Intestinal Carnitine Status and Fatty Acid Oxidation in Response to Clofibrate and Medium-Chain Triglyceride Supplementation in Newborn Pigs.

To investigate the role of peroxisome proliferator-activated receptor alpha (PPARα) in carnitine status and intestinal fatty acid oxidation in neonates, a total of 72 suckled newborn piglets were assigned into 8 dietary treatments following a 2 (±0.35% clofibrate) × 4 (diets with: succinate+glycerol (Succ), tri-valerate (TC5), tri-hexanoate (TC6), or tri-2-methylpentanoate (TMPA)) factorial design. All pigs received experimental milk diets with isocaloric energy for 5 days. Carnitine statuses were evaluated, and fatty acid oxidation was measured in vitro using [1-14C]-palmitic acid (1 mM) as a substrate in absence or presence of L659699 (1.6 µM), iodoacetamide (50 µM), and carnitine (1 mM). Clofibrate increased concentrations of free (41%) and/or acyl-carnitine (44% and 15%) in liver and plasma but had no effects in the intestine. The effects on carnitine status were associated with the expression of genes involved in carnitine biosynthesis, absorption, and transportation. TC5 and TMPA stimulated the increased fatty acid oxidation rate induced by clofibrate, while TC6 had no effect on the increased fatty acid oxidation induced by clofibrate (p > 0.05). These results suggest that dietary clofibrate improved carnitine status and increased fatty acid oxidation. Propionyl-CoA, generated from TC5 and TMPA, could stimulate the increased fatty acid oxidation rate induced by clofibrate as anaplerotic carbon sources.

Transcriptome analysis of the hypothalamus and pituitary of turkey hens with low and high egg production.

BACKGROUND: High egg producing hens (HEPH) show increased hypothalamic and pituitary gene expression related to hypothalamo-pituitary-gonadal (HPG) axis stimulation as well as increased in vitro responsiveness to gonadotropin releasing hormone (GnRH) stimulation in the pituitary when compared to low egg producing hens (LEPH). Transcriptome analysis was performed on hypothalamus and pituitary samples from LEPH and HEPH to identify novel regulators of HPG axis function. RESULTS: In the hypothalamus and pituitary, 4644 differentially expressed genes (DEGs) were identified between LEPH and HEPH, with 2021 genes up-regulated in LEPH and 2623 genes up-regulated in HEPH. In LEPH, up-regulated genes showed enrichment of the hypothalamo-pituitary-thyroid (HPT) axis. Beta-estradiol was identified as an upstream regulator regardless of tissue. When LEPH and HEPH samples were compared, beta-estradiol was activated in HEPH in 3 of the 4 comparisons, which correlated to the number of beta-estradiol target genes up-regulated in HEPH. In in vitro pituitary cell cultures from LEPH and HEPH, thyroid hormone pretreatment negatively impacted gonadotropin subunit mRNA levels in cells from both LEPH and HEPH, with the effect being more prominent in HEPH cells. Additionally, the effect of estradiol pretreatment on gonadotropin subunit mRNA levels in HEPH cells was negative, whereas estradiol pretreatment increased gonadotropin subunit mRNA levels in LEPH cells. CONCLUSIONS: Up-regulation of the HPT axis in LEPH and upstream beta-estradiol activation in HEPH may play a role in regulating HPG axis function, and ultimately ovulation rates. Thyroid hormone and estradiol pretreatment impacted gonadotropin mRNA levels following GnRH stimulation, with the inhibitory effects of thyroid hormone more detrimental in HEPH and estradiol stimulatory effects more prominent in LEPH. Responsiveness to thyroid hormone and estradiol may be due to desensitization to thyroid hormone and estradiol in LEPH and HEPH, respectively, due to up-regulation of the HPT axis in LEPH and of the HPG axis in HEPH. Further studies will be necessary to identify possible target gene desensitization mechanisms and elicit the regulatory role of the HPT axis and beta-estradiol on ovulation rates in turkey hens.

MicroRNA-30c Modulates Type I IFN Responses To Facilitate Porcine Reproductive and Respiratory Syndrome Virus Infection by Targeting JAK1.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen and has evolved several mechanisms to evade IFN-I responses. We report that a host microRNA, miR-30c, was upregulated by PRRSV via activating NF-κB and facilitated its ability to infect subject animals. Subsequently, we demonstrated that miR-30c was a potent negative regulator of IFN-I signaling by targeting JAK1, resulting in the enhancement of PRRSV infection. In addition, we found that JAK1 expression was significantly decreased by PRRSV and recovered when miR-30c inhibitor was overexpressed. Importantly, miR-30c was also upregulated by PRRSV infection in vivo, and miR-30c expression corresponded well with viral loads in lungs and porcine alveolar macrophages of PRRSV-infected pigs. Our findings identify a new strategy taken by PRRSV to escape IFN-I-mediated antiviral immune responses by engaging miR-30c and, thus, improve our understanding of its pathogenesis.

