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The objective of this study was to determine fattening performance data for Charolais, Limousin and Blonde d’Aquitaine beef cattle and associate these data with NPR2 gene 8:g.59961937 T>C (rs208158047) mutation. Experiments were conducted with 176 beef cattle (77 Charolais, 66 Limousin and 33 Blonde d’Aquitaine) at nine months of age. Experiments lasted for 9 months and animals were slaughtered at the age of 18 months. Cattle body weights were determined at four different periods: beginning of fattening (d0), 60th day of fattening (d60), 120th day of fattening (d120) and at the end of fattening (sw). In terms of rs208158047 mutation of Charolais, Limousin and Blonde d’Aquitaine breeds, TT and CT genotypes were identified, and CC genotype was not encountered. The association of average daily gain (ADG) in d0-d60, d0-d120 and d0-sw periods with the genotypes of rs208158047 mutation was found to be significant (Prs208158047-CT genotypes compared to rs208158047-TT genotypes. These results indicate that the selection of bovine NPR2 gene could be used to ensure the breeding direction for growth related traits of the beef cattle.

Maternal nutrition during pregnancy is one of the major intrauterine environmental factors that influence fetal development by significantly altering the expression of genes that might have a consequence on the physiological, morphological, and metabolic performance of the offspring in the postnatal period. The impact of maternal dietary protein on the expression of genes in sheep fetal skeletal muscle development is not well understood. The current study aims to investigate the impact of high and low maternal dietary protein on the holistic mRNA expression in the sheep fetal skeletal muscle. Dams were exposed to an isoenergetic high-protein diet (HP, 160-270 g/day), low-protein diet (LP, 73-112 g/day), and standard protein (SP, 119-198 g/day) diets during pregnancy. Fetal skeletal muscles were obtained at the 105th day of pregnancy and mRNA expression profiles were evaluated using Affymetrix GeneChip (TM) Ovine Gene 1.0 ST Array. The transcriptional analysis revealed a total of 323, 354, and 14 genes were differentially regulated (fold change > 2 and false discovery rate

Paratuberculosis (pTB), also known as Johne's disease (JD), is a contagious, chronic, and granulomatous inflammatory disease of the intestines of ruminants which is caused by Mycobacterium avium subsp. paratuberculosis (MAP) infection, resulting in billions of dollars in economic losses worldwide. Since, currently, no effective cure is available for MAP infection, it is important to explore the genetic variants that affect the host MAP susceptibility. The aim of this study was to analyze a potential association between EDN2 synonymous gene mutations (rs110287192, rs109651404 and rs136707411), that modifies susceptibility to pTB. EDN2 rs110287192, rs109651404 and rs136707411 mutations were genotyped in 68 infected and 753 healthy animals from East Anatolian Red crossbred, Anatolian Black crossbred and Holstein breed cattle by using Custom TaqMan SNP Genotyping Assays. For pTB status, serum antibody levels S/P ≥ 1.0 were assessed in carriers of the different EDN2 genotypes. EDN2 rs110287192 mutation showed a significant association with bovine pTB (adj. p < 0.05). For rs110287192 locus, the odd ratios for GG and TG genotypes versus TT genotypes were 1.73; (95% CI = 0.34–8.59) and 0.53 (95% CI = 0.12–2.37) respectively, which indicated that proportion of TG heterozygotes were significantly higher in control animals as compared to pTB animals. On the other hand, while rs136707411 mutation showed a suggestive association with pTB status in the examined cattle population (nominal p < 0.05); no association was detected between rs109651404 genotypes and pTB status. Selecting animals against rs110287192-GG genotype may decrease the risk of pTB in cattle of the Bos taurus taurus subspecies.

This study was conducted to identify gene polymorphisms and to investigate the relationships ofpolymorphisms with yield and composition of milk from Holstein cows. Significant relationships ofgrowth hormone (GH), leptin (LEP) and myogenic factor 5 (MYF5) gene polymorphisms with 305-day milk yield were observed (P < 0.01). Signal transducers and activators of transcription 5A(STAT5A) gene polymorphism did not show any significant relationships with investigated traits(P > 0.05). With regard to the 305-day milk yield, GH-LV, LEP-AA and AB and MYF5-GG genotypes were found to be superior over the other genotypes. It was concluded based on present findings that GH, LEP and MYF5 genes could be used as candidate genes for milk yield of Holsteincows.

Scrapie is a transmissible spongiform encephalopathy of sheep and goats, and scrapie eradication programs in many parts of the world rely on strong genetic resistance to classical scrapie in sheep. However, the utility of putative resistance alleles in goats has been a focus of research because goats can transmit scrapie to sheep and may serve as a scrapie reservoir. Prior work showed that disease-free survival time was significantly extended in orally inoculated goats singly heterozygous for prion amino acid substitutions S146 or K222, but average durations were only around 3 years post-inoculation. The aim of this study was to investigate whether extended survival would exceed 6 years, which represents the productive lifetimes of most commercial goats. While all control homozygotes were clinically affected by an average of

In order to identify the effects of nutrients on gene expression and to assess the interactions between genes and nutrition by means of various cutting-edge technologies, the interdisciplinary branch ‘Nutrigenomics’ was created. Therefore, nutrigenomics corresponds to the use of knowledge and techniques of nutrition, genomics, transcriptomics, proteomics, epigenomics, and metabolomics to seek and explain the cross-talk between nutrition and genes in molecular level. Macronutrients are important dietary signals that control metabolic programming of cells and have important roles in maintaining cellular homeostasis by influencing specific gene expression. Recent advancements in molecular genetics studies, for instance, use of next-generation sequencing, microarray and qPCR array to investigate the expression of transcripts, genes, and miRNAs, has a crucial impact on understanding and quantitative measurement of the impact of dietary macronutrients on gene function. This review will shade a light on the interactions and mechanisms how the dietary source of macronutrients changes the expression of specific mRNA and miRNA. Furthermore, it will highlight the exciting recent findings in relation to animal performance characteristics which eventually help us to identify a dietary target to improve animal production.

Despite its essential role in ovulation, oxidative stress (OS) has been found to be cytotoxic to cells, while microRNAs (miRNAs) are known as a major regulator of genes involved in cellular defense against cytotoxicity. However, a functional link between OS and miRNA expression changes in granulosa cells (GCs) remains to be investigated. Here, we investigate the OS modulation of apoptosis-associated miRNAs and their biological relevance in bovine GCs. Following the evaluation of cell viability, accumulation of reactive oxygen species (ROS), cytotoxicity and mitochondrial activity, we used a ready-to-use miRNA PCR array to identify differentially regulated miRNAs. The results showed that exposure to 150 mu M H2O2 for 4 h creates remarkable signs of OS in GCs characterized by more than 50% loss of cell viability, higher nuclear factor erythroid 2-related factor 2 (NRF2) nuclear translocation, significantly (p < 0.05) higher abundance of antioxidant genes, significantly (p < 0.001) higher accumulation of ROS, lower mitochondrial activity and a higher (p < 0.001) number of apoptotic nuclei compared to that of the control group. miRNA expression analysis revealed that a total of 69 miRNAs were differentially regulated in which 47 and 22 miRNAs were up- and downregulated, respectively, in stressed GCs. By applying the 2-fold and p < 0.05 criteria, we found 16 miRNAs were upregulated and 10 miRNAs were downregulated. Target prediction revealed that up- and downregulated miRNAs potentially targeted a total of 6210 and 3575 genes, respectively. Pathway analysis showed that upregulated miRNAs are targeting the genes involved mostly in cell survival, intracellular communication and homeostasis, cellular migration and growth control and disease pathways. Our results showed that OS modulates the expression of apoptosis-associated miRNAs that might have effects on cellular or molecular damages.

