From 2020, Korean Animal and Plant Quarantine Agency has reset the withdrawal time (WT) for veterinary drugs typically used in livestock in preparation for the introduction of positive list system (PLS) program in 2024. This study was conducted to reset the MRL for tiamulin (TML) in broiler chickens as a part of PLS program introduction. Forty-eight healthy Ross broiler chickens were orally administered with TML at the concentration of 25 g/L (TML-1, n=24) and 50 g/L (TML-2, n=24) for 5 days through drinking water, respectively. After the drug treatment, tissue samples were collected from six broiler chickens at 1, 2, 3 and 5 days, respectively. According to the previously established analysis method, residual TML concentrations in poultry tissues were determined using LC-MS/MS. In TML-1, TML in all tissues was detected less than LOQ at 2 days after drug treatment. In TML-2, TML in liver and kidney was detected more than LOQ at 2 days after treatment. According to the European Medicines Agency’s guideline on determination of withdrawal periods, withdrawal periods of TML-1 and TML-2 in poultry tissues were established to 0 and 2 days, respectively. In conclusion, the estimated WT of TML in poultry tissues is shorter than the current WT recommendation of 5 days for TML in broiler chickens.

This study was conducted to reset the maximum residue limit (MRL) for didecyldimethylammonium chloride (DDAC) in broiler chickens. The disinfectant containing DDAC (10%, w/w) was diluted 160 times and evenly sprayed on the bodies of twenty-four broiler chickens at a rate of 15 mL per day per bird for 7 days. After the disinfectant treatment, tissue samples were collected from six broiler chickens at 0.25, 1, 3 and 5 days, respectively. Residual DDAC concentrations in poultry tissues were determined using LC-MS/MS. Correlation coefficient (0.99 >), the limits quantification (LOQ, 2.0~10.0 μg/kg), recoveries (86.9~118.6%), and coefficient of variations (<19.98%) were satisfied the validation criteria of Korean Ministry of Food and Drug Safety. In all tissues except for liver, DDAC was detected more than LOQ at 5 days after the disinfectant treatment. In liver tissues, DDAC was detected more than LOQ at 3 days after treatment. According to the European Medicines Agency’s guideline on determination of withdrawal periods, withdrawal period of DDAC in poultry tissues was established to 26 days. In conclusion, the developed analytical method is sensitive and reliable for detecting DDAC in poultry tissues. When DDAC disinfectant is sprayed on a poultry house in the presence of broiler chickens, it is necessary to keep the disinfectant from contacting the body of the livestock.

The honey bee, Apis mellifera, has a defense system, including detoxification, antioxidation, and immunity pathways, against external stimulation such as chemicals, stress, and pathogens. However, pesticides, particularly neonicotinoids and butenolids, have been recently reported to alter physiological changes in honey bee. In this study, we investigated the expression levels of eight genes categorized into detoxification (CYPQ3), antioxidation (CAT and SOD2), and immune system (Abaecin, Apidaecin, Defensin1, Defensin2, and Hymenoptaecin), in five tissues (Head, Thorax, Gut, Fat body, and Carcass) of honey bee treated with three pesticides (Acetamiprid, Imidacloprid, and Flupyradifurone) using quantitative real-time PCR. Gene expression patterns was varied depending on the type of pesticides and tissues. However, among eight genes, the expression levels of CYPQ3 was notably induced, but those of AMPs were generally reduced by all pesticides tested in this study in five tissues. These suggest that CYPQ3-mediated detoxification pathway is induced, but AMP-mediated immune system might be disrupted when honey bee is exposed to neonicotinoids and butenolid.

This study investigated ethopabate (EPB) residues in edible tissues of broiler chickens given in drinking water and established the withdrawal time (WT) of EPB in poultry tissues. Twenty-four healthy Ross broiler chickens were orally administered with EPB at the concentration of 3.8 mg/L for 14 days (EPB-1, n=24) and 15.2 mg/L for 7 days (EPB-2, n=24) through drinking water, respectively. After the drug treatment, tissue samples were collected from six broiler chickens at 0, 1, 3, and 5 days, respectively. EPB residue concentrations in poultry tissues were determined using LC-MS/MS. Correlation coefficient values ranged from 0.9980 to 0.9998, and the limits of detection and quantification (LOQ) were 0.03~0.09 and 0.1~0.3 μg/kg, respectively. Mean recoveries in muscle, liver, kidney and skin/fat tissues were 95.9~109.8, 108.7~115.3, 89.9~96.6 and 86.7~96.8%, respectively, and coefficient of variations were less than 17.11%. At the end of the drug-administration period (0 day), EPB was detected at levels under the LOQ in all tissues from both the EPB-1 and EPB-2 groups. According to the results of EPB residue in Ross broiler tissues, withdrawal periods of both EPB-1 and EPB-2 in poultry tissues were established to 0 day. In conclusion, the developed analytical method is suitable for the detection of EPB in poultry tissues, and the estimated WT of EPB in poultry tissues will contribute to ensuring the safety of Ross broiler chickens.

