Influenza A viruses (IAVs) are members of the family Orthomyxoviridae and genus Orthomyxovirus. Avian and mammalian species are the host of IAVs, which includes humans and dogs. Canine influenza virus (CIV) is an emerging pathogen that causes severe and acute respiratory diseases in dogs. This study monitored the antigen and antibody against CIV in dogs in the Republic of Korea (ROK) from 2016 to 2021. One thousand and seventy-two nasal swabs and 1,545 blood samples were collected from animal hospitals and animal shelters. Five nasal swabs in 2017 and seven in 2018 from stray dogs were positive for CIV according to RT-PCR. The prevalence of H3N2 CIV ranged from 9.5% to 24.8%, according to the hemagglutination inhibition (HI) assay. On the other hand, none of the serum samples from 2018 to 2021 showed seropositivity against the avian H5, H7, and H9 viruses. The HI titers for H3N2 ranged from 16 to 512. The distribution of HI titer 16–32 was 57.6% in seropositive samples. The pet dogs were vaccinated against CIV, but the stray and military dogs were unvaccinated. In 2017 and 2018, the seroprevalence of CIV in stray dogs was higher than in the other years, and viral RNA was detected in nasal swabs. It may mean previous exposure of stray dogs to CIV. With the increasing number of pet dogs and the close contact between humans and dogs, canines could serve as an intermediate host for transmitting IAVs to humans. Therefore, continuous surveillance of CIV is needed for public health and the potential emergence of novel zoonotic viruses.
This study used adult wistar-based rats to observe the sexual cycle as a morphological characteristic of vaginal epithelial cells by vaginal smearing, and investigated the fetal number through mating with male rats of the same strain. The target animal was a 12 to 13-week-old Wistar-based mature unlighted rat (weight 220 g to 240 g), room temperature 23 ± 2℃, 14 hours artificial lighting (05:00 to 19:00 hours), 10 hours Adapted individuals were used for rearing for at least 2 weeks under the conditions of the darkroom (19:00 to 05:00). The feed was managed for free feeding of pellet feed for animals and water. The vaginal smearing method was used for the experiments by observing the sexual cycle every morning and confirming that the normal sexual cycle of 4 or 5 days was repeated at least 2 cycles or more. As a result, the proestrus was found to have few red blood cells, the cells and nuclei were rather large and round, and many nucleated cells were identified. In the case of the estrus, the cells were large and the nuclei were not stained, and most of the keratinocytes were found. In addition, in the metestrus and diestrus, there were many white blood cells, and it was confirmed that nucleated epithelial cells and keratinocytes were significantly reduced. The pregnancy period was 21 ± 1.8 days, and the number of live births per delivery was 11.9 on average. The number of fetuses on the 8th and 10th days of pregnancy were 15.2 ± 0.4 and 15.4 ± 0.3, respectively. On the contrary, the number of fetuses on the 12th day of pregnancy was 12.9 ± 0.6, which was significantly (p < 0.05) decreased compared to the 10th day of pregnancy, and the number of fetuses was similar until delivery. As a result of investigating the change of body weight according to the birth weight and growth stage after delivery, the birth weight of female and male was 9.2 ± 2.0 g and 9.8 ± 2.5 g, respectively. After that, until the 16th day, the female and the male showed similarly moderate weight gain, and then showed a rapid weight gain until the 21st day of lactation. With reference to the results of this study, it is expected to be used as basic data for determining the mating time of rodents and controlling pregnancy and fetal number.
The present study investigated the participation of D-serine and NR2 in antinociception produced by blockade of central erythropoietin-producing hepatocellular carcinoma (Eph) A4 (EphA4) signaling in rats with trigeminal neuropathic pain. Trigeminal neuropathic pain was modeled in male Sprague-Dawley rats using mal-positioned dental implants. The left mandibular second molar was extracted under anesthesia, and a miniature dental implant was placed to induce injury to the inferior alveolar nerve. Our current findings showed that nerve injury induced by malpositioned dental implants significantly produced mechanical allodynia; additionally, the inferior alveolar nerve injury increased the expression of D-serine and NR2 subunits in the ipsilateral medullary dorsal horn (trigeminal subnucleus caudalis). Intracisternal administration of EphA4-Fc, an EphA4 inhibitor, inhibited nerve injury-induced mechanical allodynia and upregulated the expression of D-serine and NR2 subunits. Moreover, intracisternal administration of D-amino acids oxidase, a D-serine inhibitor, inhibited trigeminal mechanical allodynia. These results show that D-serine and NR2 subunit pathways participate in central EphA4 signaling after an inferior alveolar nerve injury. Therefore, blockade of D-serine and NR2 subunit pathways in central EphA4 signaling provides a new therapeutic target for the treatment of trigeminal neuropathic pain.