Follicular development in the mammalian ovary is a complex process which is potentially regulated by an orchestrated action of the pituitary gonadotropins, e.g., follicle stimulating hormone (FSH) and luteinizing hormone (LH), and local ovarian factors, such as peptide growth factors and steroids. Along with hormonal activation it is necessary to have tight coordination of expression of genes during follicular development. This review highlights the structure and function of ovary, follicle and follicular development. This review also confirms the temporal and spatial expression of the specific genes and miRNAs and their involvement in different modulators, the synthesis of active factors, their interactions, and the dynamics of their receptors on the follicular cell surface may be the ultimate determinants of cellular events which are crucial to coordinated growth and differentiation of follicular cells leading to folliculogenesis and ovulation. DOI: http://dx.doi.org/10.3329/ralf.v1i1.22355 Res. Agric., Livest. Fish.1(1): 47-60, Dec 2014

Sulforaphane (SFN) has received a great deal of research attention because of its ability to induce the production of a battery of antioxidant enzymes in certain concentrations through the activation of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathway, which may effectively neutralize reactive oxygen species (ROS) induced oxidative stress. This study was conducted to investigate the potential of different concentrations of SFN in inducing antioxidative and apoptotic effects in granulosa cells (GCs). For this purpose, bovine GCs were collected from preovulatory antral follicles and cultured with different concentrations of SFN (0-80 μM) and based on phenotypic evaluation three concentrations were selected: 2 μM (low), 10 μM (medium), and 20 μM (high) for further investigations. The results showed that there was a dramatic loss of cell viability and higher cytotoxic effects of SFN on GCs at higher concentrations (15 μM). The expression of NRF2 increased significantly (p 0.05) with fold change ranged 3-8 in SFN treated GCs, whereas Kelch Like ECH Associated Protein 1 (KEAP1) expression was either downregulated or similar as control group under the same conditions. Moreover, the relative expression of the genes (PRDX1, CAT, TXN1and SOD1) downstream to NRF2 activation was found to be highly expressed (fold change ranged from 2 to 5, p 0.05) in SFN treated GCs compared to the untreated control. In addition, ROS accumulation was higher in GCs treated with 20 μM SFN which in turn results in a higher accumulation of lipid droplets. Compared to control, no changes in the mitochondrial activity was observed at 2 and 10 μM SFN concentrations; however, significantly lower mitochondrial activity was found at high concentration (20 μM). The results of this study clearly showed that 10 μM SFN concentration played a crucial role in activating Nrf2 pathway without inducing apoptotic characteristics and this concentration may have beneficial effects in boosting the production of phase II antioxidant enzymes in GCs. However, at high concentration (20 μM), SFN may generate excessive ROS that causes mitochondrial dysfunction and induces cellular stress and eventually leads to apoptosis. These data strongly suggest a concentration dependent antioxidative and apoptotic effects of SFN on GCs.

To evaluate associations of glucose-6-phosphate dehydrogenase (G6PDH) activity in sheep oocytes with cytoplasmic lipid content, maturational competence, developmental competence to the blastocyst stage, and gene expression of certain molecular markers., Purpose To evaluate associations of glucose-6-phosphate dehydrogenase (G6PDH)activity in sheep oocytes with cytoplasmic lipid content, maturational competence,developmental competence to the blastocyst stage, and gene expression of certainmolecular markers.Methods Before brilliant cresyl blue (BCB) staining test, oocytes were classified ashigh, middle, and low cytoplasmic lipid content (HCLC, MCLC, and LCLC) and after thetest as having low or high G6PDH-activity (BCB+ and BCB-, respectively). Aftermaturation in vitro, a group of oocytes were subjected to IVF followed by in vitroembryo culture and another group was used for evaluation of expression of candidategenes.Results The cleavage and blastosyst rates were lowest (P < 0.05) in LCLC group,intermediate (P < 0.05) in MCLC group and highest (P < 0.05) in HCLC group. More(P < 0.05) oocytes in HCLC group were BCB+, and higher (P < 0.05) maturation,cleavage, and blastocyst rates were seen for BCB+ oocytes than the BCB- oocytes.Our gene expression data indicated that mRNA transcript abundance of ITGB2, pZP3,BMP15, and GDF9 genes were similar between BCB oocytes groups. However, theexpression of ATP1A1 was higher (P < 0.05) for BCB+ oocytes compared to BCBoocytes.In addition, BAX transcript abundance was similar (P > 0.05) among BCB+,BCB-, and control groups, before and after maturation in vitro.Conclusion Activity of G6PDH in sheep oocytes is highly associated with lipid content,and compared with the morphological parameters, might be a more precise andobjective predictor for subsequent developmental competence in vitro.

Toll-like receptors (TLRs), known as the pathogen recognition receptors, recognize the microbial components. The details expression kinetics of TLRs may help to understand the immune responsiveness of pigs, but the expression patterns of all TLRs (TLR1–10) have not yet been studied in pigs. Therefore, this study unravels the expression patterns of the TLR family set of genes (TLR1–10) in different lymphoid tissues collected from pigs at different ages. A total of nine clinically healthy Pietrain pigs at three age groups were selected for this experiment. Each age group consisted of three animals. Cervical lymph node (CLN), thymus, liver, spleen, lung, heart, skin tissue and peripheral blood mononuclear cells (PBMC) were collected for both mRNA and protein isolation. The mRNA expression of TLRs (1−10) was quantified in all tissues. TLR3 mRNA was the most abundant in all tissues. It was expressed highly in thymus, kidney, lungs and liver tissues. Western blot and immunofluorescence was performed for protein expression and localization of selected TLRs (TLR2, 3 and 9) in selected tissues (CLN, spleen and lungs). The Western blot results of TLR2, 3 and 9 in selected tissues appeared to be consistent with the mRNA expression results. Cells in lungs, spleen and CLN were positively immunostained for TLR2, 3 and 9. This study sheds light on the expression patterns of TLR (1–10) genes in important lymphoid tissues in pigs of different ages. The heterogeneous expression of TLRs with ages may contribute to the altered immune responsiveness of pigs with ages.

The aim of the present study was to investigate the age-related production variation of T helper (Th)-type cytokines (IL-2, IL-4, IFN-gamma and IL-10), granulocyte macrophage-colony stimulating factor (GM-CSF) and nitric oxide (NO) by lipopolysaccharide (LPS)-stimulated porcine alveolar macrophages (AMs) in a time-dependent manner. For this purpose, AMs were isolated from 5-days (newborn), 40-days (post-weaned) and 120-days (young) old pigs. Cells were incubated for 24 h in the absence or presence of increasing concentrations of LPS (0.0, 0.01, 1.0, 5.0 and 10.0 mu g/mL)., The aim of the present study was to investigate the age-related production variation of T helper (Th)-type cytokines (IL-2, IL-4, IFN-γ and IL-10), granulocyte macrophage-colony stimulating factor (GM-CSF) and nitric oxide (NO) by lipopolysaccharide (LPS)-stimulated porcine alveolar macrophages (AMs) in a time-dependent manner. For this purpose, AMs were isolated from 5-days (newborn), 40-days (post-weaned) and 120-days (young) old pigs. Cells were incubated for 24 h in the absence or presence of increasing concentrations of LPS (0.0, 0.01, 1.0, 5.0 and 10.0 μg/mL).IL-10, IFN-γ and GM-CSF mRNA expression was upregulated in a dose-dependent manner for all age groups (P < 0.05). Age-related differences included a significantly increased IL-10 mRNA and protein production in newborn piglets compared to post-weaned and young pigs. IL-10 production pattern was similar with a higher peak between 12-36 h post-induction in all age groups. In contrast, IFN-γ mRNA and protein level was significantly elevated in young pigs 12 h and 24 h post-induction, respectively, while the time course production of IFN-γ was mostly consistent in newborn and post-weaned piglets. GM-CSF mRNA expression was significantly lower in newborn piglets than in post-weaned and young pigs. The kinetic GM-CSF expression peaked at 12 h in young and post-weaned pigs and at 24 h in newborn piglets. IL-4 mRNA levels were very low and no apparent change of IL-2 expression was observed following LPS stimulation in all age groups. Only very low levels of NO were detected in the cell supernatants of young pigs. Collectively, these studies suggest age-related differences in time-dependent production of IL-10, IFN-γ and GM-CSF by porcine AMs with potential immunoregulatory consequences to be explored further.

