Microbial signature inferred from genomic breeding selection on milk urea concentration and its relation to proxies of nitrogen-utilization efficiency in Holsteins.

Increasing the nitrogen-utilization efficiency (NUE) of dairy cows by breeding selection would offer advantages from nutritional, environmental, and economic perspectives. Because data collection of NUE phenotypes is not feasible in large cow cohorts, the cow individual milk urea concentration (MU) has been suggested as an indicator trait. Considering the symbiotic interplay between dairy cows and their rumen microbiome, individual MU was thought to be influenced by host genetics and by the rumen microbiome, the latter in turn being partly attributed to host genetics. To enhance our knowledge of MU as an indicator trait for NUE, we aimed to identify differential abundant rumen microbial genera between Holstein cows with divergent genomic breeding values for MU (GBVMU; GBVHMU vs. GBVLMU, where H and L indicate high and low MU phenotypes, respectively). The microbial genera identified were further investigated for their correlations with MU and 7 additional NUE-associated traits in urine, milk, and feces in 358 lactating Holsteins. Statistical analysis of microbial 16S rRNA amplicon sequencing data revealed significantly higher abundances of the ureolytic genus Succinivibrionaceae UCG-002 in GBVLMU cows, whereas GBVHMU animals hosted higher abundances of Clostridia unclassified and Desulfovibrio. The entire discriminating ruminal signature of 24 microbial taxa included a further 3 genera of the Lachnospiraceae family that revealed significant correlations to MU values and were therefore proposed as considerable players in the GBVMU-microbiome-MU axis. The significant correlations of Prevotellaceae UCG-003, Anaerovibrio, Blautia, and Butyrivibrio abundances with MU measurements, milk nitrogen, and N content in feces suggested their contribution to genetically determined N-utilization in Holstein cows. The microbial genera identified might be considered for future breeding programs to enhance NUE in dairy herds.

Brain Transcriptome Responses to Dexamethasone Depending on Dose and Sex Reveal Factors Contributing to Sex-Specific Vulnerability to Stress-Induced Disorders.

BACKGROUND: Glucocorticoid (GC) receptor (GR) signaling in the hypothalamus (Hyp) and in the superordinate limbic structures, such as the hippocampus (Hip), conveys feedback regulation of the neuroendocrine stress response and acts upon other neurobiological functions that ultimately influence mental health. These responses are strongly influenced by sex, but the molecular causes are still largely unexplored. METHODS: To investigate GR targets and their GC sensitivity in the Hyp and Hip, we treated juvenile male and female piglets with 10 (D10) or 60 (D60) µg/kg dexamethasone (DEX), a selective GR agonist, and analyzed transcriptome responses compared to a saline control group using RNA sequencing. RESULTS: Both doses influenced similar biological functions, including cellular response to lipid and immune cell-related functions, but the transcriptional response to D10 was considerably weaker, particularly in the Hip. Weighted Gene Co-expression Network Analysis revealed a network of genes coordinately regulated by DEX in both structures, among which the alpha-arrestin ARRDC2 takes a central position. Distinct functional groups of genes were differentially regulated by DEX between sexes depending on the dose; at D10, these included particularly mitochondrial genes, whereas at D60 interferon signaling and lipid homeostasis genes were enriched. The general and sex-specific transcriptional responses to DEX highlight microglia as the prominent target. Several key marker genes of disease-associated microglia were regulated by DEX depending on sex, such as TREM2 and LPL. CONCLUSION: The discovered expression signatures suggest that DEX induced a dysfunctional state of microglia in males, while in females microglia were primed, which could entail predisposition for different mental disorders.

Genetic background and production periods shape the microRNA profiles of the gut in laying hens.

