Efficacy of fucoxanthin extract from Sargassum horneri on 3T3-L1 pre-adipocyte differentiation.

Obesity, a chronic disease characterized by excessive body fat accumulation, is associated with significant health risks. The state of being overweight or obese leads to a number of chronic diseases, including cardiovascular disease, type 2 diabetes, cancer, and osteoarthritis. Accordingly, the regulation of adipocyte proliferation and differentiation has been the focus of many studies. The goal of the present study was to investigate the function of fucoxanthin, extracted from Sargassum horneri, in adipocyte (3T3-L1 cells) differentiation. A quantitative real-time polymerase chain reaction was conducted to investigate the mRNA expression levels of adipocyte differentiation-related genes under fucoxanthin stimulation. All adipocyte-related genes responded to PIC stimuli. Additionally, using western blotting, we confirmed that fucoxanthin reduced adipocyte differentiation. These results indicate that fucoxanthin extracted from Sargassum horneri can regulate adipogenesis. Further studies are needed to reveal the signaling pathways that lead to reduced adipocyte differentiation induced by fucoxanthin.

Production of chickens with green fluorescent protein-knockin in the Z chromosome and detection of green fluorescent protein-positive chicks in the embryonic stage.

OBJECTIVE: The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, which is the most efficient and reliable tool for precisely targeted modification of the genome of living cells, has generated considerable excitement for industrial applications as well as scientific research. In this study, we developed a gene-editing and detection system for chick embryo sexing during the embryonic stage. METHODS: By combining the CRISPR/Cas9 technical platform and germ cell-mediated germline transmission, we not only generated Z chromosome-targeted knockin chickens but also developed a detection system for fluorescence-positive male chicks in the embryonic stage. RESULTS: We targeted a green fluorescent protein (GFP) transgene into a specific locus on the Z chromosome of chicken primordial germ cells (PGCs), resulting in the production of ZGFP-knockin chickens. By mating ZGFP-knockin females (ZGFP/W) with wild males (Z/Z) and using a GFP detection system, we could identify chick sex, as the GFP transgene was expressed on the Z chromosome only in male offspring (ZGFP/Z) even before hatching. CONCLUSION: Our results demonstrate that the CRISPR/Cas9 technical platform with chicken PGCs facilitates the production of specific genome-edited chickens for basic research as well as practical applications.

Deoxynivalenol Induces Apoptosis via FOXO3a-Signaling Pathway in Small-Intestinal Cells in Pig.

Deoxynivalenol (DON) is a mycotoxin that is found in feed ingredients derived from grains such as corn and wheat. Consumption of DON-contaminated feed has been shown to cause damage to the intestine, kidneys, and liver. However, the molecular mechanism by which DON exerts its effect in the small intestine is not completely understood. As a result, we profiled gene expression in intestinal epithelial cells treated with DON and examined the molecular function in vitro. We hypothesized that DON could induce apoptosis via the FOXO3a-signaling pathway in intestinal epithelial cells based on these findings. DON induced the apoptosis and the translocation of FOXO3a into the nucleus. Moreover, the inhibiting of FOXO3a alleviated the apoptosis and expression of apoptosis-related genes (TRAL, BCL-6, CASP8, and CASP3). ERK1/2 inhibitor treatment suppressed the translocation of FOXO3a into the nucleus. Our discovery suggests that DON induces apoptosis in intestinal epithelial cells through the FOXO3a-signaling pathway.

Multi-omics approaches for comprehensive analysis and understanding of the immune response in the miniature pig breed.

The porcine immune system has an important role in pre-clinical studies together with understanding the biological response mechanisms before entering into clinical trials. The size distribution of the Korean minipig is an important feature that make this breed ideal for biomedical research and safe practice in post clinical studies. The extremely tiny (ET) minipig serves as an excellent model for various biomedical research studies, but the comparatively frail and vulnerable immune response to the environment over its Large (L) size minipig breed leads to additional after born care. To overcome this pitfall, comparative analysis of the genomic regions under selection in the L type breed could provide a better understanding at the molecular level and lead to the development of an enhanced variety of ET type minipig. In this study, we utilized whole genome sequencing (WGS) to identify traces of artificial selection and integrated them with transcriptome data generated from blood samples to find strongly selected and differentially expressed genes of interest. We identified a total of 35 common genes among which 7 were differentially expressed and showed selective sweep in the L type over the ET type minipig breed. The stabilization of these genes were further confirmed using nucleotide diversity analysis, and these genes could serve as potential biomarkers for the development of a better variety of ET type pig breed.