The aim was to study the expression pattern of the porcine TLR family (TLR1-10) genes in gut-associated lymphoid tissues (GALT) of varying ages. A total of nine clinically healthy pigs of three ages group (1 day, 2 months and 5 months old) were selected for this experiment (three pigs in each group). Tissues from intestinal mucosa in stomach, duodenum, jejunum and ileum and mesenteric lymph node (MLN) were used. mRNA expression of TLRs (1-10) was detectable in all tissues and TLR3 showed the highest mRNA abundance among TLRs. TLR3 expression in stomach, and TLR1 and TLR6 expression in MLN were higher in adult than newborn pigs. The western blot results of TLR2, 3 and 9 in some cases, did not coincide with the mRNA expression results. The protein localization of TLR2, 3 and 9 showed that TLR expressing cells were abundant in the lamina propria, Peyer's patches in intestine, and around and within the lymphoid follicles in the MLN. This expressions study sheds the first light on the expression patterns of all TLR genes in GALT at different ages of pigs. (c) 2013 Elsevier Ltd. All rights reserved., The aim was to study the expression pattern of the porcine TLR family (TLR1-10) genes in gut-associated lymphoid tissues (GALT) of varying ages. A total of nine clinically healthy pigs of three ages group (1day, 2months and 5months old) were selected for this experiment (three pigs in each group). Tissues from intestinal mucosa in stomach, duodenum, jejunum and ileum and mesenteric lymph node (MLN) were used. mRNA expression of TLRs (1-10) was detectable in all tissues and TLR3 showed the highest mRNA abundance among TLRs. TLR3 expression in stomach, and TLR1 and TLR6 expression in MLN were higher in adult than newborn pigs. The western blot results of TLR2, 3 and 9 in some cases, did not coincide with the mRNA expression results. The protein localization of TLR2, 3 and 9 showed that TLR expressing cells were abundant in the lamina propria, Peyer's patches in intestine, and around and within the lymphoid follicles in the MLN. This expressions study sheds the first light on the expression patterns of all TLR genes in GALT at different ages of pigs.

The objective of the present study was to investigate LPS and lipoteichoic acid (LTA)-induced TLRs, associated signaling molecules and inflammatory mediators, as well as to compare their combined effect in porcine alveolar macrophages. Macrophages were incubated for 24h with various concentrations of LPS, LTA, LPS+LTA or control. Multiple concentrations of LPS elicited marked up-regulation in mRNA for TLR2 and TLR4, CD14, MD2, MyD88, IRAK-4 and TRAF6 compared with the control. LTA had no effect on TLR4 and MD2; only higher doses up-regulated TLR2, CD14, MyD88, IRAK-4 and TRAF6 mRNA. LPS-activated cells released IL1-, IL12-, TNF-, IL-6, IL-8, IFN- and IL-10 in a dose-dependent manner, while LTA had no effect on IL-1, IL-6 and IFN-. Higher doses of LTA induced IL-12, TNF-, IL-8 and IL-10. Combined stimulation augmented TLR2, CD14 and MyD88 mRNA, and subsequently produced elevated levels of IL-6, TNF- and IL-8 when compared with LPS and LTA alone. Additionally, phagocytosis of macrophages was significantly increased following low concentration of LPS treatment. Only low levels of NO (nitric oxide) were detected in the LPS group. Overall, compared with LPS, LTA was a relatively weak inducer, and co-stimulation accelerated gene and cytokine production associated with pulmonary innate immune function., The objective of the present study was to investigate the lipopolysaccharide (LPS) and lipotechoic acid (LTA)-induced TLRs, associated signaling molecules and inflammatory mediators, as well as to compare their combined effect in porcine alveolar macrophages. Macrophages were incubated for 24 h with various concentrations of LPS, LTA, LPS plus LTA or control. Multiple concentrations of LPS elicited the marked up-regulation in mRNA for TLR-2 and-4, CD-14, MD-2, MyD-88, IRAK-4, TRAF-6, as compared to control. LTA had no effect on TLR-4 and MD-2; only higher doses up-regulated TLR-2, CD-14, MyD-88, IRAK-4 and TRAF-6 mRNA. LPS-activated cells released IL1-ß, IL12-ß, TNF-?, IL-6, IL-8, IFN-? and IL-10 in a dose-dependent manner while LTA had no effect on IL-1ß, IL-6 and IFN-?. Higher doses of LTA induced IL-12ß, TNF-?, IL-8 and IL-10. Combined stimulation augmented TLR-2, CD-14 and MyD-88 mRNA, and subsequently produced elevated levels of IL-6, TNF-? and IL-8 when compared to LPS and LTA alone. Additionally, phagocytosis of macrophages was significantly increased following low concentration of LPS treatment. Only low levels of nitric oxide were detected in LPS group. Overall, compared to LPS, LTA was a relatively weak inducer, and co-stimulation accelerated genes and cytokines production associated with pulmonary innate immune function.

The aim of the present study was to determine the age-related changes of phagocytic capacity and the kinetic production of cytokines in lipopolysaccharide-stimulated porcine alveolar macrophages. For this purpose, AMs were isolated from 5 (newborn), 40 (post-weaned) and 120 (young) day old pigs. Results of phagocytosis assay showed that AMs from newborn piglets had less phagocytic capacity than those of young pigs (P < 0.05). For the kinetics study, cells and supernatant were collected at 1, 6, 12, 24, 36 and 48 h after LPS stimulation for quantification of cytokine mRNA and protein by quantitative real-time PCR and ELISA, respectively. The kinetics results showed that AMs from newborn piglets were significantly less capable of producing IL1 beta, IL6, IL2 beta, TNF alpha and IL8 than post-weaned piglets or young pigs. IL18 mRNA did not show significant differences between ages. MIP2 and MCP1 mRNA was higher in young pigs. Hence, higher production of cytokines by AMs may be the surfactant factors in the pulmonary host defense system. These results indicate that AMs from newborn piglets might be functionally immature, which may lead to increased susceptibility to lung infections. Future studies of cytokine kinetics in more animals are clearly needed to confirm these results across a wider age range. (C) 2012 Elsevier Ltd. All rights reserved., The aim of the present study was to determine the age-related changes of phagocytic capacity and the kinetic production of cytokines in lipopolysaccharide-stimulated porcine alveolar macrophages. For this purpose, AMs were isolated from 5 (newborn), 40 (post-weaned) and 120 (young) day old pigs. Results of phagocytosis assay showed that AMs from newborn piglets had less phagocytic capacity than those of young pigs (P