There is growing evidence of the importance of miRNAs for intestinal functional properties and nutritional uptake. Comparative miRNAs profiles of the jejunal mucosa were established against two genetic backgrounds (Lohmann Brown-Classic (LB) and Lohmann LSL-Classic (LSL), which are similar in egg production but differ in physiological traits including mineral utilization, along the production periods of laying hens. The target genes of miRNAs higher expressed in LB vs. LSL (miR-126-3p, miR-214, miR-24-3p, miR-726-5p, miR-29b-3p) were enriched for energy pathways at all stages. The target genes of the miRNAs higher in LSL (miR-1788-5p, miR-103-3p, miR-22-5p, miR-221-3p, miR-375) were more enriched for immune and the bone signalling pathways. The most prominent expression differences were between 16 and 24 weeks of age before and after onset of laying. Our results evidence central roles of intestinal miRNAs as regulators of gene expression, influencing intestinal homeostasis and adaptation to environment in different strains and production phases.

Transcriptional responses in jejunum of two layer chicken strains following variations in dietary calcium and phosphorus levels.

BACKGROUND: Calcium (Ca) and phosphorus (P) are essential nutrients that are linked to a large array of biological processes. Disturbances in Ca and P homeostasis in chickens are associated with a decline in growth and egg laying performance and environmental burden due to excessive P excretion rates. Improved utilization of minerals in particular of P sources contributes to healthy growth while preserving the finite resource of mineral P and mitigating environmental pollution. In the current study, high performance Lohmann Selected Leghorn (LSL) and Lohmann Brown (LB) hens at peak laying performance were examined to approximate the consequences of variable dietary Ca and P supply. The experimental design comprised four dietary groups with standard or reduced levels of either Ca or P or both (n = 10 birds per treatment group and strain) in order to stimulate intrinsic mechanisms to maintain homeostasis. Jejunal transcriptome profiles and the systemic endocrine regulation of mineral homeostasis were assessed (n = 80). RESULTS: Endogenous mechanisms to maintain mineral homeostasis in response to variations in the supply of Ca and P were effective in both laying hen strains. However, the LSL and LB appeared to adopt different molecular pathways, as shown by circulating vitamin D levels and strain-specific transcriptome patterns. Responses in LSL indicated altered proliferation rates of intestinal cells as well as adaptive responses at the level of paracellular transport and immunocompetence. Endogenous mechanisms in LB appeared to involve a restructuring of the epithelium, which may allow adaptation of absorption capacity via improved micro-anatomical characteristics. CONCLUSIONS: The results suggest that LSL and LB hens may exhibit different Ca, P, and vitamin D requirements, which have so far been neglected in the supply recommendations. There is a demand for trial data showing the mechanisms of endogenous factors of Ca and P homeostasis, such as vitamin D, at local and systemic levels in laying hens.

Ablation of Red Stable Transfected Claudin Expressing Canine Prostate Adenocarcinoma and Transitional Cell Carcinoma Cell Lines by C-CPE Gold-Nanoparticle-Mediated Laser Intervention.

Claudin (CLDN) proteins are commonly expressed in cancers and targeted in novel therapeutic approaches. The C-terminal of Clostridium perfringens enterotoxin (C-CPE) efficiently binds several claudins. In this study, recombinant C-CPE conjugated to gold nanoparticles (AuNPs) has been used for prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) cell killing in vitro using gold-nanoparticle-mediated laser perforation (GNOME-LP). A PAC and TCC cell lines, as well as red fluorescence variants, allowing deep tissue imaging, were used. CLDN-3, -4, and -7 expression was confirmed by qPCR and immunofluorescences. The binding of C-CPE-AuNPs complexes on the cell surface was examined by scanning electron microscopy (SEM). Further, transcriptome analysis was carried out to evaluate the effect of C-CPE binder on the biological response of treated cells. Directed C-CPE-AuNP binding verified the capability to target CLDN receptors. Transcriptome analysis showed that C-CPE binding may activate immune and inflammatory responses but does not directly affect cell survival. Cancer cells ablation was demonstrated using a combination of GNOME-LP and C-CPE-AuNPs treatment reducing tumor cell viability to less than 10% depending on cell line. The fluorescent cell lines and the verified proof of concept in vitro provide the basis for perspective xenograft studies in an animal model.

Transcriptome analyses of liver in newly-hatched chicks during the metabolic perturbation of fasting and re-feeding reveals THRSPA as the key lipogenic transcription factor.

