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Cluster of differentiation 14 (CD14) is the pattern recognition receptor (PRR) involved in the recognition of bacterial component lipopolysaccharide (LPS) through the MyD88-dependent and TRIF pathway of innate immunity. It may be a modulator to prevent and mitigate the LPS-induced lung inflammation in pigs. However, reports on CD14 activation induced by LPS in TRIF pathway are controversial. Furthermore, the gene expression regulation of CD14 by the epigenetic factor sulforaphane (SFN) is still poorly understood. To identify the epigenetic changes of CD14 mediated with SFN in LPS-induced TRIF pathway, the PAMs model in vitro was investigated. For this, the mRNA expression of CD14 and downstream genes of TRIF pathway were quantified using qPCR. The cytokine levels of tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) were measured by enzyme-linked immunosorbent assay (ELISA). The gene expression of the epigenetic enzymes DNA methyltransferase-1 (DNMT1) and DNMT3a were quantified. The protein level of NF-κB was analyzed by western blot. Furthermore, the DNA methylation alterations of CD14 at promotor and gene-body (CDS region) were analyzed using bisulfite sequencing in SFN- and LPS-treated PAMs. It was shown that CD14 gene expression was induced by 5 µg/ml LPS in time-dependent manner. At time point 12 h, the gene expression of CD14 and downstream genes in TRIF pathway, including TRIF, TRAF6, NFγB, TRAF3, IRF7, and cytokines, such as TNF-α, IL-1β, IL-6, and IFN-β, were significantly induced by LPS. The LPS-induced gene expression was suppressed by SFN in a dose-dependent manner. The LPS-induced cytokine levels, including TNFα, IL-1β, and NF-κB levels, were also inhibited by SFN. Similarly, the DNMT3a mRNA expression was increased by LPS and downregulated by SFN at a dose of 5 µM. Furthermore, the bisulfite sequencing results showed that gene body methylation of CD14 was positively associated with gene expression of LPS-treated PAMs and this methylation status was inhibited by SFN in a dose-dependent manner. This in vitro study suggests that CD14 is involved in TRIF pathway, including TRIF-TRAF6 and TRIF-TRAF3 pathway, by LPS induction. Furthermore, this LPS-CD14 activation was suppressed by SFN via the epigenetic regulation of CD14 gene body methylation associated with DNMT3a. This study provided novel insights into SFN-mediated epigenetic downregulation of CD14 gene in LPS-induced TRIF pathway inflammation and may open new avenues for approaches to prevent and mitigate LPS-induced inflammation in pigs.

Systematic concern for animal welfare with regard to piglet castration without anesthesia is one challenge in European pig production. For instance, in 2013 the German Federal Council passed the law that non-anesthetized surgical castration will be prohibited by 2019. One alternative is the fattening of entire boars which is controversially discussed, because the risk of tainted meat has to be minimized. One possibility is to include selection for a low incidence of boar taint in breeding programs.In this study, beside androstenone (And) and skatole (Ska), boar taint perception was recorded by a 10-member trained sensory panel (SENS) established by Mörlein and colleagues. Compiling data from four research projects, in total 4,000 records for And and Ska, and 1,016 records for SENS were available.Genetic marker effects of 45,645 SNPs on And, Ska and SENS scores were estimated for 1,240 crossbred boars and 976 purebred Pietrain boars. These crosses and purebred boars reflect the heterogeneous population-structure of the Pietrain breed in Germany. Genomic BLUP was used to estimate genomic breeding values for animals in calibration and validation sets. Reliability of genomic prediction was assessed as correlation between genomic breeding value and conventional breeding values. Several scenarios were investigated to evaluate the effectiveness of genomic prediction.Heritabilities for And, Ska and SENS were 0.64, 0.48 and 0.36, respectively. In a first scenario genomic prediction was evaluated using a five-fold cross validation within all crossbred animals and showed reliabilities ranging from 0.33 to 0.49. Based on a forward prediction, using crossbred boars as calibration set and Pietrain boars as validation set, reliabilities between 0.15 and 0.50 for all boar taint traits were observed.Our analysis showed that genomic selection against boar taint is promising. It was possible to reach adequately high reliabilities, even in small populations.The project is supported by funds of the Federal Ministry of Food and Agriculture (BMEL) based on a decision of the Parliament of the Federal Republic of Germany via the Federal Office for Agriculture and Food (BLE) under the innovation support program. Grant no.: 313–06.01–28–1-68.024–11.