Identification of microRNAs controlling hepatic mRNA levels for metabolic genes during the metabolic transition from embryonic to posthatch development in the chicken.

BACKGROUND: The transition from embryonic to posthatch development in the chicken represents a massive metabolic switch from primarily lipolytic to primarily lipogenic metabolism. This metabolic switch is essential for the chick to successfully transition from the metabolism of stored egg yolk to the utilization of carbohydrate-based feed. However, regulation of this metabolic switch is not well understood. We hypothesized that microRNAs (miRNAs) play an important role in the metabolic switch that is essential to efficient growth of chickens. We used high-throughput RNA sequencing to characterize expression profiles of mRNA and miRNA in liver during late embryonic and early posthatch development of the chicken. This extensive data set was used to define the contributions of microRNAs to the metabolic switch during development that is critical to growth and nutrient utilization in chickens. RESULTS: We found that expression of over 800 mRNAs and 30 miRNAs was altered in the embryonic liver between embryonic day 18 and posthatch day 3, and many of these differentially expressed mRNAs and miRNAs are associated with metabolic processes. We confirmed the regulation of some of these mRNAs by miRNAs expressed in a reciprocal pattern using luciferase reporter assays. Finally, through the use of yeast one-hybrid screens, we identified several proteins that likely regulate expression of one of these important miRNAs. CONCLUSIONS: Integration of the upstream regulatory mechanisms governing miRNA expression along with monitoring the downstream effects of this expression will ultimately allow for the construction of complete miRNA regulatory networks associated with the hepatic metabolic switch in chickens. Our findings support a key role for miRNAs in controlling the metabolic switch that occurs between embryonic and posthatch development in the chicken.

Effect of dietary manganese on antioxidant status and expressions of heat shock proteins and factors in tissues of laying broiler breeders under normal and high environmental temperatures.

To investigate the effect of Mn on antioxidant status and on the expressions of heat shock proteins/factors in tissues of laying broiler breeders subjected to heat challenge, we used a completely randomised design (n 6) with a factorial arrangement of 2 environmental temperatures (normal, 21±1°C, and high, 32±1°C)×3 dietary Mn treatments (a Mn-unsupplemented basal diet (CON), or a basal diet supplemented with 120 mg Mn/kg diet, either as inorganic Mn sulphate (iMn) or as organic Mn proteinate (oMn)). There were no interactions (P>0·10) between environmental temperature and dietary Mn in any of the measured indices. High temperature decreased (P<0·003) Mn content, and also tended (P=0·07) to decrease Cu Zn superoxide dismutase (CuZnSOD) activity in the liver and heart. However, an increased Mn superoxide dismutase (MnSOD) activity (P<0·05) and a slight increase in malondialdehyde level (P=0·06) were detected in breast muscle. Up-regulated (P<0·05) expressions of heat shock factor 1 (HSF1) and HSF3 mRNA and heat shock protein 70 (HSP70) mRNA and protein were found in all three tissues. Broiler breeders fed either iMn or oMn had higher tissue Mn content (P<0·0001), heart MnSOD and CuZnSOD activities (P<0·01) and breast muscle MnSOD protein levels (P<0·05), and lower (P<0·05) breast muscle HSP70 mRNA and protein levels compared with those fed CON. Broiler breeders fed oMn had higher (P<0·03) bone Mn content than those fed iMn. These results indicate that high temperature decreases Mn retention and increases HSP70, HSF1 and HSF3 expressions in the tissues of laying broiler breeders. Furthermore, dietary supplementation with Mn in either source may enhance the heart's antioxidant ability and inhibit the expression of HSP70 in breast muscle. Finally, the organic Mn appears to be more available than inorganic Mn for bone in laying broiler breeders regardless of environmental temperatures.

Effect of dietary manganese on antioxidant status and expression levels of heat-shock proteins and factors in tissues of laying broiler breeders under normal and high environmental temperatures.

