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.
2013-06-21 | E-GEOD-48136 | biostudies-arrayexpress