BACKGROUND: The fasting-refeeding perturbation has been used extensively to reveal specific genes and metabolic pathways that control energy metabolism in the chicken. Most global transcriptional scans of the fasting-refeeding response in liver have focused on juvenile chickens that were 1, 2 or 4 weeks old. The present study was aimed at the immediate post-hatch period, in which newly-hatched chicks were subjected to fasting for 4, 24 or 48 h, then refed for 4, 24 or 48 h, and compared with a fully-fed control group at each age (D1-D4). RESULTS: Visual analysis of hepatic gene expression profiles using hierarchical and K-means clustering showed two distinct patterns, genes with higher expression during fasting and depressed expression upon refeeding and those with an opposing pattern of expression, which exhibit very low expression during fasting and more abundant expression with refeeding. Differentially-expressed genes (DEGs), identified from five prominent pair-wise contrasts of fed, fasted and refed conditions, were subjected to Ingenuity Pathway Analysis. This enabled mapping of analysis-ready (AR)-DEGs to canonical and metabolic pathways controlled by distinct gene interaction networks. The largest number of hepatic DEGs was identified by two contrasts: D2FED48h/D2FAST48h (968 genes) and D2FAST48h/D3REFED24h (1198 genes). The major genes acutely depressed by fasting and elevated upon refeeding included ANGTPL, ATPCL, DIO2, FASN, ME1, SCD, PPARG, SREBP2 and THRSPA-a primary lipogenic transcription factor. In contrast, major lipolytic genes were up-regulated by fasting or down-regulated after refeeding, including ALDOB, IL-15, LDHB, LPIN2, NFE2L2, NR3C1, NR0B1, PANK1, PPARA, SERTAD2 and UPP2. CONCLUSIONS: Transcriptional profiling of liver during fasting/re-feeding of newly-hatched chicks revealed several highly-expressed upstream regulators, which enable the metabolic switch from fasted (lipolytic/gluconeogenic) to fed or refed (lipogenic/thermogenic) states. This rapid homeorhetic shift of whole-body metabolism from a catabolic-fasting state to an anabolic-fed state appears precisely orchestrated by a small number of ligand-activated transcription factors that provide either a fasting-lipolytic state (PPARA, NR3C1, NFE2L2, SERTAD2, FOX01, NR0B1, RXR) or a fully-fed and refed lipogenic/thermogenic state (THRSPA, SREBF2, PPARG, PPARD, JUN, ATF3, CTNNB1). THRSPA has emerged as the key transcriptional regulator that drives lipogenesis and thermogenesis in hatchling chicks, as shown here in fed and re-fed states.

Reduced phosphorus intake throughout gestation and lactation of sows is mitigated by transcriptional adaptations in kidney and intestine.

BACKGROUND: The environmental impact of pig farming need to be reduced, with phosphorus (P) being of particular interest. Specified dietary regimens and management systems contribute to meet environmental concerns and reduce economic constrains. However, pregnant and lactating sows represent vulnerable individuals, whose reproductive potential and metabolic health status relies on adequate supply of macro- and micronutrients. The aim of this study was to investigate, whether sows fed with a dietary P content that is below or above current recommendations are capable to maintain mineral homeostasis during the reproduction cycle and which endogenous mechanisms are retrieved therefore in kidney and jejunum. Nulliparous gilts were fed iso-energetic diets with recommended (M), reduced (L), or high (H) amounts of mineral P supplements throughout gestation and lactation periods. Blood metabolites and hormones referring to the P homeostasis were retrieved prior to term (110 days of gestation) and at weaning (28 days of lactation). Transcriptional responses in kidney cortex and jejunal mucosa were analyzed using RNA sequencing. RESULTS: The variable dietary P content neither led to an aberration on fertility traits such as total weaned piglets nor to an effect on the weight pattern throughout gestation and lactation. Serum parameters revealed a maintained P homeostasis as reflected by unaltered inorganic P and calcium levels in L and H fed groups. The serum calcitriol levels were increased in lactating L sows. The endocrine responses to the dietary challenge were reflected at the transcriptional level. L diets led to an increase in CYP27B1 expression in the kidney compared to the H group and to an altered gene expression associated with lipid metabolism in the kidney and immune response in the jejunum. CONCLUSIONS: Our results suggest that current P requirements for gestating and lactating sows are sufficient and over supplementation of mineral P is not required. Shifts in renal and jejunal expression patterns between L and H groups indicate an affected intermediate metabolism, which long-term relevance needs to be further clarified.