To investigate the effect of Mn on antioxidant status and expression levels of heat-shock proteins/factors in tissues of laying broiler breeders subjected to heat challenge, we used a completely randomised design (n 6) with a factorial arrangement of 2 environmental temperatures (normal, 21 (sem 1)°C and high, 32 (sem 1)°C)×3 dietary Mn treatments (an Mn-unsupplemented basal diet (CON), or a basal diet supplemented with 120 mg Mn/kg diet as inorganic Mn sulphate (iMn) or organic Mn proteinate (oMn)). There were no interactions (P>0·10) between environmental temperature and dietary Mn in all of the measured indices. High temperature decreased (P<0·003) Mn content, and also tended (P=0·07) to decrease copper zinc superoxide dismutase (CuZnSOD) activity in the liver and heart. However, an increased manganese superoxide dismutase (MnSOD) activity (P<0·05) and a slight increase of malondialdehyde level (P=0·06) were detected in breast muscle. Up-regulated (P<0·05) expression levels of heat-shock factor 1 (HSF1) and HSF3 mRNA and heat-shock protein 70 (HSP70) mRNA and protein were found in all three tissues. Broiler breeders fed either iMn or oMn had higher tissue Mn content (P<0·0001), heart MnSOD and CuZnSOD activities (P<0·01) and breast muscle MnSOD protein levels (P<0·05), and lower (P<0·05) breast muscle HSP70 mRNA and protein levels than those fed CON. Broiler breeders fed oMn had higher (P<0·03) bone Mn content than those fed iMn. These results indicate that high temperature decreases Mn retention and increases HSP70 and HSF1, HSF3 expression levels in tissues of laying broiler breeders. Furthermore, dietary supplementation with Mn in either source may enhance heart antioxidant ability and inhibit the expression of HSP70 in breast muscle. Finally, the organic Mn appears to be more available than inorganic Mn for bone in laying broiler breeders regardless of environmental temperatures.

Effects of acute and chronic heat stress on plasma metabolites, hormones and oxidant status in restrictedly fed broiler breeders.

Heat tolerance can be improved by feed restriction in broiler chickens. It is unknown whether the same is true for broiler breeders, which are restrictedly fed. Therefore, the current study was conducted to study the effects of heat stress on plasma metabolites, hormones, and oxidative status of restricted fed broiler breeders with special emphases on the temperature and latency of heat exposure. In trial 1, 12 broiler breeders were kept either in a thermoneutral chamber (21°C, control, n = 6) or in a chamber with a step-wise increased environmental temperature from 21 to 33°C (21, 25, 29, 33°C, heat-stressed, n = 6). Changes in plasma total cholesterol, glucose, and triiodothyronine (T3) were closely related to the environmental temperature. When the temperature reached 29°C, plasma T3 (P < 0.05) was significantly decreased in acute heat-stressed birds, whereas plasma glucose (P < 0.001) and cholesterol (P = 0.002) increased only when the temperature reached 33°C. Plasma triglyceride (P = 0.026) and creatine kinase (CK, P = 0.018) were lower in heat-stressed birds than controls regardless of the temperatures applied. In Trial 2, 24 broiler breeders were divided into 2 groups and raised under 21°C and 32°C for 8 weeks, respectively. Total cholesterol was increased in chronic heat-stressed broiler breeders after 4 weeks. Plasma lactate dehydrogenase (LDH, P = 0.047) and glutamic-oxaloacetic transaminase (GOT, P = 0.036) was up-regulated after 6 weeks of thermal treatment, whereas plasma CK (P = 0.009) was increased at the end of thermal treatment. Plasma malonaldehyde, protein carbonyl content, activity of total superoxide dismutase (SOD), and corticosterone content were not altered after acute and prolonged heat challenges. Taken together, acute heat stress primarily resulted in disturbance of plasma metabolites, whereas chronic heat stress caused tissue damage reflected by increased plasma LDA, GOT, and CK. During acute heat stress, plasma metabolites were minimally disturbed in broiler breeders until the environmental temperature reached 33°C.

Zinc alleviates thermal stress-induced damage to the integrity and barrier function of cultured chicken embryonic primary jejunal epithelial cells via the MAPK and PI3K/AKT/mTOR signaling pathways.