본 연구는 한우 개체식별 및 친자감별에 있어 기존의 di-nucleotide repeat microsatellite marker 사용 시 발생했던 stutter로 인한 대립유전자 판별 오류 등의 문제들을 극복하고, 분석결과의 신뢰도와 정확도를 높이기 위해 tri-, tetra-, penta-, hexa-nucleotide repeat microsatellite 좌위들로 이루어진 새로운 개체식별 마커 13 종(BTRC6_01, BTRC19_02, BTRC11_03, BTRC16_05, BTRC9_07, BPC19_08, BTEC17_09, BPC21_10, BTEC4_11, BPC7_12, BPC1_13, BHXC29_14, BPC1_15)을 개발하였다. 선발된 13개의 좌위를 가지고 소 1,530두에 microsatellite typing을 실시한 결과, 총 61개에 대립유전자가 발견되었으며, 좌위별로 평균 4.69개의 대립유전자를 가지는 것으로 확인되었다. 마커의 다형성과 정보력의 척도인 PIC (Polymorphism Information Contents)값은 0.25(BTRC9_07)~0.59(BTEC17_09)로 나타났으며 BHXC29_14, BPC1_13, BTEC17_09, BTRC16_05, BTRC19_02, BTRC6_01 좌위들은 PIC 0.5 이상 그리고 나머지 좌위들 모두 PIC 0.25 이상의 값을 가지는 것으로 확인되어 마커로서 다형성이 있음이 검증되었다. 개발한 마커를 활용하여 한우(영암, 장흥)와 유럽우 7종 (Brown Swiss, Limousin, Angus, Simmental, Hereford, Charolais, Holstein)의 유전적 특성을 분석하였으며, 총 9개 집단의 Heterozygosity와 FIS (inbreeding coefficient) 값을 측정하였다. 기대이형접합율은 0.451(BS)~0.605(AG) 범위 내로, 한우는 0.532(영암), 0.545(장흥)의 값을 가지는 것으로 나타났다. 한우(영암, 한우)와 7종의 유럽우들 간의 유연관계 분석은 특정 대립유전자 빈도를 근거로 한 유전적 거리의 추정으로 이루어졌다. 한우집단과 Simmental 간의 유전적 거리(0.1848)가 가장 가깝고 비교적으로 Brown Swiss와의 유전적 분포(0.3352)가 가장 먼 것으로 나타났으며, 계통발생학적으로 유전적 분화 양상을 확인함으로써 본 마커의 한우 유전적 다양성 및 유연관계 분석에 활용 가능성을 제시하였다.
Sestrin-2 (SESN2) as a stress-metabolic protein is known for its anti-oxidative effects as a downstream factor of PERK pathways in mammalian cells. However, the expression patterns of SESN2 in conjunction with the UPR signaling against to ER stress on porcine oocyte maturation in vitro, have not been reported. Therefore, we confirmed the expression pattern of SESN2 protein, for which to examine the relationship between PERK signaling and SESN2 in porcine oocyte during IVM. We investigated the SESN2 expression patterns using Western blot analysis in denuded oocytes (DOs), cumulus cells (CCs), and cumulus-oocyte complexes (COCs) at 22 and 44 h of IVM. As expected, the SESN2 protein level significantly increased (p < 0.01) in porcine COCs during 44 h of IVM. We investigated the meiotic maturation after applying ER stress inhibitor in various concentration (50, 100 and 200 μM) of tauroursodeoxycholic acid (TUDCA). We confirmed significant increase (p < 0.05) of meiotic maturation rate in TUDCA 200 μM treated COCs for 44 h of IVM. Finally, we confirmed the protein level of SESN2 and meiotic maturation via regulating ER-stress by only tunicamycin (Tm), only TUDCA, and Tm + TUDCA treatment in porcine COCs. As a result, treatment of the TUDCA following Tm pre-treatment reduced SESN2 protein level in porcine COCs. In addition, SESN2 protein level significantly reduced in only TUDCA treated porcine COCs. Our results suggest that the SESN2 expression is related to the stress mediator response to ER stress through the PERK signaling pathways in porcine oocyte maturation.
