Additional file 19:List of transcripts that are differentially expressed between GV and MII CCs. (XLS 870 KB)

Additional file 4:List of transcripts that are expressed in both GV oocytes and CCs. (XLS 1 MB)

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.

In the pork industry, it is common to castrate male piglets in order to prevent boar taint. Consumers’ concern for pig (animal) welfare emerges as the castration is predominantly done without analgesia and/or anesthesia. Today, there are three alternatives; all of which has pros and cons: castration with pain relief, production of non-castrated or entire male pigs (EM) which has the risk of boar taint, and immunocastration (IC) which involves a vaccine against GnRH to prevent boar taint. Studies on these methods focus on economic (production) and social (animal welfare) aspects. However, the success of the method finally depends on consumer acceptance. For example, the possible boar taint resulting from EM may lower pork quality, while the idea of a vaccine (IC) may be rejected by consumers. Before applying EM or IC methods, stakeholders are eager to estimate customer acceptance/market acceptance. Questions such as, how do meat consumers experience animal welfare concerns, perceive new way of animal farming, evaluate product quality and price and make purchasing decisions, remain to be answered. This project will develop a multi-dimensional consumer profile and look for (and seek to explain) differences of profiles between social groups, and thus help to get a grip on the view of European market and consumers in accepting EM and IC practices. Key concept: moral dilemma in food choice Literature shows that consumers’ perception of product quality involves four dimensions: taste, health, convenience, and process characteristics including natural production, animal welfare, GMO, etc. Together with product price, opposing evaluations of these dimensions can create a dilemma in food choice. For example, people who strongly embrace animalfriendly values are confronted with the higher price of the product. Those who preferred relatively traditional farms are worried about the treatment of animals in modern production. Such dilemma does not imply an outright rejection of meat consumption, but rather an unpredictable choice. Some consumers would give up the “health and taste” to exchange it with “convenience”. Some are willing to pay more for a better farming process in considering better animal welfare and natural production. Our research is particularly interested in consumers’ moral dilemma in meat choice. Consumers for example show moral concern about the living conditions of animals and meanwhile prefer a lower price for the animal-friendly product. Our first task is to investigate the choice dilemma residing in meat consumption, with emphasis on the interaction between perceptions of farming process characteristics (animal-friendly vs. animal-unfriendly; naturalness vs. unnaturalness) and other perceptual factors, including price, health and taste, and convenience. To promote EM and IC methods, it is important to study how consumers resolve this dilemma. Literature shows resolutions of moral dilemmas related to animal welfare are avoided, dissonance resolved (e.g. denial of the animal mind), or lead to behaviour changes (e.g. reduction in meat consumption). Our second task is thus to examine how meat consumers deal with moral dilemmas. Resolve dilemma Avoid Denial Reduction ... Consumer perceptions Willingness to buy Dilemma in food choice Health and taste Convenience Farming process Price

Low cryotolerance is considered as the major drawback of in vitro-produced bovine embryos and is frequently associated with a triad encompassing increased cytoplasmic lipid accumulation, enhanced levels of reactive oxygen species (ROS) and mitochondrial dysfunction. The aim of the present study was to explore the role of the AMP-activated protein kinase (AMPK) pathway in the process resulting such phenotypes. Comparative analysis under different environmental conditions revealed downregulation of AMP-activated protein kinase cytalytic subunit 1alpha (AMPKA1), peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1A) and carnitine palmitoyltransferase 1 (CPT1) genes and upregulation of acetyl-CoA carboxylase α (ACC). In contrast, the presence of fatty acids within the culture medium resulted in a distinct molecular profile in the embryo associated with enhanced levels of ROS, mitochondrial dysfunction and elevated lipid accumulation in bovine embryos. Because AMPKA1 regulates PGC1A, CPT1 and ACC, the results of the present study reveal that AMPK in active its form is the key enzyme promoting lipolysis. Because AMPK1 activity is, in turn, controlled by the AMP : ATP ratio, it is possible to speculate that excessive uptake of exogenous free fatty acids could increase cellular ATP levels as a result of the disturbed β-oxidation of these external fatty acids and could therefore bypass that molecular feedback mechanism. Subsequently, this condition would cause enhanced generation of ROS, which negatively affect mitochondrial activity. Both enhanced generation of ROS and low mitochondrial activity are suggested to enhance the accumulation of lipids in bovine embryos.

