Longitudinal blood transcriptomic analysis to identify molecular regulatory patterns of bovine respiratory disease in beef cattle.

Bovine respiratory disease (BRD) is the most common disease in beef cattle and leads to considerable economic losses in both beef and dairy cattle. It is important to uncover the molecular mechanisms underlying BRD and to identify biomarkers for early identification of BRD cattle in order to address its impact on production and welfare. In this study, a longitudinal transcriptomic analysis was conducted using blood samples collected from 24 beef cattle at three production stages in the feedlot: 1) arrival (Entry group); 2) when identified as sick (diagnosed as BRD) and separated for treatment (Pulled); 3) prior to marketing (Close-out, representing healthy animals). Expressed genes were significantly different in the same animal among Entry, Pulled and Close-out stages (false discovery rate (FDR) < 0.01 & |Fold Change| > 2). Beef steers at both Entry and Pulled stages presented obvious difference in GO terms (FDR < 0.05) and affected biological functions (FDR < 0.05 & |Z-score| > 2) when compared with animals at Close-out. However, no significant functional difference was observed between Entry and Pulled animals. The interferon signaling pathway showed the most significant difference between animals at Entry/Pulled and Close-out stages (P < .001 & |Z-score| > 2), suggesting the animals initiated antiviral responses at an early stage of infection. Six key genes including IFI6, IFIT3, ISG15, MX1, and OAS2 were identified as biomarkers to predict and recognize sick cattle at Entry. A gene module with 169 co-expressed genes obtained from WGCNA analysis was most positively correlated (R = 0.59, P = 6E-08) with sickness, which was regulated by 11 transcription factors. Our findings provide an initial understanding of the BRD infection process in the field and suggests a subset of novel marker genes for identifying BRD in cattle at an early stage of infection.

Level of dietary energy and 2,4-thiazolidinedione alter molecular and systemic biomarkers of inflammation and liver function in Holstein cows.

BACKGROUND: The objective of the study was to evaluate the effect of overfeeding a moderate energy diet and a 2,4-thiazolidinedione (TZD) injection on blood and hepatic tissue biomarkers of lipid metabolism, oxidative stress, and inflammation as it relates to insulin sensitivity. RESULTS: Fourteen dry non-pregnant cows were fed a control (CON) diet to meet 100% of NRC requirements for 3 wk, after which half of the cows were assigned to a moderate-energy diet (OVE) and half of the cows continued on CON for 6 wk. All cows received an intravenous injection of 4 mg TZD/kg of body weight (BW) daily from 2 wk after initiation of dietary treatments and for 2 additional week. Compared with CON cows and before TZD treatment, the OVE cows had lower concentration of total protein, urea and albumin over time. The concentration of cholesterol and tocopherol was greater after 2 wk of TZD regardless of diet. Before and after TZD, the OVE cows had greater concentrations of AST/GOT, while concentrations of paraoxonase, total protein, globulin, myeloperoxidase, and haptoglobin were lower compared with CON cows. Regardless of diet, TZD administration increased the concentration of ceruloplasmin, ROMt, cholesterol, tocopherol, total protein, globulin, myeloperoxidase and beta-carotene. In contrast, the concentration of haptoglobin decreased at the end of TZD injection regardless of diet. Prior to TZD injection, the mRNA expression of PC, ANGPTL4, FGF21, INSR, ACOX1, and PPARD in liver of OVE cows was lower compared with CON cows. In contrast, the expression of HMGCS2 was greater in OVE compared with CON cows. After 1 wk of TZD administration the expression of IRS1 decreased regardless of diet; whereas, expression of INSR increased after 2 wk of TZD injection. Cows fed OVE had lower overall expression of TNF, INSR, PC, ACOX1, FGF21, and PPARD but greater HMGCS2 expression. These differences were most evident before and after 1 wk of TZD injection, and by 2 wk of TZD differences in expression for most genes disappeared. CONCLUSIONS: Based on molecular and blood data, administration of TZD enhanced some aspects of insulin sensitivity while causing contradictory results in terms of inflammation and oxidative stress. The bovine liver is TZD-responsive and level of dietary energy can modify the effects of TZD. Because insulin sensitizers have been proposed as useful tools to manage dairy cows during the transition period, further studies are required to investigate the potential hepatotoxicity effect of TZD (or similar compounds) in dairy cattle.