Dynamic profile of EVs in porcine oviductal fluid during the periovulatory period.

In mammals, around the time of ovulation, the hormonal profile dynamically changes in synchrony with reproductive events occurring in the oviduct, that is, sperm arrival, fertilization, and early embryo development. Extracellular vesicles (EVs) have been recently recognized as key components of the embryonic milieu; however, composition and function of oviductal EVs during this crucial period remains to be further explored. Therefore, we initially characterized EVs from porcine oviductal fluid specifically around the critical ovulation window: that is, estrus (E), late estrus (LE, day of expected ovulation), post ovulation (PO), and additionally diestrus (D). Total EV numbers gradually rose from D to E, LE and PO (P < 0.05), which corresponded to the total EV protein amount (P < 0.05). Strikingly, the mean size of EVs in PO was significantly smaller than in E and LE groups, which also had a lesser proportion of small EVs (P < 0.05). The EV protein cargoes during the periovulatory period were further analyzed by mass spectrometry. Qualitative analysis detected 1118 common proteins, which are most enriched in the cellular component of EVs/exosomes. Hierarchical clustering indicated similar protein profile within the biological replicates, but large discrepancy among stages. Further quantitative analysis discovered 34 and 4 differentially expressed proteins in the comparison between E and PO and in the comparison between E and LE, respectively. The dynamic EV protein profile together with the quick adaption in EV size and quantity suggests that porcine oviductal EV secretion are under the hormonal influence during the estrus cycle.

A natural Ala610Val substitution causing glucocorticoid receptor hypersensitivity aggravates consequences of endotoxemia.

Despite the crucial role of glucocorticoid receptor (GR) in proper immune responses, the effect of GR hypersensitivity on inflammation is rarely reported. To fill this knowledge gap, we exploited the natural gain-of-function substitution in the porcine glucocorticoid receptor (GRAla610Val) and challenged pigs carrying normal or hypersensitive GR using 50 µg/kg lipopolysaccharide (LPS) following pretreatment with either saline or single bolus of 60 µg/kg dexamethasone (DEX). The GRAla610Val substitution reduced baseline cortisol, adrenocorticotropic hormone (ACTH), and triglyceride concentration and granulocyte proportion whereas baseline platelet counts were elevated. Val-carriers, i.e. AlaVal as well as ValVal pigs, showed less LPS-induced cortisol rise but the cortisol fold change was similar in all genotypes. Differently, ACTH response to LPS was most significant in GRAla610Val heterozygotes (AlaVal). LPS-induced disorders, including sickness behaviors, anorexia, thrombocytopenia, cytokine production, and metabolic alterations were more intense in Val-carriers. On the other hand, Val-carriers were more sensitive to DEX effect than wild types (AlaAla) during endotoxemia, but not under unchallenged conditions. This is the first report revealing aggravated responses to endotoxemia by GR gain-of-function. Together, these results imply that GR hypersensitivity is difficult to diagnose but may represent a risk factor for endotoxemia and sepsis.

Profiling of circulating microRNA and pathway analysis in normal- versus over-conditioned dairy cows during the dry period and early lactation.