Satellite cells, the muscle stem cells, play a critical role in muscle growth, maintenance, and regeneration. A lot of muscle diseases result from defective function of satellite cells. Porcine satellite cells are a good model for studying the role of satellite cells in muscle development. Sulforaphane (SFN), a natural molecule rich in cruciferous vegetables, is a potent inducer for the NF-E2-related factor 2 (Nrf2) signaling and also inhibits the activity of histone deacetylases (HDAC). Our previous study found that SFN epigenetically suppressed the transcription of myostatin in porcine satellite cells. However, the effects of SFN on the proliferation of porcine satellite cells and the related mechanisms are far from understood. In the present study, we report that SFN enhanced the proliferation of the porcine satellite cells and modified the expression myogenic regulatory factors. SFN altered the expression of HDACs and inhibited the activity of HDACs. The activity of TGF-β signaling was suppressed by SFN treatment, which was accompanied with up-regulated Smad7, an endogenous suppressor of TGF-β signaling. Furthermore, we found that SFN increased the mRNA expression of Smad7’s transcription factors and decreased the expression of miRNAs targeting Smad7. The DNA methylation of a studied fragment in Smad7 promoter was not influenced by SFN treatment. SFN has received substantial attention because of its potential application in cancer therapy. The present study, for the first time, investigated the effects of SFN on the proliferation of porcine satellite cells and the underlying mechanism. We found that both mRNA and protein level of Smad7 were greatly increased by SFN. Thus, besides reducing TGF-β1 protein abundance, SFN also inhibits the activity of TGF-β signaling by increasing expression of Smad7. It has been shown that overexpression of Smad7 led to enhanced skeletal muscle differentiation and cellular hypertrophy. In summary, our studies state that SFN enhances the proliferation of porcine satellite cells by suppressing TGF-β signaling through activation of Smad7.

In genetic analyses new types of functional traits can be used to increase the information density between genes and phenotypes. The consideration of different omics levels, like protein and metabolite abundance allows us to elucidate the black box of phenotype expression and to identify potential candidate genes. Aim of this study was to integrate the data of the genome, proteome and metabolome to characterize underlying functional pathways, biochemical processes and corresponding candidate genes of the meat quality parameter drip loss in pigs. Generally, this trait is strongly influenced by environmental effects and therefore our hypothesis was that metabolites and proteins, significantly associated with drip loss, are more accurate and reliable phenotypes than drip loss itself. We applied an untargeted (holistic) metabolomics approach and a targeted protein profiling to determine and quantify the metabolite and protein profiles in Musculus longissimus dorsi(LD). Therefore, LD samples of 97 Duroc × Pietrain pigs were collected and phenotypes as well as genotypes were recorded. In total, 126 and 40 KEGG annotated metabolites and proteins were identified in the tissue samples. An enrichment analysis resulted in 206 detected pathways and 10 pathways showed a significant meaning for drip loss. Besides drip loss itself, 18 metabolites and four proteins that belong to the significant metabolic pathways, namely sphingolipid metabolism, pyruvate metabolism, glycolysis/gluconeogenesis and methane metabolism were analyzed as phenotypes within a genome-wide association study (GWAS). For drip loss, two proteins and 11 metabolites we detected in total 430 significantly associated genetic markers (SNPs) located on Sus scrofachromosomes (SSC) 1, 2, 3, 4, 6, 7, 8, 10, 13, 14, 16, 17, and 18. The functional annotation of these SNPs allowed discovering promising genomic regions as well as potential candidate genes. As one interesting example, on SSC 18 the genes PTN, CREB3L2 and LRGUK were identified based on significant SNPs for muscle physiology associated with drip loss, protein phosphoglycerate mutase 2 (PGAM2) and metabolite glycine. These genes are involved in the homoeostasis of muscle energy metabolism and metabolic disorders. Because of the increased information density due to the consideration of proteome and metabolome, our results lead to the conclusion that a combined omics analysis has an advantage compared to ‘classical’ GWAS. Therefore, we expect that GWAS approaches based on metabolic traits induce a smaller percentage of false-positive results and contribute to identify reliable candidate genes.