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Jejunal haemorrhage syndrome (JHS) is a sporadic and fatal enterotoxaemic disease in dairy cows associated with acute development and poor prognosis despite treatment. A 5-year-old Holstein cow with no reported pregnancy, three calving numbers, and 303 days in milk presented with hypothermia, discomfort, and inappetence. Anaemia, dehydration, faeces with blood clots, and absence of rumen and bowel movements were observed. We identified the presence of neutrophilia, hyperglycaemia, hypoproteinaemia, azotaemia, hyperlactatemia, hypocalcaemia, hypermagnesemia, hypokalaemia, and hypochloraemia through blood analyses. Necropsy and histopathologic examination revealed a dilated bluish-purple jejunum, blood clots within the jejunum, neutrophil infiltration into the submucosa of the jejunum, and vascular necrosis. Retrospective examination revealed extraordinary patterns of rumination time, activity, rumen mobility, and rumen temperature using biosensors and decreased milk yield. The abnormalities in the affected cow were detected before recognition by farm workers. To the best of our knowledge, this is the first report to examine data from biosensors in a cow with JHS. Our findings suggest that using biometric data may help understand the development of JHS. [ABSTRACT FROM AUTHOR]

In mammals, genomic imprinting operates via gene silencing mechanisms. Although conservation of the imprinting mechanism at the H19/IGF2 locus has been generally described in pigs, tissue-specific imprinting at the transcript level, monoallelic-to-biallelic conversion, and spatio-temporal chromatin reorganization remain largely uninvestigated. Here, we delineate spatially regulated imprinting of IGF2 transcripts, age-dependent hepatic mono-to biallelic conversion, and reorganization of topologically associating domains at the porcine H19/IGF2 locus for better translation to human and animal research. Whole-genome bisulfite sequencing (WGBS) and RNA sequencing (RNAseq) of normal and parthenogenetic porcine embryos revealed the paternally hypermethylated H19 differentially methylated region and paternal expression of IGF2. Using a polymorphism-based approach and omics datasets from chromatin immunoprecipitation sequencing (ChIP-seq), whole-genome sequencing (WGS), RNAseq, and Hi-C, regulation of IGF2 during development was analyzed. Regulatory elements in the liver were distinguished from those in the muscle where the porcine IGF2 transcript was monoallelically expressed. The IGF2 transcript from the liver was biallelically expressed at later developmental stages in both pigs and humans. Chromatin interaction was less frequent in the adult liver compared to the fetal liver and skeletal muscle. The duration of genomic imprinting effects within the H19/IGF2 locus might be reduced in the liver with biallelic conversion through alternative promoter usage and chromatin remodeling. Our integrative omics analyses of genome, epigenome, and transcriptome provided a comprehensive view of imprinting status at the H19/IGF2 cluster. [ABSTRACT FROM AUTHOR]

