Transcriptomic analysis reveals metabolic changes in the liver, subcutaneous adipose, and mammary gland tissues during the process of conjugated linoleic acid–induced milk fat depression in dairy cows

Conjugated linoleic acid, a long-chain fatty acid, reduces milk fat content in dairy cows, which may alleviate the negative energy balance during the early lactation period by reducing lactation energy output and thereby decreasing the occurrence of ketosis and fatty liver due to a negative energy balance. Although research has revealed the milk fat inhibition mechanism, the mechanisms underlying the metabolic interactions among the liver, subcutaneous fat, and mammary gland tissues during this period remain unclear. In this study, the molecular mechanisms underlying CLA-induced milk fat depression in the liver, subcutaneous white adipose tissue, and mammary gland tissues of dairy cows were investigated. Chinese Holstein dairy cows at 30 d postpartum were fed a control diet (6 cows) or a CLA-supplemented diet (6 cows) for 7 d, and all cows were fed the control diet only for another 7 d. Tissue samples from the liver, subcutaneous white adipose tissue, and mammary gland were collected on d 0 (before CLA supplementation) and d 7 (after supplementation) for RNA sequencing analysis. In addition, blood samples were collected from control and CLA-treated cows on d 0, 4, 7, and 14 (post-treatment) to measure the concentrations of nonesterified fatty acids (NEFA) and β-hydroxybutyric acid (BHBA). Furthermore, from d 0 to 14, milk composition and milk yield were measured. The results of lactation performance indicated that supplementation with CLA led to a significant reduction in milk fat percentage, decreasing from 4.0% to 3.5%, and no other changes in the lactation performance of the CLA group were observed. With respect to the indicators of excessive fat mobilization and circulating ketone bodies, the CLA group presented significantly lower serum NEFA concentrations than did the control group, with levels decreasing from 0.70 to 0.55 mmol/L between one day before CLA supplementation (d 0) and d 7 of CLA supplementation (d 7), whereas the BHBA concentration tended to decrease over this period. Transcriptomic analysis of mammary tissue identified 674 differentially expressed genes (DEG). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the downregulated DEG in the CLA group were involved mainly in lipid biosynthetic processes, fatty acid biosynthesis, the p53 signaling pathway, and steroid biosynthesis. Transcriptomic analysis of subcutaneous white adipose tissue revealed 614 DEG between the CLA and control groups. These DEG were significantly enriched in pathways related to fatty acid metabolism, fatty acid transport, fatty acid β-oxidation, lipid metabolism, the peroxisome proliferator-activated receptor (PPAR) signaling pathway, and AMP-activated protein kinase (AMPK) signaling pathway. Transcriptomic analysis of liver tissue identified 479 DEG, which were enriched in pathways related to fatty acid metabolism, the PPAR signaling pathway, lipid transport activity, the AMPK signaling pathway, the phosphoinositide 3-kinase-protein kinase B (PI3K-AKT) signaling pathway, the insulin signaling pathway, and the insulin resistance pathway. Collectively, these findings indicate that CLA successfully induced milk fat depression in postpartum dairy cows. Based on the up- and downregulation of genes within fatty acid metabolism, fatty acid β-oxidation, and lipid metabolism pathways in adipose tissue, CLA may promote lipid synthesis while inhibiting fatty acid oxidation and lipolysis in subcutaneous white adipose tissue. Additionally, gene expression patterns related to lipid transport activity, fatty acid metabolism, PPAR, and AMPK pathways in liver tissue suggest that CLA may increase lipid transport capacity while reducing hepatic lipid accumulation. These findings provide theoretical evidence in support of the regulation of energy balance in early-lactating cows through the inhibition of milk fat synthesis by CLA, which may be used as a strategy for preventing type I ketosis.