Increase of bovine embryos produced by in vitro fertilization (IVF) has been seen. The main reason for producing in vitro fertilized embryos in Korea has been to utilize the genetics of cows with higher carcass grade. Ovaries are collected from the cows in the slaughter house and the information on the carcass grade of the cow can be traced. Embryos produced from cows with higher carcass grade have been favored by the farmers. PCR has been one of the main techniques for sex determination of embryos targeting various genes. Bovine sex determining region Y (SRY) is specific to Y chromosome. However, it requires a control gene for PCR, if the embryo is female. In comparison to SRY, amelogenin can be amplified from male or female embryos with different fragment sizes due to differential splicing in all bovidae. The goal of this study was to determine whether there are any differences in the sex ratio of embryos produced in vitro and to compare the efficiency of sex determination using PCR. Ovaries of Hanwoo were collected and transported to the laboratory in thermal bottles. For in vitro maturation, oocytes were collected from the follicles with less than 8 mm of diameter and placed in either the Brackett & Oliphant media (BO), Tissue culture medium-199 (TCM-199), or IVMD101 media, containing 3% fetal bovine serum (FBS), 0.5 mg/ml FSH, 0.5 mg/ml LH, and 1 mg/ml estradiol-17β. For IVF, frozen sperm from Hanwoo bulls were used. After 22-24h IVF, embryos were transferred and cultured either in BO or TCM-199 with 10% FBS until the embryos were hatched. Hatched blastocysts were stored in PBS frozen, and later thawed and treated with embryo lysis buffer. After isolating genomic DNA, it was used for PCR using primers for casein beta (CSN2), as PCR control, or for male specific SRY primers. Alternatively, primers for amelogenin were used. Sex of embryos was determined and the sex ratio was analyzed. Out of 94 embryos, sex of 83 embryos (88.3%) was determined and there were 40 male embryos (48.2%) and 43 female embryos (51.8%). Sex of 31 embryos was determined using both SRY and amelogenin. Among those, 17 embryos were determined as having identical sex, while 1 embryo was determined as having different sex, and the sex of 11 and 2 embryos were determined only by amelogenin or SRY primers, respectively. In conclusion, the success of determining the sex of embryos by PCR was relatively high. Using amelogenin primer for PCR tends to be more efficient than SRY primer in determining the sex. Slightly higher ratio of female embryos was different from previous years and the cause for the difference may require further investigation.

The tyrosinase (TYR) genes have been accepted as major genes involved in the plumage pigmentation of chickens. Tyrosinase (TYR) gene is located on chromosome 1 in chicken and it is composed of five exons and four introns. TYR gene is described as a key enzyme in melanin biosynthesis. Especially, most examples of color patterns in chicken have been due to differential in the tyrosinase gene. This study was conducted to the association of feather color and sequence polymorphism in the Tyrosinase(TYR) gene was investigated using Korean native chickens(red plumage, red-line plumage, Ogol = KNC) and white leghorn(WL). From WL and KNC breed analyses, 232 differential SNPs were detected in 4th exon and 4th intron of TYR gene respectively. The genotype frequencies for 50 SNPs were compared between KCR, KCRD and KCO represented homozygous SNP types in all the analyzed SNP positions while KNC displayed various SNP types. In this study, we conclude that the variation of a wild type sequence in intron 4 of the tyrosinase gene is pigmentation of the original native chickens in korean. This work was supported by a grant from the “Livestock Preservation of Genetic Resources", Rural Development Administration, Republic of Korea.

Prolonged communication between oocytes and the surrounding somatic cells is one of the unique reproductive physiology in canine. Paracrine Kit ligand (KITL) signaling is a well-known communication between granulosa cells and the oocyte. KITL is a cytokine growth factor secreted by granulosa cells that signals via the c-kit receptor expressed by oocytes. Paracrine factors, including growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), exert their effects by binding with the kinase receptors expressed on the granulosa cells. However, the regulations of GDF9 and BMP15 in the canine KITL expression are currently poorly understood. Therefore, we investigated the effects of GDF9 and BMP15 on the expression of KITL in canine ovarian granulosa cells in vitro. In Annexin V assay recombinant GDF9 and BMP15 did not induce apoptosis in the cultured ovarian granulosa cells. When treated, FSH significantly increased KITL expression, and hCG suppressed its expression. When both FSH and hCG were treated, the expression of KITL was affected by GDF9 and BMP15 in dose and time dependent manner in the luteal granulosa cells. GDF9 (10 ng/mL) significantly decreased KITL expression after12 h. BMP15 (10 ng/mL) significantly also decreased KITL expression after 24 h. Western blot and immunochemistry results indicate that GDF9 activated Smad2/3. After blocking ALK 4/5/7 receptors by SB, GDF9 failed to activate Smad2/3, also BMP15 did not activate Smad1/5/8 after blocking ALK 2/3/6 receptors by DM. So GDF9 exerts its effects via using ALK 4/5/7 receptors to activate SMAD2/3 signaling, and BMP15 binds ALK 2/3/6 receptors to activate SMAD1/5/8 signaling. The expression of KITL was not changed by SB or DM treatment. However, the effect of GDF9 and BMP15, which decreased the expression of KITL, was suppressed by SB or DM treatment. In conclusion, this study provides the first evidence that recombinant GDF9 and BMP15 decrease KITL expression in canine ovarian granulosa cells.