The objective of this study was to determine the circulating microRNA (miRNA) profile in over-conditioned (HBCS) versus normal-conditioned (NBCS) dairy cows in combination with pathway enrichment analyses during the transition period. Thirty-eight multiparous Holstein cows were selected 15 wk before anticipated calving date based on their current and previous body condition scores (BCS) for forming either a HBCS group (n = 19) or a NBCS group (n = 19). They were fed different diets during late lactation to reach the targeted differences in BCS and backfat thickness until dry-off. A subset of 15 animals per group was selected based on their circulating concentrations of nonesterified fatty acids (on d 14 postpartum) and ß-hydroxybutyrate (on d 21 postpartum), representing the greater or the lower extreme values within their BCS group. Blood serum obtained at d -49 and 21 relative to parturition (3 pools with 5 cows per each group and time point) were used to identify miRNA that were differentially expressed (DE) between groups or time points using miRNA sequencing. No DE-miRNA were discovered between NBCS versus HBCS. Comparing pooled samples from d -49 and d 21 resulted in 7 DE-miRNA in the NBCS group, of which 5 miRNA were downregulated and 2 miRNA were overexpressed on d 21 versus -49. The abundance of 5 of these DE-miRNA was validated in all individual samples via quantitative PCR and extended to additional time points (d -7, 3, 84). Group differences were observed for miR-148a, miR-122 as well as miR-455-5p, and most DE-miRNA (miR-148a, miR-122, miR-30a, miR-450b, miR-455-5p) were downregulated directly after calving. Subsequently, the DE-miRNA was used for bioinformatics analysis to identify putative target genes and the most enriched biological pathways. The most significantly enriched pathways of DE-miRNA were associated with cell cycle and insulin signaling as well as glucose and lipid metabolism. Overall, we found little differences in circulating miRNA in HBCS versus NBCS cows around calving.

Does Maternal Stress Affect the Early Embryonic Microenvironment? Impact of Long-Term Cortisol Stimulation on the Oviduct Epithelium.

Maternal stress before or during the sensitive preimplantation phase is associated with reproduction failure. Upon real or perceived threat, glucocorticoids (classic stress hormones) as cortisol are synthesized. The earliest "microenvironment" of the embryo consists of the oviduct epithelium and the oviductal fluid generated via the epithelial barrier. However, to date, the direct effects of cortisol on the oviduct are largely unknown. In the present study, we used a compartmentalized in vitro system to test the hypothesis that a prolonged stimulation with cortisol modifies the physiology of the oviduct epithelium. Porcine oviduct epithelial cells were differentiated at the air-liquid interface and basolaterally stimulated with physiological levels of cortisol representing moderate and severe stress for 21 days. Epithelium structure, transepithelial bioelectric properties, and gene expression were assessed. Furthermore, the distribution and metabolism of cortisol was examined. The polarized oviduct epithelium converted basolateral cortisol to cortisone and thereby reduced the amount of bioactive cortisol reaching the apical compartment. However, extended cortisol stimulation affected its barrier function and the expression of genes involved in hormone signaling and immune response. We conclude that continuing maternal stress with long-term elevated cortisol levels may alter the early embryonic environment by modification of basic oviductal functions.

Ileal Transcriptome Profiles of Japanese Quail Divergent in Phosphorus Utilization.

Phosphorus (P) is an essential component for all living beings. Low P diets prompt phenotypic and molecular adaptations to maintain P homeostasis and increase P utilization (PU). Knowledge of the molecular mechanisms of PU is needed to enable targeted approaches to improve PU efficiency and thus lower P excretion in animal husbandry. In a previous population study, Japanese quail were subjected to a low P diet lacking mineral P and exogenous phytase. Individual PU was determined based on total P intake and excretion. A subset of 20 extreme siblings discordant for PU was selected to retrieve gene expression patterns of ileum (n = 10 per PU group). Sequencing reads have been successfully mapped to the current Coturnix japonica reference genome with an average mapping rate of 86%. In total, 640 genes were found to be differentially abundant between the low and high PU groups (false discovery rate ≤ 0.05). Transcriptional patterns suggest a link between improved PU and mitochondrial energy metabolism, accelerated cell proliferation of enterocytes, and gut integrity. In assessing indicators of the efficient use of macro- and micronutrients, further research on turnover and proliferation rates of intestinal cells could provide an approach to improve P efficiency in poultry species.

Identification of the Key Molecular Drivers of Phosphorus Utilization Based on Host miRNA-mRNA and Gut Microbiome Interactions.

