Background: The molecular events associated with regulation of milk fat synthesis in the bovine mammary gland remain largely unknown. Our objective was to study mammary tissue mRNA expression via quantitative PCR of 45 genes associated with lipid synthesis (triacylglycerol and phospholipids) and secretion from the late pre-partum/non-lactating period through the end of subsequent lactation. mRNA expression was coupled with milk fatty acid (FA) composition and calculated indexes of FA desaturation and de novo synthesis by the mammary gland.

The relationship between inflammation and general health conditions in dairy cows and the link between inflammation, liver function, and fertility are poorly understood. To clarify these relationships, 120 multiparous dairy cows were followed throughout an entire lactation. Blood samples were collected during the first month of lactation for a metabolic profile, and milk yield, disease occurrence, and fertility parameters were monitored during the entire lactation. Twenty-four cows were culled, and another 19 were excluded because they had serious problems after 30 d in milk (DIM) and before the first insemination. The remaining 77 cows were pregnant at the end of lactation and were retrospectively grouped into quartiles based on liver activity index (LAI), which is based on plasma negative acute phase proteins. Cows in the lower (LO) and intermediate lower (INLO) quartiles of LAI had more severe inflammations with high concentrations of haptoglobin (0.77 and 0.61 g/L) and globulin (42.5 and 39.0 g/L), respectively, during the first week of lactation compared with cows in the upper (UP) and intermediate upper (INUP) quartiles of LAI (haptoglobin: 0.28 and 0.45 g/L, and globulin: 34.2 and 36.9 g/L, respectively). At 7 DIM, the cows in LO and INLO had greater bilirubinemia (8.7 and 10.5 vs. 6.3 microM/L in UP) and lower blood urea (3.5 and 3.7 vs. 4.1 mM in UP). The INLO group exhibited more days open (139 vs. 93) and services per pregnancy (2.68 vs. 1.65), but lower milk yield (38.3 vs. 40.8 kg/d at 28 DIM) compared with UP. The LO group did not have a significantly lower fertility status, but presented the lowest milk yield (34.1 kg/d at 28 DIM). Our data suggest that cows with lower LAI scores had a more pronounced inflammatory status during the first month of lactation, an impairment of usual hepatic functions (e.g., bilirubin clearance), and a larger negative energy balance. The same cows had poorer performance (lower milk yield and fertility) than cows with higher LAI scores. Overall data suggest that any effort to avoid the acute phase response in the transition period would be useful for optimizing the productive and reproductive performance of high-yielding dairy cows.

Paraoxonase (PON) is a liver protein with hydrolase activity that is released into the blood stream. Paraoxonase may serve as an index of liver function because it is drastically reduced in chronic liver damage. Sixty-seven periparturient dairy cows were used to evaluate the relationship between plasma PON, health problems, inflammatory conditions, and liver function. Baseline plasma PON concentrations during the first 30 d in milk (DIM) were retrospectively used to group cows into quartiles. Metabolic profile, lipid metabolites (e.g., nonesterified fatty acids, beta-hydroxybutyrate), inflammatory indices (haptoglobin, ceruloplasmin), low and high density lipoprotein cholesterol, vitamin A, vitamin E, reactive oxygen metabolites, total antioxidants, and PON in plasma were measured 2 wk before to 8 wk after calving. Weekly milk yield, body condition score, and all health problems were recorded. After parturition (7 DIM), cows in the lower PON group had the lowest plasma concentrations of negative acute phase proteins compared with the higher PON group for retinol binding protein (23.2 +/- 2.86 vs. 36.0 +/- 2.96 microg/dL of vitamin A), albumin (31.6 +/- 0.73 vs. 33.9 +/- 0.75 g/L), total cholesterol (2.04 +/- 0.30 vs. 2.45 +/- 0.42 mmol/L), and the highest concentrations of haptoglobin (0.67 vs. 0.24 +/- 0.03 g/L; positive acute phase protein) and globulins (37.2 vs. 32.3 +/- 1.4 g/L). Plasma bilirubin was highest in the cows (10.1 vs. 6.2 +/- 0.6 micromol/L) in the lowest PON quartile. Plasma PON was negatively correlated with haptoglobin (r = -0.39) and bilirubin (r = -0.42) and positively correlated with retinol binding protein (r = 0.54), albumin (r = 0.38), and cholesterol (r = 0.55) fractions. A total of 82.3% of cows in the lower quartile and no cows in the upper quartile experienced serious inflammation. Lower quartile cows produced 28.1 +/- 10.3 kg of milk/d; whereas upper quartile cows produced 38.3 +/- 7.7 kg of milk/d during the first 30 DIM. A reduction in the ability of the liver to cope with the increased metabolic demand near parturition in dairy cows can be diagnosed using changes in baseline plasma PON.