The cancer and Parkinson's disease associated protein DJ-1 is multifunctional protein that involves in diverse cellular process. DJ-1 protein has a cellular protective role and promoted cell survival under an oxidative stress. However, the cellular protective mechanism of DJ-1 is not fully understand, and we needs to be further study their functions in novel organisms. In the present study, we investigated the protective role of DJ-1 against induced oxidative stress in canine cell line. On the basis of these experiments, canine DJ-1 overexpressing and null cell lines were established. The stable overexpression and down regulation of DJ-1 efficiency confirmed by the western blot analysis. Subsequently, the DJ-1 gene transfected cell lines and control cells were subjected to induced the oxidative stress, and then cell viability, cell proliferation assay, cellular apoptosis detection analysis (Annexin V and TUNEL assay), intracellular ROS and mitochondrial activity were measured appropriately. The results showed that DJ-1 overexpressed cells were up-regulated cell viability under oxidative stress conditions induced by the rotenone and hydrogen peroxide (H2O2), whereas loss of DJ-1 cells were down-regulated the cell survival activity. Additionally, overexpression of DJ-1 cells increased cell resistance to oxidative stress and inhibited the elevation of cell death and cellular ROS induced apoptosis. Moreover, DJ-1 overexpressed cells was increased mitochondrial functions by using confocal microscopy with MitoTracker staining. On the contrary to this, DJ-1 null cells show defective cellular protection and mitochondria activity against oxidative stress conditions. Our data indicate that canine DJ-1 protein attenuates cellular apoptosis and ROS generation, enhances the cellular survival activity and promote mitochondrial function under the oxidative stress, likewise other mammalian cells. Importantly, DJ-1 overexpression may be an important part of a protective strategy as a sensor for oxidative stress.

Interferon tau (IFNT), produced by the mononuclear trophectoderm, signals the process of maternal recognition of pregnancy in ruminants, but its expression in vivo is not well characterized. Objectives of this study were to determine IFNT gene isoforms expressed in the bovine uterus, and to identify differences in promoter sequences of IFNT genes that differ in their expression. Through the RNA-seq analysis of bovine conceptuses on days 17, 20 and 22 (day 0 = day of estrus), the expression of only two IFNT transcripts, IFNT1 and IFNTc1, were found, which were indeed classified into the IFNT gene clade. IFNT mRNAs were highest on day 17, and then decreased on days 20, and 22, which were also supported by the results of quantitative RT-PCR. Bovine ear-derived fibroblast (EF) cells were then cotransfected with luciferase reporter constructs carrying 5‘-upstream (positions -1000 to +51) regions of IFNT1 or IFNTc1 and various transcription factor expression plasmids. CDX2, either alone or with other Ap-1, ETS2 and/or CREBBP transcription factors, was found to increase luciferase activity approximately 10 and 18 fold more than twice of those cotransfected with bIFNT1, c1-Luc construct. Furthermore, The degree of transcriptional activation by a combination of the AP1, ETS2, CREBBP and/or CDX2 expression vectors was similar to that of CDX2 along plasmid. However, expression patterns of these Luc activity differented. Whereas bIFNTc1-Luc showed lowest antivity had than bIFNT1-Luc reports. Although, lowest antivity had of the bIFNTc1 –Luc report, cotransfected with the bIFNTc1-Luc construct and AP1(JUN) or/and ETS2 expression plasmid, Luc activity was enhanced approximately 2 and 4-fold more than the bIFNT1-Luc. Furthermore, along CDX2 expression factor had high effect on activity of bIFNT1-Luc reporter than the c1 gene in EF cells. These results suggest that two forms of IFNT genes are expressed in utero and their transcriptional regulations differ.