The aim of this study was to elucidate the underlying biochemical processes to identify potential key molecules of meat quality traits drip loss, pH of meat 1 h post-mortem (pH1), pH in meat 24 h post-mortem (pH24) and meat color. An untargeted metabolomics approach detected the profiles of 393 annotated and 1,600 unknown metabolites in 97 Duroc × Pietrain pigs. Despite obvious differences regarding the statistical approaches, the four applied methods, namely correlation analysis, principal component analysis, weighted network analysis (WNA) and random forest regression (RFR), revealed mainly concordant results. Our findings lead to the conclusion that meat quality traits pH1, pH24 and color are strongly influenced by processes of post-mortem energy metabolism like glycolysis and pentose phosphate pathway, whereas drip loss is significantly associated with metabolites of lipid metabolism. In case of drip loss, RFR was the most suitable method to identify reliable biomarkers and to predict the phenotype based on metabolites. On the other hand, WNA provides the best parameters to investigate the metabolite interactions and to clarify the complex molecular background of meat quality traits. In summary, it was possible to attain findings on the interaction of meat quality traits and their underlying biochemical processes. The detected key metabolites might be better indicators of meat quality especially of drip loss than the measured phenotype itself and potentially might be used as bio indicators.

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.

Among other microRNA clusters, we previously showed that the miR-183~96~182 cluster (miR-183, miR-96, and miR-182) is abundantly expressed in bovine granulosa cells (bGC) of preovulatory dominant follicles obtained at the follicular phase of the bovine oestrous cycle. Moreover, this miRNA cluster are validated to coordinately target the Fork head O1 (FOXO1), a subfamily of transcription factors that regulate genes involved in cell proliferation, apoptosis, cell cycle arrest, and metabolism. However, the functional involvement of miR-183~96~182 cluster in bGC function by regulation of FOXO1 is not yet determined. Here, we aimed to investigate the function of miR-183~96~182 cluster in bGC using in vitro cell culture model. For this, bGC were aspirated from ovarian follicles (Ø 3–5 mm) obtained from local abattoir. Cells were plated in 24-well plate (2.5 × 105 cells well–1) in DMEM/F-12 (Sigma, Germany) supplemented with 10% FBS (GIBCO, Grand Island, NY) and 1% penicillin/streptomycin (GIBCO) and incubated at 37°C in 5% CO2. Transfection of bGC with miRNA mimics, inhibitors, FOXO1-siRNA, and appropriate controls (Exiqon, Vedbæk, Denmark) was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Cell proliferation was determined using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technology, Kumamoto, Japan). Cell cycle distribution was determined with flow cytometric analysis. Total RNA was isolated using miRNeasy mini kit (Qiagen, Hilden, Germany), quantification of target gene was performed using qPCR, and data were analysed using ΔΔCT method. Differences in the mean expression values between treatments were analysed with two-tailed Student’s t-test and statistical significance was defined at P ≤ 0.05. Results showed that a sponge effect was observed upon inhibition in individual miRNA of the cluster, which could be attributed to the partial sequence similarity among cluster members. Both FOXO1 mRNA and protein expression were significantly reduced upon transfection of bGC with miR-183~96~182 cluster mimics, while miR-183~96~182 cluster inhibition increased both FOXO1 mRNA and protein expression. Transfection of bGC with miR-183~96~182 mimics promoted cell proliferation, while inhibition tends to slow down proliferation. Furthermore, the proportion of bGC under G0/G1 arrest markedly declined (P < 0.05), while the S and G2/M phases increased in response to miR-183~96~182 mimicking. Selective knockdown of FOXO1 with FOXO1-siRNA significantly reduced FOXO1 mRNA and protein expression. Interestingly, knockdown of FOXO1 showed similar phenotypic effects such as that of miR-183~96~182 mimics transfection, which resulted in elevated bGC proliferation and reduction in the proportion of cells under G0/G1 arrest. In conclusion, overexpression of miR-183~96~182 cluster promote bGC proliferation and G0/G1 to S and G2/M cell cycle transition through coordinated regulation of genes in the FOXO1 signaling axis.

