ABSTRACT:The objective of the present study was to refine a previously developed method to isolate primary 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 composition 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.

The objective of the study was to investigate selected key regulatory pathways of milk protein biosynthesis in primary bovine mammary epithelial cells (MECs) of dairy cows during the first 155 days of lactation. In addition, cows were exposed to feed restriction for a short period (FR) during different stages of lactation (week 4 and 21 pp) to study adjustment processes of molecular protein biosynthesis to metabolic challenge. Morning milk samples from twenty-four Holstein-Friesian cows were collected throughout the experimental period (n = 10 per animal). MEC from raw milk were purified using an immunomagnetic separation technique and used for real-time quantitative PCR analyses. As was seen in transcript abundances of all major milk proteins, mRNA levels of E74-like factor 5 (ELF5), an enhancer of signal transducer and activator of transcription (STAT) action, concomitantly decreased towards mid-lactation. Expression of ELF5 as well as of all milk protein genes showed a similar increase during FR in early lactation. Occasional changes in expression could be seen in other Janus kinase (JAK)/STAT factors and in mammalian target of rapamycin (mTOR) pathway elements. Amino acid transfer and glucose transporter and the β-casein expression were also partially affected. In conclusion, our findings suggest a pivotal role of the transcription factor ELF5 in milk protein mRNA expression with complementary JAK/STAT and mTOR signalling for the regulation of protein biosynthesis in the bovine mammary gland.

BackgroundCortisol is secreted into blood in reaction to acute stress, but also in phases of diminished feed intake and changed animal behavior. As cows do not always show clear signs of discomfort, reliable diagnostic markers could be used to provide information regarding individual cows’ distress. The objective of this study was to establish an ether free immunoassay for the detection of cortisol and to determine values during the first 22 weeks of lactation. Furthermore, the response in milk cortisol levels was assessed during times of metabolic stress and pain associated symptoms of lameness.MethodsMilk yield and composition, blood serum glucose, NEFA and BHBA as well as milk cortisol were determined in 24 multiparous Holstein-Friesian cows over the course of the first 22 weeks of lactation. Animals were further checked for signs of clinical diseases on a daily basis. Two feed restrictions over three days (FR; 70 % of precious ad libitum intake) were performed during the 4th wk and the 21st wk, respectively. An ELISA for cortisol measurement in easily accessible bovine skimmed milk was established and applied.ResultsOn the last day of FR in early lactation, a reduction in milk yield and changes in serum metabolites compared to respective previous values were detected. The FR in mid-lactation resulted in no changes in milk production and serum metabolites. Milk cortisol was highest during first wk of lactation and remained on comparable levels thereafter. Milk yield and composition were not influenced by FR. Lameness resulted in enhanced milk cortisol levels.ConclusionMilk cortisol could be used as an indicator of painful symptoms such as lameness. Higher values of milk cortisol levels during first wk of lactation should be taken into account for interpretation.

BackgroundConjugated linoleic acid (CLA) is a collective term for isomers of octadecadienoic acid with conjugated double-bond system. Thus, it was the objective to investigate whether milk composition and metabolic key parameters are affected by adding CLA to the diet of dairy cows in the first four weeks of lactation.MethodsA study was carried out with five primiparous cows fed a CLA supplemented diet compared to five primiparous cows without CLA supplementation. CLA supplemented cows received 7.5 g CLA/day (i.e. 50% cis(c)9,trans(t)11- and 50% t10,c12-CLA) starting two weeks before expected calving and 20 g CLA/day (i.e. 50% c9,t11- and 50% t10,c12-CLA) throughout day 1 to 28 of lactation.ResultsThe CLA supplement was insufficiently accepted by the animals: only 61.5% of the intended amount was ingested. Fed CLA were detectable in milk fat, whereas contents of c9,t11-CLA and t10,c12-CLA in milk fat were higher for CLA supplemented cows compared to the control group. On average over the entire treatment period, there was a decrease of saturated fatty acids (FA) in milk fat of CLA supplemented cows, combined with a higher content of monounsaturated and trans FA.Our study revealed no significant effects of c9,t11- and t10,c12-CLA supplementation either on milk yield and composition or on metabolic key parameters in blood. Furthermore the experiment did not indicate significant effects of c9,t11- and t10,c12-CLA-supplementation on gene expression of peroxisome proliferator-activated receptor-alpha (PPARα), PPARγ, sterol regulatory element-binding protein-1 and tumor necrosis factor-alpha in liver tissue.ConclusionsFeeding c9,t11- and t10,c12-CLA during the first weeks after calving did not affect metabolic key parameters of blood serum or milk composition of fresh cows. Milk fatty acid composition was changed by feeding c9,t11- and t10,c12-CLA resulting in higher contents of these isomers in milk fat. High contents of long chain FA in milk fat indicate that CLA supplementation during the first four weeks of lactation did not affect massive peripheral lipomobilization.

The objective of this experiment was to study milk productivity, metabolic adaptation and effect of a short-term feed restriction (FR) on key performance indicators during early lactation in cows classified according to energy-corrected milk (ECM) yield and milk protein concentration. Twenty-three multiparous Holstein-Friesian cows were categorized in four groups according to respective averaged values on Days 23-25 postpartum: high ECM yield and high protein concentration; low ECM yield and low protein concentration; high ECM yield and low protein concentration and low ECM yield and high protein concentration. Dry matter intake was reduced to 68.3% for three subsequent days. Our results showed that short-time FR in early lactation succeeded in enhancing energy deficit of cows in all groups. Milk fat, milk protein and lactose concentrations as well as milk fat yield were not influenced by FR. Several hepatic genes encoding for enzymes involved in catabolism of amino acids, β-oxidation, gluconeogenesis and ketogenesis as well as mRNA encoding for insulin receptor showed increased transcript abundances after FR, primarily in cows with high milk yield and low milk protein concentration.