Background: Aflatoxin B1 (AFB1) is a toxic metabolite generated by Aspergillus species and is commonly detected during the processing and storage of food; it is considered a group I carcinogen. The hepatotoxic effects, diseases, and mechanisms induced by AFB1 owing to chronic or acute exposure are well documented; however, there is a lack of research on its effects on the intestine, which is a crucial organ in the digestive process. Dogs are often susceptible to chronic AFB1 exposure owing to lack of variation in their diet, unlike humans, thereby rendering them prone to its effects. Therefore, we investigated the effects of AFB1 on canine small intestinal epithelial primary cells (CSIc). Methods: We treated CSIc with various concentrations of AFB1 (0, 1.25, 2.5, 5, 10, 20, 40, and 80 μM) for 24 h and analyzed cell viability and transepithelial-transendothelial electrical resistance (TEER) value. Additionally, we analyzed the mRNA expression of tight junction-related genes (OCLN, CLDN3, TJP1, and MUC2), antioxidant-related genes (CAT and GPX1), and apoptosis-related genes (BCL2, Bax, and TP53). Results: We found a significant decrease in CSIc viability and TEER values after treatment with AFB1 at concentrations of 20 μM or higher. Quantitative polymerase chain reaction analysis indicated a downregulation of OCLN, CLDN3, and TJP1 in CSIc treated with 20 μM or higher concentrations of AFB1. Additionally, AFB1 treatment downregulated CAT , GPX1, and BCL2. Conclusions: Acute exposure of CSIc to AFB1 induces toxicity, and exposure to AFB1 above a certain threshold compromises the barrier integrity of CSIc.

본 연구는 과체중과 정상체중의 비글견 사이에서 미네랄 소화율의 차이를 평가하기 위해 수행되었다. 중성화 된 건강한 비글견 11마리(47.7개월령 ±0.14)의 Body condition score (BCS)를 기준으로 Normal BCS 그룹(BCS≤5, n=5)과 High BCS 그룹(BCS ≥6, n=6)의 두 그룹에 배치하였다. 시험사료는 반려견의 영양소 요구량을 충족하도록 제조하여 개체 별 에너지요구량에 맞춰 일일 2회에 나누어 14일 동안 급여하였다. 미네랄의 외관상 전장 소화율은 0.5% 산화크롬(Cr2O3)을 이용한 지시제법을 이용하여 평가되었다. 사료와 분변 내 Macro 미네랄(K, Mg, P, Na, Ca)과 Micro 미네랄(Se, Fe, Zn, Cu, Mn)의 함량은 유도결합플라즈마 분광광도계를 이용하여 분석하였다. 체중과 BCS는 Normal BCS 그룹 보다 High BCS 그룹이 유의하게 높았으며(p<0.01), 시험기간 동안 두 그룹 내 각각의 체중과 BCS는 통계적으로 유의한 변화는 관찰되지 않았다(p>0.05). 미네랄 소화율을 평가한 결과, Macro 미네랄 중에서는 마그네슘, 인, 칼슘의 소화율이 High BCS 그룹에서 높은 경향으로 관찰되었으며(p<0.1), Micro 미네랄 중에서는 High BCS 그룹에서 망간의 소화율이 유의하게 높았고(p<0.05), 셀레늄과 아연의 소화율은 높은 경향(p<0.1)을 나타내었다. 또한, 통계적인 유의성과 관계없이 분석한 모든 미네랄 지표에서 High BCS 그룹이 Normal BCS 그룹보다 높은 소화율의 결과를 보여 주었다. 본 연구의 결과는 비글견에서 과체중 또는 비만이 미네랄 소화율의 변화를 유도할 잠재적 가능성을 보여주었다.

