Effect of short-term feed restriction on temporal changes in milk components and mammary lipogenic gene expression in mid-lactation Holstein dairy cows.

Investigations of the temporal changes in mammary gene expression that occur during sudden diet change have been limited by the use of mammary tissue as the source of RNA because of the invasive nature of mammary biopsy procedures. However, the cytosolic crescent, present in 1% of the largest milk fat globules, contains mammary epithelial cell RNA that has become trapped between the inner and outer milk fat globule membranes during final formation and secretion of milk fat into the lumen of the mammary alveoli. We hypothesized that cytosolic crescent RNA extracted from milk fat could be used as an alternative source of mammary epithelial cell RNA to measure the immediate temporal changes in gene expression as a result of changes in diet. In this experiment, feed restriction was used to mimic the state of negative energy balance observed in early lactation and induce a rapid change in milk fat yield and lipogenic gene expression. Ten multiparous Holstein dairy were fed a basal diet ad libitum during a 14-d preliminary period followed by a 4-d experimental period where 5 cows remained on ad libitum feeding and 5 cows were fed at 60% of their d 8-14 intakes (restricted) on d 15 to 18 and then returned to ad libitum feeding on d 19 to 21. Milk samples were collected from each milking on d 13 to 20 and the milk fat was immediately isolated, mixed with Trizol LS, and stored at -80°C for subsequent extraction of RNA that was used for measurement of gene expression. Feed restriction tended to increase milk fat percentage. However, total milk and milk fat production were reduced by 21 and 18%, respectively. Consistent with increased use of body fat for milk synthesis, serum nonesterified fatty acids increased 6-fold (0.78 mEq/L in the feed restriction vs. 0.13 mEq/L ad libitum group), whereas the milk fatty acids

Inhibition of stearoyl-CoA desaturase 1 reduces lipogenesis in primary bovine adipocytes.

The objectives were to determine the effect of stearoyl-CoA desaturase (SCD1) inhibition on adipocyte proliferation, differentiation and cellular lipid metabolism in bovine primary adipocytes. Inhibition of SCD1 activity by sterculic acid (SA) or conjugated linoleic acid, trans-10 cis-12 isomer, (t10, c12-CLA) did not alter adipocyte cellular proliferation, viability or differentiation. In 1,2-[(13)C]-acetate supplemented cells, the mass isotopomer distribution analysis showed that the fractional synthesis rate of [(13)C]-16:0 was reduced (P < 0.01) in SA and t10, c12-CLA treatments compared to control. Of the lipogenic genes, t10, c12-CLA treatment decreased (P < 0.05) the expression of SCD1, acetyl-CoA carboxylase (ACC), fatty acid synthase; whereas SA supplementation decreased (P < 0.05) the expression of ACC. Both SA and t10, c12-CLA increased (P < 0.05) the expression of hormone-sensitive lipase and carnitine palmitoyl transferase involved in lipolysis and oxidation. Inhibition of SCD1 in bovine adipocytes decreases de novo fatty acid synthesis by down-regulating genes involved in lipogenesis and up-regulating genes involved in lipolysis and oxidation.

Cis-9, trans-11 conjugated linoleic acid is endogenously synthesized from palmitelaidic (C16:1 trans-9) acid in bovine adipocytes.