Zinc (Zn) could alleviate the adverse effect of high temperature (HT) on intestinal integrity and barrier function of broilers, but the underlying mechanisms remain unclear. We aimed to investigate the possible protective mechanisms of Zn on primary cultured broiler jejunal epithelial cells exposed to thermal stress (TS). In Exp.1, jejunal epithelial cells were exposed to 40℃ (normal temperature, NT) and 44℃ (HT) for 1, 2, 4, 6, or 8 h. Cells incubated for 8 h had the lowest transepithelial resistance (TEER) and the highest phenol red permeability under HT. In Exp.2, the cells were preincubated with different Zn sources (Zn sulfate as iZn and Zn proteinate with the moderate chelation strength as oZn) and Zn supplemental levels (50 and 100 µmol/L) under NT for 24 h, and then continuously incubated under HT for another 8 h. TS increased phenol red permeability, lactate dehydrogenase (LDH) activity and p-PKC/PKC level, and decreased TEER, cell proliferation, mRNA levels of claudin-1, occludin, zona occludens-1 (ZO-1), PI3K, AKT and mTOR, protein levels of claudin-1, ZO-1 and junctional adhesion molecule-A (JAM-A), and the levels of p-ERK/ERK, p-PI3K/PI3K and p-AKT/AKT. Under HT, oZn was more effective than iZn in increasing TEER, occludin, ZO-1, PI3K, and AKT mRNA levels, ZO-1 protein level, and p-AKT/AKT level; supplementation with 50 µmol Zn/L was more effective than 100 µmol Zn/L in increasing cell proliferation, JAM-A, PI3K, AKT, and PKC mRNA levels, JAM-A protein level, and the levels of p-ERK/ERK and p-PI3K/PI3K; furthermore, supplementation with 50 µmol Zn/L as oZn had the lowest LDH activity, and the highest ERK, JNK-1, and mTOR mRNA levels. Therefore, supplemental Zn, especially 50 µmol Zn/L as oZn, could alleviate the TS-induced integrity and barrier function damage of broiler jejunal epithelial cells possibly by promoting cell proliferation and tight junction protein expression via the MAPK and PI3K/AKT/mTOR signaling pathways.

Current state of Marek's disease virus microRNA research.

MicroRNA (miRNA) is a major family of small RNAs that posttranscriptionally regulate gene expression. Small RNA profiling studies have revealed that some viruses, particularly large DNA viruses, such as Marek's disease virus (MDV), encode their own set of miRNAs. There are currently 406 viral miRNAs in miRBase, of which 392 are encoded by herpesviruses. To date, 26 MDV-1 miRNAs, 36 MDV-2 miRNAs, and 28 herpesvirus of turkeys miRNAs have been identified. Interestingly, herpesvirus miRNAs appear to have spatial conservation, located in clusters within repeat regions, but lack sequence conservation. Two clusters of MDV-1 miRNA have been identified, one located near the MEQ gene and one within the latency-associated transcript (LAT). miRNA profiling studies have shown that MDV miRNA are differentially expressed between strains and stages of infection. For example, mdv1-miR-M4 and mdv1-miR-M2-3p are three- and sixfold higher, expressed, respectively, in vv+ strains compared to vv strains. A recent study found that deletion or seed region mutation of mdv1-miR-M4 reduces viral oncogenicity, suggesting a link between mdv1-mir-M4 and lymphoma development in MDV-infected birds. Taken together, current research suggests that viral miRNAs are a key component of MDV pathogenesis.

The interplay between MDV and HVT affects viral miRNa expression.

It is well established that herpesviruses encode numerous microRNAs (miRNAs) and that these virally encoded small RNAs play multiple roles in infection. The present study was undertaken to determine how co-infection of a pathogenic MDV serotype one (MDV1) strain (MD5) and a vaccine strain (herpesvirus of turkeys [HVT]) alters viral miRNA expression in vivo. We first used small RNA deep sequencing to identify MDV1-encoded miRNAs that are expressed in tumorigenic spleens of MDV1-infected birds. The expression patterns of these miRNAs were then further assessed at an early time point (7 days postinfection [dpi]) and a late time point (42 dpi) in birds with and without HVT vaccination using real-time PCR (RT-PCR). Additionally, the effect of MDV1 co-infection on HVT-encoded miRNAs was determined using RT-PCR. A diverse population of miRNAs was expressed in MDV-induced tumorigenic spleens at 42 dpi, with 18 of the 26 known mature miRNAs represented. Of these, both mdv1-miR-M4-5p and mdv1-miR-M2-3p were the most highly expressed miRNAs. RT-PCR analysis further revealed that nine MDV miRNAs were differentially expressed between 7 dpi and 42 dpi infected spleens. At 7 dpi, three miRNAs were differentially expressed between the spleens of birds co-infected with HVT and MD5 compared with birds singly infected with MD5, whereas at 42 dpi, nine miRNAs were differentially expressed. At 7 dpi, the expression of seven HVT-encoded miRNAs was affected in the spleens of co-infected birds compared with birds only receiving the HVT vaccine. At 42 dpi, six HVT-encoded miRNAs were differentially expressed between the two groups. Target prediction analysis suggests that these differentially expressed viral miRNAs are involved in regulating several cellular processes, including cell proliferation and the adaptive immune response.

