The objective of this study was to evaluate the effects of the different ratios of unsaturated fatty acids (UFAs) (oleic acid, linoleic acid, and linolenic acid) on the cell viability and triacylglycerol (TAG) content, as well as the mRNA expression of the genes related to lipid and protein synthesis in bovine mammary epithelial cells (BMECs). Primary cells were isolated from the mammary glands of Holstein dairy cows and were passaged twice. Afterward, the cells were randomly allocated to six treatments, five UFA-treated groups, and one control group. For all of the treatments, the the fetal bovine serum in the culture solution was replaced with fatty acid-free BSA (1 g/L), and the cells were treated with different ratios of oleic, linoleic, and linolenic acids (0.75:4:1, 1.5:10:1, 2:13.3:1, 3:20:1, and 4:26.7:1) for 48 h, which were group 1 to group 5. The control culture solution contained only fatty acid-free BSA without UFAs (0 μM). The results indicated that the cell viability was not affected by adding different ratios of UFAs, but the accumulation of TAG was significantly influenced by supplementing with different ratios of UFAs. Adding different ratios of UFAs suppressed the expression of ACACA and FASN but had the opposite effect on the abundances of FABP3 and CD36 mRNA. The expression levels of PPARG, SPEBF1, CSN1S1, and CSN3 mRNA in the BMECs were affected significantly after adding different ratios of UFAs. Our results suggested that groups 1, 2, and 3 (0.75:4:1, 1.5:10:1, and 2:13.3:1) had stronger auxo-action on fat synthesis in the BMECs, where group 3 (2:13.3:1) was the best, followed by group 4 (3:20:1). However, group 5 (4:26.7:1) was the worst. Genes related to protein synthesis in the BMECs were better promoted in groups 2 and 3, and group 3 had the strongest auxo-action, whereas the present study only partly examined the regulation of protein synthesis at the transcriptional level; more studies on translation level are needed in the future. Therefore, when combining fat and protein synthesis, group 3 could be obviously fat and protein synthesis in the BMECs concurrently. However, further studies are necessary to elucidate the mechanism for regulating fat and protein synthesis in the BMECs.

ABSTRACT:The objective of this study was to evaluate the effects of the different ratios of acetate and β-hydroxybutyrate (BHBA) on cell viability, triacylglycerol (TAG) content, and mRNA expression of the genes related to lipid and protein synthesis in bovine mammary epithelial cells (BMECs). Primary cells were isolated from the mammary glands of Holstein dairy cows and were passaged twice. Then, the cells were cultured with different ratios of acetate and BHBA (1 : 3, 1 : 2, 1 : 1, 2 : 1, 3 : 1, 4 : 1, and 1 : 1, Group 1 to Group 7, respectively) for 48 h, and the fetal bovine serum in the culture media was replaced with fatty acid-free bovine serum albumin (BSA) (1 g/l). The control culture media contained only fatty acid-free BSA without unsaturated fatty acids (0mM). Cell viability was not affected by adding different ratios of acetate and BHBA, but TAG accumulation was significantly influenced by supplementing the culture media with different ratios of acetate and BHBA. The expression levels of genes related to milk fat (FASN, ACACA, CD36, SCD, FABP3, LPL, PPARG, and SPEBF1) and milk protein-related genes (CSN1S1, CSN3, mTOR, 4E-BP1, S6KB1, STAT5, JAK2, and LEPTIN) were significantly affected by the addition of different ratios of acetate and BHBA to the BMECs. Our results suggested that Groups 3 and 4 (1 : 1 and 2 : 1) had a stronger acceleration of milk fat synthesis, and Group 4 (2 : 1) had the strongest effect. The expression of the CSN1S1 and LEPTIN mRNAs was more effectively promoted in Groups 3 and 4 (1 : 1 and 2 : 1), and Group 3 (1 : 1) had the strongest acceleration. Expressions of genes related to milk protein synthesis (mTOR, 4E-BP1, S6KB1, JAK2, and STAT5) were up-regulated using a ratio of acetate and BHBA of 2 : 1. Taken together, the 2 : 1 ratio of acetate and BHBA had the best effect for both the milk fat synthesis and milk protein synthesis genes. However, further studies are necessary to elucidate the mechanism for regulating milk fat and protein synthesis by different ratios of acetate and BHBA.Keywords: ratio of short chain fatty acids; milk fat precursor; dairy cow; milk fat; milk protein; gene expression List of abbreviations: BHBA = β-hydroxybutyrate, TAG = triacylglycerol, BMECs = bovine mammary epithelial cells, FBS = fetal bovine serum, UFA = unsaturated fatty acids, BSA = bovine serum albumin, FAs = milk fatty acids, GAPDH = glyceraldehyde 3-phosphate dehydrogenase, FASN = fatty acid synthase, ACACA = acetyl-CoA carboxylase, SCD = stearoyl-CoA desaturase, CD36 = cluster of differentiation 36, FABP3 = fatty acid-binding protein 3, LPL = lipoprotein lipase, PPARG = peroxisome proliferator-activated receptor γ, SREBF1 = sterol regulatory element binding transcription factor 1, CSN1S1 = αs1-casein, CSN3 = κ-casein, mTOR = mammalian target of rapamycin, 4EBP1 = eukaryotic translation initiation factor 4E, RPS6KB1 = ribosomal protein S6 kinase 1, STAT5 = signal transducer and activator of transcription 5, JAK2 = Janus kinase 2, LEPTIN = leptin, PBS = phosphate buffer saline, DMEM/F12 ...