Hepatic Gene Expression Profiles of Growing versus Finished Beef Steers
ABSTRACT: Growing and finishing phases are two important animal production stages, which differ fundamentally in compositional growth. However, the physiological mechanisms altered concomitantly with the shift in whole-body compositional gain as cattle fatten (growing vs. finished steers), are poorly understood. Microarray analysis using the Bovine Gene 1.0 ST Array was conducted to determine shifts in hepatic genomic expression profiles of growing vs. finishing beef steers. The specific overall hypothesis tested was that genes involved in amino acid, carbohydrate and lipid metabolism, antioxidant capacity and immune responses were differentially expressed in growing vs. finishing steers. Overall design: Sixteen weaned Angus steers (BW = 209 ± 29.4 kg) were randomly assigned (n = 8) to develop through growing (final BW = 301 kg) or finished (final BW = 576 kg) growth phases and individually fed enough of a cotton seed hull-based diet to achieve a constant ADG (1.5 kg/d). Steers had ad libitum access to fresh water and an industry standard vitamin-mineral supplement. Liver samples were collected for RNA extraction and microarray analysis.
INSTRUMENT(S): [BovGene-1_0-st] Bovine Gene 1.0 ST Array [transcript (gene) version]
Project description:The goal of this study was to test the hypothesis that sodium selenite (ISe), SEL-PLEX (OSe), vs. a 1:1 blend (MIX) of ISe and OSe in a basal vitamin-mineral (VM) mix would differetianlly alter pituitary transcriptome profiles in growing beef steers commonly grazing an endophyte-infected tall fescue pasture. Predominately-Angus steers (BW = 183 ± 34 kg) were randomly selected from herds of fall-calving cows grazing E+ pasture and consuming VM mixes that contained 35 ppm Se as ISe, OSe, and MIX forms. Steers were weaned, depleted of Se for 98 d, and subjected to summer-long common grazing of an E+ pasture (0.51 ppm total ergovaline per ergovalinine; 10.1 ha). Steers were assigned (n = 8 per treatment) to the same Se-form treatments upon which they were raised. Selenium treatments were administered by daily top-dressing 85 g of VM mix onto 0.23 kg soyhulls, using in-pasture Calan gates. We collected pituitary samples and examine for changes in global expression pattern by microarray analysis. Overall design: 20 total samples were analyzed (n = 6 for ISe, and n=7 for both OSe and MIX).
Project description:The bovine transcriptome dynamics in logissimus muscle (LM) during the post-natal growth remain unknown. Biopsies of LM from Angus steers were harvested at 0, 60, 120, and 220 d from early-weaning. A 13,153 bovine oligonucleotide array was used for transcript profiling. Functional analysis of microarray data was performed using the Dynamic Impact Approach (DIA) by means of KEGG and DAVID databases. During the growing phase, most of the highly-impacted pathways (e.g. Ascorbate and aldarate metabolism, Drug metabolism - cytochrome P450 and Retinol metabolism) were inhibited. The finishing phase, was characterized with the most striking differences with 3,784 differentially expressed genes (DEG; FDR <0.01). The functional analysis of those DEG revealed that the most-impacted KEGG canonical pathway was “Glycosylphosphatidylinositol (GPI) - anchor biosynthesis”. The inhibition of this pathway suggested a unique role of GPI-anchor proteins in intracellular trafficking during the finishing phase. A mechanism regulating muscle growth during the finishing phase was uncovered in which inhibition of calpastatin and activation of tyrosine aminotransferase ubiquitination promote proteasomal degradation, while the concurrent activation of ribosomal proteins promotes protein synthesis, thus, the balance of these processes likely results in a steady state of protein turnover during the finishing phase. Overall, results underscored the importance of transcriptome dynamics in LM to support key biological functions during rapid growth. The study utilized a subset of 14 animals from a larger study encompassing 32 purebred Aberdeen Angus steers from the University of Illinois beef cattle herd. Steer calves were early-weaned (134 ± 10 d age) and after weaning were placed on a diet of 85% corn silage and 15% wet distiller’s grains (as-fed basis) for 3 wk. Subsequently, one half of Angus (n = 16) steers was randomly assigned to a high-byproduct or high-grain diet for a 112 d growing phase. The high-byproduct diet contained (dry matter basis) 35% corn silage, 20% corn gluten feed, 38% soyhulls, 3% cracked corn, and 3% soybean meal (49% crude protein). The high-grain diet contained 20% corn silage, 68% cracked corn, and 11% soybean meal (49% crude protein). Both diets contained 1% limestone/dicalcium phosphate/mineral/vitamin/urea/dry molasses mixture. Calculated NEG for the high-byproduct diet was 1.19 Mcal/kg and 1.43 Mcal/kg for the high-grain diet. At the end of the growing