Objective: Transgenic hens hold a great promise to produce various valuable proteins. Through virus transduction into stage X embryo, the transgene expression under the control of constructed chicken ovalbumin promoters has been successfully achieved. However, a validation system that can evaluate differently developed ovalbumin promoters in in vitro, remains to be developed. Methods: In the present study, chicken oviduct epithelial cells (cOECs) were isolated from oviduct tissue and shortly cultured with keratinocyte complete medium supplemented with chicken serum. The isolated cells were characterized with immunofluorescence, western blot, and flow cytometry using oviduct-specific marker. Chicken mutated ovalbumin promoter (Mut-4.4-kb-pOV) was validated in these cells using luciferase reporter analysis. Results: The isolated cOECs revealed that the oviduct-specific marker, ovalbumin protein, was clearly detected by immunofluorescence, western blot, and flow cytometry analysis revealed that approximately 79.40% of the cells contained this protein. Also, luciferase reporter analysis showed that the constructed Mut-4.4-kb-pOV exhibited 7.1-fold (p<0.001) higher activity in the cOECs. Conclusion: Collectively, these results demonstrate the efficient isolation and characterization of cOECs and validate the activity of the constructed ovalbumin promoter in the cultured cOECs. The in vitro validation of the recombinant promoter activity in cOECs can facilitate the production of efficient transgenic chickens for potential use as bioreactors. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the pancreatic differentiation potential of α-1,3-galactosyltransferase knockout (GalTKO) pig-derived bone marrow-derived mesenchymal stem cells (BM-MSCs) using epigenetic modifiers with different pancreatic induction media. Methods: The BM-MSCs have been differentiated into pancreatic β-like cells by inducing the overexpression of key transcription regulatory factors or by exposure to specific soluble inducers/small molecules. In this study, we evaluated the pancreatic differentiation of GalTKO pig-derived BM-MSCs using epigenetic modifiers, 5-azacytidine (5-Aza) and valproic acid (VPA), and two types of pancreatic induction media - advanced Dulbecco's modified Eagle's medium (ADMEM)-based and N2B27-based media. GalTKO BM-MSCs were treated with pancreatic induction media and the expression of pancreas-islets-specific markers was evaluated by real-time quantitative polymerase chain reaction, Western blotting, and immunofluorescence. Morphological changes and changes in the 5'-C-phosphate-G-3' (CpG) island methylation patterns were also evaluated. Results: The expression of the pluripotent marker (POU class 5 homeobox 1 [OCT4]) was upregulated upon exposure to 5-Aza and/or VPA. GalTKO BM-MSCs showed increased expression of neurogenic differentiation 1 in the ADMEM-based (5-Aza) media, while the expression of NK6 homeobox 1 was elevated in cells induced with the N2B27-based (5-Aza) media. Moreover, the morphological transition and formation of islets-like cellular clusters were also prominent in the cells induced with the N2B27-based media with 5-Aza. The higher insulin expression revealed the augmented trans-differentiation ability of GalTKO BM-MSCs into pancreatic β-like cells in the N2B27-based media than in the ADMEM-based media. Conclusion: 5-Aza treated GalTKO BM-MSCs showed an enhanced demethylation pattern in the second CpG island of the OCT4 promoter region compared to that in the GalTKO BM-MSCs. The exposure of GalTKO pig-derived BM-MSCs to the N2B27-based microenvironment can significantly enhance their trans-differentiation ability into pancreatic β-like cells. [ABSTRACT FROM AUTHOR]

Mitochondria are necessary for the transition from oocyte to embryo and for early embryonic development. Mitofusin 1 is the main mediator of mitochondrial fusion and homeostasis. We investigated Mitofusin 1 expression levels in porcine somatic cell nuclear transfer (SCNT) embryos. The rate of blastocyst formation in SCNT embryos was reduced significantly compared with that of parthenogenetic activation embryos. SCNT embryos showed significantly decreased Mitofusin 1 expression and mitochondrial membrane potential, while exhibiting increased reactive oxygen spe-cies and apoptosis. Mitochondrial functional changes were observed in the SCNT embryos and may be correlated with low levels of Mitofusin 1 to negatively affect development. [ABSTRACT FROM AUTHOR]

It is very difficult to get the information about semen quality analysis in transgenic pigs because of limited numbers and research facilities. Therefore, in the present study, we analyzed the semen quality of transgenic boars generated for xenotransplantation research. Briefly, the semen samples were collected from 5 homozygous α1,3-Galactosyltransferase knock-out (GalT-/-) transgenic boars and immediately transported to the laboratory. These semen samples were decupled with DPBS and conducted to analyze semen parameters by a computer-assisted semen analysis (CASA) system. The boar semen were examined all 12 parameters such as total motility (TM), curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), and hyperactivated (HYP), etc. In results, among the 5 GalT-/- boars, three boars (#134, 144, and 170) showed normal range of semen parameters, but #199 and 171 boars showed abnormal ranges of semen parameters according to standard ranges of semen parameters. Unfortunately, #171 boar showed azoospermia symptom with rare sperm counts in the original semen. Conclusively, assessment of semen parameters by CASA system is useful to pre-screening of reproductively healthy boar prior to natural mating and artificial insemination for multiplication and breeding. [ABSTRACT FROM AUTHOR]

