Project description:The objective of this study was to examine changes in muscle gene expression of growing steers during a period of dietary energy restriction followed by a period of realimentation. Crossbred Aberdeen Angus x Holstein Friesian (n = 24) steers were assigned to one of two feeding treatments. Over a 99 d period, 1 group (n=12) was offered a high energy control diet consisting of concentrates ad libitum and 7 kg of grass silage per head daily. The second group (n=12) was offered an energy restricted diet consisting of grass silage ad libitum plus 0.5 kg of concentrate per head per day. From the end of the differential feeding period (99 d), both groups of animals were offered a total mixed ration (grass silage:concentrate ratio of 80:20). This period, which lasted 200 d, was known as the realimentation period. All animals were slaughtered on d 299 of the study. Muscle biopsies were collected at 2 time points (end of the differential feeding period (d 99) and during the realimentation period (d131). RNA was extracted and muscle gene expression was examined using RNA-seq technology and bioinformatic analysis. During the differential feeding period, 17 over-represented pathways were identified, including the peroxisome proliferator activated receptor signalling, glycolysis/ gluconeogenesis and metabolic pathways controlling the metabolism of lipids and lipoproteins which indicate reduced energy intake and fat tissue accumulation occurring in muscle tissue during the restriction phase. During the realimentation period, 164 differentially expressed genes were annotated to 9 over-represented pathways including starch and sucrose metabolism, carbohydrate digestion and absorption and TGF-β signalling pathway. It is hypothesised that the signalling effects of the TGF-β pathway were reduced thereby promoting accelerated cell growth and proliferation in muscle tissue of animals experiencing compensatory growth. This information can be exploited in genomic breeding programmes to assist selection of cattle with a greater ability to compensate following a period dietary restriction.

Project description:The objective of this study was to examine changes in muscle gene expression of growing bulls during a period of dietary energy restriction followed by a period of subsequent realimentation and compensatory growth. Purebred Holstein Friesian bulls (n=20) were assigned to one of two feeding treatments (i) restricted feed allowance for 125 days (n=10) followed by ad libitum access to feed for a further 55 days or (ii) a control group with ad libitum access to feed through out the 180 days trial (n=10). The first 125 days of the trial were denoted as Peirod 1, during which treatment groups were fed differentially. The subsequent 55 days, denoted as Period 2 during which all bulls were fed ad libitum. All bulls received the same diet of 70% concentrate 30% grass silage through out the experimental trial,with the amount of feed provided different dependnet on each treatment group. Muscle biopsies were collected at 2 time points (end of the differential feeding in Period 1 (d 120) and during the realimentation phase in Period 2 (d 15 of re-alimentation). RNA was extracted and muscle gene expression was examined using RNAseq technology and bioinformatic analysis. During the differential feeding period, over-represented pathways including fatty acid beta oxidaiton, oxidative phosphorylation and TCA cyclewere identified, which indicate utilisation of lipid sotes for energy utilisaiton and also alterations in energy produciton during dietary restriciton in muscle. During the realimentation period, pathways involved in energy produciton were over-represented, with the direction of fold changes opposite to that of these pathways in Period 1. additionally, a number of genes involved in cell division and cellular proliferation were up-regulated during compensatory growth in re-alimentation, thereby promoting accelerated cell growth and proliferation in muscle tissue of animals experiencing compensatory growth. This information can be exploited in genomic breeding programmes to assist selection of cattle with a greater ability to compensate following a period dietary restriction. 40 muscle RNA samples were analysed in total. 10 samples were from muscle biopsies collected at the end of a period of dietary restriction (d 120) and 10 samples were from muscle biopsis collected during the initial stages of compensatory growth (d 15 of re-alimentation). In addition, RNA was also anlaysed from 10 samples collected from animals fed a libitum at each of these two timepoints.

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:Growing ruminants maintained under dietary restriction for extended periods will exhibit compensatory growth when reverted to ad libitum feeding. This period of compensatory growth is associated with increased feed efficiency, lower basal energy requirements, and changes in circulating concentrations of metabolic hormones. To identify genetic mechanisms contributing to these physiological changes, 8 month-old steers were fed either ad libitum (control; n = 6) or 60-70% of intake of control animals (feed-restricted; n=6) for a period of 12 weeks. All steers were then fed ad libitum for the remaining 8 weeks of the experiment (realimentation period). Liver was biopsied from each animal at days -14, +1 and +14 relative to realimentation for RNA extraction and gene expression analysis by microarray hybridization. Steers were assigned randomly to one of two treatment groups, control or feed-restricted, and housed indoors in individual pens. Steers were acclimated to their pens for 5 d prior to starting the experimental treatments. Feed was offered once daily between 0630 and 0930 and orts from the previous day's feeding were collected and weighed to estimate actual intake. Control animals were fed ad libitum throughout the 20-wk experimental period. Feed-restricted steers were offered 60-70% of intake of control animals for 12 wks to target a limited rate of gain of approximately 0.5 kg/d. Restricted steers were then fed ad libitum for the remaining 8 wks of the experiment (realimentation period). During the first 3 d of realimentation, feed offered to both treatment groups was divided into two equal rations to gradually adjust restricted animals to full intake. Water was offered ad libitum throughout the experimental period. Approximately 200 mg of liver tissue was collected from each steer by needle biopsy using a Tru-Cut biopsy needle at -14, +1, +14 d relative to realimentation. Liver samples were immediately frozen in liquid nitrogen and stored at -80C until RNA isolation. Total RNA was isolated from 36 liver samples using TRIZOL Reagent (Invitrogen Corp., Carlsbad, CA). Samples were DNase-treated using the TURBO DNA-free kit (Ambion, Inc., Austin, TX) according to manufacturerâ??s instructions, followed by column purification using the RNeasy Mini Kit (Qiagen, Valencia, CA). Quality and concentration of RNA were assessed using a 2100 Bioanlayzer (Agilent Technologies, Palo Alto, CA) and ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Probe labeling, hybridizations of probes to the oligo microarray, and array scanning were performed by the Roche NimbleGen Systems, Inc. Microarray Core Facility in Reykjavik, Iceland according to standard procedures (Madison, WI; http://www.nimblegen.com).

