Madin-Darby Bovine Kidney cells cultured with 150 mM of Wy-14 643 (WY, PPARa agonist) or twelve long-chain fatty acids (LCFA; 16 : 0, 18 : 0, cis-9 -18 : 1, trans-10-18 : 1, trans-11-18 : 1, 18 : 2n-6, 18 : 3n-3, cis-9, trans-11 -18 : 2, trans-10, cis-12-18 : 2, 20 : 0, 20 : 5n-3 and 22 : 6n-3) were used to uncover PPAR-a target genes and determine the effects of LCFA on expression of thirty genes with key functions in lipid metabolism and inflammation. Among fifteen known PPAR-a targets in non-ruminants, ten had greater expression with WY, suggesting that they are bovine PPAR-a targets. The expression of SPP1 and LPIN3 was increased by WY, with no evidence of a similar effect in the published literature, suggesting that both represent bovine-specific PPAR-a targets. We observed the strongest effect on the expression of PPAR-a targets with 16 : 0, 18 : 0 and 20 : 5n-3.When considering the overall effect on expression of the thirty selected genes 20 : 5n-3, 16 : 0 and 18 : 0 had the greatest effect followed by 20 : 0 and c9t11-18 : 2. Gene network analysis indicated an overall increase in lipid metabolism by WY and all LCFA with a central role of PPAR-a but also additional putative transcription factors. A greater increase in the expression of inflammatory genes was observed with 16 : 0 and 18 : 0. Among LCFA, 20 : 5n-3, 16 : 0 and 18 : 0 were the most potent PPAR-a agonists. They also affected the expression of non-PPAR-a targets, eliciting an overall increase in the expression of genes related to lipid metabolism, signalling and inflammatory response. Data appear to highlight a teleological evolutionary adaptation of PPAR in ruminants to cope with the greater availability of saturated rather than unsaturated LCFA.

Dietary lipid supplements affect mammary lipid metabolism partly through changes in lipogenic gene expression. Quantitative PCR (qPCR) is a sensitive, reliable, and accurate technique for gene expression analysis. However, variation introduced in qPCR data by analytical or technical errors needs to be accounted for via normalization using appropriate internal control genes (ICG). Objectives were to mine individual bovine mammary microarray data on >13,000 genes across 66 cows from 2 independent studies to identify the most suitable ICG for qPCR normalization. In addition to unsupplemented control diets, cows were fed saturated or unsaturated lipids for 21 d or were infused with supplements (butterfat, conjugated linoleic acid mixture, long-chain fatty acids) into the abomasum to modify milk fat synthesis and fatty acid profiles. We identified 49 genes that did not vary in expression across the 66 samples. Subsequent gene network analysis revealed that 22 of those genes were not co-regulated. Among those COPS7A, CORO1B, DNAJC19, EIF3K, EMD, GOLGA5, MTG1, UXT, MRPL39, GPR175, and MARVELD1 (sample/reference expression ratio = 1 +/- 0.1) were selected for PCR analysis upon verification of goodness of BLAT/BLAST sequence and primer design. Relative expression of B2M, GAPDH, and ACTB, previously used as ICG in bovine mammary tissue, was highly variable (0.9 +/- 0.6) across studies. Gene stability analysis via geNorm software uncovered MRPL39, GPR175, UXT, and EIF3K as having the most stable expression ratio and, thus, suitable as ICG. Analysis also indicated that use of 3 ICG was most appropriate for calculating a normalization factor. Overall, the geometric average of MRPL39, UXT, and EIF3K is ideal for normalization of mammary qPCR data in studies involving lipid supplementation of dairy cows. These novel ICG could be used for normalization in similar studies as alternatives to the less-reliable ACTB, GAPDH, or B2M.

Long-term mammary expression patterns of lipogenic gene networks due to dietary lipid remain largely unknown. Mammary tissue was biopsied for transcript profiling of 29 genes at 0, 7, and 21 days of feeding cows saturated lipid (EB100) or a blend of fish/soybean oil (FSO) to depress milk fat. Milk fat yield decreased gradually with FSO and coincided with lower molar yield of fatty acids synthesized de novo, stearic acid, and oleic acid. The PPARγ targets LPIN1 and SREBF1 along with ACSS2, ACACA, FASN, and LPL increased by day 7 of feeding EB100, but differences between diets disappeared by day 21. Expression of SCAP increased markedly over time with FSO and differed from EB100 by approximately sevenfold on day 21. Expression of THRSP decreased by day 7 with both diets and returned to basal levels by day 21. SCD expression increased linearly through 7 days and remained elevated with both diets, a likely mechanism to ensure the proper level of endogenous oleic acid via desaturation of dietary stearate (EB100) or via more SCD protein to account for the reduction in stearate supply from the rumen (FSO). Despite this response, endogenous oleate was insufficient to restore normal milk fat synthesis. Only 2 of 29 genes differed in expression between diets on day 21, suggesting that transcriptional control mechanisms regulating fat synthesis were established as early as 7 days post-feeding. Gene expression reflected vastly different physiological responses by mammary tissue to adjust its metabolism to the influx of saturated fatty acids, trans10-18:1, and/or to the lack of stearic acid.