Global gene expression analysis of the turkey hen hypothalamo-pituitary-gonadal axis during the preovulatory hormonal surge.

The preovulatory hormonal surge (PS) consists of elevated circulating luteinizing hormone (LH) and progesterone levels and serves as the primary trigger for ovarian follicle ovulation. Increased LH and progesterone, produced by the pituitary and the granulosa layer of the largest ovarian follicle (F1), respectively, result from hypothalamic stimulation and steroid hormone feedback on the hypothalamo-pituitary-gonadal (HPG) axis. The hypothalamus, pituitary, F1 granulosa, and granulosa layer of the fifth largest follicle (F5) were isolated from converter turkey hens outside and during the PS and subjected to RNA sequencing (n = 6 per tissue). Differentially expressed genes were subjected to functional annotation using DAVID and IPA. A total of 12, 250, 1235, and 1938 DEGs were identified in the hypothalamus, pituitary, F1 granulosa, and F5 granulosa respectively (q<0.05, |fold change|>1.5, FPKM>1). Gene Ontology (GO) analysis revealed key roles for metabolic processes, steroid hormone feedback, and hypoxia induced gene expression changes. Upstream analysis identified a total of 4, 42, 126, and 393 potential regulators of downstream gene expression in the hypothalamus, pituitary, F1G, and F5G respectively, with a total of 63 potential regulators exhibiting differential expression between samples collected outside and during the PS (|z-score|>2). The results from this study serve to increase the current knowledge base surrounding the regulation of the PS in turkey hens. Through GO analysis, downstream processes and functions associated with the PS were linked to identified DEGs, and through upstream analysis, potential regulators of DEGs were identified for further analysis. Linking upstream regulators to the downstream PS and ovulation events could allow for genetic selection or manipulation of ovulation frequencies in turkey hens.

Proteomic and phosphoproteomic analysis of chicken embryo fibroblasts infected with cell culture-attenuated and vaccine strains of Marek's disease virus.

Vaccination is an effective strategy to reduce the loss of chickens in the poultry industry caused by Marek's Disease (MD), an avian lymphoproliferative disease. The vaccines currently used are from attenuated serotype 1 Marek's disease virus (MDV) or naturally nononcogenic MDV strains. To prepare for future immunity breaks, functional genomic and proteomic studies have been used to better understand the underlying mechanisms of MDV pathogenicity and the effects induced by the vaccine viruses. In this study, a combined approach of quantitative GeLC-MSE and qualitative ERLIC/IMAC/LC-MS/MS analysis were used to identify abundance changes of proteins and the variations of phosphorylation status resulting from the perturbations due to infection with an attenuated oncogenic virus strain (Md11/75C) and several nononcogenic virus strains (CVI988, FC126 and 301B) in vitro. Using this combined approach, several signal transduction pathways mapped by the identified proteins were found to be altered at both the level of protein abundance and phosphorylation. On the basis of this study, a kinase-dependent pathway to regulate phosphorylation of 4E-BP1 to modulate assembly of the protein translation initiation complex was revealed. The differences of 4E-BP1 phosphorylation patterns as well as the measured abundance changes among several other proteins that regulate host transcriptional and translational activities across the virus strains used in this study provide new insight for future functional and biochemical characterization of specific proteins involved in MDV pathogenesis.

Integrated analysis of microRNA expression and mRNA transcriptome in lungs of avian influenza virus infected broilers.