Effect of Application Amounts on In Vitro Dermal Absorption Test Using Caffeine and Testosterone.

Dermal absorption of chemicals is a key factor in risk assessment. This study investigated the effects of different amounts of application on dermal absorption and suggested an appropriate application dose for proper dermal absorption. Caffeine and testosterone were chosen as test compounds. An in vitro dermal absorption test was performed using a Franz diffusion cell. Different amounts (5, 10, 25, and 50 mg (or µL)/cm2) of semisolid (cream) and liquid (solution) formulations containing 1% caffeine and 0.1% testosterone were applied to rat and minipig (Micropig®) skins. After 24 h, the concentrations of both compounds remaining on the skin surface and in the stratum corneum, dermis and epidermis, and receptor fluid were determined using LC-MS / MS or HPLC. Dermal absorption of both compounds decreased with increasing amounts of application in both skin types (rat and minipig) and formulations (cream and solution). Especially, dermal absorptions (%) of both compounds at 50 mg (or µL)/cm2 was significantly lower compared to 5 or 10 mg (or µL)/cm2 in both rat and minipig skins. Therefore, a low dose (5 or 10 mg (or µL)/cm2) of the formulation should be applied to obtain conservative dermal absorption.

N-acetylcysteine modulates cyclophosphamide-induced immunosuppression, liver injury, and oxidative stress in miniature pigs.

Cyclophosphamide, a cytotoxic anticancer agent, induces immunosuppression and has several adverse effects. N-acetylcysteine alleviates oxidative stress, liver injury, and intestinal tissue damage. The present study examined whether N-acetylcysteine modulates the adverse effects of cyclophosphamide in pigs. Miniature pigs (n = 15) were used as an experimental model to evaluate the effects of N-acetylcysteine treatment on immune reactions, liver injury, and oxidative stress after cyclophosphamide challenge. Corn-soybean meal based dietary treatments were as follows: control diet with either saline injection, cyclophosphamide injection, or 0.5% N-acetylcysteine and cyclophosphamide injection. N-acetylcysteine increased the number of immune cells and decreased TNF-α production after cyclophosphamide injection and decreased TNF-α, IFN-γ, NF-κB, and IL-8 expression and increased IL-10 expression in peripheral blood mononuclear cells. Serum levels of alanine transaminase and aspartate aminotransferase decreased, superoxide dismutase activity increased, and malondialdehyde activity decreased following N-acetylcysteine treatment after cyclophosphamide injection. N-acetylcysteine decreases immunosuppression, liver injury, and oxidative stress in cyclophosphamide-challenged miniature pigs. The present study suggests that N-acetylcysteine has therapeutic application in livestock for modulating immune reactions, liver injury, and oxidative stress.

Whole-genome resequencing analysis of 20 Micro-pigs.

BACKGROUND: Miniature pigs have been increasingly used as mammalian model animals for biomedical research because of their similarity to human beings in terms of their metabolic features and proportional organ sizes. However, despite their importance, there is a severe lack of genome-wide studies on miniature pigs. OBJECTIVE: In this study, we performed whole-genome sequencing analysis of 20 Micro-pigs obtained from Medi Kinetics to elucidate their genomic characteristics. RESULTS: Approximately 595 gigabase pairs (Gb) of sequence reads were generated to be mapped to the swine reference genome assembly (Sus scrofa 10.2); on average, the sequence reads covered 99.15% of the reference genome at an average of 9.6-fold coverage. We detected a total of 19,518,548 SNPs, of which 8.7% were found to be novel. With further annotation of all of the SNPs, we retrieved 144,507 nonsynonymous SNPs (nsSNPs); of these, 5968 were found in all 20 individuals used in this study. SIFT prediction for these SNPs identified that 812 nsSNPs in 402 genes were deleterious. Among these 402 genes, we identified some genes that could potentially affect traits of interest in Micro-pigs, such as RHEB and FRAS1. Furthermore, we performed runs of homozygosity analysis to locate potential selection signatures in the genome, detecting several loci that might be involved in phenotypic characteristics in Micro-pigs, such as MSTN, GDF5, and GDF11. CONCLUSION: In this study, we identified numerous nsSNPs that could be used as candidate genetic markers with involvement in traits of interest. Furthermore, we detected putative selection footprints that might be associated with recent selection applied to miniature pigs.