Morphology of cumulus-oocyte-complexes (COCs) at germinal vesicle (GV) stage as one of the evaluation criteria for oocyte maturation quality after in vitro maturation (IVM) plays important roles on the meiotic maturation, fertilization and early embryonic development in pigs. When cumulus cells of COCs are insufficient, which is induced the low oocyte maturation rate by the increasing of reactive oxygen species (ROS) in porcine oocyte during IVM. The ROS are known to generate including superoxide and hydrogen peroxide from electron transport system of mitochondria during oocyte maturation in pigs. To regulate the ROS production, the cumulus cells is secreted the various antioxidant enzymes during IVM of porcine oocyte. Our previous study showed that Mito-TEMPO, superoxide specific scavenger, improves the embryonic developmental competence and blastocyst formation rate by regulating of mitochondria functions in pigs. However, the effects of Mito-TEMPO as a superoxide scavenger to help the anti-oxidant functions from cumulus cells of COCs on meiotic maturation during porcine oocyte IVM has not been reported. Here, we categorized experimental groups into two groups (Grade 1: G1; high cumulus cells and Grade 2: G2; low cumulus cells) by using hemocytometer. The meiotic maturation rate from G2 was significantly (p < 0.05) decreased (G1: 79.9 ± 3.8% vs G2: 57.5 ± 4.6%) compared to G1. To investigate the production of mitochondria derived superoxide, we used the mitochondrial superoxide dye, Mito-SOX. Red fluorescence of Mito-SOX detected superoxide was significantly (p < 0.05) increased in COCs of G2 compared with G1. And, we examined expression levels of genes associated with mitochondrial antioxidant such as SOD1, SOD2 and PRDX3 using a RT-PCR in porcine COCs at 44 h of IVM. The mRNA levels of three antioxidant enzymes expression in COCs from G2 were significantly (p < 0.05) lower than COCs of G1. In addition, we investigated the anti-oxidative effects of Mito-TEMPO on meiotic maturation of porcine oocyte from G1 and G2. Meiotic maturation and mRNA levels of antioxidant enzymes were significantly (p < 0.05) recovered in G2 by Mito-TEMPO (0.1 μM, MT) treatment (G2: 68.4 ± 3.2% vs G2 + MT: 73.9 ± 1.4%). Therefore, our results suggest that reduction of mitochondria derived superoxide by Mito-TEMPO may improves the meiotic maturation in IVM of porcine oocyte.
Ganglioside GM3 is known as an inhibition factor of cell differentiation and proliferation via inhibition of epidermal growth factor receptor (EGFR) phosphorylation. Our previous study showed that the exogenous ganglioside GM3 reduced the meiotic maturation of porcine oocytes and induced apoptosis at 44 h of in vitro maturation (IVM). However, the role of ganglioside GM3 in the relationship between EGFR signaling and apoptosis during porcine oocyte maturation has not yet been studied. First, porcine cumulus-oocyte complexes (COCs) were cultured in the NCSU-23 medium with exogenous ganglioside GM3 according to maturation periods (non-treated, only IVM I: 0 - 22 h, only IVM II: 22 - 44 h and IVM I & II: 0 - 44 h). We confirmed that the proportion of germinal vesicle breakdown (GVBD) increased significantly in the IVM I treated group than in the control group. We also confirmed that the meiotic maturation until M II stage and polar body formation decreased significantly in the only IVM I treated group. Cumulus cell expansion and mRNA levels of the expansion-related factors (HAS2, TNFAIP6 and PTX3) decreased significantly in the IVM I treated group than in the control group. Protein levels of EGFR, p-EGFR, ERK1/2, and p-ERK1/2 decreased significantly in the GM3-treated groups, during the IVM I period. In addition, cellular apoptosis, determined using TUNEL assay, and protein levels of Cleaved caspase 3, were increased significantly in the GM3-treated COCs during the IVM I period. Based on these results, ganglioside GM3 exposure of porcine COCs during the IVM I period reduced meiotic maturation and cumulus cell expansion via inhibition of EGFR activity in pigs.
