EFFECT OF CREEP-FEEDING ASSOCIATED WITH INTENSIVE FEEDLOT ON DNA METHYLATION IN THE SKELETAL MUSCLE OF NELORE CATTLE

• Grant number: 24/22290-5
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: May 01, 2025
• End date: February 29, 2028
• Field of knowledge: Agronomical Sciences - Animal Husbandry - Genetics and Improvement of Domestic Animals
• Principal Investigator: Guilherme Luis Pereira
• Grantee: Gustavo Lucas Bezerra Tinoco
• Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil

Abstract

DNA methylation is an important epigenetic mechanism that regulates gene expression without modifying the DNA sequence. It is known that environmental factors, including diet, can influence methylation patterns, impacting various aspects of animal development and phenotype. Thus, this study will aim to characterize and identify gene regions and regulatory elements linked to differentially methylated cytosines (5'mC) in the Longissimus thoracis (LT) muscle, at two time points - weaning and slaughter - of Nelore cattle subjected to supplementation in the creep-feeding phase, followed by the intensive rearing and finishing phases on pasture. In addition, using a multi-omics approach, genes directly and indirectly regulated by methylation will be identified, as well as those with different methylation patterns related to quantitative carcass and meat quality characteristics. To this end, two experimental groups of male Nellore cattle were established: treatment - creep-feeding (CF) associated with intensive feedlot, consisting of 20 calves at pasture receiving concentrate supplementation during the rearing phase and, subsequently, protein-energy supplementation during rearing and finishing phase; and control - no creep-feeding (SCF) associated with intensive feedlot, consisting of 20 calves at pasture receiving mineral salt (ad libitum) during the rearing phase and, subsequently, protein-energy supplementation during rearing and finishing phase. Muscle tissue biopsies were taken at two different times: T1 - at weaning (232 days of age); T2 - at pre-slaughter (554 days of age). The total DNA extracted from the muscle aliquots will be used to build genomic libraries for bisulphite sequencing. Therefore, 12 samples (six from each group) will be used in T1 and T2, totaling 24 libraries. The data obtained will be analyzed for quality, aligned to the reference genome (ARS-UCD2.0) and converted into a matrix containing the proportion of methylated cytosines at each position in the genome using the Bismark program. Gene annotation and identification of 5'mC (5'-methylcytosine) and differentially methylated regions between CF and SCF will be carried out using the methylKit package in the R environment for T1 and T2. Analyses to identify relationships between differentially methylated genes and global gene expression in skeletal muscle will be carried out using the supervised integrative multivariate technique based on MB-sPLSDA (multi block sparse partial least square discriminant analysis) known as DIABLO (Data Integration Analysis for Biomarker discovery using Latent variable approaches for Omics studies) implemented in the R package mixOmics, also for T1 and T2. Finally, in order to identify the relationships between epigenetic marks, different expression profiles and carcass and meat quality characteristics, unsupervised integrative multivariate analysis based on MB-sPLS will be carried out using the R mixOmics package, in which matrices containing the proportion of 5'mC and gene expression given in counts for all annotated genes and matrices containing the quantitative values of carcass characteristics obtained by ultrasound at T1 and T2, as well as meat quality obtained immediately after T2, will be related. Therefore, the genomic data will be analyzed in combination with the quantitative phenotypic data at each of the times, and meat quality will be analyzed together with the genomic data from T2. By exploring the interface between early nutrition and epigenetic regulation, it will be possible to identify probable associations between differential methylation patterns and relevant phenotypic traits, such as carcass and meat quality, expanding knowledge about the molecular mechanisms underlying animal productivity and the quality of end products.