Age-related compositional and functional changes in micro-pig gut microbiome.

Age-related changes in human gut microbiota composition have been reported, and such changes might be influenced by the intake of nutrients or diets. To investigate the effects of aging on the gut microbiota independent of nutrient effects, we analyzed the gut microbiomes of 126 micro-pigs at a wide range of ages from newborns to 10 years old. The micro-pigs were reared in a constantly controlled environment. The diversity of the gut microbiome was found to continuously change with age. We also found associations between age and specific members and functions of the gut microbiome. Consistent with previous studies on the human gut microbiome, beneficial microbes including probiotic bacteria and short-chain fatty acid-producers decreased in older pigs, whereas Bacteroides increased with age. Based on the correlation network, Bacteroides seemed to have an important role in determining the relative abundances of other beneficial microbes. Our results suggest that maintaining beneficial gut microbes at a specific ratio corresponding to a certain age might contribute to a younger gut microbiome-age. Furthermore, due to similarities with the human system, micro-pigs are a useful animal model to elucidate the links between aging and the microbiome.

Omega-3 fatty acids modulate cyclophosphamide induced markers of immunosuppression and oxidative stress in pigs.

Immunosuppression directly correlates with economic benefits in livestock. Although omega-3, known as an energy source, is used as a pharmaceutical molecule, it remains unknown whether dietary supplementation with omega-3 can alleviate cyclophosphamide-induced immunosuppression in pigs. Omega-3 treatment increased the number of white blood cell, lymphocytes, and monocytes and decreased tumor necrosis factor (TNF)-α production under CTX challenge. In addition, we confirmed that omega-3 decreased the expression of nuclear factor (NF)-κB, TNF-α, interferon (IFN)-γ, and interleukin (IL)-8 in peripheral blood mononuclear cells. Additionally, omega-3 alleviated the activities of liver injury markers (alanine transaminase [ALT] and aspartate transaminase [AST]) and modulated oxidative stress markers (superoxide dismutase [SOD], malondialdehyde [MDA], and glutathione peroxidase [GPx]) in the blood serum after the CTX challenge. Based on these results, we suggest that omega-3 treatment modulates CTX-induced immunosuppression and oxidative stress in pigs. These results may have important implications in the development of new therapeutic approaches to improve immunosuppression, hepatic injury and dysfunction, and oxidative stress in pigs.

N-acetylcysteine modulates lipopolysaccharide-induced intestinal dysfunction.

The gastrointestinal epithelium functions in nutrient absorption and pathogens barrier and its dysfunction directly affects livestock performance. N-Acetylcysteine (NAC) improves mucosal function, but its effects on intestinal functions at the molecular level remain unclear. Here, we performed gene expression profiling of the pig small intestine after dietary NAC treatment under LPS challenge and investigated the effects of NAC on intestinal epithelial cells in vitro. Dietary NAC supplementation under LPS challenge altered the small intestine expression of 959 genes related to immune response, inflammatory response, oxidation-reduction process, cytokine-cytokine receptor interaction, and the cytokine-mediated signalling, Toll-like receptor signalling pathway, Jak-STAT signalling pathway, and TNF signalling pathway. We then analysed the expression patterns of the top 10 altered genes, and found that NAC markedly stimulated HMGCS3 and LDHC expression in IPEC-J2 cells. In vitro, NAC pre-treatment significantly reduced TNF-α and NF-κB, TNF-α, IFN-γ, and IL-6 expression in LPS-induced IPEC-J2 cells. NAC treatment also significantly reduced oxidative stress in LPS-induced IPEC-J2 cells and alleviated intestinal barrier function and wound healing. Thus, NAC as a feed additive can enhance livestock intestinal health by modulating intestinal inflammation, permeability, and wound healing under LPS-induced dysfunction, improving our molecular understanding of the effects of NAC on the intestine.