From 2020, Korean Animal and Plant Quarantine Agency has reset the withdrawal time (WT) for veterinary drugs typically used in livestock in preparation for the introduction of positive list system (PLS) program in 2024. This study was conducted to reset the MRL for amprolium (APL) in broiler chickens as a part of PLS program introduction. Forty-eight healthy Ross broiler chickens were orally administered with APL at the concentration of 60 mg/L (APL-1, n=24) for 14 days and 240 mg/L (APL-2, n=24) for 7 days through drinking water, respectively. After the drug treatment, tissue samples were collected from six broiler chickens at 0, 1, 3 and 5 days, respectively. Residual APL concentrations in poultry tissues were determined using LC-MS/MS. Correlation coefficient (0.99 >), the limits quantification (LOQ, 0.3~5.0 μg/kg), recoveries (81.5~112.4%), and coefficient of variations (<15.5%) were satisfied the validation criteria of Korean Ministry of Food and Drug Safety. In APL-1, APL in all tissues except for kidney was detected less than LOQ at 3 days after drug treatment. In APL-2, APL in liver and kidney was detected more than LOQ at 5 days after treatment. According to the European Medicines Agency’s guideline on determination of withdrawal periods, withdrawal periods of APL-1 and APL-2 in poultry tissues were established to 3 and 2 days, respectively. In conclusion, the developed analytical method is sensitive and reliable for detecting APL in poultry tissues. The estimated WT of APL in poultry tissues is longer than the current WT recommendation of 2 days for APL in broiler chickens.

양돈산업은 돼지고기 내 웅취발생 예방을 위한 방법으 로 수컷 자돈에게 물리적 거세를 관행적으로 실시해왔다. 그러나 동물복지에 대한 관심이 고조됨에 따라 일부 국가 들에서는 고통 최소화 물리적 거세법 권장 또는 대체 방 법에 대해 연구를 수행하고 있다. 따라서 본 연구에서는 비거세돈(EM; entire male pigs) 및 거세돈(CM; castrated male pigs)에서 이눌린의 급여가 지방조직 내 웅취물질 농도에 미치는 영향을 구명하고자 실시하였다. 시험동물 은 총 26두의 3원 교잡돈(EM, n=18; CM, n=8)을 사용하 였다. 시험 처리구는 3% 이눌린의 급여와 비급여를 설정 하여 총 4개 처리구(EM0, EM3, CM0 및 CM3)로 구성하 였다. 웅취분석을 위해 지방조직은 등지방, 목지방 및 삼 겹지방을 수집하였다. 등지방 내 인돌 농도는 EM0에 비 해 CM3에서 감소하였고(p<0.05) 목지방 및 삼겹지방에서 는 처리구간 차이가 없었다. 지방조직 내 평균 인돌 농도 는 EM0에 비해 EM3, CM0 및 CM3에서 감소하였다 (p<0.05). 등지방 내 스카톨 농도는 CM0, CM3에서 EM0 보다 감소하였다(p<0.05). 또한, 목지방 내 스카톨 농도 및 지방조직 내 평균 스카톨 농도는 CM3에서 EM0과 EM3 보다 감소가 확인되었다(p<0.05). 그러나 안드로스테논 농 도는 분석된 지방조직들 모두에서 처리구간 통계적인 차 이가 없다. 이러한 결과들은 이눌린 급여에 의해 비거세 돈의 지방 내 웅취물질 저감에 부분적인 효과를 나타냈 을 뿐만 아니라 거세돈에서도 웅취물질 감소에 긍정적인 영향을 미쳤음을 시사한다. 따라서, 본 연구결과는 물리적 인 거세가 전면 금지된다면 동물복지형 사양관리 기술개 발의 기초자료로 활용할 수 있을 것이다.

