Retrospective study using biosensor data of a milking Holstein cow with jejunal haemorrhage syndrome.

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.

A functional regulatory variant of MYH3 influences muscle fiber-type composition and intramuscular fat content in pigs.

Muscle development and lipid accumulation in muscle critically affect meat quality of livestock. However, the genetic factors underlying myofiber-type specification and intramuscular fat (IMF) accumulation remain to be elucidated. Using two independent intercrosses between Western commercial breeds and Korean native pigs (KNPs) and a joint linkage-linkage disequilibrium analysis, we identified a 488.1-kb region on porcine chromosome 12 that affects both reddish meat color (a*) and IMF. In this critical region, only the MYH3 gene, encoding myosin heavy chain 3, was found to be preferentially overexpressed in the skeletal muscle of KNPs. Subsequently, MYH3-transgenic mice demonstrated that this gene controls both myofiber-type specification and adipogenesis in skeletal muscle. We discovered a structural variant in the promotor/regulatory region of MYH3 for which Q allele carriers exhibited significantly higher values of a* and IMF than q allele carriers. Furthermore, chromatin immunoprecipitation and cotransfection assays showed that the structural variant in the 5'-flanking region of MYH3 abrogated the binding of the myogenic regulatory factors (MYF5, MYOD, MYOG, and MRF4). The allele distribution of MYH3 among pig populations worldwide indicated that the MYH3 Q allele is of Asian origin and likely predates domestication. In conclusion, we identified a functional regulatory sequence variant in porcine MYH3 that provides novel insights into the genetic basis of the regulation of myofiber type ratios and associated changes in IMF in pigs. The MYH3 variant can play an important role in improving pork quality in current breeding programs.

Transdifferentiation of α-1,3-galactosyltransferase knockout pig bone marrow derived mesenchymal stem cells into pancreatic ß-like cells by microenvironment modulation.

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 ß-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 ß-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 ß-like cells.

Exogenous Expression of an Alternative Splicing Variant of Myostatin Prompts Leg Muscle Fiber Hyperplasia in Japanese Quail.

Myostatin (MSTN) negatively regulates muscle growth and development through inhibiting myoblast proliferation and differentiation. Five alternative splicing isoforms of MSTN (MSTN-A to MSTN-E) have been discovered in domestic avian species. MSTN-A has high expression in skeletal muscle and encodes the full-length peptide with anti-myogenic activity. Another isoform, MSTN-B, is also highly expressed in skeletal muscle and encodes a truncated peptide that has pro-myogenic capabilities in vitro, which include promoting the proliferation and differentiation of quail muscle precursor cells. The objective of this study was to investigate overexpression of MSTN-B in vivo by using two independent lines of transgenic Japanese quail with expression directed in the skeletal muscle. Unexpectedly, the chicken skeletal muscle alpha actin 1 (cACTA1) promoter resulted in restricted exogenous MSTN-B protein expression to certain skeletal muscles, such as the gastrocnemius and tibialis anterior, but not the pectoralis major muscle. Gastrocnemius weight as a percentage of body weight in transgenic quail was increased compared to non-transgenic quail at posthatch day 21 (D21) and posthatch D42. An increase in the size of the gastrocnemius in transgenic quail was attributed to an increase in fiber number but not fiber cross-sectional area (CSA). During embryonic development, paired box 7 (PAX7) expression was prolonged in the transgenic embryos, but other myogenic regulatory factors (MRFs) were unchanged after MSTN-B overexpression. Taken together, these data provide novel insights into the regulation of skeletal muscle development by alternative splicing mechanisms in avians.

Immune molecular profiling of whole blood drawn from a non-human primate cardiac xenograft model treated with anti-CD154 monoclonal antibodies.