BACKGROUND: Avian influenza virus (AIV) outbreaks are worldwide threats to both poultry and humans. Our previous study suggested microRNAs (miRNAs) play significant roles in the regulation of host response to AIV infection in layer chickens. The objective of this study was to test the hypothesis if genetic background play essential role in the miRNA regulation of AIV infection in chickens and if miRNAs that were differentially expressed in layer with AIV infection would be modulated the same way in broiler chickens. Furthermore, by integrating with parallel mRNA expression profiling, potential molecular mechanisms of host response to AIV infection can be further exploited. RESULTS: Total RNA isolated from the lungs of non-infected and low pathogenic H5N3 infected broilers at four days post-infection were used for both miRNA deep sequencing and mRNA microarray analyses. A total of 2.6 M and 3.3 M filtered high quality reads were obtained from infected and non-infected chickens by Solexa GA-I Sequencer, respectively. A total of 271 miRNAs in miRBase 16.0 were identified and one potential novel miRNA was discovered. There were 121 miRNAs differentially expressed at the 5% false discovery rate by Fisher's exact test. More miRNAs were highly expressed in infected lungs (108) than in non-infected lungs (13), which was opposite to the findings in layer chickens. This result suggested that a different regulatory mechanism of host response to AIV infection mediated by miRNAs might exist in broiler chickens. Analysis using the chicken 44 K Agilent microarray indicated that 508 mRNAs (347 down-regulated) were differentially expressed following AIV infection. CONCLUSIONS: A comprehensive analysis combining both miRNA and targeted mRNA gene expression suggests that gga-miR-34a, 122-1, 122-2, 146a, 155, 206, 1719, 1594, 1599 and 451, and MX1, IL-8, IRF-7, TNFRS19 are strong candidate miRNAs or genes involved in regulating the host response to AIV infection in the lungs of broiler chickens. Further miRNA or gene specific knock-down assay is warranted to elucidate underlying mechanism of AIV infection regulation in the chicken.

Alterations in hepatic mitotic and cell cycle transcriptional networks during the metabolic switch in broiler chicks.

During embryonic life, chicks mainly derive energy from hepatic oxidation of yolk lipids. After hatch, chicks must rely on carbohydrate-rich feed to obtain energy. This requires an abrupt and intensive switch of metabolic processes, particularly in the liver. We recently identified a number of transcriptional and post-transcriptional regulatory networks that work concordantly to tune metabolic processes during the metabolic switch. Here, we used delayed feeding post-hatch (48 h) to impede the metabolic switch in broilers. We used RNA-seq to identify hepatic transcriptome differences between late stage embryos (E18) and two-day-old chicks (D2), which were either fed-from-hatch (FED) or not fed (DLY). Between FED and E18, 2,430 genes were differentially expressed (fold-change≥ 2; FDR p-value 0.05), of these 1,237 were downregulated in FED birds and 1,193 were upregulated. Between DLY and E18, 1979 genes were differentially expressed, of these 1,043 were downregulated and 936 were upregulated in DLY birds. Between DLY and FED, 880 genes were differentially expressed, of these 543 were downregulated and 337 were upregulated in DLY birds. We found that in addition to disturbances in a number of metabolic pathways, unfed chicks had a widespread suppression of gene networks associated with cell proliferation, cell cycle progression and mitosis. Expression patterns suggest that hepatocytes of delayed-fed birds have abnormal mitosis and increased polyploidization. This suggests that post-hatch feed consumption maintains the rate and integrity of liver growth immediately, which in turn, likely helps facilitate the appropriate programming of hepatic metabolic networks.

Remodeling of Hepatocyte Mitochondrial Metabolism and De Novo Lipogenesis During the Embryonic-to-Neonatal Transition in Chickens.

Embryonic-to-neonatal development in chicken is characterized by high rates of lipid oxidation in the late-term embryonic liver and high rates of de novo lipogenesis in the neonatal liver. This rapid remodeling of hepatic mitochondrial and cytoplasmic networks occurs without symptoms of hepatocellular stress. Our objective was to characterize the metabolic phenotype of the embryonic and neonatal liver and explore whether these metabolic signatures are preserved in primary cultured hepatocytes. Plasma and liver metabolites were profiled using mass spectrometry based metabolomics on embryonic day 18 (ed18) and neonatal day 3 (nd3). Hepatocytes from ed18 and nd3 were isolated and cultured, and treated with insulin, glucagon, growth hormone and corticosterone to define hormonal responsiveness and determine their impacts on mitochondrial metabolism and lipogenesis. Metabolic profiling illustrated the clear transition from the embryonic liver relying on lipid oxidation to the neonatal liver upregulating de novo lipogenesis. This metabolic phenotype was conserved in the isolated hepatocytes from the embryos and the neonates. Cultured hepatocytes from the neonatal liver also maintained a robust response to insulin and glucagon, as evidenced by their contradictory effects on lipid oxidation and lipogenesis. In summary, primary hepatocytes from the embryonic and neonatal chicken could be a valuable tool to investigate mechanisms regulating hepatic mitochondrial metabolism and de novo lipogenesis.

Development and application of a phosphoproteomic method using electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), IMAC, and LC-MS/MS analysis to study Marek's Disease Virus infection.