Intermuscular fat is essential for enhancing the flavor and texture of cultured meat. Mesenchymal stem cells derived from intermuscular adipose tissues are a source of intermuscular fat. Therefore, as a step towards developing a platform to derive intermuscular fat from mesenchymal stem cells (MSCs) for insertion between myofibrils in cultured beef, an advanced protocol of intermuscular adipose tissue dissociation effective to the isolation of MSCs from intermuscular adipose tissues was developed in cattle. To accomplish this, physical steps were added to the enzymatic dissociation of intermuscular adipose tissues, and the MSCs were established from primary cells dissociated with physical step-free and step-added enzymatic dissociation protocols. The application of a physical step (intensive shaking up) at 5 minutes intervals during enzymatic dissociation resulted in the greatest number of primary cells derived from intermuscular adipose tissues, showed effective formation of colony forming units-fibroblasts (CFU-Fs) from the retrieved primary cells, and generated MSCs with no increase in doubling time. Thus, this protocol will contribute to the stable supply of good quality adipose-derived mesenchymal stem cells (ADMSCs) as a fat source for the production of marbled cultured beef.

Tuberculosis is a potentially deadly infectious disease caused by the Mycobacterium tuberculosis (M. tuberculosis). Tuberculosis is diagnosed by proving the M. tuberculosis in sputum samples based on the results of acid-resistant staining, culture, and nucleic acid amplification tests. However, there is a report that the detection rate of M. tuberculosis is low in acid-resistant staining using tissue specimens. It has been suspected that the cause is a potential loss of acid resistance by the organic solvents used for tissue specimen preparation. Therefore, this study was pursued to find out if Gram staining and fluorescent staining in addition to acid-resistant staining would be helpful in diagnosing tuberculosis. We used four tissue (lung, small intestine, large intestine, and lymph node) samples with chronic granulomatous inflammation observed in HE staining and positive results in real-time PCR. These detection rates and staining properties were investigated through microscopic examination using the Ziehl-Neelsen, Gram, and Auramin rhodamine staining. In this studies, M. tuberculosis were observed by Ziehl-Neelsen, Gram, and Auramin rhodamine staining in all four samples. In the evaluation of clinical microbiology proficiency testing (CMPT), the Ziehl-Neelsen and Gram staining were the same result, but the Auramin rhodamine staining was relatively low. These data indicated that Gram staining is useful for detecting M. tuberculosis in formalin-fixed tissue specimens. Therefore, if the Ziehl-Neelsen and Gram staining are combined as the M. tuberculosis staining method in tissue specimens, a better direction may be provided for tuberculosis diagnosis.

This study aims to establish a modified analytical method with sensitivity and reliability for streptomycin (STP) and dihydrostreptomycin (DHS) of residues level in pig tissues, plasma and urine by LC-MS/MS on the basis of previous studies. The mass parameters of quantitative and qualitative ions for STP and DHS were optimized using multiple reaction monitoring in positive mode. The separation of compounds was conducted using BEH Amide column according to material’s characteristics. The analytes in plasma were extracted with only organic solvents. In muscle and kidney, KH2PO4 buffer solution containing 2% CCl3COOH and EDTA-Na was used as extraction solvent. The WCX cartridege was selected as SPE cartridge in considering high recoveries for STP and DHS. The analytes in urine were extracted by organic solvents with acid and addition of EDTA. The limits of detection (LODs) in STP and DHS ranged 0.45~3.66 μg/kg and 0.22~0.78 μg/kg, respectively. The limits of quantification (LOQs) were 1.35~11.10 μg/kg in STP and 0.66~2.36 μg/kg in DHS. The recoveries (%) were 94.29~104.5% in STP and 92.32~108.45% in DHS except for plasma with lower values (61.45/68.5%, respectively). In the precision evaluation, the coefficient of variation (CV, %) of STP showed <10.50% on intra-day and <18.04% on inter-day. The CV (%) of DHS showed <8.42% on intra-day, whereas <17.98% on inter-day. The modified method is reliable for continuous residual monitoring in pig to ensure food safety for consumer’s health. In addition, this method could be used in study relation to residue depletion and pharmacokinetics of veterinary drug.

Cryopreservation of porcine ovarian tissue by vitrification method is a promising approach to preserve genetic materials for future use. However, information is not enough and technology still remains in a challenge stage in pig. Therefore, the objective of present study was to determine possibility of vitrification method to cryopreserve porcine ovarian tissue and to confirm an occurrence of cryoinjuries. Briefly, cryoinjuries and apoptosis patterns in vitrified-warmed ovarian tissue were examined by histological evaluation and TUNEL assay respectively. In results, a damaged morphology of oocytes was detected among groups and the rate was significantly (p < 0.05) lower in vitrification group (25.8%) than freezing control group (67.7%), while fresh control group (6.6%) showed significantly (p < 0.05) lower than both groups. In addition, cryoinjury that form a wave pattern of tissues around follicles was found in the frozen control group, but not in the fresh control group as well as in the vitrification group. Apoptotic cells in follicle was observed only in freezing control group while no apoptotic cell was found in both fresh control and vitrification. Similarly, apoptotic patterns of tissues not in follicle were comparable between fresh control and vitrification groups while freezing control group showed increased tendency. Conclusively, it was confirmed that vitrification method has a prevention effect against cryoinjury and this method could be an alternative approach for cryopreservation of genetic material in pigs. Further study is needed to examine the viability of oocytes derived from vitrified-warmed ovarian tissue.