Most studies of xenografts have been carried out with complex immunosuppressive regimens to prevent immune rejection; however, such treatments may be fatal owing to unknown causes. Here, we performed immune molecular profiling following anti-CD154 monoclonal antibody (mAb) treatment in heterotopic abdominal cardiac xenografts from α-1,3-galactosyltransferase-knockout pigs into cynomolgus monkeys to elucidate the mechanisms mediating the undesirable fatal side effects of immunosuppressive agents. Blood samples were collected from healthy monkeys as control and then at 2 days after xenograft transplantation and just before humane euthanasia; 94 genes related to the immune system were analyzed. The basic immunosuppressive regimen included cobra venom factor, anti-thymocyte globulin, and rituximab, with and without anti-CD154 mAbs. The maintenance therapy was followed with tacrolimus, MMF, and methylprednisolone. The number of upregulated genes was initially decreased on Day 2 (-/+ anti-CD154 mAb, 22/13) and then increased before euthanasia in recipients treated with anti-CD154 mAbs (-/+ anti-CD154 mAb, 30/37). The number of downregulated genes was not affected by anti-CD154 mAb treatment. Additionally, the number of upregulated genes increased over time for both groups. Interestingly, treatment with anti-CD154 mAbs upregulated coagulation inducers (CCL2/IL6) before euthanasia. In conclusion, immunosuppressive regimens used for cardiac xenografting affected upregulation of 6 inflammation genes (CXCL10, MPO, MYD88, NLRP3, TNFα, and TLR1) and downregulation of 8 genes (CCR4, CCR6, CD40, CXCR3, FOXP3, GATA3, STAT4, and TBX21).

Ectopic Overexpression of Porcine Myh1 Increased in Slow Muscle Fibers and Enhanced Endurance Exercise in Transgenic Mice.

Myosin heavy chain (MyHC) isoforms consist of Myh7, Myh2, Myh1, and Myh4, which are expressed in skeletal muscle tissues during postnatal development. These genes influence the contraction⁻relaxation activity in skeletal muscles and are involved in determining muscle composition such as the proportion of fast-to-slow and/or slow-to-fast fiber types. Among them, Myh1 is associated with skeletal muscle contraction and is involved in both slow-to-fast and fast-to-slow transition. However, the muscle transition mechanism is not well understood. For this study, we first produced porcine Myh1 transgenic (TG) mice to study whether the ectopic expressed porcine Myh1 gene had any effects on muscle composition, especially on slow-type muscle components. Our results showed that the factors associated with slow muscles, such as Myh7, Myoglobin, Troponin (slow-type units), and cytochrome C, were highly expressed in the quadriceps muscles of Myh1 transgenic mice. Furthermore, the ectopic porcine MYH1 protein was located only in the slow-type muscle fibers of the quadriceps muscles in Myh1 transgenic mice. In physical endurance tests, Myh1 transgenic mice ran longer and further on a treadmill than wild-type (WT) mice. These data fully supported our hypothesis that Myh1 is associated with slow muscle composition, with overexpression of Myh1 in muscle tissues possibly being a new key in modulating muscle fiber types. Our study provides a better understanding of muscle composition metabolism, physical mobility, and genetic factors in muscle fatigue.

Aberrant PTEN expression in response to progesterone reduces endometriotic stromal cell apoptosis

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 progesterone-induced 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.

Generation of α-1,3-galactosyltransferase knocked-out transgenic cloned pigs with knocked-in five human genes.

Recent progress in genetic manipulation of pigs designated for xenotransplantation ha6s shown considerable promise on xenograft survival in primates. However, genetic modification of multiple genes in donor pigs by knock-out and knock-in technologies, aiming to enhance immunological tolerance against transplanted organs in the recipients, has not been evaluated for health issues of donor pigs. We produced transgenic Massachusetts General Hospital piglets by knocking-out the α-1,3-galactosyltransferase (GT) gene and by simultaneously knocking-in an expression cassette containing five different human genes including, DAF, CD39, TFPI, C1 inhibitor (C1-INH), and TNFAIP3 (A20) [GT-(DAF/CD39/TFPI/C1-INH/TNFAIP3)/+] that are connected by 2A peptide cleavage sequences to release individual proteins from a single translational product. All five individual protein products were successfully produced as determined by western blotting of umbilical cords from the newborn transgenic pigs. Although gross observation and histological examination revealed no significant pathological abnormality in transgenic piglets, hematological examination found that the transgenic piglets had abnormally low numbers of platelets and WBCs, including neutrophils, eosinophils, basophils, and lymphocytes. However, transgenic piglets had similar numbers of RBC and values of parameters related to RBC compared to the control littermate piglets. These data suggest that transgenic expression of those human genes in pigs impaired hematopoiesis except for erythropoiesis. In conclusion, our data suggest that transgenic expression of up to five different genes can be efficiently achieved and provide the basis for determining optimal dosages of transgene expression and combinations of the transgenes to warrant production of transgenic donor pigs without health issues.