Phosphorus is an essential mineral for all living organisms and a limited resource worldwide. Variation and heritability of phosphorus utilization (PU) traits were observed, indicating the general possibility of improvement. Molecular mechanisms of PU, including host and microbial effects, are still poorly understood. The most promising molecules that interact between the microbiome and host are microRNAs. Japanese quail representing extremes for PU were selected from an F2 population for miRNA profiling of the ileal tissue and subsequent association with mRNA and microbial data of the same animals. Sixty-nine differentially expressed miRNAs were found, including 21 novel and 48 known miRNAs. Combining miRNAs and mRNAs based on correlated expression and target prediction revealed enrichment of transcripts in functional pathways involved in phosphate or bone metabolism such as RAN, estrogen receptor and Wnt signaling, and immune pathways. Out of 55 genera of microbiota, seven were found to be differentially abundant between PU groups. The study reveals molecular interactions occurring in the gut of quail which represent extremes for PU including miRNA-16-5p, miR-142b-5p, miR-148a-3p, CTDSP1, SMAD3, IGSF10, Bacteroides, and Alistipes as key indicators due to their trait-dependent differential expression and occurrence as hub-members of the network of molecular drivers of PU.

DNA methylation analysis of porcine mammary epithelial cells reveals differentially methylated loci associated with immune response against Escherichia coli challenge.

BACKGROUND: Epigenetic changes such as cytosine (CpG) DNA methylations regulate gene expression patterns in response to environmental cues including infections. Microbial infections induce DNA methylations that play a potential role in modulating host-immune response. In the present study, we sought to determine DNA methylation changes induced by the mastitis causing Escherichia coli (E. coli) in porcine mammary epithelial cells (PMEC). Two time points (3 h and 24 h) were selected based on specific transcriptomic changes during the early and late immune responses, respectively. RESULTS: DNA methylation analysis revealed 561 and 898 significant (P < 0.01) differentially methylated CpG sites at 3 h and 24 h after E. coli challenge in PMEC respectively. These CpG sites mapped to genes that have functional roles in innate and adaptive immune responses. Significantly, hypomethylated CpG sites were found in the promoter regions of immune response genes such as SDF4, SRXN1, CSF1 and CXCL14. The quantitative transcript estimation indicated higher expression associated with the DNA CpG methylation observed in these immune response genes. Further, E. coli challenge significantly reduced the expression levels of DNMT3a, a subtype of de novo DNA methylation enzyme, in PMEC indicating the probable reason for the hypomethylation observed in the immune response genes. CONCLUSIONS: Our study revealed E. coli infection induced DNA methylation loci in the porcine genome. The differentially methylated CpGs were identified in the regulatory regions of genes that play important role in immune response. These results will help to understand epigenetic mechanisms for immune regulation during coliform mastitis in pigs.

Epigenome-wide skeletal muscle DNA methylation profiles at the background of distinct metabolic types and ryanodine receptor variation in pigs.

BACKGROUND: Epigenetic variation may result from selection for complex traits related to metabolic processes or appear in the course of adaptation to mediate responses to exogenous stressors. Moreover epigenetic marks, in particular the DNA methylation state, of specific loci are driven by genetic variation. In this sense, polymorphism with major gene effects on metabolic and cell signaling processes, like the variation of the ryanodine receptors in skeletal muscle, may affect DNA methylation. METHODS: DNA-Methylation profiles were generated applying Reduced Representation Bisulfite Sequencing (RRBS) on 17 Musculus longissimus dorsi samples. We examined DNA methylation in skeletal muscle of pig breeds differing in metabolic type, Duroc and Pietrain. We also included F2 crosses of these breeds to get a first clue to DNA methylation sites that may contribute to breed differences. Moreover, we compared DNA methylation in muscle tissue of Pietrain pigs differing in genotypes at the gene encoding the Ca2+ release channel (RYR1) that largely affects muscle physiology. RESULTS: More than 2000 differently methylated sites were found between breeds including changes in methylation profiles of METRNL, IDH3B, COMMD6, and SLC22A18, genes involved in lipid metabolism. Depending on RYR1 genotype there were 1060 differently methylated sites including some functionally related genes, such as CABP2 and EHD, which play a role in buffering free cytosolic Ca2+ or interact with the Na+/Ca2+ exchanger. CONCLUSIONS: The change in the level of methylation between the breeds is probably the result of the long-term selection process for quantitative traits involving an infinite number of genes, or it may be the result of a major gene mutation that plays an important role in muscle metabolism and triggers extensive compensatory processes.