phase, steers on each group were fed “step-up diets” for 10 d. Step-up diet for high-byproduct-fed steers contained 1.37 Mcal NEG/kg. Step-up diet for high-grain-fed steers contained 1.43 Mcal NEG/kg but contained distiller’s dried grains (16% of dry matter). After the “step-up” period, all steers were finished on a common high-grain finishing diet containing 1.44 Mcal NEG/kg (15% corn silage, 58% cracked corn, 25% dried distiller’s grains, 1% limestone, and 1% urea/mineral/vitamin mixture). All diets were offered on an ad libitum basis. Steers had an individual electronic identification ear tag and individual feed intake data were collected using the GrowSafe system (GrowSafe Systems Ltd., Alberta, Canada). Cattle were harvested at approximately 13 mo age. A bovine oligonucleotide (70-mers) microarray with >13,000 annotated sequences developed at the University of Illinois, was used for transcript profiling. Details on the development, annotation, and use of this microarray have been reported previously by Loor et al., 2007 (http://physiolgenomics.physiology.org/content/32/1/105.abstract). Methods for microarray hybridization and scanning were as reported by Loor et al. (2007). Briefly, slides were hydrated, dried, and placed in a UV Stratalinker 1800 (Stratagene, La Joya, CA) for ~5 min. Slides were washed with 0.2% SDS solution, rinsed with MilliQ (Millipore) H2O, and placed in warm prehybridization soln for 45 min at 42 C. The same amount of Cy3- or Cy5-labelled cDNA from muscle and a reference standard RNA pool (made of different bovine tissues) were co-hybridized using a dye-swap design (i.e., two microarrays per sample). Slides were incubated for 48 h at 45 C prior to scanning. Criteria for evaluation of slide quality included: identification of number of spots with a minimum median signal intensity of 3 SD above background; keeping slides with a minimum of 20,000 spots with minimum median signal intensity of 3 SD above background in both Cy3 and Cy5 channels; and keeping slides with a minimum mean intensity of 400 relative fluorescent units in both Cy3 and Cy5 channels across the entire slide. Data from a total of 112 microarrays were normalized for dye and microarray effects (i.e., Lowess normalization and microarray centering) and used for statistical analysis. Data were analyzed using the Proc MIXED procedure of SAS (SAS, SAS Inst. Inc., Cary, NC). Fixed effects were treatment (high-byproduct, high-grain) and time (0, 56, 120, and 220 d), and dye. Random effects included steer and microarray. A covariate adjustment was used to assess treatment and treatment × time interactions, but time effects on gene expression were assessed without covariate adjustment. Raw P values were adjusted using Benjamini and Hochberg’s false discovery rate (FDR). Differences in relative expression due to treatment × time interactions were considered significant at an FDR-adjusted P < 0.25 or at P < 0.01 for time effects. qPCR data were normalized using the median of 4 suitable internal control genes, and were analyzed using the same statistical model described above. Differences were considered significant at P-value of 0.05.
Project description:Benzo(a)pyrene is a well-established human carcinogen in humans and rodents. In the present study, we sought to determine the dose- and time-dependent changes in gene expression upon oral exposure to benzo(a)pyrene. Adult male B6C3F1 mice were exposed to four doses of benzo(a)pyrene or vehicle control for three days and sacrificed 4 or 24 hours after the final exposure. This experiment examined the hepatic transcriptional response of male mice exposed to BaP for 3 days at four different doses, including D1 (300 mg/kg BW/day), D2 (150 mg/kg BW/day), D3 (50 mg/kg BW/day) and D4 (5 mg/kg BW/day), and one control. Each dose group was further examined at 2 time points, 4 hours and 24 hours, following the final exposure. Each dose group and time point had 4-5 biological replicates. There were a total 46 samples (arrays) included in the final analysis using a two-colour reference design.
Project description:Benzo(a)pyrene is a well-established human carcinogen in humans and rodents. In the present study, we sought to determine the dose- and time-dependent changes in gene expression upon oral exposure to benzo(a)pyrene. Adult male MutaTMMouse were exposed to three doses of benzo(a)pyrene or vehicle control (olive oil) for 28 days and sacrificed three days after the final exposure. This experiment examined the forestomach transcriptional response of male mice exposed to BaP for 28 days at three different doses, including D1 (25 mg/kg BW/day), D2 (50 mg/kg BW/day), and D3 (75 mg/kg BW/day) and vehicle control. Each dose group was examined 72 hours following the final exposure. Each dose group and time point had 4-5 biological replicates. There were a total 17 samples (arrays) included in the final analysis using a two-colour reference design.