Identification of the MUC2 Promoter as a Strong Promoter for Intestinal Gene Expression through Generation of Transgenic Quail Expressing GFP in Gut Epithelial Cells.

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.

Molecular immunology profiles of monkeys following xenografting with the islets and heart of α-1,3-galactosyltransferase knockout pigs.

Effective immunosuppression strategies and genetically modified animals have been used to prevent hyperacute and acute xenograft rejection; however, the underlying mechanisms remain unknown. In this study, we evaluated the expression of a comprehensive set of immune system-related genes (89 genes, including five housekeeping genes) in the blood of cynomolgus monkeys (~5 yr old) used as graft recipients, before and after the xenografting of the islets and heart from single and double α-1,3-galactosyltransferase (GalT) knockout (KO) pigs (<6 weeks old). The immunosuppressive regimen included administration of cobra venom factor, anti-thymocyte globulin, rituximab, and anti-CD154 monoclonal antibodies to recipients before and after grafting. Islets were xenografted into the portal vein in type 1 diabetic monkeys, and the heart was xenografted by heterotopic abdominal heart transplantation. Genes from recipient blood were analyzed using RT(2) profiler PCR arrays and the web-based RT(2) profiler PCR array software v.3.5. Recipients treated with immunosuppressive agents without grafting showed significant downregulation of CCL5, CCR4, CCR6, CD4, CD40LG, CXCR3, FASLG, CXCR3, FOXP3, GATA3, IGNG, L10, IL23A, TRAF6, MAPK8, MIF, STAT4, TBX21, TLR3, TLR7, and TYK2 and upregulation of IFNGR1; thus, genes involved in protection against viral and bacterial infection were downregulated, confirming the risk of infection. Notably, C3-level control resulted in xenograft failure within 2 days because of a 7- to 11-fold increase in all xenotransplanted models. Islet grafting using single GalT-KO pigs resulted in upregulation of CXCL10 and MX1, early inflammation, and acute rejection-associated signals at 2 days after xenografting. We observed at least 5-fold upregulation in recipients transplanted with islets grafts from single (MX1) or double (C3, CCR8, IL6, IL13, IRF6, CXCL10, and MX1) GalT-KO pigs after 77 days; single GalT-KO incurred early losses owing to immune attacks. Our results suggest that this novel, simple, non-invasive, and time-efficient procedure (requiring only 1.5 ml blood) for evaluating graft success, minimizing immune rejection, and blocking infection.

HIGH INCIDENCE OF POLYSPERMIC FERTILIZATION IN BOVINE OOCYTES MATURED IN VITRO AFTER CRYOTOP VITRIFICATION.

BACKGROUND: Vitrification with the Cryotop device is the most promising technique for oocyte cryopreservation, but the high post-warming morphological survival of bovine oocytes does not guarantee high developmental competence after in vitro fertilization (IVF). OBJECTIVE: This study was designed to examine achievement of normal fertilization in bovine oocytes vitrified-warmed with the Cryotop device. MATERIALS AND METHODS: Oocytes were matured in vitro and vitrified-warmed after complete removal of the cumulus layers. Distribution of cortical granules (CGs) was assessed by Lens culinaris agglutinin (LCA) lectin staining. Ten hours after IVF, presumptive zygotes were analyzed for pronuclear formation. Day-8 blastocysts were harvested and stained with Hoechst-33342 for total cell counting. RESULTS: Both yield and mean cell number of the blastocysts were impaired by Cryotop vitrification. Incidence of polyspermic fertilization was three-times higher in vitrified oocytes compared to fresh oocytes. No difference in CG distribution was found between vitrified and fresh oocytes. CONCLUSION: Polyspermic fertilization induced in vitrified-warmed bovine oocytes may be one of the possible causes responsible for their low developmental potential.