In some human cancer cells, PTEN (phosphatase and tensin homolog deleted on chromosome 10) is known to regulate autophagy induction positively through the inhibition of PI3K/AKT pathway, leading to the activation of mTOR, a major negative regulator of autophagy. Recent studies reported that PTEN expression is abnormally decreased in endometriotic lesions. In endometriosis, abnormal PTEN expression may contribute to the alteration of endometrial cell autophagy, which may affect apoptosis because endometrial cell autophagy is directly involved in the regulation of apoptosis. To test this hypothesis, we evaluated the involvement of PTEN in the regulation of autophagy induction in human normal endometrial stromal cells (NESCs). In addition, we sought to determine whether aberrant PTEN expression in endometriotic cyst stromal cells (ECSCs) is associated with autophagy dysregulation, and a subsequent decrease in apoptosis. Our results show that PTEN expression was enhanced by progesterone treatment in NESCs. Subsequently, autophagy and apoptosis induction increased through the inhibition of AKT and mTOR activity. This progesteroneinduced increase in apoptosis was reversed by the inhibition of autophagy induction using either mifepristone (progesterone receptor modulator) or PTEN inhibitor. In contrast, progesterone had no significant effects on PTEN expression, AKT, mTOR activity, autophagy or apoptosis in ECSCs. Furthermore, in contrast to normal eutopic endometrium, endometriotic tissues have constant PTEN expression, autophagy and apoptosis throughout the menstrual cycle. In conclusion, our results suggest abnormal PTEN expression in response to progesterone was observed in ECSCs, which led to the dysregulation of autophagy induction via AKT/mTOR signalling and a subsequent decrease in apoptosis. [ABSTRACT FROM AUTHOR]

Identification of tissue- and stage-specific gene promoters is valuable for delineating the functional roles of specific genes in genetically engineered animals. Here, through the comparison of gene expression in different tissues by analysis of a microarray database, the intestinal specificity of mucin 2 (MUC2) expression was identified in mice and humans, and further confirmed in chickens by RT-PCR (reverse transcription-PCR) analysis. An analysis of cis-acting elements in avian MUC2 gene promoters revealed conservation of binding sites, within a 2.9 kb proximal promoter region, for transcription factors such as caudal type homeobox 2 (CDX2), GATA binding protein 4 (GATA4), hepatocyte nuclear factor 4 α (HNF4A), and transcription factor 4 (TCF4) that are important for maintaining intestinal homeostasis and functional integrity. By generating transgenic quail, we demonstrated that the 2.9 kb chicken MUC2 promoter could drive green fluorescent protein (GFP) reporter expression exclusively in the small intestine, large intestine, and ceca. Fluorescence image analysis further revealed GFP expression in intestine epithelial cells. The GFP expression was barely detectable in the embryonic intestine, but increased during post-hatch development. The spatiotemporal expression pattern of the reporter gene confirmed that the 2.9 kb MUC2 promoter could retain the regulatory element to drive expression of target genes in intestinal tissues after hatching. This new transgene expression system, using the MUC2 promoter, will provide a new method of overexpressing target genes to study gene function in the avian intestine. [ABSTRACT FROM AUTHOR]

CD26, also known as Dipeptidyl peptidase IV (DPP-4), is a cell surface glycoprotein that belongs to the serine protease family and has wide spread organ distribution throughout the body. CD26 was previously characterized in immune cells but also has important metabolic functions which are not yet fully understood. Thus, we investigated the effect of CD26 in porcine parthenogenetic embryos. We attempted CD26 downregulation of porcine embryos by siRNA, and evaluated CD26 suppression of developmental competencies. Although the porcine embryos injected with CD26 siRNA were able to develop to the early stage, these embryos were decreased to form blastocysts. Our results indicated that CD26 is one of factors for the regulation of development of porcine embryos. [ABSTRACT FROM AUTHOR]

Although piglets have been delivered by embryo transfer (ET) with in vitro produced (IVP) embryos and blastocysts, a success rate has still remained lower level. Unlike mouse, human, and bovine, it is difficult to a production of piglets by in vitro fertilization (IVF) because of an inappropriate in vitro culture (IVC) system in pig. Therefore, the present study was conducted to investigate whether minimized exposure time in IVC can improve the pregnancy and delivery rates of piglets. Immediately after IVM, the oocytes were denuded and co-incubated with freshly ejaculated boar semen for 3.5 to 4 hours at 38.5 ˚C under 5% CO2 in air. To avoid long-term exposure to in vitro state, we emitted IVC step after IVF. After that the presumptive zygotes were transferred into both oviducts of the surrogate on the same day or 1 day after the onset of estrus. Pregnancy was diagnosed on day 28 after ET and then was checked regularly every month by ultrasound examination. The 3 out of 4 surrogates were determined as pregnant (75%) and a total of 5 piglets (2 females and 3 males) were delivered at 118.3 ± 2.5 days of pregnancy period. In conclusion, a short-term exposure time may be an important factor in the production of IVP-derived piglets. It can be apply to the in vitro production system of transgenic pig by IVF, cloning, and pronuclear microinjection methods. [ABSTRACT FROM AUTHOR]