Project description:To investigate effects of long-term intake of RPS on gene expression in the colon and liver of pigs,thirty-six Duroc × Landrace × Large White growing barrows were randomly allocated to corn starch (CS) and RPS groups. Each group consisted of six replicates (pens), with three pigs per pen. Pigs in the CS group were offered a corn/soybean-based diet, while pigs in the RPS group were put on a diet in which 230 g/kg (growing period) or 280 g/kg (finishing period) purified corn starch was replaced with purified RPS during a 100-day trial. Liver transcriptomic results showed that the expression of CD36, CPT1B and ACADM was down-regulated, while AGPAT4, GPAT, FABP1 and FABP3 were up-regulated by the RPS diet, indicating a decrease in fatty acid intake and synthesis, and an increase in fatty acid oxidation and glycerophospholipid synthesis.Analysis of the colonic transcriptome profiles revealed that the RPS diet changed the colonic expression profile of the host genes mainly involved in immune response pathways. RPS significantly increased proinflammartory cytokine IL-1β gene expression and suppressed genes involved in lysosome. Thirty-six Duroc × Landrace × Large White growing barrows (70 days of age, 23.78 ± 1.87 kg) were randomly allocated to two groups, each group consisting of three pigs per pen, and six replicates. Pigs in the control group were offered a corn/soybean-based diet, while 230 g/kg purified corn starch (CS) was replaced with purified RPS in the RPS diet group. Diets were formulated according to the nutrient requirements of the National Research Council (1998). When animals reached the age of 120 days, diets were adapted to the nutrient requirements of the animals (finishing diet) and the amount of purified starch increased to 280 g of CS or RPS per kilogram of feed. Pigs had unlimited access to feed and water throughout the experimental period, which consisted of two 50-day trials in which the pigs consumed the growing diet (days 0-50) and finishing diet (days 51-100), respectively. On day 100, one pig from each replicate that met the target slaughter weight (105 to 110 kg) was slaughtered. The liver and colonic mucosa tissues were collected and preserved in liquid nitrogen for gene expression analysis.
Project description:The objective of this study was to identify differentially expressed genes in the liver of steers with divergent Residual Feed Intake (RFI). Methods:In total 50 purebred Angus, 48 purebred Charolais and 158 Kinsella Composite breed steers were tested for individual feed intake using the GrowSafe system for an average period of 70 to 73 days. During the feedlot test animals were fed at ad libitum with a finishing diet composed of 75% barley grain, 20% barley silage and 5% rumensin pellet. Body weight of each animal was measured at an interval of 28days and ADG for each animal was obtained from a linear regression of serial body weight (BW) measurements (Kgs) on time (days). MWT was calculated as midpoint BW0.75, where midpoint BW was computed as the sum of initial BW of the animal and the product of its ADG multiplied by half the number test days. DMI of each animal was calculated as the average daily feed intake of the animals for the time during the feedlot test (days). The expected DMI for each animal was predicted using the regression intercept and regression coefficients of ADG and MWT on actual DMI, and RFI was computed as the difference between the standardized daily DMI and the expected DMI. At the end of the test, animals were slaughtered and liver tissue was collected immediately after slaughter separately bagged in plastic bags, labeled and flash frozen in liquid nitrogen. The frozen samples were transferred to the laboratory on ice and stored at -80C until RNA extraction. From the frozen samples, 20 samples from each breed were selected for total RNA extraction including six samples with extreme high and six samples extreme low-RFI phenotypes for each breed. Complementary DNA (cDNA) libraries were constructed for each of the 60 animals and consequently single end sequenced using Illumina Hiseq 2500 sequencer. The raw reads were aligned and mapped to the bovine reference genome UMD 3.1 using Tophat2 aligner with default alignment parameters. Thereafter, reads aligned uniquely to each annotated transcript of gene in the bovine genome were counted using HTSeq-count package with default parameters. Read counts from HTSeq-count, sample information (sample id, sire, feed efficiency group and sequencing mode) and gene annotation table from ENSEMBL Biomart were used for differential expression analysis within each breed using the edgeR package in R for the six extreme low-RFI and six extreme high-RFI samples. Results: At a false discovery rate of 0.05 and fold change > 2 , we identified 72, 41 and 175 differentially expressed genes for Angus, Charolais and KC RFI divergent steers respectively. Overall design: RNA-seq of Angus, Charolais and Kinsella Composite male liver with either low or high Residual Feed Intake (RFI).