IMPROVED SURVIVAL AND DEVELOPMENTAL RATES IN VITRIFIED-WARMED PIG OOCYTES AFTER RECOVERY CULTURE WITH COENZYME Q10.

BACKGROUND: The primary problems with porcine oocyte vitrification are their low viability and development; both need improvement. OBJECTIVE: This study was designed to improve the survival and developmental rates in vitrified-warmed porcine oocytes. MATERIALS AND METHODS: Porcine oocytes matured in vitro were vitrified-warmed with Cryotop. Then the oocytes were supplemented with Q10 during recovery culture. RESULTS: The survival rates immediately after warming were 92.9% by morphological inspection and 39.3% by fluorescein diacetate (FDA) assay. The group of recovery culture with Q10 (VC+Q10) showed significantly higher viability compared to the group of recovery culture without Q10 (VC+) analyzed by morphology and the FDA. The VC+Q10 group showed a low Bax/Bcl-xl ratio and a high expression of MAP3K12 and TGFB3 compared to the VC+. The cleavage rate did not differ in both groups but, blastocyst yield was higher in VC+Q10 than the VC+ group. CONCLUSION: Supplementation of Q10 during recovery culture led to a higher blastocyst yield by increasing survival rates and regulating mRNA expressions.

A comprehensive proteomic approach to identifying capacitation related proteins in boar spermatozoa.

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.

ER stress-inducible ATF3 suppresses BMP2-induced ALP expression and activation in MC3T3-E1 cells.

Endoplasmic reticulum (ER) stress suppresses osteoblast differentiation. Activating transcription factor (ATF) 3, a member of the ATF/cAMP response element-binding protein family of transcription factors, is induced by various stimuli including cytokines, hormones, DNA damage, and ER stress. However, the role of ATF3 in osteoblast differentiation has not been elucidated. Treatment with tunicamycin (TM), an ER stress inducer, increased ATF3 expression in the preosteoblast cell line, MC3T3-E1. Overexpression of ATF3 inhibited bone morphogenetic protein 2-stimulated expression and activation of alkaline phosphatase (ALP), an osteogenic marker. In addition, suppression of ALP expression by TM treatment was rescued by silencing of ATF3 using shRNA. Taken together, these data indicate that ATF3 is a novel negative regulator of osteoblast differentiation by specifically suppressing ALP gene expression in preosteoblasts.

Long-term efficacy of encapsulated xenogeneic islet transplantation: Impact of encapsulation techniques and donor genetic traits.

AIMS/INTRODUCTION: To investigate the long-term efficacy of various encapsulated xenogeneic islet transplantation, and to explore the impact of different donor porcine genetic traits on islet transplantation outcomes. MATERIALS AND METHODS: Donor porcine islets were obtained from wild-type, α1,3-galactosyltransferase knockout (GTKO) and GTKO with overexpression of membrane cofactor protein genotype. Naked, alginate, alginate-chitosan (AC), alginate-perfluorodecalin (A-PFD) and AC-perfluorodecalin (AC-PFD) encapsulated porcine islets were transplanted into diabetic mice. RESULTS: In vitro assessments showed no differences in the viability and function of islets across encapsulation types and donor porcine islet genotypes. Xenogeneic encapsulated islet transplantation with AC-PFD capsules showed the most favorable long-term outcomes, maintaining normal blood glucose levels for 180 days. A-PFD capsules showed comparable results to AC-PFD capsules, followed by AC capsules and alginate capsules. Conversely, blood glucose levels in naked islet transplantation increased to >300 mg/dL within a week after transplantation. Naked islet transplantation outcomes showed no improvement based on donor islet genotype. However, alginate or AC capsules showed delayed increases in blood glucose levels for GTKO and GTKO with overexpression of membrane cofactor protein porcine islets compared with wild-type porcine islets. CONCLUSION: The AC-PFD capsule, designed to ameliorate both hypoxia and inflammation, showed the highest long-term efficacy in xenogeneic islet transplantation. Genetic modifications of porcine islets with GTKO or GTKO with overexpression of membrane cofactor protein did not influence naked islet transplantation outcomes, but did delay graft failure when encapsulated.