Palmitelaidic (C16:1 trans-9) acid has been suggested to have beneficial effects on human health, including reduced adiposity. Objectives of this research were to quantify the amounts of palmitelaidic acid in beef samples and determine the effect of palmitelaidic acid supplementation on lipogenesis in bovine preadipocytes and adipocytes in vitro. For the first objective, palmitelaidic acid content of LM samples from steers finished on forage or concentrate systems was determined. Palmitelaidic acid in LM samples from forage-finished beef ranged from 10 to 17 mg/100 g of muscle corresponding to 0.52% to 0.65% of total fatty acids. Forage species grazed during finishing, and animal age at harvest also altered palmitelaidic acid concentrations and contents in the LM of forage-finished beef. Palmitelaidic acid concentration of concentrate-finished beef was lower (P < 0.05; 0.25% vs. 0.56%); however, because of increased (P < 0.05) total fatty acid content with concentrate finishing, amount of palmitelaidic acid was similar (P > 0.05) to beef from steers finished on pearl millet and greater (P < 0.05) than those finished on alfalfa. For the second objective, undifferentiated preadipocytes and differentiated adipocytes were supplemented with 0 to 300 µM of palmitelaidic acid. Palmitelaidic acid supplementation reduced (P < 0.05) cell viability of undifferentiated preadipocytes at greater levels (150 and 300 µM) but did not affect (P > 0.05) the viability of differentiated adipocytes. In preadipocytes, palmitelaidic acid increased (P < 0.05) palmitelaidic and trans-11 vaccenic (C18:1 trans-11) acids at high levels of supplementation (300 µM). In adipocytes, palmitelaidic acid supplementation increased (P < 0.05) palmitelaidic acid, trans-11 vaccenic acid, and total fatty acid content. In addition, cis-9, trans-11 CLA also increased (P < 0.05) with palmitelaidic acid supplementation in adipocytes. These results indicate that palmitelaidic acid can be elongated in both preadipocytes and adipocytes and desaturated in adipocytes to generate trans-11 vaccenic acid and cis-9, trans-11 CLA, respectively. Beef products are a source of palmitelaidic acid in the human diet, which can be elongated and desaturated to produce trans-11 vaccenic acid and cis-9, trans-11 CLA.

Palmitoleic (16:1 cis-9) and cis-vaccenic (18:1 cis-11) acid alter lipogenesis in bovine adipocyte cultures.

Our objectives were to: (1) confirm elongation products of palmitoleic acid (16:1 cis-9) elongation in vitro using stable isotopes and (2) evaluate if exogenous supplementation of palmitoleic acid, elongation products, or both are responsible for decreased desaturation and lipogenesis rates observed with palmitoleic acid supplementation in bovine adipocytes. Stromal vascular cultures were isolated from adipose tissue of two beef carcasses, allowed to reach confluence, held for 2 days, and differentiated with a standard hormone cocktail (day 0). On day 2, secondary differentiation media containing 1 of 4 fatty acid treatments [0 µM fatty acid (control), or 150 µM palmitic (16:0), palmitoleic, or cis-vaccenic (18:1 cis-11)] was added for 4 days. On day 6, cells were incubated with [(13)C] 16:1, [(13)C] 2, or [(13)C] 18:0 to estimate elongation, lipogenic, and desaturation rates using gas chromatography-mass spectrometry. Enrichment of [(13)C] 18:1 cis-11 confirmed 18:1 cis-11 is an elongation product of 16:1. Additionally, [(13)C] label was seen in 20:1 cis-13 and cis-9, cis-11 CLA. Synthesis of [(13)C] 16:0 from [(13)C] 2 was reduced (P < 0.05) in palmitoleic acid and cis-vaccenic acid-treated compared with control cells following 36 h incubation. By 12 h of [(13)C] 18:0 incubation, cells supplemented with palmitoleic acid had reduced (P < 0.05) [(13)C] 18:1 cis-9 compared with all other treatments. Gene expression and fatty acid results support isotopic data for lipogenesis and desaturation. Therefore, palmitoleic acid is actively elongated in vitro and its elongation product, cis-vaccenic acid, can also reduce lipogenesis. However, inhibition of desaturation can be directly attributed to palmitoleic acid and not its elongation products, 18:1 cis-11 or 20:1 cis-13.

Peroxisome proliferator-activated receptor-gamma activation and long-chain fatty acids alter lipogenic gene networks in bovine mammary epithelial cells to various extents.

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.

Identification of internal control genes for quantitative polymerase chain reaction in mammary tissue of lactating cows receiving lipid supplements.

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.

Abomasal infusion of butterfat increases milk fat in lactating dairy cows.