To compensate for the critical shortage of human organs for allotransplantation, xenotransplantation studies using genetically modified pigs are being performed in Korea. Two types of pigs that are used are α1,3-galactosyltransferase gene knockout (GalT KO) pigs and GalT KO+hCD46 (human complement regulatory protein) pigs. The present study measured the gestation time, birth weight, daily growth rate, and heart weight of both kinds of transgenic minipigs. The gestation period for both types of pigs was 117∼119 days. There was no difference in the body weight of GalT KO (—/+) and GalT KO (—/—) piglets, but GalT KO+hCD46 (—hCD46+/+) pigs were significantly heavier at birth than were GalT KO+hCD46 (—hCD46+/—hCD46+) pigs. During the first 10 weeks of life, the daily weight gain of GalT KO+hCD46 (—hCD46+/—CD46+) piglets, which are considered the optimal type for xenotransplantation, was 0.19 kg. The weight of hearts from GalT KO piglets up to two months of age was affected more by body weight than by age. Transgenic pigs showed no differences in gestation period or reproductive ability compared with normal pigs. These results comprise basic data that may be used in xenotransplantation studies and transgenic animal production in Korea. [ABSTRACT FROM AUTHOR]

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas9) system can be applied to produce transgenic pigs. Therefore, we applied CRISPR/Cas9 system to generate FoxN1-targeted pig parthenogenetic embryos. Using single guided RNA targeted to pig FoxN1 genes was injected into cytoplasm of in vitro matured oocyte before electrical activation. In results, regardless of the concentrations of vector, the cleavage rate were significantly (p<0.05) decreased (4 ng/μl, 51.24%; 8 ng/μl, 40.88%; and 16 ng/μl; 45.22%) compared to no injection group (70.44%). The blastocyst formation rates were also decreased in vector injected 3 groups (4 ng/μl, 7.96%; 8 ng/μl, 6.4%; and 16 ng/μl; 9.04%) compared to no injection group (29.07%). In addition, the blastocyst formation rates between sham injected group (13.51%) and no injection group (29.07%) also showed significant difference (p<0.05). The mutation rates were comparable between groups (4 ng/μl, 18.4%; 8 ng/μl, 12.5%; and 16 ng/μl; 20.0%). The sequencing analysis showed that blastocysts derived from each group were successfully mutated in FoxN1 loci regardless of the vector concentrations. However, the deletion patterns were higher than the patterns of point mutation and insertion regardless of the vector concentrations. In conclusion, we described that cytoplasmic microinjection of FoxN1-targeted CRISPR/Cas9 vector could efficiently generate transgenic pig parthenogenetic embryos in one-step. [ABSTRACT FROM AUTHOR]

Background Somatic cell cloning by nuclear transfer (SCNT) in pig is clearly of great benefit for basic research and biomedical applications. Even though cloned offspring have been successfully produced in pig, SCNT is struggling with the low efficiency. Results In the present study, we investigated differentially expressed proteins of the extraembryonic tissue from pig SCNT fetus compared to control (normal) fetus. We obtained the extraembryonic tissue from embryos at day 35 of pregnancy and examined the protein expression profiles using two-dimensional electrophoresis (2-D) and Western blotting. The extraembryonic tissue of fetus in control pregnancy was compared to the extraembryonic tissue of SCNT fetus, which showed an abnormally small size and shape as well as exhibited abnormal placental morphology compared to control fetus. A proteomic analysis showed that the expression of 33 proteins was significantly increased or decreased in the extraembryonic tissue of SCNT fetus compared to control fetus. The differentially expressed proteins in the extraembryonic tissue of SCNT fetus included ATP or lipid binding proteins, antioxidant proteins, translation elongation factors, and transcription factors. Western blotting analysis indicated that antioxidant enzymes and anti-apoptotic proteins were down-regulated; however, the expression levels of apoptotic proteins, Bax and Hsp27, were increased in the extraembryonic tissue of SCNT fetus. Moreover, immunohistochemical analysis also showed that the expression of the catalase or GPX genes was decreased in the extraembryonic tissue with SCNT fetus compared to those with control fetus. In addition, we observed a significant decrease in DNA methytransferase1 (Dnmt1) expression in SCNT extraembryonic tissue, and the expression levels of Dnmt3a and Dnmt3b were abnormally higher in SCNT fetus compared to control fetus. Moreover, a marked increase in the frequency of TUNEL-positive cells was observed in the extraembryonic tissue in SCNT fetus. Conclusion These results demonstrated that pig SCNT fetus showed abnormal protein expression in the extraembryonic tissue, and extensive apoptosis occurred in the extraembryonic tissue of the SCNT fetus due to an increase in apoptotic protein expression or a decrease in antioxidant protein expression. [ABSTRACT FROM AUTHOR]