Marek's Disease (MD) is an avian neoplastic disease caused by Marek's Disease Virus (MDV). The mechanism of virus transition between the lytic and latent cycle is still being investigated; however, post-translational modifications, especially phosphorylation, have been thought to play an important role. Previously, our group has used strong cation exchange chromatography in conjunction with reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the changes in global proteomic expression upon MDV infection (Ramaroson , M. F.; Ruby, J.; Goshe, M. B.; Liu , H.-C. S. J. Proteome Res. 2008, 7, 4346-4358). Here, we extend our study by developing an effective separation and enrichment approach to investigate the changes occurring in the phosphoproteome using electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) to fractionate peptides from chicken embryo fibroblast (CEF) digests and incorporating a subsequent IMAC enrichment step to selectively target phosphorylated peptides for LC-MS/MS analysis. To monitor the multidimensional separation between mock- and MDV-infected CEF samples, a casein phosphopeptide mixture was used as an internal standard. With LC-MS/MS analysis alone, no CEF phosphopeptides were detected, while with ERLIC fractionation only 1.2% of all identified peptides were phosphorylated. However, the incorporation of IMAC enrichment with ERLIC fractionation provided a 50-fold increase in the percentage of identified phosphopeptides. Overall, a total of 581 unique phosphopeptides were identified (p < 0.05) with those of the MDV-infected CEF sample containing nearly twice as many as the mock-infected control of which 11% were unique to MDV proteins. The changes in the phosphoproteome are discussed including the role that microtubule-associated proteins may play in MDV infection mechanisms.

Centennial Review: Metabolic microRNA - shifting gears in the regulation of metabolic pathways in poultry.

Over 20 yr ago, a small noncoding class of RNA termed microRNA (miRNA) that was able to recognize sequences in mRNAs and inhibit their translation was discovered in Caenorhabditis elegans. In the intervening years, miRNA have been discovered in most eukaryotes and are now known to regulate the majority of protein-coding genes. It has been discovered that disruption of miRNA function often leads to the development of pathological conditions. One physiological system under extensive miRNA-mediated regulation is metabolism. Metabolism is one of the most dynamic of biological networks within multiple organs, including the liver, muscle, and adipose tissue, working in concert to respond to ever-changing nutritional cues and energy demands. Therefore, it is not surprising that miRNA regulate virtually all aspects of eukaryotic metabolism and have been linked to metabolic disorders, such as obesity, fatty liver diseases, and diabetes, just to name a few. Chickens, and birds in general, face their own unique metabolic challenges, particularly after hatching, when their metabolism must completely transform from using lipid-rich yolk to carbohydrate-rich feed as fuel in a very short period of time. Furthermore, commercial poultry breeds have undergone extensive selection over the last century for more desirable production traits, which has resulted in numerous metabolic consequences. Here, we review the current knowledge of miRNA-mediated regulation of metabolic development and function in chickens.

Expression Signatures of microRNAs and Their Targeted Pathways in the Adipose Tissue of Chickens during the Transition from Embryonic to Post-Hatch Development.

As the chick transitions from embryonic to post-hatching life, its metabolism must quickly undergo a dramatic switch in its major energy source. The chick embryo derives most of its energy from the yolk, a lipid-rich/carbohydrate-poor source. Upon hatching, the chick's metabolism must then be able to utilize a lipid-poor/carbohydrate-rich source (feed) as its main form of energy. We recently found that a number of hepatically-expressed microRNAs (miRNAs) help facilitate this shift in metabolic processes in the chick liver, the main site of lipogenesis. While adipose tissue was initially thought to mainly serve as a lipid storage site, it is now known to carry many metabolic, endocrine, and immunological functions. Therefore, it would be expected that adipose tissue is also an important factor in the metabolic switch. To that end, we used next generation sequencing (NGS) and real-time quantitative PCR (RT-qPCR) to generate miRNome and transcriptome signatures of the adipose tissue during the transition from late embryonic to early post-hatch development. As adipose tissue is well known to produce inflammatory and other immune factors, we used SPF white leghorns to generate the initial miRNome and transcriptome signatures to minimize complications from external factors (e.g., pathogenic infections) and ensure the identification of bona fide switch-associated miRNAs and transcripts. We then examined their expression signatures in the adipose tissue of broilers (Ross 708). Using E18 embryos as representative of pre-switching metabolism and D3 chicks as a representative of post-switching metabolism, we identified a group of miRNAs which work concordantly to regulate a diverse but interconnected group of developmental, immune and metabolic processes in the adipose tissue during the metabolic switch. Network mapping suggests that during the first days post-hatch, despite the consumption of feed, the chick is still heavily reliant upon adipose tissue lipid stores for energy production, and is not yet efficiently using their new energy source for de novo lipid storage. A number of core master regulatory pathways including, circadian rhythm transcriptional regulation and growth hormone (GH) signaling, likely work in concert with miRNAs to maintain an essential balance between adipogenic, lipolytic, developmental, and immunological processes in the adipose tissue during the metabolic switch.

