Transcriptome Profiling of Milk Somatic Cells in Holstein, Simmental, Simmental × Holstein Crossbreed and Podolica Cattle at Two Lactation Stages and Production Systems

Lactation is a dynamic process characterised by a production peak at 6–8 weeks, followed by a steady decline. To understand the molecular drivers of these phases and the influence of production systems, this study aims to provide a transcriptomic characterisation of bovine milk somatic cells (BMSCs) in Holstein (HO), Simmental (SM), Simmental × Holstein crossbreed (SM × HO), and Podolica (POD) cows at 60 and 120 days in milk (DIM). Total RNA was sequenced at high coverage, and differential expression and functional enrichment analyses were performed. While a core set of milk protein and fatty acid genes was identified, breed-specific analysis showed SM × HO had the highest variation (677 differentially expressed genes, DEGs). Genes upregulated at 120 DIM involved mitochondrial metabolism and oxidative phosphorylation, while downregulated genes were associated with nuclear transcriptional regulation. At 60 DIM, SM × HO vs. HO showed 66 DEGs, with upregulated genes linked to chromatin remodelling and immune regulation. Comparing production systems, 28 DEGs between POD and HO/SM highlighted differences in mitochondrial activity and transcriptional regulation. This study bridges a knowledge gap by profiling the milk transcriptome of unexplored cattle breeds, providing novel insights into the molecular regulation of lactation.