Background: Mammalian spermatozoa must undergo capacitation, before becoming competent for fertilization. Despite its importance, the fundamental molecular mechanisms of capacitation are poorly understood. Therefore, in this study, we applied a proteomic approach for identifying capacitation-related proteins in boar spermatozoa in order to elucidate the events more precisely. 2-DE gels were generated from spermatozoa samples in before- and after-capacitation. To validate the 2-DE results, Western blotting and immunocytochemistry were performed with 2 commercially available antibodies. Additionally, the protein-related signaling pathways among identified proteins were detected using Pathway Studio 9.0. Result: We identified Ras-related protein Rab-2, Phospholipid hydroperoxide glutathione peroxidase (PHGPx) and Mitochondrial pyruvate dehydrogenase E1 component subunit beta (PDHB) that were enriched before-capacitation, and NADH dehydrogenase 1 beta subcomplex 6, Mitochondrial peroxiredoxin-5, (PRDX5), Apolipoprotein A-I (APOA1), Mitochondrial Succinyl-CoA ligase [ADP-forming] subunit beta (SUCLA2), Acrosin-binding protein, Ropporin-1A, and Spermadhesin AWN that were enriched after-capacitation (>3-fold) by 2-DE and ESI-MS/MS. SUCLA2 and PDHB are involved in the tricarboxylic acid cycle, whereas PHGPx and PRDX5 are involved in glutathione metabolism. SUCLA2, APOA1 and PDHB mediate adipocytokine signaling and insulin action. The differentially expressed proteins following capacitation are putatively related to sperm functions, such as ROS and energy metabolism, motility, hyperactivation, the acrosome reaction, and sperm-egg interaction. Conclusion: The results from this study elucidate the proteins involved in capacitation, which may aid in the design of biomarkers that can be used to predict boar sperm quality. [ABSTRACT FROM AUTHOR]

Background: Somatic cell nuclear transfer (scNT)-derived piglets have high rates of mortality, including stillbirth and postnatal death. Here, we examined severe malformed umbilical cords (MUC), as well as other organs, from nine scNT-derived term piglets. Results: Microscopic analysis revealed complete occlusive thrombi and the absence of columnar epithelial layers in MUC (scNT-MUC) derived from scNT piglets. scNT-MUC had significantly lower expression levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) and angiogenesis-related genes than umbilical cords of normal scNT piglets (scNT-N) that survived into adulthood. Endothelial cells derived from scNT-MUC migrated and formed tubules more slowly than endothelial cells from control umbilical cords or scNT-N. Proteomic analysis of scNT-MUC revealed significant down-regulation of proteins involved in the prevention of oxidative stress and the regulation of glycolysis and cell motility, while molecules involved in apoptosis were significantly up-regulated. Histomorphometric analysis revealed severe calcification in the kidneys and placenta, peliosis in the liver sinusoidal space, abnormal stromal cell proliferation in the lungs, and tubular degeneration in the kidneys in scNT piglets with MUC. Increased levels of apoptosis were also detected in organs derived from all scNT piglets with MUC. Conclusion: These results suggest that MUC contribute to fetal malformations, preterm birth and low birth weight due to underlying molecular defects that result in hypoplastic umbilical arteries and/or placental insufficiency. The results of the current study demonstrate the effects of MUC on fetal growth and organ development in scNT-derived pigs, and provide important insight into the molecular mechanisms underlying angiogenesis during umbilical cord development. [ABSTRACT FROM AUTHOR]

Evaluates the biological significance of phospholipase c β1 gene mutation in mouse sperm in the acrosome reaction, fertilization and embryo development. Reduction in acrosome reaction rate, fertilization rate and embryo development rate; Increase in the intracellular calcium concentration; Requisite of acrosomal exocytosis.

Examines the expression pattern of cyclin D2 and pseudogene cyclin D2 in mRNA in the human ovary with age. Reverse transcription-polymerase chain reaction technique; Association of increased expression of pseudogene cyclin D2 mRNA with aging.

Investigates the efficacy of electric stimulation before and/or after intracytoplasmic sperm injection on bovine oocyte activation and embryo development. Collection and maturation of oocytes; Preparation of cryopreserved semen; Treatment for oocyte activation; Assessment of pronucleus formation; In vitro development.