Transcriptome analysis of adipose tissue from pigs divergent in feed efficiency reveals alteration in gene networks related to adipose growth, lipid metabolism, extracellular matrix, and immune response.

Adipose tissue is hypothesized to play a vital role in regulation of feed efficiency (FE; efficiency in converting energy and nutrients into tissue), of which improvement will simultaneously reduce environmental impact and feed cost per pig. The objective of the present study was to sequence the subcutaneous adipose tissue transcriptome in FE-divergent pigs (n = 16) and identify relevant biological processes underpinning observed differences in FE. We previously demonstrated that high-FE pigs were associated with lower fatness when compared to their counterparts. Here, ontology analysis of a total of 209 annotated genes that were differentially expressed at a p < 0.01 revealed establishment of a dense extracellular matrix and inhibition of capillary formation as one underlying mechanism to achieve suppressed adipogenesis. Moreover, mechanisms ensuring an efficient utilization of lipids in high-FE pigs might be orchestrated by upstream regulators including CEBPA and EGF. Consequently, high-FE adipose tissue could exhibit more efficient cholesterol disposal, whilst inhibition of inflammatory and immune response in high-FE pigs may be an indicator of an optimally functioning adipose tissue. Taken together, adipose tissue growth, extracellular matrix formation, lipid metabolism and inflammatory and immune response are key biological events underpinning the differences in FE. Further investigations focusing on elucidating these processes would assist the animal production industry in optimizing strategies related to nutrient utilization and product quality.

Deep sequencing of small non-coding RNA highlights brain-specific expression patterns and RNA cleavage.

With the advance of high-throughput sequencing technology numerous new regulatory small RNAs have been identified, that broaden the variety of processing mechanisms and functions of non-coding RNA. Here we explore small non-coding RNA (sncRNA) expression in central parts of the physiological stress and anxiety response system. Therefore, we characterize the sncRNA profile of tissue samples from Amygdala, Hippocampus, Hypothalamus and Adrenal Gland, obtained from 20 pigs. Our analysis reveals that all tissues but Amygdala and Hippocampus possess distinct, tissue-specific expression pattern of miRNA that are associated with Hypoxia, stress responses as well as memory and fear conditioning. In particular, we observe marked differences in the expression profile of limbic tissues compared to those associated to the HPA/stress axis, with a surprisingly high aggregation of 3´-tRNA halves in Amygdala and Hippocampus. Since regulation of sncRNA and RNA cleavage plays a pivotal role in the central nervous system, our work provides seminal insights in the role/involvement of sncRNA in the transcriptional and post-transcriptional regulation of negative emotion, stress and coping behaviour in pigs, and mammals in general.

Cross-talk between energy metabolism and epigenetics during temperature stress response in C2C12 myoblasts.

Objective: Environmental stress induces disturbances in cell energy metabolism and may cause epigenetic modifications. This study aimed to understand the possible impact of temperature stress (35 °C, 39 °C and 41 °C, compared to control 37 °C) on energy metabolism and epigenetic modifications, such as DNA methylation and histone H4 acetylation, as well as its effects on the expression of genes responsible for epigenetic changes, in mouse skeletal myoblasts (C2C12 cells). Methods: The results showed significantly reduced maximal respiration and spare respiratory capacity under heat stress (39 °C and 41 °C), suggesting that mitochondrial functions were compromised under these conditions. The glycolytic capacity and glycolysis markedly increased following low-temperature stress (35 °C). The results suggested that, under cold stress, cells prefer glycolysis as a rapid compensatory mechanism to meet energy requirements for adaptive thermogenic response. Results: Epigenetic changes (histone H4 acetylation and global DNA methylation) were observed under both heat and cold stress. Among the genes coding for DNA methyltransferases, the Dnmt3a was significantly increased under high-temperature conditions (39 °C and 41 °C), while Dnmt1 expression was significantly increased at low temperature (35 °C), indicating that under these conditions the cells preferred maintenance of methylation to de novo methylation activity. An expression pattern similar to Dnmt3a was observed for Gcn5, encoding for a histone acetyltransferase. The study revealed that temperature stress induced changes in the metabolic profiles, as well as epigenetic modifications, including the dynamics of the key enzymes. Conclusion: The results indicated the existence of crosstalk mechanisms between energy metabolism and epigenetics during cell stress response.

