233. Neonatal Vitamin A Supplementation Modulates DNA Methylation in Longissimus Thoracis Muscle in Beef Cattle.

Abstract Intramuscular fat deposition is regulated by myogenesis and adipogenesis during the marbling window (MW), which begins in the last months of gestation and extends through the first 200 days of postnatal life. Vitamin A (VA) supplementation during the MW has been shown to influence the expression of genes associated with adipogenic differentiation, thereby modulating intramuscular fat development. The main objective of this study was to evaluate epigenetic mechanisms underlying intramuscular fat deposition, with a focus on DNA methylation (DNAm) patterns in Angus × Nellore cattle that did or did not receive VA injections after birth. A total of 16 calves (8 males and 8 females) were allocated (balanced by sex) to either a control group (CON; n = 8; no VA injection) or a VA-treated group (VAcalf; n = 8). Animals in the VAcalf group received two injections of 200,000 IU of VA at birth and at 60 days of age. At 211 days of age, Longissimus thoracis muscle biopsies were collected for DNAm analysis. Genomic DNA was extracted, and whole-genome bisulfite sequencing was performed on all samples. Sequencing reads were subjected to quality control (FastQC v0.11.9) and adapter trimming using Trim Galore (v0.6.10). Subsequently, reads were aligned to the Bos taurus reference genome (ARS-UCD2.0) using Bismark (v0.24.2) with Bowtie 2 (v2.5.4). Deduplication and methylation extraction were performed following the Bismark workflow. DNAm analysis was conducted using the methylKit R package to identify differentially methylated loci (DMLs). Alignment metrics were consistent across all samples, with an average alignment rate of 83.08 ± 1.24%. Global CpG methylation levels were similar between treatments (CON: 68.23 ± 1.70%; VAcalf: 68.48 ± 1.88%). After applying a minimum read coverage of 10× and filtering for autosomal CpG islands, 21,691 loci were retained across all 29 autosomes. Differential methylation analysis identified 890 DMLs (q < 0.05; ≥ 10% methylation difference), of which 695 were hypomethylated and 195 were hypermethylated in the VAcalf group. Functional annotation revealed 149 genes associated with these DMLs. Among these, genes related to intramuscular fat metabolism and muscle development were identified, as shown in Table 1, including TMEM135 (fatty acid transport), FZD3 (preadipocyte differentiation), PEX5L (fatty acid oxidation), GNAQ (muscle hypertrophy), and SP1 (a transcription factor that regulates genes such as MyoD, MyoG, PPARG, and C/EBP). This study indicates that vitamin A supplementation may modulate DNAm patterns in the Longissimus thoracis muscle across different genomic regions, highlighting the hypomethylation in the promoter region of SP1. These findings suggest that vitamin A acts as an epigenomic modulator in beef cattle muscle. This study was supported by the FAPEMIG, CAPES, and CNPq, Brazil. For image description, please refer to the figure legend and surrounding text.