The aim of this study was to integrate multi omics data to characterize underlying functional pathways and candidate genes for drip loss in pigs. The consideration of different omics levels allows elucidating the black box of phenotype expression. Metabolite and protein profiling was applied in Musculus longissimus dorsi samples of 97 Duroc × Pietrain pigs. In total, 126 and 35 annotated metabolites and proteins were quantified, respectively. In addition, all animals were genotyped with the porcine 60 k Illumina beadchip. An enrichment analysis resulted in 10 pathways, amongst others, sphingolipid metabolism and glycolysis/gluconeogenesis, with significant influence on drip loss. Drip loss and 22 metabolic components were analyzed as intermediate phenotypes within a genome-wide association study (GWAS). We detected significantly associated genetic markers and candidate genes for drip loss and for most of the metabolic components. On chromosome 18, a region with promising candidate genes was identified based on SNPs associated with drip loss, the protein “phosphoglycerate mutase 2” and the metabolite glycine. We hypothesize that association studies based on intermediate phenotypes are able to provide comprehensive insights in the genetic variation of genes directly involved in the metabolism of performance traits. In this way, the analyses contribute to identify reliable candidate genes.

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.

Normal follicular development, especially from the preantral stage until ovulation, is the critical to ensure the release of a developmentally competent oocyte. We have previously shown that among several clusters of microRNAs, microRNA-17-92 cluster (miR-17-5p, miR-19a, miR-20a, and miR-92a) is differentially expressed between bovine granulosa cells (bGC) derived from preovulatory dominant and subordinate follicles. Here, we aimed to investigate the regulatory role of microRNA-17-92 cluster in bGC function. Among the target genes predicted by the miRWalk database, BMPR2 and PTEN genes were experimentally validated using the pmirGLO Dual Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA). The bGC were aspirated from ovaries obtained from a local slaughterhouse. After determining cell viability and concentration using the trypan blue exclusion method, a total 2.5 × 105 bGC per well were seeded into CytoOne 24-well plate in DMEM/F12-Ham medium (Sigma Aldrich Chemie GmbH, Munich, Germany) supplemented with 10% FBS (Gibco BRL USA, Grand Isalnd, NY, USA) and 1% penicillin/streptomycin (Gibco BRL USA). Then, the bGC were cultured at 37°C with 5% CO2 and O2. To investigate the role of microRNA-17-92 cluster in bGC function, 100 nM of individual and cluster of microRNA-17-92 mimic, inhibitor, and negative controls were transfected into subconfluent-cultured bGC. The bGC were harvested 48 h post-transfection and used for RNA isolation and subsequent cDNA synthesis and expression analysis of candidate genes using real-time qPCR. Data analysis was performed using the comparative cycle threshold (Ct) method. A cell proliferation assay was performed using CCK-8 kit (Dojindo EU GmbH, Munich, Germany). Based on the cell diameter measurement done using ImageJ 1.48v software (National Institutes for Health, Bethesda, MD, USA), those bGC with diameter >14 µm were categorized as differentiated cells, whereas those with diameter = 14 µm were considered as undifferentiated cells. MicroRNA-17-92 cluster overexpression on bGC reduced both mRNA and protein expression of BMPR2 and PTEN genes, whereas inhibition of microRNA-17-92 cluster increased their expression. Bovine GC transfected with microRNA-17-92 cluster mimic showed higher proliferation activity and decreased rate of differentiation. The opposite phenotype was observed in bGC transfected with microRNA-17-92 cluster inhibitor. Similarly, miRNA-17-92 cluster mimic transfection increased the expression of markers of proliferation, CCND2 and PCNA, and resulted in down-regulation of CYP11A1 and STAR genes as markers of differentiation. The opposite expression pattern was observed after transfection of miRNA-17-92 cluster inhibitors. In conclusion, the miRNA-17-92 cluster members coordinately regulate bGC proliferation and differentiation by targeting the expression of BMPR2 and PTEN genes.

Fat samples from roughly a thousand pig carcasses were collected and subjected to a thorough sensory evaluation and quantification using a panel of 10 trained assessors on a sensory score scale ranging from 0 = ‘untainted’ to 5 = ‘strongly tainted’. The following variables are available in the dtR.csv data set: rater: the ID of the panelist/assessor product: the product ID dev: deviation from normal smell according to the respective rater, ordinal score between 0 = ‘untainted’ and 5 = ‘strongly tainted’ (see above) andro: the product’s androstenone content (in ppm) skatol: the product’s skatole content (in ppm) The following variables are available in the (aggregated) dtM.csv data set: product: the product id mdev: mean deviation from normal smell (across panelists) of the respective product andro: the product’s androstenone content (in ppm) skatol: the product’s skatole content (in ppm) If using the data, please cite: Daniel Mörlein, Johanna Trautmann, Jan Gertheiss, Lisa Meier-Dinkel, Jochen Fischer, Hans-Jörg Eynck, Lourens Heres, Christian Looft, and Ernst Tholen (2016). Interaction of skatole and androstenone in the olfactory perception of boar taint. Journal of Agricultural and Food Chemistry, 64, 4556-4565 Available at: https://pubs.acs.org/doi/10.1021/acs.jafc.6b00355