Project description:Beef represents a major diet component and source of protein in many countries. With an increment demand for beef, the industry is currently undergoing changes towards natural produced beef. Consumers not only concern about product quality, but also for the well-being of animals. Therefore, the consumption of grass-fed meat is continuously growing. However, the nutritional true differences between feeding systems are still unclear. The aim of this study was to examine latissimus dorsi muscle quality and animal welfare by transcriptome and metabolome profiles, and to identify biological pathways related to the differences between grass- and grain-fed Angus steers. By RNA-Seq analysis of latissimus dorsi muscle, we have recognized 241 differentially expressed genes (FDR < 0.1). The metabolome examination of muscle and blood revealed 163 and 179 altered compounds in each tissue (P-value < 0.05), respectively. Accordingly, alterations in glucose metabolism, divergences in free fatty acids and carnitine conjugated lipid levels, and altered β-oxidation, have been observed. In summary, this study demonstrates a unique transcriptomic and metabolic signature in the muscle of grain and grass finished cattle. Results support the accumulation of anti-inflammatory n3 polyunsaturated fatty acids in grass finished cattle, while higher levels of n6 PUFAs in grain finished animals may promote inflammation and oxidative stress. Furthermore, grass-fed animals produce tender beef with lower total fat and higher omega3/omega6 ratio than grain fed animals, which could potentially benefit consumer health. Finally, blood cortisol levels strongly indicate that grass fed animals experience less stress than the grass fed individuals The steers came from a closed Wye Angus herd with very similar genetics. The grass-fed group was comprised of steers that received alfalfa and orchard grass hay, clover and orchard grass pasture, or orchard grass and alfalfa pasture. The grass-fed individuals consumed grazed alfalfa upon availability and bales during winter and were not exposed to any corn, any form of grain or feed by-products. The alfalfa and grass hay were harvested from land that has had minimal fertilizer and no application of pesticides or inorganic chemicals. The control group was fed a conventional diet consisting of corn silage, soybean, shelled corn and minerals. The pastures were managed as organic lands–without fertilizers, pesticides or any chemical additives. At the slaughter plant, 10 ml whole blood sample from the jugular vein was collected in EDTA tubes and directly storage at -80°C. Then, a small piece of longissimus dorsi muscle was obtained from each hot carcass at the level of the 12th intercostal space and immediately frozen in dry ice for posterior analysis.
Project description:Furan is a mouse and rat hepatocarcinogen. We sought to determine the dose-dependent changes in gene expression upon exposure of B6C3F1 mice to furan in order to better understand furan’s mode of action. Adult female B6C3F1 mice were exposed to 1, 2, 4 or 8 mg/kg bw furan or vehicle control (corn oil) for three weeks and sacrificed four hours after the final exposure. In this study we examined the transcriptional response in liver tissue of female B6C3F1 mice exposed to furan for 3 weeks at four different doses: 1, 2, 4 or 8 mg/kg bw furan (or vehicle control) and sacrificed four hours after the final exposure. Each dose group had 4-5 biological replicates. There were a total of 25 samples included in the final analysis. We used a two-colour reference design and SurePrint G3 Mouse GE 8x60K microarrays (Agilent).
Project description:Mechanism-based toxicogenomics (tgx) is used as a tool to identify markers reflective of the onset and progression of cholestasis in C57BL/6 mice using Cyclosporin A (CsA) as a model compound. Critical doses for tgx analysis were derived from a dose range finding study in which increase of serum cholesterol, total bile acids, and total bilirubin as well as induction of hepatocyte vacuolization 25 days upon repeated CsA administration through oral gavage were considered as critical effects. For tgx analysis to find early markers, livers of mice repeatedly treated with 3 mg/kg BW, 8.9 mg/kg BW, and 26.7 mg/kg BW for one, four, and eleven days were collected. 60 samples are analyzed; per treatment duration (1, 4, 11 days), time-matched vehicle (olive oil) controls and three dose groups (3, 8.9, 26.7 mg/kg BW) were included; each group consisted of 5 replicates; 3 arrays were excluded, 2 because of quality control restrictions, 1 because of outlier properties. 2 that failed QC are omitted. Final data consists of 58 CEL files.