Essential amino acid ratios and mTOR affect lipogenic gene networks and miRNA expression in bovine mammary epithelial cells.

BACKGROUND: The objective of this study was to study how changing the ratio of Lys to Thr, Lys to His, and Lys to Val affects the expression of lipogenic genes and microRNA (miRNA) in bovine mammary epithelial cells. RESULTS: Triplicate cultures with the respective "optimal" amino acid (AA) ratio (OPAA = Lys:Met 2.9:1; Thr:Phe 1.05:1; Lys:Thr 1.8:1; Lys:His 2.38:1; Lys:Val 1.23:1) plus rapamycin (OPAARMC; positive control), OPAA, Lys:Thr 2.1:1 (LT2.1), Lys:Thr 1.3:1 (LT1.3), Lys:His 3.05:1 (LH3.0), or Lys:Val 1.62:1 (LV1.6) were incubated in lactogenic medium for 12 h. The expression of 15 lipogenic genes and 7 miRNA were evaluated. Responses to LT2.1, LT1.3, LH3.0, and LV1.6 relative to the control (OPAARMC) included up-regulated expression of ACSS2, FABP3, ACACA, FASN, SCD, LPIN1, INSIG1, SREBF1, PPARD, and NR1H3 (commonly known as LXR-α). Furthermore, LV1.6 up-regulated expression of ACSL1, DGAT1, and RXRA and down-regulated PPARG expression. Although no effect of OPAA on expression of PPARG was observed, compared with the control, OPAA up-regulated expression of the PPAR targets ACSS2, FABP3, ACACA, FASN, SCD, LPIN1, INSIG1, and SREBF1. Compared with the control, the expression of the anti-lipogenic MIR27AB was down-regulated by OPAA, LT2.1, LT1.3 and LH3.0. In contrast, compared with the control, the expression of the pro-lipogenic MIR21 was up-regulated by LT2.1, LT1.3, LH3.0, and LV1.6. CONCLUSIONS: The observed up-regulation of lipogenic gene networks and the changes in expression of key miRNA involved in the control of lipogenic balance are indicative of a potentially important role of EAA ratios and mTOR signaling in the regulation of milk fat synthesis.

Digital Cushion Fatty Acid Composition and Lipid Metabolism Gene Network Expression in Holstein Dairy Cows Fed a High-Energy Diet.

The hoof digital cushion is a complex structure composed of adipose tissue beneath the distal phalanx, i.e. axial, middle and abaxial fat pad. The major role of these fat depots is dampening compression of the corium underneath the cushion. The study aimed to determine expression of target genes and fatty acid profiles in the hoof of non-pregnant dry Holstein cows fed low (CON) or high-energy (OVE) diets. The middle fat pad of the hoof digital cushion was collected soon after slaughter. Despite the lack of effect on expression of the transcription regulators SREBF1 and PPARG, the expression of the lipogenic enzymes ACACA, FASN, SCD, and DGAT2 was upregulated with OVE. Along with the upregulation of G6PD and IDH1, important for NADPH synthesis during lipogenesis, and the basal glucose transporter SLC2A1, these data indicated a pro-lipogenic response in the digital cushion with OVE. The expression of the lipid droplet-associated protein PLIN2 was upregulated while expression of lipolytic enzymes (ATGL, ABDH5, and LIPE) only tended to be upregulated with OVE. Therefore, OVE induced lipogenesis, lipid droplet formation, and lipolysis, albeit to different extents. Although concentration of monounsaturated fatty acids (MUFA) did not differ, among the polyunsaturated fatty acids (PUFA), the concentration of 20:5n3 was lower with OVE. Among the saturated fatty acids, 20:0 concentration was greater with OVE. Although data indicated that the hoof digital cushion metabolic transcriptome is responsive to higher-energy diets, this did not translate into marked differences in the fatty acid composition. The decrease in concentration of PUFA, which could contribute to synthesis of inflammatory molecules, in OVE-fed cows indicated that feeding higher-energy diets might be detrimental for the mediation of inflammation in digital cushion. This effect could be further exacerbated by physiologic and endocrine changes during the peripartal period that favor inflammation.

