Effect of Interaction Between Vitamin A and Acetic Acid on the Expression of Genes Related to Milk Composition Synthesis in Bovine Mammary Epithelial Cells

Our previous research found that vitamin A (VA) has a significant effect on promoting the expression of genes related to milk fat and milk protein synthesis in bovine mammary epithelial cells (BMECs). Based on this, this experiment was conducted by adding acetic acid (AA) to explore whether there is an interaction between VA and AA in the expression of genes related to milk composition synthesis, then gaining a more systematic understanding of the regulatory mechanism of VA involvement in milk fat and protein synthesis, this study would provide scientific basis for the rational addition of VA to feed for dairy cows and the improvement of milk quality.

In this experiment, cells were cultured using digestive method of collagenase. Two factor completely randomized trial design was used in this study. The third generation BMECs was randomly divided into 6 treatment groups, with 6 replicates in each group. After starvation treatment of serum-free medium for 24 hours, medium with VA and AA at different concentrations was added, respectively. The six treatment groups were sequentially named as control group (0 mg·mL^-1VA+0 mol·L^-1 AA), AA treatment group 1 (0 mg·mL^-1VA+0.006 mol·L^-1 AA), AA treatment group 2(0 mg·mL^-1VA+0.01 mol·L^-1 AA), VA treatment group(0.001 mg·mL^-1VA+0 mol·L^-1 AA), VAAA1 group(0.001 mg·mL^-1VA+0.006 mol·L^-1 AA), and VAAA2 group(0.001 mg·mL^-1VA+0.01 mol·L^-1 AA). After 24 hours of continuous culture, the cells and culture medium were collected as required, and the cell proliferation rate was measured using MTT method; the content of triglycerides and the activities of key enzymes in milk fat and protein synthesis were measured using kits. The relative expression of genes related to milk fat and milk protein synthesis was measured and calculated by real-time quantitative PCR.

The results showed that adding VA significantly increased the cell proliferation rate (P<0.001), but no significant effect on the cell proliferation rate was found followed by addition of AA or VA and AA (P>0.05). There was an interaction effect between VA and AA on milk fat synthesis. AA increased the triglyceride (TG) content in BMECs (P=0.01), but the addition of VA inhibited TG synthesis in the meantime (P=0.01). AA up-regulated the activities of fatty acid synthase (FASN) and acetyl CoA carboxylase (ACACA) as well as peroxisome proliferator-activated receptor γ (PPARG) gene expression in BMECs (P<0.05; P<0.05; P<0.001), while significant inhibitory effect of VA was observed (P<0.001; P=0.01; P<0.01). AA significantly increased the content of mammalian rapamycin target protein (mTOR) (P=0.05), ribosome S6 protein kinase (S6K1) enzyme activity (P<0.001), and the gene expression of CSN3 in BMECs (P=0.02). VA and AA had an interaction effect on the gene expression of mTOR and CSN2, because the simultaneous addition of VA and AA attenuated the down-regulatory effect on mTOR and CSN2 gene expression after addition of VA or AA alone.

To sum up, AA treatment group 2 had a better promoting effect on milk fat synthesis, while VAAA2 group had a weaker effect; AA treatment group 1 had a better promoting effect on milk protein synthesis, while VAAA1 group had a weaker effect. Therefore, 0.01 mol·L^-1 of AA alone is better for promoting milk fat synthesis, while 0.006 mol·L^-1 of AA is more conducive to milk protein synthesis.