An association study between polymorphisms of six genes and boar taint related compounds androstenone, skatole and indole was performed in a boar population (n=370). Significant association (P<0.05) was detected for SNP of FMO5 (g.494A>G) with all boar taint compounds, SNP of CYP21 (g.3911T>C) with skatole and indole, and SNP of ESR1 (g.672C>T) with androstenone and indole. mRNA expression of CYP21 and ESR1 was higher in CAB (castrated boar) compared to non-castrated boars; whereas, the expression of FMO5 and ESR1 was higher in LBT (low boar taint) compared to HBT (high boar taint) in liver tissue. FMO5, CYP21 and ESR1 proteins were less detectable in HBT compared with LBT and CAB in liver tissues. These findings suggest that FMO5, CYP21 and ESR1 gene variants might have effects on the boar taint compounds.

The aim of the study was to investigate single nucleotide polymorphisms (SNPs) and expression of SOX-6 to support its candidacy for growth, carcass, and meat quality traits in pigs. The first SNP, rs81358375, was associated with pH 45 min post mortem in loin (pH1L), the thickness of backfat and side fat, and carcass length in Pietrain (Pi) population, and related with backfat thickness and daily gain in Duroc × Pietrain F2 (DuPi) population. The other SNP, rs321666676, was associated with meat colour in Pi population. In DuPi population, the protein, not mRNA, level of SOX-6 in high pH1L pigs was significantly less abundant compared with low pH1L pigs, where microRNAs targeting SOX-6 were also differently regulated. This paper shows that SOX-6 could be a potential candidate gene for porcine growth, carcass, and meat quality traits based on genetic association and gene expression.

BACKGROUND:Despite its role in increasing the number of offspring during the lifetime of an individual animal, controlled ovarian hyperstimulation (COH) may have detrimental effects on oocyte development, embryo quality and endometrial receptivity. Circulating miRNAs in bio-fluids have been shown to be associated with various pathological conditions including cancers. Here we aimed to investigate the effect of COH on the level of extracellular miRNAs in bovine follicular fluid and blood plasma and elucidate their mode of circulation and potential molecular mechanisms to be affected in the reproductive tract.METHOD:Twelve simmental heifers were estrous synchronized and six of them were hyperstimulated using FSH. Follicular fluid samples from experimental animals were collected using ovum pick up technique at day 0 of the estrous cycle and blood samples were collected at day 0, 3 and 7 of post ovulation. The expression profile of circulatory miRNAs in follicular fluid and blood plasma were performed using the human miRCURY LNA™ Universal RT miRNA PCR array system. A comparative threshold cycle method was used to determine the relative abundance of the miRNAs.RESULTS:A total of 504 and 402 miRNAs were detected in both bovine follicular fluid and blood plasma, respectively. Of these 57 and 21 miRNAs were found to be differentially expressed in follicular fluid and blood plasma, respectively derived from hyperstimulated versus unstimulated heifers. Bioinformatics analysis of those circulating miRNAs indicated that their potential target genes are involved in several pathways including TGF-beta signaling pathway, MAPK signaling pathway, pathways in cancer and Oocyte meiosis. Moreover, detail analysis of the mode of circulation of some candidates showed that most of the miRNA were found to be detected in both exosomal and Ago2 protein complex fraction of both follicular fluid and blood plasma.CONCLUSION:Our data provide the consequence of hyperstimulation induced changes of extracellular miRNAs in bovine follicular fluid and blood plasma, which may have a potential role in regulating genes associated not only with bovine ovarian function but also involved in altering various physiological in bovine oocytes, embryos and modulating reproductive tract environment.

