Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptomic profiling of spleen in grass-fed and grain-fed Angus cattle

ABSTRACT: The grass-fed cattle obtain nutrients directly from pastures containing limited assimilable energy but abundant amount of fiber; by contrast, grain-fed steers receive a diet that is comprised mainly of grains and serves as an efficient source of high-digestible energy. Besides energy, these two types of diet differ in a large number of nutritional components. Additionally, animals maintained on rich-energy regimen are more likely to develop metabolic disorders and infectious diseases than pasture raised individuals. Thus, we hypothesize that spleenâ??the main immune organâ??may function differently under disparate regimes. The objective of this study was to find the differentially expressed genes in the spleen of grass-fed and grain-fed steers, and furtherly explore the potential involved biopathways. Through RNA sequencing (RNA-Seq), we detected 123 differentially expressed genes. Based on these genes, we performed an Ingenuity Pathway Analysis (IPA) and identified 9 significant molecular networks and 13 enriched biological pathways. Two of the pathways, Nur77 signaling in T lymphocytes and calcium-induced T lymphocyte apoptosis which are immune related, contain a pair of genes HLA-DRA and NR4A1 with dramatically altered expression level. Collectively, our results provided valuable insights into understanding the molecular mechanism of spleen under varied feeding regimens. We collected spleen samples from two randomly chosen animals per group, totaling four samples. The animals were born and raised at the Wye Angus farm, which has produced genetically similar progenies. The genetic resemblance among individuals permitted us to better control the variation between experimental individuals, constituting an excellent resource to perform scientific research. All animals included in this study received the same diet until weaning. Next, we assigned the animals to one certain diet at random, and exclusively raised them under that regimen until termination. The diet of grain-fed group consisted of soybean, shelled corn, corn silage and trace minerals. The grass-fed steers normally received alfalfa harvested from land without any fertilizers, pesticides or other chemicals; during wintertime, bailage was supplied. Grass-fed individuals ate no animal, agricultural or industrial byproducts and never consumed any type of grain. Grain-fed animals reached the market weight around 14 month-old; however, grass-fed steers needed approximately 200 additional days to achieve the same weight. Immediately after termination at the Old Line Custom Meat Company (Baltimore, MD), a small piece of spleen was incised, washed and frozen at -80Â°C for posterior processing.

ORGANISM(S): Bos taurus

SUBMITTER: Jose Carrillo

PROVIDER: E-GEOD-67018 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Beef represents a major diet component and one of the major sources of protein in human. The beef industry in the United States is currently undergoing changes and is facing increased demands especially for natural grass-fed beef. The grass-fed beef obtained their nutrients directly from pastures, which contained limited assimilable energy but abundant amount of fiber. On the contrary, the grain-fed steers received a grain-based regime that served as an efficient source of high-digestible energy. Lately, ruminant animals have been accused to be a substantial contributor for the green house effect. Therefore, the concerns from environmentalism, animal welfare and public health have driven consumers to choose grass-fed beef. Rumen is one of the key workshops to digest forage constituting a critical step to supply enough nutrients for animals’ growth and production. We hypothesize that rumen may function differently in grass- and grain-fed regimes. The objective of this study was to find the differentially expressed genes in the ruminal wall of grass-fed and grain-fed steers, and then explore the potential biopathways. In this study, the RNA Sequencing (RNA-Seq) method was used to measure the gene expression level in the ruminal wall. The total number of reads per sample ranged from 24,697,373 to 36,714,704. The analysis detected 342 differentially expressed genes between ruminal wall samples of animals raised under different regimens. The Fisher’s exact test performed in the Ingenuity Pathway Analysis (IPA) software found 16 significant molecular networks. Additionally, 13 significantly enriched pathways were identified, most of which were related to cell development and biosynthesis. Our analysis demonstrated that most of the pathways enriched with the differentially expressed genes were related to cell development and biosynthesis. Our results provided valuable insights into the molecular mechanisms resulting in the phenotype difference between grass-fed and grain-fed cattle. Ruminal wall samples from two randomly chosen animals per group were obtained, totaling four samples. The animals were born, raised and maintained at the Wye Angus farm. This herd, which has been closed for almost 75 years and yielded genetically similar progenies, constitutes an excellent resource to perform transcriptomic analysis. The genetic resemblance among individuals permits us to better control the cause of variation between experimental clusters and individuals. The randomly chosen pairs of animals were part of larger sets of steers that received a particular treatment. All animals received the same diet until weaning. The grain group received conventional diet consisting of corn silage, shelled corn, soy bean and trace minerals. The grass fed steers consumed normally grazed alfalfa; during wintertime, bailage was utilized. The alfalfa has been harvested from land without any fertilizers, pesticides or other chemicals. The steers ate no animal, agricultural or industrial byproducts and never receive any type of grain. Then, the calves were randomly assigned to one diet and exclusively received that regimen until termination. Grain–fed animals reached the market weight around the age of 14 month-old, however, grass-fed steers required approximately 200 additional days to achieve the same weight. Immediately after termination at the Old Line Custom Meat Company (Baltimore, MD) a small piece of ruminal wall was excised, cleaned and preserved at -80°C for posterior processing.

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 Î²-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 landsâwithout 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 -80Â°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: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 M-bM-^@M-^\Glycosylphosphatidylinositol (GPI) - anchor biosynthesisM-bM-^@M-^]. 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 M-BM-1 10 d age) and after weaning were placed on a diet of 85% corn silage and 15% wet distillerM-bM-^@M-^Ys 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 M-bM-^@M-^\step-up dietsM-bM-^@M-^] 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 distillerM-bM-^@M-^Ys dried grains (16% of dry matter). After the M-bM-^@M-^\step-upM-bM-^@M-^] 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 distillerM-bM-^@M-^Ys 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 M-CM-^W time interactions, but time effects on gene expression were assessed without covariate adjustment. Raw P values were adjusted using Benjamini and HochbergM-bM-^@M-^Ys false discovery rate (FDR). Differences in relative expression due to treatment M-CM-^W 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.

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Transcriptomic profiling of spleen in grass-fed and grain-fed Angus cattle

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