Muscle differentiation induced up-regulation of calcium-related gene expression in quail myoblasts.

OBJECTIVE: In the poultry industry, the most important economic traits are meat quality and carcass yield. Thus, many studies were conducted to investigate the regulatory pathways during muscle differentiation. To gain insight of muscle differentiation mechanism during growth period, we identified and validated calcium-related genes which were highly expressed during muscle differentiation through mRNA sequencing analysis. METHODS: We conducted next-generation-sequencing (NGS) analysis of mRNA from undifferentiated QM7 cells and differentiated QM7 cells (day 1 to day 3 of differentiation periods). Subsequently, we obtained calcium related genes related to muscle differentiation process and examined the expression patterns by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). RESULTS: Through RNA sequencing analysis, we found that the transcription levels of six genes (troponin C1, slow skeletal and cardiac type [TNNC1], myosin light chain 1 [MYL1], MYL3, phospholamban [PLN], caveolin 3 [CAV3], and calsequestrin 2 [CASQ2]) particularly related to calcium regulation were gradually increased according to days of myotube differentiation. Subsequently, we validated the expression patterns of calcium-related genes in quail myoblasts. These results indicated that TNNC1, MYL1, MYL3, PLN, CAV3, CASQ2 responded to differentiation and growth performance in quail muscle. CONCLUSION: These results indicated that calcium regulation might play a critical role in muscle differentiation. Thus, these findings suggest that further studies would be warranted to investigate the role of calcium ion in muscle differentiation and could provide a useful biomarker for muscle differentiation and growth.

Function of capric acid in cyclophosphamide-induced intestinal inflammation, oxidative stress, and barrier function in pigs.

The small intestine is not only critical for nutrient absorption, but also serves as an important immune organ. Medium-chain fatty acids have nutritional and metabolic effects and support the integrity of the intestinal epithelium. However, their roles in intestinal immunity in pigs are not fully understood. We investigated the effects of a medium-chain fatty acid, capric acid, on intestinal oxidative stress, inflammation, and barrier function in porcine epithelial cells and miniature pigs after treatment with the immune suppressant cyclophosphamide. Capric acid alleviated inflammatory cytokine production (TNF-α and IL-6) and related gene expression (NF-κB, TNF-α, IFN-γ), alleviated oxidative stress (GSSG/GSH ratio, H2O2, and malondialdehyde), and increased oxidative stress-related gene expression (SOD1 and GCLC) in cyclophosphamide-treated IPEC-J2 cells. The permeability of FD-4 and expression of ZO-1 and OCLN in cyclophosphamide-treated IPEC-J2 cells were reduced by capric acid. Dietary capric acid reduced TNF-α, IL-6, and MDA levels and increased SOD, GPx, and the expression of genes related to pro-inflammatory, oxidative stress, and intestinal barrier functions in cyclophosphamide-treated miniature pigs. These results revealed that capric acid has protective effects against cyclophosphamide-induced small intestinal dysfunction in pigs.

Fertilisation of cryopreserved sperm and unfertilised quail ovum by intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is an important technique in animal biotechnology for animal cloning and conservation of genetic resources, but has been a challenge for avian species. In the present study, we investigated the ability of cryopreserved quail spermatozoa to achieve fertilisation and embryo development. Female quail were killed 70-120min after previous oviposition to collect unfertilised oocytes from the oviduct. Fresh or cryopreserved-thawed spermatozoa were injected into the cytoplasm of unfertilised oocytes, and the manipulated oocytes were incubated in quail surrogate eggshells. Injection of fresh spermatozoa supplemented with inositol 1,4,5-trisphosphate (IP3) resulted in a significantly increased rate of embryo development compared with injection of fresh spermatozoa alone (90% vs 13%, respectively). Although >80% of embryos stopped cell division and development before Hamburger and Hamilton (HH) Stage 3, approximately 15% of embryos from the fresh sperm injection developed to past HH Stage 4, and one embryo survived up to HH Stage 39 (11 days of incubation). In the case of cryopreserved spermatozoa, the embryo development rate was 30% after ICSI, and this increased significantly to 74% with IP3 supplementation. In conclusion, cryopreserved spermatozoa combined with ICSI followed by surrogate eggshell culture can develop quail embryos.