Pulmonary alveolar macrophages (AMs) are important in defense against bacterial lung inflammation. Cluster of differentiation 14 (CD14) is involved in recognizing bacterial lipopolysaccharide (LPS) through MyD88-dependent and TRIF pathways of innate immunity. Sulforaphane (SFN) shows anti-inflammatory activity and suppresses DNA methylation. To identify CD14 epigenetic changes by SFN in the LPS-induced TRIF pathway, an AMs model was investigated in vitro. CD14 gene expression was induced by 5 mu g/ml LPS at the time point of 12 h and suppressed by 5 mu M SFN. After 12 h of LPS stimulation, gene expression was significantly up-regulated, including TRIF, TRAF6, NF-B, TRAF3, IRF7, TNF-, IL-1, IL-6, and IFN-. LPS-induced TRAM, TRIF, RIPK1, TRAF3, TNF-, IL-1 and IFN- were suppressed by 5 mu M SFN. Similarly, DNMT3a expression was increased by LPS but significantly down-regulated by 5 mu M SFN. It showed positive correlation of CD14 gene body methylation with in LPS-stimulated AMs, and this methylation status was inhibited by SFN. This study suggests that SFN suppresses CD14 activation in bacterial inflammation through epigenetic regulation of CD14 gene body methylation associated with DNMT3a. The results provide insights into SFN-mediated epigenetic down-regulation of CD14 in LPS-induced TRIF pathway inflammation and may lead to new methods for controlling LPS-induced inflammation in pigs., Pulmonary alveolar macrophages (AMs) are important in defense against bacterial lung inflammation. Cluster of differentiation 14 (CD14) is involved in recognizing bacterial lipopolysaccharide (LPS) through MyD88-dependent and TRIF pathways of innate immunity. Sulforaphane (SFN) shows anti-inflammatory activity and suppresses DNA methylation. To identify CD14 epigenetic changes by SFN in the LPS-induced TRIF pathway, an AMs model was investigated in vitro CD14 gene expression was induced by 5 µg/ml LPS at the time point of 12 h and suppressed by 5 µM SFN. After 12 h of LPS stimulation, gene expression was significantly up-regulated, including TRIF, TRAF6, NF-κB, TRAF3, IRF7, TNF-α, IL-1β, IL-6, and IFN-β. LPS-induced TRAM, TRIF, RIPK1, TRAF3, TNF-α, IL-1β and IFN-β were suppressed by 5 µM SFN. Similarly, DNMT3a expression was increased by LPS but significantly down-regulated by 5 µM SFN. It showed positive correlation of CD14 gene body methylation with in LPS-stimulated AMs, and this methylation status was inhibited by SFN. This study suggests that SFN suppresses CD14 activation in bacterial inflammation through epigenetic regulation of CD14 gene body methylation associated with DNMT3a. The results provide insights into SFN-mediated epigenetic down-regulation of CD14 in LPS-induced TRIF pathway inflammation and may lead to new methods for controlling LPS-induced inflammation in pigs.

Hydrogen peroxide (H2O2) and sulforaphane (SFN) are classical reagents to study oxidative stress and antioxidant activity in vitro, respectively. Micromole of H2O2 is sufficient to induce oxidative stress. Similarly, micromole of SFN is able to induce protective activity of cells against oxidative stress. However, higher or lower concentrations of both H2O2 and SFN may lead to aberrant results. Therefore, current study aimed to find out the appropriate concentrations of H2O2 and SFN for granulosa cells in vitro. For this, granulosa cells were collected and cultured with DMEM-F12 media at 37◦ C in a humidified environment until 60–70% confluence was achieved. After that different concentrations of H2O2 (0–400 M) and SFN (0–80 M) were added to the culture media and continue culture for 24 h. The results showed that cell viability decreased in a concentration dependent manner for both H2O2 and SFN treated cells. 50–75 MH2o2 are able to induce morphologically visible stress on cells, whereas 10–15 M SFN showed no effects on cellular morphology and viability. Analysis of candidate transcripts showed that, in H2O2 groups, 50–75 M create significantly higher expression of NRF2 & CASP3 and lower expression of KEAP1. On the other hand, 10–15 M SFN exhibited significant higher expression of NRF2 and lower expression of KEAP1, while expression of CASP3 remains unchanged. It is clear from the morphologic and genetic observations that 50–75 MH2o2 and 10–15 M SFN showed excellent response and are recommended concentrations for granulosa cells to study oxidative stress in vitro.

ESR2 is involved in oestrogen-related apoptosis in cell cycle spermatogenesis but their effects have not yet confirmed in pig. Therefore, this study was aimed to investigate the association of ESR2 polymorphism with sperm quality and boar fertility traits and to analyse the ESR2 mRNA and protein expressions in boar reproductive tissues. DNA samples from 203 Pietrain (PI) and 100 Pietrain × Hampshire (PIHA) pigs with records of sperm quality [sperm concentration (SCON), motility (MOT), semen volume (VOL), plasma droplet rate (PDR) and abnormal spermatozoa rate (ASR)] and fertility [non-return rate (NRR) and number of piglet born alive (NBA)] traits were available. A SNP in coding region of ESR2 g.35547A>G in exon 5 was associated with MOT and PDR in the PI and with SCON, VOL, MOT and PDR in PIHA population. For mRNA and protein expression study, a total of six boars were divided into two groups with group I (G-I) and group II (G-II) where G-I characterized for relatively a better sperm quality according to the mean of two groups. mRNA expression was higher in brain and testis than that in all parts of epididymis. Both qRT-PCR and western blot analysis revealed that the ESR2 gene expression and protein expression were significantly higher in testis collected from G-II compared with that of G-I boars. Moreover, ESR2 protein localization in germ cell, Leydig and Sertoli cells, epithelial cells and spermatozoa was remarkable, which indicated the important role of ESR2 in spermatogenesis process. These results might shed new light on the roles of ESR2 in spermatogenesis as candidate for boar fertility, but still the lack of association across populations should be considered.

SummaryMicroRNAs (miRNAs) are small endogenous molecules that are involved in a diverse of cellular process. However, little is known about their abundance in bovine oocytes and their surrounding cumulus cells during oocyte development. To elucidate this situation, we investigated the relative expression pattern of sets of miRNAs between bovine oocyte and the surrounding cumulus cells duringin vitromaturation using miRNA polymerase chain reaction (PCR) array. Results revealed that a total of 47 and 51 miRNAs were highly abundant in immature and matured oocytes, respectively, compared with their surrounding cumulus cells. Furthermore, expression analysis of six miRNAs enriched in oocyte miR-205, miR-150, miR-122, miR-96, miR-146a and miR-146b-5p at different maturation times showed a dramatic decrease in abundance from 0 h to 22 h of maturation. The expression of the same miRNAs in preimplantation stage embryos was found to be highly abundant in early stages of embryo development and decreased after the 8-cell stage to the blastocyst stage following a typical maternal transcript profile. Similar results were obtained by localization of miR-205 in preimplantation stage embryos, in which signals were higher up to the 4-cell stage and reduced thereafter. miR-205 and miR-210 were localizedin situin ovarian follicles and revealed a spatio-temporal expression during follicular development. Interestingly, the presence or absence of oocytes or cumulus cells during maturation was found to affect the expression of miRNAs in each of the two cell types. Hence, our results showed the presence of distinct sets of miRNAs in oocytes or cumulus cells and the presence of their dynamic degradation during bovine oocyte maturation.