No long-term feeding toxicities on the health status in rats fed with cloned Korean native beef cattle (Hanwoo) meat.

This study was designed to undertake a risk assessment to identify the health status of rats fed with somatic cell nuclear transfer (SCNT)-cloned Korean native beef cattle (Hanwoo) meat for 26 weeks. The rats were randomly divided into 5 groups, each consisting of 12 male (142.6 ± 5.23 g) and 12 female (113.7 ± 6.31 g) rats each. The animals were fed commercial pellets (control), pellets containing 5% (N-5) and 10% (N-10) of normal cattle meat, and diets containing 5% (C-5) and 10% (C-10) of cloned cattle meat. The mortality; clinical signs; body weight; food consumption; urinary, hematology, blood biochemistry, and histopathological analyses; and absolute and relative organ weights were analyzed and compared. During the 26-week test period, health status-related factors of the rats fed on cloned Hanwoo meat were found to have no test substance-related toxicities. The only difference was the increased uterus weight in female C-10 rats as compared to their counterparts counterparts (p < .05). On the basis of these health status results, it can be postulated that no food consumption risks might arise from the long-term feeding of cloned cattle meat in rats.

Proteomic analysis of cloned porcine conceptuses during the implantation period.

Differentially regulated proteins within porcine somatic cell nuclear transfer (SCNT)-derived conceptuses were compared with conceptuses that were derived from natural matings on day 14 of pregnancy. Proteins that were expressed prominently on day 14 were identified in SCNT-derived conceptuses using 2-D PAGE and MALDI-TOF MS. Sixty eight proteins were identified as being differentially regulated in the SCNT-derived conceptuses. Among these, 62 were down-regulated whereas the other six proteins were up-regulated. Glycolytic proteins, such as pyruvate dehydrogenase, malate dehydrogenase and lactate dehydrogenase, were down-regulated in the SCNT-derived conceptuses whereas apoptosis-related genes as annexin V, Hsp60, and lamin A were up-regulated. Thus, apoptosis-related genes are expressed at significantly higher levels in the SCNT-derived conceptuses than in the control conceptuses, whereas metabolism-related genes are significantly reduced.

Imprinting at the KBTBD6 locus involves species-specific maternal methylation and monoallelic expression in livestock animals.

BACKGROUND: The primary differentially methylated regions (DMRs) which are maternally hypermethylated serve as imprinting control regions (ICRs) that drive monoallelic gene expression, and these ICRs have been investigated due to their implications in mammalian development. Although a subset of genes has been identified as imprinted, in-depth comparative approach needs to be developed for identification of species-specific imprinted genes. Here, we examined DNA methylation status and allelic expression at the KBTBD6 locus across species and tissues and explored potential mechanisms of imprinting. RESULTS: Using whole-genome bisulfite sequencing and RNA-sequencing on parthenogenetic and normal porcine embryos, we identified a maternally hypermethylated DMR between the embryos at the KBTBD6 promoter CpG island and paternal monoallelic expression of KBTBD6. Also, in analyzed domesticated mammals but not in humans, non-human primates and mice, the KBTBD6 promoter CpG islands were methylated in oocytes and/or allelically methylated in tissues, and monoallelic KBTBD6 expression was observed, indicating livestock-specific imprinting. Further analysis revealed that these CpG islands were embedded within transcripts in porcine and bovine oocytes which coexisted with an active transcription mark and DNA methylation, implying the presence of transcription-dependent imprinting. CONCLUSIONS: In this study, our comparative approach revealed an imprinted expression of the KBTBD6 gene in domesticated mammals, but not in humans, non-human primates, and mice which implicates species-specific evolution of genomic imprinting.