Production of Interspecific Germline Chimeras via Embryo Replacement.

In avian species, primordial germ cells (PGCs) use the vascular system to reach their destination, the genital ridge. Because of this unique migratory route of avian germ cells, germline chimera production can be achieved via germ cell transfer into a blood vessel. This study was performed to establish an alternative germ cell-transfer system for producing germline chimeras by replacing an original host embryo with a donor embryo, while retaining the host extraembryonic tissue and yolk, before circulation. First, to test the migratory capacity of PGCs after embryo replacement, Korean Oge (KO) chick embryos were used to replace GFP transgenic chick embryos. Four days after replacement, GFP-positive cells were detected in the replaced KO embryonic gonads, and genomic DNA PCR analysis with the embryonic gonads demonstrated the presence of the GFP transgene. To produce an interspecific germline chimera, the original chick embryo proper was replaced with a quail embryo onto the chick yolk. To detect the gonadal PGCs in the 5.5-day-old embryonic gonads, immunohistochemistry was performed with monoclonal antibodies specific to either quail or chick PGCs, i.e., QCR1 and anti-stage-specific embryonic antigen-1 (SSEA-1), respectively. Both the QCR1-positive and SSEA-1-positive cells were detected in the gonads of replaced quail embryos. Forty percent of the PGC population in the quail embryos was occupied by chick extraembryonically derived PGCs. In conclusion, replacement of an embryo onto the host yolk before circulation can be applied to produce interspecies germline chimeras, and this germ cell-transfer technology is potentially applicable for reproduction of wild or endangered bird species.

Molecular responses to the influenza A virus in chicken trachea-derived cells.

The influenza A virus infects a broad range of species and spreads easily through the respiratory tract. Because of these characteristics, the influenza A virus has caused pandemic disease in humans and livestock. To investigate the early molecular responses after influenza A virus infection in chickens, we infected tracheal epithelial cells derived from 20-day-old chick embryos with influenza A virus (H1N1). The gene expression patterns of the infected tracheal epithelial cells were analyzed via DNA microarray at different time points (0, 6, 12, 24, and 36 hr) after viral infection. Differentially expressed genes were identified at 6, 12, 24, and 36 hours post infection. A total of 1,936, 2,168, 3,670 and 2,894 genes were upregulated (≥2-fold, P<0.05), whereas 884, 592, 1,503 and 1,925 genes were downregulated at the respective time points (≤0.5-fold, P<0.05). When the differentially expressed genes were functionally categorized, immune-related and defense response gene ontology terms were detected in 12, 24, or 36 hours post infection. Interestingly, in the defense response, most of the gallinacin (GAL) genes were rapidly induced within 24 hr post infection. Subsequently, we predicted transcription factor binding sites within promoters of the GAL gene family, and analyzed the gene expression pattern for the common GAL gene regulatory factors to identify the viral infection-induced immune mechanism. Our results might contribute to an understanding of early host responses and regulatory mechanisms for host defense peptide induction against viral infections in chicken.

Spatial and temporal action of chicken primordial germ cells during initial migration.

In most animals, primordial germ cells (PGCs) originate from an extragonadal region and migrate across the embryo to the gonads, where they differentiate and function. During their migration, PGCs move passively by morphogenetic movement of the embryo or move actively through signaling molecules. To uncover the underlying mechanism of first-phase PGC migration toward the germinal crescent in chickens, we investigated the spatial and temporal action of PGCs during primitive streak formation. Exogenously transplanted PGCs migrated toward the anterior region of the embryo and the embryonic gonads when they were transplanted into the subgerminal cavity, but not into the posterior marginal zone, in Eyal-Giladi and Kochav stage X embryos. These results indicate that for passive migration toward the anterior region the initial location of PGCs should be the central region. Notably, although PGCs and DF-1 cells migrated passively toward the anterior region, only PGCs migrated to the germinal crescent, where endogenous PGCs mainly reside, by active movement. In a live-imaging experiment with green fluorescence protein-expressing transgenic embryos, exogenous PGCs demonstrated markedly faster migration when they reached the anterior one-third of the embryo, while somatic cells showed epiblast movement with constant speed. Also, migrating PGCs exhibited successive contraction and expansion indicating their active migration. Our results suggest that chicken PGCs use sequential passive and active forces to migrate toward the germinal crescent.