In general, the shape of cumulus-oocyte-complexes (COCs) at germinal vesicle (GV) stage is important roles on meiotic maturation of porcine oocyte during in vitro maturation (IVM). Then, mitochondria produce reactive oxygen species (ROS) such as superoxide from electron transport system during oocyte maturation. ROS levels on oocytes are regulated by various antioxidant enzymes in cumulus cells (CCs). However, the effect of mitochondria derived superoxide production from CCs during IVM of porcine oocyte has not been reported. Firstly, we divided groups according to large number of CCs (Grade 1: G1) and small number of CCs (Grade 2: G2). Then, we counted cumulus cells of G1 and G2 oocyte by using haemocytometer. The oocyte maturation rate was significant decreased (p < 0.05) in G2 oocytes than that of G1 oocytes. We measured mitochondria derived superoxide in G1 and G2 COCs by using Mito-SOX staining. Mitochondrial superoxide was higher in G2 COCs than G1 COCs. Then, the mRNA expression levels of antioxidant enzymes (SOD1, SOD2 and PRDX3) in G2 COCs were decreased compared to G1 COCs. To reduce mitochondria derived superoxide, we used Mito-TEMPO as mitochondrial superoxide scavenger. Oocyte maturation rates in both G1 and G2 groups treated with Mito-TEMPO were increased than that of non-treated groups. Mitochondrial superoxide was lower in G1 and G2 groups treated with Mito-TEMPO than that of non-treatment groups. The mRNA expression levels of antioxidant enzymes in G1 and G2 COCs treated with Mito-TEMPO were increased compared to non-treated groups. Based on these findings, we suggest that reduction of mitochondria derived superoxide by Mito-TEMPO assists maturation competence in porcine oocytes.
Mitochondrion is an organelle for regulating calcium (Ca2+) homeostasis. Mitochondrial Ca2+ plays important roles on oocyte maturation, fertilization and embryonic development for ATP production. Low quality oocytes have mitochondrial dysfunction, which lead to overloaded Ca2+ in mitochondria. Recently, Rhod-2 is well known as a mitochondrial derived Ca2+ indicator. However, the changes of Rhod-2 in matured or fertilized porcine oocytes have not been reported. Therefore, the aim of study was to identify the effects of mitochondrial Ca2+ using Rhod-2 on quality assessment of matured oocyte and zygotes in pigs. Thus, we classified two groups (group 1: G1, compact COCs and group 2: G2, uncompact COCs) according to differences of cumulus cells amount and cytoplasm morphology in germinal vesicle (GV) stage of porcine COCs. Therefore, we investigated number of Rhod-2 spots in matured and fertilized oocytes from G1 and G2 groups. The Rhod-2 spot numbers were separated into four parts; n<10, 10≤ n < 20, 20 ≤ n < 30, and 30 < n. The Rhod-2 spots number of G2 group had greater than G1 group in part of 20 ≤ n. Additionally, we investigate mean number of Rhod-2 spots from G1 and G2 groups in matured and fertilized oocytes. As a result, we confirmed that average number of Rhod-2 spots in G2 group increased than that of G2 group. Finally, we also measured the Rhod-2 intensity in matured and fertilized oocytes of G1 and G2 groups. Interestingly, the Rhod-2 intensity in G2 group was higher than that of G1 group. (oocyte: p < 0.001 and fertilized oocyte: p < 0.05). These results demonstrated that changes in Rhod-2 spots and intensity were increased in low quality of matured and fertilized oocytes. Therefore, our results suggest that the differences in mitochondrial calcium level are associated with morphological quality of porcine COCs.
This study aimed to investigate the effect of using corn flakes, produced by pressurized steam chamber, on nutrient disappearance rate and energy value in three Hanwoo and three Holstein cows, implanted with a ruminal fistula. Corn flakes were categorized in 2 groups based on the chamber type: control (corn flakes produced using a steam chamber) and treatment (corn flakes produced using a pressurized steam chamber). Dry matter (DM) disappearance rate was 5.17% higher in treatment than in control (p<0.01). Starch disappearance rate was also higher in the treatment than in the control (p<0.01). Nitrogen free extract (NFE) and non-fiber carbohydrate (NFC) disappearance rates were 6.08 and 5.71% higher in treatment compared to control, respectively (p<0.01). In comparison by breed and incubation time, DM, starch, NFE, and NFC disappearance rates were higher in treatment than in control. The mean total digestible nutrients (TDN) was higher in treatment than in control (p<0.05). In comparison between Hanwoo and Holstein, TDN of corn flakes was slightly but not significantly higher in Holstein than in Hanwoo. Thus, these results indicate that the use of the pressurized steam chamber is recommended to increase the nutrient (starch, NFE, NFC etc.) disappearance rate and TDN.