Multiple interferon tau (IFNT) genes exist in bovine. An antiluteolytic substance secreted by the bovine conceptus and primarily responsible for maternal recognition of pregnancy is bovine trophoblast protein 1 (bIFNT1), one of new type I interferon tau (IFNT) genes. The objectives of this research were to investigate whether multiple, distinct gene encode bIFNT1 and other type I bIFNT gene in the bovine genome or not and to examine the expression of bIFNT1 and other bIFNTc1 mRNAs during conceptus development. The transcription of these genes could be regulated through caudal-related homeobox-2 (CDX2) and ETS2 and/or AP1(JUN) expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. The presence of mRNAs encoded by bIFNT1 and type I bIFNTc1 genes were examined quantitatively via reverse transcription-polymerase chain reaction (RT-PCR) analysis of total cellular RNA (tcRNA) extracted from on the days 17, 20 and 22 bovine conceptuses. bIFNT1 was highly expressed on the day 17 and transcripts were gradually and weakly detectable on the days 20 and 22. However, the other bIFNTc1 gene examined transcripts was highly expressed on the day 20 and transcripts were weakly detectable on the days 17 and 22 bovine conceptuses. Furthermore, human choriocarcinoma JEG3 was co-transfected with an -1kb-bIFNT1/c1-Luc constructs and several transcription factor expression plasmids. Compared to each -1kb-bIFNT1/c1-Luc increased when this constructs were co-transfected with, ETS2, AP1(JUN), CREBBP and/or CDX2. Also, bIFNTc1 gene was had higher effect on activity by alone ETS2, and AP1(JUN) expression factors in choriocarcinoma JEG3 cell. However, bIFNT1 gene expression of the upstream region was not idented. These results demonstrate that these genes display differential, tissue-specific expression and developmental regulation during pregnancy.

Cryopreservation of miniature pig sperm is essential because of high demand of organ transplant in mass production. However, miniature pig sperm are vulnerable to oxidative stress more than other mammals. Erythritol is a naturally occurring sugar alcohol with powerful antioxidant property. Thus, the aim of our study is to verify if erythritol could reduce lipid peroxide and enhance viability of frozen thawed miniature pig sperm. Ejaculated semen samples were frozen with cryoprotectant subjected to erythritol treatment (0, 10, 100, 500 mM). After frozen thawed, spematozoa viability were examined using the computer assisted sperm analysis (CASA) system. The product of lipid peroxidation, malondialdehyde (MDA) were quantified using spectrophotometer with DPPH and ABTS assays as ROS scavenger markers. Our result showed that erythritol enhanced sperm viability (p<0.05), reduced lipid peroxides significantly (p<0.05), proving the concentration of 100 mM erythritol to be an effective for lowing oxidative damage. Data from our study suggest that erythritol exhibits significant lipid peroxidation scavenging characteristics which may prevent oxidative damage, enhance viability of frozen thawed sperm and thus could be a effective additive as cryoprotectant.

Reprogramming is a process in which a developmentally determined cell fate is re-established to another fate by artificial modifications. Reprogramming to pluripotent state has been studied extensively in somatic cell nuclear transfer and induced pluripotent stem cells (iPSCs). Interestingly, recent studies showed that expression of a set of transcription factors also can induce transdifferentiation, also known as direct conversion or direct reprogramming. There are two major approaches in direct reprogramming: one is target cell-specific factor-mediated direct reprogramming and the other is epigenetic flexibility-inducing factor-mediated direct reprogramming. We are interested in generating induced neural cells via direct reprogramming by using pluripotency factors as epigenetic flexibility inducers and understanding the mechanistic basis of the process. We hope that these reprogramming technologies will provide a new paradigm of research in regenerative medicine as well as disease study and drug development.

Zika virus, mosquito-borne disease, caused by mosquitoes has been increased the importance. From March to September, twice a month from 7 different points (3 residential areas, 3 migratory bird sanctuary and 1 cattle shed) were collected using BG trap and BL trap. After identifying the mosquitoes collected, we confirmed the virus infection. Total 26,531 mosquitoes (6 genus 9 species) were collected, virus has been detected from the 3 species (Aedes vexans, Cuilex tritaeniorhynchus, and Amigeres subalbatus) of mosquitoes of them. It showed the highest peak in August, and then gradually decreased. The most common mosquito species was collected Aedes vexan (16,637) in the cattle shed.

