Conventional Culture Conditions Limit the Ability of Bovine Mammary Epithelial Cells to Produce Milk Fat.

Bovine milk fat is distinguished by a high proportion of short and medium chain fatty acids, a profile that mammary epithelial cells (MEC) fail to reproduce in vitro. When considering culturing MEC as a source for alternative dairy products, this failure limits the ability of replicating the functionality of milk fat. Conventional media used to grow the cells is designed to mimic monogastric animal plasma, disregarding the profound metabolic differences compared with ruminant plasma. Therefore, we hypothesized that the inability to reproduce milk fat composition in vitro reflects substrate composition rather than an intrinsic limitation of the cells. To test this, we gradually modulated MEC media composition towards a ruminant-like substrate balance, lowering glucose and supplementing acetate. Pathway analysis revealed that high glucose media enhanced TCA cycle without activating de novo lipogenesis, while acetate increased lipid accumulation in both glucose backgrounds. Caprylic acid (C8:0), a medium-chain fatty acid characteristic of bovine milk fat, increased with acetate supplementation and peaked under low glucose media with acetate, consistent with acetate increasing lower molecular weight triglycerides. These results indicate that the limited capacity of MEC cultures to generate milk-like fatty acid profiles is substantially influenced by the substrate environment, and that a ruminant-like shift in substrate availability partially, but not completely, redirects fatty acid output towards the milk fat profile.