Among modern western pigs, Duroc (high meat fat ratio) and Pietrain (low meat fat ratio) breeds extensively utilized in commercial pork production differ extremely for their muscle phenotypes. The molecular mechanism, especially the epigenetic mechanism, underlying these breed-specific differences is poorly known. Myogenic factor 6 (MYF6) is the most abundantly expressed myogenic factor in adult muscle. Moreover, MYF6 tends to be expressed more highly in muscle tissue of the lean selection line and is supposed to be one promising candidate gene for growth- and meat quality-related traits in adult pigs. Six months old female Duroc and Pietrain pure breed pigs were used in this study. Protein and mRNA levels of MYF6 in loin eye muscle were determined by Western blotting and quantitative Real-time reverse transcription PCR (qRT-PCR), respectively. The DNA methylation status of the MYF6 5'-regulatory region was determined by bisulfite sequencing PCR (BSP). The global Histone 4 acetylation at lysines 5 (H4K5) and 8 (H4K8) were examined by Western blotting. Pietrain pigs exhibited significant higher expression of MYF6 and hypermethylated E2F1 binding element within MYF6 5'-regulatory region as compared with Duroc pigs. Significant elevation in DNA methyltransferase 1 (DNMT1) expression was observed in Pietrain pigs which are in agreement with hypermethylation of MYF6. Histone acetylation level at neither H4K5 nor H4K8 is significant between two breed pigs. Nevertheless, mRNA and protein expression of E2F1 were significantly elevated in the Pietrain breed. It is thus conceivable that the upregulation of MYF6 transcription in postnatal Pietrain pigs is not associated with cis-activation by epigenetic modification of MYF6 5'-regulatory region, but may be attributed to trans-activation through enriched expression of E2F1. (C) 2011 Elsevier B.V. All rights reserved.

Cytochrome P450 aromatase is the key enzyme in estrogen biosynthesis, encoded by the CYP19 gene. However, little is know about the CYP19 roles in boar spermatogenesis. Therefore, the aim of this work was to investigate the mRNA and protein expression of CYP19 in boar reproductive tissues from boars with different sperm quality. For mRNA and protein expression study, a total of six boars were divided into two groups with Group I (G-I) and Group II (G-II), where G-I is characterized for a relatively better sperm quality. For the expression study between reproductive and non-reproductive tissues by semi-quantitative PCR study, mRNA from all six boars was pooled together according to the tissues. On the other hand, mRNA and protein expression study in different reproductive tissues from two divergent groups of animals were performed by semi-quantitative PCR, qRT-PCR and western blot, respectively. Due to the limitations of fresh samples from G-I and G-II boars, different fresh testis from a healthy breeding boar was collected after slaughtering for protein localization by immunofluorescence. The remarkable CYP19 mRNA expression was detected only in testis. The mRNA expression of CYP19 was not detectable in other reproductive tissue (epididymis and accessory glands) and non-reproductive tissue (brain, liver and muscle) by semi-quantitative PCR.

The aim of this research was to screen for polymorphism and to perform an association study of IFI6 with meat and carcass quality traits. A SNP (g.370A>G) was detected which was associated (P < 0.05) with meat colour, pH 24 h post mortem (p.m.) in ham, conductivity 45 min p.m. in loin and conductivity 24 h p.m. in ham, drip loss and carcass length in Duroc × Pietrain and with meat colour, muscle area and ham percentage in the Pietrain population. Highest expression of IFI6 mRNA was detected in skeletal muscle (longissimus dorsi) by qRT-PCR comparing different tissues. Both qRT-PCR and western blot revealed that the IFI6 gene and protein expressions were significantly (P < 0.05) higher in skeletal muscle with low drip loss compared to that of high drip loss. IFI6 protein was localized in the myocytes membrane. Results suggested that IFI6 might play roles in meat and carcass quality and is a potential positional, physiological and functional candidate gene for improving meat quality traits in pigs.

Phospholipase C zeta (PLCz) and cyclooxygenase isoenzyme type 2 (COX-2) are important in spermatogenesis, but their effect has not yet confirmed in pigs. Therefore, this study was aimed to analyse their association with sperm quality and fertility and to identify the mRNA and protein expression in boars reproductive tissues. DNA samples from 231 Pietrain (PI) and 109 Pietrain × Hampshire (PIHA) pigs with records of sperm quality [sperm concentration (SCON), motility, semen volume, plasma droplet and abnormal spermatozoa rate] and fertility (non-return rate and number of piglet born alive) traits were available. A SNP in non-coding region of PLCz g.158 A > C was associated with SCON (p A in 3' UTR was not associated with any traits. For mRNA and protein expression study, a total of six boars were divided into two groups with G-I and G-II, where G-I was characterized for relatively better sperm quality. Both genes expressed higher in reproductive tissues compared with non-reproductive tissues. Phospholipase C zeta mRNA expressed higher in testis (p < 0.01), all parts of epididymis and spermatozoa from G-I, while COX-2 expressed higher in testis (p < 0.05), head and body of epididymis (p < 0.01), and spermatozoa from G-II boar. Both proteins were localized in Leydig cells and spermatozoa. These results might shed light on roles of these genes in spermatogenesis as candidate for boar sperm quality and fertility, but still the lack of association across populations should be considered.

Osteochondrosis (OC) or leg weakness is an economically important disease of young fast growing pigs and is a concern of animal welfare. The etiology and pathogenesis of osteochondrosis is not fully understood yet, but any abnormalities in the formation of hypertrophic chondrocytes and disrupted blood supply to the growth cartilage are very important predisposing factors. Matrix gla protein (MGP) as a potential calcification inhibitor of extracellular matrix might contribute to the development of OC. Molecular characterization, polymorphisms analysis, methylation at promoter region and expression of MGP gene and protein were performed in both healthy and OC cartilage collected from a Duroc x Pietrain resource population. The porcine MGP gene consists of 4 exons and 3 introns. The full-length MGP cDNA isolated from articular cartilage consists of 606 bp with a 69-bp 5' UTR, a 312-bp open reading frame with a start codon, a 225-bp 3' UTR. Three single-nucleotide polymorphisms (SNP) were detected in the intron 1 (A-115G, C-1073T and C-1135A) and one in the 3'UTR (C-3767T). The relative abundance of MGP mRNA was lower (P

The purpose of the present study was to determine the Glutathione Peroxidase 1 (GPX 1) gene expression by Real Time PCR in the liver of healthy and diabetic rats treated with capsaicin. Twenty Sprague-Dawley rats were used in our study. Rats were divided into four groups: Group I: diabetic rats (n=5), Group II: capsaicin injected rats (n=5), Group III: Capsaicin injected diabetic rats (n=5), and Group IV: control rats (n=5). Capsaicin injection began 72 h after streptozotocin (STZ) injection. Capsaicin (1 mg/kg) was prepared in 10% ethanol, 1% Tween 20, and 80% sterile water and subcutaneously injected daily in both Groups II and III for two weeks. The results of RT-PCR conducted to determine GPX 1 gene expression indicated that capsaicin treated diabetic rats had higher GPX 1 expression compared to all groups including control (P

To study the effect of alpha-linolenic acid (ALA) on meiotic maturation, mRNA abundance of apoptosis-related (Bax and Bcl-2) molecules, and blastocyst formation in ovine oocytes., Purpose To study the effect of α-linolenic acid (ALA) on meiotic maturation, mRNAabundance of apoptosis-related (Bax and Bcl-2) molecules, and blastocyst formation inovine oocytes.Methods A preliminary experiment was conducted to analyze the concentration of ALAin "small" (≤2 mm) and "large" (≥6 mm) follicles using gas chromatography/massspectrometry analysis. The concentration of ALA in small and large follicles wasdetermined to be in a range of 75.4 to 125.7 μM, respectively. In vitro maturation (IVM)of oocyte was then performed in presence of 0 (control), 10 (ALA-10), 50 (ALA-50),100 (ALA-100), and 200 (ALA-200) μM of ALA. Meiotic maturation and mRNAabundance of Bax, and Bcl-2 genes was evaluated after 24 h of IVM. The embryoniccleavage and blastocyst formation following parthenogenetic activation were alsodetermined for each group.Results The highest concentration of ALA (ALA-200) decreased the oocyte maturationrate compared with the control group. Analysis of apoptosis-related genes in oocytesafter IVM revealed lesser transcript abundances for Bax gene, and higher transcriptabundances for Bcl-2 gene in ALA-treated oocytes as compared with the controloocytes. In term of cleavage rate (considered as 2-cell progression), we did notobserve any differences among the groups. However, ALA-100 group promoted moreblastocyst formation as compared with the control group.Conclusion Our results suggested that ALA treatment during IVM had a beneficial effect on developmental competence of ovine oocytes by increasing the blastocystformation and this might be due to the altered abundance of apoptosis-regulatorygenes.