Kinetics of Physiological and Behavioural Responses in Endotoxemic Pigs with or without Dexamethasone Treatment.

Although dexamethasone (DEX) is a widely used immunoregulatory agent, knowledge about its pharmacological properties in farm animals, especially pigs, is insufficient. Previous studies suggest that compared to other species, pigs are less sensitive to the immunosuppression conferred by DEX and more sensitive to the threat of bacterial endotoxins. However, there is a paucity of studies examining DEX immunomodulation in endotoxemia in this species. In this study, a porcine endotoxemia model was established by lipopolysaccharide (LPS) and the effect of DEX-pretreatment on the magnitude and kinetics of neuroendocrine, metabolic, hematologic, inflammatory, and behavioural responses were examined. DEX decreased cortisol, adrenocorticotropic hormone (ACTH), red blood cell, hemoglobin, hematocrit, and lymphocyte whereas glucose concentration was increased under both normal and endotoxemic conditions. By contrast, DEX decreased triglyceride, lactate, and IL-6 concentrations and increased platelet count only under an endotoxemic condition. DEX also reduced the frequency of sickness behaviour following LPS challenge. PCA showed that glucose and triglyceride metabolism together with red blood cell count mainly contributed to the separation of clusters during DEX treatment. Our study demonstrates that DEX protects pigs from inflammation and morbidity in endotoxemia, in spite of their less sensitivity to DEX. Moreover, its considerable role in the regulation of the metabolic and hematologic responses in endotoxemic pigs is revealed for the first time.

Transcriptional profiling of liver in riboflavin-deficient chicken embryos explains impaired lipid utilization, energy depletion, massive hemorrhaging, and delayed feathering.

BACKGROUND: A strain of Leghorn chickens (rd/rd), unable to produce a functional riboflavin-binding protein, lays riboflavin-deficient eggs, in which all embryos suddenly die at mid-incubation (days 13-15). This malady, caused by riboflavin deficiency, leads to excessive lipid accumulation in liver, impaired ß-oxidation of lipid, and severe hypoglycemia prior to death. We have used high-density chicken microarrays for time-course transcriptional scans of liver in chicken embryos between days 9-15 during this riboflavin-deficiency-induced metabolic catastrophe. For comparison, half of rd/rd embryos (n = 16) were rescued from this calamity by injection of riboflavin just prior to incubation of fertile eggs from rd/rd hens. RESULTS: No significant differences were found between hepatic transcriptomes of riboflavin-deficient and riboflavin-rescued embryos at the first two ages (days 9 and 11). Overall, we found a 3.2-fold increase in the number of differentially expressed hepatic genes between day 13 (231 genes) and day 15 (734 genes). Higher expression of genes encoding the chicken flavoproteome was more evident in rescued- (15 genes) than in deficient-embryos (4 genes) at day 15. Diminished activity of flavin-dependent enzymes in riboflavin-deficient embryos blocks catabolism of yolk lipids, which normally serves as the predominant source of energy required for embryonic development. CONCLUSIONS: Riboflavin deficiency in mid-stage embryos leads to reduced expression of numerous genes controlling critical functions, including ß-oxidation of lipids, blood coagulation and feathering. Surprisingly, reduced expression of feather keratin 1 was found in liver of riboflavin-deficient embryos at e15, which could be related to their delayed feathering and sparse clubbed down. A large number of genes are expressed at higher levels in liver of riboflavin-deficient embryos; these up-regulated genes control lipid storage/transport, gluconeogenesis, ketogenesis, protein catabolism/ubiquitination and cell death.