Integrating transcriptomic and metabolomic profiles of primiparous Holstein cows across multiple lactation periods reveals the regulatory mechanism underlying milk component traits

To investigate the regulatory mechanisms underlying milk production traits, transcriptional sequencing (RNA-seq) and metabolomic analysis with liquid chromatography-MS were implemented in blood and serum samples from 25 lactating Holstein cows across multiple lactation stages. We identified 16,147, 15,859, and 16,010 expressed genes (fragments per kilobase million >1) and 160, 332, and 142 differentially expressed genes (DEG) during early, peak, and late lactation, respectively; the DEG were significantly enriched in adipocytokine, PPAR, and cAMP signaling and influenced cholesterol, vitamin B6, and fat metabolism; 9 candidate genes were identified by comparing QTLs related to milk production, reproduction, and disease resistance traits. Additionally, 303 metabolites in 22 categories were detected, in which 19, 22, and 32 metabolites displayed differential abundance in the early versus peak, early versus late, and peak versus late comparison groups, respectively. Such differentially abundant metabolites (DAM) were associated with fatty acid biosynthesis of unsaturated fatty acids; valine, leucine, and isoleucine; and degradation. Pearson correlation analysis between DEG and DAM identified significant associations (|r value| > 0.6; P-value <0.05); all DEG-DAM pairs were visualized in a 9-quadrant plot. Six fatty acids-caprylic acid, heptadecanoic acid, methyl hexadecanoic acid, oleic acid, palmitic acid, and stearic acid-showed significant associations with DEG across all pairwise comparisons. Subsequently, overlapping regions between DEG and QTLs were identified, revealing 21 DEG that co-localized with milk production-related QTLs.