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The objective of the present study was to refine a previously developed method to isolate pri- mary bovine mammary epithelial cells (pBMEC) from fresh milk. Using this method, it was tested whether the number of pBMEC and the relation of recovered pBMEC to total somatic cell count vary within the individual lactation stages. Furthermore, the expression levels of the milk protein genes during the first twenty weeks of lactation were determined by quantitative PCR method. A total number of 152 morning milk samples were obtained from twenty-four Holstein-Friesian cows during the first 20 weeks of lactation (day 8, 15, 26, 43, 57, 113, and 141 postpartum). Numbers of extracted pBMEC were consistent at all time-points (1.1 ± 0.06 to 1.4 ± 0.03 ×10 3 /ml) and an average value of RNA integrity number (RIN) was 6.3 ± 0.3. Percentage of pBMEC in relation to total milk cells (2.0 ± 0.2 to 6.7 ± 1.0%) correlated with milk yield. Expression patterns of the casein genes alpha (α) S1 , (α) S2 , beta (β), and kappa (κ) (CSN1S1, CSN1S2, CSN2, CSN3, respectively) and the whey protein genes α-lactalbumin (LALBA) and progestagen-associated endometrial protein (PAEP; known as β-lactoglobulin) were shown to be comparable, i.e. transcripts of all six milk protein genes were found to peak during the first two weeks of lactation and to decline continuously towards mid lactation. However, mRNA levels were different among genes with CSN3 showing the highest and LALBA the lowest abundance. We hypothesized that milk protein gene expression has a pivotal effect on milk protein com- position with no influence on milk protein concentration. This paper is the first to describe milk protein gene expression during lactation in pBMEC collected in milk. Future studies will be needed to understand molecular mechanisms in pBMEC including regulation of expression and translation throughout lactation.

Gene expression of six major milk proteins in primary bovine mammary epithelial cells isolated from milk during the first twenty weeks of lactation