Insulin Sensitivity in Adipose and Skeletal Muscle Tissue of Dairy Cows in Response to Dietary Energy Level and 2,4-Thiazolidinedione (TZD).

The effects of dietary energy level and 2,4-thiazolidinedione (TZD) injection on feed intake, body fatness, blood biomarkers and TZD concentrations, genes related to insulin sensitivity in adipose tissue (AT) and skeletal muscle, and peroxisome proliferator-activated receptor gamma (PPARG) protein in subcutaneous AT (SAT) were evaluated in Holstein cows. Fourteen nonpregnant nonlactating cows were fed a control low-energy (CON, 1.30 Mcal/kg) diet to meet 100% of estimated nutrient requirements for 3 weeks, after which half of the cows were assigned to a higher-energy diet (OVE, 1.60 Mcal/kg) and half of the cows continued on CON for 6 weeks. All cows received an intravenous injection of TZD starting 2 weeks after initiation of dietary treatments and for an additional 2 weeks, which served as the washout period. Cows fed OVE had greater energy intake and body mass than CON, and TZD had no effect during the administration period. The OVE cows had greater TZD clearance rate than CON cows. The lower concentration of nonesterified fatty acids (NEFA) and greater concentration of insulin in blood of OVE cows before TZD injection indicated positive energy balance and higher insulin sensitivity. Administration of TZD increased blood concentrations of glucose, insulin, and beta-hydroxybutyrate (BHBA) at 2 to 4 weeks after diet initiation, while the concentration of NEFA and adiponectin (ADIPOQ) remained unchanged during TZD. The TZD upregulated the mRNA expression of PPARG and its targets FASN and SREBF1 in SAT, but also SUMO1 and UBC9 which encode sumoylation proteins known to down-regulate PPARG expression and curtail adipogenesis. Therefore, a post-translational response to control PPARG gene expression in SAT could be a counteregulatory mechanism to restrain adipogenesis. The OVE cows had greater expression of the insulin sensitivity-related genes IRS1, SLC2A4, INSR, SCD, INSIG1, DGAT2, and ADIPOQ in SAT. In skeletal muscle, where PPARA and its targets orchestrate carbohydrate metabolism and fatty acid oxidation, the OVE cows had greater glyceroneogenesis (higher mRNA expression of PC and PCK1), whereas CON cows had greater glucose transport (SLC2A4). Administration of TZD increased triacylglycerol concentration and altered expression of carbohydrate- and fatty acid oxidation-related genes in skeletal muscle. Results indicate that overfeeding did not affect insulin sensitivity in nonpregnant, nonlactating dairy cows. The bovine PPARG receptor appears TZD-responsive, with its activation potentially leading to greater adipogenesis and lipogenesis in SAT, while differentially regulating glucose homeostasis and fatty acid oxidation in skeletal muscle. Targeting PPARG via dietary nutraceuticals while avoiding excessive fat deposition might improve insulin sensitivity in dairy cows during times such as the peripartal period when the onset of lactation naturally decreases systemic insulin release and sensitivity in tissues such as AT.

Nicotinic acid increases adiponectin secretion from differentiated bovine preadipocytes through G-protein coupled receptor signaling.

