발현하였다. 모가 흑모색이고, E+E+, C/T인 개체와 부가 호반모색이며, E+E+, C/C인 개체를 교배한 경우, 자손 1두가 호반모 색(E+E+, C/C)을 발현하였다. 모가 흑모이며, E+E+, C/T인 개체와 부가 호반모이며, E+e, C/C인 개체의 교배 시, 자손 4두 중 2두는 호반모이며, 2두는 흑모(C/T)였다. 모가 흑모 이며, E+e와 C/T인 개체와 부가 호반모이며, E+E+, C/T인 개체를 교배한 경우, 자손 1두 가 황모(E+e, C/C)를 발현하였다. 본 연구 결과, 전체 8종의 교배조합 중 7종에서 일부 또는 전체 자손들이 호반모를 발현하였고, 18두 중 10두(55.5%)의 자손들이 호반모를 발 현함으로써 칡소 중 황모와 흑모인 암소를 번식에 공여하여 호반모 자손을 생산할 수 있 는 가능성을 확인하였다. 따라서, MC1R SNP 유형은 다양한 모색의 칡소를 종축으로 활 용하는 데 보완자료로 사용될 수 있을 것이다.

To elucidate a possible role for FGF9 (fibroblast growth factor9) in these events, porcine preimplantation stage embryos were characterized for the presence of FGF9 gene and protein. The objective of this study were to investigate the effects of FGF9 on hormone-stimulated porcine preimplantation stage embryos. Each embryos were collected from non-synchronized (Control; n=5) and after synchronized AI (regumate, PMSG/ hCG; n=5) sows and qPCR analyzed for FGF9 mRNA levels. The synchronization AI embryos had higher (p<0.05) FGF9 mRNA levels than that non-treated group embryos. At these stages of preimplantation development were also analyzed for immunoreactive FGF9 expression. Synchronization AI embryos were strong expression of FGF9. In contrast, non-treated embryos were remained constant during development stage. These results demonstrate the temporal variation of FGF9 mRNA abundance in hormonal stimulation and of immunoreactive FGF9 with the developmental processes occurring in the preimplantation development.