The environmental friendly control of Platypus koryoensis (Murayama), a vector of oak wilt disease, was investigated using citral. To develop an effective attracted trap was performed in Yeoju (semi-field), Gunpo (field) and Cheongju (field). Using a ratio of four attractant (ethanol 100%, ethanol+citral (90:10, 95:5), ethanol+citral+hexane extract of Quercus mongolica (90:5:5)) was investigated attractive effects for the Platypus koryoensis. Most P. koryoensis were attracted to mixture of ethanol+citral (95:5). As a result, when using a citral in a certain concentration is would be able to develop an effective and environmental friendly control agent.

Pine wilt disease (PWD) is one of the most important forest tree diseases, especially in the East Asian countries of Japan, China, and Korea. The Japanese pine sawyer, Monochamus alternatus Hope, is the PWN vector for Japanese red pine and Japanese black pine while Monochamus saltuarius Gebler is the vector for Korean white pine. Various control methods, such as aerial pesticide applications, biological control using parasitoids and fumigant such as methyl bromide are used. But the PWD still has spread. Therefore, we were selected effective aerial insecticides. Susceptibility of M. saltuarius and M. alternatus adults were investigated using 9 insecticides which are available in the market in Korea. And then, we tested them to the Apis mellifera to vertify the environmental impact.

In 2015, we reported a viral disease extremely fatal to Allomyrina dichotoma larvae spread in the majority of the larva-rearing farms in Korea. Currently, the virus-infected larva is diagnosed by PCR-based amplification but this requires laboratory equipment and agarose gel electrophoresis. Loop-mediated isothermal amplification (LAMP), a highly sensitive DNA amplification method, uses DNA polymerase isolated from Bacillus stearothemophilus and a set of six primers. It has great potential for field use because DNA is amplified under single temperature and the overall reaction completes in 30 min without laboratory equipments. Here, we report the development of on-site diagnosis method for Korean horn beetle larva infected by Allomyrina dichotoma Nudivirus.

The number of pest detection in wooden pellets has increased since wooden pellets as biomass-solid refuse fuel (Bio-SRF) was allowed an import in Korea. In this study, we analyzed an insecticidal effect on pupal and adult stages of red flour beetle (Tribolium castaneum) and maize weevil (Sitophilus zeamais) in wooden pellets using two fumigants, methyl bromide (MB) and phosphine (PH3), for establishing a guideline on fumigation. MB and PH3 both showed a dose-dependent manner in insect mortality after fumigation under 60% filing rate (FR) of wooded pellets in desiccator. In MB fumigation to T. castaneum, adults and pupae showed 100% mortality at 128 mg/L and 161 mg/L in 10°C at 24 h post treatment and pupae showed 100, 74, and 8% mortality against 99 mg/L of MB in 20, 40, and 40% FR, respectively. In PH3 fumigation, S. zeamais adults showed 92% mortality to 4 mg/L, meanwhile T. castaneum adults showed 100% mortality at 120 h post treatment.

The occurrence pattern of lepidopteran adults in a paddy-upland rotation field and an organic upland field for 8 maize and 7 sorghum varieties was surveyed from May 8 to September 27, 2016. In addition, their damage ratio and the invasive pores by Ostrinia furnacalis larvae as well as harvest yields were monitored. The density of the lepidopteran adults in two fields showed similar pattern, although a little different pattern in Heliothis assulta. The damage ratio of maize and sorghum varieties by O. furnacalis at harvest periods was depended on both varieties and cultivated environments. A middle negative linear correlation was observed in damage ratios vs yields, the number of invasive pores vs yields, and the number of larvae in corn ears vs yields, but there was not a specific correlation between the damage ratio vs the number of invasive pores. These results may be useful for the establishment of a management strategy to control oriental corn borer in paddy-upland rotation or organic upland fields for maize and sorghum.

Myeloid differentiation factor 88 (MyD88) is an intracellular adaptor protein involved in Toll pathway. In this study, we monitored the response of 4 key genes of the insect immune system against Beauveria bassiana JEF-007 in Tenebrio molitor using RT-PCR. To better understand the roles of Toll pathway in mealworm immune system, TmGPR and TmMyD88 was knocked down by RNAi silencing. Target gene expressions were decreased at 6 days post-dsRNA injection. Therefore, mealworms were compromised by B. bassiana JEF-007 at 6 days post-dsRNA injection. Silencing of the TmMyD88 and TmGPR resulted in reducing the resistance of the host to fungal infection. However, only dsTmMyD88 showed significant difference with dsEGFP by statistical analysis, which may be due to partial gene knock down of dsGPR. These results indicate that TmMyD88 is required in mealworms for survival against B. bassiana JEF-007.