국내외에서 80여 종이 넘는 담수 및 해산어류를 감염시켜 대량폐사를 발생시키는 바이러스성 출혈성 패혈증 바이러스 (VHSV) 진단검사를 위해 넙치의 여러 조직의 바이러스 발현량에 대한 정량적 데이터를 시간순서에 따라 분석하였다. 무작위 선별된 넙치에 3.0E+07 TCID50 per 0.1 mL per fish의 VHSV를 복강 주사하여 시간순서 (0시간, 6시간, 12시간, 1일, 2일, 3일, 5일, 7일)에 따라 조직 (아가미, 간, 신장, 비장, 근육)을 채취하였다. Real-time PCR 법을 통해 상대 정량한 결과 5일차 아가미, 간, 신장, 비장에서 바이러스의 발현량이 가장 높게 나타났다. 이번 연구를 통해 감염 초기단계에서 비장이 VHSV 확정진단을 위한 적정조직임을 입증하였으며, 국내 법정전염병 진단에 중요한 정보를 제공할 것이다.

With the aim of developing region specialized crops, this study was conducted to clarify effects of variant and cultivation region on antioxidative activities in various black soybean (Glycine max L.) seed tissues. Three black soybean varieties (SCEL-1, Wonheug, and Cheongja 3) were each cultivated in 4 different regions (Jeonju, Pyeongchang, Paju, and Cheonan). Harvested seeds were used to assess DPPH and ABTS radical scavenging activity, and total polyphenol, flavonoid and anthocyanin content. SCEL-1 soybean hull contained higher DPPH and ABTS radical scavenging activity (61% and 85% respectively) compared to Wonheug (40% and 50% respectively). SCEL-1 cultivated in Pyeongchang displayed the highest total polyphenol and flavonoid content (1,189 mg GAE/ 100g sample and 951 mg CTE/ 100g sample, respectively). Total anthocyanin content was ranked in the following order: SCEL-1>Wonheug>Cheongja 3. All black soybeans showed much higher antioxidant activity in the soybean hull than in the dehulled soybean. The antioxidant activity of black soybeans cultivated at high latitudes was high. These results suggest that the best black soybean variant for high beneficial biological activities is the SCEL-1 variant. For a complete understanding of the potential of black soybean as functional foods, we plan to further analyze their antioxidant activities in future studies.

Quantitative real-time polymerase chain reaction (RTqPCR) is a rapid and precise method of analysis to quantify the level of gene expression and is widely used in the diagnosis of diseases and quantitative analysis of genes. In RT-qPCR analysis, a reference gene (or housekeeping gene) is used for normalization of experimental results. Since this method of analysis detects a small quantity of the product, it is highly sensitive and it is important for the accuracy and reproducibility of the experiment to select a reference gene suitable for gene expression studies. As the expression levels of the reference gene are affected under different conditions, in order to determine the suitability of the housekeeping gene used as the reference gene, it is necessary to verify the expression stability. In the current study, the stability of the expression of 11 housekeeping genes (B2M, SDHA, GAPDH, RPL13, VIM, EEF1A1, HPRT1, GUSB, RPL19, ACTB, and ABL1) was investigated in the tissues of long-tailed chickens (heart, thigh, and breast). Expression stability evaluation was analyzed with four software: BestKeeper, NormFinder, geNorm, and RefFinder. In our study, GAPDH in heart tissue, HPRT1 in thigh tissue, and RPL13 in breast tissue were selected as the most stable reference genes. Evaluation of the expression stability of housekeeping genes can provide important data in gene expression studies by selecting an appropriate reference gene according to various conditions.

A 210-day experiment was conducted to examine the effects of starvation on survival, the gonadosomatic index (GSI), hepatosomatic index (HSI), and the intestinosomatic index (ISI), and histological changes in the renal tubule epithelium, midgut epithelium, and hepatocytes in Far Eastern catfish (Silurus asotus). The survival rate decreased to 92.2±0.47% in the fed group and 74.4±2.59% in the starved group during the 210-day experimental period. GSI, HSI, and ISI were lowest in the starved group (p<0.05). The hepatocyte nuclear area, hepatocyte cell area, the nuclear height of the midgut epithelium, and the nuclear height of the kidney were highest in the fed group (p<0.05). The hepatocyte nuclear area, nuclear height of the midgut epithelium, and nuclear height of the kidney were lowest in the starved group (p<0.05). The numbers of melano-macrophages (MMs) found in the kidney cells increased during starvation in this species. This suggests that thinner body cavity regions, the contraction of hepatocyte nuclear sites, and the spreading of kidney cell MMs in this species could be used as alternative indicators for identifying starvation conditions. Therefore, the results from our study provide accurate indications of the nutritional status of Far Eastern catfish.