Project description: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.

Project description:The objective of this study was to examine changes in muscle gene expression of growing bulls during a period of dietary energy restriction followed by a period of subsequent realimentation and compensatory growth. Purebred Holstein Friesian bulls (n=20) were assigned to one of two feeding treatments (i) restricted feed allowance for 125 days (n=10) followed by ad libitum access to feed for a further 55 days or (ii) a control group with ad libitum access to feed through out the 180 days trial (n=10). The first 125 days of the trial were denoted as Peirod 1, during which treatment groups were fed differentially. The subsequent 55 days, denoted as Period 2 during which all bulls were fed ad libitum. All bulls received the same diet of 70% concentrate 30% grass silage through out the experimental trial,with the amount of feed provided different dependnet on each treatment group. Muscle biopsies were collected at 2 time points (end of the differential feeding in Period 1 (d 120) and during the realimentation phase in Period 2 (d 15 of re-alimentation). RNA was extracted and muscle gene expression was examined using RNAseq technology and bioinformatic analysis. During the differential feeding period, over-represented pathways including fatty acid beta oxidaiton, oxidative phosphorylation and TCA cyclewere identified, which indicate utilisation of lipid sotes for energy utilisaiton and also alterations in energy produciton during dietary restriciton in muscle. During the realimentation period, pathways involved in energy produciton were over-represented, with the direction of fold changes opposite to that of these pathways in Period 1. additionally, a number of genes involved in cell division and cellular proliferation were up-regulated during compensatory growth in re-alimentation, thereby promoting accelerated cell growth and proliferation in muscle tissue of animals experiencing compensatory growth. This information can be exploited in genomic breeding programmes to assist selection of cattle with a greater ability to compensate following a period dietary restriction.

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:Two-stage two-phase biogas reactor systems consisting each of one batch downflow hydrolysis reactor (HR, vol. 10 L), one process fluid storage tank (vol. 10 L), and one downstream upflow anaerobic filter reactor (AF, vol. 10 L), were operated at mesophilic (M, 37 °C) and thermophilic (T, 55 °C) temperatures and over a period of > 750 d (Figure 1, Additional file 1). For each reactor system and for each process temperature, two replicates were conducted in parallel, denominated further as biological replicates. Further process details were as previously published. Start-up of all fermenters were performed using liquid fermenter material from a biogas plant converting cattle manure in co-digestion with grass and maize silage and other biomass at varying concentrations and at mesophilic temperatures. Silage of perennial ryegrass (Lolium perenne L.) was digested as sole substrate in batches of varying amounts with retention times of 28 d (storage of bale silage at -20 °C, cutting length 3 cm, volatile substances (VS) 32 % of fresh mass (FM), total Kjeldahl nitrogen 7.6 g kgFM-1, NH4+-N 0.7 g kgFM-1, acetic acid 2.6 g kgFM-1, propionic acid < 0.04 g kgFM-1, lactic acid 2.6 g kgFM-1, ethanol 2.2 g kgFM-1, C/N ratio 19.3, chemical oxygen demand (COD) 357.7 g kgFM-1, analysis of chemical properties according to [6]. No spoilage was observed in the silage. Biogas yields were calculated as liters normalized to 0 °C and 1013 hPa (LN) per kilogram volatile substances (kgVS). For chemical analysis, samples were taken from the effluents of HR and AF. For sequencing of 16S rRNA gene amplicon libraries, microbial metagenomes, and microbial metatranscriptomes, samples were taken from the silage digestate in the HR digested for 2 d. At this time point, high AD rates were detected as indicated by the fast increase of volatile fatty acids (VFA), e.g., acetic acid. Sampling was performed at two different organic loading rates (OLR), i.e., batch-fermentation of 500 g (denominated as “low OLR”, samples MOLR500 and TOLR500) and 1,500 g silage (denominated as “increased OLR”, samples MOLR1500 and TOLR1500).

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RNA-seq analysis of differential gene expression in muscle from angus beef steers undergoing diet restriction and compensatory growth

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