Dendritic cell (DC) subsets form a remarkable cellular network that regulate innate and adaptive immune responses. Although pigs are the most approximate model to humans, little is known about the regulation of monocyte-derived DCs (moDCs) and splenic DCs (SDCs) in the initiation of immune responses under inflammatory conditions. We investigated the activation and maturation of porcine moDC and SDC subpopulations following LPS stimulation. Porcine monocytes that would differentiate into moDCs were isolated. SDCs were isolated directly from the porcine spleen. Following LPS stimulation, phagocytosis activity, TLR4/MyD88-dependent gene expression, co-stimulatory molecule, and pro-inflammatory cytokine (TNF-α, IL-1β) and chemokine (IL-8) expressions were increased in both cell subsets. Furthermore, moDCs showed higher levels of gene and protein expression compared with SDCs. Interestingly, moDCs were found to be more responsive via the TLR4/TRAF-dependent signalling pathway of activation. Only SDCs expressed higher level of IL-12p40 gene and protein, whereas, IFN-γ gene and protein expression were likely to be unchanged after LPS stimulation in both cell subtypes. These data demonstrate that porcine moDCs display a greater ability to initiate innate immune responses, and could be used as a model to investigate immune responses against Ags.

Osteochondrosis (OC) is a joint disorder that frequently causes leg weakness in growing pigs, resulting in welfare problems and economic losses. We aimed to detect molecular pathways relevant to the emergence of the disease and to identify candidate genes for the liability to the disorder. Therefore, we compared microarray-based expression patterns of articular cartilage with ( n = 11) and without ( n = 11) histologically diagnosed OC lesions obtained from discordant sib-pairs. A total of 1,564 genes were found with different transcript abundance [differentially expressed (DE) genes] at q ≤ 0.05. To further identify candidate genes, we integrated data from quantitative trait loci (QTL) and genome-wide association (GWA) studies with the expression analysis. We detected 317 DE genes within the QTL confidence intervals, of which 26 DE genes also overlapped GWA regions. Ingenuity Pathway Analysis suggests a pathogenic role of immune response, angiogenesis, and synthesis of extracellular matrix pathways for OC. These processes could facilitate the emergence of defects. But they may also promote the degradation of articular cartilage and the worsening of the disease. A functional network was derived that comprised genes with functional and positional clues of their role in bone and cartilage metabolisms and development, including extracellular matrix genes (e.g., LOX, OGN, and ASPN), angiogenesis genes (e.g., ANGPTL4 and PDGFA), and immune response genes (e.g., ICAM1, AZGP1, C1QB, C1QC, PDE4B, and CDA). The study identified molecular processes linked to OC and several genes with positional, genetic-statistical, and functional evidence for their role in the emergence of articular cartilage lesions and the liability to OC.

To associate glucose-6-phosphate dehydrogenase (G6PDH) activity in goat oocytes with intracellular glutathione (GSH) content, meiotic competence, developmental potential, and relative abundance of Bax and Bcl-2 genes transcripts., Purpose To associate the glucose-6-phosphate dehydrogenase (G6PDH) activity inimmature goat oocyte with intracellular glutathione (GSH) content, developmentalcompetence after parthenogenetic activation and relative abundance of Bax and Bcl-2genes transcripts using .Methods Goat oocytes were exposed to brilliant cresyl blue (BCB) staining andcategorized into BCB+ (blue cytoplasm) and BCB− (colorless cytoplasm) groups. Agroup of oocytes were not exposed to BCB stain and was considered as a control.After maturation in vitro, a group of oocytes were used for determination of nuclearstatus and intracellular GSH content and another group were subjected toparthenogenetic activation followed by in vitro embryo culture.Results We found that BCB+ oocytes yielded a significantly higher maturation rate thanthe BCB− and control oocytes. Moreover, BCB+ oocytes showed increased inintracellular level of GSH than BCB− and control oocytes. Furthermore, BCB+ oocytesproduced more blastocysts than BCB− and control oocytes. The expression of antiapoptoticgene Bcl-2 and pro-apoptotic gene Bax in mature oocyte, their surroundingcumulus cells and blastocyst was affected by BCB staining test.Conclusions These results strongly suggest that BCB+ oocytes have a higher meioticand cytoplasmic maturity (as determined by intracellular GSH content), and that BCBstaining can be used to select developmentally competent oocytes for in vitro embryoproduction.

The liability to lesions of dysfunctions of bone and joints in pigs, summarized as leg weakness and mostly expressed as osteochondrosis, is an animal welfare and economic issue in pig production. The objective of this study was to identify polymorphisms in the functional and positional candidate genes keratin 8 (KRT8), Fas-associated factor 1 (FAF1) and parathyroid hormone type I receptor (PTH1R) and to evaluate their association with leg weakness traits. Therefore, osteochondrosis lesions were scored in animals of a Duroc x Pietrain F2 population (DuPi; n = 310) and commercial herds of the breed Large White (n = 298). In addition, bone mineralization traits were observed in DuPi population. SNPs were identified in genes KRT8 (g.8,039G > A), FAF1 (g.380,914T > C) and PTH1R (c.1,672C > T). KRT8 showed significant association with bone mineral density and content (P a parts per thousand currency sign 0.05). FAF1 was association with OC lesions score of all joints inspected (P a parts per thousand currency sign 0.05). PTH1R showed significant dominance effects on OC lesion scores of the distal femur articular cartilage (P = 0.01) and epiphysis of the distal ulna (P = 0.05) as well as sums of scores of all joints (OCsum, P = 0.04) and assignment to groups of either severely or gently affected animals (OCcat, P = 0.01). This study reveals clear genetic-statistical evidence for a link of KRT8, FAF1 and PTH1R with some of leg weakness related traits in pigs., The liability to lesions of dysfunctions of bone and joints in pigs, summarized as leg weakness and mostly expressed as osteochondrosis, is an animal welfare and economic issue in pig production. The objective of this study was to identify polymorphisms in the functional and positional candidate genes keratin 8 (KRT8), Fas-associated factor 1 (FAF1) and parathyroid hormone type I receptor (PTH1R) and to evaluate their association with leg weakness traits. Therefore, osteochondrosis lesions were scored in animals of a Duroc × Pietrain F2 population (DuPi; n = 310) and commercial herds of the breed Large White (n = 298). In addition, bone mineralization traits were observed in DuPi population. SNPs were identified in genes KRT8 (g.8,039G > A), FAF1 (g.380,914T > C) and PTH1R (c.1,672C > T). KRT8 showed significant association with bone mineral density and content (P ? 0.05). FAF1 was association with OC lesions score of all joints inspected (P ? 0.05). PTH1R showed significant dominance effects on OC lesion scores of the distal femur articular cartilage (P = 0.01) and epiphysis of the distal ulna (P = 0.05) as well as sums of scores of all joints (OCsum, P = 0.04) and assignment to groups of either severely or gently affected animals (OCcat, P = 0.01). This study reveals clear genetic-statistical evidence for a link of KRT8, FAF1 and PTH1R with some of leg weakness related traits in pigs.

