Effect Of 18-Carbon Unsaturated Fatty Acids Supplementation On Milk Composition Synthesis In Two And Three-Dimensional Cultured Bovine Mammary Epithelial Cells

The aim of this study was to determine the effect of supplementation of oleic acid, linoleic acid, linolenic acid and mixed addition of these three fatty acids on milk fat and protein synthesis in bovine mammary epithelial cells, so as to find suitable supply mode of 18 carbon unsaturated fatty acids for improving milk components synthesis. This study contained two trials. In trial one the effect of supplementation of different concentration of oleic acid (0μmol/L,50μmol/L, 100μmol/L,200μmol/L,400μmol/L), linoleic acid (0μmol/L,20μmol/L,40μmol/L,80μmol/L,120μ.mol/L), linolenic acid (Oμmol/L, 0.5μmol/L, 1μmol/L,2μmol/L,4μmol/L) and different ratio of these three unsaturated fatty acids (2:13.3:1,9.6:7.4:1,17.2:1.4:1) on cell viability, triglyceride content (TAG), as well as mRNA expression of genes involved in milk fat and protein synthesis in two dimensional cultured BMECs were investigated. In trial two the effect of oleic acid, linoleic acid, linolenic acid and different ratio of these three fatty acid on intracellular TAG content, mRNA expression of genes related to milk fat and milk protein synthesis in three dimensional cultured BMECs were stuied. The results showed that:(1) 100μmol/L and 200μmol/L oleic acid significantly promoted TAG synthesis in BMECs (P<0.05). Gene expression of SCD, FABP3 and SREBF1 were significantly supressed by addition of oleic acid (P<0.05), however, DGAT2 gene expression was increased. With addition of 50μmol/L oleic acid, gene expression of STAT5, mTOR, AKT and 4EBP1 were higher than those of control group. Gene expression of CSN3, CSN1S1, STAT5, mTOR,4EBP1 and AKT were decreased by adding 200μmol/L and 400μmol/L oleic acid.(2) TAG content in BMECs was significantly higher 40μmol/L and 80μmol/L linoleic acid supplementation groups than that of control group (P<0.05). DGAT2 gene expression was increased by addition of 20μmol/L-120μmol/L linoleic acid. Gene expression of ACACA, FASN and SREBF1 were significantly decreased by addition of 20μmol/L-80μmol/L linoleic acid (P<0.05). Gene expression of CSN1S1, CSN3, STAT5 and mTOR were significantly increased by addition of 20μmol/L linoleic acid (P< 0.05).(3) Supplementation of 0.5-2μmol/L linolenic acid promoted the TAG synthesis inBMECs. ACACA and FASN gene expression were higher in 0.5μmol/L linolenic acid group than those of control group. As compared with control group, DGAT2 gene expression was significantly increased by addition of 1μmol/L linolenic acid (P<0.05). Gene expression of STAT5 and mTOR were increased by supplementation of 0.5-2μmol/L linolenic acid.(4) TAG content in BMECs, was significantly higher in 2:13.3:1 and 9.6:7.4:1 mixed addition groups than those of 17.2:1.4:1 group (P<0.05). The mRNA expression of FASN, SCD, FABP3 and SREBF1 decreased with mixed addition of three unsaturated fatty acids, however, the reverse tendency was observed for DGAT2 expression (P<0.05). Gene expression of CSN1S1, CSN3, STAT5, mTOR and 4EBP1 were significantly increased when the mixed addition ratio was 2:13.3:1 (P<0.05).(5) TAG content of oleic acid supplementation group (Oμmol/L,80μmol/L, 100μmol/L) in three-dimensional cultured BMECs was higher than that of two-dimensional cultured BMECs. The mRNA expression of ACACA, DGAT2 and PPARG in 80μmol/L and 100μmol/L oleic acid addition groups were significantly higher than those of control group (P<0.05). Gene expression of SREBF1 was increase with addition of 100μmol/L oleic (P<0.05). Gene expression of CSN1S1, CSN3, STAT5 and PRS6K1 were higher in oleic acid supplementation groups than those of control group.(6) With addition of linoleic acid (0μmol/L,40μmol/L,80μmol/L), TAG content in three-dimensional cultured BMECs was significantly higher than that of two-dimensional cultured BMECs (P<0.05). The expression of DGAT2 and PPARG in three-dimensional cultured BMECs were increased by adding 40μmol/L linoleic acid. The gene expression of CSN3, mTOR and 4EBP1 were higher in 80μmol/L linoleic acid addition group than those of control group.(7) With addition of linolenic acid (0μmol/L,0.5μmol/L and 1μmol/L), TAG content in three-dimensional cultured BMECs was higher than that of two-dimensional cultured BMECs. As compared with control group, the expression of DGAT2 and PPARG in three-dimensional cultured BMECs were significantly increased by adding 0.5μmol/L linolenic acid. In addition, the mRNA expression of STAT5, mTOR,4EBP1 and PRS6K1 were significantly higher in 0.5μmol/L and 1μmol/L linolenic acid addition groups than those of control groups (P<0.05).(8) With mixed addition of oleic acid, linoleic acid and linolenic acid, TAG content in three-dimensional cultured BMECs was significantly higher than that of the two-dimensional cultured BMECs. The expression of DGAT2, PPARG, CSN3, STAT5, mTOR and AKT were higher in mixed addition group (2:13.3:1) than those of control group (P<0.05).In summary, under two-dimensional culture models, the synthesis of milk fat was increased by adding 50～100μmol/L oleic acid, CSN3 expression was decreased with addition of 100μmol/L oleic acid.80μmol/L linoleic acid had better promoting effect on milk fat synthesis, but significantly inhibited the expression of CSN1S1 and CSN3. Milk fat and milk protein synthesis were increased by adding 0.5～1μmol/L linolenic acid, the same results were observed when the supplementation ratio of oleic acid, linoleic acid and linolenic acid was 2:13.3:1. Under three-dimensional culture model, suitable concentration for promoting milk fat and milk protein synthesis of oleic acid, linoleic acid and linolenic acids were 100μmol/L,80μmol/L and 1μmol/L respectively. The promotion effect of 18 carbon unsaturated fatty acids on milk fat and protein synthesis in three-dimensional cultured BMECs was better than that of two-dimensional cultured BMECs.