427 Impact of late gestation slow-release nitrogen-enriched diets on energy metabolism in calf skeletal muscle: A proteomic and transcriptomic approach

Abstract The current study aimed to determine the enriched biological processes, through proteomic and transcriptomic data, associated with maternal slow-release nitrogen diets received during late gestation and their effects on the skeletal muscle of offspring. From d 180 to d 268 of gestation, a total of 16 pregnant Brahman cows were divided into two groups: a control (CON, n = 12) low crude protein (CP) basal diet (6% of CP, ad libitum) plus mineral mixture (130 gּ cow-1ּ d-1), or the CON treatment supplemented with a slow-released N (SRN, n = 9) based protein concentrate supplement [40% CP, fed 2 g/kg of body weight (BW)/d in the morning) composed by corn, soybean meal, urea and a SRN source (Timafeed Boost, Roullier Group, Saint-Malo, France). Muscle biopsies were performed on d 45 of age in calves for RNA sequencing (RNA-seq) and proteomic (HPLC-MS/MS) analyses. The quality control of RNAseq raw data was assessed using FASTQc software, with low-quality reads trimmed by Trimmomatic. Reads were mapped against the Bos taurus reference genome using STAR. Differentially expressed (DE) genes and isoforms, significant at an adjusted P-value ≤ 0.05, were identified using the limma package and Cuffdiff tool, respectively, in the R environment. Proteomic data was processed in MaxQuant against the Bos taurus reference proteome, with statistical analysis performed using the MSqRob package in R. Network analyses identified significant biological processes (adjusted P-value ≤ 0.05) among differentially abundant proteins (DAPs). Notably, while the experimental treatment did not affect transcript abundance, protein-level differences were observed. Enriched biological processes in SRN group skeletal muscle of calves included acetyl-CoA biosynthesis from pyruvate, mitochondrial respiratory chain complex assembly, regulation of calcium ion transmembrane transport, and several others related to energy and nitrogen metabolism. Protein-protein interaction network analyses revealed key processes such as ATP and glucose metabolism, tricarboxylic acid cycle, and sarcomere organization. Overall, our findings underscore the beneficial impact of slow-release nitrogen-enriched diets during late gestation on energy metabolism in calf skeletal muscle.