Several long-chain fatty acids (LCFA) are natural ligands of nonruminant peroxisome proliferator-activated receptor-gamma (PPARG), which, along with its lipogenic target genes, is upregulated in bovine mammary tissue during lactation. Thus, PPARG might represent an important control point of bovine milk fat synthesis. We tested lipogenic gene network expression via quantitative PCR of 19 genes in bovine mammary epithelial cells cultured with 16:0, 18:0, cis-9 18:1, trans-10 18:1, trans-10,cis-12 18:2 [t10c12 conjugated linoleic acid (CLA)], 20:5, ethanol (control), and the PPARG agonist rosiglitazone (ROSI). Triplicate cultures were maintained for 12 h with 50 muM ROSI or 100 muM LCFA. Responses common to 16:0 and 18:0 relative to the control included significantly greater expression of INSIG1 (+298%, +92%), AGPAT6 (+137%, +169%), FABP3 (+755%, +338%), and FABP4 (+171%, 157%). These were coupled with greater intracellular lipid droplet formation and mRNA of ACSS2, LPIN1, SCD, and SREBF2 in response to 16:0, and greater DGAT1 and THRSP with 18:0. Trans-10 18:1 and t10c12 CLA reduced expression of FASN (-60%, -31%), SCD (-100%, -357%), and SREBF1 (-49%, -189%). Furthermore, t10c12 CLA downregulated ACSS2, FABP3, INSIG1, SREBF2, and THRSP expression. Expression of SREBF1 was lower with cis-9 18:1 (-140%) and 20:5 (-125%) compared with the control. This latter LCFA also decreased SCD, SREBF2, and LPL expression. No effects of LCFA or ROSI on PPARG were observed, but ROSI upregulated (+39% to +269%) expression of ACACA, FASN, LPIN1, AGPAT6, DGAT1, SREBF1, SREBF2, and INSIG1. Thus, these genes are putative PPARG target genes in bovine mammary cells. This is the first report showing a direct effect of trans-10 18:1 on bovine mammary cell lipogenic gene expression. The coordinated upregulation of lipogenic gene networks in response to ROSI and saturated LCFA offers support for PPARG activation in regulating bovine milk fat synthesis.

Achieving greater understanding of the genomic influence on milk synthesis in dairy cows represents a daunting challenge. Bovine-specific microarrays have allowed for high-throughput gene expression analysis of the mammary transcriptome. However, real-time PCR (qPCR) still represents the method of choice for accurate expression profiling of small numbers of genes and verification of key microarray relationships. This method is extremely sensitive but requires data normalization to account for analytical errors. Ideally, expression of genes used as internal controls should not be affected by specific treatments or physiological state. Mammary biopsies were collected from five cows each at -15, 1, 15, 30, 60, 120, and 240 days relative to parturition for gene expression profiling. We evaluated expression of nine genes (RPS9, ACTB, GAPD, GTP, ITGB4BP, MRPL39, RPS23, RPS15, and UXT) that could serve as internal controls in mammary tissue using qPCR. Due to gradual increases in mammary RNA concentration (mug/mg tissue) over lactation, all genes investigated experienced a dilution effect. We used pairwise comparison of expression ratios to analyze the reliability of these genes as internal controls. UXT, RPS9, and RPS15 had the most stable expression ratios across cow and time. We also assessed co-regulation among genes through network analysis. Network analysis suggested co-regulation among most of the genes examined, with MYC playing a central role. Pairwise comparison was suitable for finding appropriate internal controls in mammary gland tissue. Results showed that the geometrical average of UXT, RPS9, and RPS15 expression could be used as internal control for longitudinal mammary gene expression profiling.