This study aimed to produce high-quality blastocysts and establish appropriate microinjection conditions for the introduction of target gene. First, we identified embryo development to the blastocyst stage after microinjection using the CRISPR/Cas9 system on the Cas9 protein or mRNA. As a result, we confirmed that blastocyst development in the Cas9 mRNA injected group significantly increased when compared to the Cas9 protein injected group (p<0.05). However, the blastocyst gene targeting rate increased in the Cas9 protein injected group when compared to the Cas9 mRNA injected group (p<0.05). Next, we treated the injection medium with 10 μg/ml of cytochalasin B (CB), and the microinjected embryos were cultured in CR1-aa medium supplemented with 0.1 μM of melatonin (Mela). Consequently, the blastocyst formation rate significantly increased in the CB treated group (p<0.05). After microinjecting embryos with the CB treated injection medium, we investigated blastocyst formation and quality via Mela treatment. Consequently, the Mela treated group demonstrated significantly increased blastocyst formation rates when compared to the non-treated group (p<0.05). Furthermore, immunofluorescence assay using RAD51 (DNA repair detection protein) and H2AX139ph (DNA damage detection protein) showed an increase in RAD51 positive cells in Mela treated embryos. Therefore, we verified the improvement in knock-in efficiency in microinjected bovine embryos using Cas9 protein. These results also demonstrated that the positive effect of the CB and Mela treatments improved the embryonic developmental competence and blastocyst qualities in genetically-edited bovine embryos.
The objectives of this study was to investigate the properties and stability of the wet noodles added to nanoemulsion as a industrial model system, and in so doing, survey practical applicability in the food industry. In order to test out these objectives, the characteristics and stability of the wet noodles added to nanoemulsion were investigated and their cooking characteristics and capsaicinoids loss were examined. As a result, the test results showed that the findings indicated that the post-cooking loss of capsaicinoids in the wet noodles added to double-layer nanoemulsion covered with chitosan was less than the losses in the wet noodles added to any other noodles. More especially, this demonstrates that the noodle added to double-layer nanoemulsion covered with chitosan scored significantly higher than the others with reference to their cooking properties, color, texture, stability for storage stability, and sensory evaluation. These results show that the findings of this study demonstrated that the noodles added to nanoemulsions could be produced as a food-grade merchandise because they could provide enhanced encapsulation capacity of capsaicinoids and higher acceptability.
본 연구는 의사결정 전에 행복을 느끼는 소비자와 슬픔을 느끼는 소비자 간에 자기타당화 정도의 차이와 규제초점의 차이 그리고 자기타당화 정도의 차이가 규제초점과 정보왜곡에 미치는 영향을 탐색하였다. 연구 결과, 첫째, 사전에 행복을 느낀 소비자는 사전에 슬픔을 느낀 소비자보다 자기타당화 정도가 크고, 촉진초점을 강하게 갖는다. 둘째, 사전에 느낀 슬픔감정과 비교하여 사전에 느낀 행복감정이 자기타당화를 매개로 촉진초점에 부분적으로 정적인 영향을 미치고, 자기타당화를 매개로 정보왜곡에 완전히 정적인 영향을 미친다. 본 연구는 현재의 의사결정시점 이전에 느낀 행복감정과 슬픔감정 간에 촉진초점 정도와 자기타당화 정도의 차이, 자기타당화의 정보왜곡 효과를 탐색하여 이론적으로 기여하고 있다. 이에 따라 마케팅 관리자는 소비자의 자사의 제품에 대한 사고 확신성을 높여 소비자의 자기타당화를 촉진하고, 또 촉진초점을 유도하기 위해 사전에 소비자가 행복감정을 느끼도록 해야 함을 관리적 시사점으로 제시하였다.