Abstract
The concentration of bovine milk fat changes regularly with lactation stages. In particular, milk fat percentage is higher in late lactation than mid lactation. Furthermore, milk fat composition is highly subject to a few genes. Thus, transcriptome sequencing was performed to explore the expression patterns of differentially-expressed genes (DEGs) in the parenchymal mammary gland of Holstein dairy cows between mid and late lactation. The 725 DEGs were screened (fold change > 2 and p-value < 0.05), and the peroxisome proliferator-activated receptor (PPAR) signaling pathway associated with lipid synthesis had a significant variation between the two periods (p-value < 0.05). The activation of the PPAR signal pathway may a key factor in the increasing of milk fat content in late lactation compared to mid lactation. Acyl-CoA synthetase long-chain family member 4 (ACSL4), a member of the PPAR signaling pathway, was upregulated in late lactation compared to mid lactation (p < 0.05). ACSL4 catalyzes the activation of long-chain fatty acids for cellular lipid synthesis. However, it remains uncertain that the molecular mechanism of milk fat synthesis is regulated by ACSL4 in dairy cows. Subsequently, the function verification of ACSL4 was performed in bovine mammary epithelial cells (BMECs). The upregulated expression of ACSL4 was accompanied by the increase of the concentration of intracellular triglycerides, whereas knockdown of ACSL4 decreased the concentration of intracellular triglycerides, which demonstrated that ACSL4 plays an important role in modulating milk fat synthesis. In conclusion, the results displayed that ACSL4 expression regulates triglyceride metabolism in ruminant mammary cells.
Highlights
Bovine milk is consumed globally by reason of its rich nutrients such as protein, fat, carbohydrate, and mineral contents [1].The milk fat concentration changes regularly with different lactation stages.In particular, milk fat percentage is higher in late lactation than in mid lactation [2]
Libraries were established using the RNA extracted from the Holstein dairy cow mammary glands six cDNA libraries were established using the RNA extracted from the Holstein dairy cow mammary during mid and late lactation, generating approximately 72.61GB and 72.21GB raw reads for each glands during mid and late lactation, generating approximately 72.61GB and 72.21GB raw reads for library, respectively (Table S6)
Acyl-CoA synthetase long-chain family member 4 (ACSL4) was overexpressed and knocked down (Figure pcDNA3.1-ACSL4 was significantly higher than that in the bovine mammary epithelial cells (BMECs) transfected with the empty vector (p < 0.05) and ACSL4 expression levels in the BMECs transfected with siRNA was significantly lower than that in the BMECs transfected with siRNA negative control
Summary
Bovine milk is consumed globally by reason of its rich nutrients such as protein, fat, carbohydrate, and mineral contents [1].The milk fat concentration changes regularly with different lactation stages. Previous studies of milk fat synthesis have focused on gene function verification or transcriptome analysis to screen milk fat synthesis-related genes in mammary glands [3,4,9]. All fatty acids synthesize triglycerides (TG) through activation, synthesis, transport, and other enzymatic actions, subsequently, TG are released from cells to form milk fat with the help of lipoproteins [11,12,13]. In milk-fat synthesis, the key step is the fatty acid activation via the catalytic action of acyl-CoA synthetase (ACS), a key milk fat synthesis enzyme [15]. The role of the ACSL family in fat synthesis has been reported gradually, but the molecular mechanism of the acyl-CoA synthetase long-chain family member 4 (ACSL4) regulating milk fat synthesis remains uncertain. The aim was to analyze the transcriptome profiles in the Holstein dairy cow mammary gland during mid and late lactation and explore the effect of ACSL4 on triglyceride accumulation in bovine mammary epithelial cells (BMECs)
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