MicroRNA (miRNA) are small regulatory RNA molecules that are implicated in regulating and controlling a wide range of physiological processes including cell division, differentiation, migration, apoptosis, morphogenesis, and organogenesis. The aim of this study was to determine the expression pattern of 32 miRNA and 18 miRNA processing machinery genes during somite formation in quail embryos. The embryos were collected at stages HH (Hamburger and Hamilton) 4, 6, and 9 of embryo development (19, 24, and 30 h of incubation, respectively). Total RNA including miRNA was isolated from 4 groups of embryos (each group consisting of 6 to 8 embryos) were collected at each of the 3 stages (19, 24, and 30 h). The expression pattern of candidate miRNA and miRNA processing machinery genes was performed using quantitative real-time PCR. The results demonstrated that 7 miRNA (let-7a, mir-122, mir-125b, mir-10b, P < 0.01; let-7b, mir-26a, and mir-126, P < 0.05) were differentially expressed during early quail embryo development. In addition, the expression profile of 18 miRNA processing machinery genes was not significantly increased at 30 h of incubation compared with both 19 and 24 h. Our results suggest that machinery genes for miRNA biogenetic pathways are functional, and hence, miRNA may be involved in the regulation of early quail development. These 7 differentially expressed miRNA are suggested to play critical roles in quail embryo somite formation., MicroRNAs (miRNA) are small regulatory RNA molecules that are implicated in regulating and controlling a wide range of physiological processes including cell division, differentiation, migration, apoptosis, morphogenesis, and organogenesis. The aim of this study was to determine the expression pattern of 32 miRNAs and 18 miRNA processing machinery genes during somite formation in quail embryos. The embryos were collected at stages HH (Hamburger and Hamilton) 4, 6 and 9 of embryo development (19, 24 and 30h of incubation, respectively). Total RNA including miRNA was isolated from four groups of embryos (each group consisting of 6-8 embryos) were collected at each of the three stages (19 h, 24 h and 30 h). The expression pattern of candidate miRNAs and miRNA processing machinery genes was performed using quantitative Real-Time polymerase chain reaction (qPCR). The results demonstrated that 7 miRNAs (let-7a, mir-122, mir-125b, mir-10b, P < 0.01; let-7b, mir-26a and mir-126, P < 0.05) were differentially expressed during early quail embryo development. In addition the expression profile of 18 miRNA processing machinery genes was not significantly increased at 30 h of incubation compared to both 19 and 24 h. Our results suggest that machinery genes for miRNAs biogenetic pathways are functional and hence, miRNAs may be involved in the regulation of early quail development. These seven differentially expressed miRNAs are suggested to play critical roles in quail embryo somite formation.

An impaired uterine environment triggered by the incidence of subclinical endometritis often compromises fertility in the bovine. The uterus is a dynamic organ with tight regulation of specific genes at the transcriptional and translational levels. Herein, we hypothesised that subclinical endometritis alters the expression of uterine microRNAs (miRNAs), which may result in the dysregulation of corresponding target genes and biological pathways. To test this hypothesis, we used a genome-wide RT2 (Exiqon, Vedbaek, Denmark) miRNA PCR array consisting of 354 miRNA primers and analysed miRNA expression in uterine cytobrush samples taken from cows with and without subclinical endometritis. The results revealed aberrant expression of 23 miRNAs in cows with subclinical endometritis compared with healthy cows. Furthermore, we designed an in vitro endometrial cell culture model challenged by lipopolysaccharide (LPS) to validate the differential regulation of miRNAs in cytobrush samples. Interestingly, we observed similar expression miRNA patterns in cytobrush samples taken from cows with or without subclinical endometritis and in vitro cultured endometrial cells challenged by LPS. To trace signalling pathways and biological functions potentially controlled by the aberrantly expressed miRNAs, we filtered high-ranking target genes from miRBase and analysed them using ingenuity pathway analysis. The gene networks, canonical pathways and biological functions strikingly converged to signalling pathways that mediate inflammatory responses, cellular proliferation, cell movement, the cell cycle and apoptosis in the bovine endometrium. In addition, expression analysis of key genes from the gene networks confirmed their presence and the potential regulation of these genes by uterine miRNAs. Furthermore, luciferase assay data substantiated the primary information from bioinformatic prediction that generated potential target genes for the dysregulated miRNAs in subclinical endometritis. Together, these data suggest the potential regulatory role of uterine miRNAs in the development and progression of bovine subclinical endometritis.

Osteochondrosis (OC) is an orthopedic syndrome of the joints that occurs in children and adolescents and domestic animals, particularly pigs, horses, and dogs. OC is the most frequent cause of leg weakness in rapidly growing pigs causing animal welfare issues and economic losses. In this study, a genomewide association study (GWAS) was performed using the Porcine 60k SNPChip in animals of the breed Large White (n=298) to identify chromosome regions and candidate genes associated with OC lesion scores. A total of 19 SNPs on chromosomes (SSC) 3, 5, 8, 10, 14 and 18 were significantly associated with OC lesion scores (p-values ≤ 10-5). The SNPs MARC0098684, MARC00840086, MARC0093124 and ASGA0062794 at SSC14 36.1 to 38.2 Mb encompass a region of six linkage disequilibrium (LD) blocks. The most significant SNP ASGA0062794 is located in a LD block spanning 465 kb and covering the gene encoding T-box transcription factor 5 (TBX5). A SNP (c.54T>C) identified in TBX5 was significantly associated with OC lesions scores in a single marker analysis. TBX5 c.54T>C showed highest linkage disequilibrium with ASGA00627974 (r2=0.96) and superior association with OC lesion scores over other SNPs when included in the genome scan, whereas its treatment as an additional fixed effect in the GWAS statistical model led to a drop of significance of nearby markers. Moreover, real time PCR showed different transcript abundance of TBX5 in healthy and defect cartilage. The results imply that the association signal obtained on SCC14 is largely attributable to TBX5 c.54T>C likely to be in linkage disequilibrium with a regulatory polymorphism of TBX5. The transcription factor TBX5 interacts with GJA5 and MEF2C both being involved in vascularization. This study provides evidence for epistatic interaction of TBX5 and MEF2C, thus supporting deficiency of blood supply to growth cartilage as being fundamental for the initiation of osteochondrosis.

Satellite cells function as skeletal muscle stem cells to support postnatal muscle growth and regeneration following injury or disease. There is great promise for the improvement of muscle performance in livestock and for the therapy of muscle pathologies in humans by the targeting of myostatin (MSTN) in this cell population. Human diet contains many histone deacetylase (HDAC) inhibitors, such as the bioactive component sulforaphane (SFN), whose epigenetic effects on MSTN gene in satellite cells are unknown. Therefore, we aimed to investigate the epigenetic influences of SFN on the MSTN gene in satellite cells. The present work provides the first evidence, which is distinct from the effects of trichostatin A (TSA), that SFN supplementation in vitro not only acts as a HDAC inhibitor but also as a DNA methyltransferase (DNMT) inhibitor in porcine satellite cells. Compared with TSA and 5-aza-2'-deoxycytidine (5-aza-dC), SFN treatment significantly represses MSTN expression, accompanied by strongly attenuated expression of negative feedback inhibitors of the MSTN signaling pathway. miRNAs targeting MSTN are not implicated in posttranscriptional regulation of MSTN. Nevertheless, a weakly enriched myoblast determination (MyoD) protein associated with diminished histone acetylation in the MyoD binding site located in the MSTN promoter region may contribute to the transcriptional repression of MSTN by SFN. These findings reveal a new mode of epigenetic repression of MSTN by the bioactive compound SFN. This novel pharmacological, biological activity of SFN in satellite cells may thus allow for the development of novel approaches to weaken the MSTN signaling pathway, both for therapies of human skeletal muscle disorders and for livestock production improvement.

