Research on the mechanisms that regulate milk fat synthesis in dairy cows is essential to identify potential molecular targets that in the long term can help develop appropriate molecular breeding programs. Although some studies have revealed that microRNA (miRNA) affect lipid metabolism by targeting specific genes, joint analysis of miRNA and target mRNA data from bovine mammary tissue has revealed few clues regarding the underlying mechanisms controlling milk fat synthesis. The objective of the present study was to use high-throughput sequencing and bioinformatics analysis to identify miRNA and mRNA pairs and explore further their potential roles in regulating milk fat synthesis. A total of 233 pairs of negatively associated miRNA and mRNA pairs were detected. Among those, there were 162 pairs in which the miRNAs were down-regulated and the target mRNAs were up-regulated. Among the identified miRNA, miR-106b can bind the 3′-UTR of the ATP binding cassette subfamily A member 1 (ABCA1), a gene previously identified as having a positive association with bovine milk fat synthesis. The overexpression of miR-106b in bovine mammary epithelial cells caused a decrease in triglyceride and cholesterol content while the inhibition of miR-106b increased triglyceride and cholesterol content, confirming its role in lipid metabolism. The present study allowed for the construction of a miR-106b-ABCA1 regulatory network map, thus providing a theoretical basis to target this gene in the molecular breeding of dairy cows.

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.

This study determined the associations of FADS2 c.1571G>A with milk FAs content and revealed that cows with the GG genotype had improved levels of delta-6 desaturase substrates (linoleic acid, C18:2n-6; p < 0.001) and decreased levels of desaturase products (gamma-linolenic acid, C18:3n-6; p < 0.001), indicating a reduction in FADS2 expression or delta-6 desaturase activity caused by this polymorphism. Computer alignment demonstrated that c.1571G>A occurred within a potential miR-744 binding site. When the c.1571G allele was present, the luciferase activity of reporter constructs was significantly suppressed by miR-744, while no such effect was observed with the A allele. Overexpression of miR-744 in bovine mammary epithelial cells (with the 1571GG genotype) downregulated FADS2 expression at both mRNA and protein levels. In contrast, inhibition of endogenous miR-744 with a specific inhibitor dramatically upregulated FADS2 expression. Taken together, these lines of evidence indicated that the c.1571A minor allele abolished the ability of miR-744 to bind FADS2, with a consequent increase in FADS2 expression levels and synthesis of omega-6 LC-PUFAs.

The objective of this research was to explore the effect of metformin on the lipoteichoic acid (LTA)–induced mastitis model using isolated primary bovine mammary epithelial cells (PBMECs). The PBMECs were exposed to either 3 mM metformin for 12 h as a metformin group (MET) or 100 μg/mL LTA for 6 h as LTA group (LTA). Cells pretreated with 3 mM metformin for 12 h followed by washing and 100 μg/mL LTA exposure for 6 h served as the MET + LTA group. Phosphate-buffered saline was added to cells as the control group. PBMECs pretreated with different metformin doses were analyzed by a flow cytometry (annexin V–fluorescein isothiocyanate assay) to detect the cell apoptotic rate. We performed quantitative reverse transcriptase–polymerase chain reaction and Western blot analysis to evaluate the inflammatory and oxidative responses to metformin and LTA by measuring cellular cytotoxicity, mRNA expression, and protein expression. Immunofluorescence was used to evaluate nuclear localization. The results showed that the gene expression of COX2, IL-1β, and IL-6 significantly increased in the cells challenged with LTA doses compared to control cells. In inflammatory PBMECs, metformin attenuated LTA-induced expression of inflammatory genes nuclear factor κB (NF-κB) p65, tumor necrosis factor α, cyclooxygenase 2, and interleukin 1β, as well as the nuclear localization and phosphorylation of NF-κBp65 protein, but increased the transcription of nuclear factor erythroid 2–related factor 2 (Nrf2) and Nrf2-targeted antioxidative genes heme oxygenase-1 (HO-1) and Gpx1, as well as the nuclear localization of HO-1 protein. Importantly, metformin-induced activation of Nrf2 is AMP-activated protein kinase (AMPK)–dependent; as metformin-pretreated PBMECs activated AMPK signaling via the upregulation of phosphorylated AMPK levels, cell pretreatment with metformin also reversed the translocation of Nrf2 that was LTA inhibited. This convergence between AMPK and Nrf2 pathways is essential for the anti-inflammatory effect of metformin in LTA-stimulated PBMECs. Altogether, our results indicate that metformin exerts anti-inflammation and oxidative stress through regulation of AMPK/Nrf2/NF-κB signaling pathway, which highlights the role of AMPK as a potential therapeutic strategy for treatment of bovine mastitis.