The transition period in dairy cows (3 weeks prepartum until 3 weeks postpartum) is associated with substantial mobilization of energy stores, which is often associated with metabolic diseases. Nicotinic acid (NA) is an antilipolytic and lipid-lowering compound used to treat dyslipidaemia in humans, and it also reduces non-esterified fatty acids in cattle. In mice the G-protein coupled receptor 109A (GPR109A) ligand NA positively affects the secretion of adiponectin, an important modulator of glucose and fat metabolism. In cattle, the corresponding data linking NA to adiponectin are missing. Our objective was to examine the effects of NA on adiponectin and AMPK protein abundance and the expression of mRNAs of related genes such as chemerin, an adipokine that enhances adiponectin secretion in vitro. Differentiated bovine adipocytes were incubated with pertussis toxin (PTX) to verify the involvement of GPR signaling, and treated with 10 or 15 µM NA for 12 or 24 h. NA increased adiponectin concentrations (p ≤ 0.001) and the mRNA abundances of GPR109A (p ≤ 0.05) and chemerin (p ≤ 0.01). Pre-incubation with PTX reduced the adiponectin response to NA (p ≤ 0.001). The NA-stimulated secretion of adiponectin and the mRNA expression of chemerin in the bovine adipocytes were suggestive of GPR signaling-dependent improved insulin sensitivity and/or adipocyte metabolism in dairy cows.

Increasing gene discovery and coverage using RNA-seq of globin RNA reduced porcine blood samples.

BACKGROUND: Transcriptome analysis of porcine whole blood has several applications, which include deciphering genetic mechanisms for host responses to viral infection and vaccination. The abundance of alpha- and beta-globin transcripts in blood, however, impedes the ability to cost-effectively detect transcripts of low abundance. Although protocols exist for reduction of globin transcripts from human and mouse/rat blood, preliminary work demonstrated these are not useful for porcine blood Globin Reduction (GR). Our objectives were to develop a porcine specific GR protocol and to evaluate the GR effects on gene discovery and sequence read coverage in RNA-sequencing (RNA-seq) experiments. RESULTS: A GR protocol for porcine blood samples was developed using RNase H with antisense oligonucleotides specifically targeting porcine hemoglobin alpha (HBA) and beta (HBB) mRNAs. Whole blood samples (n = 12) collected in Tempus tubes were used for evaluating the efficacy and effects of GR on RNA-seq. The HBA and HBB mRNA transcripts comprised an average of 46.1% of the mapped reads in pre-GR samples, but those reads reduced to an average of 8.9% in post-GR samples. Differential gene expression analysis showed that the expression level of 11,046 genes were increased, whereas 34 genes, excluding HBA and HBB, showed decreased expression after GR (FDR <0.05). An additional 815 genes were detected only in post-GR samples. CONCLUSIONS: Our porcine specific GR primers and protocol minimize the number of reads of globin transcripts in whole blood samples and provides increased coverage as well as accuracy and reproducibility of transcriptome analysis. Increased detection of low abundance mRNAs will ensure that studies relying on transcriptome analyses do not miss information that may be vital to the success of the study.

Transition period-related changes in the abundance of the mRNAs of adiponectin and its receptors, of visfatin, and of fatty acid binding receptors in adipose tissue of high-yielding dairy cows.

Adipose tissue expresses adipokines, which are involved in regulation of energy expenditure, lipid metabolism, and insulin sensitivity. To adapt for the transition from pregnancy to lactation, particularly in high-yielding dairy cows, adipokines, their receptors, and particular G-protein coupled receptors (GPRs) are of potential importance. Signaling by GPR 41 stimulates leptin release via activation by short-chain fatty acids; GPR 43/109A inhibits lipolysis, and GPR 109A thereby mediates the lipid-lowering effects of nicotinic acid and beta-hydroxybutyrate. The aim of this study was to compare the mRNA expression of adiponectin and visfatin, adiponectin receptors 1 and 2 (AdipoR1/2), leptin receptor (obRb), insulin receptor as of the aforementioned GPRs during the transition period in high-yielding dairy cows. Biopsies from subcutaneous fat and blood samples were obtained from 10 dairy cows 1 week before and 3 weeks after calving. For AdipoR1 and AdipoR2 mRNA abundance as well as for leptin concentrations in plasma, a reduction (P