The objective of this study was to compare the effects of abomasal infusion of butterfat containing all fatty acids (FA) present in milk, including the short- and medium-chain FA, with infusion of only the long-chain FA (LCFA) present in milk, on the FA composition and milk fat yield in lactating dairy cows. Eight rumen-fistulated Holstein cows, in early lactation (49 +/- 20 days in milk) were used in a replicated 4 x 4 Latin square design. Treatments were abomasal infusion of the following: 1) no infusion (control), 2) 400 g/d of butterfat (butterfat), 3) 245 g/d of LCFA (blend of 59% cocoa butter, 36% olive oil, and 5% palm oil) providing 50% of the 16:0 and equivalent amounts of C18 FA as found in 400 g of butterfat, and 4) 100 g/d of conjugated linoleic acid (CLA, negative control), providing 10 g of trans-10, cis-12 CLA. Fat supplements were infused in equal portions 3 times daily at 0800, 1400, and 1800 h during the last 2 wk of each 3-wk experimental period. Daily dry matter intake and milk production were unaffected by the infusion treatments. Butterfat infusion increased milk fat percentage by 14% to 4.26% and milk fat yield by 21% to 1,421 g/d compared with controls (3.74% and 1,178 g/d). Milk fat percentage and fat yield were decreased by 43% by CLA. Milk protein percentage was higher (3.70%) in CLA-infused cows than in control (3.30%), butterfat (3.28%), or LCFA (3.27%) treatments. Although LCFA had no effect on fat synthesis, abomasal infusion of butterfat increased milk fat percentage and yield, suggesting that the availability of short- and medium-chain FA may be a limiting factor for milk fat synthesis.

Principal component and multivariate analysis of milk long-chain fatty acid composition during diet-induced milk fat depression.

The objective of this study was to assess the relationship between individual milk fatty acids (FA) and diet-induced milk fat depression (MFD) using principal component analysis (PCA) and multivariate analysis (MA). Cow treatment observations (n = 63) from 3 published feeding experiments with lactating dairy cows were used in the analyses. In the PCA, principal component loading plots 1 (PC1) and 2 (PC2) described 55.9% of the total variation in milk FA and fat concentrations. Saturated FA (14:0, 16:0, and 17:0) and milk fat percentage showed negative loading for PC1. Trans-18:1 isomers (trans-6+7+8 to trans-15), trans-7, cis-9 conjugated linoleic acid (CLA), and trans-10, cis-12 CLA showed positive (opposite) loading, suggesting a negative relationship between these isomers and milk fat percentage. Cis-11, trans-13 CLA and cis-9, trans-11 CLA were associated with the PC2 axes (neutral), indicating that they were not associated with MFD. Multivariate analysis with milk fat percentage as the dependent variable and individual PC1 positive loading variables showed a breakpoint relationship for trans-6+7+8-, trans-9-, trans-10-, and trans-13+14-18:1 and a linear relationship for trans-11-, trans-12-, trans-15-18:1, trans-10, cis-12 CLA, and trans-7, cis-9 CLA. Subsequent MA was conducted on 41 treatment means from 12 independent experiments from the literature, in which concentrations of trans-6+7+8-, trans-9-, trans-10-, and trans-11-18:1, and cis-9 trans;-11, and trans-10, cis-12 CLA were reported. Significant negative effects of trans-9-18:1, trans-10-18:1, and trans-10, cis-12 CLA on milk fat percentage were observed. In this study, the PCA and MA showed that among trans-18:1 isomers, trans-10-18:1 was the most negatively correlated to milk fat percentage. However, the threshold concentration related to maximum MFD indicated that the relative potency was greatest for trans-6+7+8- and lowest for trans-10-18:1. These results suggested that trans-6+7+8-18:1 might be more important than trans-10-18:1 in MFD. Principal component analysis also showed that trans-10, cis-12 and trans-7, cis-9 CLA were the isomers most negatively correlated to milk fat percentage, implying a possible role of trans-7, cis-9 CLA in MFD. Additional experiments are needed to establish whether trans-7-18:1 is involved in MFD or that its effects are mediated via the endogenously synthesized trans-7, cis-9 CLA.