본 연구는 위장 단계의 소화과정에 관여하는 Gastric lipase (GL)를 반려견을 위한 정적 체외 소화모델(Static in vitro digestion model)에 적용을 검토하기 위하여 실시되었다. GL의 첨가가 체외 소화과정 동안 건물(Dry matter; DM), 조단백질(Crude protein; CP) 그리고 조지방(Ether extracts; EE) 소화율에 미치는 영향을 평가하였다. GL은 위장 소화단계에서 첨가되었다. 위장(39℃, 2 hr.)과 소장(39℃, 4 hr.) 소화 후에 비소화 분획을 분리하였다. 그리고 실험사료와 분리된 비소화 분획에서 DM, CP 그리고 EE 수준을 측정하고 각각의 소화율을 계산하였다. 위장과 소장 소화단계에서 측정된 DM, CP 그리고 EE 소화율은 Control과 GL 그룹 사이에서 통계적으로 유의한 차이는 관찰되지 않았다(p>0.05). 결과적으로 우리의 체외 소화모델에서 GL의 첨가는 DM, CP 그리고 EE의 소화율에는 영향을 미치지 않는 것으로 나타났다. 따라서 이와 같은 결과는 정적 체외 소화모델을 이용한 소화율의 평가에 있어서 GL의 역할은 다소 제한적일 수 있다는 것을 시사한다.

The mesenchymal stem cells (MSC) has been investigated as a source of stem cell therapy to replace and treat damaged cells. Human endometrial epithelial and stromal cells was isolated from hysterectomy tissue and the direct evidence of stem/progenitor cells in the human endometrium was identified. Endometrium derived stem cells (EnMSCs) are known to have a high proliferative ability, genetic stability, lack of tumorigenicity and low immnunogenicity during long-term cultivation. Here, we aimed to identify MSC in canine endometrium and characterize its potential to differentiate into decidua cells. EnMSCs were isolated from thrown-away spayed uterus of adult canine depending on their estrus cycle, and identified by flow cytometry, immunocytochemistry and flow cytometry with MSC specific markers. We then characterized the ability of EnMSCs by the doubling-time analysis, colony-forming units and MSC differentiation assays. Isolated EnMSCs expressed stem cell specific genes (Sox2, Oct4, Nanog, MCAM, Endoglin, Susd2 and IGTB) and MSC surface markers (CD90, CD44 and CD117). EnMSCs are also differentiated into adipogenic, osteogenic and chondrogenic cells morphologically under modified conditions with the expression of lineage specific genetic markers. EnMSCs showed higher proliferation ability than canine amniotic fluid derived MSCs which were used as a positive control. EnMSCs were cultured at low density (10, 20, cells/cm2) and initiated to form small colonies of loosely-arranged cells and gradually formed large colonies of densely-packed cells which underwent self-renewal with high proliferative potential which is similar to the clonogenicity feature of human endometrium-derived stem cells. EnMSCs were then induced to differentiate into decidua cells with 0.5 mM dbcAMP. After 14 days, EnMSCs changed their morphology into the elongated and rounded shape. The induced decidual cells expressed PRL and IGFBP1 which are typically expressed in decidua cells. In conclusion, we successfully isolated and characterized MSC in the canine endometrium which differentiated into decidua cells. These results showed that endometrium may be a promising source of stem cells, and furthermore raise the possibility of canine EnMSCs as a novel hypothetical decidualisation model of infertility associated with decidualisation insufficiency and implantation failure.

Somatic cell nuclear transfer (SCNT) has been considered for preserving genetically valuable or endangered animals. Sapsaree is a Natianal Monument in Korea to maintian a pure pedigree. The aim of this study was to produce azoospermia Sapsaree using SCNT and identify normal reproductive ability of cloned azoospermia Sapsaree. Ear skin biopsy was performed on a thirteen-year-old azoospermia Sapsaree and ear skin fibroblasts were isolated for SCNT as donor cells. The fibroblasts were injected into enucleated in vivo matured oocytes, the couplets were electrical fused by two pulse of direct current (55 V for 15 μs) using titanium and platinum fusion needle and activated by calcium ionophore. Cloned embryos were surgically transferred into oviducts of natuarally estrus cycle synchronized recipient dogs. The fusion rate of platinum needle was 70%, which was higher than those of titanium needle (64.1%). Developmental rate to the 8 cells and 10 cell stages was higher in platinum needle group (24% and 16%, respectively) than those of platinum needle group (14.8% and 3.1%, respectively). Total 35 SCNT embryos were transferred into oviducts of 3 recipient dogs and one recipient finally delivered a puppy by caesarean section. As results, this study demonstrated that platinum fusion needle could be successfully make the reconstructed embryos and improve the efficiency of canine SCNT. Cloning azoospermia Sapsaree may contribute to conserve genetically valuable and unique pedigree. And further study should be confirm whether cloned live dog is azoospermia.

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.