해산송사리, Oryzias dancena 2배체와 유도 3배체 각 조직에서의 세포주기를 비교, 분석하였다. G1, S, G2+M기의 분포 빈도에서 꼬리지느러미인 경우 2배체에서는 85.8%, 7.6% 및 6.9%이었으며 유도 3배체에서는 91.2%, 3.6% 및 5.2%이었다. 간 조직인 경우 2배체에서는 78.4%, 10.6%, 및 11.0%이었으며 유도 3배체는 86.2%, 5.9% 및 7.9%이었다. 아가미 조직인 경우 2배체에서는 79.3%, 9.4% 및 11.3%이었으며 유도 3배체는 85.7%, 5.4% 및 8.9%이었다. 2배체와 유도 3배체에서 조직 간 세포 주기 빈도에서 유의한 차이가 있었다 (p<0.05). Mitosis (체세포분열)은 유도 3배체에 비해 2배체가 더욱 활성이 있었으며, 이러한 체세포분열은 2배체와 유도 3배체 모두에서 꼬리지느러미 조직 보다는 간 조직과 아가미 조직에서 더욱 왕성하였다.

Mesenchymal stem cells (MSCs) are multipotent cells capable of replicating as undifferentiated cells, and have the potential of differentiating into mesodermal lineages. Goats are commonly used as animal models for bone tissue engineering to test the potential of stem cells for bone regeneration. Goat MSCs isolated from bone marrow (BM) or adipose tissue (AD) should be evaluated using in vitro assays, prior to their application in a tissue engineering project. In this study, we compared the stem cell properties of MSCs derived from goat AD, BM and ear skin tissue (ESK). As results, BM and ESK-MSCs exhibited a spindle-shaped morphology comparable to that of AD-MSCs. Especially, BM-MSCs could be cultured for significantly longer periods and exhibited the greatest expansion capacity, whereas AD-MSCs had the shortest culture time and lowest growth rate. Also, we compared differentiation potentials of AD, BM and ESK-MSCs into adipogenic, chondrogenic, and osteogenic lineages through specific staining and quantitative real-time RT-PCR. Collectively, we successfully isolated ESK-MSCs from goat for the first time. This study suggests that adult skin tissue of goat could be used as a source of goat MSCs. Further studies are needed to show the more information for establishment and fully characterization of goat ESK-MSCs.

본 연구는 조직들의 손상과 질병이 어떤 관계에 있지를 탐구하는 글이다. 7개 조직은 인체의 모든 구조를 이루고 있고 여러 기관과 체계와 급소들의 기능을 유지시킨다 또한 조직은 인체의 발달과 영양 상태를 유지하는 데에 매우 중요한 역할을 담당하며 소화의 불의 도움을 받아 조직들의 면역기체를 형성한다. 7개 조직 가운데 하나가 손상되면 이어지는 그 다음 조직에 영향을 미치므로 7개 조직은 순차적인 질서를 가지고 있다 음식물을 소화시켰을 때 얻은 영양 원형질에 모든 조직을 위한 필수 영양물질이 포함되어 있다. 이 영양 원형질은 각 조직에 있는 조직의 소화의 불의 도움으로 변형되어 각 조직에 영양을 공급해 준다. 7개 조직의 변형의 순서는 원형질 조직 적혈구 조직 근육 조직 지방 조직 뼈 조직 골수 조직 생식 조직의 순서이다 이와 같은 변형은 세 가지 기본과정을 통해 이루어진다. 7개 조직의 손상은 인체의 질병의 원인으로 작용한다 조직의 손상은 도샤의 손상과 더불어 인체 건강을 악화시키는데 원형질 조직은 금식 적혈구혈 조직은 제1부 24장에 설명되어 있고 근육 조직은 뜸질 등 제1부 14장에, 지방 조직은 제1부 21장에, 뼈 조직은 5가지 정화요법 골수 조직과 생식 조직은 적절한 양의 도샤와 식이 요법으로 치료할 수 있다. 그러나 본 논문에서 조직들이 손상되었을 때 구체적인 치료법은 지면의 문제로 다루지 않았다 이것은 다음 연구에 기대한다.