The AIRN lncRNA is imprinted and paternally expressed in pigs.

Genomic imprinting plays critical roles during the development of mammalian species and underlying epigenetic mechanisms frequently involve long non-coding RNAs (lncRNAs). The paternal transcription of the antisense Igf2r RNA noncoding (Airn) is responsible for paternal silencing of the mouse insulin-like growth factor 2 receptor (Igf2r) gene and maternal Igf2r expression. Although the corresponding maternal DNA methylation imprint is conserved in humans and pigs, the orthologous AIRN lncRNA has been identified in humans but not in pigs. Here, we aimed to examine imprinted allelic expression of the porcine AIRN lncRNA along with a corresponding differentially methylated region (DMR) and to analyze allelic expression of AIRN and IGF2R in pigs. By comparing parthenogenetic and control porcine embryos, we identified a maternally methylated DMR and a significantly higher expression of AIRN lncRNA in control embryos (P < 0.05) indicating its paternal expression. Further analyses revealed that the expression of AIRN lncRNA was enriched in the pig brain and its subregions, and it was monoallelically expressed; whereas, IGF2R was expressed biallelically suggesting an absence of allele-specific transcriptional regulation. Our findings will lead to further investigations into the role of the imprinted porcine AIRN lncRNA during pig development.

Genomic imprinting is important for the development of mammals and long non-coding RNAs are often involved in the imprinting process. In mice, Airn encodes a long non-coding RNA that is imprinted, and therefore, transcribed only from the paternal allele. This paternal transcription of Airn interferes with the adjacent Igf2r promoter, leading to maternal expression of Igf2r. In pigs, the orthologous AIRN has not been identified as well as its imprinting. In the current study, we report porcine AIRN and allelic expression of both AIRN and IGF2R using our parthenogenetic embryo models and various normal pig tissues.

Effects of heat stress on conception in Holstein and Jersey cattle and oocyte maturation in vitro.

Korea, located in East Asia in the northern hemisphere, is experiencing severe climate changes. Specifically, the heat stress caused by global warming is negatively affecting the dairy sector, including milk production and reproductive performance, as the major dairy cattle Holstein-Friesian is particularly susceptible to heat stress. Here, we collected artificial insemination and pregnancy data of the Holstein and the Jersey cows from a dairy farm from 2014 to 2021 and analyzed the association between the conception rate and the temperature-humidity index, calculated using the data from the closest official weather station. As the temperature-humidity index threshold increased, the conception rate gradually decreased. However, this decrease was steeper in the Holstein breed than in the Jersey one at a temperature-humidity index threshold of 75. To evaluate the effects of heat stress on the oocyte quality, we examined the nuclear and cytoplasmic maturation of Holstein (n = 158, obtained from six animals) and Jersey oocytes (n = 123, obtained from six animals), obtained by ovum pick-up. There were no differences in the nuclear maturation between the different conditions (heat stress: 40.5°C, non- heat stress: 37.5°C) or breeds, although the Holstein oocytes seemed to have a lower metaphase II development (p = 0.0521) after in vitro maturation under heat stress conditions. However, we found that the Holstein metaphase II oocytes exposed to heat stress presented more reactive oxygen species and a peripheral distribution of the mitochondria, compared to those of the Jersey cattle. Here, we show that weather information from local meteorological stations can be used to calculate the temperature-humidity index threshold at which heat stress influences the conception rate, and that the Jersey cows are more tolerant to heat stress in terms of their conception rate at a temperature-humidity index over 75. The lower fertility of the Holstein cows is likely attributed to impaired cytoplasmic maturation induced by heat stress. Thus, the Jersey cows can be a good breed for the sustainability of dairy farms for addressing climate changes in South Korea, as they are more resistant to hyperthermia.