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.

Muscle regulatory factors (MRFs) play a major role in muscle growth and development. Thus, they are considered as candidate genes for meat production traits in pigs. These basic helix-loop-helix (bHLH) and paired box transcription factors together with Bone morphogenetic proteins, regulate myogenesis: they initiate the formation of muscle fibers and regulate the transcription of muscle specific genes. The objective of this study was to investigate the mRNA abundance of transcription factor (TF) genes (MYOD, MYF5, MYOG, PAX3, PAX7, BMP2 and BMP4) in the adult skeletal muscle (longissimus dorsi) of divergent Duroc (high meat fat ratio) and Pietrain (low meat fat ratio) pig breeds. For this purpose, purebred Duroc and Pietrain animals (for each breed n = 5) were used for mRNA expression study. Highest gene expression was observed for MYOG, followed by BMP2. The lowest gene expression was observed for PAX3. Moreover, we statistically investigated differences in the expression profiles of these genes between the Duroc and Pietrain breeds. Among seven genes MYF5 and PAX3 showed different expression (P

The aim of the present study was to determine the age-related kinetic changes of Toll-like receptors (TLRs) and downstream genes expression, and secretion of cytokine in lipopolysaccharide (LPS) stimulated porcine alveolar macrophages (AM). For this purpose, AMs were isolated from 5-day-old newborn piglets and 120-day-old young pigs. mRNA expression and cytokine measurement was determined by quantitative real-time PCR and ELISA, respectively. First, AMs were incubated for 24 h in the absence or presence of increasing concentrations of LPS. Results showed the up-regulation of TLRs 2, 4, 5 and 9 mRNA from all concentrations of LPS used, as compared to non-stimulated cells, and TLR4 was the highest expression in both ages (P < 0.05). Furthermore, quantitative analysis demonstrated increased expression of mRNAs encoding TLRs 2, 4, 5 and 9, LBP, CD14, MD2, MyD88, IRAK4 and TRAF6 in both ages in a time-dependant manner (P < 0.05). Overall, LPS inducible mRNA for TLR4, LBP, CD14 and MyD88 had higher expression in newborn piglets compared with those of young pigs (P < 0.05). The level of cytokine protein IL6 and TNF alpha in supernatant fluid significantly varied with time of incubation and age of animals. Their concentration increased immediately at 1 h after LPS stimulation and remained significantly higher up to 48 h in both ages. Production of pro-inflammatory cytokine protein IL6 and TNFa in supernatant was significantly higher in young pigs than those of piglets. This study suggests that differential age-related changes in the expression of TLRs and downstream genes, and pro-inflammatory cytokine could contribute to a different age-related innate immune response during pulmonary infection. Further investigation is warranted to determine the precise effects of LPS on porcine AMs by means of a functional study across a wider age range. (C) 2012 Elsevier B.V. All rights reserved.

The Toll-like receptor (TLR)4 is critical for the recognition of Gram-negative bacterial lipopolysaccharide (LPS) but in porcine peripheral blood mononuclear cells (PBMCs) it may cooperate with other TLRs and lead to the production of inflammatory cytokines. Therefore, we analyzed TLR1-10 mRNA expression in porcine PBMCs stimulated with LPS over time (1-48 h) by using quantitative real-time PCR and cytokine proteins level by ELISA in culture supernatant. TLR1-10 mRNA was detectable in porcine PBMCs. When compared with the control (non-stimulated), TLR1 mRNA were increased (p < 0.05) at 3 h after challenge with 1 mu g/ml LPS, whereas TLR1 and TLR2 mRNA were increased. (p < 0.01) at 6 h after challenge with 10 mu g/ml LPS. TLR4 increased (p < 0.001) at 3 h after challenge with LPS and remained constant. TLR5 and TLR6 mRNA increased (p < 0.05) at 9 h and 1 h after of LPS stimulation, respectively. The mRNA of CD14 and MD2 were increased (p < 0.001) at 1 h after LPS stimulation. Additionally, at most of the time analyzed, the mRNA expression increased with the dose of LPS. The LPS concentration had influence (p < 0.05) on all the TLRs expression except TLR10; whereas time had effect (p < 0.05) on all TLRs expression except TLR2, 3,6 and 10. When compared to the control, the cytokines IL1b, IL8 and TNF alpha proteins were increased (p < 0.001) immediately at 1 h after LPS stimulation and remained constant till 48 h. IL12b was increased (p < 0.001) 12 h after challenge with 10 of LPS. Although IL8 level was the highest, the higher (p < 0.05) expression of all these inflammatory cytokines indicate that upon interacting with TLRs, LPS exerted inflammatory response in PBMCs through the production of Th1 type cytokines. The production of cytokines was influenced (p < 0.001) by both the dose of LPS and the stimulation time. Hence, the porcine PBMCs are likely able to express all members of TLRs. (C) 2012 Elsevier B.V. All rights reserved.

Background Gene expression analysis using real-time RT-PCR (qRT-PCR) is increasingly important in biological research due to the high-throughput and accuracy of qRT-PCR. For accurate and reliable gene expression analysis, normalization of gene expression data against housekeeping genes or internal control genes is required. The stability of reference genes has a tremendous effect on the results of relative quantification of gene expression by qRT-PCR. The expression stability of reference genes could vary according to tissues, age of individuals and experimental conditions. In the pig however, very little information is available on the expression stability of reference genes. The aim of this research was therefore to develop a new set of reference genes which can be used for normalization of mRNA expression data of genes expressed in varieties of porcine tissues at different ages. Results The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined in varieties of tissues collected from newborn, young and adult pigs. geNorm, NormFinder and BestKeeper software were used to rank the genes according to their stability. geNorm software revealed that RPL4, PPIA and YWHAZ showed high stability in newborn and adult pigs, while B2M, YWHAZ and SDHA showed high stability in young pigs. In all cases, GAPDH showed the least stability in geNorm. NormFinder revealed that TBP was the most stable gene in newborn and young pigs, while PPIA was most stable in adult pigs. Moreover, geNorm software suggested that the geometric mean of three most stable gene would be the suitable combination for accurate normalization of gene expression study. Conclusions Although, there was discrepancy in the ranking order of reference genes obtained by different analysing software methods, the geometric mean of the RPL4, PPIA and YWHAZ seems to be the most appropriate combination of housekeeping genes for accurate normalization of gene expression data in different porcine tissues at different ages.

Background Leg weakness issues are a great concern for the pig breeding industry, especially with regard to animal welfare. Traits associated with leg weakness are partly influenced by the genetic background of the animals but the genetic basis of these traits is not yet fully understood. The aim of this study was to identify quantitative trait loci (QTL) affecting leg weakness in pigs. Methods Three hundred and ten F2 pigs from a Duroc × Pietrain resource population were genotyped using 82 genetic markers. Front and rear legs and feet scores were based on the standard scoring system. Osteochondrosis lesions were examined histologically at the head and the condylus medialis of the left femur and humerus. Bone mineral density, bone mineral content and bone mineral area were measured in the whole ulna and radius bones using dual energy X-ray absorptiometry. A line-cross model was applied to determine QTL regions associated with leg weakness using the QTL Express software. Results Eleven QTL affecting leg weakness were identified on eight autosomes. All QTL reached the 5% chromosome-wide significance level. Three QTL were associated with osteochondrosis on the humerus end, two with the fore feet score and two with the rear leg score. QTL on SSC2 and SSC3 influencing bone mineral content and bone mineral density, respectively, reached the 5% genome-wide significance level. Conclusions Our results confirm previous studies and provide information on new QTL associated with leg weakness in pigs. These results contribute towards a better understanding of the genetic background of leg weakness in pigs.