Current genomic editing approaches in avian transgenesis.

The chicken was domesticated from Red Jungle Fowl over 8000years ago and became one of the major food sources worldwide. At present, the poultry industry is one of the largest industrial animal stocks in the world, and its economic scale is expanding significantly with increasing consumption. Additionally, since Aristotle used chicken eggs as a model to provide remarkable insights into how life begins, chickens have been used as invaluable and powerful experimental materials for studying embryo development, immune systems, biomedical processes, and hormonal regulation. Combined with advancements in efficient transgenic technology, avian models have become even more important than would have been expected.

Cryopreservation of Korean Oge chicken semen using N-methylacetamide.

The importance of genetic resource preservation has been highlighted in the literature as a means of maintaining genetic diversity. Among the various methods of preserving such resources, semen cryopreservation can be advantageous because it reduces the time of restoring genetic resources and is less technique-dependent. The Korean Oge (KO) chicken is a Natural Monument and is recognized as an important genetic resource in Korea. However, successful cryopreservation methods for KO chickens have yet to be reported. Therefore, we completed cryopreservation methods in KO chickens using N-methylacetamide (MA) as a cryoprotectant. Also we performed additional experiments to identify whether fertility and hatchability are affected by long-term storage. Finally, we examined sperm viability in the cryopreserved semen. Our results suggest that the cryopreservation method using MA can be applied to KO chickens regardless of storage period and could be a useful tool for the preservation the endangered avian species.

The distribution of neuron-specific gene family member 1 in brain and germ cells: Implications for the regulation of germ-line development by brain.

Vesicular acidification at early endosomes dissociates endocytosed receptor-ligand complexes. The ligands, receptors, or both are then directed to late endosomes for degradation or recycled back to the plasma membrane. Of neuron-specific gene (NSG) family members, early endosomal protein neuron-specific gene family member 1 (NSG1) is the most important in receptor recycling. In this study, we characterized chicken NSG1 (cNSG1). We found several functional sites related to endocytotic machinery in cNSG1 that were highly conserved with most other vertebrate NSG1 proteins. We examined the tissue and duration specificity and the temporal and spatial patterns of cNSG1 expression. cNSG1 expression was preferentially located in all regions of the brain, neuroendocrine glands, and spinal cord. Unexpectedly, cNSG1 expression was strongly detected during male and female germ-line development. Expression of NSG1 in two apparently unrelated cell types such as neurons and germ cells suggests NSG1 roles in neurons and germ-cells chemotaxis and endocytotic machinery.

Neuronal genes for subcutaneous fat thickness in human and pig are identified by local genomic sequencing and combined SNP association study.

Obesity represents a major global public health problem that increases the risk for cardiovascular or metabolic disease. The pigs represent an exceptional biomedical model related to energy metabolism and obesity in humans. To pinpoint causal genetic factors for a common form of obesity, we conducted local genomic de novo sequencing, 18.2 Mb, of a porcine QTL region affecting fatness traits, and carried out SNP association studies for backfat thickness and intramuscular fat content in pigs. In order to relate the association studies in pigs to human obesity, we performed a targeted genome wide association study for subcutaneous fat thickness in a cohort population of 8,842 Korean individuals. These combined association studies in human and pig revealed a significant SNP located in a gene family with sequence similarity 73, member A (FAM73A) associated with subscapular skin-fold thickness in humans (rs4121165, GC-corrected p-value  = 0.0000175) and with backfat thickness in pigs (ASGA0029495, p-value  = 0.000031). Our combined association studies also suggest that eight neuronal genes are responsible for subcutaneous fat thickness: NEGR1, SLC44A5, PDE4B, LPHN2, ELTD1, ST6GALNAC3, ST6GALNAC5, and TTLL7. These results provide strong support for a major involvement of the CNS in the genetic predisposition to a common form of obesity.