Characterization of hypothalamo-pituitary-thyroid axis gene expression in the hypothalamus, pituitary gland, and ovarian follicles of turkey hens during the preovulatory surge and in hens with low and high egg production.

Dysregulation of the preovulatory surge (PS) leads to lowered egg production. The hypothalamo-pituitary-thyroid (HPT) axis has been shown to influence plasma progesterone levels and follicle ovulation. The presence of thyroid hormone receptors (THR) in the reproductive axis suggests possible effects of thyroid hormone. To further understand the potential role of thyroid hormone on the PS, HPT axis plasma hormone concentrations and gene expression were characterized surrounding the PS in average egg producing hens (AEPH), low egg producing hens (LEPH), and high egg producing hens (HEPH) (n = 3 hens/group). Data were analyzed using the mixed models procedure of SAS, with significance indicated at P < 0.05. Average egg producing hens and HEPH displayed lower levels of triiodothyronine (T3) and higher levels of thyroxine (T4) inside of the PS, whereas LEPH showed inverse T3 and T4 levels relative to the PS. Expression of mRNA for hypothalamic thyrotropin-releasing hormone (TRH), pituitary thyrotropin (TSHB), and the main thyroid hormone metabolism enzyme (DIO2) were downregulated during the PS in AEPH and HEPH. Low egg producing hens displayed higher expression of mRNA for hypothalamic TRH as well as pituitary TSHB and DIO2 compared with HEPH. Average egg producing hens expression of THR mRNAs was upregulated during the PS in the hypothalamus but downregulated in the pituitary. High egg producing hens showed decreased expression of THR mRNAs in both the hypothalamus and pituitary when compared with LEPH. In ovarian follicles, THR mRNAs were more prevalent in the thecal layer of the follicle wall compared with the granulosa layer, and expression tended to decrease with follicle maturity. Minimal differences in follicular THR expression were seen between LEPH and HEPH, indicating that THR expression is unlikely to be responsible for steroid hormone production differences occurring between LEPH and HEPH. Generally, downregulation of the HPT axis was seen during the PS in AEPH and HEPH, whereas upregulation of the HPT axis was seen in LEPH. Further studies will be required to clarify the role of the HPT axis in the regulation of ovulation and egg production rates in turkey hens.

Transcriptome Analysis During Follicle Development in Turkey Hens With Low and High Egg Production.

Low and high egg producing hens exhibit gene expression differences related to ovarian steroidogenesis. High egg producing hens display increased expression of genes involved in progesterone and estradiol production, in the granulosa layer of the largest follicle (F1G) and small white follicles (SWF), respectively, whereas low egg producing hens display increased expression of genes related to progesterone and androgen production in the granulosa (F5G) and theca interna layer (F5I) of the fifth largest follicle, respectively. Transcriptome analysis was performed on F1G, F5G, F5I, and SWF samples from low and high egg producing hens to identify novel regulators of ovarian steroidogenesis. In total, 12,221 differentially expressed genes (DEGs) were identified between low and high egg producing hens across the four cell types examined. Pathway analysis implied differential regulation of the hypothalamo-pituitary-thyroid (HPT) axis, particularly thyroid hormone transporters and thyroid hormone receptors, and of estradiol signaling in low and high egg producing hens. The HPT axis showed up-regulation in high egg producing hens in less mature follicles but up-regulation in low egg producing hens in more mature follicles. Estradiol signaling exclusively exhibited up-regulation in high egg producing hens. Treatment of SWF cells from low and high egg producing hens with thyroid hormone in vitro decreased estradiol production in cells from high egg producing hens to the levels seen in cells from low egg producing hens, whereas thyroid hormone treatment did not impact estradiol production in cells from low egg producing hens. Transcriptome analysis of the major cell types involved in steroidogenesis inferred the involvement of the HPT axis and estradiol signaling in the regulation of differential steroid hormone production seen among hens with different egg production levels.