A major problem of embryos cultured in vitro with serum is cytoplasmic lipid accumulation resulting in lower cryotolerance compared with those derived from in vivo or in the absence of serum. AMPK is known as a master regulator of lipid, glucose, and protein metabolism in mammalian cells. Moreover, it has been reported as controller of acetyl-CoA carboxylase α (ACC), the gene responsible for lipid synthesis, and associated with mitochondrial biogenesis and activities in response to oxidative stress. In the present study we aimed to investigate the regulation of AMPK during serum supplementation in vitro. For this, bovine embryos were produced in vitro in SOF media supplemented with oestrous cow serum or fatty acid–free BSA as a system without serum. Triplicate pools (each 10 blastocysts) from each group were used for RNA isolation using Arcturus®PicoPure®RNA Isolation Kit (Life Technologies, USA). Reverse transcription was performed using a combination of Oligo(dT)23 and random primers. Quantification of AMPK catalytic α1 (AMPKA1), ACC, peroxisome proliferator-activated receptor gamma coactivator 1 α (PGC1A), and sterol regulatory element binding transcription factor 2 (SREBP2) transcripts were performed using ABI PRISM® 7000 SDS system (Applied Biosystems, Foster City, CA, USA) using GAPDH as internal control. Normalized log-transformed transcript amount data were statistically analysed using t-test. In addition, AMPK protein was detected by immunofluorescence, mitochondrial activity by MitoTracker® Red (Invitrogen, Carlsbad, CA, USA), and reactive oxygen species by H2DCFDA molecular probe (Life Technologies, USA), and fluorescent intensity signals were visualised under confocal laser scanning microscopy LSM 710 (Carl Zeiss, Germany). Results showed that the expression of AMPKA1, PGC1A, a mitochondrial biogenesis protein, and SREBP2, a regulator of lipid oxidation, were found to be lower (0.4-, 0.2-, and 0.7-fold, respectively; P < 0.05) in blastocysts derived from cultured with serum compared to without serum. By contrast, ACC was up-regulated in blastocysts cultured with serum by 1.8-fold (P < 0.05) compared to without serum. In comparison to blastocyst cultured without serum, a reduced fluorescent intensity was observed in AMPKA1 protein and mitochondrial activity in blastocyst cultured with serum. The presence of serum was also found to be involved in increasing reactive oxygen species accumulation in embryos cultured with serum. The reduced level of AMPK leads to increased ACC and subsequently enhanced conversion of fatty acids into lipid, which is associated with reduced mitochondrial biogenesis protein, elevated reactive oxygen species level, and reduced lipid oxidation by suppression of SREBP2. In conclusion, the presence of serum in in vitro culture environment affected the AMPK activity and thereby genes associated with lipid metabolism in early bovine embryos.

In the present study, we used an in vitro model to investigate the response of the oviduct with respect to inflammatory mediators and their regulatory microRNAs in case of bacterial infection and subsequent association with embryo survival. For this, we conducted two experiments. In the first experiment, cultured primary bovine oviductal cells (BOEC) were challenged with lipopolysaccharide (LPS) for 24h and the temporal expression pattern of inflammatory mediators and their regulatory microRNAs were measured at 0, 3, 6, 12, 24 and 48h after LPS treatment. Intriguingly, the temporal patterns of all miRNAs except miR-21 were significantly up-regulated at 6h after LPS treatment. Whereas, we observed significant overexpression of pro-inflammatory mediators as tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL1β) after LPS challenge for 24h. On the other hand, the expression level of essential elements like oviductal glycoprotein 1 (OVGP1) and insulin-like growth factor 2 (IGF2) was significantly decreased in challenged groups compared with control. Moreover, miR-155, miR-146a, miR-223, miR-21, miR-16 and miR-215 have shown a clear suppression in challenged group after LPS treatment. In the 2nd experiment there were four groups of blastocysts produced, namely embryo+LPS free media, embryo+LPS, BOEC+embryo and BOEC+embryo+LPS. The suboptimal oviduct environment due to LPS challenge is found to have a significant influence on the expression of inflammatory response genes (TNFα and CSF1), stress response genes (SOD and CAT), mitochondrial activity, reactive oxygen species (ROS) accumulation and apoptotic level either in cultured or co-cultured blastocysts. Collectively, LPS challenge led to aberrant changes in oviductal transcriptome profile, which could lead to a suboptimal environment for embryo development.

Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY), 4-cell (4C) or 16-cell (16C) were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP). Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO) using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic imprinting and chromosome segregation in IVP blastocyst groups. Furthermore, 1.6, 3.4, 3.9 and 9.4% of the differentially methylated regions that were overlapped to the transcriptome profile data were negatively correlated with the gene expression patterns in ZY, 4C, 16C and IVP blastocyst groups, respectively. Therefore, this finding indicated that suboptimal culture condition during preimplantation embryo development induced changes in the DNA methylation landscape of the resulting blastocysts in a stage dependent manner and the altered DNA methylation pattern was only partly explained the observed aberrant gene expression patterns of the blastocysts.